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The effect of chromatin structure on P element-induced male recombination in Drosophila melanogaster Fitzpatrick, Kathleen Anne 1985

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THE  EFFECT OF CHROMATIN  STRUCTURE ON P ELEMENT-  INDUCED MALE RECOMBINATION IN DROSOPHILA  MELANOGASTER BY  KATHLEEN ANNE FITZPATRICK B.Sc,  University  of B r i t i s h Columbia,  1980  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Z o o l o g y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e required standard  THE  UNIVERSITY OF BRITISH COLUMBIA  September, 1985  ©  Kathleen  Fitzpatrick,  1985  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  Zoo^O^Y  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  ii  ABSTRACT Dysgenic  male r e c o m b i n a t i o n  T-007 and OKI r a r e l y , of  chromosome  of  heterochromatic  chromatin  two.  in this  chromatin  involving with  two  region.  different  were  variegation  that  causes  neither  to i n f l u e n c e  some  alterations  t h e OKI  meiotic  i s the f i r s t  known  recombination  MR.  Analogous  chromosome  suppressor  Neither  suppressor  MR,  of b u t y r a t e .  strain.  suggesting  "factor"  similarly.  One o f t h e meiotic  to  that  the  from,  and  mutations  recombination  i n euchromatic  The o t h e r  nor d y s g e n i c  crosses  can i n f l u e n c e t h e  and hence  third  of  i s consisten  Su(var) genes i s d i f f e r e n t  i s thought  affects  condensed  levels  finding  structure  any h e t e r o c h r o m a t i c  involving  dysgenic  chromatin  This  mutations.  crosses  325  P strains.  (Su(var))  than,  frequencies,  the l a c k  m o d i f i e r of  significant  using  more  which  (a s u s p e c t e d  of P e l e m e n t s  of t h e s e  (325)  induced  that  mode o f a c t i o n specific  i s due t o t h e h i g h l y  performed  induced  heterochromatin  explanation i s that  Butyrate  strains  i n d y s g e n i c h y b r i d s d e r i v e d from  and e x c i s i o n  experiments  mutation  possible  exchange  the h y p o t h e s i s  insertion  of  One  MR  by t h e P  i f ever, occurs i n the  structure)  heterochromatic  (MR) i n d u c e d  MR i n  mutation,  recombination. influence  3_18,  Su(var)  meiotic  and  iii  TABLE OF CONTENTS  ABSTRACT LIST LIST  i i  OF  TABLES  OF  FIGURES  i  i  i  iv  ACKNOWLEDGEMENTS  v  INTRODUCTION  1  MATERIALS  AND METHODS  16  RESULTS  24  DISCUSSION  39  CONCLUSIONS  53  LITERATURE APPENDIX  I  CITED  55 64  iv  LIST OF TABLES  TABLE 1.  TABLE 2.  TABLE 3.  TABLE 4.  Recombination data, i n each genetic i n t e r v a l , f o r c o n t r o l dysgenic crosses i n v o l v i n g P s t r a i n s T-007 and OKI ....  Page 25  Recombination data, i n each g e n e t i c i n t e r v a l , f o r butyrate t r e a t e d dysgenic c r o s s e s i n v o l v i n g P s t r a i n s T-007 and OKI  29  Recombination data, i n each genetic i n t e r v a l , f o r dysgenic c r o s s e s i n v o l v i n g P s t r a i n s T-007 and OKI, to which the mutant Su(var)325 has been introduced  33  Recombination data, i n each genetic i n t e r v a l , f o r dysgenic crosses i n v o l v i n g P s t r a i n s T-007 and OKI, to which the mutant Su(var)318 has been introduced  35  V  L I S T OF FIGURES Page FIGURE FIGURE  1. 2.  FIGURE 3.  FIGURE 4.  M u l t i p l y marked s e c o n d chromosome used i n a l l e x p e r i m e n t s  18  M a t i n g scheme used i n u n t r e a t e d and b u t y r a t e t r e a t e d ( a ) and S u ( v a r ) (b) e x p e r i m e n t s  19  Second chromosome maps d e r i v e d from r e c o m b i n a t i o n d a t a of d y s g e n i c c r o s s e s i n v o l v i n g t h e P s t r a i n T-007  37  Second chromosome maps d e r v i e d from r e c o m b i n a t i o n data of d y s g e n i c c r o s s e s . i n v o l v i n g t h e P s t r a i n OKI  38  vi  ACKNOWLEDGEMENTS  I  wish  Grigliatti,  to  suggestions.  about  science),  Hansen,  my  Kafer,  b e a r a b l e one.  thesis  For i l l u m i n a t i n g I am g r a t e f u l  friends:  supervisor,  o f , and i n t e r e s t  was an u n f a i l i n g  a n d D r . A. R u d d e l l .  imagined), Kathy  my  f o r h i s support  Dr. D.A.R. S i n c l a i r and  thank  source  in,  my  Dr.  T.A.  research.  