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Gene expression in and development of trisomies of Drosophila melanogaster Devlin, Robert Harry 1984

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GENE EXPRESSION  IN  AND D E V E L O P M E N T O F  TRISOMIES  OF  DROSQPHILA MELANOGASTER  by  ROBERT B.Sc.p  HARRY  University  A THESIS  of  SUBMITTED  British  Columbia,  IN P A R T I A L  THE REQUIREMENTS  FOR  DOCTOR OF  In  DEVLIN  the  1978  FULFILLMENT  THE DEGREE  OF  PHILOSOPHY  Department of  Zoology  We  accept to  this  the  thesis  required  THE UNIVERSITY  OF  Robert  Harry  conforming  standard  BRITISH  December, (c)  as  COLUMBIA  1984 Devlin,  1984  OF  In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission.  Department of  /  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  )E-6 (3/81)  Ou^  5-\ , IS %>^T  ii  ABSTRACT  Drosophila for  an  entire  melanoqaster  individuals  chromosome  arm c a n  development.  To examine  gene  the  levels  -five  the  left  arm o f  aneuploid activity  o-f  and  duplications three  observed for  Analysis these  close  of  to  compensated  levels  compensation  expression these  of  the  gene  observations,  was  peculiar  the  X,  for  to  2R,  the  in  necessarily  loci  that  all  of  (metafemales)  some  the  variants three  appears  to  be  repressed determine  arm o f  3L  also  were  also  trisomies  is  operates  two,  compensation  in  of  in  on 3 L ,  were  the  extend  compensation  trisomies  dosage  euploid  To  whereas  in  exclusively  X trisomies  genes,  for  Compensating and  and autosomal  between  one  either  not.  autosomal  Dosage compensation may o c c u r  for  most  examined.  found  strains.  discrete:  chromosome were  the  of  expressed  For  it  if  enzyme  displayed  very or  levels  that  were  genes  of  segmental  diploid  alleles  on  in  2I_ t r i s o m i e s ,  enzyme a c t i v i t y .  is  small  revealed  pupal  located  both  levels  showed compensated in  are  examined  in  o-f  hyperploids,  genes  possessing  X-chromosomal  analagous.  been  However,  stages  in these  Elevated  observed  left  loci  mechanism t h a t  However,  genes.  X-chromosomal  suggests  trisomies  larvae  and t o  and f o r  non-compensating examined  have  Two p r o x i m a l l y l o c a t e d  dose-dependent autosomal  in  late  structural  strains.  that  loci  expression  two  electrophoretic  trisomies.  the  these  d i s t a l l y mapping  expression  This  diploid  survive to  whose  chromosome  in  were  enzymes  trisomic  all  loci  compensated. trisomies  are  not  X-chromosomal or  males  partially and  may o c c u r  by  by  females.  Ill  regulatory  controls  compensation expression complete  of  gene  LSP-la  other  expected to  be  transposed a  similar  regulation that and  some  hyperploid The  those  escape  case;  compensation by  that  to  the  gene)  in  metafemales  males  and  regulatory  to  locus  females,  had  Thus,  some in  systems  reside  this  was  the  chromosome, This  expressed  females.  appeared was  to  X-linked  operating  In  compensated  loci  in  at  be  not  metafemales.  analagous,  on  been  and  but  dosage  metafemales.  X chromosome  compensation  are  in  of  escape  It  is  possible  X—chromosomal  and r e f l e c t  a  common  form  compensation. at  which compensation  whole-arm  trisomies.  compensation  quantities  for  on of  salivary the  For  protein  post—transcriptionally  regulated.  compensation Adh gene  regulatory  the  most  mediated  fully  c o n t r o l l i n g male-female  X-chromosomal  measuring  for  was  second  trisomies  level  synthesis  an  trisomies.  the  Possibly,  The  was  transposed  by dosage  autosomal  larvae.  males,  been  between  of  diploid  which have  (per  X-chromosomal  escapes  n o r m a l l y do not  native  compensated  normally  that  from the  a  results.  loci  to  the  level  addition,  in  than  but  in  dosage  following  that  individuals  As s u c h ,  X chromosome,  appears  gene  compensation  these  compensation.  be  a  T  from male-female  by t h e  in trisomic-X  in  mechanisms  might  indicated  dosage  compensated this  as  distinct  genes  of  occured  was  investigated  RNA p r o d u c e d b y s e v e r a l  the  heat-shock  levels  appeared  regulated.  to  However,  gland polytene compensation  gene,  hsp  in  85.  be measurements  chromosomes  was  genes  by  revealed  transcriptionally  o f RNA that  Dosage c o m p e n s a t i o n on t h e existence control  of  gene  a  system that  autosomes  normally  e x p r e s s i o n by n e g a t i v e  probably  operates  reflects  in diploids  regulation.  to  the  V  TABLE  OF CONTENTS  PAGEABSTRACT  i i  T A B L E OF CONTENTS LIST  OF T A B L E S  LIST  OF F I G U R E S .  v vii «  ACKNOWLEDGEMENTS  xii  GENERAL  INTRODUCTION  CHAPTER  1.  MEASUREMENTS O F G E N E PRODUCT L E V E L S TRIS0MIC-2L LARVAE  IN  I.  •  II. III. IV.  CHAPTER  2.  II. III. IV.  3.  II. III. IV.  4.  Materials  and  Methods  19 20  Results  28  Discussion  39  IN  Introduction Materials  and  47 Methods  49  Results  60  Discussion  79  I N F L U E N C E O F T R I S O M Y ON T H E E X P R E S S I O N O F L O C I MAPPING O U T S I D E OF T H E D U P L I C A T E D R E G I O N S I.  CHAPTER  Introduction  MEASUREMENTS O F G E N E PRODUCT L E V E L S T R I S D M I C - 3 AND T R I S O M I C - X L A R V A E I.  CHAPTER  1  Introduction Materials  and  93 Methods  96  Results  101  Discussion  130  EFFECT  OF E X T E N S I V E  ANEUPLOIDY  ON  VIABILITY  vi AND P H E N O T Y P E I. II. III. IV.  CHAPTER  5.  Materials  and  •  II. III. IV.  6.  155  Results  158  Discussion..  217  IS  Introduction Materials  and  228 Methods  II. III. IV.  BIBLIOGRAPHY  231  Results  236  Discussion.....  258  REGULATION OF DOSAGE COMPENSATION METAFEMALES I.  151  Methods..  COMPENSATION IN 2 L T R I S O M I E S TRANSCRIPTIONALLY REGULATED I.  CHAPTER  Introduction  Introduction Materials  and  IN  265 Methods  267  Results  271  Discussion  288  293  vii  LIST  QF T A B L E S  TABLE  PAGE  1.  Biochemical  loci  examined  in  this  2.  Enzyme l e v e l s f o r c o m p e n s a t i n g trisomic-2L larvae.  study  loci  in  27 diploid  and 30  3.  Enzyme l e v e l s f o r n o n c o m p e n s a t i n g trisomic-2L larvae  4.  Enzyme l e v e l s f o r two u n l i n k e d trisomic-2L l a r v a e . . . . . . .  5.  Dose dependence of aneuploids in late  6.  Enzyme l e v e l s f o r c o m p e n s a t i n g trisomic-3L larvae  7.  Enzyme l e v e l s f o r n o n c o m p e n s a t i n g trisomic-3L larvae  B.  Enzyme l e v e l s  in diploid  9.  Enzyme l e v e l s  in  10.  H e a t - s h o c k p r o t e i n 68 a n d 83 s y n t h e s i s i n d i p l o i d a n d trisomic—3L s a l i v a r y g l a n d s . . . . . . . . . . . . . . . . . . . . . . . . . . . .  73  11.  Small heat-shock p r o t e i n s y n t h e s i s trisomic-3L salivary glands  75  12.  Heat-shock gl ands.  13.  14.  A c t i v i t y of trisomies  third  in diploid  levels  and  loci  loci  fat  and  and  and  in diploid  36  • and  larvae  diploid  bodies  and  62  63 64  metafemales  in  31  32  segmental  diploid  trisomic-2R  females,  in  in  and .-»  69  and  salivary  chromosome  enzyme  loci  in  2L 103  Enzyme a c t i v i t y trisomies  for  A c t i v i t y of trisomies  second  16.  A c t i v i t y of trisomies  third  A c t i v i t y of trisomies  a  A c t i v i t y of metaf emales  second  18.  diploid  77  15.  17.  loci  in  enzyme l e v e l s i n s m a l l third instar larvae  males,  RNA 8 3  loci  a  2R-1inked  enzyme  locus  in  2L 104  chromosome  chromosome  enzyme  enzyme  loci  loci  in  in  3L  106  3L 107  2L-1inked  enzyme  locus  in  2R 108  chromosome  enzyme  loci  in  110  viii 19.  A c t i v i t y of metafemales  20.  X-linked  enzyme  levels  i n Oregon R i s o - X  21.  X - l i n k e d enzyme iso-X strain  levels  in  22.  X-linked F(3R>e j e ,  third  chromosome  enzyme l e v e l s iso-X strain  enzyme  loci  in  I l l  strains  C<2L>1t;F<2R>bw/F<2R>bwj  i n C(3L)ru  st*  113  F<3R)e / r a  114  23.  X - l i n k e d enzyme l e v e l s i n C < 2 L > l t / + / F ( 2 R ) b w ; trisomic-2L individuals  24.  X - l i n k e d enzyme l e v e l s i n C ( 3 L ) r u iso-X trisomic-3L individuals  st/+/F<3R>e |  25.  X-linked larvae  enzyme  levels  in diploid  and  trisomic-2L  26.  X-linked 1arvae  enzyme  levels  in diploid  and  trisomic-3L  27.  Quantity of from female  LSPs i n d i p l o i d l a r v a e . . . . . . . . .  and t r i s o m i c - 3 L  28.  Quantity of from female  LSPs i n d i p l o i d larvae..  and t r i s o m i c - 2 L  29.  Quantity of LSPs i n males, metafemales.  30.  Expression in diploid  o f Adh f r o m v a r i o u s chromosomal and t r i s o m i c - 2 L l a r v a e  31.  Summary o f trisomies  regulatory  iso-X  115  s  115  116  117  hemolymph 120  females,  effects  hemolymph  and  on enzyme  122  124 positions  loci  126  in 132  32.  Embryonic v i a b i l i t y  33.  Inferred v i a b i l i t y of s e l e c t e d crosses i n Table 3 2 . . . . . . . .  34.  V i a b i l i t y of double t r i s o m i e s f o r one autosomal arm  35.  Inferred v i a b i l i t i e s of crosses i n Table 33... of  of  autosomal  whole arm t r i s o m i e s . . .  162  aneuploids from  and t r i p l o i d s  selected  the  deficient  aneuploids from  Survival  174  the  monosomies  37.  Number o f trisomies  sex-comb  E f f e c t of autosomal  da and F l on t h e embryonic trisomies  teeth  163  175  36.  38.  113  i n normal  177 and  sex-transformed 182  viability  of 187  ix  39.  Survival to late heterozygous for of d a *  pupae of autosomal trisomies F l and p o s s e s s i n g d i f f e r e n t doses  40.  Expression  of  dominant markers  41.  Expression  of  Bar  42.  Expression  of  sc  43.  Enzyme l e v e l s i n f a t b o d i e s f r o m d i p l o i d trisomic-2R female larvae  44.  L i v e w e i g h t , p r o t e i n c o n t e n t a n d DNA c o n t e n t f e m a l e d i p l o i d and t r i s o m i c l a r v a e . . . .  45.  E s t i m a t e s of c e l l s i z e i n d i p l o i d and t r i p l o i d , and trisomic p u p a e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  216  46.  ^H-uridine incorporation chromosomes from d i p l o i d  240  47.  Repression on  grain  of  variegator  over  48.  Amount o f  LSP-lb  49.  X - 1 i n k e d Adh gene  in trisomic-2L  in aneuploids for  v 2  grain  counts  in t r i s o m i e s . . . . . . . . . . . .  190  2L  into regions and t r i s o m i c  counts  over  males.  and 3L and  regions  198 in  212  of  2L  61A-68C  244  a n d ADH mRNAs r e l a t i v e activity  i n males,  to  LSP-lg  females,  275  50.  Relative  amounts  of  LSPs i n e u p l o i d  51.  Relative  amounts  of  LSPs i n aneuploid s t r a i n s  52.  Filter  53.  Relative  54.  Mapping  h y b r i d i z a t i o n of amounts 1  254  and  metaf emales. . .  LSP-l -  194 196  of p o l y t e n e larvae  2L o r  192  6 9 3 5  of  LSP-1  s t r a i n s . . . . . . . . . . . .  RNA  LSPs i n L82  278 279 281  strain  284  by r e c o m b i n a t i o n . . . . .  286  X  LIST  OF F I G U R E S  FIGURE  PAGE  1.  Mating  procedure  utilized  to  2.  L e v e l s o f GPDH a n d A D H e n z y m e i n d i p l o i d s a n d t r i s o m i e s p o s s e s s i n g t h e same a c t i v e a l l e l e s f o r G p d h a n d Adjh  34  3.  Electrophoretic analysis trisomic—2L larvae  38  4.  Summary b a r relative to  5.  S t r a t e g y employed t o produce f o r t h e t h i r d chromosome  of  g r a p h of enzyme diploids.  produce  GPDH i n  trisomic  diploid •  quantities  C y t o l o g i c a l p r e p a r a t i o n s of chromosome i n d i p l o i d and t r i s o m i c l a r v a e . . . . . . .  7.  Electrophoretic analysis t r i s o m i c - 3 L pupae  8.  Autoradiograms of salivary glands  9.  Summary b a r relative to  10.  11.  trisomies  42  chromosomes  F(3R)VDle  PGM i n d i p l o i d  heat-shock  protein  e  and  a n d RNA f r o m  g r a p h of enzyme q u a n t i t i e s d i p l o i d s . . . . . . . . .  Electrophoretic analysis cellulose-acetate strips gels  •  22  51  6.  of  and  i n 2L  compound-free  larvae...  i n 3L  52  67  72  trisomies 82  o f l a r v a l serum p r o t e i n s on and i n S D S - p o l y a c r y l a m i d e  100  R e l a t i o n s h i p b e t w e e n ADH e n z y m e a c t i v i t y i n d i p l o i d s and 2L t r i s o m i e s b e a r i n g t r a n s f o r m e d Adh g e n e s . . . .  129  12.  Summary o f t h e e x p r e s s i o n l e v e l o f g e n e s X-chromosomal and autosomal t r i s o m i e s  in  135  13.  Number o f  trisomies  loci  14.  Viability trisomies  to late f o r 2L  15.  Post embryonic v i a b i l i t i e s of arm t r i s o m i e s  16.  17.  V i a b i l i t y of temperatures  2L  affecting  third-instar  and 3L  Sex r a t i o o f a u t o s o m a l developmental stages  individual larvae  the  trisomies  five  of  138  partial  major  160  whole 166  at  different 170  trisomies  at  different 173  18.  Phenotypes  19.  Sex-comb morphology of d i p l o i d and t r i s o m i c pupae e i t h e r p o s s e s s i n g sex t r a n s f o r m i n g m u t a t i o n s or n o t . . . .  185  20.  Comparison of from i s o l a t e d  200  21.  K i n e t i c measurements trisomies  22.  R e l a t i o n s h i p between P6I a c t i v i t y f e m a l e and male 2L t r i s o m i e s  23.  R e l a t i o n s h i p b e t w e e n GPDH a c t i v i t y f e m a l e and male 2L t r i s o m i e s  24.  R e l a t i o n s h i p b e t w e e n ADH a c t i v i t y a n d f e m a l e and male 2L t r i s o m i e s . . . . . . . .  25.  E l e c t r o n micrographs of eye f a c e t s t r i p l o i d s , and t r i s o m i e s . . . .  26.  T r a n s c r i p t i o n autoradiogram of chromosomes f r o m a t r i s o m i c - 2 L  27.  R e l a t i o n s h i p between i n c o r p o r a t i o n of H-uridine i n t o 2L v s r e g i o n 61A-68C of 3L i n d i p l o i d s and 2L trisomies.  243  28.  Transcription in four approximately of 2L i n d i p l o i d s and 2L t r i s o m i e s  248  29.  Quantites trisomies  30.  R e l a t i o n s h i p between in three s t r a i n s . . . .  31.  Q u a n t i t y of M a l i c enzyme and i n metaf e m a l e s  32.  of  trisomies  180  enzyme q u a n t i t e s f a t body  from whole  larvae  o n GPDH f r o m d i p l o i d s and  and  larval  and  2L  size  larval  larval  from  and  size  size  203 in  in  in  diploids,  salivary gland female larvae  polytene  206  208  210  214  238  3  of  LSP genes  equal  subdivisions  a n d mRNA i n d i p l o i d s  ADH e n z y m e a c t i v i t y  in diploid  A n a l y s i s of a mutation t h a t L S P - l a p r o t e i n a n d RNA  and  252  a n d ADH mRNA  males  and  256  females 273  reduces  the  amount  of  283  XII  ACKNOWLEDGEMENTS  I  would  Dave Holm, calmly this  support,  they,  and  and wisdom f o r  discussions  am m o s t  A special She has  about  thank  I  have  provided a  from.  I  Johnson  unfold,  Pogson,  advice,  Throughout the  much o f  to  and  and  this  must for  and  and  for  course source  of of  especially  providing  research.  Many  others  noteably  Karen Morin,  Hugh  Ruth and  Budd  for  Devlin,  appreciative.  support  manuscripts.  draw  and C a r e y  research  contributed  emmotional  me t o  Cserjesi  Tom G r i g l i a t i i  encouragement  Don S i n c l a i r ,  Tom Mommson, G r a n t I  my s u p e r v i s o r s ,  my i d i o s y n c r a c i e s .  allowed t h i s  Brock,  thank  their  Don, Peter  day-to-day  which  to  tolerating  knowledge  have  for  study,  thank  like  you to  and  is  this  deserved work  b y my w i f e ,  immeasureably  by h e l p i n g w i t h t h e  c o u l d not  have  completed  Drew  by  providing  production  this  work  Devlin.  of  the  without  her.  General  Introduction  The number members  of  of  the  same sex  same o r g a n i s m . important  d i s j u n c t i o n of are  karyotypic  variability  associated  with  Several within  a  genetic  the  appear  evolutionary  introduce  into  a  of  aneuploidy. adding  or  to  dose  chromosomes The l a t t e r  is  or  of  with  the  This  appears  a  quantity to  be  and  Monod,  1961;  and  Ramakrishnan,  Ferguson-Smith and  hetzenberg  of  gene  true  Grell,  et  1964;  1962;  of  is  and  content  new p o t e n t i a l in  the  chromosomes the  1975;  O'Brien  is  regions.  of  of  and  balance  correlated in  1963;  the  Prittard  1971;  and M c l n t y r e ,  1976;  for  cell.  (Jacob  1975;  gene  by  Alterations  Tedesco,  a  in  termed  organisms  Gerbaud and G u e r i n e a u ,  a v a i l a b i l i t y of  for  number  genetic  Somerville,  Prittard,  Thus,  i n gene  Garen and G a r e n ,  Alledice  1981).  to  events  which accumulates  1973;  1979;  changes  e u p l o i d y w h i l e changes  wide v a r i e t y  Stetson  ensures  usually directly  a l . ,  and C h ia,  species,  Changes  chromosome  gene  a  an  considerable  variation  termed  product  in  be  the  equivalent  fundamental  c o n d i t i o n changes  structural  to  of  evolutionary  introduce  parts  removing p a r t i c u l a r  appears  because  population.  nucleus  cells  process.  diversity  per  between  machinery  that  be  vary  between  and  between  to  alterations  number  a l . ,  However, observed  can  or  material  such  such  the  et  is  not  and m i t o t i c  species;  sets  Chandler  genetic  meiotic  size  processes  chromosome  the  and  species  developmental  maintained.  structure  a  chromosomes  karyotypes  genome  of  for  Remarkable  u s u a l l y does  within  Constancy  prerequisite  stability. proper  chromosomes  1980;  Fasiolo  transcription  appears In  to  be a l i m i t i n g  many s p e c i e s ,  males.  This  expression  between  revealed  studies  the existence  Individuals  heterozygous  duplication  of  respectively, in  normal  The p u r p o s e large  degrees  of t h i s  of  biochemical  are trisomic  controls. products in  gene  These  compensation occur  attempted  to:  compensation expression between  i t  (1980),  study  loci  studies  dosage  gene  have  compensation. a  produce, of  gene  was t o d e t e r m i n e  have  at  2)  product  found  determine  and sex dosage  by Lucchesi Mukherjee  a n d 3)  at  what  examine  chromosomal  compensation  (1973;  (1982),  t o which  1978;  increase  but can thesis  also has  autosomal  the  of  gene  relationship compensation.  i n Drosophila  1983),  and Baker  this  level  dosage  an  gene  dosage  chromosomes, As such,  diploid  many  despite  i n Drosophila,  the extent  individuals  arm and i n  in trisomies,  t o t h e sex  of  in  quantified in  quantities  Thus,  the effect  products  chromosome  aneuploids.  i s regulated  autosomal  end, the been  diploid  document  occurs,  expression  showed t h a t ,  autosomal 1)  one o r two X  t h e amount  To t h i s  from 2 to 3.  Male-female reviewed  times  i s not limited  i n large  gene  a deficiency or  autosomal  f o r an e n t i r e  are observed dosage  equalizes  dosage  on a n e u p l o i d y i n metazoans  o f a n e u p l o i d y on gene  melanoqaster.  that  by  as  individuals.  Drosophila several  i s rectified  possessing  for either  and 1.5  diploid  content  expression.  a s many X c h r o m o s o m e s  o f autosomal  a particular 0.5  twice  mechanism t h a t  individuals  Previous  c o n t r o l l i n g gene  have  i n gene  a regulatory  chromosomes. not  females  imbalance  compensation,  -factor  Stewart  and B e l o t e  has been  and Merriam  (1983).  The  3  remainder  of  this  observations material  i n t r o d u c t i o n summarizes  made o n d o s a g e  are discussed  The r e a d e r  familiar at  experimental  section  Naturally in  some  exists  haploid  Compensation  DNA r e p l i c a t i o n  to  occur  surprising that  for  differences  determination in the  Drosophila, male  which  i n gene  dose,  one  genome  (McKnight,  thesis. the  i n gene  1974).  for  individuals  et  is  this  are  nuclei  an e x t r a  a l . ,  et  1943).  in polytenic  evolved  since  set  (Berlowitz  (Whiting,  (Rasch  dosage  Consequently,  compensate  dose  observed  chromosome  haploid  female  have  number  systems  to  contains  1939).  1983).  It  is  compensate of  with  Both  also  possesses of  genes,  Drosophila  f o r up t o  Nontheless,  a Y  and i s not this  percent  the levels  whereas  chromosome  species,  sixty  sex  i n man a n d  two X chromosomes,  a paucity  I n some  difference  i s associated  of  of  round of  1977).  the consequences  (Bull,  possesses  (the male  for survival).  in a ploidy  are  per organism  i n gene  organisms  the female  result  dosage  this  severe.  i n some  i n Drosophila  required  are  i n males  i n chromosome  has only  and t o  (wasps),  organisms  are often  Variation  the  i s accomplished by a l l o w i n g  not  aneuploidy  to  difference  an e n t i r e  are present  for differences  individuals  i n gene a  in size,  while diploids  the  can leave  directly  (Chandra and Brown,  In H a b r o b r a c o n  males  bugs,  functional:  cells  of  of  one.  differences  In mealy  parts  genetics  and proceed  pertinent  Many a s p e c t s  i n relevant  chapter  are reduced  more  1968).  these  of  i s only  cells  fertile  point  i n one sex  difference, al.,  this  organisms.  inactivated  further  occurring  but i t  compensation.  with Drosophila  introduction  some  of  gene  can the  products produced from most l o c i  l o c a t e d on the sex chromosomes  are e q u i v a l e n t between the sexes, a phenomenon termed dosage compensation.  T h i s p a r t i c u l a r c o n d i t i o n , b o t h i n terms of  v i a b i l i t y and gene e x p r e s s i o n , appears t o d e f y the normal responses  t o aneuploidy.  One  can r e a d i l y a p p r e c i a t e the  importance of such a r e g u l a t o r y mechanism f o r m a i n t a i n i n g balance of gene e x p r e s s i o n w i t h i n the c e l l . metabolic p r o c e s s e s a r e i d e n t i c a l  Presumably most  between males and females  a r e optimum when a s p e c i f i c l e v e l of product compensation i s found kingdom (Lyon,  1972;  and A l i ,  1982)  i s present.  i n several d i f f e r e n t phyla i n the M u l l e r e t a l . , 1931;  Meneely and Wood, 1984;  the  Hodgkin, 1983;  Casartelli,  A r o r a and Rao,  and  Dosage animal  1968; 1979;  Rao  and o c c u r s v i a d i f f e r e n t mechanisms.  In e u t h e r i a n mammals, the mechanism of dosage compensation i n v o l v e s i n a c t i v a t i o n of one of the X chromosomes i n the c e l l s of the females condensation  ( G a r t l e r and Riggs,  1983).  somatic  Random  of t h i s chromosome i n t o a h e t e r o p y c n o t i c element  o c c u r s d u r i n g e a r l y development (Kozak, 1975)  and produces a  mosaic of c l o n a l l y i n h e r i t e d t i s s u e with one or the other of the X chromosomes i n a c t i v e i n gene e x p r e s s i o n . reduces the female c e l l  t o an X/0  This e f f e c t i v e l y  c o n d i t i o n , and e q u a l i z e s the  X-1inked e x p r e s s i o n between male and female c e l l s . c o n t r o l s condensation  i n e u t h e r i a n s i s not known.  What The use of  X:Autosome t r a n s l o c a t i o n s has r e v e a l e d t h a t , i n mouse, t h e mechanism a c t s subchromosomally s i n c e both p i e c e s of such t r a n s l o c a t i o n can condense (Cattanach, i n a c t i v a t i o n c e n t r e i s favoured  1975). In man,  ( G a r t l e r and Riggs,  Maintenance of the i n a c t i v a t e d s t a t e may  a  a single 1983).  be mediated p a r t l y  by  5 methyl a t i o n reactivate 1981).  of  cytosine  genes  since  from a condensed  Marsupials also  difference  from the eutherian inactivated  is  that  interesting  (Tagaki  and  and S a s a k i , may e x i s t  This  may e x i s t  the Barr  that  i n marsupial  continued  responsible  Brown,  f o r condensation  1922,  soma.  selection activity  It  mark  fetus  to  i s easy  this  in a  analagous  to  imagine  favored  paternal  the  remove  i s found  somewhat  In e u t h e r i a n s ,  those  X chromosomes  female  may h a v e of  the  in  It  imprinting  to  1974),  X i s  1975).  occurs to  t h e X chromosome  the X after  factors  presumably c o u l d d i f f u s e randomly  and p a t e r n a l l y  Bridges  observed  of  males.  dosage  of  autosomes  contain  between  males  differences  female  a cell's  derived  that  X's  (Chandra and  the differences  and females  arising  are the  to  determined of  fate  to  male  become  X chromosome  direct  contain  determining m o d i f i e r genes, of  i n form and  from the haplo-X  was c o n s i d e r e d  a preponderance  on t h e r a t i o  autosomes.  suggested  T h e X chromosome  abundance  depend  an  a l . ,  major  1974).  function  such,  gene  the zygote.  maternally  In  result  to  of  also  i n the eutherian  (Lifschytz,  evolution,  et  rise  germ c e l l s  body i n t h e female  repression  transmission  between  form  suggests  but a  the paternal  <Cooper  do not g i v e  i n t h e male  inactive  with  in  that  In mammalian s p e r m a t o c y t e s  completely  i s that  i n females  1975).  a countersystem  it.  system  can  (Mohandes e t a l . ,  compensation,  non-random i n a c t i v a t i o n  embryonic c e l l s  mechanism  with 5-azacytidine  X chromosome  d i s p l a y dosage  preferentially  eutherian  treatment  material  I n a 2X c o n d i t i o n t h e b a l a n c e  is  or to  an  whereas  determining male  condition  loci.  female that  tipped  the  of  As  would the  i n favor  of  female his  while i n the  idea  sets,  1925).  the  ratio  diploid  X:Autosome .5  was  or  that It or  determination binary  a  mode o f  normal  within  the  female  genome,  are  levels.  Bridges  not  to  a n d was  (ie.  of  the  no d i f f e r e n t  very  highly  modifiers located X-chromosome  the  that  3X:2A  1.5).  The  and  the  of  female. of  .66,  accordingly,  cells  name,  can  be  which suggests  that  a  limited  to  that a  the  balance  individuals  of  modifiers  in p r i n c i p l e to  the  chromosomally  individuals).  characteristics  suggested  and  ratio  d i s p l a y e d by h a p l o - X  changing  at  .33) =  a  were  (hence t h e i r  that  suggested  haplo-4  both  a  level,  female  note  of  yields a  have  intermediate,  Bridges  of  or  »  differentiation is  phenotype  similar,  had a  in  male  autosome  determination  above  and f e m a l e  of  that  ratio  sex  karyotype  (this  individuals  phenotype d i s p l a y e d by o t h e r  organism  variation  but  action.  The m a j o r i t y  negative  male  to  ratio  the  1 or  3X:3A  number  metafemales,  male,  subsequent  male  metamales,  2X:3A  the  1X:3A  control  important  individuals  chromosome  a  consequence  characteristic haploid  to  either  is  and  characteristic was  are  1>.  (termed  appeared  between  •  termed  female  possessing  male  ratio  (often  and t h e  He s u p p o r t e d  heteroploids  designated  virtually identical  below y i e l d s a  intersexes). either  1),  (2X:2A,  ratio  cells  female  X/A -  intermediate have  on whole chromosome  A triploid  were  Individuals  i n v a r i a b l y male.  X chromosomes  male  individuals  is  number of  female  invariably  c e l l —  X it  with observations  (Bridges, denotes  haplo  in  the  male  morphological  and  and b i o c h e m i c a l  nonsex-determining  balanced  on t h e  set  of  have  loci  positive  same c h r o m o s o m e ,  number would  female  l i t t l e  and  on t h e  and thus  effect.  X  1 Muller  et  brought  a l .  about  inhibitory directly would  by X - l i n k e d  i n nature.  with  originated  exist  examine dosage  these  i n X-linked  "dosage  gene  i s  dependent and  males  vary  and females  expression.  compensation"  hypothesized  to  describe  would loci  work  mutant  be c o n t r o l l e d located  contains  diplo—X  determine female.  doubled  pigment  percent  increase  allele, pigment  confirmed pigments  evidence  i t  the  that X-linked  genome.  i s now p e r t i n e n t  to  confirming the existence Muller  (1950)  of  described  some  fraction  of eye pigment of pigment  and enables  was o b s e r v e d  i n males,  i n the female. possesses  between  the female.  but resulted  These  using spectrophotometric been  extended  male and  euploid  locus i n only  Clearly then,  twice  one t o  i n a haplo-X  A d u p l i c a t i o n f o r t h e w*  levels  and have  differences  i n d i r e c t l y by  only a small  No d i f f e r e n c e  t h e male  as does  i n mind,  could  modulation of  throughout  the levels  and females.  compensation  the hypomorphic eye mutation  the wild-type quantity  visually  Thus,  in Drosophila.  utilizing  dosage  and p h y s i o l o g i c a l  hypotheses  compensation  that  modifiers but instead  the sexes.  the experimental  This  males  a r e dose  the inhibitory modifiers  by genetic  between  expression  With  of  (1956)  from developmental  sex-determining  w_f^.  that  compensation  o f t h e X chromosome,  t h e term  not be mediated  normally  early  dosage  phenomenon.  result  gene  that  modifiers  Since  the ploidy  Goldschmidt need  postulated  d i s p l a y no net d i f f e r e n c e  Muller this  (1931)  for  a  fifty  each  a s much observations  have  quantitation  of extracted  w i t h measurements  been  using  wild  eye  8 type  alleles  Komma  (1966),  (1973)  Df t h e w h i t e Seecof  demonstrated  et  locus  a l .  that  females.  product loci,  of  Observing  a gene  maximum r e a c t i o n  i s subject  DNA r e p l i c a t i o n  synthesis  on l a r v a l  equivalent strongly  between  suggests  c o u l d be mediated  in  males  or  protein  with  other  (1965)  male that  fRoehrdanz  activites  appear  demonstrated  and L u c c h e s i ,  it  seems e v i d e n t  that  to  be t h e a v a i l a b i l i t y of  a factor  processes  measurements  o f enzyme a c t i v i t i e s  Using  of  appear  demonstrated  active  s i m u l t a n e o u s l y and t h a t  equivalent.  grounds;  type.)  If  alleles dosage  transcriptional  both  product  at  then  i t  appears  Other Thus, the  rate  coding f o r i t .  o f 6PGD,  X chromosomes the level  two X - 1 i n k e d l o c i  level,  aneuploids,  limiting.  Kazazian et of  a l .  the female  of expression  simultaneously heterozygous  compensation  enzyme  i n d i r e c t l y represent gene  is  also  levels  ( T h i s was p r e v i o u s l y s u s p e c t e d  individuals  type  that  Since  not t o be r a t e  variants  (1966)  evidence  for transcription.  the structural  electrophoretic  X chromosomes  i n segmental  limiting  a gene  t o t a l RNA  transcriptionally  1977).  dependent  intermediate  transcription  that  Other  t h e phenomenon i s  ranging  modifications.  gland polytene  and female.  t o be dose  by processes  translational  salivary  regulated  wild  velocities  t o many i n t e r a c t i o n s  to post  a n d Beerman  was  G6PDH a n d  and c o u l d be m i s l e a d i n g .  Mukherjee  of  1969).  a n d Bowman a n d S i m p s o n  t h e phenotypic end product  Dosage compensation from  (1969),  and L u c c h e s i ,  t h e two X - 1 i n k e d enzymes  6PGDH d i s p l a y e d i d e n t i c a l and  (Smith  on  of  at  i s possible that  each  genetic  f o r mutant and  are phenotypically  i s controlled  were  wild  the either  the  male  X chromosome female  i s hyperactived  X's i s being  difference  or that  supressed.  Offerman  i n the cytological  X chromosomes.  staining  and swollen i n t h i c k n e s s  autosomal  cytologically whether an  this  more  the  male  et  a l .  (1973)  (1954)  Ej_ c o l i Bogdanova  higher vitro.  I  (Khesin  a l . , 1974;  et  Mukherjee,  1981).  et  et  was n o t known being i n aspect  observed  condition,  a l .  of  that  in  have  only  one h a l f  richer found  polytene  no d i f f e r e n c e  X chromosomes  Leibovitch  that  t h e male  polytene  1M s a l t  X f o r exogenous  to  X possesses  histone  a  HI i n  f o r Ej_ c o l i  on a male  X than  on a  a l . , 1976;  female's  C h a t t e r j e e and  can be a b o l i s h e d by  wash p r i o r  a l . , 1981)  t h e DNA  in proteins.  However,  difference  staining  demonstrated  I.  in vitro  and that  autosomes.  (1980)  (1976)  or female  and p a l e r  or the  a l .  Leibobvitch et  with a  appeared  an important  i s ten percent  as high  This  (Chatterjee  reflected  X contains  the female  i s twice  chromosomes  analysis  found  Template a c t i v i t y  polymerase  the  polytene  RNA p o l y m e r a s e  of the  o f chromosomes  X chromosomes  of t h e male  a f f i n i t y than  It  paler  hybrids,  and L e i b o v i t c h et  (1983)  regions.  i n the asynapsed  i t  t o be  X's  D o b z h a n s k y (1957)  and L a i r d  but that  the capacity  the female  a  and female  the size  was i n v a r i a b l y l a r g e r  the female's  the female,  bind  remain  indeed t h e male  Khesin  and  arms  X chromosome  either  Aronson  in  it  x D_j_ t r o p i c a l i s  chromosome  that  or i f  observed  X appeared  t o almost  was t h e r e s u l t  X—chromosome s t r u c t u r e .  all  than  autosomal  difference  insularis  of  like  from the  of t h e male  male  In c o n t r a s t ,  unpaired condition,  male  The s i n g l e  arms.  (1936)  properties  polytene  paired  transcription  to  suggesting  treating  transcription that  a  IO  non-covalently  bound c h r o m a t i n  hyperactivation. on  t h e male  evidence  DNA r e p l i c a t i o n  X chromosome  implies that  chromatin  of  chromosomes  and n o t i n g  less  gain  tightly  access  mediated the  gene  deficiency  X—chromosome  DNAse  genes  a differential  i n EJ_ p s e u d o o b s c u r a  compensated  region  1971;  1973).  response  of  rate  of  on  fixed  of  t h e male X  The e f f e c t  since  to may b e  a deficiency  results  proteins)  increases  (Khesin  for  i n an the  rate  and L e i b o v i t c h ,  Eissenberg  sensitivity  and Lucchesi  of  to digestion  the  X-linked  by  i s not affected  when a n  X-chromosome  inserted  (Seecof  B a i l l i e and C h o v n i c k ,  Similarly,  a  large  i s  insensitive  (Roehrdanz  et  a l . ,  compensation that  of  t h e genome  chromosome  flies  i n the  f o r polymerase  a gene.  that  this  cytological  easier  in vivo  should be noted  i n a surrounding piece  another al.,  h is t o n e  exists  the chromatin  presumably  rate  I.  The gene  it  d i d not detect  shock  of  faster  i s observed  density,  (which  transcription  However,  (1983) heat  cluster  that  for  Taken t o g e t h e r ,  mentioned  transcribe  packing  a  an increased  making i t  actively  by nucleosome  h istone  1978).  i s possible  at  difference  effect  t h e above  compacted,  or to  intracellular of  it  occurs  1966).  allows  Considering this  characteristics, is  (Berendes,  X that  i s responsible  also  a structural  t h e male  transcription.  factor  1977).  f o r t h e enzyme  had been  1971;  autosomal to  et  a l . ,  segment  transformed  with  Tobler  transposed  compensation  (1975)  tryptophan  1969;  into et  Bowman a n d S i m p s o n ,  the dosage  Tobler  is  X-linked  investigated  pyrolase  to  mechanism dosage  i n stocks  w i l d - t y p e DNA.  the X  These  of  II  stocks the  were  mutant  enyzme,  position  dosage  (Fox e t  compensation.  mediated  transformation  a  examination  closer  (Rubin  of t h i s  question.  compensate  ( S p r a d l i n g and Rubin,  genes less  occurs than  t h e X chromosome  personal  al»,  (19B4)  the  X-linked  structural  white  gene.  although  the female  has found  that  gene  and Evans-Roberts,  Roberts,  1982)  element course  has been  damaged  the a b i l i t y of  compensate  P-element 1982)  has allowed genes  Scholnick et  expression.  a l . ,  1983; these  somewhat et  mediating hyperacti vation  of  within 1950)  Ota et  represent  (1981)  dosage  Hazelrigg  12kb s u r r o u n d i n g t h e and w i l d  types  a l . , 1981;  cases  o r has n o t been  Korge  flies  Hyperactivation of  of  (Muller, 1979;  in a  the a b i l i t y to  which do not dosage  exceptions  of e v o l u t i o n .  regulating dosage  a r e known  these  level  are located  Mutations  These  t h e m a g n i t u d e may b e  sequences  (Roberts  Presumably  1983;  coding for  inserted  Autosomal  can acquire  communication).  i n males,  two f o l d  1971).  (  and S p r a d l i n g ,  to  Posakony,  a l .  gene  sequence  More r e c e n t l y ,  transposed  J.  the X-linked  b u t had t h e w i l d - t y p e gene  new c h r o m o s o m a l displayed  -for v e r m i l l i o n ,  where  acquired  gene  Brock and  compensate. the  Sas-4  i s recombinationally separable  regulatory  during  has demonstrated  the X-linked  genes  the  that  a  to from the  structural  gene.  These r e s u l t s  imply  that  propagated  f r o m many r e g u l a t o r y  sites  d i s t r i b u t e d throughout  X chromosome, chromatin As  and that  activation  compensation  is a cis-acting  i s  alteration  structure.  previously discussed,  compensation differences  site  c o u l d be mediated between  the sexes.  Goldschmidt f e l t  that  dosage  by t h e normal p h y s i o l o g i c a l However,  Muller  (1950),  Komma  the to  II  (1966),  and Smith and Lucchesi  expression  o f t h e X chromosome  which  phenotypically transform  males  into  intersexes.  observation actually  had n o t changed  transcription  behaved dosage was  autonomously. compensation,  a cellular  culture  cells  identical 1969;  designed  gene  a l . , 1971;  (1975),  compensation  was o n l y r e p o r t e d  chromosome.  If  expression  o f enzyme  Furthermore, for  a fraction  an enzyme  had l i t t l e  concluded  of  i n females  present recently  that  observations  that  which do support  had no e f f e c t the  a l l  tissue  express  and Kakpakov,  1981).  and Gvozdev  hypothesis.  with changes  involving  t o be dose outside  Muller's  on  Dosage a whole X  dependent. the structural Thus,  inhibit  i n t h e male Birchler  form o n l y ,  loci  was a d d e d ,  on i t s e x p r e s s i o n .  found  on  regulation  inhibitory control  However,  i n abstract  types  (Kakpakov e t a l . ,  m o d i f i e r s which c o u l d  to the level  haplo X  by M u l l e r * s  appeared  effect  only  a l l cell  that  o f t h e chromosome  on t h e X chromosome. described,  this  some  and F a i z u l l i n  d u p l i c a t i o n of a region  authors loci  loci  product  or  examined  X chromosomes  to detect  as predicted  sex,  aneuploid Drosophila  Nikoshkov  experiments  X chromosome,  possessing  Indeed,  X-linked  and Merriam  mutations  individuals  (1969)  was c o n c l u d e d  from one t o s i x  of  Gvozdev et  (1973) the  with  rebutted  transformed  and found  the  the opposite  The sex o f t h e o r g a n i s m and i t  by  i n a physiological sense,  glands,  phenomenon.  levels  Stewart  into  (1956)  individuals  salivary  that  unaffected  L a k h o t i a and Mukherjee  i n female  in their  females  these  sex  demonstrated  remained  Goldschmidt  by c l a i m i n g that  morphologically.  cells  (1968)  some  gene these  expression  were n o t  (1980)  has  preliminary  hypothesis.  He f i n d s  13  specific inverse  regions effect  region.  the  loci  dosage  X chromosome  awaits  itself,  between  Rawls,  1973a;  1973a)  or  al.,  with  cells  identical  only  compensate whereas have  a  clear  a l . ,  triploids f i f t y  then  effect  on  product  X-linked  per  present,  the  eel1  but  is  rather  transcriptional the  number  of  gene  triploid et  et  not  Lucchesi  and  Ananiev  times  as  are  Plaut,  t r i p l o i d s have  product  been  individuals  have  Lucchesi  and R a w l s ,  1973b)  a  metamale  1974;  of  in  metafemales Stewart  the  ploidy  sets  the  and  (1X:3A)  has the  a  X's)  level  autosomes. is  number  It  is  profound  of  gene  of  activity.  number  on t h e  both  1975),  on t h e  X-linked  and  and M e r r i a m ,  autosomes  the  a l . ,  dosage  gene  Thus,  et  (3X:2A)  same number  X-linked  dependent  an  triploid  as  examined  (Seecof  than  et  larger  much g e n e  3X:3A  no  either  1973b;  (3X:3A>  the  o f DNA  and P l a u t ,  expression.  autosome  1969;  unit  and  (possessing  of  per  intersexes  a l . ,  rate  product  Maroni  Moreover,  on t h e  s h o u l d have  2X:3A  increase  gene  regions  1973;  1.5  product  ploidy  actual  by m o d i f i e r s w i t h i n  a l . ,  cells  as  1974;  an  aneuplaid  individuals,  X chromosomes;  (3X:3A)  percent  that  autosomal  Autosomal  1977).  (Lucchesi  reflect  (Maroni  s l i g h t l y more et  regions  Gvozdev,  product  a l . ,  these  controlled  and c o n t a i n  of  the  have  experimentation.  triploid  three  levels  (Lucchesi  on  to  outside  (Seecof and  (2X:2A).  Ananiev et  have  the  Faizullin  i n dose,  loci  X-linked and  levels  1929)  from one  1969;  diploid  However,  (Dobzhansky, diploid  in  transcription  1974).  is  altering  is  enzyme  of  further  compensation  No d i f f e r e n c e  monitoring  when v a r i e d  on whether  effect. found  X that  on e x p r e s s i o n  A decision  regulatory If  of  of  X  of  X-linked  chromosomes However,  dependent of  other  both X  14-  chromosomes  within  the c e l l .  In o t h e r  transcription  i s proportional  chromosomes.  This  transcription  i s dependent  which  exists  Plaut, this  X-linked  i s that  genes.  utilizing be  Schwartz,  hypothesis  triploids, cell  quantity 1973;  at  the activator  the total  by f i f t y  X chromosome  amount  percent,  directly amount 2X:3A 1.5  of  X-linked  individuals  i n the latter,  concentration  but since  set.  diploids  of regulatory  Cell  to  be  would  and each X.  In  be increased  per  increase  size  i s  in  were  volume.  chromosome  f o r X-chromosome  result.  be  t h e same  by  i n the Thus,  both  are compatible  transcription Maroni  would  increased  reduction  of r e g u l a t i o n  dosage  i n 2X:2A and  volume i s  would  (per  X-linked  F o r example,  one-third  molecules  molecules  regulating  be produced  the nuclear  by  increased  dependent  from t h i s  would  However,  i s altered  of regulatory  transcription  theories  and t r i p l o i d s .  X would  a proportional  concentration  a concomitant  obtained  data  If  repressor  the  would  of  i n action  t h e two X ' s  t h e male  with nuclear  and negative  the single  when t h e X / A r a t i o  autosome  positive  be r e s t r i c t e d  of  the expression  correlated  A requirement  rate.  X-chromosome then  activator  (Maroni and  the rate  be reduced.  compensation,  produced  1977).  of activator  to X  X-chromosome  activator  gene)  of  that  of  and t h e c o n c e n t r a t i o n  repressors  sets  quantity  accordingly would  o-f a u t o s o m e  the cell  cell,  which produces  transcription  an a d d i t i o n a l  gene  In t h e f e m a l e ,  one—half  A complication exists adding  male  f o r t h e same only  within  Lucchesi,  a l l the activator.  express  X-linked  on an a u t o s o m a l l y  In a d i p l o i d  i n competition  would  to the ratio  prompted t h e h y p o t h e s i s  in limited  1973b;  words,  between  and Lucchesi  (1980)  with  15  tested ratio  t h e autosomal with  possessed two  X-chromosome a diploid  set  X's transcribed  p.ossessing in  1.5  diploids  at  observations  of  the rate  made  X-chromosome  of  a male,  .66  individuals  while  t h e male cell  dependent  those  rate.  size)  Thus,  females  even  X—chromosome  on t h e X / A r a t i o .  in diploid  fragments  these  Individuals possessing  w i t h t h e same  was i n v e r s e l y been  at  by v a r y i n g t h e X/A  A l l of  autosomes.  X's transcribed  have  hypothesis  aneuploids.  .5  (presumably  transcription  additional  activation  Similar  possessing  ( W i l l i a m s o n and B e n t l e y ,  1983). Baker  and Ridge  differentiation organismal cells  (1980)  i n Drosophila  process.  Thus,  which are e i t h e r  their  X chromosomes  If  cells  can transcribe  are  or female expected  to  X-chromosome intersexes, This be  rate,  is  Maroni  transcription  suggests  controlled  that  by separate  many s e p a r a t e  subjected either  to  intermediate  or female  between  genes  levels rate,  the two.  of  and female.  at  from  either  and dosage  averages  measured 2X:3A  of  of the each  chromosome,  regulation,  the average  nuclei  c o m p e n s a t i o n may  Measurements  on each  of  Alternatively,  the  activities.  a n d many c o p i e s  genes  expression  (1973b)  nuclei  X chromosome  individual  intermediate  t h e male  only  and P l a u t  mechanisms.  expression  i s possible that  male  an  possess  and low e x p r e s s i n g  determination  on t h e p o l y t e n e  It  between  in individual  transcription of  (2X:3A)  a unimodal d i s t r i b u t i o n of  sex  than  The average  X-chromosomes high  sex  rather  intersexes  female.  both  that  a cellular,  intermediate  be found.  and found  is  or  their  then  demonstrated  triploid  male  of  male  have  gene. when  are expressed which dosage  is  at  lb  compensation levels  of  may b e  flexible  expression,  phenotype  is  limited  However, locus  a  (Cline  whereas either  genetic 1978, and  foundation.  The e x p r e s s i o n  and  to  prevent  Skripsky,  development this  gene  this  locus  control  of  and  be  are  to  and  1984)  of  this of  the  since  hyperactivation activity  state  identified,  although  autoregulation  however,  this  process  gene,  (da).  locus, for  in  the  mothers product  that  in  The w i l d  addition to  die  as  of  embryos  (Cline,  hyperactivation  been the  at  wild  type  from a  in  of  sexual  Necessarily, mutants die  The s i g n a l s  not  females  (Lucchesi  individuals  at  if which  been  been  plays  an  implied.  It  important  action  of  the  is  part  in  daughterless  da  requirements,  daughters of  dosage  regulatory  state  1983).  