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A novel requirement for the X-chromosome in P-M hybrid dysgenesis and the interaction of the garnet and… Wennberg, Richard Arnold 1989

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A NOVEL REQUIREMENT FOR THE X-CHROMOSOME IN P - M HYBRID DYSGENESIS AND THE INTERACTION OF THE GARNET AND ENHANCER OF GARNET LOCI IN DROSOPHILA MELANOGASTER by RICHARD ARNOLD WENNBERG B.Sc,  The U n i v e r s i t y o f B r i t i s h Columbia, 1983  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Genetics) We accept t h i s t h e s i s as conforming to t h e r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA December, 1988 (c)  R i c h a r d A r n o l d Wennberg, 1989  In  presenting  degree  at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  Zoology  (Genetics)  The University of British Columbia Vancouver, Canada  Date  DE-6  (2/88)  December. 1988  ABSTRACT  The  discovery  o f extreme  mutability  around the g. locus o f  D r o s o p h i l a melanogaster i n a c e r t a i n l a b s t r a i n , investigation  o f the nature o f t h i s  phenomenon  S6-1, l e d t o an and any p o s s i b l e  r e l a t i o n s h i p i t had w i t h the w e l l d e s c r i b e d P-M h y b r i d dysgenesis mutator  system. Southern g e l a n a l y s i s  showed the S6-1 s t r a i n t o  c o n t a i n P element homology and dysgenic c r o s s e s i n v o l v i n g t h i s Ps t r a i n showed m u t a b i l i t y i n the germline o f t h e F-^. An unexpected finding  was  the requirement  f o r the presence  o f the  S6-1  X-  chromosome i n F-^ dysgenic males i n order t o produce the germline mutability. the  S6-1  which  In s i t u h y b r i d i z a t i o n t o p o l y t e n e chromosomes showed  strain  were  indicated  t o have a number  on t h e X-chromosome  that  the  only  of P  sites,  the m a j o r i t y  (app. 20/33) . These  complete  P  element  or  of  results  elements  were  c o n f i n e d t o the X-chromosome. The presence o f an unexpected t h i r d class  o f orange-eyed males i n the F 2 progeny  males  x  g_  garnet  locus,  X-chromosome.  females l e d t o the i s o l a t i o n (e(qj), This  l o c a t e d i n the d i s t a l  study  included  o f the c r o s s :  S6-1  o f an enhancer  of  r e g i o n o f the S6-1  an i n v e s t i g a t i o n  into the  nature o f the i n t e r a c t i o n between the e(g_) l o c u s and the g; locus and other autosomal l o c i . to  the g_ l o c u s ,  alleles  and, more  The enhancer was specifically,  l o c a t e d t o one end o f the f i n e  description  i s given  o f the i s o l a t i o n  found t o be  to certain  spontaneous  s t r u c t u r e g_ l o c u s map. A and p r e p a r a t i o n  induced garnet mutation f o r m o l e c u l a r a n a l y s i s .  ii  specific  o f a P-  TABLE OF CONTENTS  Page Abstract  i i  L i s t o f Tables  iv  L i s t of Figures  v  Acknowledgements  Chapter 1.  vi  X-Chromosome Mutation Induction A s s o c i a t e d  with  P-M Hybrid Dysgenesis and t h e I s o l a t i o n o f t h e enhancer o f garnet  Locus  Introduction  2  M a t e r i a l s and Methods  5  Results  7  Discussion  Chapter 2.  19  The I n t e r a c t i o n Between t h e garnet of garnet  and enhancer  Loci  Introduction  23  M a t e r i a l s and Methods  28  Results  34  Discussion  41  Summary  44  Bibliography  46  Appendix  51  iii  L I S T OF T A B L E S  Table  Page  Chapter 1 1.1  X-chromosome mutation i n d u c t i o n i n the germline o f the F-j_ and F 2 male progeny  o f the c r o s s : S6-1 females  x  o g. males l.II  9  X-chromosome mutation i n d u c t i o n i n the germline o f the F-^ and F 2 male progeny  o f the c r o s s : g_  females  x  S6-1 males  11  l . I I I X-chromosome mutation i n d u c t i o n i n the germline o f the F-j_ male progeny  o f the c r o s s : S6-1 males  x  attached-X females 1.IV  13  a) X-chromosome mutation i n d u c t i o n i n the germline o f the F-^ male progeny o f the c r o s s : Oregon R males x attached-X  females  b) X-chromosome mutation i n d u c t i o n i n the germline o f S6-1 males taken d i r e c t l y from stock  iv  15  L I S T OF FIGURES  Figure  Page  Chapter 1 1.1  Photomicrograph of p o l y t e n e from S6-1  chromosome p r e p a r a t i o n  stock showing i n s i t u h y b r i d i z a t i o n of  plasmid prr25.1 DNA  17  Chapter 2 2.1  C o n s t r u c t i o n of homozygous stocks mutant at the e(gj l o c u s and the g. l o c u s  2.2  30  C o n s t r u c t i o n of homozygous stocks mutant at the l o c u s and at the autosomal l o c i p_r, bw,  e(o:)  ry_ and p_,  respectively 2.3  31  Scheme f o r removing the extraneous P elements and f o r s t a b i l i z i n g the c y t o t y p i c background of a P-induced g_ mutation  2.4  32  H y b r i d dysgenesis  screen f o r r e v e r t a n t s of the gr  mutant and subsequent r e v e r t a n t stock formation 2.5  33  Southern a n a l y s i s showing h y b r i d i z a t i o n of plasmid pn25.1 DNA  to complete Bam  HI d i g e s t s of genomic  p from the g_ mutant and r e v e r t a n t stocks  v  DNA 39  ACKNOWLEDGEMENTS  I wish t o s i n c e r e l y thank: Dr. Robert D e v l i n Dr.  Donald active of t h i s  and my  Sinclair and  for his  ongoing  and w i l l i n g n e s s t o help; guidance,  encouragement  c o l l a b o r a t i o n throughout  and  the course  project;  supervisor,  aid,  f o r h i s advice  Dr. Thomas G r i g l i a t t i ,  encouragement and support.  vi  for his unstinting  CHAPTER  1  X-CHROMOSOME MUTATION INDUCTION ASSOCIATED WITH P-M HYBRID DYSGENESIS AND THE ISOLATION OF THE ENHANCER OF GARNET LOCUS  1  Introduction  Hybrid  dysgenesis  is  a  genetic  syndrome  marked  by  appearance of a v a r i e t y of aberrant t r a i t s i n the germline progeny  of  melanogaster and  certain  (28). The  chromosomal  inter-strain  of the  in  Drosophila  symptoms i n c l u d e h i g h r a t e s of  insertional  mutation,  male  crosses  recombination  and  temperature-  sensitive s t e r i l i t y .  There e x i s t at l e a s t t h r e e separate  of  i n JX. melanogaster: the w e l l - d e s c r i b e d  h y b r i d dysgenesis  and  P-M  systems  (6),  and  a  more  recently  a s s o c i a t e d w i t h the t r a n s p o s a b l e element hobo In the males  P-M  are  system, h y b r i d dysgenesis  mated  with  M-strain  r e c i p r o c a l c r o s s i s performed, to  flies  this  from one  system  element which has particular as  the  strain  i s the  cytotype)  arms,  flies of  transposase  but  (5,  high  (45),  when P - s t r a i n not  when  c a u s a t i v e agent a  DNA  element  in  transposable  are  environment, present  in  referred  P-strain  are  13).  (48; see  repeats  generally  The P  absent  M-cytotype elements  in  M-strain  i s dependent capable  of  upon  to (P-  (Mthe  producing  below).  the  P  element  surrounding  has  31  bp  f o u r open reading  perfect  inverted  frames  (38) . A l l  four open r e a d i n g frames are r e q u i r e d t o encode transposase, P  the  r a t e of t r a n s p o s i t i o n i n a  cellular  elements  full-length  Molecularly, terminal  but  other) . The  element  extremely  P  system  (55).  results  females,  I-R  f l i e s , u s u a l l y as 30-50 copies d i s p e r s e d over a l l major  chromosome  absence  P  maternally-derived  M-cytotype.  cytotype)  kb  described  systems  (and not when matings are c o n f i n e d  or the  2.9  an  the  gene product  which 2  c a t a l y z e s the  the  t r a n s p o s i t i o n of  P  elements  P  elements  w i t h coding r e g i o n d e f i c i e n c i e s are t r a n s p o s a b l e i n the  presence  of  the  and  other t r a n s p o s a b l e elements.  trans-acting  terminal element  repeats  transposase,  (27) . A  transcript  Incomplete  p r o v i d i n g they  germline-specific  i s required  possess  splice  f o r transposase  intact  of  the  activity;  P  this  forms the b a s i s f o r the r e s t r i c t i o n of P element t r a n s p o s i t i o n t o germline t i s s u e  (29).  The nature of mutation dysgenesis  has  been  isolation  of the  mutations  were caused  instability  P  extensively  element, by  the  DNA  of  insertion  fact  that was  elements  were  rearrangement reversal  of P  implicated  the  induced  from  their  a s s o c i a t e d with  of  the  faulty  recognized  insertions  (20) .  complete  i n t o a gene or d e l e t i o n s a s s o c i a t e d with resident  include  induction  of  i n or near induction  chromosome  a gene  (13, 45).  