of encouragement  d i s c u s s i o n s (some  of them  t o J . L e u n g , D r . R.H. D e v l i n , B. I n moments  Yvette  made t h e p r o s p e c t  Lloyd,  of c r i s i s Annette  of f a c i n g  ( r e a l or  Bailey,  the f l i e s  and  again a  1  INTRODUCTION  While  looking  for  segregation  c a u g h t s t r a i n s of D r o s o p h i l a noted  low  finding  but  significant  contradicted  recombination Hiraizumi's  did  of  arise.  Since  the  e t a 1.,  1973),  the  studied  and  Oster,  Kidwell  occur  reports  melanogaster  1984;  Sved,  Kidwell, 1977;  parents from  i n male from  recently  Yamaguchi,  established  recombination syndrome termed Hybrid Green,  1980;  and  In a l l c a s e s the  caught  laboratory  i s only hybrid  dysgenesis Bregliano  one  of  1977;  (reviewed  crosses  s t r a i n s and strains.  (Kidwell  1983;  wild  observed  1974;  Waddle  Mukai,  Green,  1975; and 1977;  between female  was male  parents  However, diagnostic  male of  a  e_t a l . , 1977).  in Bregliano  Kidwell,  of  recombination  several traits  dysgenesis  and  from  been  and  can  Hiraizumi  1976;Yannopou1os  Thompson,  derived  w h i c h MR  progeny  Voelker,  Cardillino  meiotic  extensive  1971;  i n d i v i d u a l s has  1975;  wild  the  under  among t h e  1976;  hybrids  that  This  m e^a. ii££ a^js _t e_ r_.  (Hiraizumi,  1974;  Woodruff  D^  prompted  investigators:  G o l u b o v s k y ^_t a l . , 1977. observed  male  immediately  initial  numerous  Pelecanos,  in  belief  special circumstances  by  and  established  wild  (1971)  of male r e c o m b i n a t i o n .  male r e c o m b i n a t i o n  Drosophila  and  levels  not  effects in  melanogaster, Hiraizumi  long  observation  investigation  caught  the  distortion  and  e_t a l . , Engels,  1980; 1983)  2 consists which  of  a  set  of  include  the  induction  mutations,  chromosome  Yannopoulos (Slatko,  genetic  et a l . ,  1978;  and of  physiological  single  site  rearrangements  1983),  Sinclair  mitotic  and  abnormalities  visible  (Berg  and  lethal  e_t a l . ,  1980;  recombination i n females  Green,  1979)  as  well  as  males,  c h r o m o s o m a l n o n - d y s j u c t i o n , u n e q u a l t r a n s m i s s i o n of homologous c h r o m o s o m e s , and  g o n a d a l d y s g e n e s i s ( G. D.) s t e r i l i t y .  abnormalities  seem  before  during meiosis.  and/or  Hybrid resulting females cross  from  matings  the t r a i t s  do  those from  if  ever produce  occurs  t o t h e germ l i n e  only  in  between m a l e s f r o m  laboratory  (laboratory  named  restricted  dysgenesis  from  display  were  t o be  strains.  males  Progeny  crossed  to  and  Thus,  occur  offspring  p o p u l a t i o n s and  from  the  reciprocal  caught  ten-fold  females) lower  than  crosses rarely  wild  laboratory  and  Fl  Intra-strain  dysgenic offspring.  P (for paternal),  wild  wild  at a frequency at l e a s t  the d y s g e n i c c r o s s .  the  These  caught  strains  strains  M (for  maternal) . Early anomalies random  included:  chromosome  effects, 1976).  speculations  and The  infectious breakage  the  etiology  agents  (Sved,  latter  1978),  i s consistent  must  be  To p r o d u c e  placed  of  the  dysgenic  s u c h as v i r u s e s c a u s i n g  chromosomal-cytoplasmic  n a t u r e of t h e s y n d r o m e . chromosomes  into  with  single  locus  interactions the  mutator (Sved,  non-reciprocal  the d y s g e n i c symptoms, P  i n an M-type c y t o p l a s m .  In t h e o r y  3  some a g e n t  (or the l a c k  with  component  some  nature there  of  the  was  hybrid  or  "agents"  product or  lambda.  Single  "mutator"  and  locus  elements  pivotal  the  was  effects  were be  ruled  and  which  agent  hybrids P  chromosomes c o u l d " c o n t a m i n a t e "  of  P-like  several  hypothesis  generations,  characteristics  introduced  back  Kidwell,  1983).  consistently alone  could  into  and  the  could  However,  in P strains not  explain  the  fact  M  to  any  promote  the  finding  that  chromosomes  chromosomes  produce strain  major  1980). to  M  when  mapped  the  M  viruses  and the  was  i . e . the  original  out  helped  between  bacteriophage  Green,  infectious  matter  viral  of  The  although  analogy  accurately  (Slatko  observation  vague,  of an  induction  not  interacts  P chromosomes.  