from the  lack  common required  has  the  Thus,  a  occur.  nonsex-specific Sxl.  that  constitutive  have  product  of  the  which are the  from  is  needed  da  Sxl"~  (Belote function  responsible,  X chromosome  genetic  which reduce  reported  type  with  revealed  1978).  The components  identified  5x1  X/A r a t i o  conjunction  expression  mutations  the  of  (Sxl)  female  these  cannot  sexual  X chromosome  the  i n males;  multiple  lethal  is  1982}  the  clear,  Sex  gene  of  female.  do have  Nothiger,  lethals,  to  has  maintain  repressed  male  or  the  determination  (Sanchez  must  differentiated  male  hyperactivation  1981)  X-chromosome  to  1983,  compensation  both  the  analysis  1979, sex  phenomenon c a p a b l e  the and of  level.  in  Lucchesi, these  males  Several  expression  of  is  have  for been  male-specific the  1980a;  genes  in part,  lethal  X chromosome  1980b).  intimately  have  Presumably involved  in  n allowing  hyperactivation  of  required  throughout  development  absence  of  lethality 1982;  these of  CIine,  male  products  Sxl null 1984).  X-linked  only  mutations  loci.  These  (Belote,  partially  products  1983).  rescues  the  The female  ( S k r i p s k y and L u c c h e s i *  are  I?  CHAPTER  MEASUREMENTS  OF  G E N E PRODUCT  ONE  LEVELS  IN  TRISOMIC—2L LARVAE  Introduction  In five  this  genes  chapter,  the  amounts of  were examined i n d i p l o i d were  expected  p r o d u c e a p p r o x i m a t e l y 1.5  product  i n t r i s o m i e s as  products  were  observed  to  experiments  be at  three  cell, the  level  of  alleles  and as of  to  of  for  one  compensated  each  the  locus are  gene  by  to  the  other  were of gene  three  were  Subsequent  compensation  active  such compensation appears e x p r e s s i o n of  amount  levels.  locus,  other  the  W h i l e two of  (compensated)  number o f a  times  from  larvae.  d u p l i c a t e d arm and  be dose dependent,  diploid  the  the  in diploids.  revealed that,  independently All  found  within  and trisomic—2L  These genes to  located  products synthesized  alleles  present.  expressed  operate  one-third.  operates  in  each  by r e d u c i n g  Materials  Genetic  and Methods  Strains  A list chapters D.  and Procedures:  of biochemical  i s presented  melanoqaster  <iv)  C ( 2 L ) V D 4 . a l Gpdh  (vi)  al  (vii)  (v)  Gpdh",,  O r e g o n R,  t a  Bl/In (2L)Cv.al  1 3  et  Gpdh .  (xii)  B  a l . , 1972),  described Grell,  of  Individuals produced  as  Grel1,  d i e during  survive  progeny  trisomic-2L Five  Holm,  b A d h ' ^ QSP o r c n .  and ( x i i i )  1  and t r i s o m i c - X  chromosomes  i n Table  alleles  arm of  a high  entire  that  and Holm, whereas  left  arms  of  is  3L have  2  were  1978).  monosomic  proportion of  the  chromosome of  comparable.  on chromosome  and o n e on chromosome  Lindsley  chromosome  the v i a b i l i t y  individuals  systems  1976$  have  1976).  revealed  In f a c t ,  5.  and chromosomes  (see F i t z e - E a r l e  studies  adults.  several  t h e Y and 2L  for the left  embryogenesis,  gene-enzyme  chromosome,  mutant  inheriting three  to pharate  T  (ix)  ( O ' B r i e n and M a c l n t y r e ,  shown i n F i g u r e  progeny  C !  between  1970J  trisomic  and c y t o l o g i c a l  C v Gpdh**.  It.  Gpdh  the other  Genetic  hybrid  C(2L)SHl+8  Cv Opdh^l  which are l i s t e d  previously  1968J  of  It.  1  Characteristics  strains  C ( 2 L ) P . b ; C ( 2 R ) 5 K l A c n bw.  °- -^/In(2LR)SMl.Cv  (xi)  (ii)  and subsequent  C(2L)VH11t;F(2R)bw/F(2R)bw.  with translocations  (Lindsley  and  <i>  in this  The f o l l o w i n g  Gpdh** B l L / I n ( 2 L + 2 R ) C v . a l  Gpdh  strains  examined  1.  Df(2L)GdhA/In(2LR)SMl.Cv  (viii)  been  used:  (iii)  F(2R)bw/F(2R)bw.  (x)  i n Table  were  F(2R)bw/F(2R)bw.  loci  2L,  been  one on t h e X analyzed  in  this  2  2\  F i g u r e 1. Mating procedure u t i l i z e d to produce t r i s o m i c larvae. In g e n e r a l a s t a n d a r d s t r a i n was c r o s s e d t o a s t r a i n b e a r i n g a compound chromosome and two f r e e chromosomal a r m s . Z y g o t e s a r e formed w h i c h a r e t r i s o m i c o r monosomic f o r t h e arm a s s o c i a t e d w i t h t h e compound c h r o m o s o m e .  X2.  \c(2L);F(2R) \aametes  \ .  2R 2R  standard  \^  gametes  2L  •  2R  > trisomy  m  2L  m o n o s o m y 2L  23  chapter.  Their  Maclntyre,  cytological  1976;  Voelker  Woodruff  and  that  activity  the  the  number  strains five  of  with  loci  generated al.,  small  1979)  each  enzyme  T(Y;2)  in  normally levels  (three 2L.  a l . ,  Table  was  1.  1979b;  To  show  proportional  were measured  doses)  for  Segmental  chromosomes  and  Voelker et  listed  enzyme  on chromosome  the  1979a;  are  duplications  trisomic or  crossing that  a  for  null  chromosome  each  to  in of  aneuploids  described  allele  were h e t e r o z y g o u s It  and e i t h e r  noncomplementing deficiency for  allele  Adh).  for  deficiency larval  null  for  homozygous  the  Gpdh  but  the were  by L i n d s l e y  Larvae  et  or  in  produced  chromosome  Df(2L)GdhA  gene)  or  to  males  null  either  were d i s t i n g u i s h e d by t h e tubules  were  the  null  presence  contrast  to  allele of  pigment  their  variants  C(2L)P.b;C(2R)SKlA.cn In(2L+2R)Cv.al  In(2L)Cv.al  6 0  Co  was  constructed  bw m a l e s  Cv Gpdh^  from a  heterozygous  females  source.  to  al  that  for by  two  Gpdh** B l  L/  had  treated  A C(2L)VD4.al  Gpdh*  C v 6 p d h " ; C ( 2 R ) S K l A . c n bw i n d i v i d u a l and  a  stock  was  Gpdh  mating  been  established.  From a  in  nonpigmented  sibs.  electrophoretic  a  (a  Gpdh)  structural  either  to  inverted  G p d h " " " V"*  carrying  locus  females  the  for  carrying  specific  noncomplementing  A compound chromosome  recovered,  a  for  allele  A d h ^ - (a  Malpighian  <l±/lt/lt>  2500 r a d s  for  2L,  C(2L)1t8F(2R)bw/F(2R)bw  In(2LR)SMl.Cv  the  al. ,  (O'Brien  (1972).  males  or  et  gene c o p i e s ,  with  deficiency  (a  of  studied  Flies  by  Ashburner,  map p o s i t i o n s  3  with  Bl /  was cross  of  a*  females  bearing  F(2R)bw/F(2R)bw  this  compound 2L chromosome  males,  with  C(2L)P.b;  C ( 2 L ) V D 4 . a l Gpdh** B l / I n ( 2 L ) C v . a l  F<2R)bw/F<2R)bw n o n d i s j u n c t i o n a l p r o g e n y  were  These  individuals,  possessing  Gpdh  allele  0  produce  when c r o s s e d  on standard  trisomies  to  second  containing  flies  Cv Gpdh";  recovered. the  chromosomes,  three  different  electrophoretic  variants. All  strains  were  cornmeal  sucrose  medium s u p p l e m e n t e d w i t h t h e mold  Tegosept  (0.22%)  and with t h e a n t i b i o t i c s  streptomycin  Enzyme  All to  at  determinations  "*C-1  in  of  <DDC),  centrifugation,  third pint  larvae except  instar  culture  standard inhibitor  tetracycline  dopa,  of  1 4  and  corrected  the presence 1973).  released  (except at  from the were  b u f f e r (pH  (dopa) was u s e d .  f o r DDC) w a s  340nm b y t h e p r o d u c t i o n was d e t e r m i n e d  from  of heat-inactivated were  <pH 7 . 2 )  DDC a c t i v i t y  f o r spontaneous  A l l assays  lOmtl p h o s p h a t e  MTris  on 50  Homogenates  dihydroxyphenylalanine  i n absorption  2  performed  collected  bottles.  enzyme a c t i v i t y  C0  were  larvae  p e r ml o f  f o r which 0.1  by t h e change  t h e amount  Konopka,  C on  each.  u t i l i z a t i o n o f NADH o r N A D P H ;  from X  late  f o r a l l enzymes  monitored or  of  f r o m 25mg  decarboxylase After  25 degrees  o f enzyme a c t i v i t y  of uncrowded h a l f  prepared 8.2)  10 m g / 1 i t e r  at  Assays:  150mg s a m p l e s  sides  reared  decarboxylation  extract  (Hodgetts and  optimized with respect  to a l l  25  components.  The c o n d i t i o n s  phosphoglycerate 7.6/0.9mM  kinase  f o r each  (PGK; EC 2 . 7 . 2 . 3  EDTA/15mM NADH/2.2mM  glycerate-3-phosphate/3.2mM glyceraldehyde-3-phosphate a-glycerol-3-phosphate 0.1  Mglycine,  cytoplasmic phosphate,  dehydrogenase 1.7g/liter, (DDC;  becquerels)  Nuclear;  3 Ci/mmol;  0 . IM T r i s and  of  0.IM Tris,  utilization  of  concentration albumin  compensation compound-free  of  dopa  of  buffer,  decarboxylase  u C i (1  Ci=3.7  x  dehydrogenase NADP/0.5mM  = i  1.1.1.42),  isocitrate;  (6PGD;  EC 1 . 1 . 1 . 4 4 ) ,  0.IM  / 3 . ImM  one u n i t  substrate  (IDH; EC  i s defined as  per min.  (Lowry,  a s u n i t s p e r mg o f  1952),  the  Protein with bovine  serum  standard. levels  were  expected  estimated  strain  R strain.  chromosome  where  alcohol  (New E n g l a n d  dehydrogenase  was d e t e r m i n e d  as the  dopa  A c t i v i t e s are expressed  1 pmol  The enzyme  Oregon  *C)  i n the extract,  acid;  50mM  O . IM g l y c i n e p h o s p h a t e  mM N A D P / 2 0 m M M g C l  6-phosphogluconate. protein  l J  1.1.1.8),  EC 1 . 1 . 1 . 3 7 ) ,  mM p h e n y l t h i o u r e a / 0 . 2  p H 8 / 5 m M Mg=<V0.05mM  pH 7 . 5 / 1 . 3  (NAD) (GPDH E C  p e r ml 5  pH 7 . 2 / 0 . 1 2 m M  isocitrate  6-phosphogluconate  Tris,  (EC 1 . 2 . 1 . 1 2 )  mM N A D / 2 % e t h a n o l ;  (l-  pH  of  NADH/5mM o x a l o a c e t i c  pyridoxal-5'-phosphate/25 10*°  0.IM Tris,  units  (cMDH;  (ADH; E C 1 . 1 . 1 . 1 ) ,  EC 4 . 1 . 1 . 2 6 ) ,  as follows:  g l y c e r o l - 3 - p h o s p h a t e / 1 0 m M NAD+J  dehydrogenase  pH 9 . 0 / 2 . 5  ),  dehydrogenase  dehydrogenase  pH 7.3/0.25mM  were  ATP/13mM  MgS0*»/2.5  pH 9.5/2.5mM  malate  assay  i n t h e absence  dosage  b y summing t h e a c t i v i t y  p l u s one h a l f  This  of  corresponds  2L t o t h e k a r y o t y p e  of  the activity  to the parental of the t r i s o m i e s .  found found  i n the i n the  contribution T h e enzyme  levels were  expected  estimated  as  noncompensated were  obtained  strains  expected  by  simply  value.  of the  error  were  compared  f o r these  f r o m t h e two  contribution to sizes.  using a t-test  for differences  compensation  expected  estimates  with their sample  dosage  i n sample  values  diploid  the  Observed and with  correction,  variance  as  suggested  (1974).  Electrophoretic  Procedures  Electrophoresis variants  Gels  both  mean a n d w i t h t h e i r  necessary,  citrate  autosomal  by p a r t i t i o n i n g t h e v a r i a n c e  values  Welch  of  two-thirds  Standard  i n accordance  estimated  were  i n the presence  o f 6PDH i n t r i s o m i e s  was p e r f o r m e d gel system,  were  stained  in a  pH 6 . 9  f o r 6PDH  12%  <wt/vol)  run at (Shaw  300V  possessing starch/0.1M  (25-35  and Prasad,  mA) f o r 1970).  three Tris 32hr.  27 Table 1 chapters  B i o c h e m i c a l l o c i examined i n t h i s and subsequent (-from T r e a t - C l e m o n s a n d D o a n e , 1 9 8 4 ) .  Gene  Product  Adh Ak Aid Aldox Cat cMdh Ddc E s t - •6 Fum Gpdh Gpt Had hsp 22 hsp 23 hsp 26 hsp 27 hsp 68 hsp 70  alcohol dehydrogenase arginine kinase aldolase aldehyde oxidase catalase cytoplasmic malate dehydrogenase dopa-decarboxylase esterase—6 -f u m a r a s e alpha-glycerophosphate dehydrogenase glutamate-pyrovate transaminase beta-hydroxyacid dehydrogenase 22 kD h e a t s h o c k p r o t e i n 23 kD h e a t s h o c k p r o t e i n 26 kD h e a t s h o c k p r o t e i n 27 kD h e a t s h o c k p r o t e i n 6 8 kD h e a t s h o c k p r o t e i n 70 kD h e a t s h o c k p r o t e i n  hsp 83 Idh LSP- l a L S P - •lb LSP- ig L S P - •2 Men Pgd  83kD h e a t s h o c k p r o t e i n i s o c i t r a t e dehydrogenase l a r v a l serum p r o t e i n - 1 a l p h a l a r v a l serum protein—1 beta l a r v a l s e r u m p r o t e i n - 1 gamma l a r v a l serum p r o t e i n - 2 m a l i c enzyme 6-phosphogluconate dehydrogenase phosphoglucoi semerase phosphoglycerate kinase phosphoglucomutase superoxide dismutase glucose-6-phosphate dehydrogenase  Pgi Pgk Pgm Sod Zw  G e n e t i c Map Position  2L-50.1 3L-25.2 3R-91.5 3R-57.2 3L-44.3 2L-37.0 2L-53.9 3L-36.8 1-19.9 2L-20.5 1-42.6 1-54.4 3L-67B 3L-67B 3L-67B 3L-67B 3R-95D 3R-87A and 87C 3L-63BC 3L-27.1 1-39.5 2L-1.9 3L—1.41 3L-37 3R-51.7 1-0.63 2R-20.5 2L-5.9 3L-43.4 3L-34.6 1-62.9  22  RESULTS  The e f f e c t  of  enzyme a c t i v i t y species.  enzyme  has  Indeed,  aneuploids or  are a  aneuploidy for been  extensively  changes  often  used  dose-dependent  (O'Brien  and  However,  i n Drosophila the  expression of  seven  Maclntyre,  is  trisomic  region,  comparing trisomic  enzyme  simple  show s t r i c t level of  three  mechanism in  this  diploid  Gpdh,  is  are  also  that  for  arm.  genes  the  gene of  an  expression  '  1974).  trisomy the  was  on  gene  expression a  accomplished  stocks  two  and  diploid  for  the  three  sum o f  by  their  parental  regions,  a  the  enzyme  a  expected  activities  of  each  compensatory  prediction  the  2-4.  would  equally,  level  of  of  the  would be  that  three  genes  that  resulting  two-thirds  activities at  Tables  previous  expression  approximately  clearly  in  in trisomies  the  the  alleles  The enzyme are  autosomal  Accordingly,  The s i m p l e s t  activity,  From  duplicated  controlling  of  level.  presented  Alternatively,  on a l l  r  This  presented.  aneuploids  dependence.  and cMdh  for  with  w i t h i n and o u t s i d e  o f f s p r i n g are  acts  noncompensated  autosomal  of  C(2L)+;F((2R>bw/F(2R)bw  gene c o p i e s .  level  its  on  number  associated  we e x a m i n e d  located  The r e s u l t s  segmental  trisomic  of  parental  may e x i s t  compensation  Pqkt  effect  would s i m p l y represent  the  in a  Rawls and L u c c h e s i ,  in diploid  levels  dose  regulating  Therefore,  trisomic-2L  prediction  locus  segments  structural  levels  their  with  the  locate  2L.  and  findings  to  chromosome  Oregon R and  enzyme  activity  systems  offspring.  Expected  examined  namely  stocks, for  genetic  i n enzyme  1976;  unknown.  gene-enzyme  small  in of  loci,  a the  2^  expected located arm.  if  located  contrast, loci,  levels  of  (Table  3).  in  occurs  i n approximately  In  even  compensation  for  the  the  activity,  is  noncompensated  trisomic  show  f  individuals  2).  two-thirds  of  gene.  absence  Thus,  capable  others  are  the  two  of  chromosome  proximally  dosage  s i g n i f i c a n t l y higher  are  loci  dose-dependent  are  it  of,  whereas  appears  to  regionally distributed along  Results  from r e c i p r o c a l  crosses  not,  are  expected,  that  some  directed  and t h i s  were  compensation  than  appears  or  compensation, be  These  produced by the  i n d i c a t i n g an  ADH a c t i v i t y a  distal  enzymes  Adh and D d c  (Table  loci  toward  difference  the  chromosome  arm.  statistically  i ndi s t i ngui s h a b l e . The s t r u c t u a l the  X chromosome  trisomy  2L  might  below the  expression the  of  same two  This  was  have  on t h e  the  active  activites  loci  diploid  in  of  what  unlinked  trisomies  level  (Table  be  made b y c o m p a r i n g t h e  i n both  diploid  on  the  diploid  for  the  Figure  2. is  strains  same C ( 2 L ) strains  chromosome enzyme For  6pdh  reduced carrying  carried  locus f  in  the  The  on  the  activity  either  of  strains. of  in  trisomic  strain a  question.  null  any,  approximately  enzyme a c t i v i t y  expressions  a  if  loci.  and t r i s o m i c - 2 L  by approximately either  trisomy  chromosome  with a  on  4).  a  can  of  was  the  gene  effect  a n d 6P6D  effect,  of  loci  alleles  trisomic  observe  3L  assessment  standard  deficiency in  these  alleles  C(2L)-bearing  shown  of  to  a c c o m p l i s h e d by comparing the  individual  which  IDH o n chromosome  monitored  expected  A direct  for  were  enzyme a c t i v i t i e s 15%  genes  null  allele  The r e s u l t s of  the  one-third  allele  in  or  a  or are  two in  the  deficiency  a  30  Table 2 Enzyme l e v e l s trisomic-2L larvae.  f o r compensating  loci  Enzyme a n d chromosome  Genotype  Pgk 22C-23E  Oregon R (diploid)  0.986  C(2L)+;F(2R)bw/ F(2R)bw (diploid)  0.831  C(2L)+/ +/F(2R)bw (CF? x OR*) (trisomic-2L)  0.973  + 0.031  0.212  O.132 ± 0.008  3.36  0.154  + 0.005  3.71  ± 0.019  3.91  1.32  Compensated Estimate  0.883  + 0.016  + 0.011  ±  0.053  +  0.294  (4)  3.73  (3)  Noncompensated Estimate  0.056  (10)  (10)  0.168  +  (10)  (14)  (10)  C(2L)+/ +/F(2R)bw (0R<? x C F ? ) (trisomic-2L)  + 0.008  cMdh 30F-31D  (14)  (11)  + 0.049  position  Gpdh 25F-26B  (IO)  + 0.019  i n d i p l o i d and  +  0.102  (11)  0.238  + 0.006  5.39  +  0.044  0.159  + 0.004  3.59  +  0.029  E n z y m e a c t i v i t i e s e x p r e s s e d a t t h e mean ± S E i n u n i t s / m g o f protein. E s t i m a t e s o f enzyme a c t i v i t i e s e x p e c t e d i n t r i s o m i e s f o r dose—dependent (noncompensated) and compensated gene expression are provided. Numbers i n b r a c k e t s r e p r e s e n t t h e sample s i z e s .  31 Table 3 Enzyme l e v e l s trisomic-2L larvae.  f o r noncompensating  Enzyme a n d chromosome Adh 35B2  Genotype  +2.4  loci  i n d i p l o i d and  position Ddc 37B10-37C7  Oregon R (diploid)  67.6  8.61+1.02  C(2L)+?F(2R)bw/ F(2R)bw (diploid)  40.9  C(2L)+/ +/F(2R)bw (CF x OR ) (trisomic-2L)  89.O +  C(2L)+/ +F(2R)bw (OR x CF ) (trisomic-2L  88.1  Noncompensated Estimate  74.7  +  1.8  15.2 +  0.552  Compensated Estimate  49.8  +  1.20  10.1  0.348  (14)  +  (16)  1.7  (14)  0.78  (18) 1.9  (10)  +  10.9 +  16.7 +  0.714  (20)  1.7  (3)  +  E n z y m e a c t i v i t i e s e x p r e s s e d a t t h e mean + S E i n u n i t s x 1 0 p e r mg o f p r o t e i n f o r A d h a n d i n u n i t s x 10** f o r D d c . E s t i m a t e s o f enzyme a c t i v i t i e s e x p e c t e d i n t r i s o m i e s f o r dose-dependent (noncompensated) and compensated gene expression are provided. Numbers i n b r a c k e t s r e p r e s e n t t h e sample s i z e s . 3  32 Table 4 trisomic—2L  Enzyme l e v e l s larvae.  f o r two u n l i n k e d  Enzyme a n d chromosome Idh 66B-67C  Genotype  Oregon R (diploid)  110+4.3  C(2L)+lF(2R)bw/ F(2R)bw (diploid)  73.3 ± 2 . 8  C(2L>+/ +/F(2R)bw (CF x OR > (trisomic-2L)  74.9 ±  C(2L)+/ +/F(2R)bw (OR x CF ) (trisomic-2L)  81.5  Estimated  mean  Repressed  Estimate  (IO)  loci  i n d i p l o i d and  position 6Pgd 2D3-2D6  36.8  +  1.2  (10)  (7)  54.0 ± 1 . 1 (10)  1.0  (10)  40.2  ±  1.6  (10)  +  4.0  91.7  ±  1.39  49.7  ±  0.576  60.6  ± 0 . 9 3  33.1  ±  0.38  (3)  E n z y m e a c t i v i t i e s e x p r e s s e d a t t h e mean ± S E i n u n i t s x 1 0 p e r mg o f p r o t e i n . E s t i m a t e s o f enzyme a c t i v i t i e s e x p e c t e d i n t r i s o m i e s f o r dose-dependent (noncompensated) and compensated gene e x p r e s s i o n a r e p r o v i d e d . Numbers i n b r a c k e t s represent the sample s i z e s . 3  33  F i g u r e 2. E x p r e s s i o n of enzyme l o c i on t h e C ( 2 L ) l t chromosome i n d i p l o i d s and i n t r i s o m i e s p o s s e s s i n g e i t h e r a n u l l a l l e l e or a d e f i c i e n c y f o r the l o c i measured. Only the genotype of the l e f t arm o f chromosome 2 i s g i v e n . A c t i v i t i e s f r o m l o c i on t h e C ( 2 L ) l t chromosome a r e g r a p h e d r e l a t i v e t o t h e d i p l o i d level. A l l s t a n d a r d e r r o r s w e r e l e s s t h a n 5X o f t h e m e a n . ( L e f t ) G p d h . (Right) Adh.  35  for  the  structural  expression  of  trisomic—2L the  gene.  this  individuals.  alleles  in  the  individuals  standard  second  response  of  For valid  of  the  strains  enzymes  mature these the  third  Small were  small  instar  strains of  with  their  three  normal  completely  first,  distinguish three  gel  that  these  expressed  all  (Figure  the  genes  the  is  be  dose the  be  a  dependent  same  structural were  to  stage genes  in of  for  performed  all  on  The enzyme a c t i v i t i e s that  is,  the  in  levels  s i g n i f i c a n t l y higher  two  variants  three 3).  may o c c u r  may b e  second,  the  electrophoresis  demonstrated were  or,  while  electrophoretic  Starch  allele  level  between  must  dependence;  compensation  repressed  that  in  than  parents.  each  diploid  loci  were  in  the  compensation  (Table 5).  doses  diploid  Simplistically, mechanisms:  dosage  and enzyme a s s a y s  larvae  on  two  alleles.  d u p l i c a t i o n s of  made,  the  reveal  d u p l i c a t i o n s at  a n e u p l o i d s showed d o s e  those  the  autosomal  compensating  with  allele  results  active  involving  unaffected  and noncompensating  number o f  observed  development. five  the  of  the  in  experiment  was  an Adh n u l l These  that  compensated  expression  chromosome  bearing  compensating  is  analogous  Adh, the  C(2L)  demonstrates  system  an  chromosome.  phenomenon,  diploid  In  locus,  trisomic  independent  clearly  gene-enzyme  noncompensating  active  This  of  other  expressed one  allele  two  are  alternatives, for this  alleles  by one  Gpdh  were  this  may  fully  two  two-thirds  of  be active.  trisomies  To  possessing  produced.  enzyme f r o m of  at  of  trisomies  compensated  gene  3b Table 5 Dose dependence o f enzyme l e v e l s aneuploids i n late t h i r d instar larvae.  i n small  segmental  Gene  Dupli— cation  Translo— cation  Pgk  22D-24A  G120  2  0.559  + 0.018  (5)  R136  2  0.535 + 0.009  (7)  DpG120-R136  3  0.714  0.033  (8)  D106  2  0 . 113 + 0 . 0 0 8  (5)  G105  2  0 . 136 + 0 . 0 0 B  <5>  DpD106-6105  3  0.207  + 0.007  (5)  L52  2  1.53  + 0.098  (3)  A162  2  1.44  + 0 . 130 (3)  DpL52-A162  3  1.91  + 0.040  (5)  G74  2  39. 1  + 0.67  (6)  P58  2  36.7  + 0.68  (6)  3  68.0  + 1.60  (6)  D219  2  11.03  0.85  (4)  H174  2  11.70  + 0.76  (4)  D219-H174 3  15.98  + 0.36  (4)  Gpdh  cMdh  Adh  25F-26B  30F-31EF  34B-35D  Dp  Ddc  36EF-37D  Dp  G74-P58  Dose  Activity  Ratio  1 .31  1 .66  1.29  1 .79  1 .41  A c t i v i t y u n i t s a r e t h e same a s i n T a b l e s 2 t o 4 . R a t i o r e p r e s e n t s t h e enzyme l e v e l s f o u n d i n d u p l i c a t i o n - b e a r i n g l a r v a e r e l a t i v e t o t h e mean o f t h a t f o u n d i n t h e i r parents.  37  F i g u r e 3. E l e c t r o p h o r e t i c a n a l y s i s o f Gpdh v a r i a n t s i n trisomies. B, A, and C r e p r e s e n t m o b i l i t y v a r i a n t s of Gpdh. Lanes B, A, and C a r e f r o m d i p l o i d homozygous i n d i v i d u a l s ; l a n e s A / B , A / C and B / C a r e f r o m d i p o i d h e t e r o z y g o t e s * lanes designated T are from t r i s o m i e s possessing a l l three variants produced from r e c i p r o c a l c r o s s e s . The l e f t l a n e of each p a i r of T r i s o m i c samples were d e r i v e d f r o m t r i s o m i c o f f s p r i n g p r o d u c e d from c o m p o u n d - f r e e males, and t h e r i g h t l a n e from c o m p o u n d - f r e e female parents. +ve, M o b i l i t y towards anode.  39 DISCUSSION  In  Drosphila,  predictable  rates  1983).  The t o t a l  nucleus  is  Thus, gene  X-linked  constant,  varies  or  twice  with  three  or at  example,  50%  greater  than  two  sets  autosomes).  et  a l . ,  relevance two  or  males  to  three or  of  females genes  expression  (Lucchesi,  contrast  autosomal  loci  constitution.  are dose Genes  of  diploid level  i s directed  individuals loci  at  t o match  that  number.  For  in individuals  possessing  (a t r i p l o i d  that  1975)  compensation  dependent  traits  levels  of  and L u c c h e s i , has been  regardless  duplicated  is  and  particular  individuals  sex-linked  i t  two X  (Lucchesi  with one, equally,  duplications or deletions  loci,  the  individuals  i s of  diploid  of  intersex)  (two X chromosomes  show d o s a g e  with  of X  on t h e i r  females  structural  X-linked  females,  Maroni  i n small  chromosomes.  of  express  X-linked  X  X-linked  For example,  d o show d o s e - d e p e n d e n t  to  of  1977;  diploid  X—linked  Whereas  1978;  a  transcribe  and Merriam,  having small  (Lucchesi,  t h e number  The observation  X chromosomes  d i f f e r e n t and  within  single  autosomes  diploid  study.  chromosome  In  sets  normal  Stewart this  to  dependent  f o r t h e X chromosome 1974;  a  the total  transcription  and t h r e e  trisomic  the rate  transcription  chromosomes  of  of  X chromosomes  and i s d i r e c t l y X-linked  at  t h e number  i n the nucleus.  In a d d i t i o n ,  chromosome-linked autosomes  rate  of  proportion  two-thirds  respectively.  transcribe  transcription  regardless  in direct  present  loci  depending on t h e genotype  the transcriptional  templates one  X-linked  of  of  the X  gene 1980).  observed the  segments  of  that  chromosomal the  4-0  autosomes,  like  dose-dependent individuals have  Gpdh  activity appears arm  levels  that  (20.5),  expected  of  product  distally  a n d cMdh  However,  show  Thus,  dose  dosage  phenomenon a c t i n g  loci  levels  2,  we  (Pok  of  a compensatory  on t h e t r i s o m i c  mechanism chromosome  loci  (Adh  dependent.  compensation  at  in  arm o f chromosome  two p r o x i m a l l y l o c a t e d  remain  autosomal  2).  mapping enzyme  Not a l l genes  since  produce  (Table  left  (37.2))  of a d i p l o i d .  and Ddc ( 5 3 . 9 ) )  all-or—none  counterparts,  for the entire  t o be a c t i n g .  Superficially,  gene  three  a r e compensated  (50.1)  X-linked  trisomic  observed  (7.6),  their  the level  appears  of the  t o b e an  individual  gene. The r e s u l t s that the  the distal proximal  that  this  either  one-third  a difference  number  melanoaaster distinct  al.,  1981).  compensation miranda  of  et  organization  (Roberts  a l . ,  One c a n  response or  to  they  of  i n D. are not  located  1979aS  for sex-linked  i n a related  in  compensation.  and E v a n s - R o b e r t s ,  regionality  while  reflects  may b e s i m p l y a n a r t i f a c t  compensated,  suggest  speculate  a difference  W h i l e two genes  observed  studied  compensates  4).  of responding  sampled.  examine loci  (Figure  regionality  has been  systems  i n t h e compensatory  In c o n t r a s t ,  (Strobel,  not  capable  loci  regions  noncompensated both  does  a r e not dosage  To f u r t h e r  for  this  gene-enzyme  o f t h e chromosome  i n gene  structure  Alternatively,  in  two-thirds  regionality  chromosome  small  of the f i v e  Ota et  dosage  species,  p.  1978).  the response  i n trisomies,  A d h a n d f o r Gtodh w e r e  of  compensated  and  t h e same t w o a c t i v e  measured  in  diploid  alleles  the  F i g u r e 4. S u m m a r y b a r g r a p h o-f e n z y m e q u a n t i t i e s i n 2 L t r i s o m i e s r e l a t i v e to diploids. Level 2 gene-dose expressed represents the expected compensated l e v e l of e x p r e s s i o n ; l e v e l 3 gene-dose expressed r e p r e s e n t s the expected noncompensated estimate.  a a  •o < a> E  S  LU  o o  (9 TJ  a O  a.  pass aid x 3 asoQ-auag  4-3  and  in  trisomic  the  standard  strains  chromosome  deletion  that  Adh,  activity  the  (Figure  and  diploid  The  identical  possessed  eliminated the of  each  strains,  as  allele  of  strains  suggests  that  between  them  minimal.  In  allele  one-third diploid  in  the  strain.  were  variants  further  To examine levels  chromosome level this  of  were  reflects  a  loci  and other  of  used)  decreases trisomy,  gender,  in  trisomies,  any  interpreted  was  in  of  3L  trisomies.  initially  as  in  the  three  that  these  2L  all  three  results  are  genetic  Bijlsma, extensive  use  each  them  1980)  15%).  (isogenic  survey  of  not  are  a  Whether genes,  a  other  unlinked  specific  that  effect  standards.  genotypes should  rather  strains  established  internal  altered  specific  X  displayed  on n o n l i n k e d  we h a v e  as  loci,  variation  enzyme a c t i v i t y locus  on u n l i n k e d  a n d 6PGD f r o m t h e  trisomy  dealing with grossly in  each  allele  possessing  IDH a n d 6PGD a c t i v i t i e s  changes  of  by  (approximately  Because  to  differences  same  trisomies,  of  simply  more  locus.  and  expression  this  For  trisomic  trisomic  reduced  a  allele.  noncompensated  trisomy  the  I960;  a  in  or  2.  expected  or  strains,  allele  third  GPDH d e m o n s t r a t e d  effect  a l . ,  awaits  in  the  the  trisomies  In  than  we h e s i t a t e  Therefore,  for  null  physiological  strain  of  trisomic  identical  contrast,  i n f l u e n c e of  general  et  of  any  expression  measured.  (Laurie-Ahlberg not  of  a  The i m p l i c a t i o n s of  lower  in  were  was  any  i n chapter  the  either  IDH f r o m c h r o m o s o m e  somewhat  differences  the  the  expressed.  discussed  the  to  Analysis  electrophoretic loci  trisomic  relative  the  Adh a l l e l e s  diploid  Gpdh  In  activity  expected  expression  is  2).  such  perhaps  than  as be  reflective  of  total  genomic  The d a t a does it  dosage  gene  existence  of  which  act  repressing  X—linked  increases,  template  presented  for  second  loci  that  activator  that  X-linked  Plaut,  number  of  1973;  activator been  presented  compensation 1973). we f o u n d (1974)  have  reported  the  repressor from  (Stewart, and Merriam,  loci  on t h e X chromosome.  for the activator  have  Increases  no e f f e c t genes  model  that  regions  Maroni  account  Indeed,  (Maroni i n the  amount  t h e amount  of  of  evidence  sex-linked  1980;  of  diffusible  on t h e t o t a l  Recently,  of  can adequately  a  or decreases  because  constant.  strains.  of  i s modulated by  compete  1973).  each  E v i d e n c e has been  genes  remains  i n trisomic-2L  of  amount  for the latter  model  by p a r t i a l l y  limited  (Maroni and L u c c h e s i ,  Either  genes  the transcription  from the X-linked  substance  the  a  Schwartz,  of  compensated,  transcription  repressor  is  A s t h e number o f X  and against  that  produce  X chromosomes  transcription  these  suggests  nonlinked  and  X-linked  of  and both  One model  postulates  (compensation).  1980)  for  on a c t i v a t i o n  not dosage  1950).  a n d t h e amount  why s h o u l d  hypothesized,  model  the concentration  of  and, second,  i s based  other-  (Muller,  (Birchler,  model  of  mechanism  transcription.  themselves  decreases  the existence  been  The f i r s t  loci,  b y what  Two m e c h a n i s m s  of  and t h e o t h e r  the activity  increases  occur  have  the level  transcription  chromosomes substance  at  X-linked  First,  aneuploids?  expression.  modulate  1975)  compensation  compensation  on r e p r e s s i o n  X-linked  The  dosage  presumed t o  based  two q u e s t i o n s .  f o r autosomal  sex-linked are  raises  autosomal  exist  activity.  has  dosage  and P l a u t ,  for the  compensation  Rawls and Lucchesi  chromosome  2L a r e  capable  H-5  of  reducing  regulatory It for  males  activity  loci  is  the  the  not  may e x i s t  females  in  generally  autosomal  dosage  autosomal dosage  would  be  of  compensation  genome.  a  even  allows in  vary  for  cope  mechanism e x i s t s  to  compensate  which occurs  organisms. i n number,  between  However, it  is  not  because o b v i o u s why  should exist.  The f a c t  occur  X chromosomal  for  both  speculation  individuals  mechanism.  diploids.  that  control  overall is  this  and  in Drosophila,  simply reflects  What  may s i m p l y b e with extreme  that  Such a  modulating the  i n normal  to  the  these  in trisomies  forced  a  negative  2L.  templates  can  homeostatic  responsible  expression,  mechanism  do not  and compensation  compensation  existence  X-linked  compensation  trisomies  indicating that  on chromosome  i n many h i g h e r  autosomes  survival  6PDH,  surprising that  imbalance  and  of  mechanism  level  of  observed normal  perturbations  the  gene  as  regulatory within  the  CHAPTER  MEASUREMENT  OF  GENE  TWO  PRODUCT  AND T R I S O M I C - X  LEVELS LARVAE  IN  TRISOMIC—3L  4-1  Introduction  When s m a l l level  of  (Grell,  most  gene  1962).  accessibility in  turn  level  of  of  gene  a  gene  to  to  loci  possessing  product  et  levels  of  genes  activity  (Chandler  mode o f g e n e  of  situation  (Rawls  activity  f o r sex  1983).  On t h e o t h e r  trisomy  i s not expected  since  hand,  chromosome  examined  showed  duplications, levels  of  three  enzyme  This  altered genes,  the  i s reminiscent  i n metafemale X-linked  genes  required  possessing  determination  of  1974).  strategy  individuals  Belote,  arm o f  arm and c o n t r o l  of  final  and T o p o l ,  by r e g u l a t o r y  trisomies  compensation  which  the expression  loci  (diploid)  f o r compensation  an e v o l u t i o n a r y  between  Parker  the  individuals  i n small  and L u c c h e s i ,  thought  chromosomes  1983;  could be caused  Dosage  expression  apparatus,  i n whole-arm t r i s o m i e s .  individuals. as  may i n f l u e n c e  influencing the  While a l l f i v e  i n autosomal  observed  of  a l . ,  i n the duplicated  Compensation  dose  we e x a m i n e d  compensated  expression  loci  w i t h t h e gene  products  factor  et  chapter  enzyme  showed  are present  the  duplications for the left  when d u p l i c a t e d  compensated  gene  a r e examined,  the transcriptional  a l . , 1982).  elevated these  directly  i n Drosophila melanogaster  various  (Devlin  varies  be an i m p o r t a n t  In t h e p r e v i o u s  several  which  products  Many r e g u l a t o r y  appears  1984).  two  duplications or d e f i c i e n c i e s  different  (Lucchesi,  1983,  compensation  t h e number  to  of  of  the  (trisomic-X) may b e equalize  gene  numbers o f X Baker and  for  autosomes  autosomal normally  T8  does  not vary  compensation  within of  autosomal  reflect  the existence  control  gene  This  to  that  found  trisomies loci  dosage  dosage  compensate  Lucchesi,  determine  if  the response  autosomal  of  loci  were  studied  located  i n h y p e r p l o i d s may d i f f e r  compensation  between  males  and f e m a l e s .  of  which r  in six In and a l l  do not  do  i n D.  P i e r c e and  suggest  compensation  occur  Four  similar We h a v e  i n metafemales  and females  These observations  i s  dependent.  on 3L,  (Abraham and L u c c h e s i . 1 9 7 4 ;  1980).  can a l s o  Dj_ m e l a n o o a s t e r  males  1961).  compensation.  two a r e dose  to  autosomal  chromosome t h r e e .  Two g e n e s  between  see Seecof,  which  loci  i n 3L t r i s o m i e s  pseudoobscura  (also, to  the other  that  normally operate  the extent  arm o f  X-linked  compensated.  compensate  which  f o r X-chromosome dosage  while  proposed  i n w h o l e a r m t r i s o m i e s may  in diploids  and t o  for the left  five  We t h e r e f o r e  processes  compensation of  compensate  contrast,  genes  examines  occurs,  observed  that  of  expression  chapter  compensation  a species.  that  regulation  f r o m X chromosome  of  dosage  M A T E R I A L S AND METHODS  Production  of  Genetic and  Trisomies  symbols  are described  the nomenclature  arms  of  i n L i n d s l e y and G r e l l  individual  compound chromosomes  i s by the alphanumeric  system  The  stock  F (3R) V D 1 . e / F ( 5 R ) V D l  was  constructed  C(3R)VKl.e of  C(3L)VG1. as  shown  females  3  ru st.  T(3;4)e/TM3.Sb ftncompletus)  i n Figure  from a  Ser males.  ri  males  T ( 3 ; 4) e / T M 3 f e m a l e s were e s t a b l i s h e d .  6  °Co  Surviving  which were  euchromatic  breakpoints  these  been  we c a n e x c l u d e  confirmed,  right  arms  of  chromosome no  associated was  crossing  large  with  replaced  Trisomies  the second,  and t h e l e f t  additional  this  with  R standard  normal  to  F(3R)  i n that  no obvious  While the  half  translocation grounds  the right Thus,  have  the left  arm o f  the  6).  regions  not and  third  we a r e c e r t a i n  euchromatic  arm (see F i g u r e st  and crossed  possessed  on g e n e t i c  of  rads  r_J_ l i n e s  arm o f t h e X.  free  that  would be  C(3L)VZl.ri  by n o n d i s j u n c t i o n .  arm o f chromosome  three  are produced by  stt  F(3R)VD1.e /F(3R)VD1.e Oregon  this  probably  C(3DVGl.ru  C(3DVGl.ru a  of  8 3  to  lines  duplications  for the left  2500  were d e t e c t a b l e .  breakpoints  (1976).  (radius  cytologically  heterochromatic  and f r e e  C(3DVZ1.rii  source  and i n d i v i d u a l  chromosomes  5:  with  were c r o s s e d  One o f  b y Holm  e  were t r e a t e d  gamma-irradiation  suggested  (1968),  B  t o an  strain.  Monosomies f o r 3L a r e p o o r l y  viable,  50  F i g u r e 5. S t r a t e g y employed to produce compound—free chromosomes f o r t h e t h i r d chromosome. Wavy l i n e s r e p r e s e n t t r e a t m e n t w i t h gamma r a d i a t i o n f r o m a C o source. In t h e s e c o n d g e n o t y p e t h a t was t r e a t e d w i t h r a d i a t i o n , the s t a n d a r d chromosome was m a r k e d w i t h t h e m u t a t i o n s r i a n d p . Genotypes at bottom of page a r e : A) C ( 3 L ) V G l , r u s t ; F(3R)VDl,e /F(3R)VDl,e and B) F(3L>VDl,ri/F<3L>VDl,ri;C(3R)VKl,e A O  f J  s  ! 3  e  52,  F i g u r e 6. C y t o l o g i c a l p r e p a r a t i o n s of chromosome F ( 3 R ) V D l e i n d i p l o i d and t r i s o m i c l a r v a e . Salivary gland polytene chromosome p r e p a r a t i o n f r o m a f e m a l e t r i s o m i c - 3 L l a r v a e . Note t h e t h i c k e n e d 3L e l e m e n t , and t h e a b s e n c e of s m a l l c h r o m o c e n t r a l duplications. B . M i t o t i c chromosome p r e p a r a t i o n f r o m trisomic-3L brain tissue. Note t h a t n i n e major autosomal arms, two X ' s and two s m a l l f o u r t h chromosomes a r e present. C. C y t o l o g i c a l v e r i f i c a t i o n of the of C<3L>VZl,riS F<3R>VDle /F(3R)VDle stock. E !  r a  f 3  3 J  5t and  are not recovered  within  t e n days  The  strain  C(3R) V K l . e pP/TM5  1 3  i n populations of t h i r d  development  25 d e g r e e s  were  gamma-radiation  as  treated  s  follows:  w i t h 2500  and crossed  F(3R)VDl.e /F(3R)VDl.e '  C.  r i  rads  of  to C(3DV61.ru sti  males and  E  F ( 3 D F (3R) / T M 5 h e t e r o z y g o u s  individuals  were  recovered.  A v i a b l e combination of F ( 3 D and F(3R)  chromosomes  was e s t a b l i s h e d .  C(3DP2.riiC(3R)VKl.e**  nondisjuctional  to  Dp(3R)173/Df(3R)a74 for  83DE;  are  produced.  The was  Keppy  stock  derived  a wild  was  introduced  standard  type  F(2D+  bearing  1979),  larvae.  a  or  to  individuals  trisomic  f o r 3R  T  chromosome was  F(2L)nub  g  and t h e C(2R)P.px  resulted  This  b pr chromosome  stock,  in fair  M o n o s o m i e s f o r 2R f a i l  to  Pom /In(3DP.Me  was c o n s t r u c t e d  2  as f o l l o w s :  when c r o s s e d  survival  F ( 3 R ) V D 1 . e ° / F (3R) VD1  strain  these  deficiency  The F(2L)pr  by n o n d i s j u n c t i o n .  The C ( 3 L ) V D 2 . L v  63  follows:  Oregon R s t r a i n ,  trisomic-2R  e  strain  By c r o s s i n g  F(2L)pr/F(2L)pr;C(2R)P.px  yjf_/yjf| as  to  1 3  recovered.  by r e c o m b i n a t i o n between  and  PgnrlJ  a standard  and Dennel,  constructed  were  (a s t r a i n  crossed  from which  C(3R>VKl.e  individuals  either  T h e s e were  males,  F(3L)VDl.ri/F(3R)VDl.ril  individuals  larvae  F <3L) V D 1 . r i / F ( 3 D V D 1 . r i I  was c o n s t r u c t e d  females  at  instar  A Lv st  hatch.  of  to  a  55  chromosome  was  recombinants These Pqm  were  constructed  from Lv st/Pgnr-  tested  for  c h r o m o s o m e was  a  a n d was  the  Pom  balanced  marker  12  were  to  C(3R)VD1.+  males.  A  and c r o s s e d  to  e  desired  stock.  Pam /TM3.Sb 1  pupae  can  Ser  Trisomies C(l)RM.y  (Kerkis,  black  mouthparts. Strains  1931),  were  cornmeal-sucrose of  streptomycin  Enzyme  1/2  on  a  stl  stl  is  for  to  possess  three  different  Pom. produced by  Oregon R males.  at  to  trisomic-3L  and metafemales  food  crossed  X—chromosome w e r e  raised  and  stock  25  were  degrees  c o n t a i n i n g 0.22%  C on  Larvae  crossing  were  identified  by  their  standard  Tegosept  and  10mg/l  each  from the  side  tetracycline.  Assays:  Wandering of  the  females  sexed  stock  which  variants  pn v / 0  this  produce  When t h i s  for  Lv  was  C(3DVGl.ru to  3  be r e c o v e r e d  electrophoretic  of  phenotypically Sb*  f  Ly  C(3DVD2.Lv  F(3R)VDl.e VF(3R)VDl.e' the  and a  C(3DVGl.ru  P g m ^ / I n ( 3 D P . M e Pam^ c h r o m o s o m e recovered  recover  over Females  and c r o s s e d  to  females.  In<3L)P.Me.Po(ir»+In<3R)C.Sb. irradiated  used  pint  nitex  third  culture screen.  instar  larvae  bottles, Larvae  washed  were  were  collected  in distilled  homogenized at  water  25mg  per  and ml  dried of  5b lOmM P D ice  linM p h e n y l t h i o u r e a ,  4 f  for  ten  minutes, 4  and  degrees  C.  pH 8 . 2 ,  then  allowed  centrifuged  minutes  at  diluted  extracts  to  extract  volume.  D u p l i c a t e measurements  f r o m w h i c h was  an  without  assay  followsNADH,  substrate.  AK: O.IM g l y c i n e ,  kinase,  pH 7 . 5 ,  NaPCU, 10~*  20u/ml  pH 6 . 8 ,  10  M eserine,  o-dianisidine malate;  lactate O.IM  0.2M  5  x  +  10-=  acid,  stop  p  0.2mM NADH,  b-HAD:  6PGD:  2mM f r u c t o s e - 6 - p h o s p h a t e , glucose-6-phosphate O.SmM N A D P ,  dehydrogenase, pH 7 . 8 ,  hypoxanthine,  PGI:  i.24mg/ml  3.3u/ml  lOu/ml  pH 8 . 5 ,  pH 7 . 5 ,  30mM  gluconic  50uM  1.3mM N A D P ,  O.IM T r i s ,  5mM M g C l - . ,  G6PDH:  6.6mM  2  dehydrogenase;  lu/ml  50mM  5mM M g C l ,  0.5mM  20mM  pH NADP,  PGM: O . I M T r i s ,  pH  7.5,  Glucose-6-phosphate  5mM g l u c o s e - l - p h o s p h a t e ;  ImM E D T A ,  20u/ml  0.5%  a-ketoglutarate;  NADP,  pH 7 . 5 ,  O.IM T r i s ,  3.ImM 6 - p h o s p h o g l u c o n a t e ;  with  pH 7.8,  lOmM  O.IM T r i s ,  2mM N a m a l a t e ;  MgCl?;,  0.2mM  CAT: O.IM  IM a l a n i n e ,  20mM MgCls,, 0 . 5 2 m M  NADP,  5mM  reaction  H 7.5,  lOmM N A D ; I D H : O . I M T r i s ,  lp/ml  as  20mM a r g i n i n e ,  dehydrogenase;  from  acetate,  acid,  MgCl-,  were  K C I , 20mM M g C l * ,  O . I M NaPCv,  GPT: O.IM NaP0„,  glucose-6-phosphate;  each  derived  conditions  on  M  57. S D S ; FUM:  pH 8 . 5 ,  from  value  ten  appropriately  dependent  w e r e made  blank  on  EST-6: O.IM  *M b-napthyl  dehydrogenase,  Tris,  7.5,  a  The assay  lactate  20mM ^ 0 2 ;  p—chloromercuribenzoic  Na  substracted  from  directly  lOmM A T P , 2mM p h o s p h o e n o l - p y r u v a t e ,  pyruvate PCU,  measurements  sit  12,OOOg f o r  Enzymes were measured  give  extract,  at  to  cytochrome-c,  xanthine  oxidase.  SOD: O . I M PO*,, 10-*M Rates  were  determined  57 by  changes  i n absorbance  IDH,  G6PD,  SOD;  and at  reduction extract and  P6I and PGM; a t  (measured  Fridovitch,  absorbance  were  240nm f o r C A T ; a t  550nm)  before  1969).  units  (1976).  Thus,  by t h e method  Expected  levels  from the parental  an expected  dose  o f enzyme  dependent  activity  found  i n Oregon R.  level  strain  A compensated  dependent  a l .  of  as changes  in  extract. (1952) in  or trisomies  chromosome  o f enzyme  plus  (McCord  is  one-half  estimate  that the  i s  estimate.  Electrophoresis  Extracts EDTA,  activity  activity  contribution  i n the compound-free  Enzyme  the a d d i t i o n of  i n the  of Lowry e t  found  the dose  o f enzyme  cytochrome-c  A c t i v i t i e s are expressed  activity  two-thirds  of  and a f t e r  protein  b-HAD,  550nm f o r E S T - 6 a n d  F o r SOD, t h e r a t e  p e r m i n p e r mg s o l u a b l e  estimated  arms.  at  340nm f o r A K , G P T , G6PDH,  to calculate  was a s s a y e d  Bradford  :  250nm f o r F U M .  was u s e d  Protein  at  pH 8 . 2  membranes,  of pupae  were  on C e l l o g e l  and s t a i n e d  r u n i n 89mM T r i s ,  (Kalex  Scientific)  f o r PGM a c t i v i t y  8 9 mM b o r a t e ,  ImM  electrophoretic  after  Shaw a n d P r a s a d  (1970).  Quantitation  For female  each  of heat-shock  sample,  third-instar  protein  salivary  larvae  synthesis:  glands  were  and incubated  at  dissected  from ten  37 d e g r e e s  C for 1  58 hour 6.8  i n Ephrussi-Beadle (PBEBS).  incubated  The glands  i n 2Qul  2Qul  were then  496  of Laemli  Laskey  days. bands  degrees  dissected  25  C.  were  laid  and counted  wandering,  2mg/ml  with  1/10  iso-amyl  a 35i s t a c k i n g  gel.  After  for fluorography X-ray  film  the gels  for  and  by s c i n t i l l a t i o n  (Bonner  seven  appropriate  counting.  larvae  were heat  F a t body and s a l i v a r y  separated  pH 4 . 5 ,  bentonite, After  shocked  glands  at  were  lOmM P 0 * ,  and f r o z e n  RNA w a s e x t r a c t e d 4ug/ml  at  -70  from t i s s u e  from  polyvinyl sulphate,  IV. S D S , 0 . I X  extracting  phenol:chloroform:iso-amyl  chlorform:  over  pH 7 . 5 ,  sample,  diethylpyrocarbonate. with  to  each  15V. S D S - a c r y l a m i d e  individuals in ice-cold  For each  heparin,  with  third-instar  C f o r one hour. from these  long,  From  10  RNA.  i n d i v i d u a l s i n 0.14M NaOAc,  50ug/ml  determined.  and exposed  ImM p h e n y l t h i o c a r b i m i d e , degrees  counts  were p r e p a r e d  o f heat—shock  Female, 37  dried  Autoradiograms  Measurement  minute  boiled for  the gels  were e x c i s e d  a 30  by s o n i c a t i o n ,  w i t h 0.38% DATD,  1974),  S-methionine  were d i s r u p t e d  cpm w e r e r u n o n a 28cm  electrophoresis, and  After  C and  s o l u t i o n was  a n d TCA p r e c i p i t a b l e  cross-linked  3 K 5  25 d e g r e e s  SDS c o m p l e x i n g  minutes  gel  20/tCi  at  (1970)  The glands  25,000  lmM P C U , p H  placed  Ci/mmol).  added.  sample  containing  PBEBS c o n t a i n i n g  (New E n g l a n d N u c l e a r incubation,  saline  alcohol  once  with  (50:50:1),  alcohol  (50:1),  t h e RNA w a s  volume of a c e t a t e  and 2.5  volumes  phenol, once  once  with  precipitated  of ethanol.  