breakage  or  at P element s i t e s of r e s i d e n c e along a chromosome, element  orientation  in a  gene,  t r a n s p o s i t i o n of other t r a n s p o s a b l e elements  the  evident  also  as the most common means of dysgenic mutation mechanisms  before  many of the  shown t o be  the imprecise e x c i s i o n of P elements  Other  Even  hybrid  (12). D e l e t i o n s induced through  M o l e c u l a r c h a r a c t e r i z a t i o n has or incomplete P elements  studied.  insertions  (19, 47) , which was  presence of the M-cytotype excision  i n d u c t i o n a s s o c i a t e d w i t h P-M  and  catalysis  of  (13, 55).  Most spontaneous mutations  i n D r o s o p h i l a are a s s o c i a t e d with  insertion  elements  spontaneous dysgenesis  of  transposable  mutations  systems they  Thus, the P-M  arise can  infrequently,  appear  general,  but  at a much h i g h e r  h y b r i d dysgenesis system  3  (30) . In  in  such  hybrid  frequency.  can be used t o screen f o r  P-induced mutants at a p a r t i c u l a r cloned  for  molecular  transposon-tagging cloning  and  gene l o c u s ,  characterization  (3) . T h i s  characterization  technique  which can then  by  the  has  method  facilitated  be of the  o f many D r o s o p h i l a genes i n recent  years. Experiments  described  in this  chapter  were  designed  to  determine the r e l a t i o n s h i p between an observed h i g h frequency o f u n s t a b l e g_ mutations of  hybrid  dysgenesis.  experiments: presence  (i) the  of the  dysgenesis  in a certain Two  novel  findings  demonstration  X-chromosome  mutation  lab s t r a i n  in a  induction;  enhancer o f garnet mutation.  4  arose  P-strain  ( i i ) the  system  from  of a requirement  certain  and  and the P-M  these  f o r the  for  isolation  hybrid of  an  Materials  1.  Culture  conditions:  and  Flies  Methods  were  maintained  and  crosses  performed i n q u a r t e r - p i n t m i l k b o t t l e s or s h e l l v i a l s at 2 2 ° C medium  containing  cornmeal,  (methyl-p-hydroxybenzoate)  sucrose,  was  included  inhibitor.  To suppress b a c t e r i a l  was  either  used  streptomycin 2.  Mutant  i n the  growth  combination  and  agar. food  ampicillin  with  Tegosept  as  a  mold  (50 m g / l i t e r )  tetracycline  or  with  (15 m g / l i t e r each).  and  descriptions Lindsley  in  yeast  on  wild-type strains:  of  most  and G r e l l  of  the  (31) . Any  See  mutations  Appendix. used  other s t r a i n s  can  Detailed  be  found  are d e s c r i b e d  in  i n the  text. 3. Mating schemes f o r h y b r i d dysgenesis i n v e s t i g a t i o n : For of  the  experiments,  s t r a i n females  P-strain  males  were  (15 p a i r s per b o t t l e ) ,  mated  female  test  offspring  crosses  s i b matings.  were  were  s c o r e d f o r mutant  performed  with  masse  to  M-  or the r e c i p r o c a l . F-^. males  from these matings were c r o s s e d t o v i r g i n Maxy the  en  each  the  a/FM7  females  induction.  F 2 male  progeny  and  Similar from  F-^  In these t e s t c r o s s e s , groups of 3 males were mated  t o harems of 10-15 were t r a n s f e r r e d  Maxy  a/FM7  females i n s h e l l  to quarter-pint  milk b o t t l e s  vials. after  The  flies  t h r e e days,  and t h e r e a f t e r t r a n s f e r r e d t o f r e s h medium i n b o t t l e s every t h r e e days  to  a  maximum  of  four  subsequently d i s c a r d e d . Mutant their  FM7/Y  crosses.  sibs,  Male  the  lethal  only  transfers.  parents  were  females were allowed t o mate w i t h  surviving  mutations  stocks w h i l e male v i a b l e mutants 5  The  male  were were  progeny  kept  as  o f the  test  FM7-balanced  crossed to v i r g i n  females  c a r r y i n g a t t a c h e d - X chromosomes 4. i n s i t u squashes and  hybridization:  (C(l)DX,y_ f/Y) t o o b t a i n a s t o c k .  Larvae  w e r e r a i s e d a t 18°C a n d  Gall  for salivary  gland  prepared according to Atherton  ( 1 ) . S q u a s h e s w e r e t r e a t e d a t 70°C i n 2xSSC f o l l o w e d  t r e a t m e n t w i t h RNase A  (100 ug/ml) a s p e r P a r d u e  Slides  according t o the procedure  al.  chromosome  were a c e t y l a t e d  (22) a n d d e n a t u r e d as p e r P a r d u e  and G a l l .  c a r r i e d o u t a t 25°C u s i n g t r i t i u m - l a b e l l e d p r o b e  and G a l l  (39).  of Hayashi  Hybridization  d e s c r i b e d by Bingham e t a l . ( 3 ) . A u t o r a d i o g r a p h y  performed  as p e r Pardue  with  Bingham e t a l . ( 3 ) . S l i d e s  were s t a i n e d  toluidine  blue  at  distilled  water,  i n 2xSSC alcohol  room  dehydrated,  was  was  the exception noted f o r 5-6 m i n u t e s  temperature, air-dried,  et  according to the  procedure  and G a l l  by  by  i n 0.4%  rinsed  with  and mounted  with  p l a s m i d PTT25.1  DNA,  c o v e r - s l i p s i n Permount. The p r o b e  f o r these h y b r i d i z a t i o n s  c o n t a i n i n g t h e complete P element translation Oakville,  (Amersham  Ont.)  with  J  Nick  H-dCTP  precursor.  6  was  sequence  Translation (Amersham  ( 3 8 ) , l a b e l l e d by n i c k K i t , Amersham  Corp.) as  a  Corp.,  radioactive  Results  1. P element for  induced X-chromosome mutagenesis:  paralysis  isolated, unusual  mutants  undertaken  d e s i g n a t e d S6-1, phenomena not  high  frequency  these  of g  mutations  involvement  of  to  our  lab,  an  EMS  one  screen  strain  was  l a t e r shown t o e x h i b i t the p a r a l y s i s  some  mutation.  the induced mutation, the appearance  mutations  proved a  which was  related  c r o s s e s designed t o map  in  In  to  was  be  noted.  highly  further  unstable,  t r a n s p o s a b l e element.  a n a l y s i s done on the S6-1  Upon  s t r a i n and on the gr  of a  study,  suggesting  Southern  In  the  hybridization  strain  (which had  been used i n the o r i g i n a l mapping experiments and had allowed f o r the  detection  of  the  spontaneous  c o n t a i n homology t o P element  DNA  g  mutations)  showed  S6-1  9  and cp t o d i s p l a y no P  to  element  9  homology. The S6-1  and gr s t r a i n s were then t e s t e d i n a s e r i e s o f  c r o s s e s designed t o determine dysgenesis  was  the  cause  i f mutation  induction  of t h i s  unexpected  results  of these  from  high  hybrid  mutability  around the g l o c u s . Before finding  describing  must  be  the  noted.  In  a  cross  the F-^ S6-l/g_  Surprisingly, coloured  eyes,  in an  S6-1/Y  one  other  x  g^/g^  males  o  9  females,  of  crosses,  and  addition  to  unexpected  eyes showed up i n the F 2  g^/Y  progeny  males third  were allowed t o mate.  with class  wild-type of  males  or with  orange  As r e v e a l e d , a mutation on the S6-1  chromosome, mapping d i s t a l l y  and  around  g e n e t i c map  i n t e r a c t s w i t h a homozygous or hemizygous g o f g a r n e t - c o l o u r e d eyes.  position  mutation,  the mutant  effect  may  not be i d e n t i c a l t o one p r e v i o u s l y d e s c r i b e d  or may  garnet-  7  This  X4,  enhancing  enhancer  locus  (42)  and  since  lost;  i t will  be  referred  to hereafter  as enhancer o f  garnet, e(g_) . (The i s o l a t i o n and c h a r a c t e r i z a t i o n o f e(g_) w i l l be further  detailed  later  i n this  chapter  the nature  of  and a l s o  i n t h e next  chapter.) To  investigate  spontaneous  g_ m u t a t i o n s ,  r e c i p r o c a l l y crossed, was  looked  outcrossing  the  the appearance p  S6-1  and  and mutation i n d u c t i o n  g  of the  strains  were  on the X-chromosome  f o r i n t h e germline o f the F-^ and F 2 male progeny by with v i r g i n  females c a r r y i n g the m u l t i p l y  marked X-  chromosome, Maxy a, and balanced over t h e m u l t i p l e i n v e r s i o n FM7. The  r e s u l t s o f these  the  existence  and  o f mutation  offspring  surprising  consistent  dysgenesis  (see Tables 1.1  interesting  system  observation  was  with  the complete  induction  i n t h e germline o f the F-^ male p p p x g /g ; i . e . , t h e g /Y males from t o Maxy a/FM7 females, produced no  progeny o f the c r o s s : S6-1/Y t h i s c r o s s , upon o u t c r o s s i n g mutant  were not e n t i r e l y  o f a P-M h y b r i d  l . I I ) . The most  absence  crosses  i n 14,784  r e s u l t given p  female  the e a r l i e r  progeny  evidence  scored,  that  a  most  S6-1 i s a P-  s t r a i n and gr i s an M - s t r a i n . A  subsequent  (C(l)DX,y_ that  cross  between  f/Y; M cytotype)  retained  S6-1/Y males  virgin  the p a t e r n a l  females  X-chromosome.  