zygotic  could  or  cytoplasm  "components"  chromosomal p o s i t i o n  A  of  some s u g g e s t i o n ( E n g e l s , 1979)  dysgenesis  single  t h e r e o f ) i n the M  in a  took  dysgenesis  furthermore,  not  on  when  (see B r e g l i a n o  were  in  and  observed  the v i r u s  theory <  non-reciprocal aspect  of  the  was  the  interaction. Despite first  known i n d i c a t o r  to the i n v e s t i g a t i o n easily nature (1977).  observed of He  aspect  that  recombination  of d y s g e n e s i s , of m u t a t i o n  induced  observed  that  (MR)  the e m p h a s i s  induction,  of d y s g e n e s i s .  dysgenically had  male  Early  mutations visible  soon  shifted  a somewhat insights came  dysgenic  more  into  from  the  Green  mutations  4  behaved  like  mutable  a h i g h degree type  or,  purely  (w)  these  mutations  genetic  observed to  that  Mutations a  found  the  in  abnormal  w i t h P chromosomes.  inserting could  occur  analysis Bingham, Rubin, w  mutations  inserted  reviewed  which  excision  been  Rubin,  of  DNA  t o be into  were  or  to c a r r y  P elements:  strains  about  chromosomes.  lack  mutation  1976). with  He  hybrid element  he  proposed  of the  element  reversions  by  subsequent  Most  and R u b i n , 1983).  was  the  molecular  Kidwell,  1982;  with  the  and  O'Hare  Dysgenically  induced  of  The  f u n c t i o n a l P elements.  found  genome, s c a t t e r e d  Because  P elements; the  by  inserted  A l l P s t r a i n s have been  strains lack  of  those  accompanied  insert.  and  presence  Reversion  invariably  30-50 p e r M  through  Rubin,  locus.  a l l of  were named P e l e m e n t s .  major  confirmed 1982;  the  unstable  most  the  that  elements  associated  elements  all  the  i n Engels,  shown  Based  events.  has  and  of  and  wild  that  (Green,  associated  loci,  Bingham, K i d w e l l ,  foreign  mutations the  hypothesis  were  to  state.  suggested  were the c o n s e q u e n c e  removal  (Collins  1983;  displayed  revert  More s p e c i f i c a l l y ,  genetic  or e x c i s i o n  1982;  they  the t r a n s p o s i t i o n of a m o b i l e  specific  upon  transposition Green's  from  mutations  into  Green  traits  associated  site  could  mutant  prokaryotes  resulted  single  stable  data,  dysgenesis  that  i n that  i n d y s g e n i c h y b r i d s were i n s e r t i o n  those  postulated  alleles  instability.  a l t e r n a t i v e l y , to  on  similar  of  white  over all M  elements  are  5  apparently strains M  non-mobile  produce  strains  The  and  system  M  Rubin,  All  1983)...  bp  reading  these  defective locus  appears encode  are  with  has  The  complete inverted  Incomplete  contain  not  the  open  r e a d i n g frames  a product required  reading  transposase these  of  function.  mutations  therefore the  frames  believed  and  the  In  kb  addition,  to  restore  that  a l l four  hybrid  (O'Hare  in  length,  P elements For  mutations in size  at from  0.5  element  pairwise this  reading  white to  so  product  i n any  function.  a " t r a n s p o s a s e " ( K a r e s s and  has  of the  abolish  are  i t  element  combinations  frames  1.6  long  Thus,  complete This  exist.  the  elements.  i n the  open  example,  are mobile only  complete  This  contains 4  Frameshift mutations  fail  p r o d u c t i o n of  i s 2.9  for transposition.  "transposase".  of  P  isolated.  deletions.  complete  P  the  element.  characterized  or d e f e c t i v e  P elements by  been  and  with  and  properties  repeats  associated  Incomplete  element  i n h y b r i d s between  element  internal  that  cytoplasmically.  of t h e  the v a r i o u s  been i s o l a t e d  accompanied  that  been t e r m e d  in  repressor  (O'Hare and R u b i n , 1 9 8 3 ) , r a n g e d  they  four  the  has  elements  kb i n l e n g t h . as  transposable  allows mobility  terminal  frames.  of  of  i t i s believed  accumulates  although a repressor  P element  31  that  the  absence  i s consistent  The  with  both  females  dysgenesis,  and  repressor  lack  repressor. males  a  i n the P s t r a i n ,  It  the of is  involved  Rubin,  1984).  6  As  noted  previously,  "repressor" The sites  function  sites  of  P  this  a  into  new  residence)  which  Lim,  The  1980).  