T h e RNA  59 was  dissolved  denatured agarose  in distilled  five  gels  minutes  containing  RNA w a s b l o t t e d hybridized hsp  (pPW229)  SDS. of  25 d e g r e e s  one hour  The b l o t s  time  linear  at  to  allow  range  translation  of  formaldehyde  1980)  et  2% f o r m a l d e h y d e ,  C, cooled (Rave e t  a n d r u n o n IX a l . ,  prehybridized,  simultaneously  a l . ,  1979).  with  The f i l t e r s  C i n two changes  to  densitometric the film. a l . ,  of  2  C i n two changes  were exposed  et  to  197*7).  The  and then  clones  for the  a n d f o r t h e h s p 7Q g e n e  65 d e g r e e s  (Rigby  made  65 d e g r e e s  nitrocellulose,  (pPW244>  (Holmgren  30 min a t for  to  (Thomas,  83 gene  at  water,  X-ray  film  scanning  of  DNA w a s l a b e l l e d 1977).  were washed  for  X S S C , 0.1% S D S , a n d of  0.1  X S S C , 0.1%  for variable t h e bands with  periods  in the  ^ P by n i c k  to RESULTS  In  the  several of  previous chapter  autosomal  chromosome  levels  loci  two.  from genes  Drosophila  of  within  i n an  Methods.  development and  for  (with  Since  individuals 3R w a s  locus. the  As i n  third  located their  for  a  deficiency  conclusions  done, our  corresponding  of  are  six  a  83DE)  for  6  both  and  in  whose  and t o  trisomic-2L  trisomies 2R  2R w e r e  for  survive  an  3R  very  analysis  limited  was  any  for  from  to  one  restricted  to  major  stage.  structural trisomic  dosage  genes  for  Estimated a  was  throughout  erroneous,  adult  and 7 ) .  dependent  of  Materials  for  for  analysis  individuals  (Tables  dose  our  the  enzymes,  enzyme  were  viability  comparison  trisomies  survive to  shown f o r  parents to  stage;  left  2L.  individuals  observed  and o b s e r v a t i o n s  larval  to  on measurements  study,  the  chromosome  Their  In  based  arm  described  v i a b i l i t i e s may b e  previous  activity  diploid  limited  third  6).  for  for  w h o l e arm t r i s o m i e s  trisomic—3L  that  of  phenomenon f u r t h e r ,  not  the  individuals.  arm r a r e l y  on 3L  to  compensation  measured.  is  (Figure  comparable  instar  chromosome The  verified  with poor  not  trisomic  Oregon R background as  without  poorly.  of  is  been  of  dosage  additional  loci  strains  trisomic-X  or  have  The s u r v i v a l  cytologically  this  three  autosomal  Compound-free  and  individuals  To examine  melanooaster  Compensation  generated  in  we o b s e r v e d  3L  are and  for  values compensated  (diploid) and  level  Methods).  Sod,  Pqm)  of  expression  Four  had  of  levels  of  individuals  consistent  (Table  Two l o c i ,  6).  dependent  levels  observations  are  approximately while  the  of  the  loci  the  were  (Est-6  was  3L  were  As  noted  of  i n the  located  limited  ordered,  above,  are  located  Curiously,  a  How d o e s individual  arm  that  two-thirds  of  for  all  3R  compensation  loci  Simplistically,  to  gene  gene  all  observed. loci  at  product  of  are  on  3 D . and  associated  measure  one  (Table  one-third  the  three  measured  (Pai)  proximal  occur  of  survive poorly,  able  This  i n the  not  two-thirds  we w e r e  of  not.  compensating  separation was  were  proximal  (although  level  loci  the  non-compensating  the  individuals.  located  was  characteristically  Nevertheless,  dosage  gene?  within  2R a n d  is  trisomies  condition  loci  both  for  too  2L:  and  proximal  regional  trisomies  dose-dependent it  a  proximal  in trisomic-2R  in  d i s t r i b u t i o n of  the  aneuploidy.  displayed  the  the  within  with locus  distal  with 2L,  chromosome  d i s p l a y phenotypes  trisomy  compensating  whereas  and  case  they  These  compensate  from dose-dependent  to  for  expression  dose  (Table 7).  loci  i n the  the  the  displayed  trisomic-2L  investigation,  less  of  dose-dependent.  compensated,  and Cat)  one-third  are  Materials  AJk,  levels  found  d i s t r i b u t i o n of  dosage-compensated as  and C a t ,  of  present  However,  Est-6  r  (see  trisomic-3L  w i t h compensated  two-thirds  loci  presented  (Idh  in  those  Genes  appears  activity  to  non-compensating  In  loci  similar  The r e g i o n a l  chromosome  six  also  enzyme a c t i v i t y  remainder  non-random.  the  are  level  of  alleles  of  8). of  2R.  the a  single,  Table 6 Enzyme l e v e l s trisomic-3L larvae.  f o r compensating  loci  Enzyme  i n d i p l o i d and  Locus  Genotype  Idh  Ak  Sod  Oregon R (diploid)  0.622  14.2  0.172  .022  ± 0 . 6  ±  (6)  C(3L)VG1, r u s t l F(3R)VDl,e / F(3R)VDl,e  ±  13.2  1.29  .009  :  -09  (4)  (6)  0.156  1 . 12  (7)  0.529  Pgm  s  s  ±  .022  .04  :  (6)  C(3L)VG1, r u s t / +/F(3R)VDl,e (trisomic-3L)  t  (6)  0.539  .006  ±  -11 (6)  (4)  0.97  0.187  15.4  s  ±  .012  :  0.6  (6)  t  (7)  .007  ±  .11  (4)  (6)  Estimates dose-dependent  0.840 ±  compensated  .012  0.560  t  .008  20.3 +  .2  0 . 107 ±  13.5 ±  .1  ±  .001  1.77 +  .06  0 . 161  1. IB  .002  • .04  A c t i v i t i e s e x p r e s s e d a s t h e Mean ( ± s t a n d a r d e r r o r ) c h a n g e i n a b s o r b a n c e p e r mg p r o t e i n p e r m i n . e x c e p t f o r S o d w h e r e t h e a c t i v i t y i s expressed as the concentration of extract protein r e q u i r e d t o g i v e a 50% r e d u c t i o n i n r e a c t i o n v e l o c i t y . Number i n p a r e n t h e s i s i s t h e sample s i z e .  fc3 Table 7 Enzyme l e v e l s trisomic-3L larvae.  f o r noncompensating  Enzyme Genotype  Est-6  Oregon R (diploid)  0.663  +  loci  i n d i p l o i d and  Locus Cat  .023  6.74  (10)  C(3L)VG1, r u s t ! F(3R)VDl,e / (diploid)  0.340  C(3L)VG1, r u s t / +/F(3R)VDl,e (trisomic-3L>  0.737  +  +  .45  (6)  .010  6.18  ±  .25  s  (10) +  (6)  .002  8.61  +  .61  s  (10)  (6)  Estimates dose-dependent  0.672  +  .008  9.55  +  .17  compensated  0.448  +  .005  6.37  +  .12  Activities absorbance the sample  e x p r e s s e d a s t h e Mean ( + s t a n d a r d e r r o r ) c h a n g e i n p e r mg p r o t e i n p e r m i n . Number i n p a r e n t h e s i s i s size.  ti-  Table  8  Enzyme l e v e l s  in  diploid  and t r i s o m i c - 2 R  Genotype  larvae. PGI  Oregon R (diploid)  2.04  +  0.08  (6)  F(2L)pr/F(2L)pr/C(2R)P, (diploid)  F(2L)pr/+/C(2R)P, (trisomic-2R)  px  2.75  +  0.23  (6)  px  3.76  +  0.19 (6)  Estimates dose-dependent  3.77  +  .12  compensated  2.51  +  .08  Activity  expressed  as  in  Table  6.  lo5  dosage  compensated  diploid  level.  expressed  allows  Alternatively,  gene, a  Pgm, e x i s t s  variants  constructed.  equivalent genes  apparently  Since  measurements  showed  diploid  at  7 clearly  one can i n f e r  gene  i n trisomic-3L  the  o f enzyme a s d o e s  X-linked  gene  activity  For  most  genes,  of  gene  product.  despite  the difference  Several  studies  1975;  Gvozdev  (metafemales) gene  products.  Faizullin loci  et  extracts in  larvae,  larvae  approximately a l l  three  level  i n these  from  o f PGM.  individuals  e a c h Pgm  produced allele  only  in a  two-thirds  diploid.  metafemales  and females  et  a l . , 1983)  However,  that  that  a l . , have  express  1974s  a n d ZJJ) p r o d u c e  equivalent  have dose  as  Stewart  diploid  that  reported dependent  that  the  sexes.  and Merriam, trisomic-X  levels  results  amount  well,  number between  revealed  contradictory  (1973)  produce  f o r X - l i n k e d genes  i n X—chromosome  individuals  different  allozymes  t h e same  i s true  (Lucchesi  and Gvozdev  (6-Pod  in  males  This  shows  PGM a c t i v i t y  structural amount  t h e s e two  equally to the total  of t o t a l  could be  form and  with  in trisomic-3L  contribute  the  t h e 3 Pgm c i s t r o n s  a l l three  Thus,  levels,  of  of  by t h e  i n a monomeric  each  possessed  amounts.  alleles  t o d i s t i n g u i s h between  Figure  individuals  two-thirds  the three  Trisomic-3L individuals  electrophoretic  these  two o f  at  T h e enzyme e n c o d e d  simple test  possibilities.  were  could be expressed  preferentially.  compensating thus  gene  of  also  X-linked exist.  two X - l i n k e d  levels  of  4fe  F i g u r e 7. Electrophoretic a n a l y s i s of t r i s o m i c - 3 L pupae. 1. d i p l o i d , Pom / T M 3 . S b S e r Pom* 2. trisomic-3L, C < 3 L ) V D 2 . L v P a m - / / I n < 3 L ) . He P o m * / PomVF(3R)VDl.e '. 3. d i p l o i d , C<3L)VD2.Lv Pom^/ In ( 3 D P . He P o m * ; F(3R)VD1.e / F(3R) V P l . e " 1  £ ;  m  1  PBM i n  diploid  and  enzyme i n metafemales.  To examine gene e x p r e s s i o n i n  metafemales, we measured gene—enzyme systems.  t h e a c t i v i t y of f i v e  In a d d i t i o n t o 6-Pod and Zw  ( G 6 P D ) , we have examined t h e e x p r e s s i o n of Fum Had, t h r e e l o c i i n metafemales.  X—linked  Bpt  T  t h a t had not been i n v e s t i g a t e d  r  previously  The data f o r males, females and metafemales a r e  presented i n T a b l e 9 .  In metafemales, 6-Pqd and Z_w_  show d i p l o i d l e v e l s of a c t i v i t y , a l t h o u g h t h e former i s s l i g h t l y higher than expected.  The t h r e e other l o c i (Fum.  Opt. Had) were a l s o compensated  i n metafemales.  The  s t r u c t u r a l genes f o r t h e s e enzymes a r e w i d e l y d i s t r i b u t e d much of t h e l e n g t h of t h e X-chromosome.  along  F o r b-HAD, d e s p i t e a  marked d i f f e r e n c e i n a c t i v i t y between males and females, an a p p r o p r i a t e compensated  response was s t i l l  would appear t h a t many l o c i  observed.  Thus, i t  on t h e X-chromosome a r e c a p a b l e of  dosage compensating i n metafemales, a s w e l l as i n males and females.  Heat-shock genes a r e d i f f e r e n t i a l l y expressed i n t r i s o m i e s .  The heat-shock genes comprise a group of c o o r d i n a t e l y c o n t r o l l e d genes t h a t a r e a c t i v a t e d i n response t o environmental stress.  A f t e r heat shock, t h e s e genes d i s p l a y an a l t e r e d  chromatin s t r u c t u r e and t r a n s c r i b e a t very r a p i d r a t e s . final  The  p r o d u c t i o n of heat-shock p r o t e i n s r e s u l t s from marked  a l t e r a t i o n s both i n t h e t r a n s c r i p t i o n a l and t r a n s l a t i o n a l processes.  In Pj_ pseudoobscura  r  a t l e a s t one of  Table  9  Enzyme 1 e v e l s  in  males,  females and metafemales. Enzyme  Locus  Genotype  Pgd  Fum  Gpt  Had  Zw  +/0 (male)  0.158  0.258  1.50  0.243  0 . 141  ±  .012  ±  (6)  C(1)RM, y pn v / Y (female)  .009  0.295 +  (6)  C(1)RM, y pn v/+ (metafemale)  .018  +  (4)  0 . 175 ±  ±  (4)  0 . 143 ±  .003  .019  ±  .014  +  .009  (5)  (6)  1.19  0 . 129  0 . 150  .07  ±  .14  .007  ±  (6)  1.45 +  .011  (6)  (6)  0.318 ±  -17  (6)  0 . 158 ±  .007  (6)  (4)  (6)  (6)  0.222  0.424  1.94  0.251  .011  0 . 147 ±  .014 (6)  Estimates dose-dependent ±  compensated  ±  0 . 148 ±  Activity  .005  expressed  .003  as  .009  ±  0.283 ±  .006  i n Table  6.  .06  ±  1.29 ±  .04  .005  0.221 ±  0.167 ±  .003  .006 0 . 147  +  .004  10  these  heat-shock  dosage  compensates  Luccchesi  chromosomal  68  70,  hsp  protein  genes  are  is  determine  whether i n 3L  synthesized  in  they  found  are  the  three  expression  affected  by a  one—fifth of  final  for  of  the  3  is  ratio  of  e f f i c i e n c i e s of  3L.  into  found  if  these  the  of  amount  This  located at  87C.  dosage of  each  37  hsp  degrees  C  and  three,  strains  70.  five  used  of Two  quite  result  similar suggests  that  not d i f f e r e n t i a l l y  is  hsp  the  heat-shock  total  approximately  70.  proportional  translation  hsp  h s p 68  the  are  Second,  equivalent  The  are  chromosome  hsp  ratios  are  68  genes  incorporation to  hsp  while  were  the  in all  h s p 68 for  950  i n c u b a t i o n at  examined.  these  only  of  the  at  proteins  arm o f  relative  directly of  located  Since a l l  right  chromosome  proteins,  87A a n d t h r e e  after  two genes  =S-Met  amount  each  be expected  similar  these  P.  production! at  and  chromosome 3.  we m e a s u r e d  First,  strains  of  product  templates would  of  70  glands  duplication  incorporation  arm o f  3L h e a t - s h o c k  h s p 68  (Pierce  same  s i n g l e gene hsp  shows t h e into  in  and  p o s i t i o n 63BC on  equivalent dosages  apparent.  all  right  on t h e  o n XR)  two heat—shock  Figure 8a).  at  10  S-methionine  between  salivary  83  and f e m a l e s  on t h e  two g e n e s the  located  Table  points  for  trisomies,  (see  are  are  here. 3 e 5  hour  (hsp  cytological  on t h e  with  compensated  genes  at  found  responsible for  To  one  males  encoded by a  two c l u s t e r s ,  for  is  f  genes  located  in  70  83  element,  are  X-linked  The homologous gene  The s t r u c t u r a l  and  is  between  1980).  p  melanooaster.  3L.  genes  Therefore, to  the  proteins.  levels were  of  the  number This  amount  of value  transcription  involved  in  the  and  11  F i g u r e 8. A u t o r a d i o g r a m s o f h e a t - s h o c k p r o t e i n a n d RNA f r o m s a l i v a r y glands. A. Heat-shock proteins 1. d i p l o i d , c o m p o u n d free stock 2. d i p l o i d , O r e g o n R 3. t r i s o m i c - 3 L B. Heat-shock RNA 1. t r i s o m i c - 3 L 2. d i p l o i d , c o m p o u n d - f r e e 3. diploid, O r e g o n R.  72,  I  73 T a b l e 10 Heat-shock trisomic-3L salivary  protein glands.  68 a n d 83 s y n t h e s i s  Heat Genotype  Shock  i n d i p l o i d and  Protein  68  Oregon-R (diploid)  0.222  83  +0.11  0.030  (11)  C(sl)VGl, ru stl F (3R) V D 1 , e / F (3R> V D 1 , e ^ (diploid)  0.233  C(3L)VG1, r u s t / + / F(3R)VDl,e (trisomic-3L)  0.224  +  +  .004  (10)  .016  0.042  +  .005  s  (10)  +  (10)  .012  0.039  +  .004  E I  (14)  (14)  Estimates dose-dependent  0.057  +  .003  compensated  0.038  +  .001  The i n c o r p o r a t i o n i n t o h s p 68 i s e x p r e s s e d r a l o n e w h i l e hsp 83 i s e x p r e s s e d r e l a t i v e t o i n t o h s p s 68 p l u s 7 0 . The values given are standard error. Numbers i n p a r e n t h e s e s a r e  elat the the the  i v e t o hsp 70 incorporation mean + sample s i z e s .  production levels the  o-f  o-f  these  production  three  strains,  observed  within  serve  internal  the  as  hsps  83  level  is  strains. trisomies  is  heat-shock the  four  four  are  observed  strain,  by of  loci  it  a  appears  small  located  for at  within  oriented  i n both  directions  a  differential  hsp in  linked,  26  and  hsp  trisomic—3L  compensated  and 23  If  clearly small  hsp's  mechanisms To  the  not could of  26,  hsp  and hsp from a  of  22  response response  for All  Although showed  dose  rates  are  over  of  not  would  partial for  hsp  these  two  on t h e  combined,  observed. known.  make a n y  of  dependent  response  recovery  is is  and 22).  showed o n l y  migration and 23  23  coding  showed an  discrete  expression  diverse  heat-shock  22  genes  Another  T h e amount  was  23  from imprecise  hsp  dose.  genes  11).  glands  However,  these  1980).  hsp  (Table  in  transcription  a l . ,  weight  similar  gene  27,  with  et  10.  trisomic-3L  contains  (hsp  salivary  of  level  show t h i s of  in  deviations  expression  was  amounts  in Table  production  region  trisomy  due t o  between  response  relative  increased  (Corces  resulted  compensated  complexity of  the  83  in  on 3L,  expression,  These  proteins  the  67B  individuals. of  70  two p r o t e i n s  shown  level  hsp  12kb  to  formed  may h a v e  heat-shock gels.  27  a  and hsp  these  also  low m o l e c u l a r  response  level  compensation. 22  the  is  proteins  encoded  tightly  that  equivalent  3L. hsp  are  68  quantify the  that  heat-shock  hsp  Because  differential  diploid  compensating  puff,  to  on  another at  a  proteins. for  we f e l t  standards  expressed Thus,  heat—shock  and because  a  encoded  The Hsp  two  SDS  a  How  the  The of  a  number  possible.  investigate  whether  the  regulation  of  hsp  production  in  15 T a b l e 11 Small heat-shock p r o t e i n trisomic—3L s a l i v a r y glands.  synthesis  Heat Genotype  27  Qregon-R (diploid)  C(sl)VGl, ru st; F(3R)VD1,e*VF(3R)VD1, i (diploid)  Protein  .004  ±  0.343  .009  ±  (11)  (11)  0.061  0.265  0.327  .018  fc . 0 2 1  0.093 ±  .026  +  ±  (10)  0.316  0.244 +  ±  .023  .005 (9) 0.088  t  .037  (10)  .034  0.064  .054  (11)  ±  22  23  0.151  (8)  C(3DVQ1, ru st/+/ B F<3R>VDl,a (tri somic-3L)  Shock 26  0.033 ±  i n d i p l o i d and  .003 (10)  0.102 +  .008  (14)  (14)  (14)  (14)  0.078  0.341  0.480  0.120  Estimates dose-dependent  t .008 compensated  0.052  t  The i n c o r p o r a t i o n p l u s 70.  ±  into  h s p 68  .005  .010  +  0.227 +  .006  i s expressed  .023  +  0.332 ±  .015  relative  .002  0.080 +  .001  t o h s p 68  lb  3L  trisomies  measured and  the  from f a t  tissues to  is  the  the  were hsp  ratio  mediated  body.  Northern blots  85  and t h e the  two  scans  data,  as  as  It  clear  well  somewhat  lower  hsp  the  than  70  the  level  obtained  trisomies  of  hsp83  diploid  levels  results  imply  same and  rate that  the  regulated that  at  the  that each  of  hsp is  83  hsp  determine  that  is  83  gene  to  affected  or  synthesis  would  measure  If  the  internal  reduced)  absolute  although  were  dose-dependent while  are  the  only  found. at  These the  number o f  templates,  protein  83  in trisomies  level.  It  is  (23  a n d 22)  although  I  have  be  a  trisomic  dose  of  hsp  Since 83  70)  also  not  level  this  both  genes  condition  dependent  observed.  levels  (hsp  of  attempted  one  product  the  hsp  is  (either of  hsp  experiment  R N A , we c a n n o t  is  possible  is  when m e a s u r i n g  transcription  standard  s i m i l a r l y by the  increased  values.  here.  another.  and t h e  of  these  a  transcribed  hsp's  A possible complication exists relative  with  Thus,  post-transcriptional  post-transcriptionally,  shows  Equivalent results  heat-shock  small  12  RNA i n t r i s o m i e s ,  regardless  the  from  in trisomies  gene  these  and dose-dependent  examined.  83  glands  corresponding  Table  consistent  we  and  determined  corresponding protein  the  for  8b),  autoradiograms.  observed  production of the  RNA f r o m  probes  (Figure  was  transcription  from s a l i v a r y  total  expression.  strain,  compensation  regulated to  in  of  RNA w e r e of  genes  expected,  level  levels  of  compensated  dosage-dependent i n both  gene  RNA i s o l a t e d  RNA l e v e l s of  o-f  simultaneously to  expected  that  83  level  of  densitometric  is  hsp  the  levels  hybridized  of  at  both 8 3 RNA did  resolve  not this  17 T a b l e 12 glands.  Heat-shock  RNA 8 3  levels  i n -Fat b o d i e s  and s a l i v a r y  Ti ssue Genotype  F a t Body  Oregon-R (diploid)  0.284  Salivary  + 0.39  0.314  <4)  C(3L)VG1, r u s t ! F(3R)VD1,e*VF (3R)VD1,e (diploid)  C(3L)VG1, r u s t / + / F(3R)VDl,e (trisomic-3L)  0.316  ±  Gland  ±  .027  (4)  .056  0.333  ±  .034  r a  (4)  0.428  ±  (4)  .062  0.457  +  .044  e i  (5)  (6)  Estimates dose-dependent  0.458  +  .029  0.490  +  .018  compensated  0.305  +  .019  0.327  ±  .012  h s RNA 8 3 l e v e l s e x p r e s s e d r e l a t i v e t o h s RNA 7 0 . Mean + standard error Numbers i n p a r e n t h e s e s a r e t h e sample s i z e s .  IS  difficulty. hsp or hsp by hsp  83  gene  responds 70 the 70  genes  However, either  are  remained  say  unaffected  similarly to  the  unaffected  observation  individuals.  is  we c a n  that  the  unchanged  hsp  that  by t h e 70  amount  transcription  trisomic  genes.  by t r i s o m y  between  the  of  for hsp  diploids  That 3L 68  is  the  condition the supported  protein  and  of  relative  trisomic—3L  DISCUSSION  Normally, dosage  a direct  and gene  Investigations that  correlation  product of  i s observed  i n a wide v a r i e t y  enzyme  levels  have  been  a r e a n e u p l o i d f o r whole chromosomes  The d i f f e r e n c e birds  and L e p i d o p t e r a n s  in  Z-linked  et  a l . ,  gene  1982).  observed 1970;  in ploidy  Scandalios,  Elevated  Tsaftaris  et  Scandalios,  levels  a l . , 1982;  Birchler  1975;  Wijsman and van den B e r g , 1975;  Magenis et  Francke,  1979;  Cox e t  1982;  1976;  K l o s e and P u t z ,  many o f  these  trisomies the  f o r gene  within  sets  a cell.  maintaining excess,  control  have  These  This  o n t h e amount implies that  particular  levels  small  changes  copies)  concentration.  of  of  gene  M c M i l l i n and and animals George and et a l . ,  1983).  dependent  In  fact,  response  indicate  in  that  transcription  gene  factors  a n d Ramage,  Epstein  and Benz,  for  been  1980;  1975;  observations  gene  Braverstock  N i e l s o n and  1981;  1980;  t h e dosage  a structural  limits  and that  used  1972;  1982)  a l . ,  a l . ,  Steinback  mapping.  (one t o t h r e e  effective  1983;  studies  a v a i l a b i l i t y of  generally  in  Kurnit,  have  of  difference  19791  Fobes,  and Newton,  (Marimo and G i a n n e l l i ,  chromosomes  (McDaniel  Carlson,  Smith and C o n k l i n ,  arms.  dependent  product  plants  1971;  species  o r chromosome  f o r t h e sex  gene  from several  1981;  organisms.  and T u r n e r ,  of  gene  made o n many  i n a dosage  (Johnson  and F r y d e n b e r g ,  1974;  exists  results  products  i n trisomies  Nielson  that  o-f  between  product  synthesized  responsible for  expression  i n the doses  of  are not s u f f i c i e n t  are  generally  t h e genes to/change  they their  30 In that, is  contrast,  this  study  i n D r o s o p h i l a , dosage  not r e s t r i c t e d  display  diploid  trisomies.  levels  of  compensated  (diploid)  gene-product  levels  been  observed  al.,  1982)  mechanism t h a t  enzyme  This  operates  activity  of  the loci  (dosage  on a l l t h r e e  revealed  that  copies  variants,  i t  this  transcriptionally  possible single by the  to  Since  cell  i n these  examining left  the activity  strains.  alleles  material  of  are expressed  variants  levels  1972;  by a  o f PGM Using copies  alleles  are active  issue  i s not  has been  which  is  molecule, cell,  with three  resolved on  in  and i f a l l  the ratios  of  (1:2:4:2:1).  separable a l l  in a  located  compensated  showed t h a t  of  trisomic  should be p r e d i c t a b l e  o f Gpdh  et  duplicated  a l l three  locus  in a single  occurs  of a  in  2 and i s dosage  strain  also  Devlin  i t  This  t h e Gpdh  bands  a trisomic-2L  electrophoretic  a l l three  of  Carlson,  enzyme,  GPDH i s a d i m e r i c  in the various  Reanalyses  active  individuals.  arm o f chromosome  trisomic-2L three  that  1971;  individuals.  that  PGM i s a m o n o m e r i c  demonstrate  has  1981;  a  of  Pearson  compensated  electrophoretic  individuals.  show  1981;  trisomies  i n trisomic-3L  were  compensation)  in  1982).  are found  locus  i n whole-arm  examined  and Newton,  i s clear  loci  individuals  (Schwartz,  i n autosomal  trisomies  Independence  and H e r e f o r d ,  Birchler  findings  autosomal  when d u p l i c a t e d  and S c a n d a l i o s ,  of genes  study  dose  1980;  Many  expression.  eukaryotes  Fobes,  i n whole arm  i n trisomic—2L  of  (Osley  McMillin  Compensation  or  on gene  and higher 1979;  9)  level  i n lower  a l . , 1982;  locus.  activity  Approximately two-thirds (see F i g u r e  Birchler,  compensation  t o t h e X—chromosome.  trisomic—3L  et  has confirmed our previous  three  F i g u r e 9. S u m m a r y b a r g r a p h o f e n z y m e q u a n t i t i e s i n 3L trisomies relative to diploids. Gene dose e x p r e s s e d 2 i n d i c a t e s a d i p l o i d l e v e l of enzyme a c t i v i t y . Gene dose e x p r e s s e d 3 i n d i c a t e s a dose-dependent quantity.  92  Q)  V) V)  O  tt  Q. H  UJ 2  tt CO  o Q i  tt c tt O  IDH  AK  SOD  EST-6  PGM  CAT  83 structural  genes  that,  though t h e expression  even  reduced the to  amount  changes  by  of  Moreover,  gene  synthetic  amount  1974).  gene  Thus,  trisomies that  does  copies  act  dose  i t  appeared  f o r 2L, to  were  observed  template  was  (compensating),  of  in  is directly  the.loci  be segregated  In c o n t r a s t ,  locus. that  for a  locus  The regulatory of  Indeed  a  particular  limitations  in diploids;  dependent  that  is,  on t h e  1972;  compensation  i n the  Bewley e t a l . ,  i n whole-arm  to transcriptional  processes  diploids.  compensating  subunits.  occurs  competition  gene  allele  measured  into  on an autosomal  t h e compensatory  on t h e w h o l e chromosome  and thus  the  third.  regulation  from  are not observed  i n trisomies,  chromosome  by one  1978).  the others.  that  i f  deficiency reveal  a compensated  likely  sensitive  in trisomies  for this  not arise  a l .  ie.  used  ( O ' B r i e n and M c l n t y r e ,  o n e - t h i r d of  not operate  trisomies  does  from a l t e r a t i o n s  not compensated  dosel  further  e q u a l l y on each  seems  normally occur  Gpdh  levels  w i t h t h e Gpdh  pathway  arises  Since are  by product  o f GPDH e n z y m e  structural  alleles  (Aragoncillo et to  We a l s o  the p o s s i b i l i t y that  in trisomies  appears  each  gene  was r e d u c e d  and i n d e p e n d e n t l y of  normal the  activity  the results  factor  cell.  individuals  structural  eliminate  individual  specific effect  Gpdh  inhibition  compensation  of  in trisomic  GPDH a c t i v i t y  feedback  i n each  enzyme p r o d u c e d r e m a i n e d d i r e c t l y  or null  results  between  of  i n the  deficiencies  These  active  b y two t h i r d s  total  amount  were  arm a s  separate  could be regulated i n 3L t r i s o m i e s  regions as  large  mechanism  a unit.  and non—compensating of  arm  In  loci the  chromosomal  compensating and  8f non-compensating o-f  t h e chromosome.  transcription trisomies regional does small  not exist heat  shock  to  However,  shock  rate,  protein  interaction  of  (Schlesinger suggested  is  found  a l . ,  gene,  h s p 8 3 mRNA may h a v e  translated  less  efficiently  than  a loci  on t h e  within  a  the  small  chromosome  partitioning  based  loci  that  do n o t . dependent  protein  83  et  shock  type  levels  synthesis  regulation  by a well  i s  occurs  implied.  processes  al.(1982a) protein  of  i n 3L  choreographed  have  may b e  regulation  h o w m u c h h s p mRNA  t h e amount  of  by autogenous  controls.  trisomies,  of 2L  could  h s p 83 p r o d u c t i o n i n  No m a t t e r  independent  as  dose  heat  of  c o u l d be r e s t r a i n e d or  at  This  levels  even  and t r a n s l a t i o n a l  the production of  individuals.  1983)  that  DiDominico  by a u t o r e g u l a t i o n .  the data  d i s t i n g u i s h between  occurs  1982).  regions,  throughout  post-transcriptional synthesis  glands  may b e h a v e  such  heat-shock  from a p a r t i c u l a r  accumulated Mackie,  et  to  and those  since  f o r compensated  trisomic—3L produced  exists  transcriptional  that  controlled account  location,  of  compensating and  no s i m p l e mechanism,  mRNA f o r h s p 8 3  a diploid  that  that  genes  are distributed  compensation  trisomies.  adjacent  regions  and noncompensating  i n 3L suggest  argues  loci  chromosomal  In a d d i t i o n ,  5).  separate  from s a l i v a r y  compensating  (12kb),  ThiB  chromosomal  Heat  for large  proteins  domain  Therefore  The  at  that,  into  measurements  chromosomes  (see Chapter  non-compensating  respond  not segregated  More i m p o r t a n t l y ,  d i s t r i b u t i o n of  independently.  arm.  were  on p o l y t e n e  indicate  chromosomal  on  loci  been  is  protein (Parsons and  After  heat  shock  degraded  more  rapidly or  h s p 70 RNA.  For  in  example,  85 DiDominico  et  70 s y n t h e s i s  a l .  <1982b)  after  heat  shock  degradation  of  that  compensation  dosage  control 1981;  h s p 7 0 mRNA.  or by s e l e c t i v e  Pearson  et  individuals  regulation,  this  other  chromosomes the  level  suggest  of  particularly the  ability  noteworthy t o dosage  EL. p s e u d o o b s c u r a evolutionary requirement In  this  normal  regard,  proposed  trisomies.  gene  at  and H e r e f o r d ,  protein  rates  f o r h s p 83 i n  whose  to explain  hypothesis  repressors  In view o f  h s p 83 gene  1980). arisen  from  shock  (Lindquist,  expression documented  can affect  they  how s e x - l i n k e d  that  the level  changes  acquired in  under  i s  brought that  i n the dose  was  dosage in  autosomal of  l i n k a g e group  (Muller  of  level  conditions.  the existance  of expression  is  1980).  compensation  influence  the  a  A simple hypothesis,  o n t h e same  at  The  i n the cytoplasm  postulates  encoded  mediated  i n Drosophila i t  during non-heat  trisomies?  polytene  the transcriptional  may h a v e  conditions  Measurements on t h e  five).  and L u c c h e s i ,  c o u l d apply t o dosage This  trans-acting  i s well  (Osley  the X-linked  h s p 83 c a n b e f o u n d  i n autosomal  compensation  It  that  interest  translational  i s t h e mechanism by which compensation  originally  genes  some  i s generally  (see chapter  for this  (non-stressful)  What about  for this  selective  by  transcription  compensate  pressure  of  of hsp  post-transcriptional  hsp production  (Pierce  by  While compensation  compensation  of  cessation  i s probably atypical.  and of  nature  can occur  may i n v o l v e  that  rapid  i s therefore  RNA d e g r a d a t i o n  transcription  autoregulatory  It  i n yeast  situation  mRNA l e v e l s  shown t h a t  i s accomplished  a l . , 1982).  trisomic-3L  of  have  et of  a l . , a  t h e gene  as  the  1931).  regulatory that  i t  8b  controls. genome,  F o r example  i n a systematic  R a w l s 8c L u c c h e s i  (1974)  observed  chromosomal |r e g i o n s ,  when d u p l i c a t e d ,  a  located  gene  enzyme s y s t e m  magnitude of conceivable regulatory same  these that  loci  nullify  since  19BI)  inversley  arm.  has been that  related  Adh-1.  is  produces  similar  doses  IL  of  hypothesis that  whose  strains  Plaut,  1973b;  19B3). products  t o the dose  levels  X-linked  limited The  X/A ratio  Maroni  activators limiting  In c o r n ,  of  they  this  to 0.33  One of  with  various  suggests  a r e encoded by  arms)  other  (Schwartz,  be c o n t i n u a l l y and 1.5  adjustable  (Maroni and and B e n t l e y ,  single  are compatible  with the  results  gene theory  from competition f o r  encoded appears  than  1971).  d i d not measure  theory  these  An a l t e r n a t i v e  19B0; W i l l i a m s o n  transcription  (1979,  were  compensation  influence  compensation  activator  IL.  be  would  enzmyes  1979).  (or chromosome  between  would  Birchler  of transcription  appears  on t h e  arm and c o n s e q u e n t l y  of dosage  studies  cells,  be located  tested.  Birchler,  they  trans—acting  i n genotypes  and L u c c h e s i ,  these  dosage  this  quite  concentration  number.  of product 1971S  i s  o-f  and the  compensation  o f chromosome  within  transcription  in single  dosage  of several  located  Although  would  the activity  on chromosomes  with  It  well  transcription  X-chromosome  33%.  often  the activity  linkage group,  particularly  rate-limiting activators  those  repressed  repressor  i n template  several  and negative  case,  f o r the regulation  located  that  In t h i s  (Schwartz,  genes  in  genes  the increased  observed  loci,  structural  the increase  hypothesis  approached  o-f t h e D r o s o p h i l a  that  o n t h e same  c o n t r o l l i n g them  chromosome  observed  changes  survey  on t h e  autosomes.  t o be l e s s  attractive  87 for  e x p l a i n i n g dosage  because For  of  the  example,  special  several  same a c t i v a t o r small  response the  so  segmental  activator  must to  that  in  the  observed  possible that  autosomal) the  Stewart  Gvozdev  et  included other had  of  the  loci  not  two  (Fum,  been  is  for  five  four  produced  of  levels  of  of  that  loci  similarities  in all  trisomies  by s i m i l a r  1975;  response.  X—linked  whose  on t h e  1973;  Williamson  with a  is  protein  for  one  very  more  strains.  It  and  However,  levels  Lucchesi  et  and B e n t l e y ,  we e x a m i n e d  of a l . , 1983;  the  These and  three  expression  metafemales.  measured.  It  is  mechanisms.  p r e v i o u s l y mentioned gene-enzymes  207.  discrete  (sex-linked  i n metafemales.  loci  single  between  and t r i s o m i c - 2 L  genes  consistent  a  in  located  X-linked  in  a  trisomy  genes  unlinked  and Gvozdev,  Had)  the  Furthermore,  reasons  Got,  to  arm f o r  these  approximately  compensated  some  by  observed  respond  For  examined  metafemales  that  conflicting reports  1983).  five  c o u l d be  predict  we n o t e d  controlled  and Merriam,  regulated  it.  3).  trisomic-X  (Faizullin  a l . ,  expression  in  be  accommodate  trisomies.  of  activation  chapter  contains  6 - P G D a n d G6PDH  would  compensation  c o u l d be  literature  1974;  autosomal  expression  (see  to  same chromosome  previous chapter,  compensation is  the  have  dependence  theory  genome;  in trisomies the  in  trisomies  required to  A l l genes  i n the  activator  elsewhere  In  dosage  observed  increase  rare  would  aneuploids.  arm would  i n autosomal  contrivances  genes  reside  be  limiting  compensation  The data  compensated A fifth product  noteworthy products  are  level  locus, than  that  we o b t a i n e d  6-Pqd  was while  observed  of  for  expression f  expected  for  a  compensated in  metafemales,  88  both  dose  been  observed  These the is  dependent  <Ananiev  observations  regulatory not  it  metafemales  is  not  the  expression  of  autosomal  i n metafemales.  that  dosage  a  complex  at  family  or  of  may b e  in male-female  recently  six).  in  does  the  is  Clearly,  compensation of  found  and f e m a l e s ,  from these  variations  precision  which  males  in  and  from  gene  resolution  it  of  we h a v e  chapter  in  a  X and  transcription  investigations  chromosome  influenced  an  dosage  number between  the  species,  major  by  number  is  several  an  element For  but  is  the  i n Dj_ p s e u d o o b s c u r a .  element  X=».  partially  This  exists neo-X  can  comprises  1952)  as  a  for  the  observed  between  sexes  is  elements  exist  either  example,  element  the  arm of  left  right  arm of  (chromosome as D  an (after  chromosome  the  Another  second  chromosome  compensated  compensation  chromosomal  X-chromosome.  and S t o n e ,  chromosome  only  three  as  X—chromosome  f  for  differs  between  apparent  lack  1974).  precision  compensation  euploid  Drosophilidae,  i n Dj. melanoaaster  miranda  is  chromosome  Between  Patterson three  in  least  autosome  It  of  females,  view,  (see  analogy  lack  X-linked  between  a l . ,  have  systems.  the  arms).  the  compensation  differences for  an  T  phenomenon and t h a t  regulatory In  LSP-la  this  hyperploids awaits  rate  and  dosage  with  a  transcription et  However,  males  i n metafemales  regarding  as  mechanism t h a t  compensate  compensated  conclusion in  by a  Consistent  completely  fully  involved. euploid  of  Lucchesi  interpreted  possible that  occurs  compensation. that  may b e  between  levels  al.,1974s  et  mechanism  observed  therefore,  and compensated  X—chromosome  appears males  to  i n D.  be  and f e m a l e s  (Strobei  et  89  al,  1978;  glimpse  at  Lucchesi dosage a  Das et  an e v o l u t i o n a r y  (1978)  compensation  behave  facilitate  hypothesis be  were  sex-linked the  Some response present have  element  dosage  If  D.  pseudoobscura  gene  on t h i s  to alterations  study  melanoaaster  pseudoobscura  dosage If  genes,  compensation  see Wright,  (RNA l e v e l s ) ,  then  by t h e a c q u i s i t i o n of  i n ploidy  when i t  trisomies  present  (Est-5  for  One o f  and females  trisomies of  1975). in D  f  and Abraham and L u c c h e s i ,  do not compensate  in  but do i n D. (Abraham a n d L u c c h e s i ,  1975;  P i e r c e and  i s  which  on  i n autosomal  males  1961,  could  and male-female  (involving  both  Est-6  such an  Three of t h e genes  are also  and between  this  problem by examining the  o r on t h e X.  in this  arms  o n t h e new X - c h r o m o s o m e .  (Abraham a n d L u c c h e s i ,  two other  then  of  compensation.  may e v o l v e  elements  compensates  P_JJ_ m e l a n o a a s t e r  a n d h s p 85  chromosome units,  arm o f t h e X i n JL. p s e u d o o b s c u r a .  of  1974)  the establishment  (X a n d a u t o s o m a l )  D i n j)^ m e l a n o a a s t e r )  pseudoobscura;  d i s c u s s e d how  dosage  of autosomal  can be shed  T  compensation.  a s two independent mechanisms,  regulatory  Idh  fortuitous  X chromsomes.  X-linked  compensation  measured  genes,  However,  become  then  trisomy  on an autosome  right  these  i f  o f t h e same  been  during  chromosomes.  arms  correct,  light  have  the a c q u i s i t i o n of s e x - l i n k e d  operate  necessary  (1978)  arisen  a  of dosage  independent regulatory  a specialized extension  compensation  D.  of sex  when t h e s e  Alternatively,  the  pair  allows  intermediate  may h a v e  inherently as  compensation  and perhaps  and Charlesworth  heteromorphic  might  a l . , 1982)  90  Lucchesi,  1980).  mRNA s y n t h e s i s trisomies, controls  during  a b i l i t y of  a gene  signals  distant gene  compensate  sequences) dosage  dosage they all  effect  located since to  with the notion  do not c l a r i f y trisomies  t h e gene  a l . ,  new s e q u e n c e s i n males  genes  occurs  i s not The  from  1981),  since  a l . ,  1983).  important  (with  for  flanking  its ability  1984).  The e v o l v i n g X  capable  of  in a  causing  In any e v e n t , Drosophilid and  by two s e p a r a t e of whether  to  fashion analogous  autosomal  to  these  species  are  male-females  mechanisms.  However,  or not compensation  and X-chromosomal)  an  dosage  retains  enhancers.  that  are  gene  in different  the issue  (autosomal  (Korge,  Scholnick et  an X—linked  transcription  compensation  gene  an autosome  transcription  on t h e X a r i s e s  1983;  near  for  chromosomes.  t h e X—chromosome c a n  (Hazelrigg et  may a c q u i r e  compensate  i n autosomes  i n sex  compensate  onto  to  Therefore,  compensate  compensation  dosage  on i n d i v i d u a l  concordant  dosage  transposed  eukaryotic  studies  to  transposed  hyper—active other  to  from the s t r u c t u r a l  compensate  chromosome  i n EL. p s e u d o o b s c u r a .  sequences  this  a q u i r e d new r e g u l a t o r y  a l l o w e d them  ( S p r a d l i n g and Rubin,  Presumably, mediating  p r o b a b l y have  f o r dosage  of  and h s p 85  i n Dj_ m e l a n o q a s t e r  evolution that  a gene  ability  autosomal  genes  number  prerequisite  protein  do not r e g u l a t e  these  chromosome the  Since Est-6  occurs  in  b y a common  mechanism. Organisms expression Rasch  et  Bicudo,  have  exploited  for variations  a l . , 1977; 1983;  Fraser  Gartler  several  i n gene  dose  and Nurse,  and Riggs,  1983;  means  to  modulate  (Casartelli 1979; Baker  Berger,  et  gene  a l . ,  1982;  and B e l o t e ,  1969; de C .  1983).  91 This  d i v e r s i t y alone  eukaryotic trisomies evolved  extensive response control  systems.  control  accomodate  find  suggests in  et  that  that  is  complexity  a  which have  i n gene (see  chapter  fundamental  reflects  some  euploidy.  1982). organisms  to  rates It  compensate  of  would  have  a  homeostatic gene  not  be  exploited  for  three)  is  of  of  this  that  in  and p e r f e c t e d occurring  system  diploids  s u r p r i s i n g though  naturally  the  gene  regulatory  expression  not  However,  property  compensation  arm  certainly  dosage.  A simple interpretation  a l . ,  of  Compensation i n whole  mechanisms  it  the  eukaryotes.  regulation  from  to  differences  maintaining balanced  (Devlin  of  testimony  and widespread e x i s t e n c e  hyperploid for  regulatory reveals  to  is  this  to form  deviations  CHAPTER  INFLUENCE  OF TRISOMY  MAPPING O U T S I D E  THREE  ON T H E E X P R E S S I O N OF  LOCI  OF T H E D U P L I C A T E D R E G I O N  0  °I3  Introduction  The  synthesis  of  functional  variety  of  pathway  has, t o varying degrees,  quantity require in  or  quality  the action  t h e genome.  affect  change the  1979;  and  1971;  gene their  product effect  (Rawls  Others  step,  (Kacser  such  act  utilizing  likely  confer  specialized  to various  (Banerji  et  al.,  et  1983;  "negative"  altering  Twardzik  a l ,  1981;  Where  the of  et King  final  a n d may t h e r e f o r e  the kinetics  exists  t o be q u i t e  sequences  response  Banerji  or  genes  to modify the  level.  1981).  et  common  exert  quantity a  f o r end product  The mechanism by which varied,  control  and  systems  can be  envisaged  1979).  Regulatory which  is  Johnson  1974a)  affects  and Burns,  which  regulatory  do not appear  the potential  "positive"  (Goldberger,  1977;  steps  are quite  1969;  the  elsewhere  m o d i f i c a t i o n or by  and L u c c h e s i ,  product  synthetic  for  modifiers  product  a  to regulate  coded  genetic  the transcriptional  regulation genes  by d i r e c t  and L u c c h e s i ,  a regulatory-gene  biosynthetic  or cofactors  gene  in a  Many o f t h e s e  (Ganschow a n d S c h i m k e ,  1983).  at  product.  active  involves  reaction  Some t r a n s - a c t i n g  product  Bewley  McDonald,  of  1980).  t h e gene  Each  trans-acting  levels  products  the potential  t h e gene  o f enzymes  in vivo stability  al.,  processes.  of  Thus,  the final  (Paigen,  of  interacting  gene  have  been  enhancement  physiological  a l . , 1981;  Dierks et  identified in of  transcription  or developmental a l . , 1981;  a l . , 1983;  Queen a n d B a l t i m o r e ,  Gillies  a l . ,  et  1983).  eukaryotes  Pelham, 1983;  in  stimuli 1982;  Chandler  et  94-  Cis-acting elements exert  enhancement  i n yeast  has been  effect  (Dubois et  its  X-chromosome  dosage  and r e q u i r e s  and  1980).  expression  transcription  shown t o a l . ,  require  1982).  compensation  transcription Lucchesi,  of  also  i n eukaryotes  of  involve large  1979,  and Tata  1981),  more  described  subtle.  one-third  seem  displayed  b y some  observation strain,  trivial  that  gene  of  of  two  examined  duplicated elsewhere families other  arm.  be r e p r e s s e d autosomal  much  of  alterations i s  activity  as  one of  stocks).  explore  the of  a  can be  well  and t o  of  as  within of  the expression  by J .  genes o f two  mediated  and t h e  transformation  P o s a k o n y who k i n d l y  that  the majority  mechanism i n a t  w h i l e few a c t i v a t i o n s  of  the  the  evolutionary origin  by P-element  We f i n d  genes  discuss  to  The previous  the expression  natural  by a t r a n s - a c t i n g  the extent  for Drosophila.  the expression  was p e r f o r m e d  trisomy,  on t h e o r d e r  individuals  in trisomies,  regulation  a r t i f i c i a l l y created  Drosophila  i s to  H e r e we e x a m i n e  genes,  (transformation the  this  regulatory  which were  paradoxical  a n d among  chapter  occurs  i n t h e genome of  more  gene  4).  this  implications  with the  the dramatic  v a r i a b i l i t y i n gene  repression  chapters  expression  Perhaps  of  (Belote  in  two c h a p t e r s ,  to  among s t r a i n s ,  (see chapter  The p u r p o s e which  compared  genes.  considerable  detected  i n gene  function  of  s e e Ashburner and Bonner  i n contrast  i n the previous  Reductions  gene  changes  (for examples,  to  Drosophila,  regulation  activity  effects  i n trans  i n v o l v e s an a c t i v a t i o n  transcriptional or Searle  products  In  trans-acting  Many e x a m p l e s  by T y l transposable  of  least  provided  loci one  expression  are  can  35  observed.  Modulation  can  regulatory  mechanisms  (such  or  developmental  genes  in  similar  cues) ways.  operate as  and does  independently of  X—chromosome d o s a g e not  other compensation  obligatorily affect  similar  Materials  Genetic  and Methods  stocks  and  Trisomies For  the  the  stacks  were  produced as  experiments  autosomal from  crosses  concerning  trisomies,  and  Oregon R stock  described X-linked  isogenic was  yield  the  chapters.  enzyme a c t i v i t y  X chromosome  introduced  by n o n d i s j u n c i o n , t o  in previous  into  in  (iso-X)  the  compound-free  following  stocks:  i s o - X / i s o - X ; C C 2 D V H 1 . It;F(2R)bw/F<2R) w and  iso-X/iso-X8C(5L)VGl.  ru  st;F<5R>VDl.e '/F<3R)VDl.e . E  This  latter  larval level  stock  of  expression  Adh  a  a  treated  osp  compound stock:  pr  males  C(2L)b  From which  these all  i n the  individual  b, A d h  stock  OSP  N : B  stock. was  bearing  of  in  null  the  2L mutations  for  cn  gamma i r r a d i a t i o n w e r e  A newly induced introduced  into  mated  C(2L)b a  produce  the  following  prsF(2R)bw  Females  an Adh gene  crosses  Adh genes  pr  involving  To measure  by n o n d i s j u n c t i o n t o  Adh"= osp  and n u l l  experiments  trisomies.  w i t h 2500 r a d s  Pin.  bearing  position,  i n 3L  chromosome  free  Pin/F(2R)bw  used  compound-free stock  compound s e c o n d  N 2 ;  also  of  was c o n s t r u c t e d .  