and a t t a c h e d - X  produced  These  F-^ males,  males upon  o u t c r o s s i n g t o Maxy a/FM7 v i r g i n s , gave r i s e t o induced mutations at  a frequency  greater  than  3x10"^  j_  scored,  a r e s u l t i n d i c a t i v e of a hybrid  l.III).  Thus  i t appeared  that  mutation  presence o f the S6-1 X-chromosome. 8  n  t h e 16, 027 o f f s p r i n g  dysgenesis c r o s s induction  (Table  required the  Table  1.1  X-chromosome mutation i n d u c t i o n  i n the germline o f the  and F 2 male progeny o f the c r o s s : S6-1 females  F  1  F2  g  p  males.  S6-1/Y progeny; 8,500 females scored over Maxy a or FM7:  Mutants N  x  (6-1)  No. _L Freo. (x!0~ ) 4  6  7.1  Mut. events j_ Freq. (x!0~ ) 4  1  1.2  p  g /Y progeny; 4, 438 females scored over Maxy a or FM7:  Mutants sn * + ' eye w  No. j_ Freer. (x!0~ ) 4  4 2 1  9.0 4.5 2.3  Mut. events j_ Freq. (x!0~ ) 4  1 1  2.3 2.3  1  2^2.  Total: 3  6.8  /Y progeny; 5,061 females scored over Maxy a or FM7: Mutants (2-D w - N (1) orange eye (1) v (s) g  No. : Freer. (xlO ) 4  2 1 1 1  4.0 2.0 2.0 2.0  2 1 1 1  Total: 5  4.0 2.0 2.0 2.0 10.0  (Cont. next page)  9  (Table 1.1 cont.)  orange eye/Y progeny; Mutants  4,962 females scored over Maxy a or FM7:  No. j_ F r e q . (x!0~^)  w - N (9-1) +' eye (1)  9 1  x  18.1 2.0  Mut. events j_ F r e q . (xlO 5 1 Total: 6  T o t a l number o f females scored, a l l c l a s s e s , i n F  10.1 2.0 12.1  2  experiment  2  experiment  14,461. T o t a l number o f m u t a t i o n a l events, a l l c l a s s e s , F 14.  (Freq. =  9.7xl0~ .) 4  1 = male l e t h a l s = sterile  10  Table  X-chromosome mutation and F  male progeny  2  g. /Y progeny; z  Mutants  + '/Y progeny;  Mutants w - N (2-1)  aorange (2-1) eye sn  w 9 . ~ sn eye - c t orange  females  x  o f the F-^  S6-1 males.  14,784 females scored over Maxy a or FM7:  0  Mut. events j_ F r e q .  0  0  0  5,701 females s c o r e d over Maxy a or FM7: No. j . F r e q . (xlO )  Mut. events j_ F r e q . (xlO  4  sn w sn  A  i n the germline  No. j_ Freq.  3. /Y progeny;  Mutants  induction  of the c r o s s : a  0  **2  l.II  3 2  4, 247  5.3 3.5  1 2 Total: 3  )  1.8 3^5. 5.3  females s c o r e d over Maxy a or FM7:  No . : F r e q . ( x l O ) - 4  2 2 2 2 1 1  4.7 4.7 4.7 4.7 2.4 2.4  Mut. events : F r e q . ( x l O  2 2 1 1 1 1 Total: 8  4.7 4.7 2.4 2.4 2.4 2.4 18.8  (Cont. next page)  11  (Table l . I I cont.)  orange eye/Y progeny;  No. i Freq.(xlO  Mutants N w  sn  4,64 9 females s c o r e d over Maxy a or  (9-1) N (6-1)  9 6 3  -  19.4 12.9 6.5  FM7:  Mut• events I. F r e q . (xlO 6.5 12.9 2.2 21.5  3 6 Total:  J L  10  T o t a l number of females scored, a l l c l a s s e s , i n F  2  experiment  2  experiment  14,597. T o t a l number of m u t a t i o n a l events, a l l c l a s s e s , F 21.  (Freq. =  1.4xl0~ .) 3  1 = male l e t h a l s = sterile  12  Table 1.III X-chromosome  mutation  male progeny o f t h e c r o s s :  induction S6-1 m a l e s  Mutants  No. : F r e a . ( x l O  orange eye a (1-1) w - N (17-1/3-s) r a s (4-1) v (4-s) sn w Y_  34 23 20 4 4 2 1 1  4  )  i n the germline x  females.  Mut . e v e n t s : F r e q . ( x l O  21.2 14.4 12.5 2.5 2.5 1.2 0.6  0.6  attached-X  of the F  Total:  21 11 15 4 3 2 1 1 58  13.1 9.4 2.5 1.9 1.2 0.6 0.6 36.2  T o t a l number o f f e m a l e s s c o r e d o v e r Maxy a o r FM7 := 16,027 (8,040/Maxy  1 = male s =  a; 42/89 m u t a n t s )  lethal  sterile  13  6.9  As c o n t r o l s , Oregon R w i l d - t y p e males males  taken  virgins  directly  and  the  from  F-^  stock  female  were  progeny  (M-strain)  crossed scored  with for  mutation i n d u c t i o n . In the Oregon R experiment, seen i n 20,332 f l i e s scored. In the S6-1 event scored  (Table 1.IV).  T h i s frequency  seen w i t h the F^ males from the cross, (Mut.  a result  consistent  S6-1  Maxy  a/FM7  X-chromosome  no mutations were  experiment,  frequency of g r e a t e r than 4x10"^ was  and  seen  a mutational  i n 18,737  i s approximately  S6-1/Y  with a P-M  x  1/10  attached-X  hybrid  that  dysgenic  dysgenesis  event = a presumed s i n g l e m u t a t i o n a l event  flies  rise  to  one mutant or a c l u s t e r of mutants i s o l a t e d from the same v i a l  or  bottle  as  progeny  of  the  3  male  parents.  See  giving  system.  Materials  and  Methods.) The  types of mutations  induced by dysgenic males i n a l l of  the c r o s s e s were c h a r a c t e r i s t i c of P-M high  frequency  from  imprecise e x c i s i o n  S6-1  strain  presumptive  of N  (see  and  w  - N  deletions,  of the P element  below),  and  a  the e(g.) mutation on the S6-1 of  chromosome, showing  a the  either  g_  ts  resident  similar  resulting  at  (total)  most garnet  were male l e t h a l ,  P element  resident  The  (mutant  frequently or  in light  3C  i n the  frequency  of loci  of the e x i s t e n c e of  orange  at  induced eyes.  29°C)  on  mutations A  the  same  were  those  minority  of  suggesting imprecise e x c i s i o n  i n the cr r e g i o n of the S6-1  at the l o c u s , 14  these of the  X-chromosome  m a j o r i t y , however, were male v i a b l e ,  f u r t h e r mutation  a  X-chromosome, as w e l l as an e a r l i e r  mutation  mutations  below).  presumably  i n s e r t i o n mutation events at a number of v i s i b l e  on the X-chromosome. I n t e r e s t i n g l y ,  finding  h y b r i d d y s g e n e s i s : e.g.,  and  s u g g e s t i n g P element  (see  subject to  insertion  or  Table  l . i v  a) X-chromosome mutation i n d u c t i o n i n the germline o f the F-^ male progeny  o f the c r o s s : Oregon R males  Mutants  No. j_ F r e q .  0  0  x  attached-X females.  Mut. events j_ F r e q .  0  0  0  T o t a l number o f females scored over Maxy a or FM7 = 20,332.  b) X-chromosome mutation males taken d i r e c t l y  Mutants  induction  from s t o c k .  No. : Freer. (xlO ) 4  w - N (3-1) a (1-1) orange eye w v (s) ras (1)  i n the germline o f S6-1  3 2 1 1 1 1  1.6 1.1 0.5 0.5 0.5 0.5  Mut.  events : F r e q . ( x l O-4.  3 2 1 1 1 1 Total: 9  T o t a l number o f females scored over Maxy a or (9,400/Maxy  1 = male  a; 5/9 mutants)  lethal  s = sterile  15  1.6 1.1 0.5 0.5 0.5 0.5 4.8  FM7 = 18,737.  alteration  of  the  resident  o r i e n t a t i o n as the 2.  In  situ  cause of the  hybridization  chromosomes:  In  situ  p r e p a r a t i o n s of S6-1 number of P s i t e s , (approximately both the  P  3C  was  site,  structure  or  mutation.  of  P  element  hybridization  to  DNA  to  polytene  i n d i v i d u a l s showed the  S6-1  polytene  chromosome  s t r a i n to have a  the m a j o r i t y of which were on the X-chromosome  20/33 per  (w - N)  There  element's  and  no  individual).  12C  There  (g_) r e g i o n s  homology  to  P  (Figure  element  chromosome p r e p a r a t i o n s of e i t h e r gr  was  P  homology  in  1.1).  DNA  seen  in  polytene  or Oregon R i n d i v i d u a l s  (not  shown). The from  in situ hybridization  the  genetic  were l i k e l y with the  due  crosses, to  P  3.  indicated  X-chromosome, and  e(g)  i n the  vicinity  the  end  the  distal  of  showed  no  duplication  (w Y +  for  the  effects  i n dysgenic  crosses,  S6-1  strain  perhaps immobilized  of p o s i t i o n  presence  phenotype  hemizygote. In a d d i t i o n , a  observed  Crude two-point g e n e t i c  X-chromosome.  (or even i n the  results  there  repeat sequence).  locus:  p l a c e d e(g)  mutation  the  element t r a n s p o s i t i o n  a mutated i n v e r t e d  I s o l a t i o n of the  mutation  that  only complete P element or elements i n the  b e i n g present on the (e.g. by  r e s u l t s , combined w i t h the  In  of  the  either  which  genetic  absence  of  heterozygous g?)  the mutant e{g) region  4 on the  mapping  as  a  at  the  g  the  2  e(g)  homozygote  phenotype was  contained  map,  an  rescued  e(gj  +  or by  allele  chromosome i n males) , s u g g e s t i n g t h a t e (g_) i s a hypomorph or  amorph.  In  homozygous g  females, resulted  heterozygous in  a mildly  16  e(g)  in  the  presence  enhanced phenotype  of  i n newly  1.1  Figure Photomicrograph o f polytene  chromosome p r e p a r a t i o n from S6-1  stock showing i n s i t u h y b r i d i z a t i o n  17  o f p l a s m i d pn25.1 DNA.  eclosed  flies,  which q u i c k l y  overlap  g a r n e t . Thus,  s t r i c t l y a recessive The (Figure  presence 1.1)  mediated  distal  P  arising  from  were  crossed  to  males  on  small  element  scored  but may  instead  (C(l)DX,y_  were  be  truncated  P  a  i n an  have  crossed  a  P  and  revertants  study  was  still  with  or a p o i n t  (e.g. P  reside  complete  l e a v i n g the d e s i r e d dysgenic F-^ males  female)  at  P  F 2 male  of  e.