insertion  site  it is can  now  well  elements  alter events two  the  are  the  of  A  influenced  P-mediated  by  by  the  exchanges). crossover  P  than  element  to  a  occurs.  mutation  is  transposable Male  known  to  Recombinant  only  one (due  studies are  and  recognition  factors  recovered  of  conserved.  unclear.  Several  the  preferred  insertion  1983).  a  insertion is  a  which  events  of  (Simmons  induced  those  and  traits  highly  frequently is  long,  contribute  remains by  specific  existence  which  (Engels,  c l u s t e r e d and  the  other  reflect  recognition  the  Although  mechanism  r e c i p r o c a l products  timing that  1983).  dysgenically  recombination  observed  evidence  might  exhibited  recombination  i s not  meiotic  premeiotic  The  p o s i t i o n at of  to  i n s e r t s i s 8 bp  controls  the  nature  cause  possible  1981).  location.  Rubin,  proposed  and  sequence f o r P element  that  understood,  recombination  non-random  e x i s t s , i t s sequence i s not  influence  Although  (thought  chromosome r e a r r a n g e m e n t s  and  likely  the  upon i n s e r t i o n o f  sequence  consensus  -GGCCAGAC- (O'Hare  are  of  possible.  Preston,  site-specificity s u c h as  been  P element  is duplicated  dysgenesis,  sequence  the  and  recognition  apparent  yet  analysis  rearrangements  chromosomal  particular  Thus,  not  e x i s t (Engels  sequence  into  has  similar  P-induced  element  "hot-spots" sequence  of  a  of  the  to  the  provide  symmetrical  7  exchanges Denell,  ( V o e l k e r , 1974;  1981;  products small  Sinclair  deletions  pattern  and  normally  residence  thus,  and  residence  of  rearrangement  elements  may  that  and  to  breaks  did  the took  not  DNA  not  Johnson  and  1985)  thus  the  of  1983).  may  be  the  related  an  and  Preston  breaks  to  the  the  Grigliatti,  i n the  place  in  verified In  disporportionate1y and  1977)  that  intervals  sites  of  that  the  centric  the  i t has  of  the  l e a d i n g to  high  levels  heterochromatin. whether  the  of  This  the  the MR  Therefore  1973;  centromere.  m a j o r i t y of  examined,  DNA  occur  heterochromatin.  case  P  events  recombinant  However,  b e c a u s e a p p r o p r i a t e l y marked each  the  1985).  recombinant  nearest  of  t e r m i n i of  h o m o l o g o u s chromosome, and  of  sites  (1984),  strand i n f i l t r a t i o n  (Sinclair  meiotic  extension  at  of  pattern  do  In  produce  the  The  as  to  pattern  reflect  to s t r a i n  Engels  strand  paired  suggestion  established  strain  The  not  o f s t u d i e s h a v e s h o w n ( H i r a i z u m i e t a1. ,  exist.  euchromatin  and  expected  not  (Engels,  i n host  Thompson,  be  does  elements.  recombination  preferentially  could  the  of t h e c l o s e l y  Woodruff  and  recombination  result  A number  led  from  hypothesis  suggested  precise  Isaakson,  duplications.  observed  differs  duplex  Grigliatti,  i s non-random,  recombination  been  and  1978;  of random b r e a k a g e w h i c h would be  recombination  of  Sved,  this  chromosomes  intervals  with  contained  both  it  recombination  could took  not  be  place  8 preferentially  i n one  Sinclair constructed dysgenic OKI,  and  or t h e  Grigliatti  second  induced  They used  and  rolled  ( r l ) to  is  entirely  involving  between  the  showed  heterochromatic  high  centric  in  contrast,  i n the e x p e r i m e n t over  patterns different Grigliatti  was  heterochromatin  that  attribute  between the s t r a i n s They a r g u e  that  recombination frequency  the  might  by  strain  result  low from  of the P r e c o g n i t i o n to i n s e r t  into  the  which  recombination  no  recombination T-007, OKI,  MR  in  but  in  However, i n  the  none JL_t  and  was and  regions actually j_l.  In  heterochromatic  low  frequency.  were s t r a i n  irradiation.  i n c i d e n c e of  and  Sinclair  and  to  differences elements.  heterochromatic  absence,  or  s e q u e n c e , or f r o m heterochromatic  The  specific  of r e s i d e n c e of P  the  T-007,  chromosome  specificities  i n the s i t e s  the e x t r e m e l y  of the P e l e m e n t steric  observed  obtained  h i 2,  of  markers l i g h t ( l t )  h-12,  at a very  specially pattern  strains:  between  involving but  of r e c o m b i n a t i o n from  of  heterochromatin  observed,  the  markers.  