females to  was  serum p r o t e i n s  trisomies, Adh  e  of  stock  transposed  mutations female  this  in the  to  is  a  to  chromosome  Adh  larvae  encoded  crossed  new  native  trisomic—2L  ADH e n z y m e a c t i v i t y  were  genes.  were  by the  selected  transposed  in gene.  Enzyme  Assays:  The in  assays  chapter  f o r PGK, GPDH,  2.  0.1M T r i s ,  The assay  pH 7 . 5 ,  dehydrogenase/ml, experiment  pH 9 . 0  Analysis  Serum  Larval  Larval  serum  For analysis  hemolymph  from wandering  experiments  PO*,  minutes,  Laemli  staining  a  with coomasie  scanned  with  amounts  of  a Beckman  individual  2mM N A D ,  were  analysed  1968).  i n two  third-instar  larvae  trisomies)  buffer.  blue  (Brock  L S P ' s were  of  females  collected  only  i n lOmM  an equal After  boiling  and R o b e r t s ,  1980). the gels  spectrophotometer.  expressed  as  for  (cross-linked  R250 a n d d e s t a i n i n g ,  DU-B scanning  samples  (using  were  r u n o n 97. S D S g e l s gel  electrophoretic  electrophoresis,  t o w h i c h was a d d e d  37. s t a c k i n g  In t h e  ADH e n z y m e  and Ursprung,  by SDS-gel  were  isomerase/ml.  Proteins.  SDS c o m p l e x i n g  t h e samples  with  3  autosomal  ImM P T U , p H 7 . 5  volume of  DATD)  with  M Na--C0 ,  (Sofer  proteins  systems.  in  triosephosphate  i n 0.5  0.275M b u t a n - 2 - o l ,  f o r A l d o l a s e (ALD)  glucose-6-phosphate  Adh t r a n s f o r m a n t s ,  was a s s a y e d  of  1 unit  10 u n i t s  involving  conditions  0 . 2 m M N A D H , ImM  fructose-l-6-diphosphate,  activity  described  1 and f o r A K , PGM,C A T , A O , M E , P G 1 , FUM, GPT a n d  G6PD i n c h a p t e r were:  ADH, IDH a n d 6-PGD a r e  10 with  After were The  the percentage  of  total  LSP i n the  measurements separated  of  by t h i s  .01%  in  method  the  pH 9 . 4  for  strips  were  stained  interest twelve  optical identify  were hours  density the  were  cut  on  SDS g e l  crosslinked  (Roberts  the  out  strips,  although  this  therfore  was  not  strips  in  not  blue,  was  destained  strips  LSP-lg, is  in  this  of  LSP-lb,  LSP-2, was  at  acetic  not  LSP-la,  acid  various  in  and  SDS  and L S P - 2 .  LSP-lb,  and  run  gels On  LSP-lg,  reproducibly resolved  system.  The  To  10b),  migration LSP-la  bands  1979a).  595nm.  (Figure  The  and t h e  SDS-complexing buffer  The o r d e r  protein  measured  into  measured  Cellogel  1979b).  and E v a n s - R o b e r t s ,  LSP on t h e  10C).  on  SOmM N a b a r b i t a l ,  and E v a n s - R o b e r t s ,  samples  migration  latter  were  for  hemolymph was  and e l e c t r o p h o r e s e d  Roberts  w i t h DATD i s  Cellogel  LSP-1,  and d i s s o l v e d i n g l a c i a l  and e l u t e d  (Figure  and L S P - l b  only  lOa).  with coomasie  particular  bands an  pH 1 0 . 0  (after  successful  ImM P T U ,  excised  of  LSP-la  cellulose-acetate  hours  was  and b s u b u n i t s of  bromophenol b l u e , Scientific)  for  a  7  3.5  analysis  (Figure  lOmM N a ? . B * 0 ,  (Kalex  of  This  L S P - 2 and L S P - l g :  To r e s o l v e collect  sample.  and  9<?  F i g u r e 10. Electrophoretic a n a l y s i s o f l a r v a l serum p r o t e i n s on c e l l u l o s e - a c e t a t e s t r i p s and i n S D S - p o l y a c r y l a m i d e g e l s . A. S e p a r a t i o n of LSP s u b u n i t s on C e l l o g e l membranes B. S D S - p o l y a c r y l a m i d e g e l of LSP s u b u n i t s . Lanes 1 and 6 are t o t a l hemolymph s a m p l e s f r o m l a t e t h i r d i n s t a r larvae. Lanes 2 t o 5 c o n t a i n LSP s a m p l e s i s o l a t e d f r o m C e l l o g e l membranes by e l u t i n g e x c i s e d b a n d s i n L a e m l i ' s SDS c o m p l e x i n g solution. C . SDS g e l o f t o t a l hemolymph f r o m : 1. O r e g o n R; 2 . 2 L trisomy; 3. compound-free d i p l o i d ; 4. Oregon R  |00  101  RESULTS  I  Enzyme  loci  Previous duplicated  investigations  have  shown t h a t  regions  o-f  w h o l e arm a u t o s o m a l  compensated  levels  of  gene  constitutes  a  outside  the  trisomic  fifteen  enzyme  the  comprehensive  loci  X—chromosome.  several  effects  Genes o u t s i d e  products.  arms. in  larvae  response  arm o p e r a t e s  structural  gene  by one  third  similar  levels  of  variation  in  X-chromosome  autosomal  genes.  by  a  mechanism as  females, located  then  in  If  trisomy  elsewhere  reduction  most  that  for  one  all  of  loci  of  of  2L,  has  genes  5).  genes  in  2R,  of  3L  and  revealed Drosophila.  within  a  of  each  M a l e s and suggesting  affect  autosomal  arm s h o u l d In  investigation  transcription  not  produce  repressed  for  operating  genome. of  be  products  number d o e s  compensation  i n the  expression  can  chapter  gene  one  the  expression  expression  observed  (see  the  analysis  by r e d u c i n g the  of  similar  for  this  duplicated region  duplicated  possess  of  can  expression  examined  a n e u p l o i d y on gene  The compensatory  the  trisomic  within  trisomies  The present  of  We h a v e  The r e s u l t s  of  the  survey  genes  between not  addition,  the  an  proportional  that  expression  genes males  affect  females  occurs and  genes  overall to  the  size  of  102  the  duplication  could  transcriptional quantities. suggests  their  all  activity  is  2)  gene  directed  trisomies,  to  to  the  Aldox.  located  enzyme  in it  activity other Pai  f  unlinked located  Therefore, is  not  the  In  2L  was  one  trisomies  to  of  genes  analysis  enzyme-loci,  of  four  present  was  gene  in  is the  (Table  13).  altered  levels  of  This  reader  six  quantities  30%  close  However, activity.  more  is  significant  trisomy  o b s e r v e d (ADH  also  gene within  that higher  by t h e  we h a v e than  to  the  in  other  The  condition  was  this  in the  trisomies.  autosomal  duplicated  performed with on t h e  of  one-third  expression  gene  expected).  trisomic-2L  The a c t i v i t y  in  occuring  The  each  of  at  are  enzyme  controls  by c l o s e  located  the  diploid  of  14).  was  are  diploids,  trisomies,  and  only^two activations  affected  reduced  larvae  activity  in diploids.  was  (Table  repression  limited  of  autosomal  locus  of  produced approximately than  chapter  by o n e - t h i r d .  in  for  limiting  next  which  expression  expression  displayed  o n 2R  A similar Seven  where  the  trisomies  2L  3R,  of  genes  These enzymes  estimates  repressed  loci  on 3R,  by an  in  and  all  incorrect.  presented.  3L  diploid  represents  gene-enzyme  was  in  in trisomic-2L  are  i n the  enzyme  expression  enzymes  of  and t h e are  no a l t e r a t i o n  12  to  present  this  P G M , C A T , a n d MEN) w e r e  two o f  since  Figure  in  shown a r e  1)  activity  observed  2L  may b e  on 2R,  represented.  <AK,  that  if:  competition  hypotheses  strains  Also  relative  graphically  six  located  expected  if  enzymes  of  strains.  that  these  parental  by genes  through  presented  of  activities  three  and  both  diploid  encoded  components  The d a t a  that  The  arise  trisomies  arm.  trisomies  autosomal  for  arms,  3L. were  103 T a b l e 13 trisomies.  A c t i v i t y of  third  chromosome  Enzyme  Genotype  y/y (diploid)  y/y8C(2L)lt, F(2R)bw/ F(2R)bw (diploid)  y/y;C(21)lt/ +/F(2R)bw (diploid)  Estimated Mean  loci  in  Pgm  Cat  Aldox  10.5  1.41  7.70  9.49  ±  .76  ±  .10  +  2L  Locus  AK  .64  ±  Men  .470  .45  .052  (5)  (5)  (5)  (5)  (5)  11.4  1.36  7.06  10.9  .395  ±  .91  .08  t  .54  ±  .86  ±  .063  (5)  (5)  (5)  (5)  (5)  10.6  1.27  8.06  13.2  .432  ±  .62  ±  .11  t  (5)  (5)  10.9  1.39  7.38  .49  :  .05  t  ±  .35  (5)  ±  Repressed Estimate  enzyme  .93  ±  (5)  (5)  -31  .050  10.21  .433  +  .034  .44  (10)  (10)  (10)  (10)  (10)  7.27  .927  4.92  6.81  .289  t  .33 (10)  .03 (10)  t  .21 (10)  ±  .29 (10)  ±  .023 (10)  Enzyme a c t i v i t y i s e x p r e s s e d as t h e change i n a b s o r b a n c e p e r m i n . p e r mg o f p r o t e i n . N u m b e r s i n p a r e n t h e s e s a r e t h e s a m p l e sizes. R e p r e s s e d E s t i m a t e i s t w o - t h i r d s t h e e s t i m a t e d mean. FF  104T a b l e 14 Enzyme a c t i v i t y 2L t r i s o m i e s . Genotype  for  a  2R-1inked  Enzyme  enzyme  locus  in  Locus  Pgi F(2L)b pr/F(2R)b F(2R)bw/F(2R)bw (diploid)  prl  7.59  +  .42  (6)  C(2L)ltlF(2R)bw/ F(2R)bw (diploid)  7.25  ±  .21  C(2L)lt/F(2L)b pr» F(2R)bw/F(2R)bw (trisomic-2L)  5.35  Estimated  Mean  7.42  +  . 12  Repressed  estimate  4.95  +  .08  (6)  +  .17  (A)  Enzyme a c t i v i t y i s e x p r e s s e d a s t h e c h a n g e i n a b s o r b a n c e p e r p e r mg p r o t e i n . N u m b e r s i n p a r e n t h e s e s a r e t h e s a m p l e s i z e s . R e p r e s s e d e s t i m a t e i s t w o - t h i r d s t h e e s t i m a t e d mean.  min  examined. seven 3L  Unlike  Gpdh.  Adh  found  the  3L.  trisomy  chromosome able  outside  trisomy  in  melanoqaster only  also  This of  a  type  arms  supportive  were  Muller's for  1931  evidence  because  not  the  to  all  has  of  on 2L,  strain major  would  are  was  was  males  the  one  that  dosage  gene  arms  on gene  by  i n D.  expression,  d u p l i c a t e d arm the  most  X-linked  has  express  but  common  one-third.  trisomic  few m o d i f i e r s o f  and f e m a l e s  we  located  trans-acting  It  arm  17).  repressors condition.  repressors  compensation,  obtained.  than  affected.  right  repressed  By f a r  if  loci  equally  by a p p r o x i m a t e l y  result  proposal  other  poorly viable  autosomal  the  that  (Men a n d  more  for  only  than  duplication  (Table  within  activity  bears  (Pgk.  The  aneuploids are  located  been  loci  in  intermediate  repress  loci  examined  Gpdh  X-chromosome  the  activity  loci  16).  b e i n g d u p l i c a t e d by the  X-chromosome  expression  in  of  of  trisomies  by the  linkage groups.  repression  genes  responsible  or  (Table  striking effects  genes  reduction  of  Since  the  have  for  the  activity  appears  trisomic-2L  on t h o s e  is  these  that  can  of  expression  gene,  trisomy  on o t h e r  effect  This  this  3L  these  the  of  these  unaffected  trisomy  Because  2R.  in  values  for  six  levels  The two o t h e r  although  measure  Clearly,  not  2L,  two.  of  15).  was  f  autosomal  to  one-third  Aldox  Four  2L,  approximately  displayed levels  for  last  produced  (Table  for  showed r e d u c e d  diploids.  estimated  gene,  Thus,  The  were  two  to  observed  enzyme a c t i v i t y  o n 3R)  autosomal  does  the  in diploids  between  of  relative  of  both  situation  systems  and P a i )  r  one-third  Ald.  the  gene-enzymes  trisomies  105  little  been  inferred  autosomal most  are  genes  gene equally.  10b T a b l e 15 trisomies.  A c t i v i t y of  second  chromosome  enzyme  loci  Genotype  Pgk  aGpdh  Adh  Oregon R (diploid)  5.17  1.64  0.388  ±  C(31_)VGl,rust/ F(3R)VDl,e / F(3R)VDl,e (diploid)  .35  +  .03  ±  .021  in  3L  Pgi  8.05 +  .52  (5)  (6)  (6)  (6)  4.39  .967  .344  6.28  e s s  C(3L)VG1,rust/ +/F(3R)VDl e (trisomic-3L) f  +  . 16  +  .021  +  .015  ±  .35  (4)  (6)  (6)  (6)  2.87  .811  .235  5.31  65  +  Estimated Mean ±  Repressed Estimate  +  .026  +  .015  +  .27  (5)  (6)  (6)  (6)  4.78  1.31  .366  7.17  .10  +  3.19 ±  Enzyme a c t i v i t y i s p e r mg o f p r o t e i n . Repressed estimate  .10  .07  .01  +  .876 +  .006  .006  +  .244 +  .004  .16  4.78 +  .10  e x p r e s s e d as t h e change i n a b s o r b a n c e per min Numbers i n p a r e n t h e s e s a r e t h e sample s i z e s . i s t w o - t h i r d s t h e e s t i m a t e d mean.  101  T a b l e 16 trisomies.  A c t i v i t y o-f  third  chromosome  enzyme  Genotype  Men  Aldox  Oregon R (diploid)  .452  5.14  ±  C(3L)VG1, r u s t / F(3R)VDl,e / F(3R)VDl,e (diploid)  .048  ±  loci  (6)  .364  5.93  3L  Aid  1.14  .48  (7)  in  ±  .06 (4) 1.25  e  S 3  C(3L)VG1, r u s t / +/F(3R)VDl,e (trisomic-3L)  +  .022  +  .46  ±  .04  (6)  (6)  (4)  .330  5.61  .983  B  +  .011  Estimated Mean ±  +  .057  (6)  (5)  .408  5.54  1.19  ±  .17  .272 ±  Enzyme a c t i v i t y i s p e r mg o f p r o t e i n . Repressed estimate  .19  (7)  -014  Repressed Estimate  +  .009  +  3.69 +  .11  .02  .797 +  .011  e x p r e s s e d as t h e change i n a b s o r b a n c e per min Numbers i n p a r e n t h e s e s a r e t h e sample s i z e s . i s t w o - t h i r d s t h e e s t i m a t e d mean.  108 Table  17  A c t i v i t y of  a  2L-1inked  enzyme  Enzyme Genotype  locus  2R  trisomies.  Locus  Gpdh  Oregon R (diploid)  .998  F(2L)pr/F(2L)pr, C(2R)px (diploid)  .646  F(2L)pr/+/ C(2R)px (trisomic-2R)  .639  Expected  .822  ±  .016  .548  ±  .010  Repressed  in  +  .028  (4)  Mean Estimate  Enzyme a c t i v i t y i s p e r mg p f p r o t e i n . Repressed estimate  ±  .059  (4)  ±  .049  (6)  expressed as the change i n absorbance per min Numbers i n p a r e n t h e s e s a r e t h e sample s i z e s . i s t w o - t h i r d s t h e e s t i m a t e d mean.  If  X-chromosomal  conditions,  then  expression  of  gene-enzyme autosomal 19).  In  arms  enzyme  in  that  not  a  Cat  two  loci  this  variant  We h a v e  autosomal  were  of  loci  in  loci  15%  males  four  major  (Tables of  were  unaffected  autosomes  diploid 2).  less  specific  do not  i n metafemales: of  serum p r o t e i n  in expression  product  also  in  (LSP-la).  i n metafemales  be  X/A r a t i o  levels  of  in  X-linked  trisomies gene  does  not  expression  noted  many  gene  displays  it  equivalent  should  includes the  on 3 L ,  Therefore,  possess It  In  repression  trisomies.  levels  This  located  females.  genes.  suppression  only  these encoding A  a  (see  section  chapter).  the  18 &  product  although  or  show t h e  autosomal  larval  LSP-la,  eight  non-isogenic condition.  repressed,  euploid  on autosomal  chapter  levels  the  r  than  escape  measured  from the  their  produced about  seen  could repress  and metafemales  despite  X and t h e  reduction  Decreasing increased  is  comparable  X chromosome  genes.  these  synthesize  (see  gene,  one-third of  loci  loci  hyperploids  related  of  Pgm,  effects  all  X-linked  the  the  are  produced s i m i l a r  few autosomal that  regulatory that  six  metafemales  (one-third) appears  loci  Pok and P o i  metafemales,  the  females  and f e m a l e s ,  However,  trisomy  with representatives  i n males,  Adh_, I d h ,  partially;  for  autosomal  these  males  Aldox).  for  some  metafemales,  (gpdh,  trisomy  systems  A l l of  between  and autosomal  result  in  II  no T a b l e 18 A c t i v i t y of metafemales.  second  chromosome  Genotype  Enzyme Pgk  Oregon +/0 (male)  R  3.48  +  .714  (6)  +  .043  .125  .006  3.89  (5)  Estimated Mean  3.36  ±  .07  .698  +  .017  .139  +  .004  3.73  +  .06  Repressed Estimate  2.24  +  .05  .465  ±  .011  .093  +  .003  2.49  +  .04  (6)  .08  .668  (6)  +  +  (6)  2.91  Enzyme a c t i v i t y i s p e r mg o f p r o t e i n . Repressed estimate  .154  .22  C(1)RM y pn v/+ (3X2A)  +  .053  +  3.24  (6)  ±  +  Pgi  C(l)RM y pn v / Y (female) f  .682  in  Adh  (5)  .15  loci  Locus  Gpdh  .23  +  enzyme  .012  3.57  (6)  .039  .162  (6)  +  +  .04  (6)  .018  3.14  (6)  ±  .40  (6)  expressed as the change i n absorbance per min Numbers i n p a r e n t h e s e s a r e t h e sample s i z e s . i s t w o - t h i r d s t h e e s t i m a t e d mean.  Ill T a b l e 19 A c t i v i t y of metafemales.  third  chromosome  Genotype  Enzyme Idh  Oregon +/0 (male)  R  .369  ±  .633  (6)  +  .049  in  Aldox  Cat  4.13  ±  .27  6 . 19  +  .25  (5)  (6)  (4)  .014  loci  Locus  Pgm  .013  +  enzyme  C(1)RM, y pn v / Y (female)  .330  C(1)RM, y pn v/+ (metafemale)  .339  Estimated Mean  .349  ±  .005  .709  +  .034  4.02  +  .10  5.B1  +  .09  Repressed Estimate  .233  +  .003  .472  ±  .023  2.68  ±  .06  3.87  +  .06  .784  (6)  +  .115  3.91  (5)  .026  .772  (6)  Enzyme a c t i v i t y i s p e r mg o f p r o t e i n . Repressed estimate  ±  ±  +  .32  5.42  3.95  (6)  +  .25  (5)  (6)  .083  ±  .38  6 . 17  (6)  ±  .33  (5)  e x p r e s s e d as t h e change i n a b s o r b a n c e per min Numbers i n p a r e n t h e s e s a r e t h e sample s i z e s . i s t w o - t h i r d s t h e e s t i m a t e d mean.  112. A fundamental male-female gene  aspect  dosage  expression  number  of  is  50X  greater  effect  are  of  is  sets.  an  in  For  a  diploid  with the  factor(s),  not  known  effect.  autosome  C o n c e i v a b l y , by  sets  males)  could occur.  To t e s t  both  for  trisomy  expression different iso-X  was  2L  was  parental  differences,  compensated amount three  of of  (Tables  6PT the  Male expression  25  autosomal  is  20  (2X2A).  Thus,  activator  model  activity It  is  this of  genes,  loci  Marked  or  Sc 2 2 ) . these  that  see  (at  to  in of  in  the  between  However, enzymes  the  stocks  variations  Zw b e t w e e n  i n males  of  least  in  is  this  activity  Fum  interesting  It  content  iso—X chromosome  3L.  dosage  manifest  autosomal  examples,  for  (2X3A)  transcription.  iso-X-chromosome  ( T a b l e 21  the  intersex  p o s s i b i l i t y , the  8c 2 1 )  in  of  required to  in  X-linked  the despite  were  dosage  note  that  than  the  females  in  stocks.  between  data  were  diploids  genotypes  Se 2 6 ) ,  (For  in  by autosomal  the  s i g n i f i c a n t l y higher  five  level  X—linked  trisomy  21-24).  and f e m a l e  made b e t w e e n (Tables  the  this  (Tables  two c o m p o u n d - f r e e s t r a i n s these  of  from t h i s  stocks.  and CF2 s t r a i n s  are  monitored  and f o r  observed  for  increasing  activation  enzymes  female  in  with changes  X-chromosome  in trisomies,  X—linked  increases  triploid  limiting  genome  four  operating  the  in a  coded  specifically required for entire  that  example, gene  are  if  is  system  correlated  X-linked  than  ie.  control  directly  consistent  compensation;  the  compensation  autosome  transcription  of  two  trisomies  of  pooled for  and t r i s o m i e s .  the  p o o l e d male  observations  failed  to  can  activate  be any  the  analysis  When a  comparison  and f e m a l e made. of  the  of is  data  First,  both  X-linked  loci  M3  Table  20  X-linked  enzyme  levels  i n Oregon R i s o - X  Enzyme Genotype  female  Pgd  .163  +  .263  (7) male  .153  male/ female ratio  Table iso-X  ±  ±  Opt  .022  .007  .253  +  .185  .143  .053  1.15  +  ±  .005  (7) .05  .118  ±  .009  (7>  <7>  (13)  1.12  .962  .935  .826  enzyme  levels  in  +  +  C(2L>11JF(2R)bw/F(2R)bw!  Locus  Fum  .008  .156  (8) female  .05  <7>  Pgd  .182  +  Zw  (7)  Enzyme  male  1.23  (7)  21 X-linked strain.  Genotype  Locus  Fum  .008  strain.  +  Gpt  .006  1.01  (8) .009  .177  +  +  Zw  .03  .162  (8) .008  1.22  +  ±  .009  (8) .04  .171  +  (8)  (8)  (8)  (8)  1.02  1.13  1.21  1.06  .006  male/ female ratio  Enzyme a c t i v i t y i s p e r mg o f p r o t e i n .  expressed as the change i n absorbance Numbers i n p a r e n t h e s e a r e t h e sample  per min sizes.  U4-  T a b l e 22 X - l i n k e d enzyme F(3R)e ;iso-X strain.  levels  i n C(3L)ru  stlF(3R>e / e  e  Enzyme Genotype  female  Pgd  .198  ±  Fum  .005  .232  (7) male  male/ female ratio  .185  +  Locus  +  .006  Gpt  1.39  (7) .006  .249  +  +  Zw  .04  .224  <7> .013  1.53  +  +  .010  (7) .04  .225  +  .013  (7)  (7>  (7)  (7)  .934  1.07  1.10  1.00  Enzyme a c t i v i t y i s p e r mg o f p r o t e i n .  expressed as t h e change i n absorbance Numbers i n p a r e n t h e s e a r e t h e sample  per min sizes.  Table iso-X  115  23 X - l i n k e d enzyme l e v e l s trisomic-2L individuals.  in  Enzyme Genotype  female  Pgd  .128  +  .119  ±  Locus  Fum  .004  .223  (8) male  C(2L>lt/+/F(2R)bw|  +  Gpt  .009  1.04  (8) .004  .232  +  +  Zw  .03  .082  ±  (8) .009  1.19  ±  .002  (8) .04  .091  +  .003  (8)  (8)  (8)  (8)  .929  1.03  1.14  1.11  male/ female ratio  Table iso-X  24 X - l i n k e d enzyme l e v e l s trisomic-3L individuals.  i n C<3L)ru  Enzyme Genotype  male  Pgd  .179  +  .194  (10) female  male/ female ratio  .139  +  +  .011  Gpt  .844  ±  (10)  .006  .174  +  .018  Q  Locus  Fum  .006  st/+/F(3R)e |  Zw  .064  .153  +  (10) .816  +  .067  (10) .134  +  (8)  (8)  (8)  (8)  .776  .897  .967  .876  Enzyme a c t i v i t y i s p e r mg o f p r o t e i n .  .007  expressed as t h e change i n absorbance Numbers i n p a r e n t h e s e a r e t h e sample  .007  per min sizes.  lib  T a b l e 25 larvae.  X-linked  enzyme  levels  in diploid  Enzyme Genotype  Pgd  iso-X Oregon R (diploid)  .15B  iso-X| C(2L)lt F(2R)bw/ F(2R)bw (diploid)  .184  +  Fum  .003  .258  (14)  +  ±  ;167  (16)  +  Gpt  .014  1.19  +  Zw  .02  .131  (14)  .003  1.12  (16)  +  1.12  .01  .167  +  .003  (16)  .229  Estimated Mean  .171  +  .001  .213  +  .003  1.16  +  .01  .149  +  .001  Repressed Estimate  .114  +  .001  .142  +  .002  .770  ±  .003  .099  +  .001  (16)  +  .003  (26)  (16)  .003  ±  iso-Xl .124 + .001 C(2L)lt/ +/F(2R)bw (16) (trisomic-2L)  Enzyme a c t i v i t y i s p e r mg o f p r o t e i n . Repressed estimate  ±  trisomic-2L  Locus  (14)  .003  and  .01  .087  (16)  +  .001  (16)  e x p r e s s e d as t h e change i n a b s o r b a n c e per min Numbers i n p a r e n t h e s e a r e t h e sample s i z e s . i s t w o - t h i r d s the compensated estimate.  Ul T a b l e 26 1arvae.  X-linked  enzyme  levels  in diploid  Enzyme Genotype  Pgd  iso-X Oregon R (diploid)  .158  iso-X? C(3L)ru stl F(3R)e / F(3R)e (diploid)  .192  +  Fum  .003  .258  (14)  +  .002  .241  +  trisomic-3L  Locus Gpt  .014  1.19  (14)  (14)  r a  +  and  +  Zw  .019  .131  (14)  .004  1.46  (14)  +  +  .003  (26)  .014  .225  (14)  +  .004  (14)  s  iso-Xl .161 + .002 C(3L)ru st/ +/F(3R)e° (18) (trisomic-3L)  -.185  +  .005  .856  (18)  +  .024  .145  (18)  +  .003  (18)  Estimated Mean  .175  +  .001  .249  +  .004  1.33  +  .01  .178  +  .001  Repressed Estimate  .122  +  .001  .166  +  .003  .883  +  .004  .119  +  -001  Enzyme a c t i v i t y i s p e r mg o f p r o t e i n . Repressed estimate  e x p r e s s e d as t h e change i n a b s o r b a n c e p e r min Numbers i n p a r e n t h e s e a r e t h e sample s i z e s . i s t w o - t h i r d s the compensated estimate.  examined.  Such  transcription they  either  many o t h e r Second, one  of  finding  activators  do not copies  all the  respond  a  four  exist  reside  for  that the  encoding  X-linked  loci  arms  elsewhere  in the  present  loci  were  repressed  autosomal  loci  autosomal  also  It  if  on t h e s e  are  trisomies.  to  suggests  would  aneuploidy in a  or  by o n e - t h i r d  appear  that  examined, if  they  do,  genome.  in  at  X-linked  s i m i l a r way a s  least  loci do  can  autosomal  loci. This  experiment  X chromosomes stock.  rather  as  homozygous inviable  those  for  that  Larval  Base  further  responds  to  the  of  the  be  applied  to  group  activated,  or  repressed.  3L  so  Oregon R  for  2L  trisomies  extremely  was p r e c l u d e d .  previous section  genes  into  expression  of  group of  position,  those  However,  expression  in trisomies.  suggest  that  condition independently.  on chromosomal  regulation  a  observed  however,  chromosome were  trisomic  function or  its  here!  were  from the  Base  genes  gene  of  chromosomes  results  analysis  on  condition  same  been performed with  Gene F a m i l i e s i n T r i s o m i e s  serum p r o t e i n  The r e s u l t s  iso-X  reported  the  E x p r e s s i o n of  initially  than  E s s e n t i a l l y the  trisomies  II.  had  must  render  which  it  Based  are  gene  either  no s i m p l e r u l e  can  unaffected,  some p r o p e r t y  To address genes  which  each  of  each  susceptible  this  question,  have  only  gene  or  to  the  recently  diverged  (Brock  and Roberts,  proteins encoded  a r e coded by a  latter  this  group  1981),  is  a l l of  (LSP-la  gene  1979b)  group  of  them  located  whereas genes  similar  o n t h e X,  has been  examined.  f o r by two major  single  Evans-Roberts, This  1983)  on 3L  LSP—1  i s of  located  LSP-lb  of  serum  LSP-2  While each  composition  and L S P - l o  is  and  i s produced by three  on d i f f e r e n t  on 2L,  genes:  (Roberts  interest.  i n sequence  are  groups  The l a r v a l  (Smith  genes.  gene  in  et a l . ,  chromosome  arms  on  3D . Measurements collected absolute live  L S P ' s were  from wandering quantitation  weight)  involved  proved  of  these  difficult  t h e amount  others,  and t o  LSP-2  Therefore,it  expression  LSP-2  absolute samples stains this  amounts taken  and i s  protein  expected  ratios  were  is  technical  relative  this  were  protein  larvae  i n column 5 of  that  the LSP-1 proteins  is  i n 3L t r i s o m i e s  genes  and that  are unaffected.  407. o v e r  to  parental levels  that  LSP-lg,  of  the  of  this  i n agreements  the expression  Relative  The  gene  The l e v e l  also  L S P - 2 / L S P - l a and L S P - 2 / L S P - l b suggest  diploid  LSP-2  by the d u p l i c a t i o n .  for  f r o m hemolymph  Elevated  (about  to  the  diploids.  Table 27.  suggesting  relative  examine  and t h e i r  p e r mg  standard  determined  in trisomies  level)  to  or  A  As an  L S P - 1 was m e a s u r e d  to  10).  problems  hemolymph.  was i m p o r t n a t  observed  to  (per l a r v a e  in trisomies  unaffected  relative  dependent LSP-lb  shown  o f hemolymph  (Figure  p r o v i d e an i n t e r n a l  from t r i s o m i c - 3 L  compensated  expression protein  of  owing t o  each  to  larvae  proteins  extracting  of  measurement. of  made f r o m s a m p l e s  third-instar  in quantitatively  alternative, the  of  LSP-2 of  is  the  dose  L S P - l a and  equivalent  110 T a b l e 27 hemolymph  Quantity of LSPs i n d i p l o i d from female larvae. LSP  and  trisomic-3L  Genotype  2/la  2/lb  lg/2  total LSP-2 protein  iso-X Oregon R (diploid)  l.BB  1.35  1.03  0.739  iso-X; C(3L)ru s t l F(3R)e /F(3R)e (diploid) e s  iso-XS C(3L)ru s t / +/F(3R)e ( t r i s o m i c—3L) E S  +  B  +  ±  Dose-dependent Estimate ±  Compensated Estimate  .11  +  .09  ±  .09  ±  .052  (8)  (8)  (11)  (8)  1.59  1.54  0.905  1.01  .08  +  .09  +  .09  ±  .07  (8)  (8)  (11)  (8)  2.65  2.65  1.07  1.29  .08  +  . 16  (8)  (8)  2.53  2.22  .05  1.69 +  Ratio  .03  Measurements i n r i g h t - m o s t C o o m a s i e B l u e a t 595nm.  +  ±  .09  ±  (16)  .05  .03 (8)  1.38 ±  .04  1.48  0.92  • .03  t -03  column  are i n units  of absorbance by  m  amounts  of  trisomic located  LSP-2 are  strain. on 3L,  elevated  in  expression trisomies The  observed  Since  we c a n  of as  the in  four  larvae  relative  L S P - 2 was  diploid  parents.  present  in  that  neither  the  LSP-lb  yet  an  Thus,  template.  is  euploids.  Thus, to  LSP-1  appear  to  the  this genes  the  is  more  less  to  both levels  the  same  are  also  the in  LSP—1 s u b u n i t s p r o d u c e d  in  these  these  three  T h e amount  trisomies  genes  by a  protein  in  closely  noteworthy  is  3L  closely  like  a  to  trisomies  the  the  LSP—1 g e n e s i n more  genes  for  X>  than  in  may  other  respond two  the  al,  X—chromosomal 6.  they  1981).  are  While of  these  otherwise.  trisomies In  the  responded  expression  suggests  of  is  than  et  per  also  other  (Smith  its  product  each  respond  i n chapter  diploids.  which  X chromosome  detail  in  genes  to  in  appear  two  related  are  However,  aneuploidy:  possible explanation, for  as  less  while the  and  2L.  trisomies  of  their  condition  observed  related  LSP-lg  in  would  ( L S P - l a on t h e 2L  of  on 3 L ,  it  trisomic  effects  that  as  d u p l i c a t i o n of  i n these  examined  product  are  genotypes,  down—regulate  seemed  discussed  the  rate  in  of  regulatory  failed  of  of  very  that  in trisomies  all  LSP-1  gene  expression be  are  at  by p r o d u c i n g one—third  even  differently  It  same  compensating  dose  one-third  similarly  in  in conclusion,  occurs  d u p l i c a t e d by the  amount  produced  unaffected.  the  affected  The other  genes  and  protein  shown i n T a b l e 2 8 .  in  is  is  gene gene  are  LSP-lg Thus,  three  Since both  gene  eqivalent  increased  that  larvae.  the  are  doses  gene  this  diploid  and L S P - 2  LSP genes  of  trisomic-2L  two  two  diploids.  quantities  to  LSP-lo  deduce  trisomic-3L  i n the  will  summary,  those  The  Ill T a b l e 28 hemolymph  Q u a n t i t y of LSPs i n d i p l o i d from female larvae. LSP  and  trisomic-2L  Ratio  Genotype  lb/2  la/2  lg/2  Oregon R (diploid)  .937  .646  1.03  C(2L)ltl F(2R)bw/ F(2R)bw (diploid)  C(2L)lt/ +/F(2R)bw (trisomic-2L>  +  .029  ±  ±  Repressed Estimate  .03 (16)  .833  .664  .827  +  .023  +  .067  (9)  (9)  (6)  .813  .424  1.02  +  .022  ±  .17  (9)  (9)  (6)  1.30  .655  .929  .03  +  .868 ±  +  (8)  .024  Dose dependent Estimate  .046  (8)  .053  +  +  .020  .012  +  .436 +  .008  .032  .619 +  .021  1X3  experiments  showed t h a t  the  compensated  i n metafemales  X—linked despite  completely  dosage  compensate  expression  of  closely  not  affected  LSP—lg even  trisomic-X levels  beyond the  whose from  the  females  to  the  to  Thus, the  two  genes  LSP1—o)  are  the  effects  of  since  many d i f f e r e n t  a  in  that  gene  genes  in  in  the  all  the  loci  show t h e  was shows  metafemales,  to  date  of  LSP-2,  in  same  is  does  similar  ways.  (LSP-la These  evolutionarily  converse  that  metafemales  family.  s i m i l a r l y to  However,  The  X chromosome  LSP's  which are  to  metafemales,  similarly  i n the  respond  can  in  examined  similar  of  gene  one-third  quantity  do respond related  that  T a b l e 29  The q u a n t i t i e s  are  this  LSP1—b.  discrete  autosomal  expression  necessarily  trisomy.  on 2 L ,  d u p l i c a t i o n of  least  suggest  do not  only  of  dramatically  for  was  and f e m a l e s .  However,  reduced  LSP—1 s u b u n i t s ,  alter  related  larvae.  gene  inability  males  gene  in diploids.  the  observations  related  markedly reduced  found  the  between  expected  the  and m a l e s .  appear  Indeed,  is  is  level  were  level  This  product  relative  and  in  protein  reduction.  not  the  LSP-la  the  closely  regulatory  obviously not  response  true  in  trisomies.  Adh  transformants  i n 2L  The p r e c e d i n g respond  that  section  d i f f e r e n t l y to  Two e x p l a n a t i o n s members  of  for the  trisomies  the  this  revealed same result  LSP gene  that  very  s i m i l a r genes  whole-arm t r i s o m i c seem  family,  plausible. while similar  can  condition. It at  is  possible  the  IZf Table  29  Quantity  of LSPB  i n males,  females LSP  and metafemales.  Ratio  genotype  LSP-lb/LSP-2  LSP-lg/LSP-2  Oregon +/0 (male)  1.06  1.04  R  C(l)RM,y (female)  +  .07  (8)  pn v / Y  0.756  +  .15  (9)  .046  1.04  (8>  +  +  ±  .08  (9)  C ( l ) R M , y pn v/+ (metafemale)  0.913  .088  0.521  Estimated  Mean  0.908  ±  .021  1.04  Repressed  Estimate  0.606  ±  .014  0.693  (7)  ±  .080  (9)  ±  .04  +  .030  1X5  structural  gene  sequences.  level,  Alternatively,  controlled  by s i m i l a r  regulation  of  these  three  arise  in  possibility possess  Adh D.  could  first,  chromosomal if  the  positions, diploids single  to  transformed  and  the  activity  was  19%  single  Adh g e n e s  chromosomal  if  it  higher  if  positions.  of  i n 2L than  the  J.  1,  the  of  that  considerably No a p p a r e n t  in  the  chromatin this  which  copies  of  and  present the  Adh  non-native  repressed,  to  determine  chromosomal  expression this,  the (the  between expression  strain  expression ie.,  the  and  and of  of  D.  1980)  diploids  in  2L  the  ADH e n z y m e  dose-dependent  shows  duplicated  Posakony the  in  in  of  could  strains  Adh; Goldberg,  trisomies; a  expression  transposed  from w i l d - t y p e  examined  the  chromosomal  to  not  in  To i n v e s t i g a t e  different  level  were  the  trisomic-2L  residing  in  same  T a b l e 30  varies  differences  expression  expression  second,  be  not  possessing  the  regulatory  modifying  objectives  i n Chapter  activated  of  of  To accomplish  strains  was  i n column 2  two  genes  gene  data  six  o r i g i n a l l y clone  A s shown  or  in different  with  trisomies.  used  gene  being examined.  when l o c a t e d  maintains  trisomies. Adh  see  positions  and  capable  strains  were  to  may  local  and p r o v i d e d by D r s .  Adh s t r u c t u r a l  Goldberg five  gene  repressed  Adh gene,  gene  Adh g e n e s  There  be  but  mode o f  constructed  synthesized  experiment:  genes  domains c o u l d a f f e c t  The d i p l o i d  Goldberg.  gene  the  functional  were  LSP-1  different  d e p e n d i n g on whether  we h a v e  locations.  very  sequences  regulatory  containing  the  An a l t e r e d  trisomies a  5"  chromatin  genes.  arm e n c o d e d domain  c o u l d have  estimate.  expression  The of  the  different  correlation  appears  to  exist  T a b l e 30 E x p r e s s i o n of Adh from v a r i o u s i n d i p l o i d and trisomic—2L larvae. karyotypic  strain  wSAdh  chromosomal position  35B  diploid  2.09  +  .16  19E  1.75  +  trisomic-2L  2.36  2.02  (4)  tAP-3  36A  0.893  ±  17C  2.29  +  .08  1.19  89A  0.339  .11  +  2.29  12A  0.333  + (4)  .17  +  .02  +  .18  (6)  .043  0.493  (4)  tAP-17  ±  ratio  1 . 13  1.15  1.33  (6)  (4)  tAP-14  .22  (6)  (4)  tAP-5  + (6)  .10  positions  condition  (4)  tAP-1  chromosomal  +  .034  (6)  .016  0.463  +  .029  1.00  1.45  1.39  (6)  Enzyme a c t i v i t y i s e x p r e s s e d a s t h e c h a n g e p e r ml o f e x t r a c t . Numbers i n p a r e n t h e s e s  i n absorbance p e r min a r e t h e sample s i z e s .  in between  gene  expression  resides.  Adh  lowest only  activity  4.8kb  genes  However,  of  i n the  Generally, diploids;  (about  the  genes  The  to  is  the  basal line Adh  level  clear  activated  between  that  i n 2L  level  of  activated  was  the  the  expressing 50%  by t h e  a  also  of of  a b i l i t y of  at  again  Figure  activation  is  these  shown i n  amount  and  Table  this  as  of  of  of  quantity to  this  gene.  in  depicts  Adh gene  the  in  in  diploids  2L  trisomies.  be  correlated that  with the  at  1.49;  this  would  predict  near  zero  level  could  be  2L.  in  showed  noteworthy  d u p l i c a t i o n of  30.  tAP-1  activated 11  of  same  trisomies  enzyme p r e s e n t  the  is  transpositions  activation  negatively It  of  the  The e x p r e s s i o n  i n an  were  amount  X axis  lowest  levels.  results  trisomies  also  intermediate,  experiments  the  both  similarly in  had t h e  diploid  2L  which d i s p l a y are  condition is  expression.  intercepts gene  of  type)  arm on w h i c h  The a c t i v i t y  varying extents.  relationship  It  DNA.  Adh t r a n s f o r m a n t s  trisomies  versus  tAP-3  d u p l i c a t i o n of  other  wild  and t A P - 1 7  i n these  1  a  15%  chromosome strains  expressed  had n e a r l y  •the Adh "" g e n e that  two  trisomic-2L  tAP-14  tAP-5  the  Adh r e g i o n  enzyme a c t i v i t y , and  and t h e  its  regression that  an  F i g u r e 11. R e l a t i o n s h i p b e t w e e n ADH e n z y m e a c t i v i t y i n d i p l o i d s and 2L t r i s o m i e s b e a r i n g t r a n s f o r m e d Adh g e n e s . T h e ADH e n z y m e a c t i v i t y i n d i p l o i d s i s p l o t t e d o n t h e Y a x i s . The r a t i o of t h e a c t i v i t y o b s e r v e d i n 2L t r i s o m i e s t o t h e a c t i v i t y o b s e r v e d i n d i p l o i d s i s p l o t t e d on t h e X a x i s . R e g r e s s i o n p a r a m e t e r s : r = - . 9 7 9 , b= - . 1 9 5 , X i n t e r c e p t = 1.49.  AjM/joy HQV  130  Discussion  In spread genes  this  chapter  effects that  of  are  summarized  in  First,  all  in  a  not  similar  an  trisomy  Figure genes  way.  polymerase,  ribosomes  expected  be  that  were  expression affected  three  one-third  in  that  Reductions  Conklin,  a  In  of  the  of  gene all  some  loci  mechanisms Second, three Z_w_ i s  degree.  trisomies,  is  but  to  in  for  be  distinct suggests  gene For  were  reduced  modify  gene  can  be  example,  all  repressed not  affected  might  others,  trisomies.  enzyme p r o f i l e s regulatory  not  particular  Gpdh  obvious.  (eg.  Since  by  i n metafemales. each  mechanisms  of  are  disturbances  products  repressed  wide  types  condition are  operate  a  and  effects  expression  but  how  immediately  gene  similar  contrast,  and 3L  these  trisomic  then  to  for  common r e g u l a t o r y  repression i n the  observed  plants  etc.)  numbers  physiological  modes  made o f  trisomy  are  The  again  responsible  for  differences.  most  one-third  been  general  unaffected,  discrete  observed The  a  given  regulatory  2L  individuality  the  in a  altered  trisomies.  argues  are  affected  is  of  Two p o i n t s  in trisomies.  Cat,  terms  12.  d i f f e r e n t l y by t h e  whereas  been  of  observed  discrete  in  has  The extent  If  for  effects  are,  affected.  responsible  to  examination  for  (McDaniel 1975;  of  amounts a  few  gene of  loci  and Ramage,  Birchler  effect  expression  gene in  observed  relative  products  yeast  1970;  and Newton,  trisomies to  Carlson,  and F r i i s ,  1972;  and  Smith  mammals  is  diploids.  in trisomies  (Gullov  1981)  in  has  also  1980), and  (Klose  and  131 Putz,  1983).  T a b l e 31  combinations by  one—third.  for  loci  differ  This  gene  resides  trisomies,  one-third the  seems  negative  only  that  (23%)  these  x  70% a r e  that  is  repressors  loci  of t h e i r  1000 b a n d s / a r m  i n the trisomic  that  measured  were  the three  expression  by  by .70)  it  and that  about  would  condition to repress  40% o f  trisomies,  may b e l o c a t e d  be t h a t  this in a l l  approximately  are not exceptional  divided  show  not repressed  i n any of  whose  expressed  loci  Since  reduced  predicted  number o f  were  A s i m p l e p r e d i c t i o n would  trisomies examined  three  loci  the trisomies.  regulators  2R o r 3 R .  of response  A large  was n o t d u p l i c a t e d  likely  level,  on t h e d u p l i c a t e d arm, and a r e  of t h i r t e e n  i n any of  genome  gene-trisomy  from the d i p l o i d  manner.  of r e g u l a t i o n !  three  of the  by concentration-dependent  a dose—dependent  type  that,  i s the type  controlled  structural in  that  shows  on  4300  need  either  bands  (3  t o be  a l l loci  that  were  examined. Thus, in  these  i t  would  studies  appear  a r e under  regulatory  gene.  controlled  i n part  produced.  A comparison  studied all  reveals  cells  As such,  that  of  gene  of  1980)  i t  would  i s doubtful  of  the functions their  but they  null  mutants  are incapable  one  repressors  negative  enzymes  Although  conditions bearing  i s  which  of t h e genes  be s u f f i c i e n t l y f i t t o s u r v i v e  h o m o z y g o u s Gpdh  laboratory  flies  least  measured  i n Drosophila  respective  laboratory that  at  expression  and are p h y s i o l o g i c a l l y v i t a l .  al.,  the  of the enzyme-loci  the control  many o f  a r e d i s p o n s i b l e under  example,  most  by the q u a n t i t i e s  loci  mutants  that  are  that  operate  some  of  (Voelker  null  were  these et  activity  in the wild.  For  can be maintained i n of  flight  owing t o  in  the  132  T a b l e 31 trisomies.  Summary o f r e g u l a t o r y  effects  Trisomic  on enzyme  loci  in  Chromosome  Regulatory Effect  2L  3L  X  unaffected  8  3  6  17  activated  1  0  1  2  total  partially  repressed  1  3  3  7  repressed  by one t h i r d  6  11  4  21  4(100%)  11  number f u l l y r e p r e s s e d on same chromosome a r m total  examined  3(507.)  16  4(36%)  17  14  47  133  inefficient  shuttling  mitochondrial  of  membrane.  show a c h a r a c t e r i s t i c development onset of  of  each  during thus  could  that  this  (studies loci  of  high  on m e t a b o l i c  enzymes  are  Thus,  repressors  i n concert  systems  of  the type  genes  bithorax  modifiers The trisomies  modulated within several  of  clearly  i n more  than  a chromosome regulatory  loci  It  i s  and likely  reasons  and  here  their  may b e  more  types.  are  appear  inducible,  t o be  of  negative  et  large,  i t  such  as  scute  dependent  1982).  regulatory  not e x i s t ,  loci  operating  by concentration  a l . ,  cues  Indeed,  c o u l d be  complex  or  negatively  by  mechanisms.  control  one t r i s o m y .  arm i s  weight  growth)  f o r other study  loci  A one r e g u l a t o r  does  the  electrophoresis).  expression  regulatory  negative  larval  independently of developmental  Botas  i s n o t known.  relationship  gene  be r e p r e s s i b l e  1978;  is  and a l s o  and t h e e x p r e s s i o n  (Lewis,  w i t h body  observed  i t  12  T h e amount  for historical  in this  and n e g a t i v e  may a l s o  number  than  measured  with other  positive  simultaneously or  of  operate  biased  as  at  1976).  a r e among t h e o l d e s t  modulation of  may a l s o  i n Figure  activity  (larval  b e mapped b y  developmentally regulated  regulated.  and  to  repression  genes  shown  "housekeeping" enzymes.  the f i r s t  several  the  in activity  of  activity  i s highly  common f o r h o u s e k e e p i n g However,  increase  reductions  metabolic  of genes  negative  t h e enzymes  i s roughly correlated  be c o n s i d e r e d set  across  ( O ' B r i e n and M c l n t y r e ,  product  a r e among  Thus,  and then  pupariation  periods  Many o f  exponential  proceeds  gene  NADH e q u i v a l e n t s  operating  t o one  since  is  are duplicated  gene  several  Because  in  loci  are  t h e number  of  possible  that  quite i n each  trisomy.  loci  Figure  /3f  F i g u r e 12. S u m m a r y o-f t h e e x p r e s s i o n l e v e l o f g e n e s i n X-chromosomal and autosomal t r i s o m i e s . On e a c h X a x i s , a v a l u e of 1.0 r e p r e s e n t s t h e d i p l o i d l e v e l of e x p r e s s i o n per gene.  156  s  o 5  U E a &  Ul  E  9  CM i  * t t  1  i  t  i * t I  f f  •D  a  13 • o  •1  t  I  l*s  k  ll m l  i  N  N  8  Q. O  11 11 HI  JL  E  il *§*  3 U.  + - t  72 SJ  i i  u a a .  0) Q.  I S * !  k  N  6  f*-  V Z X £  O E a u .  )3b  13 shows the d i s t r i b u t i o n of the number of l o c i a f f e c t e d by d i f f e r e n t numbers of t r i s o m i e s . loci  were extremely common then we  a f f e c t e d by a l l t r i s o m i e s .  which  If the  regulatory  might expect most l o c i  However, a mean number of  t r i s o m i e s a f f e c t each l o c u s f o r the t h r e e aneuploid tested.  are  to  1.38  conditions  If the whole genome were surveyed an average of  t r i s o m i e s would be expected t o a f f e c t each l o c u s . must be at l e a s t one  2.3  Since  there  r e g u l a t o r y l o c u s on each t r i s o m i c arm  for  an a f f e c t e d l o c u s , then on average, a minimum of between two t h r e e such r e g u l a t o r y l o c i  e x i s t f o r each gene.  be  and  Unless the  average s t r u c t u r a l gene can a l s o a c t as a r e g u l a t o r y gene, or t h e r e are more r e g u l a t o r y genes than s t r u c t u r a l genes, we  can  conclude t h a t each r e g u l a t o r y gene a f f e c t s the e x p r e s s i o n  of  more than one  genetic  locus.  At t h i s p o i n t , however, we  d i s t i n g u i s h between many r e g u l a t o r s o p e r a t i n g each or a few Few  loci  r e g u l a t o r s each o p e r a t i n g  on a few  cannot  genes  on many l o c i .  appear t o be a c t i v a t e d i n the t r i s o m i c  conditon.  Table 31 shows t h a t of the 47 gene—trisomy combinations examined o n l y two  r e s u l t e d i n l e v e l s of product per  s i g n i f i c a n t l y above d i p l o i d . discussed  in detail  One  i n chapter 6.  template  of these, The  other  Men,  will  locus,  be  Aldox  belongs t o a f a m i l y of genes which code f o r enzymes t h a t molybdenum as a c o f a c t o r .  Several  have been i d e n t i f i e d  c i n . l x d . and  (mal.  r e q u i r e d f o r the p r o d u c t i o n these enzymes (see Bentley loci  may  exist.  trans-acting genetic lpo) t h a t  T  bind loci  are  of normal q u a n t i t i e s of and  W i l l i a m s o n , 1979)  If these or other  loci  and  synthesize  additional  137  F i g u r e 13. Mean n u m b e r  Number o-f t r i s o m i e s a f f e c t i n g i n d i v i d u a l loci. of t r i s o m i e s a f f e c t i n g an i n d i v i d u a l gene = 1.38.  |2>3  number of trisomies  products changes this is  at  levels  in their  enzyme.  at  AO s y n t h e s i s which acetic  one of  these  result.  enzymes  acid,  (Dickinson  activity.  trisomies  is certainly  the  final  chromatin  maturation  There gene  a second  could conceivably act  correct  1983),  although  of  several  reports  1982).  The paucity  could  arise  because  genes  are themselves  regulating fashion  other  of  genes  likely,  of  loci  This  Indeed,  as  of  the basis  this  1981),  i s  the  AO i n 2 L  not  arise  regulatory  to  trans-activation  levels  (Mel1man e t  activations positive  that  in  at.,  dosage  imply  that  are present  in  level  strengthened  1964;  trisomies of  other  compensation.  are capable  the phenotypic further  of  unlinked  regulation  and d i r e c t l y  would  of  of  correlated most  of  the  excess  p o s s i b i l i t y , which has been  dominance at  and Burns,  of  autosomal  in a positive rare.  null  (Williamson and B e n t l e y ,  gene  to  2% o f  expression  increased  required for biosythesis  quantities.  cases  appeared  capable  subjected  a n d more  are extremely  components  Kacser  loci  of  have  to  molecule.  