(g_) .  an  the  No  indicate insertion  mutation. I t imprecise  element) e_(o_)  &(g)  element  in this  virgin  the  scored. T h i s may  associated  element  S6-1  g?/Y  and  a  locus. stock  or elements,  study i n c a p a b l e of  m o b i l i z i n g a mutated P element from the e(g_) s i t e .  18  element-  M-cytotype)  a s s o c i a t e d w i t h the p r e v i o u s  could  o f f the  X-chromosome  S6-l/g  F u r t h e r , the recombination event t h a t formed the may  i s almost  reversion  f/Y;  a deletion  transposable  element  S6-1  a l l o w e d t o mate  i s not  might a l s o be a d e l e t i o n of  hours t o  e(qj  represent  f o r garnet-eyed  mutation  excision  24  (from a stock formed from a s i n g l e  attached-X  e.(g)  the  of  scale  r e v e r t a n t s were i s o l a t e d i n 5,693 f l i e s that  first  the  a recombination event  the F-j_ progeny were  sites  e(g;) might  mutation. A  e(g_) gr/Y  male  offspring  modifying e f f e c t  suggested t h a t  done i n which  i n the  one.  of  insertion  females;  the  darkened  Discussion  The  lab s t r a i n  S6-1  was  c e r t a i n c r o s s e s between S6-1  shown t o behave  occurrence of dysgenic e f f e c t s such  strain  c r o s s e s . Crosses females  produced  of  S6-1  (P)  F-^ germline  males w i t h  mutation  i s a t e n - f o l d g r e a t e r frequency p  than  that  seen  crosses P-M  or S6-1  either  h y b r i d dysgenesis An  g_  intra-strain  unexpected  attached-X  of mutation  (M-strain) male  x  induction  S6-1  female  crosses, frequencies i n d i c a t i v e  of  (45).  f i n d i n g was  (M)  M-  r a t e s g r e a t e r than  the  complete  absence of  i n d u c t i o n i n the F j progeny of a c r o s s between S6-1 p homozygous gr  Mutation  i n the germline of the F-^ progeny  this  with  in  examined as an i n d i c a t o r of the  10 ; J  a P-strain  males and M - s t r a i n females.  i n d u c t i o n on the X-chromosome was  of  as  females.  This  finding  mutation  (P) males and  indicates  that  the  presence of the p a t e r n a l S6-1  X-chromosome i n the germline of the  F^  f o r mutation  progeny  crosses was  met  i s a requirement  involving i n the  the  S6-1  crosses  strain.  involving  a c c o u n t i n g f o r the h i g h mutation  induction  i n dysgenic  T h i s i s a requirement attached-X  M-strain  r a t e s observed  which  females,  i n those c r o s s e s  (as d e s c r i b e d above). In s i t u h y b r i d i z a t i o n 30  P  element  chromosome. element(s)  copies, The  evidence  capable  i s (are) l o c a t e d  the  showed  S6-1  majority suggests  of producing  of  t o possess  approximately  which  along  that  lay  the  transposase  on the X-chromosome, perhaps  only  i n the  the  complete S6-1  XP  strain  immobilized t h e r e ,  as by a mutated i n v e r t e d repeat sequence. The  nature  of the mutations  induced  along the X-chromosome  was  consistent with  20) .  A  high  observed,  system  frequency  especially  associated  a  with  of  the  frequency the  cross  and  the  cross than cross,  x  observed  somewhat h i g h e r  the  S6-1  frequencies  w  S6-1  eye/Y), element  and  g  g  germline  mutations  regions of  of the dysgenic F-^  of the  intra-strain  higher  of due  P  elements  frequency  F2  the  was  (presumably  the  level  of  the to  P  The  progeny  (P)  S6-1  x  female  element  those  S6-1  and  female  (M) x  of the  cross  frequencies  (P) m a l e  Among t h e in  x  of  loci.  attached-X  male  cross.  reciprocal  observed  portions  presumably  gy  F 2 progeny of the  was  of  19,  c r o s s e s were i n t e r m e d i a t e between  F 2 of the a  (13,  at a v a r i e t y of v i s i b l e  former c y t o t y p i c background.  substantial  - N  i n the  i n the  i n the  i n the  rate  deletion  e x c i s i o n events  i n the  seen  indicating  mutation  dysgenesis  areas), along w i t h a s i m i l a r t o t a l  frequencies  reciprocal  hybrid  putative  presumptive i n s e r t i o n mutations Mutation  P-M  around the  imprecise  resident i n those  of  were female  (M)  male  transposition in  F 2 progeny, classes  a  higher  possessing  P + '/Y,  X-chromosome  d e l e t i o n i n d u c t i o n at  orange  sites  of  P  residence. p  The  occurrence  experiments  of  ( T a b l e 1.1)  two  separate  revertants  is particularly  of  g  i n the  i n t e r e s t i n g g i v e n t h a t g_  2  i s an M - s t r a i n w h i c h shows a c o m p l e t e a b s e n c e o f P e l e m e n t s by situ  hybridization.  These  events  may  F2  represent  the  in  P-induced  t r a n s p o s i t i o n of another t r a n s p o s a b l e element a s s o c i a t e d w i t h the p p spontaneous g a l l e l e . As g i s one o f t h r e e s p o n t a n e o u s g_ a l l e l e s m o d i f i e d by e(g) , d e t e r m i n a t i o n of the molecular structure  of  these  revertants  20  may  prove  informative  in  the  e l u c i d a t i o n o f the nature o f spontaneous mutation at the g_ locus and the i n t e r a c t i o n between g_ and e(g_) (see Chapter 2 ) . The e(g.) mutation, present i n the d i s t a l r e g i o n X-chromosome, was  identified  as a r e c e s s i v e  o f the S6-1  enhancer  of the  mutant phenotype o f homozygous or hemizygous g_ mutants. The e(o_) mutant has no phenotype i n the absence o f the g  mutation, and i t  appears t o f u n c t i o n as a hypomorphic or amorphic mutation.  21  CHAPTER 2  THE INTERACTION BETWEEN THE GARNET AND ENHANCER OF GARNET LOCI  22  Introduction  Enhancer phenotypic  or  suppressor  mutations  to  modify  the  e f f e c t o f u n r e l a t e d mutations l o c a t e d at d i s t a n t s i t e s  i n the genome. S e v e r a l a l l e l e - s p e c i f i c described  i n Drosophila  particular  mutant  these  act  suppressor  which  alleles loci  suppressor  can r e v e r s e  at other  loci.  loci  have been  the phenotypes o f  In a d d i t i o n ,  some o f  have been noted t o behave as enhancers i n  i n t e r a c t i o n s with c e r t a i n o f the s u p p r e s s i b l e a l l e l e s , i n c r e a s i n g the mutant phenotype. Most  allele-specific  modifier  loci  exert  their  effects  through d i r e c t i n t e r a c t i o n s with spontaneous mutations a s s o c i a t e d with  transposable  invariably gypsy,  element  elements  copia  and  i n s e r t i o n s . The  o f the r e t r o t r a n s p o s o n 412  (14, 36,  44) ,  i n s e r t i o n s are  class,  which  specifically  are composed o f  i n t e r n a l DNA segments v a r y i n g i n s i z e from 5 t o 9 kb with long t e r m i n a l repeats  (LTRs) s e v e r a l hundred n u c l e o t i d e s  each end (15) . Other members o f t h i s of  yeast  within  (43) and the v e r t e b r a t e  the LTRs  signals.  DNA  retrotransposon tRNA  primer  transcripts products  are t r a n s c r i p t i o n sequences  site  retroviruses initiation  adjacent  have been  of  to  retroviruses,  identified  homologous t o r e t r o v i r a l  long at  f a m i l y are the Ty elements (54). Contained and the  which  and  termination Drosophila  LTRs show homology t o the p o l y p u r i n e  binding  direct  tract  polyadenylated  encode p u t a t i v e  protease,  and  gene  e n d o n u c l e a s e and  r e v e r s e t r a n s c r i p t a s e (33). There systems  have  been  described  five  retrotransposon-mediated  i n D_j_ m e l a n o g a s t e r , 23  suppressor  a l l of which  require  homozygous mutations Hairy-wing  (su(Hw))  at the  suppressor  suppresses  mutations  i n s e r t i o n s at a number of d i f f e r e n t ct,  h and bx)  the  same  f o r k e d and  are  suppressed  that  apricot  (36);  (su(w ))  loci  lozenge by  associated with ( i n c l u d i n g Hw,  (40) ;  (iii)  suppressor  mutation,  of  (including  p_r and  insertions  (46) .  