frequencies  occurred  a  in a l l strains.  3 of t h e 4 s t r a i n s ,  the  the  Male  observed  flanking  crossing  four P  heterochromatic.  experiments  each  by  examine  d e l i n e a t e a r e g i o n of  segments was  19 7 8  to  type.  utilized  the h e t e r o c h r o m a t i c  euchromatic  observed  (1985)  chromosome  recombination  1978.  other chromatin  from  the  a  low  inability  r e g i o n s due  to  constraints.  Chromatin  structure  can  influence  gene  activity.  For  9  example, t r a n s c r i p t i o n a l l y  active  chromatin  histones - especially  are  i n which  the  hyperacetylated  al.,  1979).  with  and  typical open,  The  (Davie  acetyl  thus  the  core  chromatin facilitates  DNA,  promoting  By  analogy,  influences sequences regions that  one  exist  o f t h e DNA  they  are  Therefore,  recombination condensed. alterations different  recombination been  occur  both  of t h e  be  Possibly  et  interfere  lead  in  to a  this  the more  "open"  polymerases  chromatin  to  former  may  be  so  inaccessible  expect  the  of  might so  may  P  elements.  any  dysgenic  i t is  highly is  that  allow  P insertion  alter  the Most  in heterochromatin although  condensed  hypothesis  recombination.  p r e v i o u s l y observed,  the  because  this  euchromatin,  highly  i f  also  recognition  and  to  little  structure  expected  structure  Thus, a l t h o u g h  genome and  induced would  may  heterochromatin  prediction  possible  that  heterochromatin  i n euchromatic One  in  i n chromatin  areas  this  H4  Levy-Wilson  interaction  b i n d i n g of RNA  insertion.  i n the  in  and  and  transcription.  might  One  dysgenically  had  the  H3  t h e h i s t o n e s may  structure.  essentially  one  1978;  histone-DNA  c o u l d argue  P element may  Candido,  structure  conformation thereby  and  g r o u p s on  weaken  nucleosome loose  core  genes are a s s o c i a t e d w i t h  changes  in  p a t t e r n of notably, where might  none also  intervals.  means  of  altering  chromatin  structure  is  10 through  treatment  induces  a  rapid  acetylated  and  n-butyrate. extensive  1978;  Cousens  e_t  appears  to  Candido  et  a l . , 1979; be  deacetylase  due  e_t  and  In a d d i t i o n  established  al.,  1974;  Prasad, and  have  Cox,  1980; 1977;  I and The  e t a l . , 1979;  malignant  I I , and  effects  In a d d i t i o n  HI  (Tralka 1975;  Tallman 1978;  to the  and  on  effects  cells on  that butyrate H2A  and  due  to  and  impair  can  and  1979).  butyrate  Griffen  et  1976;  e_t a l . , 1977;  Ghosh  Fallon  and  culture, inhibit  1979;  with  also  normal  Moreover,  nucleases (reviewed  Cox,  Butyrate  cells  in culture  cell  been  Sinha,  yielding  to  the  protein  Cserjesi,  characteristics.  more a c c e s s i b l e  butyrate  and  new  Hayman e t a l . , 1 9 8 0 ) .  micrococcal nuclease of  induces  Prasad  Moyzis,  biochemical  of  B o f f a e_t a l . ,  e_t a l . , 1979;  transformation,  and  have d e m o n s t r a t e d histones  and  effect  t o i n c r e a s e d r a t e s of  (Reeves  e_t a l . , 1977;  This  1978;  transcription  Leder,  Leavitt  chromatin  of  noted  and  Chou  morphological renders  been  Leder  Rubenstein reverses  patterns  of  1978;  o b s e r v a t i o n s , i t has  that butyrate treatment  the  Chalkley,  inhibition  Candido,  mRNA s y n t h e s i s i n F r i e n d c e l l s in  and  amount  e_t a l . , 19 7 8;  a l . , 1980).  to these  n-butyrate  the  Vidali  non-competitive  (Reeves  cells, in  Sealy  et a l . , 1978), r a t h e r than  acetylation.  treatment  HeLa  increase  a l . , 1978;  D'anna  to  enzyme  Vidali  Changes  In  h i s t o n e s ( R i g g s e_t a l . , 1977;  Simpson,  1978;  with  such by are  In  it  as  DNAase  Kruh,  1982).  reversible. vivo studies  p h o s p h o r y l a t i o n of  methylation  of  arginine  and  11  lysine et  residues  in  vivo  important  effects  position  other  nuclear  phenomenon r e l e v a n t  of  butyrate  effect  Position  from  i t s  others. is  Often  Most  i n the  proteins  euchromatic  gene may  (Boffa  in  of  cases  or  several  detectable,  breakpoint  are  Goldstein, locus  i s an  result  of  any  shown  by  Spofford, In  most  1967).  