duplication  Mattei,  and does  of  from the e s t a b l i s h i n g  f o r Aldox  few other  21  than  Such  i t  exist  bearing  production of  effect  any step  by gene  i n human t r i s o m y  less  AO l o c u s .  t h e enzyme  enzymes  Alternatively,  at  structure  are relatively  expression  the over  loci  acetaldehyde strains  possess  of  quantities  different  1981)  AO, then  on 2L and i f  increased  converting  1970)  of  the quantity  located  several  a regulatory  the d u p l i c a t i o n of  regulate  level,  of  Therefore  o-f s y n t h e s i s  i s  and Gaughan,  wild-type  the  genes  capable  f o r AO ( D i c k i n s o n ,  genes  would  While  mutations  from  the rate  a dose-dependent  would  produce  limit  concentration  If  expressed  that  suggested  (Muller, by the  1950;  fact  that  most  genes  dependent  on t h e i r  expression that  produce  appear  modulations  levels  dose. not  be  product  rate  limiting  induction or  controlled  mediated  by changes  in  quantities  The  expression  of  Adh i n v a r i o u s  do e x i s t ,  but  wild  cells  type  irrelevant. reduce arise has  the  In  inserted  the  two  possess  of  so  the is  not  the  least  strains,  are  perturbations  not as  is  noteworthy,  flanking  communication). as  well  are  expression  the  of  larger  on t h e  expression  is  expressed  at  wild-type level  have are  their  by h y p e r p l o i d y f o r expression  subjected  trisomies. amount  of  to  reduced  activity  suggests  functionally  2L.  of  in  that  a  (ie  this  level  phasing,  regard,  of  that  expression  DNA  these  In  by an  (J.  particular  the  trisomies  amount gene.  high  level gene  A gene only  of  45%  in  slightly  effects 2L  expression  which i s  that  which  position as  for  that  Adh genes  by chromosomal  between  may  local  However,  as  positions  This  in euploids is  increases  The r e l a t i o n s h i p activation  level  in  which Adh  Adh r e g i o n  activated  modulation  sufficient  are  insertions.  dependent  the  gene  position  lowest  b u f f e r e d from  Adh i s  is  Perhaps  is  activated  native  the  are  positions  of  into  the  suggests  gene  chromosome  as  gene  regulators.  effectors  quantities  of  rather  chromosome  synthesis  in  such,  varying degrees.  displaying of  as  but  negative  configuration  It  and,  different  Adh t o  ideal  amount  Adh  of  increased  condensation).  transformants  genes  level  of  as  of  directly  involved  positively,  positive  chromatin  personal  the  that  diploid  Posakony,  2L,  their  expression  because  degree  that  that  are  inactivations)  may n o t  provided evidence  that  components  activity  has  be  Thus,  to  (not  of  only  and  the  slightly  14-1  active locus this is  would exists  gene  active  upper  in  of  limit  expression the  may e x i s t  of  intermediate  two  alter the  level this act  with  alter  the  rate  of  experiment  monitoring  the  COS c e l l s ,  these  activated promoter at  42  to  the  were upper  not  For  either  that  and  it  these the  of  herpes that  if  The two appeared  Bienz  is  (and  a  LSP-1  affect could  In  positive  could heat  genes  can  cells  was  added of  effects  expression  respond  in  be  activation  regulatory  TK g e n e  gene  shock  the  promoter  conditions. three  could  reminiscent  growing  no f u r t h e r  the  some  (1982).  or  heat-shock  that  to  that  could  TK a c t i v i t y  enhancer  construct,  vary  processes  This  can  at  thymidine kinase  gene  the  levels  position  Both  an  the  suggests  activator  is  when  not  Adh a r e ,  initiation.  tk  because  does  chromosomal  viral  the  However,  observed.  under  fact  a  activator  way;  This  binding.  observed  fully  increase  activation  while the  the  the  However,  dependent  by Pelham and of  is  further  can  a  regulator  low e x p r e s s i o n  expression  v i c i n i t y of  C).  very  example,  described  authors  any  that  When  more  position)  activator.  transcription  additive  limit The  was  of  the  TK-enhancer element  expression  at  expression,  expression  the  degrees  Only  Adh.  Adh gene  activator  RNA p o l y m e r a s e  by p l a c i n g in  low t h e  indicate  times  Adh e x p r e s s i o n .  quantity  of  1.5  expression  condensation,  of  results  When t h e  concentration  of  competitive.  stability  an  is  affecting  chromatin  trisomies,  the  for  in a  the  These  chromosomal  gene.  levels  processes  extent,  native  increasing  activator  directly  in  in diploids.  its  expression  by SOX.  which p o s i t i v e l y r e g u l a t e s  duplicated than  (ie.  incapable  activated  on 2L  is  produced  the  be  had  an  14-Z differently regulators is  i n the different do not operate  common t o  sequence level Adh  a l l three  composition  (Powell  have  et  all  shown t h a t  different  chromatin  kb o f  these  as  to operate  function  of  these  storage  Sacchromvces the  more  as  increasing to  shown  of  i n c i s over  affecting a range  but have  structures  (see  require  trans—acting  i s to  repression  of  SIR l o c i  regulatory  signals  elements  gene  activity  does  The in  by  appear  exist  i n a concentration of  and  information  conceivably  activation  Some  switching  eliminating transcription  on hormone of  type  controlled  (silent  Enhancer  been  genetic  evidence  transcription.  are negatively  1982).  since  transcription,  regulation,  i n v o l v e d i n mating  of  were  While the wild-type  activate  positive  the  of  i n t h e mammalian genome.  introduction).  under  local  DNA s e q u e n c e  elements  either  the level  diverse  promoter  S i m i l a r elements  Studies  that  located  of the  viral  (Nasmyth,  i t .  mechanism by which  alterations several  t h e RNA  in  enhancers,  cerivisae  diffusible  manner.  A possible  in  on  entitled  genes  switches,  varies  at  elements  wild-type function  repressors)  gene  the that  similar  These  for trans-acting  cassette  are very  which are capable  genes  elements  are themselves  exist  this  positions.  apparently  function  they  of  that  expression  the studies  of  native  enhancers  genes  In a d d i t i o n ,  described  as  o f gene  1984).  expression  DNA.  discovered  discovered of  been  suggests  regulated  Recently,  expression  first  These  configuration.  level  several  genes.  i s through  have  of  on a component  could operate  elements  studied  and a r e c o o r d i n a t e l y  chromosomal  regulators  trisomies  gene  varies  or  to  act  greatly  which  respond  dependent  expression  have  with the dose  of  1+3  the  inducing agent.  Hormone—receptor  transcription  occurs  DNA s e q u e n c e s  (Chandler  genes the  by the binding  p o s s i b i l i t y that  coding  by v a r y i n g  loci.  one gene  this  i s the case,  Britten,  compensation  of  of  i s  specific  that  most  control  of  allows could  be  repressor  likely  regulatory  that  more  locus.  batteries  If  in a  (see Davidson and  1973b).  activators  of  of  where  control  substance. to  trisomies, Autosomal  chapters.  X-linked  maintain four  we d e t e c t e d trisomies  and i n males  to  0.45.  small,  the aneuploid condition s t i l l  system  f o r changes  content, Lucchesi,  rather 1980).  i n the X/A ratio  than  F o r example,  a few a u t o s o m a l l y  encoded  i t  for the  males  major  a  genes  from  changes valid the  1.0  to  are  assay autosomal  (Maroni and  i s conceivable  activator  and f e m a l e s .  activity.  by modifying content  regulatory  autosomal  i n females  provides  system  autosomally  While these  t h e X-chromosome  been  limiting  in X-linked  the X/A ratio  has  19731  could provide a  between  the four  no i n c r e a s e  from 0.5  this  should be  levels  i n two o f  change  support  compete  molecules  dosage  (Schwartz,  Trans-acting,  templates  equivalent  loci  to  transcription  activator  to regulate  in Drosophila  Evidence  X-chromosome  These  surveying  hypothesized  t h e X-chromosome  i n previous  or  it  gene  conceivable  has been  summarized  0.91  expression  by a g i v e n  regulation  quite  control  and P l a u t ,  encoded  The f i n d i n g  to  1979).  Positive  Maroni  is  of  previously,  i s controlled  manner  complex  a form of negative  the level  then  this  induction of  modulation d u r i n g development  As discussed  than  coordinate  gene  of  a l . , 1983).  i n D r o s o p h i l a a r e under  controlled  In  et  mediated  that  o n l y one  are responsible  for  IH-H-  controlling X-linked  enzymes  Therefore, must  an  if  reside  activator the  the level  only  genes  This  locus  region  may b e r e d u n d a n t  they  half  they of  decreasing  the expression  reductions loci  Further,  several  in activity  a r e dosage  two o p p o s i n g r e g u l a t o r y concert—that  is,  hyperactivation result,  affect  t h e same  elongation).  process,  any  of  the regulatory  are  this  loci  study,  was d u p l i c a t e d loci  of  different  the expression  i n two  redundant,  compensation  i n males  rather  occurs  by  than by  ( S p r a d l i n g and Rubin, t o be d i s r u p t e d i n  loci  displayed one-third  f o r 2L or males  dosage  i s not to  f o r 3L,  yet  and females.  are capable  of  acting  systems  say that  i e . RNA p o l y m e r a s e  these  Thus, in  d u p l i c a t i o n and  compensation.  two r e g u l a t o r y  (this  the activity  bearing  by t h e autosomal  by male-female  mechanisms  dosage  X-linked  systems  these  such  throughout  In t h e p r e s e n t  not appear  between  repression  implies that  independent  that  i n trisomies  compensated  they  d i d not observe  genome  i n females  does  exist,  Alternatively,  They  activator  of expression  regulation  genes  on 2L o r 3 L .  suggests  the level  trisomies.  these  trisomies.  flies  either  t h e autosomal  increasing  This  of  do not e x i s t .  If  i n 3L  examined  could activate  to reside  evidence  (1974a)  that  A c t i v a t i o n s of  and d i s t r i b u t e d  t h e genome.  suggested  or  do not appear  1983).  arms.  in strains  that  aneuploids.  Recent  activator  c o u l d be r e i t e r a t e d  finding  approximately  i n 2L o r  chromosome  f o r much o f  particular  different  a few p o s i t i v e  enzyme  duplications  transcription.  not observed  Rawls and Lucchesi  X—linked  gene.  were  on t h e other  genome.  one  o-f X - l i n k e d  This  operate  both  by  could not  i n i t i a t i o n or  If5  A parallel  conclusion  made o n X — l i n k e d differences between  loci  different  present of  operate  Genetic gene  of  (McDonald recently alter  Selection  et  a l . ,  reported  1978).  the expression  Hewitt,  1972;  McDonald  of  levels  and females.  This  argues  and r e g u l a t i o n  h a s been  of  number  of  and Ayala,  and Tanda,  Laurie-Ahlberg  et  a l . , 1981;  Hori  et  1983;  King  Miyashita in and  et  and McDonald,  a l . ,  1982;  1983;  condition of  these  i n combination with different  structural differences  gene  (or c l o s e l y  occur  locus  1980;  19811 Laurie-Ahlberg  and L a u r i e - A h l b e r g , 1984;  Considerable polymorphism  regulators allelic  exists  states  of  mapping c i s r e g u l a t o r s )  for the quantities  which  (Pipkin  Bewley and L a u r i e - A h l b e r g ,  and L a u r i e - A h l b e r g , 1984).  the a l l e l i c  have  modifiers  Bijlsma,  a l . , 1982;  Maroni  for  laboratory  enzyme  1978;  through  recognized  trans-acting  a particular  on  The p o t e n t i a l  studies  Hori  Wilton  can a r i s e  i n the  a l . , 1980;  1982$  genetic  displaying the  et  a l . ,  by  pronounced e f f e c t s  Laurie-Ahlberg  et  the  equal  demonstrated  A large  level  different  despite  of m o d i f i e r s .  changes  the existence  of  exist  processes.  and l o s s  and s u c c e s s f u l l y  genes  the strains,  for individuals  of such  Marked  X-linked  from w i l d - t y p e s t r a i n s  accumulation  significance  many y e a r s  males  compensation  observations  strains.  However,  between  between  for the  a consequence  strain.  by independent  a phenotype  differential adaptive  is  m o d i f i e r s a r e known t o h a v e  expression.  extremes  this  expression  dosage  between  of several  i n each  are observed  male-female  modifiers  and  Presumably  levels  activity  that  expression  i n the expression  strains.  modifier  of  gene  can be obtained  of enzymes  in  nature,  the large  synthesized  (over  tenfold  Several to the  of  regions  f o r Adh  these  modifier  Variations affect  the quantity  modification  of  gene  displaying  altered  regulatory  loci.  product  altered  arises these  is  evolutionary  genes time  scale  regulation  complexity  times. of  Drosophila  functionally mechanism. evolutionary  the modifier  product.  of  same  this  expression  to regulate  19691  of  the  on a developmental  and Davidson,  of  gene to  expression  or  Wilson,  allows  be s y n t h e s i z e d i t  i s an e x t r a o r d i n a r y  (monosomy v e r s u s  by the  regulatory  The d i f f e r e n t i a l  Presumably,  genes  that  compensation  constraints  strains  while i n the latter  example  of  at i n the  T h e X-chromosome  a group  of  a r e m o d u l a t e d b y a common  operates  as  which are a p p l i e d  disomy f o r sex  together  i s a common o c c u r a n c e  d i f f e r e n t i a t i o n and development.  Dosage  also  1979).  products  unrelated  could  and i n  the quantity  genes,  being  Trans-acting  could be mediated  of  gene  gene.  m o d i f i e r genes  i n trisomies  (Britten  Coordinate  related  of  dosage,  or groups  and B r i t t e n ,  appropriate  regulator  has the potential  Davidson  functionally  of  loci  gene  of  levels  variation.  of  affect  these  quantities  these  expression  As such,  structural  In t h e f o r m e r ,  regulatory of  of  they  of  regulatory  by gene  and that  levels  alleles  enzyme  from a l l e l i c  expression  1976;  of  gene  regulators  altered  i n the dose  1980).  t h e m o d i f i e r s c a n map  synthesized.  from d i f f e r e n t  from d i s t i n c t  a l . ,  shown t h a t  diffusible  Presumably,  arise  et  from the s t r u c t u r a l  may e n c o d e  transcription.  synthesized  have  enzyme m o l e c u l e s  loci  expression  studies  distant  number o f  Laurie-Ahlberg  T  a consequence to  determination  the  of  X—chromosome  purposes)  rather  1*1 than  as  a  products  system that  modulations to  cope  is  not  would loci  occur  l i t t l e  they  i n gene  w i t h any  selective  control  to  of  pressure  we o b s e r v e  that  of  autosomal  trisomies,  less  than  in  seems  However,  expression  both  of  that  trisomies  (an  should  exception  simple  was  evolutionary regulatory  terms,  genes  significance) While  this  should  compensated presented may b e  acquire  the  dosage  it  thi6  were  be  one  that  i n some c e l l  compensation  on  levels  on t h i s  arm.  repress  the  whereas  may b e  exclusively  gene,  have  to  have  normally varies these  genes  evidence  (Maroni et for  In  adaptive  has  X—chromosome d o s a g e  pressure  were  almost  advantageous  types  the  Two p o s s i b l e  and f e m a l e s ,  selective  loci  autosomal  over  repression  that  can  genes  the  Clearly,  show  located  autosomal  which  and  regulatory  located  trisomies  no p r o b l e m i f  males  genes  enigma seem p l a u s i b l e .  product  present  such  enzyme-loci  X-linked  may n o t  suggests  incomplete  Furthermore,  for  There  arm.  which  are  i n metafemales).  chromosome  between  which  and of  affect  (whose  on a  half  autosomal  evolved  and t r a n s p o s i t i o n s ,  loci  the  The  aneuploidy  arms.  regulatory  disperse  the  be r e c a l l e d ;  repressed  explanations  of  gene  not  natural  chromosome  trisomies  X-linked  X-chromosomal  LSP-1o.  all  X-chromosomal  paradoxical  for  translocations  Thus,  repressed  since  have  chromosome  same  would  arm.  trisomies  dose,  evolutionary"history,  different  developmentally related.  these  be on t h e  inversions,  aneuploid  regulates  i n autosomal  with variations  be  loci.  It  coordinate!.y  f u n c t i o n a l l y or  associated  long  in  are  which  pericentric a  which  these  low because  in  dose.  are  dosage  been  compensation  a l . ,  1974).  modifier loci their  effects  to may  be the  b u f f e r e d by other same  genes.  autosomal on  genes  product genes  are themselves  elevated  genes  type  suggest  dosage  negative  in trisomies  negative  The types will  these such  regular compensation  The few a c t i v a t i o n s  genes of  of  gene  Compensation of of  indirect  be d i s c u s s e d  of  evidence  do not c o n t r o l  regulatory  as  their  regulatory  the majority  offers  regulatory  i n metafemales  unlike  of  on  regulate  or by h y p e r p l o i d  on t h e autosomes.  regulators.  may o p e r a t e  quantities  by an e x t e n s i o n  compensation  observed  that  trisomies,  because  which act  on t h e X chromosome  compensated.  could occur  observed  expression  dosage  regulators  m o d i f i e r s which  In X-chromosomal  may n o t b e s y n t h e s i z e d  male-female  encoded  may b e e q u a l l y a b u n d a n t  counterparts,  regulatory  the  Alternatively,  the autosomes.  autosomal  autosomally  gene  to  other  mechanisms further  of  that  in  chapter  6. Previous detected appear  of Adh  the existence  to  Birchler  reports  modify  the Drosophila  analyzed  several  of  ADH p r o d u c e d .  as  a detailed  inform  us of  Regulatory  of  and L u c c h e s i ,  trans-acting  the expression  (personal  expression,  (Rawls  of  communication) genome  particular  and L a u r i e - A h l b e r g  modifier loci  of  individual  the complexity  elements  that  of  which  Recently, survey  m o d i f i e r s of (1983)  have the  quantity  two approaches,  regulatory  loci,  gene  control.  allow a particular  gene  to  m o d i f i e r s may b e f o u n d  modification  of  and i t s  by standard  flanking  in  sequences,  as  well  should  trans-acting  trans-acting  t h e gene  genes.  which a f f e c t  these  have  effects  h a s made a n e x t e n s i v e  A combination of  analysis  1974b)  regulatory  for dosage-sensitive  and Maroni genetic  1974a,  respond  to  vitro followed  by  p—element to  mediated  specific  interesting parts  of  remote  the  transformation  genetic to  conditions.  know w h e t h e r  immediate 5'  and p r o p o g a t e In  whether  the  transposed  in  chapter  Adh  this gene  in  its  this  It  the  regard,  effect it  will  control  promoter,  their  distances.  and a n a l y s i s  or  over  would  be  be  the  whether  are  they  relatively  i n 2L  be  repressed  by t r i s o m y  native  position.  for  integral are  more  long  interesting  examined  response  particularly  elements  Adh genes  can  of  to  know  trisomies 3L  as  is  the  150  CHAPTER  EFFECT  OF E X T E N S I V E  FOUR  A N E U P L O I D Y ON V I A B I L I T Y  AND P H E N O T Y P E  151 Introduction  Aneuploids viable  than  their  observation types a  that  within  balanced  fitness. exist,  i n Drosophila diploid  the karyotype  genomic  (Lucchesi,  characteristic 1922).  possible  trisomies  diploids  (Rhoades  affected  l i t t l e  to  where  genome. changes  exists  dosage  deleterious  from the d i p l o i d however,  only  1935).  For  i n Datura  phenotype  two o f  Other  the ten  change  auto  to  gene  can reproduce (Stebbins,  vegetatively  for the survival  5X)  i s i s  i s a  perhaps  of  resistant 1973)  less. quite  and  In well  particularly  because  these  and a r e s u c c e p t i b l e  to  1966).  the diploid  cytologically  (about  (Khush,  sets  kingdom  and a l l o p o l y p l o i d s ,  are  which are  balance  chromosome  The p l a n t  species  amounts  the organisms  i n number o f  Drosophila,  verified  that  for  i n gene  t h e chromosomes  number a r e p o l y p l o i d  i n many s p e c i e s .  In  minimize  f o r any of  many o f  tolerated  hybridization  suggests  prerequisite  variations  by a n e u p l o i d y f o r modest  changes  organisms  strongly  cell  aneuploidy are variable.  and M c C l i n t o c k ,  the overall  of  a n d most  are morphologically d i s t i n g u i s h a b l e from  i n chromosome  source  of  changes  contrast,  rich  to  less  1978).  In maize,  However,  consequently  species,  natural  operates  primary trisomies  (Blakeslee,  most  are invariant,  physiological effects  example,  the  cases  a mechanism o f t e n  produce  of  are  The p a r a l l e l  condition i s a necessary  In those  The  eukaryotes  counterparts.  an i n d i v i d u a l  consequences  and other  (Bridges, of  and t r i p l o i d 1921)  haploid  c o n d i t i o n have  and g e n e t i c  and t e t r a p l o i d  evidence females  been  151  (Morgan, and  1924;  Stewart  are of  aneuploidy,  t h e genome  must  duplicated  with  hyperploid  sensitive  lethal into  which,  a l l other  l i t t l e  regions  a class  abnormal  is  better  bristle  tolerated  duplicated  called  frequency,  n o t more  than  to arise  in a single  While  17.) o f t h e g e n o m e  mosaic  diploid  individual  competitive  patches  and found  disadvantage.  It  aneuploidy  exerts  i t s effects  expression  within  the cell  development. trisomies  A study  viability.  that  fall  recovered  than  3% ( a n d  recovered.  with aneuploidy Ripoll  i n an  (1980)  otherwise  t o assume  were  i n turn  affects  c o m p a r i n g t h e anatomy that  different  of  organismal  of the effects  at  a  that  by d i s r u p t i n g t h e balance  that  of Datura revealed  are  hyperploids  the aneuploid c e l l s  i s reasonable  58  characterized  are  more  males.  Hyperploidy  adult  are not  of aneuploid t i s s u e  in  loci  growth.  107. o f t h e g e n o m e  cell  phenotypic  dose,  Minutes which a r e  hypoploids involving  from reduced  induced  h a s two  approximately  The p h y s i o l o g i c a l d i s r u p t i o n s a s s o c i a t e d appear  could be  Many o f t h e s e  hypoploidy.  f o r approximately  modest  commonly  effect.  for  when d u p l i c a t e d  contain  which  one r e g i o n  in diploids  regions  morphology and delayed than  loci  (Bx a n d C o ) w h i c h c a u s e  are present  of mutations  only  T h e X chromosome  and t h e autosomes  (1972)  quantitative  that  i n two doses autosomal  a l .  Drosophila can  found  which i s l e t h a l  a phenotypic  et  o f t h e number o f  They  effect.  when t h e y  or have  by  with  and estimates  and one r e g i o n  regions  t o which adult  be present  and that  X chromosome  has provided  abnormal.  viability,  The  T h e work b y L i n d s l e y  (1973)  on t h e e x t e n t  haplo or t r i p l o  changes  1983).  and Merriam  observations withstand  Cline,  various were  gene  153 correlated 1934).  with different  This  phenotypes all  of  conclusion  aneuploid regions  i s confirmed by examining  d i s p l a y e d by primary  the primary  trisomies  (Blakeslee,  observed  i n aneuploids reflect  regions.  Datura  in similar  in  a particular  be  elongated.  reduced  a l . ,  determining  trisomic These  cell  1959)  ways;  Datura  positional  of  genes  stem,  i f  leaves  flowers  suggest  1939)  that,  are  and  elongated will  also  in addition  occur  morphogenesis  in  may b e  and seeds  d i s r u p t i o n s can also information,  Thus,  contained  characteristics  f o r example, then  parallel  modifications  (Goodspeed and A v e r y ,  observations  viability,  the  are morphologically  the action  different  et a l . ,  trisomies.  and t h e phenotypic  In N i c o t i a n a  (Avery et  affected  1922)  and t e r t i a r y  of  distinct  these  (Sinnot  in  to  processes  or  differentiation. Bridges observed from  between  differences  X chromosome  individuals caused  genes  genetic  for the fourth  cuticular  was  suggested  diploids  the relative  haploid  the  (1922)  imbalance.  chromosome.  i t  is unlikely  reverse  Indeed,  t h e sex  of  for  that  of  the difference  encoded  on t h i s  autosomal  an i n d i v i d u a l  loci  from  argued  diplo-X  that  this  i n the a c t i v i t y  While t h e phenotype  dosage,  responsible.  Individuals haploid  characteristic  difference  undoubtedly the consequence  genes  are  and Bridges  is  sex-determining  that  display  females  arise  individuals  phenotypically distinct  and f e m a l e s ) ,  differences  i n Drosophila  Thus,  from the d i p l o i d .  by the q u a n t i t a t i v e  on t h i s  the morphological  and a n e u p l o i d s  chromosome  are also  (males  that  the  of  of  males and  X-chromosome  i n product  chromosome  is  a r e known t h a t  levels  for  solely can a l t e r  ( L i n d s l e y and G r e l 1 ,  1968).  or It  /5f is  probably the  X-chromosome In  ploidy  this  individuals  functional  state  influences  chapter,  of  these  (Baker  we e x a m i n e  the  pre-adult  grossly  abnormal  material.  The r e s u l t s  suggest  all  of  sensitive  and  development, the  pupal  stage  effects  include disruptions  perhaps  sexuality.  regulatory  effect  heterozygous determination  for and  In is  a  but  one  mutation dosage  that  1980).  viabilities  of  of  chromosomal  that  inviability  is  observed  that  the  is  case,  in  genes  amounts  most  embryonic  sensitive.  in morphological  observed  other  and R i d g e ,  possessing  stages  and  in a  evidence  of  trisomic-2L gene  compensation.  stage  is  least  Phenotypic  characteristics a  at  and  cryptic  females  which regulates  which  are  both  sex  155 Materials  and Methods  Aneuploids chromosome—2L described  possessing  were  synthesized  by L i n d s l e y  et  al.  T(Y;2)B251/In(2LR)SMl.Cv different 2L  T(Y;2)s  produces  the  arm o f these  a  of  hours  development  (For  larval the  if  was  development  larval  the  the  a  carrying  (all  the  the  long of  mouthparts)  eggs  were  had been  over  thirty sex  measure  a  after  egg  determined  instar  larval  stage,  as  marker  and  the  the  twenty-four  hours  was  collected  actively  designed to  collected  serving  from  mouthparts).  females  and  2L  identification  studies,  phenotypes  in  (yellow  monitored  on  translocation.  broken  (black  were  positions  d u p l i c a t i o n of  of  experiment  eggs  of  chromosomes  in various  stages  controls  y e l l o w was  every or  several  viability  hundred larvae  and a l l o w i n g  frequencies  trisomies  of  was  were  them t o assumed  scored  bearing  a  trisomies at  different  pupate. to  was  arise  estimated stages  by  of  The d i f f e r e n c e s from  lethality.  directly. deficiency  for  the  Gpdh  as  subsequent  unsure.  development 2L  males  wandering t h i r d  Post-embryonic collecting  T(Yl2)  allowed accurate  H a t c h i n g was  at  instars  pupation  T(YS2)s  duplications  Crossing  breakpoint  period after  of  to  and development  trisomies,  Scoring  twelve  Pupal  the  diploid  hour  period.)  lay.  first  the  many e g g s .  ratio  hour  to  appropriate  viability  two  laying  females  Y chromosome)  to  (1972).  which possess  aneuploids i n the  For over  of  the  relative  sex  individuals  sized  with the  with breakpoints  heterochromatin  Selection  different  region  in  15b  (-For  kinetic  analysis  y/v;C(2L)VHl.lt. females Be*  to  were  selected  E n z y m e was  to  of  their  and  sexed  weighed.  POU,  in  assayed  their  genotype.  enzyme  measure  the  size  of  eye  with  the  cut  out  the  and  The  was  and  assayed  compared  size  had  on  larvae  water, in  were  and  20Qul  spun f o r the  enzyme  then  of  dried  lOmM  1  supernatent  was  activities. from female  stored  were  was  and  parents.  homogenized  a n d PGI  and  photographing microscope  of  weighed  in  extent  pupal  aid  described  of  the  quantity  described  group  crossing  frozen  thawed,  for  trisomic-2L  in  groups  sonicated  PGK, GPDH,  of  and  ADH, PGI,  larvae fifty  or  per  centrifuged; C A T a n d AO  activities.  To  method  individuals  distilled  microfuge,  samples was  in  dissected  parents,  supernatent  organism  The e x t r a c t  were  These  from these  effect  larva  A D H , GPDH,  diploid  by  larval  wandering third—instar  Eppendorf  bodies  wandering  compound—free  and r i n s e d  Each  an  for  Fat  the  what  ImM P T U , p H 7 . 5 .  minute  the  extracted  individual  collected,  produced  males.  at  euploid,  determine  activity,  were  F(2R)bw/F(2R)bw  stage.  To  GPDH e n z y m e )  Df(2L)GdhA/Bc Elp  progeny  that  o-f  or  a  adult  eye  DNA w a s  previous or  (195fi).  chapters. pupal  facets  from each  eye  in  a  was  cut  by t r a c i n g  the  were  diphenylamine measurements  was  estimated  scanning  250—T).  gene,  size.  Protein size  Bar  The t r a c i n g s  by t h e  Cell  eyes  the  measured  estimate  measured  (Cambridge Stereoscan  ten  was  of  eyepiece.  p r o v i d e an  by Burton  adult  variegation  micrometer  to  of  of  as  by  electron  A tracing out  were  and  outlining  weighed.  a  151 Comparison triploid  of  these  strains  quantitative.  values  suggested  between that  eyes  this  from d i p l o i d  method  was  and  reliably  158  Results  Effect  of extensive  a n e u p l o i d y on v i a b i l i t y  The c u m u l a t i v e aneuploidy (Lindsley  effects  are well et  a l . , 1972).  size  rather  than  cell  viabilities  were  examined  larval  which most  with  exceed  et  together, arises  more  and  of  None were  recovered  regions  suggest  would  that  adult  processes  is  inversely  duplications  arm a r e  and c o n c l u s i o n s examined  the  two t h r o u g h  and that  t h e chromosome  cell  various  viability  on p h y s i o l o g i c a l  over  450,000  were  affected  reached  viability.  by  Taken  inviability that  a r e common  or  trisomic-2L  monosomic-2L  or trisomic-3L  allow  such  suggesting  not arise  by a s i n g l e  zygotes  individuals that  to  lethality  from the excess  of  f o r EMS i n d u c e d survive  of  to  adult.  these  gene  product  gene.  chromosomes  allow  simple manipulation of  o f t h e D r o s o p h i l a genome.  combinations  chromosome  aneuploid  monosomic-3L  Compound  possessing  larval in  Therefore,  that  screened  does  by increases  of aneuploidy,  when t h e y  for  development.  that  encoded  14 s h o w s  segmental  i n Drosophila  i s the case  largely  arm o f  of  viability  individuals  one—half  results  mutations  individuals  Figure  than  from e f f e c t s  200,000  of  this  the size  i s unknown.  for the left  (1972)  these  We h a v e  number)  Similar effects  al  a l l stages  occurs  the extent  severely.  Lindsley  to  growth  development.  correlated  for adult  Whether  (where  duplications  increasing  documented  development  sized  of  and development.  of stocks  bearing  By c r o s s i n g  compound o r  large  various  compound—free  \51  F i g u r e 14. V i a b i l i t y to l a t e t h i r d - i n s t a r larvae of partial trisomies f o r 2L. V i a b i l i t y measured r e l a t i v e t o diploid c o n t r o l s p r o d u c e d i n t h e same c r o s s e s . Sample s i z e s are between 119 a n d 301 f o r e a c h p o i n t . T r a n s l o c a t i o n s used were: B110, A62, G74, B92, L52, A l l l , H121, H116, R136 ( L i n d s l e y e t a l . , 1972). D u p l i c a t i o n s extend f r o m t h e p r o x i m a l r e g i o n of 2L (chromosome p o s i t i o n 40) o u t t o t h e more d i s t a l chromosomal p o s i t i o n s , w h i c h a r e i n d i c a t e d on t h e X a x i s .  Ibl  chromosomes three) other in  of  any  arm.  Since the  viability  independent  of  ploidy  arm t r i s o m i e s  hatch  is  represent viable  the  better  are  trivial  Cross  arises  a  than  have  reduced  been  of  even  Crosses  about  the  compound moves  one  of  and  i n both  arms,  a  assumed  all  stage.  frequency  surviving  (ie.  all  similarly,  larval  have  are  and  that  Similarly,  These  standard  of  egg  2  as  to  5  stains.  50%  and  from  inferred;  of  egg  larvae which are  been  most  assumed  to  considerations  aneuploids possessing sets  any  presented  viability  the  the  the  of  produced  aneuploid conditions  lethality  properties;  free  as  made w h e r e  are  arm behave  double trisomies  frequency  eggs.  dosage  often  poor  arm.  far  that  are  been  other as  are  to  haploids,  reduced  or  triploids,  described.  that  meiotic  the  any  chromosome  compound-free  frequencies  has  autosomal  balanced  from embryonic  autosomal  very  double monosomies).  of  1 shows  unfertilized  of  it  the  one  aneuploid classes  classes  have  assumed  (ie.  (from  investigation  particular  develop  is  since  numbers  tetraploids)  display  it  by themselves  increased  of  one  are  dosage  aneuploid types  combination of  survive not  can  when c r o s s e s  reduced,  in this  progeny  for  the  i n d e p e n d e n t l y of  Therefore,  monosomic the  vary  Aneuploid  monosomic  frequency.  Therefore,  arm  several  individuals  whole  to  used  i n T a b l e 33.  example,  hatching  possible  The c r o s s e s  crosses,  presented  is  chromosome  T a b l e 32.  some  for  it  at the  This  well  from d e p o s i t i o n  hatch  as  examine  suggest  random.  can  hatch.  These  sexes,  wild-type strain  If  would  lines  that  the  the  the be  four  egg-hatch  possess  free  arms  similar segregate  compound p a i r e d about  25%;  of  possible  display  all  two  presumably  if  the  with  IbX  Table  32  Embryonic  Exp.  viability  of  whole arm t r i s o m i e s .  Parent Female  R  First Instars  Male  Eggs  hatch  Oregon R  384  364  .924  1  Oregon  2  y/y?C<2L)lt; F(2R)bw/F(2R)bw  y/y;C(2L)lt; F(2R)bw/F(2R)bw  1812  897  .495  3  y2/y2;F<2L)pr/ F(2L)pr;C(2R)px  y2/y;F(2L)pr/ F<2L)pr;C(2R)px  1513  782  .517  4  C(3L)ru st; F<3R)e VF<3R)e  C(3L)ru  1222  555  .454  1279  619  .484  £  5  e  st;  F<3R)  e^/FiZVe*  F(3L)ri/ F<3L)ri ;C<3R)e '  F(3L)ri/ F(3L)ri;C(3R)e  6  Oregon  C<4R)P1+  112  101  .901  7  C(l)RMy  pn v / 0  Oregon R  992  693  .699  S  C<2L)+; F(2R>bw/F<2R)bw  Oregon R  495  232  .469  9  Oregon R  C(3L)ru st| F<3R)e /F<3R>e  12B2  583  .455  e  R  ! 3  B  e j  10  F(3L)pr/F(2L)pr; C<2R)px;y/y  Oregon R  1141  522  .457  11  F<3L)ri/F(3L)ri; C(3R)e  Oregon R  1929  307  . 159  12  C<3R)e ; F(3L)ri/F(3L)ri  X Y/0? Dp T p l / D f T p l  1472  245  . 166  13  C<2L>VH1,it; C(2R)VHS7,Pin  C(2L)VHl,lt; <2R)VHS7,Pin  1010  289  .286  14  C(3L)SH2+; C(3R>SH19+  C<3L)SH2+; C(3R)SH19+  2467  645  .261  15  y2/y2;C<2R)px; F<2L)pr/F(2L)pr  C(2L)lt; C(2R)Pin+  1369  287  .209  16  F<3L)ri/F(3L)ri C<3R>«  C(3L)+;C<3R>+  815  200  .245  C(3L)ru st; F(3R)VDle / F<3R>VDle  C<3L)+;C(3R>+  2157  262  . 121  s  1 3  S  17  s  GJ  /b3  T a b l e 33 crosses in  Inferred v i a b i l i t y of T a b l e 32.  selected aneuploids  from  Exp.  karyotypic class  1  diploid  92.4  adult  2  diploid  99.0  adult  3  diploid  103.4  adult  4  diploid  90.8  adult  5  diploid  96. B  adult  6  Ts4  90. 1  adult  7  TsX  93. 1  adult  8  Ts2L  93.7  adult  9  Ts3L  91.0  adult  10  Ts2R  91.4  pigmented  11  Ts3R  31. B  second  12  T s 3 R / D f <3R>Tpl  66.6  tan  13  diploid  1.00  adult  14  diploid  1.00  adult  15  Ts2R  83.9  third  16  Ts3L  98.2  pigmented 2  17  Ts3R  48.6  first  Nondisjunctional Ts Trisomy 5 8  adults  7. v i a b i 1 i t y  also  recovered:  of  1)  16;  2)  the  latest stage development  9;  pupae  instar  pupae  3)  instar  pupae  instar  7  1  3  trivalent  moved  expected.  Thus the  compound-free disomic  for  Crosses  13  are  to  but  one  6  trisomies  chromosome  when p r e s e n t arm  of  from  causes:  chromosome  Cross  arms  locus 12  deficiency trisomic 3R  for  for  lower size  major  than  15  detrimental proceeds, late  effects  of can  and  the  the  of  33).  or  chromosome.  that  gametes  they  are  the it  is  bears  post  the  but  loci  A s was  the  are  arise  five  major  and  83DE).  on  continue  survive  the  stage.  to  3L For  of  do  and 2L  that  the  the  to  larval  was  3R.  in  refractory  2L,  complete  suggesting  discussed  for  to  individuals  viabilities  Trisomies  a  otherwise  relative  as  right  haplo—triplo  may e x i s t  However,  adult  for  the  these  pupation. to  of  all  viability  T h i s may  trisomies  embryonic  die  that  i n d i v i d u a l s with  particularly  to  of  (chromosome r e g i o n  v i a b i l i t y of  trisomy.  begin  poorly.  viabilities  inviable  clear  Trisomies  region,  other  is  on e m b r y o n i c  largest  region  for  viabilities  It  effect  very  2)  Drosophila.  stage  trisomies  stages  chromosome  of  egg  is  enchanced  triplo  shows  trisomies  and  condition.  arm  observed  Figure  nullosomic  nondisjunctional and  be  these  compound  strains  v i a b i l i t y of  However,  would  from  either  embryonic  triplo-lethal  have  other  the  the  the  few  little  proximal  that  that  32  survive  this  its  and/or  section,  three  the  the  trisomic  1)  3R,  trisomies.  has  examined,  in  shows  examine  arm  are  20%  investigations.  (Tables  the  chromosome  two  lethal  in  that  than  products  to  compound-free  12  less  gametes which  present  to  of  meiotic  demonstrate  the  Crosses  hatch  corresponding  by the  for  whole-arm  are  arm  17  a  primary  lines  the  produced  suitable  randomly,  five  previous  the development so  the  until X  trisomies  F i g u r e 15. Post embryonic v i a b i l i t i e s of the f i v e major whole arm t r i s o m i e s . D e v e l o p m e n t a l t i m e s a r e shown f o r 2L t r i s o m i e s . T h e v i a b i l i t y o f t r i s o m i c - 2 R s e c o n d a n d t h i r d i n s t a r l a r v a e was not measured. However, o b s e r v a t i o n s s u g g e s t t h a t few i n d i v i d u a l s progress past the second l a r v a l molt.  IT  Hours of  development  grown one  at  per  18  degrees  C,  this  2000 z y g o t e s .  trisomies  was  not  viability  19371  Frost,  I960;  used  in this  study,  for  The v i a b i l i t y  calculated,  adult  However,  frequency  of  metafemales Rolfes adult  another  exceeded similar  development  and  through  final  the  for  their  development the of  X axis  to  2L  stages  proceeds  Oregon R and  retarded  condition were  collected  developmental the  delay  in  first  was  far  diploids.  For  example,  as  270  measured  than  3L  low.  The  (Brehme,  For  the  one  strain  in  to  be  greater  to  200.  hours  for  2L  survival  the  that  the  since  trisomic  third  instar  trisomies  a  a  average  the  plotted  reach  population.  larger  is  on rates  possess is  linear  trisomic  similarly.  in  by t h e  which  developmental  that  stages  trisomies  at  The d e l a y  suggesting  to  3L  trisomies  diploids.  the  and  strain  represents  2L  have  pre-imaginal  better  likely  much  late  through  for  of  individuals  in  trisomies  The r a t e  suggests  experienced  population  long  for  about  development.  trisomic—2L  individuals  is  as  trisomies  developmental  individual  for  the  and t h u s  likely  less  be  adult  strains.  (r=.996>  most  development  was  1961).  show somewhat  was  relative  on t h e  healthier  stage  extremely  X-chromosomal  imago  trisomies  stage  to  considerably  A comparison  development affects  of  parent  15).  2L  but  rate  for  development  throughout  of  diploid  the  T  and 3L  pupation,  (Figure  adult  also  varies  viability  Thus,  The developmental and  is  estimated  genotype,  SX.  viabilities  to  and H o l l a n d e r ,  CCDRM.YS. In(l)EN.v/In<l)sc^ viability  but  was  The  data  particular  bias  in  Indeed,  favour the  trisomic  the  range  strains larvae a n d 290  for  than  were  the  in  observed  hours  for  3L  Ib8 trisomies, pupated  while i n the diploid  180 h o u r s  Dobzhansky metafemales Indeed,  reported  can be i n c r e a s e d of  i s negatively  survival  through  pupal  may a l s o  trisomies  high  that  Temperature genes gene  (Suzuki product  the  survive to  et  a l ,  (unless  at  genes  1976). Y  tot:>  C.  stages  of  increased  by  Surivival  through  t h e number  of  i n elevated  gene  produces  of  of  a  by genes  temperature  many  levels  Many p r o d u c t s  i n trisomies high  the  at low  on t h e e x p r e s s i o n  product).  Therefore,  shows  development.  and can r e s u l t  levels  16  are  present  on  may a c c e n t u a t e  imbalance. Elevated  of  i s  Figure  s l i g h t l y by growth  the particular  elevated  temperature.  markedly reduces  abnormal  of  f o r t h e X and f o r t h e  period  18 d e g r e e s  late  1976)  low  temperature.  marked e f f e c t s  d u p l i c a t e d arms.  this  at  at  both  to  be i n c r e a s e d  temperature-sensitive synthesized  trisomies  temperature  can have  l a r v a e had  the v i a b i l i t y  by growth  the larval  the cultures  temperature;  that  related  maintaining stage  the last  egg d e p o s i t i o n .  (1928)  the v i a b i l i t y  autosomes that  post  strain  temperature  subjected Moreover,  Su (Var) - 5  T  levels  However,  this  of  at  least  transcription  (En(Var)5  and En(Var)7)•  viability  of  Df(2R)MS210  T  variegation  to  of  (Clark  d i d not affect Similarly,  suppress  which removes  most  expression  (Spofford,  variegation,  and K i e f e r , of  1977).  variegation  the deficiencies  variegation, of  of  cause  and two enhancers  2L t r i s o m i e s . blocks  the level  one suppressor  suggested  suppressor,  heterochromatic  increases  to position-effect  has been  elevated  also  for  large  but  the heterochromatin  of  F i g u r e 16. Viability temperatures.  of  2L  and 3L  trisomies  at  different  100  • 3L •  2L  pupariation pharate adults  co 3  50  0  i 18  1  25  Temperature (°C)  r 29  Ill  2L,  d i d not  interest,  the  however,  deficiency adult  reduce  of  stage  the  at  a  is  the  has  been  shown t o  and  Leibovitch,  overall  level  2L  gene  hi stone of  of  of  about  gene  trisomies.  were  found  17..  This  and a  a l . ,  Of  which possess  causes  expression.  Since  some a  survive to  chromosome of  It  an  is  this  region  (Khesin  possible  elevation might  the  region  this  variegation  1979).  templates  to  deletion  position effect  Moore et  gene  2L  2L  cluster,  suppress  1978;  o-f  trisomies  390E r e g i o n  histone  of  that  frequency  contains  removal  viability  in  be  that  the  expected  o  to  reduce  the  explanation Wright,  et  viability  is  warranted.  a l . ,  chromosome  are  of  1976) not  is  as  2L  trisomies,  an  alternative  The d e f i c i e n c y used large  and t r i s o m i e s  severely  aneuploid as  (Df<2L)161  f  produced with are  this  whole-arm  trisomies. The v i a b i l i t i e s of are  shown i n F i g u r e  slower  than  boundary not  is  survive  table  17.  and female  While  do f e m a l e s ,  their  to  that  as  as  females  well  in Figures  haplo-X  condition is  female;  it  may b e  physiological  not  less  17a  trisomic  of  their  male  and b ) .  to  to  This to  accommodate  caused  by a n e u p l o i d y .  X-chromosome  dosage  compensation  withstand  then  these  additional  trisomies.  This  trisomies  (Tables  individuals  degrees  was  tested  34  8c 3 5 ) .  of  might  larval  the  the  males  do  (see that  the  diploid  additional  is  operating  be  expected  are  3L  pupal  development  of  by examining t h e  and  develop  However,  aneuploidy than  Two p o i n t s  2L  may i n d i c a t e  that  stress  metafemales  the  pupal  for  larvae  sisters.  through  equivalent  able  trisomies  viability  similar  inserts  If  male  fully to  in  better  autosomal  viability striking.  of  double  First,  172-  F i g u r e 17. Sex r a t i o o f a u t o s o m a l t r i s o m i e s a t d i f f e r e n t developmental stages. A. T r i s o m y 2L B. Trisomy 3L. Zero t i m e o n t h e X a x i s r e p r e s e n t s t h e t i m e when t h e f i r s t larvae began t o p u p a t e . L a r v a e were sexed and c o u n t e d as t h e y d e v e l o p e d t o the wandering l a r v a l s t a g e of development. The t a b l e inserts i n e a c h f i g u r e show t h e sex r a t i o s o f t r i s o m i e s a t t h e first i n s t a r l a r v a l s t a g e i n A . , and at t h e p h a r a t e a d u l t s t a g e i n A . and i n B.  A  300-.  0  48  96  hours  144  n+  T a b l e 34 V i a b i l i t y of double f o r one autosomal arm.  trisomies  and t r i p l o i d s  deficient  Parent Female  Exp  Male  eggs  first instars  hatch  1  Oregon R  C(2L>lt;C(2R>Pin+  1834  165  .089  2  Oregon R  C<3L)+|C<3R> +  1999  249  . 125  3  y2/y2;F<2L>pr/ F(2L)prlC(2R)px  F<3L)ri/F<31)ri| C(3R)e  1053  3  .003  4  y2/y2;F(2L>pr/ F<2L>pr|C<2R)px  C(3L)ru st| F(3R)e /F(3R)e  2394  67  .028  y/y;C<2L)lt; F(2R)bw/F(2R)bw  F<3L)ri/F(3L)ri| C(3R)e«  1512  66  .044  y/y|C(2L>lt; F(2R)bw/F<2R)bw  C(3L)ru s t l F<3R)e /F(3R>e«  1517  55  .036  C(l)RMy  C(3L)ru st| F<3R>e /F(3R)e  2044  467  .228  5  6  7  v pn/0  < 3  s  3  s  e i  3  B  8  C(l)RMy  v pn/0  C(2L>lt;F(2R)bw/ F(2R>bw|y/y  2141  558  .261  9  C(l)RMy  v pn/0  F(2L)pr/F(2L)pr| C(2R)px|y2/y  981  183  . 186  10  C(l)RMy  v pn/0  F(3L)ri/F<3L)ri! C(3R)e  666  10  .015  C(2L>lt|C(2R)Pin+  2485  39  .016  F(3L>ri/F<3L>ri| C(3R)e  C(2L>lt|C(2R)Pin+  2486  52  .021  13  y/ylC(2L)lt? F(2R)bw/F(2R)bw  C(3L)+;C<3R)+  1510  31  .021  14  y /y ;F(2L>pr/ F<2L)pr|C<2R)px  C(3L>+|C(3R>+  7895  129  .016  15  C(l>RM,y v p n / y | F<2L)pr/F(2L)pr| C(2R)px  C(3L>P2,ri| C(3R)VMK1  1040  2  s  11  C(3L)ru st| F(3R>e /F(3R>e' s  12  3  s  : 2  : 2  .001*  115  T a b l e 35 Inferred v i a b i l i t i e s of c r o s s e s i n T a b l e 34.  