sable pr  b w  )  (s_u(s_)), at  which  different  interactions:  described  i n yeast  (31);  suppressor  (43) , maize  (34)  (su(pr)) (46) ; and mutations  associated with  ( i i ) su(s.)  412  enhances  a s s o c i a t e d w i t h gypsy  ( i i i ) s u ( f ) , which enhances w  Transposon-associated  white-  are i n v o l v e d i n enhancing  (i) su(p_r) enhances Hw  (36); and  of  of p u r p l e  suppresses  t h r e e mutant a l l e l e s at the lz. and b_x l o c i insertions  insertion  insertions  loci  Three of the above suppressor l o c i  gypsy  which i s a s s o c i a t e d  (4); (iv) suppressor  w  gypsy  suppresses  by  suppresses p_r and p r ^ , a s s o c i a t e d with 412 suppressor  (su(f))  caused  a  of  f, lz_, y_/  alleles  su (Hw)  with a c o p i a i n s e r t i o n  (v)  (i) suppressor  ( i i ) suppressor of f o r k e d  suppresses the w  a  loci:  a  (18).  systems have a l s o  and mice  been  (52) . In each  case  they c o n s i s t of a t r a n s p o s a b l e r e c e p t o r element i n s e r t e d near t o , and  having  acting  a  cis-effect  upon  r e g u l a t o r y element  a  particular  which e i t h e r  gene,  alters  the  and  a  trans-  stability  of  the r e c e p t o r element or the e x p r e s s i o n of the adjacent gene. The  molecular  mechanisms  underlying  the  Drosophila  t r a n s p o s o n - a s s o c i a t e d suppressor systems have been the s u b j e c t of much i n v e s t i g a t i o n . extensively  The  nature  studied  and,  of s u p p r e s s i b l e a l l e l e s more  recently,  c h a r a c t e r i z a t i o n of the su(s) and su(w ) l o c i has begun a  24  has  been  molecular (7, 57).  There as  are  not  all  suppressible no  certain  which determine s u p p r e s s i b i l i t y ,  retrotransposon  loci  requirement  element  factors  are  for  (25) , nor  affected  the  induced  by  complete  mutant  the  suppressor  DNA  sequence  alleles  at  loci.  There i s  the  inserted  of  i s t h e r e an a b s o l u t e requirement f o r a s p e c i f i c  o r i e n t a t i o n of the element r e l a t i v e to d i r e c t i o n of t r a n s c r i p t i o n of the mutant gene Ty  element  (2, 26)  orientation  accumulated transposon  that  two  insertion  developmental time the  is  a  requirement  important with  respect  the  suppressible  to  are  coding  the  s p e c i f i c i t y ) of  alleles  at  the  into introns  would  mutation p l a c e s the  a  preliminary  f, y_ and  (17,  40,  v  forked  temporal mechanism locus.  and  copia  molecular  loci  of  I f the  (40,  suppression  suggest  by  the  mutants  that DNA  the  sequence  insertion within  analyses  indicate  forked  mRNA 41) . via  reduced l e v e l  gypsy-caused f and interference  at the  yellow  expression  of  of  an  suppressible  gypsy or  412  insertion  46).  su(Hw) and/or su(f)  gypsy,  of the  expression  Experiments i n v o l v i n g gypsy i n s e r t i o n mutations by  and  leaky  inserted into nontranslated regions.  (30) , and  which  as  transposons are  intron  56),  behave  (2,  of the w  has  location  sequences  (hypomorphs)  analysis  Evidence  a f f e c t e d gene.  alleles  53,  (43)).  factors  (and/or t i s s u e  i n s e r t e d element and The  ( c f . c e r t a i n yeast suppressors, where  to  have  This  may  recessive  yellow l o c i a  similar  form  mutation  gypsy element on  basis at  a  of  of a  distant  seen w i t h  the  t o a type of t r a n s c r i p t i o n a l the  recipient  observed i n r e t r o v i r u s e s 25  have shown pattern  the  of normal t r a n s c r i p t s  y_ mutants i s due  s i m i l a r to t h a t p r e v i o u s l y  and  suppressible  f or y_ l o c i , (10,  11),  a  decrease i n gypsy t r a n s c r i p t i o n c o u l d l e a d to an level  of  f  or  y_ mRNA. As  the  suppressible  increase  mutations  i n the are a l l  hypomorphs, an i n c r e a s e i n gene product l e v e l need not be extreme to r e v e r s e the phenotype. Thus, the wild-type and  su(f)  for  the  may  be  to  produce  t r a n s c r i p t i o n of  c o n s i s t e n t with  the  Experiments accumulation  with  of  i n i t i a t e d within  to  one  decrease  truncated efficiency  of  The  locus  This  is  of gypsy t r a n s c r i p t seen i n  mutant  a  white  have  or,  demonstrated  transcripts  copia  element  of  white  mutants  of  case,  su(w )  the  seems  a  t r a n s c r i p t s that  the  to  are  increase  rare  normal  to  the  white  (30).  mutations  element than the  or  inserted into  alternatively,  splicing  the  terminated  l o c i f u n c t i o n as  c l e a r . I t i s i n t e r e s t i n g to note, however,  enhances  one  that  associated  with  a  different  associated  with  the  mutations  suppresses. L i k e the  its  w  frequency  a  (40) .  mechanism by which the t h r e e suppressor  transposable it  element  white gene. In t h i s  correct  enhancers i s not each  level  of the  copia,  t r a n s c r i p t s found i n w  gypsy  necessary  (41).  the  LTR  the  within  the  abnormal  second i n t r o n of the act  trans-acting co-factors  decreased  homozygous su(Hw) mutants  f u n c t i o n of su(Hw)  wild-type  e.(o_) i s  an  suppressor target  mutations, e(g.) has  locus.  allele-specif ic  Rather,  no  direct  homozygous  enhancer  of  (or  certain  effect  on  hemizygous) spontaneous  mutations at the q_ l o c u s . The  garnet  genetically  at  locus the  [1/44.4] fine  26  has  been  structure  extensively level  (8,  studied 9,  24) .  Biochemically,  the eyes  of g_ mutants show d e f i c i e n c i e s f o r both  p t e r i d i n e and ommochrome pigments, i n eye d i s c t r a n s p l a n t s suggestion  and the mutation i s autonomous  (31). These  observations  have l e d t o the  t h a t o_ belongs t o a c l a s s of t r a n s p o r t  i n c l u d e s w and p_) unable t o t r a n s p o r t eye pigment cells  possessing  synthesis has  which  enzymatic  machinery  to  been  a group  precursors  complete  found  to  share  o f ATP-binding  amino  acid  sequence  b a c t e r i a l transport  thought  into  pigment  with  the  mammalian  multidrug  resistance  t o be a mutation i n a c e l l membrane t r a n s p o r t  (21) .  27  homology  proteins  had p r e v i o u s l y been shown t o share s i g n i f i c a n t  homology  (which  (51). I t i s i n t e r e s t i n g t o note t h a t the white product  recently  with  the  mutants  (23)  amino a c i d gene,  also  mechanism  M a t e r i a l s and Methods  1. C u l t u r e c o n d i t i o n s : As per Chapter 2. Mutant and w i l d - t y p e 3. C o n s t r u c t i o n for  both  involving  s t r a i n s : As per Chapter  of s t o c k s : i )  the  e(g)  mutation  P-strain  males  prevent the dysgenic  1. 1.  In c o n s t r u c t i n g stocks and  and  another  M-strain  mutant  females  homozygous  locus  were  crosses  avoided  m o b i l i z a t i o n of P elements w i t h i n the S6-1  s t r a i n , which c o n t a i n s the mutant e(g)  l o c u s . F i g u r e s 2.1  at d i f f e r e n t autosomal l o c i ii)  To c o n s t r u c t  a stock  the  attached-X dysgenic  of a P-induced g mutant  This  was  a  male  viable  females) which had y_,  cv,  v,  and  screen  f via  Southern  remaining i n t h i s stock  was  induced  g  repeatedly  used  mutation  mutation  a  the  (P)  x  was  screen  selected with  in  (g ) . p  attached-X  to M-strain  extraneous P  the  revertant  screen  carried  the  subsequent recombinational revertants. 28  out  recombinant  F 2 male  y_ QV v f/y_ cv v f demonstrated  6-7  i n the g  for  subsequently  s t a b l e M-cytotype  of the i s o l a t e d  stock  including a site  dysgenic  and  outcrossed  in a  in  a double  hybridizations  stock,  in  remove more of  (kept  s e l e c t i o n of P  and  relatively  subsequently r e c e i v e d the X-chromosome markers  o f f s p r i n g from the c r o s s : g /Y situ  locus  of the g_ mutations induced  from Chapter 1 was  mutant  2.2  ry_, PJ.) , r e s p e c t i v e l y .  (p_r, bw,  f r e e of extraneous P elements, one  P  and  i l l u s t r a t e the c o n s t r u c t i o n of such stocks mutant at the g and  to  (Figure  and 2.3).  over the  c l e a n i n g and  to  sites  of  This  the  recombined  C(l)DX,y_ f/Y v i r g i n elements  P  region.  revertants further  (M) . In  severe g  2  M-strain  and  females to  obtain  Figure  P-  2.4  the  g  p  shows  allele  and  outcrossing  4. I_n s i t u h y b r i d i z a t i o n ; A s p e r C h a p t e r 1, w i t h (by  nick  t r a n s l a t i o n ) gypsy  or copia  DNA  tritium-labelled  used  as probe  where  noted. 5.  