mosaic to  a  active  In  likely  have  loci to  be  permanent  event.  That  a l t e r a t i o n or  recombination  is  of  and  not  to  involves a  activities  the  of  rearrangement (Hartmann-  variegation mutation  a  centric  i n a c t i v a t i o n of  experiments  gene  variegate,  system  the  a  clonally  to  inactivated  In a h o m o z y g o t e , the  independent  of  variegating  and  found  to  of  piece  a  juxtaposed  closest  in  that  i n some c e l l s  where  of  melanogaster.  broken  been  cases  is  expression  a variegating  genes  segment.  are  expression  development  examined  heterochromatic loci  discussion  t r a n s c r i p t i o n a l f a t e of  loci  gene  be  the  Drosophila  i s the  early  majority  in  proximity  the  determined  propagated. and  The  to  gene  variegation  heterochromatin.  gene  on  effect variegation  resulting  be  and  a l . , 1981). An  in  histones  is  i n the  (for  each  not.the gene  review,  can see  1976). many  inactivation  respects, in  "heterochromatic  variegation  mammals.  It  is  spreading"  (first  resembles thought observed  to by  X-chromosome result Demerec  from and  12 Slizynska,  1937):  the  heterochromatinization from  in individual  between  cytological  which a p a r t i c u l a r of  mosaicism  condensation  of the c h r o m a t i n ,  the rearrangement  extent  progressive  breakpoint cells.  extending  to a  There  greater  is a  or  distally  or  lesser  good  correlation  o b s e r v a t i o n s on t h e n u m b e r  of c e l l s i n  l o c u s i s h e t e r o c h r o m a t i n i z e d and t h e d e g r e e  i n an i n d i v i d u a l ' s  phenotype  (Hartmann-Goldstein,  1967). It sodium in  w  has  been  shown  (Mottus  butyrate e f f e c t i v e l y individuals  m 4  reductions  in histone  variegation. present  and  Thus  zation)  suppresses (Moore  gene  to the extent  i n the  variegation  of  1983)  also  number  that w  +  that  suppress  of  alter  histones chromatin  (heterochromati-  i s affected.  In  summary,  changes  which  butyrate i s associated  are thought  to r e s u l t  structure.  In  addition,  inactivation  of  euchromatic  breakpoint. inability region  ^ t a l . , 1979,  o f h i s t o n e s f o r DNA that  1982),  the i n a c t i v a t i o n  multiplicity  reductions  or i n t h e a f f i n i t y  structure  (it a l . , 1980,  It i s believed  genes  that  of h e t e r o c h r o m a t i n  of  this  heterochromatin,  used  near  to a  results  over  in this  of  study  chromatin  suppress  the  of i t s c h r o m a t i n action  chemical  the  heterochromatic  effect  to "spread"  suspected  i t was  this  certain  in altered  i t i s known  (due t o t h e l o o s e n i n g  Because  with  from  the  euchromatic structure).  butyrate to t r e a t  upon  dysgenic  13  hybrids  of  two  hypothesized  P  strains.  steric  heterochromatin  If  constraints  one  would  butyrate  reduces  upon P e l e m e n t  expect  to  observe  the  insertion in  heterochromatic  male r e c o m b i n a t i o n as a r e s u l t of the b u t y r a t e t r e a t m e n t . C h e m i c a l s added produce  multiple  altered  t o the c u l t u r e medium of any  effects.  chromatin  In  structure  Variegation  s u p p r e s s o r genes,  assembly  maintenance  or  opportunity way,  to  without  disrupt the  exogenous c h e m i c a l Using isolated  w^  51  melanogaster. on  chromosomes  different  2  perhaps enzymes  thought  effects  t o be  butyric  acid,  one  of  them.  involved  i n the  structure,  which  as, an a s s a y ,  in a  may  be  offer  more  the  precise  produced  Sinclair  suppressor  by  e_t a l . (1983)  ( S u ( v a r ) ) and  m a j o r i t y of t h e S u ( v a r ) genes a r e and  3.  Su(var)  systems.  mutants Because  systems, involved  s t r u c t u r a l , non-histone assembly,  clustered  suppress of  this  chromosomal or  several apparent  i t i s argued  i n chromatin  modification  3  i n D £ £.s £ j) h. i^ 1 a.  (En(var)) mutations  o f S u ( v a r ) genes a r e  for  of  only  structure  variegation  variegating  as  be  chromatin  g e n e r a l e f f e c t on v a r i e g a t i n g products  case  may  treatments.  