Exp.  karyotypic  class  y.  selected  viability  aneuploids from  the  latest stage of development  1  Ts2L;Ts2R  35.6  first  instar  2  Ts3L5Ts3R  50.0  first  instar  3  Ts2R;Ts3R  1 . 14  first  instar  4  Ts2R;Ts3L  11.2  second  5  TS2L;TB3R  17.5  early  6  Ts2L;Ts3L  14.5  second  7  TsX;Ts3L  0  8  TSX;TS2L  8.8  9  TsXiTs2R  0  egg  10  TsX;Ts3R  0  egg  11  2X;3N-3R  12.6  second  12  2X;3N-3L  16.7  third  instar  13  2XI3N-2R  16.4  third  instar  14  2X;3N-2L  13.1  first  instar  15  3X5 3 N - 2 L  0  instar third  instar  instar*  egg  instar  egg  * c o n f i r m e d c y t o l o g i c a l l y ; one l a t e second i n s t a r larvae c o n t a i n e d t e n l a r g e chromosome arms, two f o u r t h chromosomes, and a n X a n d a Y c h r o m o s o m e . T s = T r i s o m y . 3 N «= t r i p l o i d f o r a u t o s o m a l arms e x c e p t f o r t h o s e i n d i c a t e d as m i s s i n g by a sign.  the  embryonic v i a b i l i t y  single cross one  chromosome 2)  arm  combinations (crosses  2  that  chromosome  genes  form of  positive  condition  survive not  negative  Dobzhansky,  It  autosomal  often  forming unhatched  indeed  expressed  in  3  and  on a v e r a g e ,  does more  not  to one  than  of  the  arm appear  than  a  involving  (crosses  chromosome  would  to  of  any  arising these  occur  in  X chromosome  suggesting of  should  the  to  of  be  the  the is  haploid  late  many o f at  larvae. as the  diploid  48  These hours  genes levels  the  a  viable the  a  might  arms  karyotypic  genie do  condition  after  these  hypoploid  high the  not  (see  does also  proportion  egg  individuals  i n the  of  hyperactivation  that  of  This  from  rectifies  that  capable  observations  autosomal  be noted  are  expression  monosomies d e v e l o p e d t h r o u g h  and as  possible that  Ts3R  trisomies.  level  monosomies f o r  these  is  the  compensation  consequence  1957).  occasionally,  Ts3Ll  X chromosome  many g e n e s  regulation  the  (Table 36), a  that  A corollary  dosage  However,  as  of  includes several  largest  autosomal  regulation  because  well  arise  in  Monosomy f o r  imbalance.  This  a n e u p l o i d y any  by r e d u c i n g  condition.  condition.  the  X chromosome  revealed  compensated  occurs  hyperploid  arms  chromosomes  s u r v i v e worse,  the  the  1 or  both  arms.  apparently by a  10)  for  do d o u b l e t r i s o m i e s  involving  i n v o l v e 3R, Thus,  cross  and t h i r d  trisomies.  chapters  dosage  that  to  with additional  Previous  be  7  in  than  second  (crosses  and 5 ) .  compatible  being  Ts2R  double trisomies  double autosomal  other  Ts2LS  from the  Second,  autosomal the  double trisomies  s u r v i v e much b e t t e r  arm e a c h  6).  (ie  of  stage,  hatched  fertilization.  monosomic arm  i n monosomies  of  only It  are  (compensated)  m Table  36  Survival  of  autosomal  Parent female  male  monosomic produced  monosomies. # o f 48 hr o l d eggs  # with mouth parts  'A u n h a t c h e d monosomic larvae  y/y;C<2L)lt; F(2R)bw/ F(2R)bw  Oregon R  2L  100  85  85.0  C(3L)ru st; F(2R)e / F<2R)e  Oregon R  3L  502  451  89.8  S 3 ! 9  but  the  genes.  inviability  and  effect.  produced 35).  diploids To reduce  w h i c h were  These  individuals but  relative  to  trisomies.  only  X chromosomes.  two  incomplete may h a v e  were  for and  for  consequence i n product  these  this  for  These If  i n the  one  few  unregulated  between sufficient  triploids  autosomal than  arm  (Tables  d i d the  individuals  X-chromosome (see  Baker  reduced  autosomal  dosage and  34  potential  possessed  Belote,  To t e s t  in  was  1983)  yielded triploid However,  &  diploid  compensation  viability.  arm.  for  were  l i m i t e d developmental  should have  cross  a  level  1.5,  triploid  their  made t h a t  o n l y one  to  of  may b e  survived better  possessed  to  genes  factor  individuals  contributed  deficient 34  still  i n these  crosses  Table  a  deficient  monosomies,  two  as  The t w o f o l d d i f f e r e n c e  monosomies this  arises  it  this,  females  cross  15  of  C(1)RM+/yfj  C(2L)ho/+/C(2R)px ; C(3L)ri/+/C(3R)cu C(2L)P.bi  F(2R)bw/F(2R)bw  individuals best,  triploid  were  at  males  recovered.  limited potential  compensation  females  the  no  These  by such experiments  exists  for  complete  chromosome  arm  level  suggest  that,  at  hypoploid  in Drosophila.  Phenotype  Aneuploid diploids  (see  for  example  eye  facets  revealed  individuals Figure  missing, (Figure  that  all  18).  are  The a l t e r a t i o n s  duplicated  25).  phenotypically distinct  or  very  subtle;  mispositioned hairs  between  The p r e v i o u s  trisomies  may b e  from  section  d i s p l a y common  of  this  chapter  developmental  179  F i g u r e 18. Phenotypes of trisomies. A. S D S - p o l y a c r y l a m i d e g e l s of p r o t e i n samples e x t r a c t e d from p h a r a t e a d u l t s : l e f t , O r e g o n R; m i d d l e , 2L t r i s o m y ; r i g h t , 3L t r i s o m y . Whereas q u a n t i t a t i v e d i f f e r e n c e s i n band i n t e n s i t i e s are apparent, no bands were c o m p l e t e l y l o s t i n t r i s o m i e s , nor were any u n i q u e b a n d s o b s e r v e d . B. A l a t e h a t c h i n g l a r v a e presumed t o be monosomic f o r t h e left arm of chromosome two. D e v e l o p m e n t o f s p i r a c l e s , trachea, m a n d i b u l a r h o o k s , and d e n t i c l e b e l t s were n o t e d i n t h e s e individuals. C. t o p l e f t and r i g h t , t r i s o m i c - 2 L f e m a l e s ; bottom l e f t , diploid female; bottom r i g h t , d i p l o i d male. D. S a l i v a r y g l a n d p o l y t e n e chromosomes f r o m a trisomic-3R larvae ( r a r e l y s u r v i v i n g ) , presumed t o c a r r y a d e l e t i o n f o r the h a p l o - t r i p l o l e t h a l r e g i o n . C h r o m o s o m e 3R c a n b e s e e n a s the t h i c k arm j o i n i n g t h e c h r o m o c e n t r e at s i x o'clock.  abnormalities  such  autosomal  X-chromosomal  and  disruptions The ratio this is  of  sexual  slow growth  is  to  0.5  or  or  exceeds  X/A r a t i o  of  characteristics possess  a  otherwise  diploid  genome.  X/A r a t i o  development male  is  is  large  obvious. lies  .89.  An e x a m i n a t i o n  that,  w h i l e 2L  (2L  approximately Poor  the  which  is  the  testes  of  X/A r a t i o  late-pupal  than  the  an  normal,  which f a i l e d testes  to  sexual  these ratio  for  on  was  an  for  3L  female  of  males  in  possessed head  of  threshold  elongate  required for  2L o r  for  .66  and  revealed  trisomies  characteristics  individuals  content  X/A r a t i o  both  display  Autosomal  the  3L  it  Flies  intersexes)  trisomic  sperm w i t h normal  male in  between  a n d when  no e f f e c t  that  the  When  results.  autosomal  and yet  with  sets.  occurs,  trisomic  suggests  appeared  sixty-four  lower  .89  a n d 3I_ t r i s o m i c  development  since  of  autosome  i n the  abdomens.  correlated  triploid  females  somewhere  trisomies  rudimentary  individuals  In  This  is  and f e m a l e s .  change  approximately  development  rather  males  phenotypic  development  2X3A,  Adult  and misshapen  development  female  (ie.  of  legs  number o f  male  1.0,  .66  lethality.  Drosophila  the  less,  trisomies  the  hind  phenotype of to  and  aneuploids share  gnarled  X chromosomes  equal an  well:  sexual  ratio  with  as  as  possessed many bundles  of  morphology). was  not  expected  approximately  complete  .44,  male  development. The a  sex—comb  convenient  teeth  marker  for  present  on t h e  examining sexual  example,  while diploid  males  each  (see  1 i n Table 37),  leg  (genotype  2).  genotype  forelegs  A mutation  possess  males  development.  about  which s h i f t s  of  ten  females  For  sex-comb have  development  provide  teeth  none towards  the  on  T a b l e 37 Number o f trisomies.  sex-comb  teeth  i n normal  and  sex-transformed  foreleg Genotype  #  teeth  7. g a p  n  10.2  + 0.2  0.0  22  male  0.0  + 0.0  10  female  30  male  10  female  30  reduced male  10  female  1  X/Y  2  XX O r e g o n R  3  X/YjC<2L>lt/+/ F(2R)bw  11.8  + .01  4  X/XlC<2L)lt/+/ F(2R)bw  0.0  + 0.0  5  X/Y»C(3L)ru +/F<3R>VDle  st/  6.9  + 0. 1  st/  0.0  + 0.0  6  Oregon R  of  X/X»C(3L)ru +/F(3R)VDle  10.0  3.3  phenotype  s  Q  7  X/Y;Mas/+  10.7  + 0.2  0.0  22  male  8  X/XsMas/+  7.0  + 0.1  0.0  30  intersexual  9  X/Y«C<2L>lt/+/ F<2R)bw;Mas/+  11.8  + 0.2  6.7  23  male  10  X/X;C(2L)lt/+/ F<2R)bw;Mas/+  9.0  + 0.2  16.7  27  intersexual  11  X/Y;C(3L)ru st/ Mas/F<3R)VDle »  6.8  + 0.3  25.0  12  reduced  X/X;C<3L)ru st/ Mas/F(3R)VDle  3.9  + 0.2  0.0  30  intersexual  13  X/Y;Bs dsxpP/ dsx pP  4.8  + 0. 1  26  intersexual  14  X/X;Bs dsxpP/ dsx pP  5. 1  + 0. 1  22  intersexual  15  X/YlBs C(3L)ru st/ dsx p P / F(3R)VDll,dsxpP  4.5  + 0.2  10  intersexual (1)  16  X/X;Bs (C(3L)ru dsx p P / F(3R)VDll,dsxpP  8  intersexual  mal  e  12  ( 3  (1)  basitarsis  st/  one/half  5.4  +  normal  0.3  length  with gnarled  appearance  133 male  mode o f  expression  intermediate (genotype  number o f  8).  intermediate  mutation  state  between  both  the  sexes  are  characteristic  3L  are  Table  shows more  teeth  gap  is  often  Apparently sexual  The r e d u c t i o n poor  results  shifts test  the  this  between  a  is  in  males  other  between  dramatically  suggest  be rows  of  these  number  for  for  on  in  2L  and  for  (genotype  3).  average  19A a n d B ) ,  is  in  and t h e the  (Figure  3L  the  (genotype  5,  consistent  these  we e x a m i n e d  the  bristles  the  3L  number  of  Table with  37). the  partially female.  To  interaction  trisomic number o f  Mas m u t a t i o n  the  individuals.  towards  the  a  19F).  which determines  individuals  condition increased  in  sexual  teeth  d u p l i c a t i o n of  locus  bearing  of  trisomic  observed the  the  them  disrupted slightly since  X/A r a t i o  tooth  possibility further  and f e m a l e s  increases  While morphologically  (Figures  reduced  that  transforming  males.  two  i n males  intersexual  and f r o m d i p l o i d s  to  the  development  Furthermore,  in trisomies  diploids  appears  sex-comb  and  possess  the  an  intermediate  males  as  development  sex  of  same  since  to  female  are  and  E).  i n males  sex-combs  simply  i n the  The morphology of  diploid  comb  19  an  teeth  male  and f e m a l e  teeth  than  not  is  The t r i s o m i c - 2 L in  male  14).  the  observed  testicular  These  and  i n the  (Figure  trisomic-2L  the the  teeth  both  from each  phenotype  sex-comb  these  19).  teeth  it  of  (Figure  that  of  structure  and a r e  are  development  develop  similar,  different  slightly these  13  phenotype of  37  to  observed  number o f  causes  teeth  bristles  which s h i f t s  (genotype  The  the  those  female  reduces  females  sex-comb  However,  corresponding a  ( M a s c u l i n i z e r , Mas)  condition. sex-comb  (compare  teeth  I8t  F i g u r e 19. Sex-comb morphology of d i p l o i d and t r i s o m i c pupae e i t h e r p o s s e s s i n g sex t r a n s f o r m i n g m u t a t i o n s o r n o t . A. T r i s o m i c - 2 L male B. Oregon R d i p l o i d male C. Trisomic-3L male D. d i p l o i d dsx pP f e m a l e E. d i p l o i d Mas/ Ly s t female F . T r i s o m i c - 2 L male w i t h comb-gap p h e n o t y p e .  m  genotypes obtained  7  and 8  versus  9  and  in wild-type trisomies  duplication  of  trisomic—3L  condition again  development! trisomic-3L 7  and 8  Mas)  sex-comb versus dsx  of  the  the  reduction males  and  or  in  number 16).  is  processes  and  sex-comb  3L  to  femles the  null  phenotype  requires  i n 3L not  that  the  trisomies  directly  modifications arises  involved in  and N o t h i n g e r ,  in  As  such,  da,  a  required  for  female  demonstrates trisomies from  the  d_a  experiment  II  shows  observed  that  females  X-linked  is  the  locus  of  da  was  of  activity  in diploids;  was  derived  not  from da  to  1982). on  The the  oogenesis.  lethal, I  8x1  (see in  females  i s Table  case  d i d not  mothers  for are  38)  2L  viability.  the  sexual  female  unchanged  had e x c e l l e n t  this  for  during  Experiment  to  gene,  dependent  maternal-effect  effect  males  an  required  zygotes  development.  the  whereas  The t r i s o m i c - 3 L  of  (daughterless)  from that egg  female  sex-specific  that  14  sex  (Sanchez  the  and  from d i s r u p t i o n s  proceed  of  13  suggesting  be  activity  (genotype  condition  shown t o  Sxl  in  to  (genotypes  been  of  observed  affect  (Sex-lethal),  expression  is  (allelic  The e x p r e s s i o n  to  female  Mas m u t a t i o n  differentiation.  development  The  product.  observed  has  that  slightly.  mutation  neomorphic,  was  suggests  number  the  is  result  promote  tooth  Another  possible  which are  to  bearing  mutation  Mas i s  entirely  development  sex-comb  males  This  wild—type  development  d u p l i c a t o n of  in  m o d i f i c a t i n of  therefore  appeared  12). the  A similar  and  male  females  and  whereas  dsx  It  11  tooth  IS  promotes  prevented  locus,  that  2L  versus  dsx.  r  10).  hatch  However, 3L  trisomies.  still  T a b l e 38 autosomal  E f f e c t of trisomies.  da  and F l  on t h e  embryonic v i a b i l i t y  Parents  Female  I  y/y;da/da  II  y/y;da/da  First-instar #  Male  eggs  y/y;C<2L)lt; F<2R)bw/ F(2R)bw  y+  larvae y  female  7.  male  X  C(2L)lt; F(2R)bw/F(2R)bw  733  0  0  170  93  C(3L)ru st; F(3R)e /F<3R)e  491  0  0  25  20  12  9  100  78  ! S i  III  of  of  Fl  oc  ptg  v/Y  E 3  510  IS8 completely their that  lethal  brothers a  was  One o f  mentioned  the  product,  of  What  of  of  the  are  Sxl  is  not  of  the  control  of  mode o f  possibility  was  were  <F1)  and t r i s o m i c  and  reduced to  to of  for  is  expression  prevent  (Cline, in  (constitutive a n d cja  to  1978).  males,  required  for  mutants  dosage  for  the  insensitive). i n the  this  two  2L  or  i n these  is  to  observation  3L.  Females  that  from  regions. of  allele The  ploidy the This  females of  Sxl  rational  require  the  hyperaction  essential  occur.  two  viability  lethal  for  X-chromosome  and autosomal  probably arises  female  Sx1 * p r o d u c t  allow hyperactivation by t h e  genes  follows.  product of  X-chromosomal  the  for  as  prevent  quantities  supported  is  by examining t h e  either  experiment  absence  active  compensation  specific  heterozygous  product  Sxl  gene  females  be  to  As  transcription not  is  the  Sxl  in  lethal  between  tested  that  Sxl*  suggest  the  product  i n females.  required for  viability  dosage  between  this  da-*- g e n e  X—chromosome  male  to  of  known.  interaction  for  locus  must  the  therefore  The r e l a t i o n s h i p in  the  required  gene  viability  These r e s u l t s  succeptible  characteristics. also  the  da.  X-chromosome  the  addition,  reduced.  of  is  in  males  of  Sxl  Sxl*  and male  determines  sexes  the  is  hyperactivity  locus  action  female  hyperactivation  compensation  makes  primary roles  however,  Necessarily,  but  severely  3L  previously,  expression  Sxl  also  killing  expression  since  expected,  d u p l i c a t i o n of  sex-specific  allow  as  are  Thus,  required  This some  i n males.  interpretation  complementing  is  mutations  181  at  the  5x1  Somehow,  a  quantity  of  to  manifest  this  gene  The r a t i o product  the  product  of  the  Sxl*  threshold  Sxl*  reduced  product  be to  to  encoded the  is  required to suggest  that  heterozygous females  of  drastically  reduced  viability;  type  through  development locus  reduced may h a v e da*  of  this  of  these  females  on 2L  arisen  a  this,  females  (+  possessing Control  diploids  are  data  fundamentally  crosses  unaffected  imply  I  class) three  that  involved  a  2L  only  Experiment  and at  c o u l d be  with Sxl  of  that  (Experiments locus  of  in  10%  as  viable  stage  of  would  appear  levels  the  shows  as  that  a  in  of  wild  that  relative  type  the to  allele  viability the  (Gla males,  I,  II  on 2L o t h e r  with Sxl  have  were  rescued da*  However  inviability  elevated  T a b l e 39  of  Trisomic-3L  and r e s u l t s  the  copies  not  it  the  following  viable.  Thus,  two  suggest  in  same  below  case.  larval  on 2 L ,  copies  the  The  about  trisomies  in is  the  are  instar  consequence  To t e s t  locus.  observed  constitution  they  which i n t e r a c t s  which possessed gene.  Sxl  is  that  gene  locus  be  Table 38).  da  logical  autosomal  ratio  perfectly  same  first  Since  as  class).  These  the  III,  viability.  product.  produced  to  (cross  exists  the  level  might  trisomic—2L  wild  seems  hyperactivation.  £1. are  the  It  (by m a k i n g  This  this  for  be  autosomal  reduced  elicit  females.  by an  by d u p l i c a t i n g t h i s  product  of  must  one-third  heterozygous).  experiments  survival  regulation.  should  product  females  females  females  Sxl*  c o u l d be  wild-type time  allow partial  the  monitored such  locus  3L  and than  trisomies III da  determining the  in  and  Table  is state  39).  \<\0  T a b l e 39 S u r v i v a l t o l a t e pupae of autosomal trisomies h e t e r o z y g o u s f o r F l a n d p o s s e s s i n g d i f f e r e n t d o s e s o f da-*-.  I  Parents  Trisomic—2L Progeny male  Female  Male  y/y;C(2L>lt; F(2R)bw/ F(2R)bw  II  F l oc p t g da/61 a  y;61a v/Y  y  45  Parents  78  male  C(3L)ru st; F<3R)e / F(3R)e < s  Bla  +  1  0  Trisomic—3L male  female  F l oc p t g da/61a  v/Y  female/ male ratio  female  .008  Progeny female  Bla  +  61a  +  7  2  15  3  female/ male ratio  2.0  s  III  Parents  female  +/+ (Canton  Diploid  male  S)  F l oc p t g da/61a  Bla  v/Y  115  male :  Progeny female  +  Bla  105  101  +  73  female/ male ratio  .79  141  of  dosage  compensation  regulates gene  Ill  the  product  General  Expression  expression is  not  of  can  be  dominant  1950).  In  to  1984  ).  for  three  mutations,  the  Thus, in  a  a  were of  similar  variegated variegation  metabolic expression  be  observed  all  effect  locus  its  to  not  can  of  also  in  late was  the be  gene to  monitor  (Sinclair  et  a l . ,  markers,  a  which had  were  suppressed  examined  in  trisomies  consequence  which have  trisomies,  (Muller,  used  pupae,  as  mutations  new  of  functions  although  the  (ru+)  may h a v e  been  the  expression  of  reduced. most  genes  observed.  to  heterochromatin A potent  heterochromatin. (such  level  which a r i s e  in  new a c t i v i t y  several  which a l t e r s  expression.  the  of  low or  of  A l l mutations  juxtaposed  is  with  subjected  processes  wild-type allele  way w a s  heterochromatin enhance  mutations  unaffected  one  general  Genes  to  with  expression  such  haplo-insufficiency.  expression  interacts  and genes  The dominant phenotype  (neomorphs)  or  this  Considerations  strains  the  which can  40).  gene  mutations  general  As such,  phenotypes (Table  infer  to  Sxl  Whether  variegation.  addition,  alterations  X—chromosome.  known.  phenotype of  used  the of  Physiological  position-effect  The  of  as  variegated  the  are  m o d i f i e r of Removal  of  Y chromosome)  phenotype  (ie.  subject  to  position-effect large  has  reduce  blocks  long the  been  of known  number  of  Table  40  Dominant Mutation  Expression  Location  of  dominant  typel  markers  in  trisomy examined  trisomies.  Expression  s  2L-1.3  haplo insufficient  Ts2L  fully suppressed  Sp  2L-22.0  neomorph  Ts2L  unaffected  Tft  2L-53.2  neomorph  Ts2L  unaffected  Fl  2L-54.8  neomorph  Ts2L  unaffected  L  2R-72.0  neomorph  Ts2L  unaffected  Gla  2L  neomorph  Ts2L  unaffected  ru+  3L-0.0  wild  Ts3L  reduced  R  3L-1.4  haplo insufficient  Ts3L  Ly  3L-40.5  haplo insufficient  Ts3L  D  3L-40.7  neomorph  Ts3L  variable  61  3L-41.4  neomorph  Ts3L  unaffected  Sb  3R-58.2  neomorph  Ts3L  unaffected  or  2R  type  (2)  (3)  suppressed  fully suppressed  (3) (3,4)  (2)  1 . Some n e o m o r p h s may b e a n t i m o r p h i c ( L i n d s l e y et a l . , 1972)1 2. e x p r e s s i o n r e c e s s i v e i n t r i p l o i d s ; 3. e x p r e s s i o n dominant i n t r i p l o i d s s 4. i n one c r o s s a l l u l a were p r e s e n t i n D b e a r i n g pupae, i n a n o t h e r t h e y were a b s e n t .  l°,3  cells  i n which the  heterochromatin the  to  the  gene  a d d i t i o n of expression  promoting) possess,  as  a  increase  in  condition Bar  gene  total  when i t  reduces  size  is  of  chromosome  (compare  would  42).  required V 2  on  mutation  the  the (sc*  (ajc)  normal  v s ;  )  type  the  of  thoracic  expression)  the  this  (ac''  effect  (compare this  Y  normal  Y  because  the  a  both  (sc)  genes  these  genes  development. hairs  left  are  The  present and  scutellar B and  9  arm  variegation.  thoroughly using  1,  1 and  developing  expression)  genotypes  the  was  with a  positon-effect  and  of  (B""~)  possessing  in the  10%  expression  presumably a r i s e s  and h a i r  dorsocentral  (compare  what  diploids  of  increase  a  controls  functions  suggests  trisomies  Y chromosome  scute  number o f  of  addition to  which v a r i e g a t e s  bristle  reduces  more  influence  heterochromatin  duplications for  enhance  and f o r  abdominal t e r g i t e s  number  that  examined  The w i l d  for  This  of  the  to  to  inactivated  strongly  (In(l)sc  achaete  similar  appear  in  males  of  an  Autosomal  genes,  relative  causes  block  the  the  addition  (heterochromatin  rearrangement  and 4 ) .  it  (Table  sc  2  was  genome  1974).  into  the  proposed which  inactivating  trisomic  sizes  Thus,  the  eye  decrease  To monitor  this  the  largely  observation  the  additional  inserted  is  rearrangement for  eye  two  to  d u p l i c a t e d arm)  B™ g e n e  chromosome  This  one  However,  have  of  been  on v a r i e g a t i n g  chromosome  eye.  has  diploids  Table 41).  the  A theory  chromatin.  may h a v e  the  to  chromatin  the  Conversely,  tends  (Zuckerkandl,  to  In  active).  genome  minimum,  examined..  is  by b i n d i n g  factors  (corresponding  3,  the  position effect.  that in  gene  bristles  Table  41  Expression  Genotype  3  of  Bar v a r i e g a t o r Eye SizeCum^)  ± 5 . 4  in trisomic-2L n  Oregon R (diploid)  424  C(2L)+/+/F(2R)bw (trisomic-2L)  354  ±  13.9  20  y sc/  148  ±  14.7  50  males.  trisomy/diploid  20 0.84  (diploid) 2.23 4  C(2L)+/+/F(2R)bw» +/B Y (trisomic-2L)  330  ± 7 . 9  50  S V  Because B i s a dominant mutation, i n c r e a s e d from a reduced l e v e l of expression. a  eye s i z e  arises  195 versus  2  a n d 6,  decreases with the  the  the  Table 42).  expression  classic  trisomic-3L  Strong  for  proximal and  contains  2L  of  for  variegation  al.,  Moore et  proximal  trisomies. elsewhere  It  a  large  of  isolated,  a  lack  of  suggest in  only  over  chromatin  of  content  quantities  of  duplicated  arms.  of  fifty  left  communication). observed  i n 3L  not  arise  i n the  specific  as  the a  nucleus,  gene  Moore  whole are  by  arm  located when  were  finding  i n nature) et  a l . ,  may a c c o u n t These of  consequence  of  1983b).  three for  the  results  variegated altered  from  synthesized  that  mutations  chromosome  expression  that  induced by  variegation  rather  et  (Don S i n c l a i r  trisomies.  but  2L  variegation  (Sinclair  arm o f  of  9  which  1978!  genes  amorphic  This  products  region  with the  of  genotype  the  verified  suppressor the  for  possess  as  enhancing  arm  whole-arm  enhancement  variegation  this  in  4).  of  specific  consistent of  modifications to does  38C-40  strong  ( w h i c h may b e  f e w map t o  enhancement  trisomies  is  contrast,  deficiency for  as  that  In  which  The p r o x i m a l  recently  and  region  personal  that  not  consistent  (genotype  individuals  a  3)  observed  The l e v e l s  capable  been  sulfonate  Interestingly,  (Sinclair,  1983).  suppressors  central  genes.  (Dp(2L)  10)  likely  communication)  the  (genotype  again  in  cluster,  which are  of  chromosome  (Khesin and L e i b o v i t c h ,  a l . ,  has  Y  variegation  was  arm  genotype  quite  This  ethyl-methane map t o  is  on 2L  number  genes  and  duplications are  duplicated. personal  5) this  histone-gene  the  variegating  variegaton  (genotype  suppresses 1979!  of  condition supresses  36F-40  the  these  for  duplications for  Dp(2L)  the  of  response  enhancement  trisomies  Removal  increased from  these  genes  19 b  Table  42  Expression of s c  v Z  i n a n e u p l o i d s f o r 2L and 3 L .  tergite 3  tergite 2  dorso— centrals  #  genotype  n  1  Oregon  10  119.7 + 3.0  104.5 + 2.4  4 + 0  2  V2/Y  25  42.8 +1.86  41.5 +1.84  1.2 + .16  3  V2/0  25  12.4 +1.36  13.5 +1.22  4  V2/Y C(3L)ru s t / +/F(3R)e  26  69.5 + 2.04  56.1 + 2.02  V2/Y C(2L)lt/+/ F(2R)bw  25  1.48 .40  +  6  C(2L)SHllt; F(2R)bw/ F(2R)bw/Y  50  7  C(2L>ltS F(2R)bw/ T(Y,2)B110RBS V2/Y  8  9  R  0.84 + .15  scutellar  4  + O  3.80 + .38 2.44 + .19  2.92 + . 14  +  2.96 .17  s  5  10  2.68 .82  +  1.36 .19  +  1.60 .82  44.8 +1.36  37.6 +1.23  +  3.16 .14  +  2.82 .16  25  19.2 +1.5  15.3 ± 1 . 2  3.08 ± . 7 3  +/Y;C<2L>+5 F(2R)bw/ F(2R)bw  10  113.4 + 4.5  ±  96.4 1.9  4.0 + 0  4.0 + 0  +/Y,C(2L)lt; F(2R>bw/ T<Y,2)B110RBS  10  115.8 + 3.2  101.4 + 2.4  ±  3.8 .10  4.0 ± 0  V2/YC<2L>ltS F(2R)bw? T(2?3)VZ1R/ F<3R)VDle  18  10.8 ± 1 . 2  ±  6.61 .88  ±  3.17 .17  +  C 3  Values  given  a r e means  + standard  errors.  1.48 ± . 2 5  ±  1.5 .35  m Repression  o-f  enzyme a c t i v i t y  Compensation the  organismal  changes should which  all  instar  loci  level  in tissue not  of  in specific  measured  from t r i s o m i e s  the  producers  pattern  dissected 43).  of  clearly for  a  o n 2L,  activated  Pai  and Cat  enzyme a c t i v i t y  seen all  trisomic—2L This these  (both fat  may h a v e  level  compensated  enzymes such  not as  only of  live  of have  20).  However,  the  this  fat  This  observed is  lower  dose  largely  (Table  were  than  expected  loci  not  pattern  overall  than  to  that  level  basis)  of  in  expected.  body b e i n g r e c o v e r e d  from  is  in  cannot  found  bodies  similar  a n d mg p r o t e i n  tissue  activity  ADH, which  the  very  from l e s s  since  Thus,  were  A  was  b o d y was  weight  of  dose-dependent  tested  in fat  (Figure  on a  stage  lower  Of t h e  remained u n a f f e c t e d . observed  one  made f r o m f a t  were  Aldox  third  bodies  is  P S K a n d a—GPDH  in fact  was r e p r e s s e d ,  enzymes,  i n T a b l e 43.  ADH s h o w e d  expression.  in diploids.  levels  we w o u l d  of  and  tissue  developmental  o n 2L,  effects  i n trisomic—2L  This  at  or  Such  made f r o m f a t  listed  The a c t i v i t i e s  resulted  than  same  result  quantity  Several  i n measurements  body p r e p a r a t i o n s  individuals,  trisomies  had,  located  i n whole l a r v a e enzymes  responses  in extracts  the  could  expression.  tissues.  enzymes  dose-dependent,  compensated  located  of  genes  activity.  not  the  of  trisomies  and d i p l o i d s .  observed  from larvae  Of t h e  levels  is  of  phenomenon.  in tissue  gene  display characteristic were  cellular  i n autosomal  s p e c i f i c i t y of  dissected  similar  a  from a l t e r a t i o n s  be o b s e r v e d  larvae,  major  is  in  much m o r e f r a g i l e account larval  dependent  for  the  extracts. values  produced i n the  If  it  for fat  body.  T a b l e 43 Enzyme l e v e l s i n trisomic—2L female larvae.  fat  bodies  Enzyme Pgk  Genotype Oregon R (diploid)  y/y;C(2L)lt/ F(2R)bw/ F(2R)bw (diploid)  1.B4 ±  ±  y/+;C(2L)lt/ +/F(2R)bw (trisomic— ± 2L)  .23  +  Repressed  and  Locus  aGpdh  Adh  PQi  Cat  1.49  .324  3.93  3.  ±  -17  .031  ±  .23  ±  Aldox 19  -27  1.36 ±  .09  (11)  (11)  (11)  (11)  (11)  1.84  1.56  .212  5.57  3.96  2.49  .09  ±  .09  ±  .009  ±  .46  ±  .39  ±  .37  (11)  (11)  (11)  (11)  (11)  (11)  .995  .991  .348  2.12  2.89  2.49  ±  .031  +  .019  ±  .13  ±  .20  ±  .20  (11)  (11)  (11)  (11)  (11)  (11)  2.76  2.31  .374  4.75  3.58  1.93  .07  +  1.84 +  diploid  (11)  .056  Estimate  ±  from  .05  Enzyme a c t i v i t y i s p e r mg o f protein.  .06  +  1.54 ±  ±  .249  .01  expressed  .009  +  as  .006  the  .13  3. ±  change  +  in  ±  2.38  16 .09  . 12  ±  .08  absorbance  .09 1.28  ±  .06  per  min  F i g u r e 20. C o m p a r i s o n of enzyme q u a n t i t e s ( i n 2L trisomies r e l a t i v e t o d i p l o i d s ) from whole l a r v a e and from i s o l a t e d fat bodies. L e f t b a r f o r e a c h enzyme r e p r e s e n t s d a t a f r o m whole l a r v a e , r i g h t bar f o r f a t bodies. Expression l e v e l o f 1.0 r e p r e s e n t s t h e d i p l o i d amount p e r gene.  Expression Level o  I  o i  5  b  '—3 I  J  :  b  I  Larvae Fat Body  QO~C  201 Since  both  dose-dependent  measurements)  were  fat-body,  overall  was  the  and r e p r e s s e d  affected  the  results  whole organism  is  of  serious  for  a  ways  (based  in  larval  specific  the  the  extracts  inconsistency  tissue  that  from  fat-body  However,  discrepency  on  in extracts  activity  in origin.  obtained  a  similar  reduction  probably experimental  between  in  loci  and  warrants  for  the  further  investigation. Modification reduced  to  catalytic  the  enzyme m o l e c u l e s  activity  detected.  Trisomies for  deficiency  for  the  Gpdh  same two  alleles  strain.  Enzyme was  both  genotypes  for  and  in  2L  were  locus  this  kinetic  a-glycerophosphate  substrates  (Figure  that  occurring.  were  postranslational In  addition,  the  results  differ  from those  These  authors  found  of  distal  GPDH f o r  quarter  present  study  altered of  and  Lucchesi,  and t h e  Alternatively,  the  made o n e n z y m e s obtained  results  examined  the  from adult  from l a r v a l  for  The Km's f o r  this  these  Rawls and L u c c h e s i  Two e x p l a n a t i o n s  c o u l d be  that  21A  the  of  enzymes.  are this 1).  (1974b). to  25CD  apparent  seem  Km  possible.  performed by  Rawls  fortuitous.  made b y R a w l s a n d L u c c h e s i  flies,  two  enzyme  (Chapter  region  from  to  properties  substantially  thorough* as  measurements  the  larvae  duplication for  as  a  compound-free  2L) s i g n i f i c a n t l y r e d u c e d  was n o t  only  sufficiently different  a—glycerophosphate.  The  were  of  were  for  been  third-instar  electrophoretic  These  a  parental  m o d i f i c a t i o n s of  to  that  the  and NAD.  appear  not  possessed  from l a t e  enzyme do n o t  (the  be  as  not  have  account  produced which contained  aspects  substrates  suggest  extracts  and t h u s  gene  extracted  21)  the  which would  whereas  those  Perhaps  in  this  different  were  study  regulatory  202  F i g u r e 21. K i n e t i c m m e a s u r e m e n t s o n GPDH -from d i p l o i d s a n d trisomies. L i n e w e a v e r — B u r k e p l o t s - f o r A . NAD a n d B . alphaglycerophosphate.  2L  -XJOT3  204affects  are  operating  between  Modulations  of  enzyme  activities  in  or  cell  organism  DNA  the  observations  result  response  is  cases  the  are  with  developmental  not  correlated  changes  (such  about  as  some  in  stages.  with  total  (Table  44).  show  a  non—linear  size  of  the  such  an  effect  It  Pai) in  regression 22-24. (Gjsdh,  has  be  changes  protein  is  or  responsible  and  one  extracts lines  proportional  show a to  of  which  et for  three is  not  of  relationship Estimates  third  parental that  with  (two  is  of  this  were  locus larvae  some  enzymes  changes  in  the  whether observed  which are  in  modulated  were trisomic  tests  that  the  of  larvae  levels  two  85%  instar  reduced  larvae  the  and  To t e s t  Adh)  the  processes  1979).  loci  from these  activities  size.  the  First,  trisomies  effects  activity  is  from  metabolic  diploid  a l . ,  over  in  previously  of  from s i n g l e  obtained  The enzyme Pgi)  (Clarke  activity  Trisomic  reported  in  different  discrete  their  activity  producing  then  internal  such  than  activities  is  trisomy,  in  of  disruptions.  locus  expected.  been  fly  a  enzyme  relationship  adult  the  and  examined  then  not  a  in  by d i s r u p t i o n s  would  repression  significantly  modulations  10-15% s m a l l e r  trisomies,  If in  not  proteolysis),  specificity  (Gpdh  product is  that  physiological  discrete.  less  If  suggest  general  reduction  one-third. brought  of  fairly  significantly  are  nor  two  levels.  Several not  size,  these  are  for  shown  modulated  2L.  The  in  Figures  loci  closely data  suggest  that  the  205  F i g u r e 22. f e m a l e (A) the change  R e l a t i o n s h i p between PSI a c t i v i t y and l a r v a l s i z e i n a n d m a l e (B) 2 L t r i s o m i e s . Enzyme a c t i v i t y u n i t s a r e i n a b s o r b a n c e a t 340nm p e r m i n p e r l O u l o f e x t r a c t .  Z07  F i g u r e 23. R e l a t i o n s h i p b e t w e e n BPDH a c t i v i t y a n d l a r v a l i n f e m a l e (A) a n d m a l e <B> 2L t r i s o m i e s . Enzyme a c t i v i t y a r e t h e c h a n g e i n a b s o r b a n c e a t 340nm p e r m i n p e r 5 0 u l o f extract.  size units  F i g u r e 24. R e l a t i o n s h i p b e t w e e n ADH a c t i v i t y a n d l a r v a l size i n - f e m a l e (A) a n d m a l e (B) 2 L t r i s o m i e s . Enzyme a c t i v i t y u n i t s : c h a n g e i n a b s o r b a n c e a t 340nm p e r m i n p e r 5 0 u l o f e x t r a c t .  x\o  A  .02-  weight  B  (mg)  .02-  0  .9 weight (mg)  1.8  differences  in  activity  differences  in  larval  less  than  clearly  25i f o r  increases  with  this  suggests  data  result  in  expected at  dose  an  Table trisomies  not  live  not  be  activity.  responsible  be  5  a  detected more  Alteration concentration of  measured pharate triploid  the  of  DNA p e r  adults  eye  (Figure  females  consistent  of  test  to  with the  diploid  the  found  these observed  for  change  observed  of  molecules As an in  reduced  levels  of  changes  females  observation  that  is  in  as  of  of  1.47  cell  this  genes  these.  described.  in  altered size  we  micrographs  of  size  (Table  size  In  the  cell  facet  in  enzyme  individual  and r e s u l t  The r a t i o  that  possiblity is  electron  was or  suggests  estimate  are  number  measurements  this  in  in cell  volume c o u l d change  facets  25).  Clearly  was  DNA a n d p r o t e i n  crude  regulatory  the  however,  in  of  should  mg s o l u b l e p r o t e i n  Changes  the  the  of  by such  cellular  than  a n d DNA c o n t e n t  i n p o l y t e n y of  expression.  size  lower  changes  critical  of  gene  for  slope  trisomies  result  levels  strains.  magnitude of  Of c o u r s e ,  not  levels  protein  basis,  the  is  trisomies.  Although  weight  of  individuals.  p o l y t e n i z a t i o n could account  could  chapter  values  between  However t h e  9%  are  Interpolation  ADH a c t i v i t y ,  these  for  DNA.  would  in  44  on a  difference  (Adh) whose  larvae.  approximately  in  gives  the  curve  s t r i k i n g enough t o  and d i p l o i d s .  of  size  enzyme a c t i v i t y  different  levels  not  of  from  and t r i s o m i e s  enzyme  activity  level.  levels  are  an  resulting  diploids  The other  size  the  noncompensated  in  reduced  that  weight)  between  and shows  ADH a c t i v i t y  relationships  unit  enzymes.  increasing  dependent  changes  size  both  non-linear  (per  is  in  45),  adult and  is  proportional  2\2.  T a b l e 44 Live weight, protein content f e m a l e d i p l o i d and t r i s o m i c larvae. ug p r o t e i n / mg l i v e weight  size (mg)  Genotype  Oregon R (diploid)  1.78 ±  97.6 ±  .05  (7)  (10)  C(2L)lt; F(2R)bw/ F(2R)bw/ (diploid)  1.90 ±  C(2L)lt/+/ F(2R)bw (trisomic— 2L)  C(3L)ru st; F(3R)VDle F(3R)vDie (diploid)  +  2.29  ±  ug DNA/ mg l i v e weight  .012  3.56  (5)  1.8  020  (8)  (3)  1.59  99. 1  ,349  1.3  ,013  ±  ug DNA/ protein  .347  (10)  .09  mg  in  319  106  .05  a n d DNA c o n t e n t  (10)  (8)  (3)  1.89  90.9  ,306  3.01  3.52  B s  C(3L)ru st/ +/ F(3R)VDle + (trisomic-3L) e J  ±  .02  +  1.6  012  (10)  (8)  (3)  1.69  86.2  .289  .07 (10)  ±  1-9  ,007  (10)  (3)  3.37  3.35  2i3  F i g u r e 25. triploids, c. d i p l o i d  E l e c t r o n micrographs of eye f a c e t s from d i p l o i d s , and t r i s o m i e s . a. d i p l o i d adult b. t r i p l o i d adult pupae d. t r i s o m i c - 2 L pupae e. t r i s o m i c - 3 L pupae.  2\5  to  the  level  examined and  the  similar  had  o-f  estimated  ratio in  all  (approximately increase  of  cell  the  i n many o r g a n i s m s .  Both  cell  10%  lower  size  was  size  size.  trisomic  protein,  sizes to  genotypes  10'/. i n e a c h  in cell  diploids, soluble  ploidy  It  organism (Table  45).  trisomy) would  individuals  because  only  their  are  that  smaller,  cells  are  than  diploids,  remarkably  The e x t r a  d i d not  appear  trisomies  result  DNA c o n t e n t in  an  relative  to  and have  smaller.  less  lit)  T a b l e 45 trisomic  Estimates pupae.  female genotype  of  stage  Oregon R (diploid)  adult  C ( l ) R M y w~a m/ Base 3N (triploid)  adult  cell  size  in diploid  size (arbitrary units)  167  +  246  (n)  5  +  10  7  12  and t r i p l o i d ,  size relative t o Oregon R  and  cell  larvae size  1.00  1.47  Oregon R (diploid)  pupae  180  ±  3  10  l.OO  lOl  y/y;C(2L)lt; F(2R)bw/F(2R)bw (diploid)  pupae  199  +  3  10  1.10  105  y/+!C(2L)lt/ +/F(2R)bw (trisomic-2L)  pupae  167  +  6  10  .931  106  C(3L)SH2+1 F(3R)VDle / F(3R)VDle (diploid)  pupae  C(3L)SH2+/+/ F(3R)VDle (trisomic-3L)  pupae  194  +  5  12  1.08  103  3  r a  < 3  171  +  5  12  .953  size  102  211  Discussion  This large  chapter  regions  pre-adult are  of  this  with  Since  products  i n some  possible  the effects  The c o n c l u s i o n s  t h e body of  to  we a l s o  have  of these  speculate  of aneuploidy f o r  on v i a b i l i t y and phenotype  problem i n adult  species.  is  t h e genome  Drosophila.  consistent  examined  has explored  derived  literature  from t h i s  that  aneuploids  the levels  as of  (see previous  on t h e genetic  basis  study  p r e v i o u s l y has  Drosophila as well  examined  of  of  other several  gene  chapters),  i t  these  syndromes. The the  larger  disruption observed.  duplicated, thus  more  of  aneuploidy,  As increased  gene p r o d u c t s  unbalanced)  genome. to  the degree  levels  of  loci  are synthesized  at  elevated  relative  to the majority  condition  i s exaggerated,  physiological  processes  ensue  While disruptions  pathways  i n the previous  increased  product  notable  effect,  product  levels  most  genes  product  as  are present  are recessive  suggested  and Burns  difference  chapter alone  exists  at  between  (and  i n the  disruptions  that  suggests  than that  diploid half  T h e most many  levels.  t h e normal  That amount  of  phenotype.  and elaborated  a physiologically  results  i s not  i s that  the wild-type  (1950)  diploid  i t  the  are responsible.  i n 3L t r i s o m i e s , less  are  and developmental  inviability,  suggest  to maintain  (1980),  of  that  by M u l l e r  are  loci  increased  to physiological  particularly  is sufficient  However, Kacser  levels  of  be  and v i a b i l i t y and phenotype  are undoubtedly the cause  presented  will  numbers  As t h i s  altered.  the greater  further  by  levels  of  significant  and o n e - h a l f  diploid  21? gene in  product.  the  data  expression  consistent  stage, for  Muller  large  with  of  with  half  have  product  the  limited. while  levels  usually  level  of  were  represents  system  controlling  fact, the  such  trisomic  individual  and  unlinked been  the  arm,  would  stage. (such  as  act  a  as  a  for  then  buffer  important.  of  survival  reduced  plus  expression. studies  the the This  probably even  of  as  regulatory may n o t  for  number number  an  aneuploid  linked that that  genes are  are  conclusion  on a n e u p l o i d s of  outside  the  of  the  be  In  on genes  and p h e n o t y p e  combination of  diploid  aneuploids.  responsible  proportion  natural  and  genes  when o p e r a t i n g  partially  could  that,  the  the  If  become  level,  Thus,  large  may  that  autosomal  these  gene  against  suggests  exist  individuals  present  from t h i s  of  some  processes are  can  example).  particular  product  This  regulation,  a  for  normal  dose-dependent,  the  24  presented  product  Reductions  from s i m i l a r  Many o f  gene  significant  survival  versus  The a b i l i t y t o  of  also  exists  developmental  adaptively  may b e  and Newton,  discrete  Figure  gene  d e p e n d on what  and have  (Birchler  is  may b e  remain  derived  of  can  The o v e r a l l  compensated  linked  low t h a t  compensation  negative  inviability.  are  so  one-third,  for  (see  potential  optimum.  as  a  amount  expression.  little  At  variability  We h a v e  s u f f i c i e n t l y severe,  product  the  responsible  size  in excess,  gene  view.  quantities  i n gene  considerable genes.  i n the  synthetic  Thus,  variation  this  similar  variations  that  particular  variations  organisms  these  of  noted  has  also  corn  1982).  tolerate gross  double trisomies)  aneuploidy varies  aneuploids observed were  able  to  with  developmental  in this  develop  study  through  embryogenesis and  died  (see a l s o  monosomies and to  were  be  to  first  able  to  1972).  develop instar  insensitive  products  deposited  temporarily  buffer  observations  on t h e v i a b i l i t y  discs  which a r e not r e q u i r e d  1971)  and, as such,  probably during  pupal  elevated females. system that  arises  temperature These  developmental and  complex The  trisomies autosome In the  i n pupal defects.  that  revealed were  addition,  growth  no more autosomal  X-chromosome  Thus,  are  period  abnormal the  large  (Shearn  less  most  i t  i s  caused  to  than  an a l r e a d y  also  i s  sensitive  viable  it  et a l . ,  period  However,  become  development  gene  development  during this  However, is  may  f o r the imaginal  overtax  further.  appear  Maternally  disruptions. trisomies  strained possible  by  specific  involves cell  division  movements.  examined  that  1957).  i n Drosophila  Larval  during this  Pupal  contrast  drawn f r o m  except  development  morphogenetic  studies  1983).  perhaps  viability  in  Drosophila possessing  a n d when m a l e s  conditions  and reduce  lethality  of  that  i s  against  been  aneuploid lethality  development  organogenesis  and S c h a l e t ,  development  from metabolic  moribund  i n t h e egg d u r i n g oogenesis  a l . ,  by a m i t o t i c  of  (This  aneuploidy.  conclusion has also  (Garcia-Bellido et largely  larvae.  became  autosomal  stages  development  to  This  proceeds  late  drawn b y H e r s k o w i t z  expression.  deletions  Similarly,  to  involved i n early  be r e l a t i v e l y  synthesized  Wurgler, to  the conclusion processes  from t h e e g g . but soon  able  occasionally  the to  and emerge  the v i a b i l i t i e s of  the condition  viable  than  trisomies  involving  double  t h e X p l u s an  double autosomal which were  s u r v i v e d p o o r l y compared  to  also  trisomies.  trisomic  autosomal  for  trisomic  220 males of  and f e m a l e s .  X-chromosome  triplo-X large  dosage  diploids,  degree  possibility whereas  This  suggested  compensation  the  dealt  pre-imaginal  i n chapter  viability  comparable,  from pupation to  trisomies.  Two r e a s o n s  remain  dependent  chromosome dosage  such  compensation  compensation important  during  studies  which examine  females  by somatic  viability  in  Nohtinger, during  period.  larvae  the  viability over.  and a r e Thus,  relative  metafemales  may s u r v i v e b e t t e r  most  loci  are  the  on t h e  affect  throughout  imbalance  of  less  than  that  This  study  these  are  gene  arms  products  found has die  individuals  in  their  revealed as  are  than  contrast,  embryos  that or  of  incapable of  in  poor and  X-linked  genes  of  Second, trisomies  12).  because  condition  trisomies Thus,  trisomies  the is  net much  counterparts.  monosomies first  from  produced  trisomic-X  X-chromosomal autosomal  particularly  have  autosomal  (Figure  is  viability  the  genes  X  (Sanchez  autosomal in  some  dosage  cells  trisomies.  genome in  the  autosomal  suggested  male  of  male-female  and  compensation  repressed In  the  on t h e  that  is  for  First,  These c e l l s  autosomal  X chromosome.  loci  autosomal  that  of  may e n h a n c e  metafemales  of  to  viability  i n v i a b l e i n pupae  dosage  development  This  This  the than  development  pupal  crossing  1982).  pupal  throughout  a  However,  by t h e  appears  and  six.  trisomies;  It  diplo  conditions.  this.  compensated  mechanism.  the  better  for  autosomal  may b e  operates  is  may a c c o u n t  in  genes  adult  existence  condition to  trisomic  with further is  the  haplo,  trisomic  autosomal  metafemales  dose  despite  between  X-chromosomal  p a r a l l e l e d the is  that,  instar  fully  for  all  larvae.  compensating  four  major  Thus, the  211  expression level.  This  However, which  of  a  a l l of  their  i s clearly  series  prevent  of  haploid  genes  not the case  mutations  back  well  k a r y o t y p i c a l l y monosomic f o r t h i s  pupal  these  stage)  Perhaps all  condition renders  of  than  individuals f o r autosomal  compensatory  activity  i n males.  