I s o l a t i o n o f DNA f o r S o u t h e r n g e l a n a l y s i s : The p r o c e d u r e u s e d  f o r DNA i s o l a t i o n was a m o d i f i c a t i o n o f t h a t d e s c r i b e d b y et a l . (16). Approximately microfuge  tube  with  a  c o n t a i n i n g 0.1M N a C l , mercaptoethanol  300 f l i e s w e r e h o m o g e n i z e d i n a 1.8 m l glass  0.5%  Triton  b u f f e r c o n t a i n i n g lOOmM N a C l , was  added  pestle  i n 600 u l o f a  30mM T r i s - H C l ; p H  and  and mixed  with  X-100.  single  ug/ml  RNase  gently  chloroform A  t h e homogenate.  once w i t h  70% e t h a n o l  equal  a t 42°C.  a n d 100 u g / m l a t 65°C f o r  e x t r a c t i o n was was  p r e c i p i t a t e d 2X w i t h  of a  8.5 a n d 20mM EDTA  1% SDS  The DNA  and resuspended  amount  was i n c u b a t e d  e x t r a c t i o n . RNA  f o r 1 hour  3X w i t h p h e n o l ,  An  lOOmM T r i s - H C l ; p H  1.5-2 h o u r s . A s i n g l e p h e n o l : c h l o r o f o r m a  buffer  8.5, lOmM EDTA, lOmM b e t a -  p r o t e i n a s e K were added and t h e m i x t u r e  by  Gehring  digested  followed with  was e x t r a c t e d 95% e t h a n o l ,  i n TE b u f f e r  100 very  rinsed  (lOmM  Tris-  H C l ;pH 8.0, 1 mM EDTA). 6. S o u t h e r n g e l a n a l y s i s : described plasmid  i n Maniatis  prr25.1  DNA  S o u t h e r n g e l a n a l y s i s was p e r f o r m e d a s  et a l .  (3)  (32).  labelled  The h y b r i d i z a t i o n p r o b e  by n i c k  Nick  T r a n s l a t i o n K i t , Amersham C o r p . ,  dATP  (New E n g l a n d N u c l e a r  translation  was  (Amersham  O a k v i l l e , Ont.) w i t h  3 2  p-  C o r p . , B o s t o n , Mass.) a s a r a d i o a c t i v e  precursor.  29  2.1  Figure Construction  of homozygous stocks  mutant  at the  e(gj  and. the a l o c u s .  0.(3.)  /0.(3.)  (P)  x  FM6/Y (M)  a  J e(a)/FM6  x  a*/Y  (M)  S(a)/a  x  O.R.  males  b  (M)  c  J e(a)  a /Y  (recombinant)  x  FM6/M(l)of  J e(a)  a /FM6  x  e(a)  a /Y  I STOCK N.B. a  V i r g i n females s e l e c t e d f o r each c r o s s .  e(a)/e(a)  = S6-1  stock.  b  * = a a l l e l e s 1, 2, 3, 50-e  c  O.R.  = Oregon R  wild-type.  30  and  53-d.  (backcross)  locus  Figure 2.2 Construction  o f homozygous stocks  and at t h e autosomal  mutant  at t h e e_(g_)  locus  l o c i p_r, bw, ry_ and p_, r e s p e c t i v e l y .  e (a) /e (a) , +/+  (P)  x  FM6/Y, +/+  (M)  a  J FM6/e(a), +/+  FM6/e(a), Er*/+  £(a)/£(fl)/  x  FM6/Y, r j r * / p _ r * (M)  x  e(g_)/Y, rjr*/ +  x  e_(a)/Y, pr.*/px*  I I  b  STOCK  N.B. V i r g i n females s e l e c t e d f o r each c r o s s . a  e(o:)/e(a) = S6-1 stock.  D  P r o t o c o l f o r p_r  i l l u s t r a t e d , where * = p_r a l l e l e s  Same p r o t o c o l was used f o r t h e bw locus a l l e l e s locus a l l e l e s  1 and bw.  1, 2b and 75;  1, 2, 8, 26 and 41; and p. l o c u s a l l e l e s 1 and p.  31  ry_  Figure Scheme  f o r removing  stabilizing  2.3  the extraneous  the c y t o t y p i c background  P  elements  o f a P-induced  and f o r  g_ mutation  (see M a t e r i a l s and Methods).  y_ cv v g. f/Y (P) p  x  O.R. v i r g i n  females (M)  a  J y_ cv v g_ f/+ v i r g i n p  g_  p  females  f/Y (recombinant)  x  O.R. males  x  O.R. v i r g i n  J  (M)  females (M)  J g_  p  f/+ v i r g i n  g. /Y p  females  (recombinant)  x  J  x  O.R. males  (M)  C(1)DX/Y v i r g i n  females (M)  C(1)DX/Y v i r g i n  females (M)  J a_ /Y p  X x  I STOCK  O.R. = Oregon R wild-type.  32  Figure  2.4  H y b r i d dysgenesis screen f o r r e v e r t a n t s o f the subsequent  r e v e r t a n t stock formation (see M a t e r i a l s and Methods).  y_ cv v g_ f/Y (P) p  y_ cv v g  Y_ cv v c j .  g  r  mutant and  e  v  r e v  f/Y (dysgenic)  f/g, (revertant)  f/Y (recombinant)  2  r e v  f/Y  g  r  f/Y  v  C(1)DX/Y v i r g i n females (M)  x  g /g  x  g_ /Y  J  I  2  e  x  J J J  v i r g i n females (M)  2  x  C(1)DX/Y v i r g i n females (M)  x  C(1)DX/Y v i r g i n females (M)  x  C(1)DX/Y v i r g i n females (M)  I  STOCK  N.B. F i v e separate r e v e r t a n t s  33  (1-5) were i s o l a t e d i n t h i s way.  Results  1. A n a l y s i s o f the modifying e f f e c t o f ela) loci:  To t e s t  were  the e f f e c t  mutation  on v a r i o u s g mutants,  o f e(g_)  c o n s t r u c t e d which  were  homozygous  at t h e g  and a mutation  on mutations at other  locus  f o r both  (Figure  stocks  the  e(gj  2.1; [modifying 1  effects  measured v i s u a l l y ] ) .  9. r g  and c j r  g alleles  Five  . A l l o f these  J  were used:  a r e spontaneous  7  g ,  g ,  mutations and  have been shown by i n t r a g e n i c recombination mapping t o l i e i n the f o l l o w i n g order along the X-chromosome: g 3d-g2_g50e_gl g3  ^  5  f  24) . The phenotypes the presence of g i g  o f t h e mutants g  and a_ were u n a f f e c t e d by J  o f homozygous e(g) . However, t h e mutant and gr  a l l became more severe  phenotypes  ( i . e . l e s s pigmented)  i n combination with homozygous e (g_) . As  noted  i n Chapter  1,  t h e S6-1  stock  contains  t e m p e r a t u r e - s e n s i t i v e g mutation and shows P element in  situ  hybridization  t o t h e g_ r e g i o n  a  homology by  on p o l y t e n e chromosomes.  T h i s g^- mutation i s not v i s i b l e when homozygous, but when p l a c e d a  (g ) or a d e f i c i e n c y  over a more severe a l l e l e (Df (l)g.  1  To check S6-1  f B) the mutation can be scored i n f l i e s r a i s e d a t 29°C. f o r a modifying e f f e c t  o f e(g)  X-chromosome, c o n t a i n i n g both e(g)  X-chromosomes mutations  f o r the g region  constructed to contain  o r t h e e(g)  r e g i o n . In both  mutation  on the g_ and g. , ts  both  ts  mutation, t h e  was t e s t e d  over  t h e e_(cr_) and o_  and t h e d e f i c i e n c y  2  f o r the g  i n s t a n c e s , at 2 9°C, female f l i e s showed the same  mild  garnet  phenotype  seen  Thus  there  i s no e(g_)  i n t h e absence  modifying  present i n the S6-1 s t o c k .  effect  o f homozygous on t h e g_  ts  e(g_) .  mutation  The  effect  o f e(g.) on m u t a t i o n s  at other l o c i  was t e s t e d b y  c o n s t r u c t i n g s t o c k s homozygous f o r b o t h t h e e ( g ) m u t a t i o n mutation pr,  i n question  bw, ry_ a n d p_  represented p_r , b w  bw ,  ( F i g u r e 2 . 2 ) . The a u t o s o m a l e y e c o l o u r  were t e s t e d i n t h i s spontaneous  bw ,  1  Results loci  both  bw ,  2 b  and t h e  fashion; the alleles  and x-ray  induced  ry. , ry_ , ry_ , ry_ ,  7 5  1  2  showed t h e p h e n o t y p i c  8  ry_ ,  2 6  used  mutations: p.  4 1  o f homozygous  p_r ,  a n d p_P.  1  e x p r e s s i o n o f none o f t h e s e  t o be a f f e c t e d by t h e p r e s e n c e  loci  mutant  (or hemizygous)  e(g) . 2.  In situ hybridization  whether  t h e enhanceable  associated  with  gypsy  to  either  (not  of these  garnet  or copia  p r e p a r a t i o n s from these with tritium-labelled  t o polytene  chromosomes: To  alleles  g , g^Oe  g-53d  2  insertions,  a  n  determine  d  polytene  w  e  r  e  chromosome  s t o c k s were s e p a r a t e l y h y b r i d i z e d i n s i t u  g y p s y o r c o p i a DNA. T h e r e was no h o m o l o g y  transposable  elements  i n t h e 12C  (g) r e g i o n  shown).  3. P r e p a r a t i o n o f s t o c k s f o r c l o n i n g t h e g l o c u s : To b e s t  analyse  many o f t h e q u e s t i o n s p e r t a i n i n g t o t h e i n t e r a c t i o n b e t w e e n t h e g and  e(g) l o c i  i t would be d e s i r a b l e t o o b t a i n a c l o n e  gene f o r m o l e c u l a r  study. A molecular  w o u l d a l s o be o f g r e a t in  Drosophila  genetic  be  t o be a n a l y s e d  techniques,  biochemical  strain  and  itself  a s i t was one o f t h e f i r s t structure level  i t s molecular  unelucidated.  t o determine  structure  Furthermore,  loci using and  i t would  the structure of the g locus i n the  and t o examine  associated w i t h g mutations  study o f t h e g locus  at the fine as  f u n c t i o n remain  of interest  S6-1  interest,  of the g  any a l t e r a t i o n s induced  35  a t t h e DNA  i n t h e dysgenic  screens  level from  Chapter  1. po  One allele  o f these induced a mutants  allele  i n females.  mutant  crossed with  In stock, t h i s  garnet  g /a  eyes. When the  a /e(a)  a  e(a) a -  mutation  produced  same orange-eyed  females, a l l female  mutation  wild-type i n  with  underwent  eyes. These  females,  It would seem then t h a t t h i s new a new  appeared  when h e t e r o z y g o u s  m u t a t i o n a l event producing orange  e(a)  ) , p i c k e d up over the a  p r e s e n t on the Maxy a chromosome,  hemizygous males but  when  (a  o  a  severe  a  further  orange-eyed  female  males,  progeny  with  males were c r o s s e d w i t h  offspring  had  orange  eyes.  orange mutation c o u l d r e p r e s e n t  interaction.  