enhancer The  may  chromatin  side  dominant  variegation  of  variegation  m  the  organism  that  the  structure;  proteins  maintenance  or of  chromatin. One  of  the  clusters  of  Su(var)  genes  on  the  third  14  chromosome i s l o c a t e d (Henikoff,  1979).  suppression that  of  Deletion  the  There  i s some  code  products  associated  level  s u p p r e s s i o n of t h e  i n which  level  of  heterochromatic Also, that the  presence  Biochemical  variegating  (DNAase least that  observed  above  gene  absence  of  a  could  specifically  then  type  the  For  i s absent,  nucleosome the  the  i s substantially heterochromatic; very  participate in  often  the  locus.  indicates  euchromatic  suppressor  alters  m  chromatin  mutations. that  structure  s p a c i n g ) and  chromatin  in  that  structure  at to  flies.  suppressor be  used  that  of  mutation  to a l t e r  g e n e r a l e f f e c t of  butyrate),  the  possible  effects  without of  such  chromatin  heterochromatin,  the  side  normally  in preparation) indicates  w^  and  of  or  heterochromatin.  i s more  genes r e t u r n  for wild  principle,  described (perhaps  gene  suppressor  genes  (Hayashi, unpublished)  (Ruddell,  sensitive sites  some  In  i n the  w  these  entirely  can  in  suggest  i n a c t i v a t i n g the v a r i e g a t i n g  white  evidence  results  hypomorphs  Y chromosome  which  spreading,  than  be  87B-E  i t s a b s e n c e f r e e s up an o t h e r w i s e  product  variegating  may  Su(var) mutations  c y t o l o g i c a l evidence the  the  the  sites  e_t a l . t h e r e f o r e  with  Y chromosome i s a l m o s t  the i m p l i c a t i o n i s t h a t limited  these  i n d i c a t i o n that  i n males  The  p a i r of s i t e s a t  mutations  instance  reduced.  of  Sinclair  Su(var)  amorphs.  of  an u n u s u a l  of v a r i e g a t i o n .  many  for  near  the  chemical  as  those  structure  i n contrast  to  complication  of  treatments.  15  Accordingly,  d y s g e n i c and  non-dysgenic  as i n the b u t y r a t e e x p e r i m e n t s third  chromosome  Su(var)  their  s u p p r e s s i o n of  c r o s s e s were  w i t h the a d d i t i o n  genes  variegation.  chosen  for  the  performed  of one  of  strength  two of  16 MATERIALS AND  Culture  conditions:  Flies shell  were  vials  Tegosept  reared  on  a  suppress  or  food  concentration maximize  the  delay  butyrate of  the  at  22°C.  (OKI  of  150  effect  and  mM. on  adjusted  marked  inbred  the  strains  used 1971;  laboratory in  (10  Where n o t e d ,  which  t o 5.5.  with  food either  each)  some c r o s s e s  was  added  to was  structure  while  i s observed  as  1979).  the  mg/1  concentration  chromatin  were:  In a l l c a s e s , A l l crosses  the  T-007/CyO  Hiraizumi  i n Oklahoma,  highly  used  dram  to  were  a  final  used  to  minimizing a  result  the  were  final  of pH  performed  chromosomes:  P-strains  Hiraizumi,  8  medium.  in  of a m p i c i l l i n  was  This  (Mottus,  b o t t l e s or  included  butyric acid  development  was  isolated  P  which  treatment  food  P-Strains  Texas,  in  to  was  A combination  growth.  milk  sucrose-cornmeal-agar  tetracycline  bacterial  on  The  pint  (methyl-p-hydroxybenzoate)  streptomycin  raised  in half  standard  as a mould i n h i b i t o r .  the  METHODS  Woodruff  strain control  (T-007  e t a l . , 1973) and  Oregon  Thompson, R  crosses  was  used  (Oregon  isolated and  OKI/OKI  1977).  The  i n place R  in  is  an  of M  strain). The  strain  used  to  monitor  recombination  was  an  M  strain  17 bearing 1.3),  the  second  black  narrow  (b,  48.5),  wing-dominant  107.3).  For  phenotypes, and  chromosome:  a see  Grigliatti,  divided  into  interval  light (n.w  Grell  and  The  (see  and  entirely  euchromatic.  culture  as  This  2R)  CyO  and  2  3)  and  was  the a d d i t i o n  of a t h i r d  the  gene S u ( v a r ) 3 2 5 ,  325  and  318  (described  in Sinclair  both are  maintained  in  referred  et a l . ,  the  5  multiply  hereafter  be  entirely  1 and  inverted  t o as  chromosome homozygous  or Su(var)318;  can  span  The M s t r a i n u s e d i n t h e s u p p r e s s o r c r o s s e s was with  ,  their  that  is 4  y*.  Sinclair  Note  intervals  the  (hereafter  and  also  1).  