percent  of  the diploid  i s also  affect  monosomies.  This  lesser  a b i l i t y of  affect  unlinked  The female  that  determining  factors  species  (relative that  factors  maleness sex  genes  exist  affects  much a s  when i t  i s  males  male  from the to  trisomies). some  do n o t ,  are not  The balance  not  autosomal  trisomic,  males  1980).  does  may p o s s e s s  determining sexuality  that  sixty  and L u c c h e s i ,  t o t h e autosomal  t h e genome.  of  are about  be i n f e r r e d only  trisomic-2L  1983).  the levels  males  as  the  are not a b o l i s h i n g  (Belote  trisomic-3L  whereas  suggests  w i t h sex  phenotype  other  loci  genes  unlinked  t h e X chromosome,  throughout  determination  level  (up t o  monosomy f o r t h e X c h r o m o s o m e  of  characteristics  distributed  these  female  The  (see B e l o t e ,  Indeed,  mutant  p o s s i b i l i t y can  observation  suggests  i n these  possible that  the expression  mutations  in  f u n c t i o n a l l y as  chromosome.  monosomies  lethal  enzymes  individuals  i s considerably better  specific  X-linked  It  these  t h e male  several  lethals)  from being hyperactivated  This  viability  diploid  (male-specific  males. as  the  f o r t h e X chromosome.  a r e known  t h e X-chromosome  to  evenly  theory  of  sex  determining and female  and that  the relative  d i s t r i b u t i o n of  the sexual  phenotype of  the zygote.  chromosomes,  may b e d e t e r m i n e d  the expression  by one of  t h e sex  b y t h e mammalian Y chromosome) presumably are located  of  one  sexual  chromosomes  and determinants  on t h e autosomes.  In  In  (eg, for the  1VL  Drosophila, autosomes  male  determinants  because  genotypes  shifted  towards  present  in triploids.  possessing  and as of  Whether  this  a  general  shifting arises  female-determining are  known of  set  at  (Baker  Pipkin  et  from an  female  1960)  shift  sets  experiments determinants  individuals  possessing  exist is  on t h e  not  amounts  of  female  X—chromosome, with  two  towards  but,  It  is  balance it  determinants have  X chromosomes. male  that  development,  these  less  However,  male  alone. female  Thus  because  studies  they  by  autosomal Both  that  phenotype  left  only  arm may  of  of also  in Drosophila  sex  female  by t h e  relative  males,  with only  determinants  intersexes  flies  located  and  controlled  not  phenotypes.  the  the  is  adult  determinants  between  Triploid not  are  cells  duplications  hypothesis  possible that  is  of  male  with the  female  of  mode,  extensive  towards  were  of  large  sex  Pipkin,  femaleness.  d u p l i c a t i o n of  duplication for  instead,  would  that  and t h a t  suggests  autosomes.  diploids,  intersexes,  consistent  a  male  examined  fragments  viability  the  is  been  number  influence the  X chromosome.  determined by a  determinants  In  revealed  exist,  on t h e  than  development  are  exclusively  three  rather  are  set  al.,1937s  i n the  from enhanced  In  have  X-chromosome  increase  d i d not  1959;  et  the  intersexes  phenotype towards  1983).  1937).  d i d not  chromosome  or  X chromosomes  most  on  X chromosomes  triploid  Patterson  sexual  a l . ,  regions  female  the  material  (1947$  of  trend,  and B e l o t e ,  autosomal  (Patterson  19311  genes  the  of  two  exist  a d d i t i o n a l autosome  Drosophila,  and S c h u l t z ,  capable  that  when a n  In  hypothesized to  possessing  additional pieces  (Dobzhansky 1940)  maleness  are  than  may b e  have  one  females  shifted  an a d d i t i o n a l  223 set  of  female in  autosomal  determinants,  determinants  cell  size.  chromosome possessed  It  in  has  is  other  male  the  individuals  sex  d e t e r m i n i n g gene  (by  Alternatively,  the  reduced when  the  mutation  females  Sxl  locus  However, haplo-X  (males)  be  became  a  chromosome)  would  have  their  female sexuality  facilitated  promote  X  femaleness.  correct  would  an  regions  chromosome  towards  of  become  X  of  increase  autosomal  arm p o s s e s s e d  to  diploid  (CIine, since  1983;  the  Sxl  individuals,  to  again  with  by  a  female  development  to  lethal  is  from  as  would  would  only males  that  female  by s o m a t i c  male. the  Sxl*  has  of  as  that  be  are d_a  expression  of  more  the  1982).  expressed  locus  to  in  be  activated  above  controls,  inconsequential.  should  have  shown t h a t  well  action  of  strengthen  is  they  effect  not  this  crossing  product  the  and L u c c h e s i ,  From t h e s e  development,  of  normally i s  Mosaic a n a l y s i s removed  lack  inability  and t r i s o m i c - 3 L  phenotypically interpreted  locus  suggests  The l e t h a l  Skripsky  the  femaleness  sensitized  males.  f r o m da. m o t h e r s  viabilities.  sustain  are  daughterless  than  diploid  gene  males  presumably r e s u l t s  zygotes  Shifting  Sx1  has  existing  dose  shifted  element  If  the  maintenance  this  trisomic-3L  on  and  single  X and Y chromosomes  3L  when t h i s  chromosome  then  that  species.  to  concentration  hemizygous.  female-like  in  a  the  o n e - t h i r d by the  note  then  balance  this  a b i l i t y of  That of  receiving  if  to  translocation  genes,  original  important  determinants,  chromosome  because  reduced  Drosophila  sex  determining  been  but  over  the  similar cells  develop  experiments, required as  i n which  it  has  to  regulating  dosage  been  the  22Hcompensation. t r i s o m i c —3 L  Since  males  active.  Thus,  reduce  viability  partly  female.  Cline  X-linked  it  the  (1983S  -Follows t h a t  absence  if  genes  the  1984)  of  are  the  Sxl*  dosage  Sxl  locus  product  is  in  not  could  p h y s i o l o g y of  these  has  that  suggested  compensated  individuals  the  is  interactions  o  between  c£a a n d S x l  alterations  in  the  specific  effects  levels.  During  trisomic—2L  allele  appear the  of  result  suggests  as  proposes,  another  the  of  Sx1 * p r o d u c t  would  be  zero.  the  would that  found  dosage that  that  i n the  observed females  Cline,  the  rate  of  these  and/or dosage  healthy  since  for  to  a  genes.  of  Sxl  of  needs  female  to  sexual  is  as  measured  a  reference.  i n males  would  genes. only  to  elicit  be  is  are  sterile)  If,  the  a  characteristic  The present fall the  to  male  development.  mutations  This  product  compensation  (albeit  female-specific  product  ratio two  product  that  development.  transcribed  the  gene  we n o t e d  which a c t s  this  of  Indeed,  It  one-third mode is  responsible  known w h i c h  males  data of  of  likely for  the  transform  ( L i n d s l e y and  Grell,  1984).  locus  determine  F_l_ ( a  during  Two s i m p l e p o s s i b i l i t i e s f o r First,  these  studies  reference  ratio  diploid  lethality, into  not  female  this  compensation  activation  196BS  a  transcription suggest  of  product  to  of  depend on t h r e s h o l d s  early  is  discrete  states  level  gene  ratio  of  die  Sxl  In  from  heterozygous  that  to  to  course  Sxl>  relative  arise  expression  females  lethal  Cline  do not  on 2L  the  c o u l d code  number o f  this  for  autosome  a  regulation gene  sets  product  present.  seem that 1.5  likely. is  used  times  225  more p r o d u c t not  would  compensated.  may r e p r e s s  be produced  Alternatively,  the expression  analagous  to  By e i t h e r  mechanism,  autosomal  standard  one-third. reference  repression  heterozygous threshold between  male  have  1982).  1977,  of  synthetic  has  been  a l . ,  shown i n F i g u r e to  12.  the be reduced  to  autosomal  females  that  that  of  are  the lies  possible that  cells  of  dosage  F1/+  for hyperactivation) tissue  that  autosome their  the wild—type products  male  i s noteworthy  cells  activity  of  most  i n yeast  and Lambrukos,  1980),  are  1974;  i n Drosophila  (Priest  and P r i e s t ,  et  1983).  However,  size  arm  n o r m a l l y d i e , and a r e not  (required  mammals a l . ,  on t h i s  compensation  i s also  is  these  ( S k r i p s k y and  metamales  sets,  of  are  X-chromosomes  (1X:3A),  which  also (Lucchesi,  et  alt  1983).  demonstrated  (DeMaggio  loci  per three  polyploid  the  i t  gene  i n a way  Diploid  dosage  i n some  cells  hyperactivating  Cline, Since  However,  this  locus  would  suggesting of  if  p o s s i b i l i t y , the  on 2 L .  and develop as It  one S x l gene  relative  i n the absence  lethal  may s u r v i v e  capable  et  for activation  However,  Lucchesi,  not r e s i d e  but these  loci  heterozygotes  may b e a c t i v a t e d  specific  cells  product  i n t h e second  and 0.33.  heterozygotes, observed.  t h e S x l gene  f o r F_J_ a r e v i a b l e ,  compensation  a regulatory  t h e enzyme  i n F1/+  need  ratio  0.5  of  of  Sxl*  However, genes  i n 2L t r i s o m i e s  i n autosomal  larger genes  than  has n o t been  (Cifferi Roose  et  1969),  and Rawls,  Westerveld  we d i d n o t o b s e r v e  trisomies  a l . ,  suggesting  that  et  possibly  altered.  and G o t t l i e b ,  (Lucchesi 1969;  diploids,  in  1980; 1973)  a l . ,  plants Timko  and i n  1972;  an i n c r e a s e d this  This  Chang  cell  relationship  may n o t where  hold  the  (Timko e t al.,  for  fractions  various  a l . ,  or  decreased  1980)  with  increasing  from increased  account  for  examples  These would since  the  DNA c o n t e n t .  In  size,  and  the of  protein  observed gene  next exert  genome.  of  arising  fashion  a  expression  1980)  series.  of  their  et  is  a l . ,  increased 1976;  Timko  possible that  effects  gene  expression  in  concentration-independent  to  act  a  volume u s u a l l y v a r i e s alterations  DNA c o n t e n t i n gene  can  the  not  product  do not  on gene  regulatory  et  modified  investigates effect  It  either  exist  of  have  also  (Audit  ploidy.  trisomies,  changes  are  do  quantities  nuclear  expression  chapter  of  genes  Exceptions  cell  in  levels.  at  expression.  to  be  a  size,  could  polyploid  directly  by themselves  appear level  to  molecules  with  organism  account  General  modifiers  responsible.  which autosomal  for  The  trisomies  221  CHAPTER  COMPENSATION  IN  2L  TRISOMIES  FIVE  IS  TRANSCRIPTIONALLY  REGULATED  21$  Introduction  Dosage of  the  better  expression one  compensation  to  Belote,  1983).  in  strain  each  gene  products  particular  gene  1975). level  While of  to  trisomic  the  amount  in  the  for  the  (Lucchesi  mechanisms  unresolved,  et  involved  question  is  are  rate,  whether  continuous  is  possible  present  in  possibility  transcription, levels.  Data  presented al.,1983).  to  nucleus.  would  a  In  predict  while the  a l . ,  Therefore,  1974;  the  al.,1974; this  would  open  at  the the  A fundamental,  or  rates at  the  of  for female rate  X—chromatin  levels in  the  former  of compensated  p o s s i b i l i t i e s has  Lucchesi  levels  transcription  result  these  leaves  of  that  Merriam,  1965),  (3X2A),  noncompensated  of  male  amount  metafemales  latter  and  transcription  adjustment  Of  operates  remain obscure.  and  of  observation  Stewart  the  levels  display diploid  compensation  at  at  by Baker  genotypes. is  from  transcription  equivalent  study  varied  supporting both  (Ananiev et  (reviewed  different  determined  i n response the  hence,  one  gene  chromosome  X-chromosome  whether  templates a  of  is  possessing  (Mukherjee and Beerman,  X-linked or  this  X-chromosome  s e x - l i n k e d dosage  transcription  individuals  present  the  in Drosophila  modulation of  autosomes  constants  accumulate  products  molecular yet  remains  genes  transcribe  to  The t o t a l  interest  individuals of  relative  of  Diploid  X-chromosomes  rates  X-linked  examples  i n eukaryotes.  three  different  described  for  et  al.,1974;  the  question  been'  Ovozdev of  et  whether  2l°(  or  not  dosage  mechanisms  compensation  in  addition to  compensation  between  any,  dosage  between  male-female  and/or  i n these those  males  hyperploids occurs  which mediate  and f e m a l e s .  compensation metafemale  dosage  The r e l a t i o n s h i p ,  i n autosomal  dosage  by  trisomies  compensation  is  if  and  also  uncertain. Compensation limited for  to  this  the  one  chromosome  Pearson  et  are  al.,1982).  terminal  at  the  in  The  their  trisomies chapter,  of hsp  However,  a  variety since  compensate gene  or  product  19811  these  is  of  levels  Osley  was  may a r i s e  and  have of  genes  effects  of  also  Hereford,1981I in  appears  RNA s t a b i l i t y ,  occurs  in  unknown.  found  to  yeast  to  be  or  normal  levels  limitations  behave  autosomal In  might  on gene  discretely  dependence), from c o n t r o l  a  previous  be  Aneuploidy  physiological effects  show d o s e  of  pathways.  generally  loci  arm  related  control  from  not  activity,  left  regulatory  The maintenance  regulated.  individual they  for  is  series  compensation  instead,  synthetic  compensation  post-transcriptionally have  dosage  which compensation  Drosophila 83  i n the  a n e u p l o i d s may a r i s e  normal  at  aneuploid  cit.  translation,  maturation.  in these  level  of  chromosomes  with multiple copies  a l . ,  None of  level  product  product  present  to  et  op.  of  t r a n s c r i p t i o n a l l y mediated;  operating  of  19798  sex  o n ADH e n z y m e  Adh-1 gene  strains  (Faye  to  an  effect  examples  i n yeast  on p l a s m i d s  linked  Zea mays,  the  (Birchler,  present  not  l i t t l e  of  Several  described  In  has  location  references). been  genes  Drosophila.  chromosome  despite  of  the  be  expected  expression.  (either  they  modulations  mechanisms  of  operating  250  in  normal  diploids.  undertaken autosomal (2)  the  to  T h e work d e s c r i b e d  determine  trisomies  a b i l i t y to  regionally  is  3  H-uridine  higher that  in  operates  many,  produced  was  trisomies not  from a  We h a v e on 2L  than  a l l ,  to  it  loci  compensating  is is  are  occurs  translation.  Taken  when c o m p e n s a t i o n  for  autosomal  trisomies,  transcriptional.  the  measured  revealed  level  of  (3)  genetic  measure  that  autosomal locus.  transcription  on  by  15%  diploids In  and  addition,  mRNA  a  compensation  gene  suggesting  filter  together, a  is  that  <LSP-lb)  The data  that  was  each  in  transcription,  and  compensated.  gene  was  compensation  i n normal  hybridization.  suggest  dosage  of  approximately  gene  to  (Adh)  level  found  noncompensating  prior  the  chapter  compensation  chromosome,  utilized  chromosomes.  but  the  this  dosage  independently at  incorportation 2L  at  undergo autosomal  Autoradiography polytene  (1)  regulated  distributed along  compensation  the  whether:  in  is  regulation  these  of  experiments  observed is  LSP-lb  in  generally  17>\  Materials  and Methods  Drosophila  stocks,  crosses  and c u l t u r e  Two d i p l o i d  strains  to  generate  trisomies  for the left  (Fitz-Earle  and Holm,  1978).  (Oregon  o-f D r o s o p h i l a  females  routinely.  was u s e d  d i e during some  delay  relative  sampling of  instars  were  that  pupation.  at  were  used  t h e mold  tetracycline Genetic the  between  standard  text  while trisomies  to euploid larvae  actively  i n these  males  experiments.  Tegosept  For a l l to those  i n Lindsley  (1970).  Autoradiography  12 h o u r s  third of  only  female  A l l strains  were  reared  medium s u p p l e m e n t e d  and the  lOmg/liter  and chromosome  are described  Transcription  at  (0.22%)  this  genome,  cornmeal-sucrose  and s t r e p t o m y c i n markers  diploid  from  f o r 2L develop  wandering and w i t h i n  C on s t a n d a r d inhibitor  controls.  was r e s t r i c t e d  To m a i n t a i n an o t h e r w i s e  25 d e g r e e s  with  used  arm o f chromsome two  Monosomic—2L p r o g e n y  embryogenesis,  experiments,  larvae  were  R) a n d F(2R)bw/F(2R)bw  with  mel a n o a a s t e r  A cross  y/Y-S C ( 2 L ) v » 2 . 1 t «  cross  conditions  each.  rearrangements and G r e l l  antibiotics  not c i t e d  (1967)  in  and G r e l l  Methods study.  o-f M a r o n i  Salivary  third-instar  and Plaut  glands  larvae  were d i s s e c t e d  pH 6 . 8 .  25ul  o f t h e same s a l i n e  H-uridine  were then  G l a n d s were  (45.9 fixed  incubated  Ci/mmole,  and squashed  slide  on d r y i c e , t h e c o v e r s l i p  plunged  into  ethanol-acetic ethanol with  over  t h e X-chromosome,  RNA  (diluted  developed  1:1  61A-68C  in  i n 95%  air dried, distilled minutes  the  preparation  coated water),  i n Kodak D - 1 9  toluidine blue.  arm o f chromosome and r e g i o n  and t h e  and stored  for three  i n 0.1%  freezing  were p o s t f i x e d  with  acetic  Grains  two, w i t h i n region of  the third  were IA—3C  chromosome.  Isolation  Fat P0*,  Slides  Glands i n 50%  After  was r e m o v e d  in  of  rinsed  They were then  C and s t a i n e d the left  (1:3),  f o r 10 m i n u t e s  12 h o u r s .  f o r two weeks,  19 d e g r e e s  counted of  for 8 to  (3:1)  minutes  0.luCi  technique.  100% e t h a n o l .  K o d a k NTB2 e m u l s i o n  exposed at  by standard  acid  for five  c o n t a i n i n g ImM  New E n g l a n d N u c l e a r ) .  i n aceto-methanol  this  wandering  saline  which contained  acid,  was  were m o d i f i e d f o r  from  i n Ephrussi-Beadle  PO*,  3  (1973)  bodies  were d i s s e c t e d  ImM p h e n y l t h i o u r e a ,  extracted Roberts  from t h i r d  pH 7 . 2 .  Total  acetate,  pH 4 . 5 ,  heparin,  2mg/ml  extracting  twice  larvae  used  by Brock and  The e x t r a c t i o n  buffer  1% S D S , 4>jg/ml  polyvinylsulphate,  bentonite, with  i n lOmM  RNA w a s  b y a m o d i f i c a t i o n o f t h e method  (1982).  instar  contained  0 . 1 4 M Na 5Qug/ml  a n d 0.1% d i e t h y l p y r o c a r b o n a t e .  redistilled  phenol,  with  After  233 phenol:chloroform:iso-amyl  alcohol  (50:50:1)  chloroform:isoamyl  (50:1).  t h e RNA w a s p r e c i p i t a t e d  70%  ethanol.  and  reprecipitated  resuspended  alcohol  The samples with  ethanol.  i n d i s t i l l e d water  spechtrophotometrically  Filter  were washed  at  formaldehyde  o f RNA w a s d e n a t u r e d  f o r 5 minutes,  cooled,  according  (1980).  filters  were  Three  extract.  1980),  68  C.  removal  were used probes:  pR^LSPlg:1,  at  80 d e g r e e s  and s p o t t e d  to  assayed  were  of  and averaged  for  95% c o m p l e t e  f o r subsequent 1)  SAC-1,  within three  a plasmid containing  the 5'  coding  et  a l . , 1981),  a plasmid containing  sequence. the 2.1  1981)  (Smith  into  This  latter  at  hours.  a plasmid containing 2)  p l a s m i d pUC12  degrees  T h e same using  the  the D.  sequence  of  the  p l a s m i d was c o n s t r u c t e d of  70  filters  a n d 3)  the majority  k b Bam H I f r a g m e n t  described  x S S C , 0.1%  hybridization assays  1980),  gene  as  each  from the  (Thomas,1980)  (Goldberg,  pLSPlb:B2.1,  subcloning  buffer  Thomas  out i n 0.1  of h y b r i d i z e d probes  C i n 2%  onto  t h e method  was c a r r i e d  x wash  Adh gene  the LSP-lg  (Smith,  i n 0.05  was o v e r  melanoqaster  coding  and washing  The removal  performed  following  of  acids  P r e h y b r i d i z a t i o n and h y b r i d i z a t i o n were  (Thomas,  filters  nucleic  260nm.  filters  CS  The  pH 4 . 5  and q u a n t i f i e d  nitrocellulose  was  i n O.14M Na a c e t a t e ,  in  Hybridization  A 4ug a l i q u o t  SDS a t  and with  LSP-lb by  pR^LSPIb:1  ( V i e i r a and Messing,  1982)  by  234-  standard  procedures  contains  only  because to  a  LSP-lb  small  the LSP-lb  (McClelland transcript arise  (Maniatis et  coding sequence.  nuclear  gene  a l . , 1981).  relative  loaded  onto  counts  the f i l t e r s  experiments.  The r a t i o  constant  this  over  sensitive. specific (Maniatis  et  determined strain  with  compensated  Northern  For degrees  of  that  Blot  pH 8 . 0 .  containing  20mM b o r i c  pLSP-lb:H2.1.  to  t o LSP—lg  showed  in  counts  were  translated 6 1  RNAse  to  cpm/ug  noncompensated ratio  o f RNA  remained  and n i c k IO  might  these  purified  2 x  snRNA  that  t h e amount  found  An e s t i m a t e d  blots,  C f o r 5 minutes  (1980).  this  95% o f t h e c o u n t s  v a l u e was  from the  from t h e Oregon R s t r a i n .  i s 2/3  3'  of t h e noncompensated  diploid  The  estimate.  Hybridization  northern  a l . , 1979)  of  and over  ratio  estimate  sequences  low, ambiguity  experiments  the range  LSP-lb  necessary  within  t h e amount  b y summing t h e L S P - l b / L S P - l g 1/2  was  fragment  LSP-lb:1  RNA i s v e r y  approximately  a l . , 1982).  formamide,  Thomas  t o  Although  over  P l a s m i d s were of  This  linearly related  range,  activities  pR  Control  were  This  i s present  in  to LSP-lb  from i t s presence.  hybr.idizable  et  RNA g e n e  structural  et  a l . , 1982).  5ug of  total  RNA w a s d e n a t u r e d  i n 1 x gel buffer  T h e RNA w a s s e p a r a t e d  and then  acid,  The f i l t e r  65  c o n t a i n i n g 50% o n IV. a g a r o s e  ImM E D T A a n d 3% f o r m a l d e h y d e  blotted  at  gels (Rave  t o n i t r o c e l l u l o s e as d e s c r i b e d by  was h y b r i d i z e d  to  nick-translated  235  Southern  Blot  Analysis  DNA w a s i s o l a t e d Tris,  lOOmM E D T A ,  phenol, and  once  then  with  with  17. S D S , p H 8 . 0  centrifugation,  with  0.3ml  of  solvents  as described  ethanol-precipitated and d i a l y z e d against  digested  with  nitrocellulose  alcohol volume  a 20Qug/ml  larvae  of  alcohol  solution  III,  floatation  of  by Southern  and  2.5  by  was i n c u b a t e d with  (Marusyk  f o r 30 m i n u t e s .  acids  boiled  the  lOug  i n lOmM T r i s ,  r u n o n 1.2% a g a r o s e  as described  (50:50:1)  acids  and r e p r e c i p i t a t e d .  by f i l t e r  with  Nucleic  the nucleic  DNA w a s r e s u s p e n d e d  lOOmM  3M N a a c e t a t e  T h e DNA w a s e x t r a c t e d above,  in  twice  (50:1).  and t h e mixture  t h e same b u f f e r Hind  instar  and e x t r a c t e d  collecting  RNAse was added  C f o r 3 hours.  1980)  one-tenth  After  degrees  pH 7 . 5  third  phenol:chloroform:isoamyl  of ethanol.  pancreatic  late  chloroform:isoamyl  were p r e c i p i t a t e d volumes  from  at  37  organic of  the  ImM E D T A ,  and Sergeant,  T h e DNA w a s  gels  (1975).  and b l o t t e d  to  23b Results  Transcription  Dosage  compensation  individuals Stewart  has been  and Merriam,  autosomal 1982),  Autoradiography  trisomies  although  remains  H—uridine  been  1975).  used  Baker,  for a  Transcription strain  chromosomes. chromosome wild-type the half  as  diploids. nucleus apparent  if  et  regulation  transcriptional  measure  1974;  gene p r o d u c t s  (Devlin  i n autosomal  occurs regulation  i s  trisomies,  the incorporation method  demonstrate  that  X-chromosome  regulated  from one t o t h r e e 1965;  in  a l .,  This  was e x a m i n e d  both  X  see Lucchesi,  of  has dosage  i n d i p l o i d s and  chromosomes 1983  i n two g e n o t y p e s .  and B e l o t e and  R)  strain  boundary,  as,  and have  A transcription  from a trisomic-2L grains  The d i p l o i d  a C ( 2 L ) and two F(2R)  Individuals trisomic  (Oregon  that  a l . ,  chromosomes.  two were p r o d u c e d  viable  observed  et  review).  contained  larval-pupal  trisomic-X  (Lucchesi  which such  is transcriptionally  and Beerman,  1983  control  at  to  t r i p l o i d s possessing  (Mukherjee  been  To determine  successfully  in  Compensation of  has also  polytene  genes  documented  was e m p l o y e d t o  into  compensation in  X-linked  f o r compensation  autoradiography 3  well  the level  unknown.  responsible  of  for the entire  by crossing carrying  taken  the former  standard  trisomic-2L  50% l o n g e r  is  are distributed  shown  of  arm of  strain  to  chromosomes.  individuals  autoradiograph female  left  are  At  about  to develop  than,  a salivary  gland  in Figure  predominantly  26.  over  a  It  is  237  F i g u r e 26. T r a n s c r i p t i o n autoradiogram of s a l i v a r y g l a n d p o l y t e n e chromosomes f r o m a t r i s o m i c - 2 L f e m a l e larvae. Chromosome 2L i s t h e t h i c k chromosome arm a t t h e t o p o f t h e  nucleus.  235  23q euchromatic  regions  o f t h e chromosomes  Chromosome 2 L i s t h i c k e r examination The is  reveals  X chromosome in diploid  grains  chromosome  differences  in  over  controls,  on t h e l e f t  a l l strains  normalized  than  in label  those  (3X2A) shows length  arm o f  manner,  than  that  t h e amount strain  0.174,  4,  present being then  and d i p l o i d as a f r a c t i o n been  Table 46).  corresponds i n 2L  at  i n a l l three  incorporation statistically,  arising  between  strains  diploid  standard.  incorporated  autosomal  level.  of the t o t a l  higher  trisomies  Comparisons  these this  findings fraction,  1976).  1.5  would  the ratios  and d i p l o i d s  t h e amount  If  of  bands  a l l genes  in diploids  to  measurements, trisomies  with  euchromatic  strain  Lefevre,  close  When  i n which t r a n s c r i p t i o n on  equivilant rates  although  w h i c h was  result  from  The region  t o the proportion of polytene  a trisomic/diploid ratio  However,  By  i s consistent  In t h e d i p l o i d  closely  though  controls.  nuclei  made a n d c o r r o b o r a t e  (804/5059=.159,  transcribed  is similar,  This  i n these  i t  arm o f  slightly  the transcriptional  (column  many l o c i  than  was, a g a i n ,  compensated  have  of the l e f t  of u r i d i n e  are  transcription  homologues.  thicker  as an i n t e r n a l  that  i s expressed  close  t h e mean n u m b e r o f  three,  interpretation  2L  that  between  chromosome  i n the diploids.  trisomic  i s also  of the d i p l o i d  the  between  and  synapsed  individuals  was u s e d  2L i n t h e t r i s o m i c - 2 L  at  the nucleolus.  i n c o r p o r a t i o n can be minimized.  over  found  arms,  three  varibility  involved,  in this  of  T a b l e 46  the entire  higher,  internal  61A-68C  i n metafemales  two from t r i s o m i c - 2 L  consistently using  the other  the presence  females.  observed  than  and over  were  and t r i s o m i e s , be  of  do not  incorporation  expected. uridine differ i s  24-0 T a b l e 46 "^H—uridine i n c o r p o r a t i o n i n t o r e g i o n s chromosomes from d i p l o i d and t r i s o m i c larvae. Grain Genotype  Mean g r a i n s on 2L  C(2L)ltl F(2R)bw/ F(2R)bw (diploid)  C(2L)lt/+/ F(2R)bw (trisomic-2L)  Ratio  *  Mean + SE. total grains  +  +  2L/61A-6BC  2L/total*  1.85  0.174  12.5  + 0.09  + 0.012  (28)  (27)  (9)  96.4  2.08  0.200  0.09  ±  polytene  Ratios  83.9  8.6  of  0.007  1A-3C/61A-68C  0.300 ±  0.022 (11)  0.289 ±  0.031  (43)  (37)  (23)  (19)  1 . 16  1 . 12  1. 15  0.963  over  a l l euchromatic  chromosome  regions  24-1  consistently  12 t o  observations  are consistent  study  (Devlin  et  dose—dependent measured, product,  16% h i g h e r  a l . ,  three  dose—dependent  loci,  predict  of  1.2  a  rate  times  results  27.  will  containing  two  determine this  diploid  the  of  comparable synthesis though  (1.5  the slope,  lines  slopes  These  comprised  of  of  strain  studies  on 2 L ,  for  a  given  to  synapsed  chromosome  trisomies. estimated et  which i s  at  10% f o r  a l . , 1.35  1974). times  an  occurs.  Clearly,  strains  t h e amount  2L  have  o f RNA  i n t h e two s t r a i n s  h a s 50% m o r e  even  template.  an apparent  to  However,  self-absorption,  and d i p l o i d  on 2L r e v e a l e d  of  would  have  three  and r e p r e s e n t s  equivilant  proportion  a r e shown i n  (Lucchesi  a slope  Thus,  gene  We d i d n o t a t t e m p t  has been  the trisomy  of  approximately  than  1974).  corrected  loci  on 2L r e l a t i v e  data  when n o c o m p e n s a t i o n  on 2L i s n e a r l y  previous  10%)  of  grains  females  shows  enzyme  on 2L can be  f o r autosomal  (see T a b l e 47).  the trisomic-2L  Our  27  minus  from a trisomy  regression  a l . ,  diploid  i n Figure  level  et  remain  a l l genes  beta-particles  factor  to  of  3L.  self-absorption  relative  approximation expected  more  (see Maroni  line  of  chromosome  absorb  differential  The d o t t e d  t h e number  loci  on 3L t r i s o m i e s  transcription  region  a correction  metafemales  the  finding.  A polytene  of  previous  levels This  i n trisomies  with t h i s  by p l o t t i n g  homologues  dependent.  Studies  of  some  compensated  representitive  rates  that  These  our  Of t h e f i v e  level.  on t h e 61A-68C  Figure  if  dose  transcription  consistent  The d i f f e r e n t  those  aneuploids.  fully  the diploid  demonstrated  and suggest  showed c o m p l e t e l y  and two were  strain.  with the f i n d i n g s of  1982)  i n these  i n the trisomic  regional  24-2  F i g u r e 27. R e l a t i o n s h i p between l a b e l l i n g on 2L v e r s u s l a b e l l i n g on r e g i o n 61A-68C of 3 L . The l i n e a r r e g r e s s i o n f o r the d i p l o i d s t r a i n i s r e p r e s e n t e d by t h e s o l i d l i n e and f o r the t r i s o m i c - 2 L s t r a i n by the dashed l i n e . The d o t t e d line r e p r e s e n t s an e x p e c t e d v a l u e i f no c o m p e n s a t i o n were o c c u r r i n g (1.35 x diploid). t2L: trisomy 2L.  Grains  Ol-J  o  over 2L  24-T Regression of grain T a b l e 47 o n g r a i n c o u n t s o v e r 6 1 A -- 6 8 C . Genotype  diploid  t r i somic -2L  Regression Parameter  counts over  2L or  Chromosome  r e g i o n s of  2L  Region 36BC-40  2L  21A-24F  25A-30F  31A-36BC  b  1.92  0.438  0.521  0.482  0.455  s  0.06  0.032  0.038  0.035  0.029  a  -3.2  2.8  -2.9  -2.8  2.2  r  0.969  0.819  0.818  0.821  0.855  n  27  21  21  20  20  b  1.89  0.431  0.538  0.417  0.403  s  0.07  0.025  0.025  0.029  0.032  a  7.3  5.8  4.2  2.2  3.9  r  0.919  0.839  0.824  0.776  0.749  n  37  29  29  29  29  0.980  0.984  1.03  0.865  0.886  trisomic diploid  b= r e g r e s s i o n c o e mf f i c i e n t ! s " a=» y i n t e r c e p t ! r correlation examined.  standard error of b! c o e f f i c i e n t ; n™ n u m b e r  of  nuclei  2^5  distribution three were  loci  of compensating  that  located  while  were  enzyme were  on t h e X  (Strobel  et  this  regions  t h e chromosome  trisomies for (  compensating regulatory for  trisomies  in  their  arm.  level been  genes.  ability  If  over  approximately  equal  to  loci.  large  o n chromosome regions  regions.  (Pak a n d G p d h ) .  fully  gene, locus,  compensated  A d h . The r e g i o n Ddc.  The region gene,  cMdh.  36C-40A  T h e amount  of  3  d i s t r i b u t i o n of a  responsible in  autosomal rather  than  t h e chromosome  differ  i n 2L t r i s o m i e s ,  then  by measurements We e x a m i n e d  21A-24F  of  four  transcription and  response  31A-36B  25A-30F,  i n our previous  includes  and one dose  includes the  H-uridine  distributed  regions  s u b d i v i s i o n s of 2L i n t h e  showed a compensated  one  of  the  involving  Compensation  include study  that,  that  Nevertheless,  compensation  discrete  study,  t h e 2L arm c o u l d be  of control. acting  possible  a regional  Two o f t h e r e g i o n s ,  that  h a s been  shown  was r e g i o n a l l y  autoradiographs. loci  loci  have  seemed  should be detectable  transcription  of  A regional  (1982)  In a s i m i l a r  t o undergo  differences  levels  of compensating and  in trisomies  peculiar  may h a v e  individual  a l .  Thus i t  and noncompensating  system  on  of 2L.  3 L , we d i d n o t o b s e r v e  a chromosomal  these  exist.  response  level  i n Dj. miranda  arm, d i s c r e t e loci  The  t h e chromosome a r m ,  and noncompensating  Das et  compensatory within  element  K  of  loci.  (diploid)  dose—dependent  portion  a l . , 1978).  noncompensating  a compensated  which produced  of compensating  observed  at  two-thirds  i n the proximal  distribution  within  expressed  i n the distal  t h e two l o c i  and non—compensating  dependent  dose-dependent  incorporated standard)  into  each  region  was measured  Regression  analysis  i n the trisomic—2L  of  suggest  not exist  of  large  suspect the  number o f  that  small  on 2 L .  that,  loci  X-chromosome  loci  This  i n each  an  extra  between  autosome  transcription  h a s been diploids set  from X - l i n k e d  Furthermore,  it  of  is crucial,  autosomes  accommodate  appears  variable  that  arises  the ratio  of  a  the  strain. a loci  transcription we  as an a r t e f a c t  of  chromosome.  Trisomies  the activity  sets  (Lucchesi,  results  templates  quantities  between  i n comparisons  dosage  of  transcriptional  the  and t r i p l o i d s where  since  each  and, therefore,  between  autosome  i n the latter  in  resolution,  averages  observed  detected  47.  and non-compensating  sampled on t h i s  a n d t h e number o f  that  within of  region,  exists  the  exist  level  T r a n s c r i p t i o n i n Autosomal  relationship  expression  this  technique  A striking correlation chromosome  at  i n Table  incorporation in  i s clear  regions  compensating  This  of  differences  at  the regionality  number o f  i t  i s compensated  chromosomal  d i s t r i b u t i o n of  does  the slopes  strains  no major  different  results  regional  a  regions  Furthermore,  responses These  and d i p l o i d  chromosomal  level.  of  are presented  strains.  are graphically represented  From t h e r a t i o s  these  and d i p l o i d  analysis  Figure  trisomic-2L  internal  data  of  the  the  of these  The r e s u l t s 28.  this  (normalized t o  the X  1977). gene  the presence  of  i n a 50% i n c r e a s e  (Maroni of  of  of  and P l a u t ,  X chromosomes  compensation  X-chromosome  in  1973). to  sets  can  material  as  well  an  F i g u r e 28. R e l a t i o n s h i p between l a b e l l i n g w i t h i n f o u r s e p a r a t e i n t e r v a l s o f 2 L a n d l a b e l l i n g w i t h i n r e g i o n 6 1 A - 6 8 C o f 3I_. The s o l i d l i n e r e p r e s e n t s t h e r e g r e s s i o n f o r t h e d i p l o i d s t r a i n and the dotted l i n e regression for the trisomic s t r a i n . For a l l f o u r g r a p h s , g r a i n number o v e r r e g i o n 6 1 A - 6 B C i s r e p r e s e n t e d on t h e X a x i s ; t h e Y a x i s d e p i c t s g r a i n numbers over r e g i o n 21A-24F in a), 2 5 A - 3 0 F i n b ) , 3 1 A - 3 6 B C i n c> a n d 3 6 B C - 4 0 i n d > . Shown above t h e g r a p h s a r e t h e l o c a t i o n s of t h e f i v e enzyme l o c i measured i n a p r e v i o u s study ( D e v l i n et a l . , 1982). t2L= t r i s o m y 2L.  noncompensating loci  compensating /oci Pgk 1  Gpdh .  I  25A  cMdh  »  Adh  Ddc _ L _  •  31A  36 BC diploid \2L 80-1  24-S (Maroni  and L u c c h e s i ,  examine  whether  the X,  X/A ratio  partial  incorporation  uridine into  relative  to  then  a  autosomal  material,  autosomal  loci  8898/8094  responsible  X-linked  transcription  compensation  transcribed  level. at  overcompensated,  enzyme  loci  discretely  on t h e  the X  express  If  a l l  this  autosomal  X—linked be expected  in  with the increase Alternatively,  for activating  in  i f  X-chromosome then  we d e t e c t e d  a much  larger  no i n c r e a s e  in  experiment.  experiments  in trisomic-2L  transcriptional  chromosome  alter  Genes  The a u t o r a d i o g r a p h i c  be  would  bands).  in this  would  the t i p of  region.  activating  However,  To  by the  T a b l e 46  are not randomly d i s t r i b u t e d ,  may b e e x p e c t e d .  set  compensation  1A-3C at  the control of  1983).  i n t h e trisomic—2L and  (in accordance  increase  Individual  region  10% i n c r e a s e  individuals  transcription  dosage  i n column 5 of  are equally capable  trisomic-2L  i n an autosome  transcription  The e n t r i e s  transcription,  and B e n t l e y ,  T r a n s c r i p t i o n was m e a s u r e d  of  incorporation regions  increases  X-linked  strains.  chromosome.  Williamson  sufficiently to affect  we c o m p a r e d  diploid  1980;  rates,  with the average  appearing have  individuals  Clearly,  various  (Devlin  et  either over  a l . ,  that  at  large  that  the  individual  under  genes  could  or regions  Previous  individual  1982).  the concept  occurs  however,  as compensation.  revealed  support  genes  of  studies  the with  behave  The r e l i a b i l i t y of  inferring  250  gene  expression  products  i s  by measuring  subject  to  post-transcriptional of  individual  quantities  loci  of  complications  processes.  mRNA p r o d u c e d  levels  Roberts  and Evans-Roberts,  (see  chapter  dependent third and  3)  of  sequentially  LSP-lb. LSP-lg  LSP-lo gene,  probed with  located  to  RNA l e v e l s  Drosophila  species  structural  gene  LSP-lb  as  well  considerable a  Southern  cross  blot  trisomic-2L  LSP-lo  of  with  The major  bands  1981)  labelled,  are  distinct  males  (Smith  displays from  Hind  III  LSP-lb  samples  dose  wandering  nitrocellulose  filters  to  dosage  and females  et  strains  of  1982).  a l . , 1981)  genomic that  and L S P - l g these  i n the  used for  several The L S P - l a homology  with  and Figure  29b  shows  DNA f r o m l a r v a e  of  was p r o b e d  sequences. genes  a n d when t h e t w o s t r a i n s i n t h e band r a t i o s  control  compensation  907. s e q u e n c e  digested  to  and  has been  h y b r i d i z a t i o n i s expected.  corresponding  difference  of  and Roberts,  and trisomic—3L  simultaneously  hemolymph  (Roberts  method  has approximately as  on  produce  an i n t e r n a l  the lack  (Brock  made f o r  (2-1.9,  DNA c o m p l i m e n t a r y  3  as  A similar  between  been  The  was u s e d  demonstrate  LSP-lb  RNA e x t r a c t e d  on chromosome  1979a),  response  f o r which data  Adh(2-50.1),  cloned  the  have  known t o  b o d y was b o u n d t o  investigation.  previously LSP-la  fat  further  i n trisomic-2L  a n d A d h mRNAs.  Evans-Roberts, present  protein  is  protein  from  measurements  1979a),  activity.  larvae  arising  One g e n e ,  and t h e other,  levels  instar  of  terminal  from two genes  are available.  levels  of  To examine  i n trisomies,  protein  compensated  the levels  is  (Smith  et a l . ,  a r e compared  observed.  Since  a much  25/  F i g u r e 29. Q u a n t i t e s o-f L S P g e n e s a n d mRNA i n d i p l o i d s a n d trisomies. a) N o r t h e r n b l o t o f t o t a l RNA f r o m 1) diploid compound f r e e , 2) 2 L t r i s o m y a n d 3) O r e g o n R p r o b e d w i t h L S P - l b sequences. b ) S o u t h e r n b l o t o f H i n d I I I d i g e s t e d g e n o m i c DNA f r o m 1> t r i s o m i c - 2 L l a r v a e a n d 2) t r i s o m i c - 3 L l a r v a e probed s i m u l t a n e o u s l y w i t h L S P - l b and L S P - l g sequences.  J.5L  12  k b  8.5  LSP-10  6.1  LSP-loc  2.9 LSP-17 1.5 1.3 t  153 of  the  larval  tissue  under—replication the  trisomic  from  the  preparations. restriction to  cross  specific This  In  of  LSP-lo.  cross  LSP-lb  of  The l e v e l  of  modulation  of  unknown.  LSP-lb  expressed  sequences.  sufficient in  to  T a b l e 48,  relative  where  RNA a p p e a r s  to  and  trisomic-2L  individuals.  be How  compensation  of  LSP-lb  not  with the  dosage  (Roberts  that  This  observation  compensate  and E v a n s - R o b e r t s , result  to  the  normal  1979b;  implies that  allow compensation  operate  that  between  the  X-linked  the  gene,  males  Brock  and  regulatory  in trisomies  allow compensation  the  to  LSP-lb in  affect  S u p e r f i c i a l l y , the  1982).  that  given  LSP-la  corresponding  achieved  does  those  results  not  chromosome  LSP-la  observed  to  in  inferred  is  LSP—la.  processes  probes  be  in polytene and  that  occuring  in trisomies  discordant  Roberts,  kb b a n d i s  both  not  RNA l e v e l s  appears  females  the  RNA a r e  in diploid  and  6.1  is  suggests  could only  LSP-lb  hybridization is  equivalent  is  a  the  result  sequences  arm o b s e r v e d  addition to  fragments,  of  gene  this  interpretation  chromosome  interpretation  levels  polytenized,  h y b r i d i z a t i o n of  The degree our  of  arms.  thicker  is  may d i f f e r  between  males  from  and  females. The strains good  levels differ  of  considerably  correlation  enzyme  activity  differences unknown, modifiers  but of  A d h mRNA s e e n  between i n each  (Table  the  amount  strain  i n Adh e x p r e s s i o n an  extensive  ADH e n z y m e  in  48). of  between  has  of  two  diploid  However,  (Figure  analysis  levels  the  is  a  A D H mRNA a n d t h e  level  30).  for  these linked  been  there  The r e a s o n strains  is  and  unlinked  reported  recently  of  25* Table  48  Genotype  Oregon R (diploid)  C(2L)ltl F(2R)bw/ F(2R)bw (diploid)  Amount o f L S P - l b Ratio» LSP-lb/LSP-lg  1.90+0.03  a n d ADH mRNAs r e l a t i v e Ratio* ADH/LSP-lg  0.137  (3)  1.85  +  +  0.013  to  LSP-lg.  LSP-lg cpm/filter  2167  (3)  0.19  0.079  (3)  +  O.181  0.021  1377  0.050  599 +  (3)  2.81  +  .09  0.149  Comp. Estimate  1.87  +  06  0.099  +  152  (3)  +  Noncomp. Estimate  197  (3)  (3)  C(2L)lt/+/ 1.78 + 0.03 F(2R)bw (trisomic-2L) (3)  +  (3)  +011  +  007  comp.= compensated; n o n c o m p . " noncompensated. Mean * corrected f o r d i f f e r e n t probe s p e c i f i c activities  +  SE  96  255  F i g u r e 30. R e l a t i o n s h i p b e t w e e n A D H e n z y m e a c t i v i t y a n d ADH mRNA i n t h r e e s t r a i n s . ADH e n z y m e a c t i v i t i e s a r e f r o m T a b l e 3 ; ADH RNA l e v e l s a r e f r o m t h i s c h a p t e r a n d a r e m e a s u r e d relative t o L S P - l g RNA.  100  n  • trisomic -2L  I UJ Q  • diploid (Oregon R) 50  • diploid (compoundfree)  .05  ADH mRNA  .1  257  (Maroni  and L a u r i e - A h l b e r g ,  enzyme a c t i v i t y value  (Devlin  w a s 19% h i g h e r  et  a l . , 1982).  levels  show  a  higher  than  expected.  results these  similar  obtained  data  Of  some  is  3.6  about  trisomic-2L free  than  2R d i p l o i d  strain  and probed  this  technical  is  arises (ie.  is  associated  delay  i n development.  then  could  result  trisomies  Thus,  i t  of  slow  RNA) i n  To confirm t h i s were  from  extractabi1ity  noteworthy  development levels  of  may b e t h a t  that  displaying were  occurred  time  Figure  reduced  strategy  in  of  Whether a RNA i n  t h e l o w e s t RNA the  greatest  t h e same at  observed  proteins  result,  r u n on N o r t h e r n  phenomenon o r  mRNA q u a n t i t i e s  growth.  total  a n d t h e comppound 2 L ,  i s clear  and t r a n s l a t i o n  i n elevated  to  biological  is  counts  L S P - I Q RNA  reproducible.  the strain  If  transcription.  48).  genotype  differential It  acts  is  may b e a d e v e l o p m e n t a l  consequence  of  the  importantly,  in hybridizable  intermediate.  from a  a r e 21%  support  More  (relative  DNA. I t  values  compensation  (Table  i n RNA l e v e l s  the extended  individuals.  the level  f o r each  with  and t r i s o m i e s  rate,  i s  n o t known.  level  diploids  dosage  strains  with LSP-lb  problem  trisomies)  at  t h e A D H mRNA  strongly  measurements.  prevalent  difference  difference  data  i n Oregon R i n d i v i d u a l s ,  blots  this  that  l a r v a e , ADH  dose-dependent  trisomic-2L  the difference  less  new RNA e x t r a c t s  that  gene  i s  three  29  the expected  i s clear  autosomal  the three  times  It  protein  that  concern  between  than  The present  -for e a c h  observed  In t r i s o m i c - 2 L  relationship:  with  suggest  independently  1983).  the in  in same  trisomies  these  quantities arising  as  of a  mRNA i n  secondary  25%  Di s c u s s i o n  In small  Drosophila, segmental  observed.  of  gene  developmental observed under  for  such  reasons  In  conditions  at  the  measurements  that  less  than  with  levels  Adh  produced  mRNA l e v e l s  considerable between of  Although  the  of  their  were  this  transcription  rate  autoradiographic determinations,  a  of  of  any  on 2L  are  that  in  this  transcribed  The amounts  of  products.  of  mRNA,  where  of  gene  product  existed  to the  measure  the  results  compensation  mRNA consistent  between  i n each  with  at  while  Adh,  enzyme p r o d u c e d  together  were  For  levels  correlation  that  to  strain,  protein  gene,  show  expression  terminal  experiments, suggest  gene  on 2L  one  not  Autoradiographic  rate.  attempt  are  various  The r e s u l t s  trisomic-2L  levels  levels  cues,  modulation of  loci  for  located  d i d not  the  accumulate  genes  i n the  good  that  a  is  similar  compensated  dose-dependent.  amount  study  the  by  individuals  enzymes  etc.).  the  diploid  differences  the  in  compensated  genotypes,  mRNA a n d  that  different  of  of  transcription.  two-thirds  f r o m two  LSP-lb  of  These  in viability  appear  the  product  duplications,  i n developmental  of  duplicated  whole—arm t r i s o m i e s  1982).  modulations  suggest  produced the  might  most  level  approximately one-third  a l . ,  is  i n gene  small  Levels  alterations  reveal  to  and r e d u c t i o n  post—transcriptional  occurs  et  gene  increase  autosomal  metafemales.  (i.e.  chapter  in  (Devlin delay  an  contrast  products  dose-dependent  autosomal  aneuploid,  usually some  when a n  the  for  of  exist the  amount  strain. specific the  L S P - l b RNA  differences  in  251 gene  dosage  i n autosomal  transcriptional In  f o r t h e X chromosome,  enzyme a c t i v i t i e s  Stewart and  at  using  ratios  system(s) possess  that  at  considerable  dosage  products  rate  i s set  at  compensation  i s determined dealt  (1983)  transcription controls  and Lucchesi  to those  trisomies  Measurement  of  of  measurements  et  have  1974), at X/A  regulatory  for sex-linked  (as  level  observed  genes  that  t h e male  or the  level  That  et  X-chromosome female  is  brought  dosage  or female  level  that  i s  a  by Baker and  Dose-dependent suggest  of  by Ananiev  levels  of  regulatory  mediating transcriptional  do not e x i s t  produced  operates  the  i n the reviews  would  have  suggests  t h e male  this  modulatory  t h e enzyme  (1983).  in  a l . ,  compensation  controls.  transcription  Faizullin  transcription  (Lucchesi  in their  either  length  i n metafemales  similar  autosomal  with at  at  see  a dose-dependent  at  1974}  level  implies that  a l . , 1983)  by p o s t - t r a n s c r i p t i o n a l  compensation  levels  X-linked dosage  at.,  However,  i n metafemales et  of  al.,1983;  compensation  flexibility  of  i f compensation  females  Alternatively,  and that  possibility  et  et  the transcriptional  Such a r e s u l t  which mediate  transcription  Belote  Gvozdev  Compensated  1974 a n d G v o z d e v  about  (Lucchesi  X—chromosome d o s a g e  1.0.  transcription  level,  level  i n trisomic-X  capabilities.  the  the occurance  t o determine  results.  above  at  measurement  1973 f o r an e x c e p t i o n ) .  observed  suggesting  in  1975;  i s occurring  conflicting  al.,  the protein  autoradiography  genotype  been  has demonstrated  and Merriam,  Bvozdev,  can occur  level.  trisomies  compensation  trisomies  compensation  f o r t h e X chromosome.  i n larvae  with varying  degrees  2b0  of  segmental  Lucchesi, dosage  a n e u p l o i d y f o r t h e X chromosome  1980)  compensation  templates  for a  activator  of  Schwartz,  1973).  range  of  females, capable the  of  been  possible  substance  most  If  likely  detecting  (Maroni system  trisomic—2L X-linked  females  enzyme  i n metafemales.  regulatory  quantities  factors  trisomic-2L  relative  or trisomic-3L  possible that  genes.  If  located  on 2 L .  this  10%,  If  i s the case,  of f l i e s  1974).  were  While  factor It  transcription  of t h i s  possessing  study  in Indeed, in 3).  It  is  out by one or a few do not appear  problem requires large  a  in this  control.  (see chapter  genes  genes  trisomies  not t o be a c t i v a t e d  such  is  t o be d u p l i c a t e d  autosomal  i s carried  in  the case,  t h e measurements  then  The r e s o l u t i o n  analysis  this  may h a v e  individuals  regulation  involved  of a t r a n s c r i p t i o n  to the diploid  appear  over  a r e p r o d u c e d b y many  i n X-chromosome  equally  complete  t h e genome.  also  the  Experiments  gene(s)  (see Rawls and L u c c h e s i ,  by about  loci  that  i n males and  could be detected.  no i n c r e a s e  imply  operates  p r o p o r t i o n o f t h e genome  detected  would  19738  system  significant  the X/A ratio  and P l a u t ,  this  throughout  alter  a s an  adjustable  the regulatory  an e f f e c t  X-linked  be f u l l y  distributed  before  functions  of  should  operates  limiting  X-chromosome  rate  autosomal  unsuccessful that  that  Such a r e g u l a t o r y  transcription  that  by t h e c o m p e t i t i o n  transcription  X/A ratios.  it  the hypothesis  i s mediated  limiting  production of  have  supported  X-linked  X-chromosome a  have  (Maroni and  and small  a  t o be more  autosomal  duplications. The c y t o l o g i c a l been  well  documented.  manifestation While  of dosage  compensation  t h e X-chromosomes  i n females  has are of  2fe» similar single  staining  intensity  X i n males  i s more  (Dobzhansky, increased in  In  level  vitro  1981)  1957).  than  of  of  an a l t e r e d  individuals  in diploid  gene  t h e X chromosome of  for further  discussion).  thickened  f o r t h e autosomes)  gene  the cytological  expression  compaction  greater  than  unsuitable  for correct  It  that  35-40%  produce  does  not (see  yet,  If  reflect next  i s possible observed  that  (for the X  i s not  then,  at  observable  chromosome  in diploid  functioning of the  a  t h e genes on  packing,  phenomenon  have  dosage  in trisomies  chromatin  this  many o f  the  the  Trisomic-2L individuals  by a l t e r i n g  level.  is  Clearly,  p e r homologue  repressed.  f o r downward r e g u l a t i o n ,  both  per c e l l .  2 L chromosome  arm a r e t r a n s c r i p t i o n a l l y regulates  X chromosome  i n metafemales  transcription  and Mukherjee,  p a c k i n g may e x i s t .  Nevertheless,  product  w i t h an  RNA p o l y m e r a s e  chromatin  the polytene  females.  X-linked  level  compensation  at  that  "puffed"  i s correlated  Chatterjee  reduced  least  condition  1974;  proportionally  or  puffed  the  somewhat  (Khesin and L e i b o v i t c h ,  structure  this  d i f f u s e and appears  coli  and suggests  chapter  autosomes,  by E .  same amount  the  as  transcription  trisomic-X  wider  of  This  and t h i c k n e s s  females  is  transcription  apparatus. Alternatively, trisomies not  may a r i s e  responsible  females. state  of  f o r X-chromosome  chromosome and gene  trisomies,  dosage  from a combination of  Such c o n t r o l  development autosomal  t h e mechanism of  systems  compaction. expression dosage  compensation  regulatory  regulation  may o p e r a t e In view o f  in  controls  i n males and independently of  the  the s i m i l a r i t i e s  in  d i s p l a y e d b y X—chromosome a n d  compensation  on t h e X chromosome a n d  Vol  on  t h e autosomes  may h a v e  Dosage compensation or  have  arisen  X-chromosome more  highly  originally by  the  regulatory  to repress  altering  Muller,  modifying been  the expression  Williamson  chapters, by l o c i ,  and Gethman,  1973)  region  repressing  the activity  21A-25D,  subdivision  specific  chromosome  Gpdh.  result  present  within  of t h i s  region  exert  capable  1973;  (see of  have  Rawls and  As discussed  in  could be  the duplication, of  (1974),  2L,  other  (but not  i s capable  one-third.  loci,  an e f f e c t  of  However,  completely  a l l regions  several  are not a d d i t i v e ,  made  trisomies  appeared  but rather  capable  a d u p l i c a t i o n of  o f GPDH b y a l m o s t  implies that  The e f f e c t s  been  the transcription  that  While  i n Drosophila  1983).  on chromosome  regions,  genes.  regions  have  i n autosomal  suggest  f o r t h e change,  This  of  and B e n t l e y ,  of Rawls and L u c c h e s i  chromosome  effect.  genes  genes  (1931)  e s t a b l i s h e d by  ( O ' B r i e n and Gethman,  of repressing  O'Brien  responsible  autosomal  a l .  a  dependent  Chromosomal r e g i o n s  themselves  The r e s u l t s  et  X—linked  chromosomal  specific  compensation  genes.  no s i n g l e  of  reflects  may b e c o n t r o l l e d  i n a dose  of other  that  Muller  compensation  o f some  of  previously  are capable  controls.  which act  Birchler,1980).  1974;  regulated which  reports  reflect,  and females  m o d i f i e r s has not been  the expression  Lucchesi,  may s i m p l y  possible  i n males  the expression  means,  described  previous  dosage  foundation.  i n maintaining  i s also  such  genes  of these  1950;  It  compensation  proposed that  existence  involved  homeostasis.  dosage  experimental of  systems  evolved form of  X-linked  manner  i n whole-arm t r i s o m i e s  from,  transcriptional  a common r e g u l a t o r y  or  on t h e  however,  had a  small  perhaps expression  and a  2b3 maximum r e d u c t i o n  of  one-third  The mechanism b e h i n d trisomies  is  suggesting  unknown.  that  chapters  three  of  that  copies the  a  expression  trisomies  is  et  systems  dependent  factor  a l . ,  of  length if  of  of  2L  in  a  compensation 1983; escape  a  three  that,  was  not  by the  number  of  Such a  response control  genes  of  for  a  similar  their  for  X-chromosome  and L u c c h e s i , in  the  that  present  copies  present over  locus  then  study.  for  or  on  a  The  loci  small  in  present  levels,  for  and  regulatory  study.  observed  i n males  1977),  other  enzyme  all  compensated  transciption.  by the  to  is  particular  observed  supported  which  product  locus.  active,  eliminates  of  i n hyperploids operates  on t h e  detection  gene  each  trisomies,  transcriptionally  structural  response  fashion  Roehrdanz  are  in  in  discretely  revealed  exclusively the  behave  independently at  on f e e d b a c k  the  compensation  regions  of  genes  compensation  operates  gene  19B2).  required  regionality  have  unaffected  (Devlin  competition  Individual  given  observed.  transcriptional  regulation  The p r e v i o u s  is  along  the  However, chromosome  dosage  ( S p r a d l i n g and  Rubin,  such  would  an  effect  2bH-  CHAPTER  REGULATION  OF  SIX  DOSAGE C O M P E N S A T I O N  IN  METAFEMALES  24,5 Introduction  Dosage compensation number  occurs  (Mukherjee  i n Drosophila  and Beerman,  X-chromosome not  affect  genes  material  the total  chromosome  -For d i f f e r e n c e s  by t r a n s c r i p t i o n a l  1965).  autosomes.  with a constant amount  to  "compete"  Males,  for a factor  transcribe  their  triploid  intersexes  (2X:3A),  express  their  (Maroni  and P l a u t ,  inversely  1973).  correlated  That  variety  of  single,  continuously adjustable  recent  aneuploids implies that  genetic  compensation prevention Lucchesi,  studies  activation  1980?  Individuals produce as  capacity in  to  males  of  t h e same  (3X.2A)  twice  at  of  ratio  system.  19781  genes  in a is  a  dosage  i n males and B e l o t e and  1982).  of  (metafemales)  X-linked  also  gene  demonstrate  Simplistically,  this  formed from t h e X - l i n k e d  may r e f l e c t  levels  However,  hyperacti vation  amount  and  X-linked  compensation  (CIine,  as  X/A ratio,  intermediate  autosome  genes  the rate,  t h e mechanism f o r  and thus  compensate.  product  X-linked  f o r t h e X chromosome  be accomplished by one of  metafemales  that  i n females  and females  dosage  t h e amount  could  trisomic  at  dosage  Skripskey & Lucchesi,  approximately  euploid  suggest  X-linked  produced by the  regulatory  may b e b i ' f u n c t i o n a l :  of  Consequently,  transcription  with the X to  does  this  with an intermediate on average  of  background  from  t h e number o f  X chomosomes  X chromosomes  modulation  autosomal  1980).  w i t h one—half  chromosome  t h e amount  of t r a n s c r i p t i o n  females,  is  Varying  (Maroni and L u c c h e s i ,  appear  i n sex  two mechanisms. an e x t e n s i o n  products the  reduction  templates Compensation i n  of the  regulatory  2bb mechanisms males  that  responsible for  and f e m a l e s .  compensation  genes  trisomic  for  female  the  capable  either  the  arise  compensation  male-female  of  male  setting  or  =  a  transcription  level. 1.5)  consequence  in  of  of  Individuals  would  and compensation  as  between  dosage  the  female  (X/A r a t i o  expression  then  dosage  be  set  at  these  additional  controls.  Several  However,  be  X chromosome  must  regulatory  of  at  mode o f  hyperploids  levels  Alternatively,  may o n l y  X—linked  the  are  studies  X-linked dosage  have  revealed  enzymes  are  compensation  that  compensated  produced  in  (diploid)  trisomic-X  i n whole-arm t r i s o m i e s  strains.  is  not o  limited  to  strains  trisomic  either  product  as  in  genes  the  found  in trisomies  trans-acting and  X chromosome.  Lucchesi,  Muller  et  al.  chromosome.  can  be  regulators 1974a). (1931)  in and  chapter,  the  is  controlled  activity and  addition to,  of  was  the  can  occur  male-female  presence  of  compensation  compensation question  (Rawls  in  examined.  hypothesis  by mechanisms  autosomal  whether  genes  that  by  on t h e  the  mechanisms.  X—linked were  of  of  autosomal  d u p l i c a t e d arm  by s i m i l a r  dosage  for  o r i g i n a l l y proposed  dosage  several  with the  the  in  same amount  compensation  i n metafemales  consistent strains  raises  duplicated  3 L make t h e  within  for  loci  by t h e  theory  account  trisomies  females,  trisomic—X in  for  The p a r a l l e l i s m s between  phenomenon c o u l d be  expression  the  Dosage  encoded  to  two  and  or  explained  Such a  X-chromosomal  males  2L  euploids.  and  this  for  Many o f  in  In  euploid  Their  compensation  independent  compensation.  of,  X  24,1 Materials  Genetic  In  and Methods  stocks  general,  attached-X male  metafemales  strain  strain  (Kerkis, The  and c r o s s e s  w/0  used  were  generated Adh" gene  involving  by c r o s s i n g  pr cn females  2  on t h e i r  chromosome.  19E o f  tAP-17  The males were for were  of  and n u l l used  the  attached-X  v  (1968).  metafemales  In t h e  were  an Adh  alleles i n these  on t h e  second  crosses  were  tAP-1  t h e Adh (11.8kb)  insertion  of  Adh i n t o  listed  i n T a b l e 51  structural  (Goldberg et  personal  and females  produced by mating  v/Q females.  <V " ) .  and G r e l l  possessing  strains  Posakony,  I  size  C ( 1 ) R M v p n v /Y_:b_  d e r i v e d from Oregon R stock experiment  gonad  Genetic  Adh (Table 4 9 ) ,  t h e X chromosome  (J.  of  pr cn and  3  i s a 4.8kb  chromosome  in Lindsley  Adh'"-> p r c n .  an i n s e r t i o n  into  ( D E N ,v / p .  an  standard  on t h e b a s i s  v pn v / Q . C(1)RM.  t o males  The male  a  O r e g o n R a n d C a n t o n S*  X chromosome  tAP-1/Y; b AdrV"^ tAP-17/Y;b  were  Y S . In  (y_) t o  of mouthparts  C(1)RM.  are described  experiments  selected  and c o l o u r  and C(1)RM.  markers  is  were  wild-type strains  strains  produced by c r o s s i n g  marked w i t h y e l l o w  and they  1931)  were  a l . ,  12A o f  region  1983). the X  communication). i n experiment cultures.  I of  T a b l e SO  The genotypes  and f o r t h e experiment  Oregon R males  The genotypes  gene  with C(1)RM.  shown f o r e x p e r i m e n t  III  listed  i n Table v pn in  52  2bS Table  51  C(1)RM.  were YS.  genotypes derived  d e r i v e d from  In ( D E N ,  listed  from  experiments  for  experiment between  The genotypes were  between  v / q females.  crosses  w/Q f e m a l e s .  crosses  siblings;  Canton S males  The  IV i n T a b l e  51  Oregon R males listed  thus,  and  i n each  they  have  represent  and C(1)RM. set  of  the  same  progeny v  autosomal  background. A duplication T< 185) v 11B12  K 7 A c :  .  This  r e g i o n of  heterochromatin balanced  over  and  the  X.  When t h i s  is  of  LSP-la  rearrangement  the of  to  the  third  stock  Tb)  or  which  are  LSP-la  variegation  was  examined.  alleles  balanced LSP-la  i e )  of  over  either LSPl-a  translocation L.  with  a wild-type  a  supressor  from  a  stock  of  bearing  low  LSPl-a  were  attached-X  chromosome and  TM6B.Tb  cv  (v  Tubby,  The  to  and t h e  TM6B.Tb  larvae  crossed  to  females  Su(Var)A154  v  -/Y;  Dp ( 1 ; 3) v * ~ ~ / T M 6 B . T b m a l e s  x  P(l)DX yf/Y;Su(Var)A154/TM6B.Tb) t  females. analysis.  Wild-type  was  Craymer)  (phenotypically  (wild-type).  Males  over  female  diploid  duplication  LSP-la  balanced an  the  t  the  possessing  This  X chromosome r e p l a c e d  duplicated for  duplication  chromosome.  into  to  (Dp ( 1 ; 5) v * ™ V T H 6 B , T b )  of  producing  9E3-4  inserted  C(1)RM v pn v / g f e m a l e s ,  be r e c o v e r e d  response  the  from  In(3LR)TM6B.Tb ( p r o v i d e d by D r .  can  are  has  X chromosome  deletion bearing  crossed  was c o n s t r u c t e d  (non  Tubby)  male  larvae  were  selected  for  2bS  Enzyme  Assays:  Enzyme a s s a y s instar of  larvae  Tris,  performed  which were  lOmM N a P C U ,  enzyme  were  homogenized at  ImM p h e n y l t h i o u r e a ,  (ME; E C . N o . 1 . 1 . 1 . 4 0 ) pH 7 . 5 ,  Alcohol were:  ImM M n C l ,  50mM N a C 0 , a  Larval  h a s been  expressed  Serum P r o t e i n  Larval heamolymph membranes eluted  pH 9 . 0 ,  3  (Kalex  in glacial  were  LSP  p e r ml  c o n d i t i o n s were:  0.1M  2mM N a m a l a t e .  a s t h e mean +  separated  acetic  as a fraction  Quantitation  weight  third  For malic  No. 1.1.1.1)  (LSPs)  acid  at  assay  conditions  butan-2-ol.  standard  of LSP-1  from l a t e  error.  third  instar  by e l e c t r o p h o r e s i s  stained  on C e l l o g e l  with Coomasie Blue  R-250,  and q u a n t i f i e d  595nm  and Evans-Roberts,  expressed  live  2mM N A D , 0 . 2 7 5 M  Scientific),  spectrophotometrically Roberts  of wandering  Analysis  serum p r o t e i n s samples  the assay  (ADH:E.C.  25mg  pH 7 . 5 .  2mM N A D P ,  H  dehydrogenase  Activity  on e x t r a c t s  ( m o d i f i e d f r o m t h e method o f  1979a).  of LSP-lb  Amounts o f L S P - l a  are  or LSP-2.  RNA  RNA w a s m e a s u r e d  i n males,  females  and metafemales  by  2.10  filter in  hybridization  chapter  five.  Total  probed  sequentially  (Rigby  et  a l . ,  entire  coding  and  pLSPlb:B2.1 gene  Analysis by  described  LSP-1 a'-  were  first  e i s  and  autoradiograms  the  filters  these  genomic allele  6  prodecures probed  LSP-la  3 : 2  with  containing  containing  the majority  to  either  to  inserted  five. from  3 = :  X-ray  of  1981). the  was  performed  R (wild-type)  or  Filters  L S P - l a DNA probe  was r e m o v e d ,  and  P-labelled film.  Comparison  an e s t i m a t e  of  of  the quantity  of  L S P - l b RNA. pattern  of  the  following  the  i n chapter  into  a l . ,  DNAs  RNA f o r t h e  Oregon  This  provides  was e x a m i n e d  a portion  blots  variant).  with  restriction  P-labelled  et  and  1984).  hybridized  described  with  subclone of  were  RNA r e l a t i v e  LSP-la*-- ^  a plasmid  ^P-labelled  DNA a n d r e - e x p o s e d  The  translated  were p r o d u c e d .  two autoradiograms  LSP-la  nick  i n chapter  to  described  nitrocellulose  (Smith  (low p r o d u c i n g  hybridized  t h e methods  for LSP-la  a l . ,  was e x t r a c t e d  from  LSP-lb  et  to  o f L S P - 1 RNA o n N o r t h e r n  procedures  measurements  pR~LSPla:l,  sequence  (Devlin  using  the following  a plasmid  f  1980)  RNA w a s b o u n d  with  1977):  the  LSP-lb  (Thomas,  five.  plasmid of  pLSP-laB2.9,  the 3'  plasmic  Southern  coding  pUC12.  a  blots  were  2.9kb  and f l a n k i n g  sequences  11 \  Results  The h y p o t h e s i s occurs  makes  X-linked  females, failing  genes,  could to  be dose  dosage  Some g e n e s  are dose  gene was  that  between  locus  a l . ,  Second,  genes  and f e m a l e s ,  might  male  suggests  trisomic-X  that  apparently Malic  of  region  contains enzyme  enzyme a c t i v i t y  31).  autosomal  this  a  the region of  i n metafemales.  by  regulatory  trisomic—X was o b s e r v e d appears  to  t h e amount  of  was 30-40% h i g h e r  counterparts.  to  activity  locus  In c o n t r a s t ,  strains  (BD10-12  enzyme a c t i v i t y  and two d i f f e r e n t  the expression  i s not compensated  malic  and t h e r e f o r e  (Figure  is  Williamson and  increases  i n malic  for a duplication  enzyme  an X-chromosome  females  and female  strains.  for malic  1981),  region  and f e m a l e s ,  i n both  gene  t h e Men l o c u s .  i n males,  euploid  trisomy  This  compensated  ME a c t i v i t y  obtained  40%.  activates  males  males  of  males and  i n metafemales.  in trisomic-X  described  No d i f f e r e n c e  dosage  their  have  et  when d u l i c a t e d ,  examined  strains.  be  Voelker  (1983)  approximately  dosage  the expression  between  i n euploid  dependent  the structural  3-51.7,  that,  dependent  strains  metafemales.  Although  9A1-2)  First,  compensated  compensate  in  Bentley  while  i n trisomic—X  from male-female  two p r e d i c t i o n s .  compensate  (Men.  compensation  by a mechanism s e p a r a t e  compensation some  that  The s i m i l a r  than effects  and f o r a whole arm t h e Men r e g u l a t o r y Each  template  appears  272  F i g u r e 31. Q u a n t i t y o-f M a l i c e n z y m e i n d i p l o i d m a l e s a n d -females and i n m e t a f e m a l e s . 1. a) m a l e s a n d b) f e m a l e s f r o m Oregon R s t r a i n ; 2 . a) f e m a l e s a n d b) m e t a f e m a l e s (3X2A) f r o m t h e c r o s s Oregon R male X C(1)RM. v w/Q f e m a l e ; 3 . a) f e m a l e s a n d b) m e t a f e m a l e s f r o m t h e c r o s s O r e g o n R m a l e X C<1)RM. y pn v / Q . The arrows < < ) i n d i c a t e t h e exptected l e v e l of a c t i v i t y i f complete dosage compensation were o c c u r i n g ( c a l c u l a t e d f r o m t h e e x p e c t e d c o n t r i b u t i o n o f a c t i v i t y from the p a t e r n a l X chromosome). The numbers t o t h e r i g h t o f b a r s 2b a n d 3b r e p r e s e n t t h e r e s p e c t i v e r a t i o s o f enzyme a c t i v i t y o b s e r v e d t o t h a t e x p e c t e d i f d o s a g e c o m p e n s a t i o n were c o m p l e t e . Enzyme a c t i v i t y u n i t s a r e t h e change i n absorbance at 340nm/min/ml of e x t r a c t . Standard error bars are shown. n £ 5 extracts for a l l genotypes.  CNJ  *~  AjjAjioy  auiAzuj  214-  to  be e x p r e s s e d To  lines  of  in  gene  is  individuals, gene  a l . ,  1983).  left  expression  when i t  is  Therefore,  the the  X-linked 49.  not  strain in  in  per  Thus,  acquired  template  the  the  number  tAP-17  For  both  was  the  while the  between  in trisomic-X  mechanism r e g u l a t i n g  is  males  normal  (Woodruff  and  shown t h a t :  1)  does  females  males  second  are  able  trisomic—X  contained  shown  null  from  in  Adh  gene  i n males. to  these  expression  per  found  affect  chromosome.  represents  The  dosage  while strain genes  tAP-1  examined, and  the  diploid  tAP-17  compensate  and f e m a l e s ,  individuals.  dosage  in  experiments  i n the  dosage  the  not  from  and  same i n m e t a f e m a l e s  to  for  chromosomal  ADH p r o d u c e d  transposed  Adh gene  ability  have  activity  activity  gene  trisomic—2L  on t h e  the  in euploid  gene  and  by  at two  The r e s u l t s  column, to  of  observed  only.  strain  compensate.  compensation the  relative  partially  ADH a c t i v i t y females.  last  Adh gene  compensate does  the  its  several  The s t r u c t u r a l  X chromosome  in these  Adh g e n e s  ADH a c t i v i t y  Adh g e n e s  In  expressed  native  in  males  used  in  the  amount  in euploid  A l l genotypes for  X-linked  experiments  for  used  X chromosome  located  compensate  the  we h a v e  chromosome  trisomy  genes  is  arm of  dosage  transposed  Table  normally  Previous  not and  is  females.  Adh  (Goldberg et  We m e a s u r e d  the  i n which the  2)  females.  further,  the  position.  alleles  question  in diploid  into  1979).  does  as  inserted  35B2—3 o n t h e  gene  Adh  this  EL. m e l a n o a a s t e r  Ashburner, Adh  same r a t e  Dj_ m e l a n o a a s t e r  transformation  band  the  investigate  structural  Adh  at  This  compensation  for  differences  it  escapes  is  not  expected  i n normal  males  if and  215 T a b l e 49 X-linked met a f e m a 1 e s . Exp.  II  Adh gene  Genotype  activity  Sex  i n males,  females,  ADH A c t i v i t y l  and  ADH A c t i v i t y per gene2  tAP-1/Y  male  1.41  +  .06  (6)  1.00  tAP-1/tAP-1  female  2.49  +  .14  (6)  0.88  tAP-1/Y  male  1.41  .06  (6)  1.00  tAP-1/  1.34  .08  (6)  0.95  C<l)RM,y  pn v  meta— female  C<l)RM,y  pn v / Y  female3  O.Ol  <2>  tAP-17/Y  male  0.367  +  .011  (14)  1.00  tAP-17/tAP-17  female  0.518  ±  .016  (14)  0.71  tAP-17/Y  male  0.500  .018  (8)  1.00  .021  (9)  0.75  tAP-17/ C(l)RM,y  pn v  meta— female  0.373  C(l)RM,y  pn v / Y  female3  0.008  (2)  1. ADH a c t i v i t y e x p r e s s e d a s t h e c h a n g e i n a b s o r b a n c e a t 3 4 0 n m p e r m i n p e r mg l i v e w e i g h t . Numbers i n p a r e n t h e s e s a r e t h e number o f e x t r a c t s . 2 . ADH a c t i v i t y i s e x p r e s s e d p e r g e n e c o p y r e l a t i v e t o t h e a c t i v i t y found i n males. If complete dosage compensation o c c u r r e d t h e n t h e ADH a c t i v i t y p e r g e n e i n f e m a l e s s h o u l d b e 0.5 t h a t of males; i f no compensation o c c u r r e d then i t should be 1.0 3 . T h e a b s e n c e o f ADH a c t i v i t y i n t h i s g r o u p o f f e m a l e s demonstrates that t h e Adh-P element construct has not transposed t o t h e autosomes d u r i n g t h e course of t h e s e experiments.  11Io  females  also  functions  The two  genes  metafemales. level.  Their  is  X chromosome. majority  of  loci  an  obligatory to  levels  of  1975;  et  1983).  apparent  at  the  that  male-female controls model, the  loci  euploid  which  female  dosage  operate  the  to  repress  levels  mechanism  to  metafemales, compensated  then  Compensation dosage  (downward  in  in  their  in  expect  all  (Lucchesi  genes  is  1983;  the  Gvozdev if  one  are  set  In  repression  by  and t h u s  produce  expression  in  that  some  which  loci,  could  be  normally i s  not  the  are  not  compensated  regulation).  metafemales  examined  this  regulatory  gene  (activation),  of  regulatory  further.  second  the  19741  control  additional  metafemales a  a l . ,  i n metafemales  escape  on t h e '  above,  resolved  under  in  show  et  and B e n t l e y ,  in  female  compensation  discussed  expression  If  the  metafemales  and t h a t  X-linked  males  that  expression,  product.  might  set  can  occur  independent  of  male-female  compensation.  We h a v e that  one  in euploid  metafemales  of  mechanism  repress  at  genes  compensated  and Adh l o c i  of  be  contradiction  compensation,  dose-dependent  in  level  regulatory  acts  two  Williamson  are  ME a c t i v a t o r  second  the  metafemales. compensation  condition for  expression  and Merriam, This  to  in  dosage  conclude  p r e v i o u s l y examined  Stewart  assumes  escape  appears  we c a n  contrast  dosage-compensated  a l . ,  transcription  above  expression results  not In  reduce  discussed  From t h e s e  metafemales  to  expression  dosage  of  LSP-la.  a  gene  compensated  between  males  and  211  females dosage  (Roberts  compensation  metafemales, in  males,  found  then  male-female  trisomic-X  diploid used  located  from the  the  remain  diploid  (Table  50)  LSP-la  gene  However, that  the  between  than  operating  c o u l d be  of  was  LSP-la compared  previous  normal  of  dosage  females gene  amounts (see  does  males  and  The d a t a  not  fully  female,  for  it  that  to  a/b  that  expression  III  of  strains  and  found  LSP-2 genes  are  and  I  are  and  It  II  the  in  in  males.  T a b l e 51 produced  a/2).  show in  Therefore,  compensate  fully  regulatory  male-female  in  respectively,  dosage is  of  amount  Experiments  LSP-la  columns  suggest  the  of  in  LSP-lb  compensation I  the  structural  observation  i n Experiments  equal  repress  examined.  of  in  amount  with the  strains  all  the  does  that  compensated  their  it  compensation  produced  T h e amount  third  as  than  then  in  responsible to  mechanism,  gene  in  independent  chromosomes  LSP-la  those  mechanism  if  and  and  metafemales.  LSP-la  second  results  euploid  of  since  approximately  while  by a  male-female  regulation,  standards  escapes  metafemales  escape  amount  If  transcription  Alternatively,  LSP-la  confirm,the  the  larger  females.  internal  on t h e  reduce  T h e amount  and  1979a).  should  X chromosome  males as  a  to  compensation  strains.  for  gene  operates  dosage  transcription  were  this  females.  strains  trisomic  operates  and produce  in diploid  trisomic  in  and E v a n s - R o b e r t s ,  compensated controls  in other  dosage  compensation  LSP-la  in  are  metafemales. Translational larval  development  responsible  for  control (Powell  LSP-la  affects  LSP s y n t h e s i s  et  1984)  a l . ,  compensation  during  and c o u l d  i n metafemales  normal  be (which  11$  Table  50  Exp.  I  Relative  Genotype  amounts  LSP-la/ LSP-lb  Sex  strains. LSP-la/ LSP-2  +/Y  (Oregon  R)  male  0.446  +  .034  (10)  0.429  ±  .032  (10)  +/+  (Oregon  R>  female  0.743  +  .070  (11)  0.630  ±  .052  (11)  male/female ratio  II  of LSPs i n euploid  0.60  0.68  iso-x  male  0.578  ±  .033  (6)  0.508  +  .015  (6)  iso-x  female  0,749  +  .038  (6)  0.794  +  .045  (6)  male/female ratio  0.77  Q u a n t i t i e s o f L S P s a r e e x p r e s s e d a s t h e mean Numbers i n p a r e n t h e s i s a r e t h e sample s i z e s .  0.64  ( + standard  error).  Table  Exp. I  51  Relative  Genotype  amounts  Sex  LSP-la  LSP-lb  LSP-2  0.482 + .016  (8)  0.521 + .034  (8)  C(1)RM, y pn v / Y  female  0.827 + .042  (8)  0.647 + .024  (8)  C(1)RM, y pn v/+  3X2A  0.861 + .  106 (7)  0.770 + .099  (7)  .873  + .022  .779  + .  .021  +/0 ( C a n t o n S>  male  0.405 + .017  (12)  0.537 + .025  (12)  C(1)RM.YS, In ( D E N , y / Y  female  0.373 + .017  (10)  0.679 + .046  (12)  C(1)RM.YS, In ( D E N , y/+  3X2A  0.489 + .023  (12)  0.852 + .059  (12)  0.519 + 019  0.811 + .026  +/0 ( O r e g o n R )  male  0.467 + .025  (8)  0.441 + .030  (8)  C(DRM,  y w/Y  female  0.757 + .027  (8)  0.402 + .038  (8)  C(DRM,  y , w/+  3X2A  0.728 + .064  (8)  0.394 + .043  (8)  0.816 + 018  Compensated Est1mate  IV  LSP-la  male  Compensated Estimate  III  strains.  +/0 ( O r e g o n R )  Compensated Estimate II  of LSPs i n aneuploid  0.562 + .024  Dp(113)LSP-la/ +|C(1)RM, y pn v / Y  female  0.995 + .063  (3)  1.13 ± 0.11 (3)  TM6B,Tb/+| C(1)RM, y pn v / Y  female  0.824 + .019  (3)  0.831 + .049  Ratio  1.21  Q u a n t i t i e s o f L S P s a r e e x p r e s s e d a s t h e mean Numbers i n p a r e n t h e s i s a r e t h e s a m p l e s i z e s .  (3)  1.36  ( + standard  error).  2%0  display at  an  altered  which L S P - l a  measured mRNA  amounts  is  suggest  The  because  transcribed. hyperaction activating  situation  to  partially  be  was  isolated  ten I,  in  sequences genes of  in  2L  protein  LSP-la This  testing,  suggest  percent  of  the  T a b l e 53  to  Experiment  for  was  appeared  called  in  have  LSP-lb  females, This  to  while  data  is  be  have  X  an  allele  0 2 1  this  St I I ,  of  males  eliciting  capable  further, may b e  ie.  of  the  analagous  to  Adh  expression  the  gene  if  foreign  of  in  rapidly  capable  where  an  amount I  very  the  the  was  chromosomal  third  chromosome, included  LSP-la reduced  in  was levels  of  LSP-la,  chromosome  product  that  LSP-la  fortuitously  LSP-la*-  diploid  of  trisomies  expression  significantly less was  level  mRNA i n  level.  may n o t  three  stock  which possessed  mutation  found  to  i n metafemales  by r e s i d i n g i n a  stock  subsequent  LSP-la  normally i s  chapter  activated  This  upon  LSP-la  saturated.  i n which the  and  total  gene  b y gamma r a d i a t i o n ,  examined.  that  compensation  A new f r e e - a r m  experiments  produced  of  X-linked  is  repressed  of  relative  compensated  transcription unit  gene  we  the  translation.  this  described  position.  fully  dosage  most  the  only  two-thirds  To determine  i n metafemales  amount  The r e g u l a t o r y  transcription  induced  to  occurs  the  compensation  incomplete  may a r i s e  could  Again,  prior  rate).  mRNA f r o m t h i s  produced a  that  controlled  of  of  approximately  metafemales  This  compensation  (Table 52).  males  developmental  of  than  .  LSP-la  that  wild-type  (Figure  32a).  Measurements allele  of  produces  LSP-la  Table 50).  approximately  (compare This  Experiment  mutation  was  231 Table  52  Filter  h y b r i d i z a t i o n of  Genotype  +/0  ( O r e g o n R)  L S P - 1 RNA.  Sex  XLSP-la  cpm  LSP-la/LSP-lb  male  3726  1.83  + . 12  (3)  C(l>RM,y  pn v / Y  female  3112  2.67  + .35  (4)  C(l>RM,y  pn v/+  3X2A  2319  3.06  + .02  (4)  3.00  + .014  Compensated  Estimate  Values represent in parentheses.  the  mean  +  standard  error.  Samples  sizes  are  F i g u r e 32. A n a l y s i s o f a m u t a t i o n t h a t r e d u c e s t h e amount o f L S P - l a p r o t e i n and RNA. A . L S P ' s s e p a r a t e d o n C e l l o g e l m e m b r a n e s . L e f t , O r e g o n RJ r i g h t , LSP-1 ° = . B . N o r t h e r n b l o t a n a l y s i s o f RNA f r o m O r e g o n R ( l a n e s 1 a n d 3> a n d f r o m L S P - l ( l a n e s 2 and 4 ) . The l e f t a u t o r a d i o g r a m i s f r o m a f i l t e r h y b r i d i z e d t o L S P - l a DNA. The r i g h t a u t o r a d i o g r a m i s f r o m t h e same f i l t e r w h i c h h a d b e e n washed f r e e of t h e L S P - l a p r o b e , and t h e n r e h y b r i d i z e d w i t h L S P - l b DNA. T h e a m o u n t o f L S P - l a RNA ( r e l a t i v e t o L S P - l b RNA) i s m u c h l o w e r in the LSP-l s t r a i n than i n the w i l d - t y p e Oregon R s t r a i n . C . R e s t r i c t i o n e n z y m e d i g e s t i o n o f DNA i s o l a t e d f r o m O r e g o n R ( l a n e s 1, 4 a n d 7 ) , from LSP-l ( l a n e s 2, 5 and 8>, and f r o m C a n t o n S ( l a n e s 3 and 9 ) . Lane 6 contained incompletely d i g e s t e d DNA. T h e e n z y m e s u s e d w e r e E c o R I ( l a n e s 1, 2 a n d 3 ) , Hind III ( l a n e s 4 and 5 ) , a n d Bam H I ( l a n e s 7 , 8 a n d 9). 1  1  L  0 3  o : E  L  o : H  2?f Table  Exp.  I  53  Relative  Quantititation  Total Protein Levels  amounts  Sex  °S-Met Pulse Labelling 3  male/female ratio  LSP-la  LSP-la  LSP-lb  LSP-2  male  0.064  +  .010  (6)  0.053  +  .009  (6)  female  0.080  +  .008  <5>  0.068  ±  .009  (5)  male/female ratio  II  of LSPs i n LB2 s t r a i n .  0.79  0.78  male  0.295  +  .033  (6)  female  0.362  ±  .057  (6)  0.81  Q u a n t i t i e s o f L S P s a r e e x p r e s s e d a s t h e mean Numbers i n p a r e n t h e s i s a r e t h e sample s i z e s .  ( + standard  error).  IS 5 mapped b y r e c o m b i n a t i o n t o in  close  agreement  39.8  with the  mapped b y e l e c t r o p h o r e t i c  on t h e  position  variants  1979b).  Further  characterization  abnormal  genomic  DNA r e s t r i c t i o n  quantitiy to  of  LSP-lb)  level  (Figure  32b),  females  remained stock this of  from t h i s  incomplete.  by p u l s e  LSP-la  predicted  from the  expressed  at  its  hyperactivation To  were  level,  T(1S 3)y+ (9E3-4  to  is  levels than  of  from  an  LSP-la  of  to  even  into  the  of  (Experiments  males  compensation  synthesis  higher  LSP p r o t e i n .  when t h e  LSP-la  it  does  still  levels  in  in  this  the  X  gene  is  levels would of  be  this  not  respond  raised  this  '  fully  to  above  structural  the gene  of  chromosome  this  of  although  third show  elevated  somewhat  The reduced  gene  This  the  region  Table 51).  variegation.  than  Analysis  not  be  of  segregant  the  males.  can  copies  duplicated LSP-la  position-effect  relative  t h e DNA  although  were  controls,  IV,  an  The  between  dosage  LSP-la  heterochromatin  to  of  quantities that  of  duplicated for  relative  this  consequence  had  32c).  diploid  LSP-lb  with three  insertion  gene  from the  compensation  LSP-la  (Figure  Table 53),  of  The h y p e r p l o i d  Females  expected  expression  that  larvae  11B12)  chromosome.  that  this  S-methionine confirmed  II,  quantities  by dosage  produced.  3 C 5  maximum r a t e  demonstrate  diploid  with  Evan-Roberts,  (measured  LSP—la  Measurements  relative  revealed  a  54)  as  mutation  revealed  (Experiment  synthesis  has  of  mutant  labelling  observation  mutant  presumably as  A comparison  and  showed t h a t pattern  (Table  o-f L S P - l a  (Roberts  s i g n i f i c a n t l y reduced  rearrangement. and  (39.5)  mRNA p r o d u c e d f r o m t h i s  was  X chromosome  may h a v e  was  tested  level  arisen by  less of  Table  54  Mapping  LSP- 1  0 : 2  by  recombination. LSP-la al l e l e  recombinant chromosome  n  v  g  12  0  12  y  cv  17  17  0  g  f  19  7  12  12  y  cv  16  9  7  9  f  v  182  recombinants between v and L S P - l a  +  35  21  T a b l e l i s t s F -» p r o g e n y -from a c r o s s b e t w e e n y c v v f f e m a l e s a n d LSP-la'males. Map d i s t a n c e f r o m v t o g i s 1 1 . 4 . Map d i s t a n c e f r o m v t o L S P - l a l 8 2 i s 21/35 x 11.4 = 6.84. Map p o s i t i o n o f v i s 33.0. T h e r e f o r e map p o s i t i o n o f L S P - l a i s 33.0 + 6.84 =» 3 9 . 8 . s  e 2 :  L  6 3 2  231  introducing  this  duplication into  L S P - 1 a*- -- a l l e l e .  Figure  61  duplicated than  a  LSP-la  single  activated  by  supressess (Figure  gene  stock  More  in  active  (or  female  metafemales  accumulate  more  it  is  the  which  we c a n  was  observed  in  only a  probably d i d not  arise  product  the  in  that  proportion of  only  a  o-f  LSP-la  only  cells). are  compensation  from an  its  duplication  d u p l i c a t i o n was  the  is  communication)  conclude in  gene  strongly  inactivated  increases  Therefore,  less  personal  being  in this  the  The d u p l i c a t e d  Thus,  dependent  larvae.  LSP-la.  male.  mutation  gene  active  dose  bearing  that  Don S i n c l a i r ,  LSP-la  importantly, in  (a  in LSP-la  the  shows  euploid  position.  elevation  stock  indeed synthesizing  indicating that  because  observed  an  variegation;  32d)  partially  in  is  Su(Var)A154  heterochromatic partial  gene  32d  a  inability  observed to  1W  Discussion  The and in  s i m i l a r i t y between  metafemales a l l trisomies  autosomal (Devlin  diploid exists  this  a l .  levels  of  could  The that  level  one-third  data  while  separable  t h e Adh gene  acquire  male-female  i n autosomal LSP—la  between  gene  males  trisomies.  into  gene  to  dosage  rates  at  the  mechanism  f o r m a t i o n by an  trisomies  discussion,  see  the p o s s i b i l i t y  occurs  by a  compensation.  For  example,  t h e X chromosome c a n  o n t h e X chromosome  The r e l a t e d  X chromosomes  disclose  compensation,  but i t  dosage  1983).  and i n X-chromosomal  and females,  trisomies  compensation  chapter  from male-female  dosage  templates  and f u r t h e r  and B e l o t e ,  inserted  from those  of  product  i n X-chromosomal  produce mechanism  t h e male—female  (for reviews,  of  a single  transcription  X-linked  in this  regulated  with the male-female  and t h e h y p e r p l o i d  presented  compensation  If  When t h e r a t i o  and Baker  Compensation i n  f o r X-chromosome  1.5,  compensation  trisomies  rates  i n concert  to repress  1983,  mechanism  then  is  that  trisomies  the observation  X-chromosomal  mechanism c o u l d s e t  operate  Lucchesi,  parallels  transcripts.  of autosomes  female  additional  work  speculate mechanisms.  transcription  mechanism.  compensation diploid  that  i n autosomal  transcriptionally  and thus  i n a l l trisomies,  sets  to  via similar  X-linked  mechanism must  complete  tempting  largely  (1974)  for decreasing  compensation  both  is  a l . , 1984)  et  duplicated  i t  occurs  trisomies  et  Lucchesi  the  makes  compensation  i t  trisomies.  escapes  is fully  LSP-lb.  fails  to  compensate  Furthermore,  compensation compensated  which i s  located  in X on  2<?9 the  left  arm of  when t h i s  chromosome  arm i s t r i s o m i c  observations  also  are operating  chromosome  i n euploid  is  t h e same  size  as  prevent  dosage  hyperactivation  this  of  unit  cross—sectional Nevertheless, chromosome  area  i n metafemales  The to  proof  male-female  could the As  LSP-la  that  such,  these  template  this  rate  intriguing,  the LSP-la  supports occur  LSP-la It  these  at  i n X—chromosomal investigations  that  an L S P - l a  subject  that  variant  However,  metafemales transcription  from  possible.  i n males  However,  in  the  processes. in  do not  mechanisms.  i s not  t h e maximum r a t e  trisomies.  level  observations  the notion  c o u l d not be i n c r e a s e d  is,  from the X  but i s compensated  c o u l d be argued  occurs  that  counterpart.  the diploid  i s compensated  with  the  synthesized  by separate  single X  paired X  in sizel  times  gene  a  Thus,  the three  female  to  which  i s correlated  compensatory  compensation,  responses  be f o r t u i t o u s .  reduced of  dosage  observation  i s reduced  that  individuals,  compensatory  product  two s e p a r a t e  observation  trisomic-X  the  of  1980).  of  and  females.  mutations  are not diminished 1.5  1957)  i n euploid  the size  In c o n t r a s t ,  of  Although  to  i n males  its diploid  t h e amount  (compensated). constitute  of  (Dobzhansky,  possessing  i s approximately  Cytological  compensatory  X homologues  genes  compensation  The s i n g l e X  and L u c c h e s i ,  width.  i n metafemales  synapsed  "puffed"  i s reduced  X—linked  of  a l . , 1984).  two s e p a r a t e  i n males  (Belote  chromosome  homologues  is  compensation  i n females  et  capable  i n metafemales.  the paired  t h e X chromosome  increased  that  males  Indeed,  homologue  (Devlin,  suggest  mechanisms  two, i s a l s o  but c o u l d be  during  a l l e l e was  the  course  discovered amount  of  that  produces  LSP-la  less  than  ten percent  mRNA a n d p r o t e i n .  This  variant  behaves  i n a manner  similar  that  escapes  full  compensation  i t  females. to  In t h i s  be s a t u r a t e d ,  occur  i n these It  should  metafemales (for  case,  appears  presented some  be noted  even  (Brock in this  compensation  separable  above  1.0  fe-Pqd) Such  are  components the  products  1980), The  of  are also  issue  have  of  does  not  not  1983).  of  male  on t h e X i t  on t h e X  need  the  results  i n aneuploids with  Indeed,  in individuals genes  with male-female It  upward r e g u l a t i o n lethal  genes  how c o m p e n s a t i o n  about  by  W i l l i a m s o n and  with  X/A ratios  (Zw a n d  i n X-chromosome  i n metafemales.  X/A ratios  may b e b r o u g h t  two X - l i n k e d  specific  that  not be mutually  content.  dosage  i s n o t known i n males, (Belote  r e q u i r e d f o r downward r e g u l a t i o n  exactly  time,  Nonetheless,  mechanism.  i s consistent  loci  i s a c c o m p l i s h e d b y two  by v a r i a t i o n s  mediate  modulation i n  are regulatory  arrival  compensation  shown t h a t  operating  that  still  appear  comply with t h e i n t e r p r e t a t i o n  compensation  affected  compensation  a recent  i s possible that  the expression  an e f f e c t  would  In e v o l u t i o n a r y  Such mechanisms  some  they  arrived  i n metafemales  and 1.5,  (1983)  i s  chapter  between  male-female  unit  in  males and  which escape  either  recently  and Roberts,  It  Bentley  the loci  i n that  LSP-la  exclusive.  the  that  have  mechanisms.  1.0  between  hyperactivation  (Adh t r a n s f o r m a n t s ) .  chromosome  the wild-type a l l e l e  males.  are peculiar  that  t h e normal  L S P - l a low  the transcription  yet complete  Men), or they  chromosome  dosage  to  of  i s brought  in  for  i f example  and L u c c h e s i , metafemales.  about  is  •further of  complicated  X—linked  al.,  1974;  that  dosage  by s t u d i e s  transcription Gvozdev  et  post-transcriptionally LSP-la this  If  the  occurs  i n these  female  may o p e r a t e gene,  that  Thus,  which  that  dosage  As mentioned adjustable  gene  i n many c e l l s  organism)  or  measurement individual for  a heat-shock  gene  and L u c c h e s i ,  species  that  P-element  of  have  of for  1980),  abnormal  mediated  an e x t e n s i o n in  of  diploid  t h e male  male—female  dosage  compensation  For each  cell,  on o r o f f .  the evidence  previously  cited  introduction,  t h e amount many g e n e s  While  perhaps  This  transcription  the expression of  of  of  within  this  a  a  might  single  enzyme p e r a single  chromosomes).  rate  or  or  may b e c o n t i n u o u s l y  X-chromosome  in euploid  mechanism  either  on t h e p o l y t e n e  i s required.  (Pierce  of  of  the transcription  cells  demand  at  examining  the expression  of  only  i n the general  ( f o r example  (as f o r t r a n s c r i p t i o n  then,  compensation  rates  from data  to  by a  compensation  i s either  be f i t t e d  interpreted  et  i s  regulation  occurs  mechanism.  must  be  situation  that  dosage  euploid  interpretation  continously  would  compensation  hyperactivation  adjustable.  This  i n metafemales  by a s w i t c h i n g  suggests  (Ananiev  Our measurements  suggest  levels  i n metafemales  may b e d e t e r m i n e d  rate.  dose-dependent  transcription.  suggests  and females  report  individuals  regulated.  from male-female  hypothesis  each  at  compensation  distinct  males  1963).  mRNA i n X t r i s o m i e s  gene  this  products  a l . ,  compensation  that  single  Clearly,  gene  h a s been  cell  in  accomplished  Dj_ p s e u d o o b s c u r a  producing  X/A ratios  transformation  individuals i s difficult.  i n Dj_  in  this The advent  2c\l  melanoaaster reality.  makes c o n s t r u c t i o n  of  appropriate strains  a  293  BIBLIOGRAPHY Abraham I , L u c c h e s i J C (1974) Dosage C o m p e n s a t i o n o f g e n e s t h e l e f t a n d r i g h t arms o f t h e X chromosome o f D r o s o p h i l a pseudoobscura and w i l l i s t o n i . 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