T h i s orange mutation, w h i l e i n stock, subsequently underwent a  further  m u t a t i o n a l event  p r o d u c i n g a a mutation  both males and females. Crossed w i t h both g / g 2  expressed i n  e(a) a/s.(a) 2  and  2  o  a  females  the  hybridization lines  to  female  progeny  polytene  t o have P element  were  chromosomes  homology  garnet-eyed. showed  i n the a  a b i l i t y t o f o l l o w t h i s m u t a t i o n a l sequence  In  a l l three  region.  situ mutant  Clearly,  an  at the molecular l e v e l  would be i n v a l u a b l e i n uncovering the nature of mutation at the a locus  and  the  mechanism  of  interaction  between  the  e(a)  of  the  and  a  loci. The region  mutant was  isolated was  chosen  f o r the  a presumptive  i n the attached-X  P  molecular  element  study  insertion  screen from Chapter  i s o l a t e d over the F M 7 chromosome and was  a male  stock mated  to  Methods, Chapter 1). g  r  C(l)DX,y_  f/Y  1.  garnet  mutation This  (a ) P  mutation  subsequently kept as  females  (see M a t e r i a l s  males c r o s s e d with homozygous a  and  females  p  produced  female progeny  w i t h garnet eyes, a 36  males c r o s s e d with  and g  females homozygous f o r both e(g_) with  a mildly  eclosion,  which  enhanced  garnet  quickly  darkened  gave r i s e  phenotype  to  daughters  ( i . e . orange)  t o garnet w i t h i n  the  9  hours.  This  i s the  same r e s u l t  as  conclude t h a t e (g) ,  (see R e s u l t s , Chapter 1) ; one may the S6-1  first  24  9  g/e(g)  seen w i t h  upon  of-  flies  p r e s e n t on  and, t h e r e f o r e , g_ X-chromosome, has no m o d i f y i n g e f f e c t p  p on the g_ mutation. The i n i t i a l  removal o f the m a j o r i t y o f extraneous P  element  p sites  from the g  stock was  double recombinant cross  involving  gf  y_ cv v  f/Y male i n the F  males  M a t e r i a l s and Methods). was  accomplished w i t h the s e l e c t i o n of a  and  offspring  2  homozygous y_ cv v  T h i s new  strain,  f  containing  of a  females 6-7  P  (see  sites,  kept as a male stock mated t o attached-X females. Males  from  this  s t r a i n were used i n a dysgenic screen f o r r e v e r t a n t s o f the  a_  mutation  p  isolated of  (g  these  XXJ  (Figure  - ) J  2.4).  from f i v e  strains  were  Five  such  independent  formed  as  revertants  were  r e v e r s i o n events. Stocks  diagrammed  i n Figure  2.4,  c r o s s i n g o f f the X-chromosome d i s t a l t o the g locus i n an attempt to  further  remove extraneous  P  elements,  and  outcrossing  three  times t o M - s t r a i n attached-X females, both t o remove P s i t e s  and  t o e s t a b l i s h the r e v e r t a n t s i n a s t a b l e M-cytotype. Eye c o l o u r i n each  of the r e v e r t a n t s was  visually  undifferentiable  from  wild-  type i n e i t h e r hemizygous males or heterozygous females over the 9  g_  allele. To remove the remaining extraneous P element  y_ cv  v  cytotype,  a  P  f/Y flies  strain, from t h i s  as  well  as  to  establish  stock were s u c c e s s i v e l y 37  sites a  from the stable  M-  outcrossed to  M-strain  Oregon  selected  on  R  wild-type  e i t h e r side  generations  flies,  of the  (see F i g u r e  2.3).  single  g_ l o c u s  In  situ  recombinants  i n the  third  being  and  h y b r i d i z a t i o n to  fifth  polytene  p  chromosome p r e p a r a t i o n s PTT25.1 DNA  as probe, showed two  s t r o n g s i t e i n the bands proximal present,  to  12C  of  i t on  strains,  gel  with  the  size,  element  with  in situ  v g.  of  end  of  sequence amount  a  the  gel.  band,  probe  bound  rehybridizing  element level  kb  the  one  was  DNA,  i n the  one two  site  the  Hind  III  g^  was X-  one  may  co-migrates with  g  lane.)  38  a;  (Figure  the  12-13  other  so  for  of  kb  with  off  that  the  in  the  site  resides  non-P-specific 17C,  d i s p r o v e d by washing a  fragment  same  seen  element  detection  1.8  in  fragment  P  minimal  t h a t one  the  kb  site  HI  gel-purified, containing  12-13  kb  at  +R  2.5) .  as t o have run  h y b r i d i z a t i o n only  The  five  the  extent  be  (50)], was  restriction  detecting  as  from the c y t o l o g i c a l r e g i o n  filter  and  its  e i t h e r i n a Bam  site  HI  and  band,  or i s so small  Bam  strain  probe  other p o s s i b i l i t y ,  same  complete  well  as  insufficient  on the prr25.1 plasmid  translated,  DNA  showed only  [which c o r r e l a t e s with DNA  present  as  A l t e r n a t i v e l y , the  at  which  above  strain,  hybridization resides  fragment  latter  on  homology. Presumably,  Southern a n a l y s i s . (The in  the  pn-25.1  strain  homology  of  performed  f  p  which co-migrates at 12-13 the  plasmid  another, weaker, s i t e  X-chromosome. T h i s  from  labelled  Surprisingly, P  using  1.1).  DNA  y_ cv  the  with  the  strain,  s i t e s of P element homology:  a n a l y s i s was  genomic  progenitor,  g  (g_) r e g i o n , and  chromosome ( c f . F i g u r e Southern  new  homology i n the g r e g i o n , on the o r i g i n a l S6-1  as was  digests  from t h i s  the  band was  and nick  solely 12-13  P kb  present  Figure  Southern  analysis  showing  2.5  hybridization  o f plasmid  pr[25.1  p  DNA t o complete Bam HI d i g e s t s o f genomic DNA from the g and r e v e r t a n t  mutant  stocks. 1  2  3  4  5  6  7  S i z e markers were Hind I I I fragments from phage lambda DNA. a  Hybridization  t o DNA  from  cytological  region  17C  sequences f l a n k i n g complete P element i n pn-25.1. Lane 1 = q_ . Lanes 2-6 = g _ p  + R l  39  ~ . 5  Lane 7 = y_ cv v a; f . p  by  DNA  in  y_ cv v gf  the  homology present The  f  i n that  revertant  examination  lane,  lanes  band present  strain.  i n the  g?  showed m u l t i p l e absent  These r e s u l t s strain  (with P element i n s e r t i o n ) revertants  the  other  sites  of P  upon  close  strain.  h y b r i d i z a t i o n was  each r e v e r t a n t  the  i n a d d i t i o n to  excision  the  but  12-13  indicate that  represents  and t h a t  represents  at  bands,  DNA  kb  level  in  the  12-13  kb  from the  g  region  the absence of t h i s band i n of  P  element  DNA  as  the  region  DNA  mechanism of r e v e r s i o n . The  determination  of  fragment  i n a complete Bam  one  remove  to  construction T h i s has i n our  of  this a  the  size  HI  digest  fragment  fragment-enriched  f a c i l i t a t e d the recent  l a b by  from  of  the  of g_  p  a  garnet  genomic DNA  gel  plasmid  for DNA  use  enables in  library  the (37) .  c l o n i n g of the g l o c u s , performed  D.A.R. S i n c l a i r , u s i n g the technique of P element  transposon-tagging  (3).  40  Discussion  The modifier  enhancer locus.  o f garnet was shown t o be an a l l e l e - s p e c i f i c Homozygous  (or hemizygous)  e(g) enhances t h e  phenotypes o f the spontaneous garnet mutations g , gp® z  i  but  does not enhance the spontaneous a l l e l e s g  mutations, g  and g?^^,  e  ^  and g  J  or two new  and g .  Most spontaneous mutations i n D r o s o p h i l a are a s s o c i a t e d w i t h the  insertion  of transposable  t r a n s c r i p t i o n regulatory sequences  elements  sequences  (30, 40). A l s o ,  (30), e i t h e r  into  5'  (35, 49), or i n t o RNA coding  i t i s transposon  induced mutations  which have been shown t o be m o d i f i a b l e by r e c e s s i v e mutations a t distant l o c i  (36, 40) .  Similarities described  between  the e(g)-g  interaction  and t h e w e l l -  transposon-mediated suppressor systems  i n Drosophila  were sought. When t e s t e d w i t h other l o c i , modify the phenotype o f mutant and p i n k l o c i associated  (including  with  phenotype  a l l e l e s at the p u r p l e , brown, rosy  t h e p_r and p r  412 i n s e r t i o n s  su(s_) ) . The mutations of c e r t a i n  su(p_r), mutant  e(g_) was shown not t o  mutations, which are  b w  and s u p p r e s s i b l e su(s) and su(f_)  alleles  by su(p_r) and  can enhance the  associated  with  gypsy  or  c o p i a i n s e r t i o n s , however, i n s i t u h y b r i d i z a t i o n demonstrated t h e absence o f gypsy or c o p i a DNA sequences the  g_ a l l e l e s enhanceable by e(g) . The  recessive  interaction this  i n the garnet r e g i o n o f  with  nature  spontaneous  of  the  e(g)  enhanceable  mutation  alleles  and i t s  suggest  that  system may be analogous t o t h e transposon-mediated a l l e l e -  specific  s u p p r e s s o r systems 41  described  previously  (36, 40) .  However, the mechanism by which the e(g)-g not  clear.  Because i t i s an enhancing  transcriptional  interference  interaction  interaction,  or r e d u c t i o n  occurs i s  release  i n abnormal  from  transcript  accumulation are not a p p r o p r i a t e mechanisms. That w i l d - t y p e e(g.) c o u l d produce a gene product which a c t s d i r e c t l y upon g sequences regulating  transcription,  some necessary c o - f a c t o r  or a l t e r n a t i v e l y ,  w i l d - t y p e garnet Albeit  invoke  a  located  possible  o f e(g) and i t s lack  i s unlikely  of effect  on  locus.  i n the absence o f molecular data,  i t i s tempting t o  t ransposon-mediated  for  interaction. be  i t c o u l d produce  f o r g gene product a c t i v i t y ,  g i v e n the a l l e l e - s p e c i f i c i t y the  that  Given that  mechanism  e.(g_)-cf  the enhanceable g mutations are known t o  at one end o f the f i n e  mechanism  the  i s suggested:  structure  Transposable  g  locus  element  map,  insertion  i n t o the promoter or 5 ' r e g i o n o f the g gene would d i s r u p t transcription  (essentially  eliminating  transcription  a  normal  from  the  normal s t a r t s i t e ) . The g gene a c t i v i t y which i s observed i n the hypomorphic  enhanceable mutants may  read-through element  initiated  and c o n t i n u i n g  from into  result  a promoter  the g  gene.  from  transcriptional  within In t h i s  an  insertion  s c e n a r i o , the  mutant garnet phenotype r e f l e c t s l e s s e f f e c t i v e t r a n s c r i p t i o n o f the  g locus,  or the p r o d u c t i o n  transcription  o f the i n s e r t i o n  type a c t i v i t y  o f the e(g) gene  mutations  at e(g_) would b l o c k  o f a m o d i f i e d gene product. I f  elements were dependent  on  wild-  (analogous t o su(Hw) and gypsy), read-through  into  the g gene and  r e s u l t i n the appearance o f an enhanced mutant phenotype. To  determine  the true  42  nature  of t h i s  system  of  gene  interaction mutant was  a  molecular  analysis  i s needed.  (o. ) i s o l a t e d i n the experiments p  described  of g w i l l  enable  the  elucidation  of the  l o c a t i o n o f the p u t a t i v e  i n s e r t i o n elements  mutants,  facilitate  and  informative a  g  e(g_)  also  sequence  of s u c c e s s i v e  P-induced  g  the  g_  i n Chapter  1  mutant  alleles  g locus  i n the  presence  and  (.g  of  (see  seen  R e s u l t s ) . cDNA  of the t r a n s c r i p t s produced absence  of  e.(a_)  will  be  structure  of  and the nature o f the i n t e r a c t i o n between the g  43  g  a potentially  i n the d e t e r m i n a t i o n of the m o l e c u l a r  loci.  and  i n the spontaneous  study  )  The  structure  mutations at the g locus  and Northern g e l a n a l y s i s  informative the  will  different  isolation by  P-induced  prepared f o r the m o l e c u l a r a n a l y s i s of the garnet l o c u s .  cloning  in  A  and  SUMMARY  The nature o f extreme m u t a b i l i t y lab  strain  around the g l o c u s  i n the  S6-1 was determined t o be a s s o c i a t e d w i t h P-M  hybrid  d y s g e n e s i s . S6-1 i s a P - s t r a i n : males o f t h i s stock, when c r o s s e d to M - s t r a i n v i r g i n  females, g i v e r i s e t o F-^ progeny  showing the  germline m u t a b i l i t y t y p i c a l o f the P-M system. When t e s t e d over a m u l t i p l y marked X-chromosome, mutation a number  o f X-chromosome  loci.  i n d u c t i o n was observed at  The p r e p o n d e r a n c e  of induced  mutations a s s o c i a t e d w i t h the g_ locus may be accounted f o r by the presence o f a P element  s i t e o f r e s i d e n c e i n the cr r e g i o n i n the  S6-1  by  strain,  chromosome element  as shown  p r e p a r a t i o n s . New  insertion  element,  in situ  e.g.  or a  hybridization  mutations  may  change  associated  of  orientation,  reversal  thus with  to polytene arise  from  P  the r e s i d e n t  P  rearrangement,  or  imprecise e x c i s i o n . A novel s i t u a t i o n was the requirement f o r the presence o f the S6-1 X-chromosome i n F-^ dysgenic males i n order to produce only  the germline m u t a b i l i t y . The r e s u l t s  complete  P  element  or  elements,  suggest t h a t the  ( i . e . those  capable  of  a l l o w i n g t r a n s p o s i t i o n ) , are c o n f i n e d t o the X-chromosome i n the S6-1  strain. The e x i s t e n c e o f a mutation, present i n the d i s t a l r e g i o n of  the S6-1 X-chromosome, which enhances c e r t a i n mutations locus  i s reported. This  identical garnet  modifier,  phenotype all  with  e(g) mutation,  a previously acts  as  a  o f the spontaneous  which  d e s c r i b e d , and recessive  may  at the g  or may  subsequently  mutation  o f which map t o one end o f the f i n e s t r u c t u r e  lost,  t o enhance the  garnet mutants g , g ^ 2  not be  5  g  e  and g ^ , 5  d  locus map.  e(g_) d i d not modify a P-induced g_ mutant, a t e m p e r a t u r e - s e n s i t i v e g_ mutant, or the spontaneous garnet a l l e l e s g_ was  shown  to  have  mutant  alleles  system  may  at  no  effect  the  loci  represent a  transposon-mediated  on  the  J  phenotype  p_r, bw,  variation  and g_ . A l s o , e(g_)  ry_ and  on  the  s u p p r e s s i o n systems  of  a  number  of  p_. T h i s  enhancing  previously  described  i n D_s_  melanogaster.  C e r t a i n suppressor mutations i n D r o s o p h i l a have been r e p o r t e d t o enhance mutations at d i s t a n t  loci  insertions.  situ  However,  in  a s s o c i a t e d w i t h gypsy hybridization  to  or c o p i a polytene  chromosomes from the t h r e e enhanceable g. mutations shows them not to be a s s o c i a t e d w i t h gypsy or c o p i a As a f i r s t interaction,  a  insertions.  step t o understanding the mechanism o f the e(g,)-g; P-induced  garnet  mutant  (g_ ) p  was  isolated  and  prepared f o r the molecular study of the g_ l o c u s . The i s o l a t i o n o f revertants  of  this  mutation  and  the  removal  of  extraneous  P  elements from the stock has f a c i l i t a t e d the recent c l o n i n g o f the g_ l o c u s .  45  BIBLIOGRAPHY  Atherton, D. and G a l l , J . (1972) S a l i v a r y g l a n d squashes for in situ nucleic acid hybridization studies. D r o s o p h i l a Inform. Serv. 49:131-133. 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Marked w i t h t h e r e c e s s i v e  Of  sn  2.  and  B.  v i a b l e but s t e r i l e . a:  alleles  Such  attached-X  females  produce  and a p a t e r n a l  Y.  that  C(l)DX/0 i s  a l l e l e s y_ a n d f .  inversion  w i t h markers  y3  l d  s_c®  w  a  4  F  Maxy  Monocentric  females.  attached-X's  B a l a n c e r X-chromosome  yO  X.  a p a t e r n a l X and m a t e r n a l Y, and d a u g h t e r s  inherit  FM7:  (see (31) o r  descriptions):  f.: Compound  chromosomes sons  chromosomes a n d m u t a t i o n s  Multiply y_  c4  sc. s c j 8  Male v i a b l e Suppresses  marked 5 1  and f e r t i l e ;  crossing  homozygous  female  o v e r i n t h e X-chromosome.  X-chromosome  bearing  pjn w e c r b cm c t ^ s n ^ r a s  2  the  g_  2  f  recessive  os a n d c a r .  Homozygous o r h e m i z y g o u s Maxy a n o t v i a b l e .  bw '  2  bw '  2  1  1  b  b  '  7  5  :  Recessive  = spontaneous;  ^x,  J, j u e ,  locus  [1/44.4],  JOU.  alleles bw  7 5  at the  = x-ray  Spontaneous  Localized  to  brown  locus  [2/104.5].  induced.  recessive  polytene  alleles  at the garnet  chromosome  region  12B9-  Notch  locus  12C7.  N:  Dominant  mutation  (wing  t i p phenotype)  at  the  [ 1 / 3 . 0 ] . L o c a l i z e d t o p o l y t e n e chromosome b a n d 3C7.  P; Spontaneous r e c e s s i v e a l l e l e s  px ' 1  b w  :  Spontaneous  recessive  at the pink  alleles  at  locus  the  [3/48.0]  purple  locus  [2/54.5].  rv ' 1  ry ' x  w  2  '  8  '  2 6  '  4 1  : Recessive  a l l e l e s at the rosy locus  ^ = spontaneous/ ry°'  and w : a  Spontaneous  yj  - = x-ray  recessive  [1/1.5]. L o c a l i z e d t o polytene  52  [3/52.0]  induced.  alleles  at the white  chromosome band 3C2.  locus  

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