chromosome with  55.1),  chromosome  number  intervals  heterozygote (2L,  marked  Figure  euchromatin,  (rl,  mutations  (1968; see  r\L ( i n t e r v a l  whereas  rolled  position  Pin-yellow t i p (Pin  these  multiply  intervals  heterochromatic,  In:  and  Lindsley  b e t w e e n Jl_t and  chromosome:  , 83.0)  (JS, g e n e t i c  55.0),  of  five  a  (l_t,  description  1985).  heterochromatin  TJ  Star  CyO).  t h e same, for  either  referred  to  as  1983).  Genetic crosses: The crosses (a) were  procedure i s outlined  Standard crossed  bottles  the  on  to  untreated  and  butyrate  treated  in Figure 2(a).  recombination  Approximately milk  for  experiments:  multiply  25 f l i e s normal  of each f o o d and  marked sex  T-007 M  or  strain  were p l a c e d i n  allowed  t o l a y eggs  OKI  males  females. half-pint for three  18  K  3 C  H 4b  1.3 2  Figure  1.  M u l t i p l y marked experiments. a b c  Pin  nw 83.0  .Yt  1  5  a  107.3  b  second  chromosome used  i n t e r v a l e n t i r e l y euchromatic i n t e r v a l spans b o t h e u c h r o m a t i n and heterochromatin i n t e r v a l e n t i r e l y heterochromatic  in a l l  19  Untreated and butyrate treated crosses P  S b It rl nwD PinYt ^ ^ ^ CyO  T-007 CyO  oV  m or OK1 OreR 0  o r  r  e  R  Half the parents lay eggs on standard medium,the other half on butyrate treated medium II  F2  B  S b It rl nwD Pin* T-007 or OK1 or OreR Progeny scored for recombination on chromosome 2  Suppressor crosses P  FI  F2  Figure  S b It rl nwD PinYt CyO  Su(var) Su(var) H  V  S b It rl nwD PinYt _ Su(var) * * T-007 or OK1 or OreR ' + ^ O  CyO  bJUL It rl Itrl  OK1  OreR  $8 T  T  Progeny scored for recombination on chromosome 2  2.  M a t i n g scheme used i n u n t r e a t e d and b u y r a t e t r e a t e d (A) and S u ( v a r ) (B) e x p e r i m e n t s . Except where n o t e d , a l l c r o s s e s were r a i s e d on s t a n d a r d medium.  a  O  20  days.  The  allowed  parents  to  l a y eggs  discarded. strains. second were  At l e a s t  transferred  f o r three 5 bottles  collected  females.  In each i n 8 dram  then  back  3 males  shell  vials.  milk  5 more d a y s .  three  replicates  cross.  Progeny  for a total from  on t h e second  parents  were  introduced  control  f o r these  except  that  strain  were  (b)  into  the  of a P s t r a i n ,  10  _r_l  virgin 50  t o new  vials  were t r a n s f e r r e d t o  of at l e a s t  chromosome  _b l_t  at l e a s t  to continue  crosses  strains  to l a y eggs  twice  to provide  150 v i a l s were  examined  f o r 21  bottles  males  f o r each  days  or  from  after  vials. was  for the As  a  followed  the Oregon R  used.  Butyrate  used  females  marked  t o l a y eggs f o r  c r o s s e s , t h e same p r o c e d u r e  instead  recombination  these ^experiments those  cross  was r e p e a t e d  these  recombination  with  then  of the P  the P  transferred  and a l l o w e d  This process  mated  and  were  virgin  were a l l o w e d  The o r i g i n a l  bottle  to  For each  d a y s and t h e m a l e s w e r e t h e n  for  The p a r e n t s  were  three  a half-pint  and  from  crossed  case  females.  bottles  the m u l t i p l y  chromosome  w e r e s e t up.  10 new  days,  bearing  vials  with  fresh  w e r e s e t up f o r e a c h  and a second and  to  more  F l h y b r i d male p r o g e n y  chromosome  females  were  were  experiments:  obtained  i n the a f o r e m e n t i o n e d  were p e r f o r m e d  essentially  from study.  The  t h e same  parents f o r cultures  The b u t y r a t e  as d e s c r i b e d above,  except  as  crosses that the  21  FI  individuals  were  grown  on  medium  containing  butyrate  (150mM).  (c)  Suppressor  experiments:  The  protocol  c r o s s e s i s g i v e n i n F i g u r e 2(b). tested  for their  described  effect  above.  Oregon R males marked second  on MR  In t h i s  one  of  two  of the group cross.  was  Thus,  same c u l t u r e s  possible  the  for  propagated  as  used  control)  b e a r i n g the  multiply  mutations:  third  divided  in half  i n the 3 2 5 c r o s s  t h o s e used  either  p o p u l a t i o n of e i t h e r  the p a r e n t s i n t h e 325  exchange  the  through Both  progeny,  or  product  may  f o r t h e 318  large be  observed.  represented  single  individual  occur  of  subsequent  numbers  is  events  products  p r o d u c t s of  exchange F2  or ( f o r a  and  the P  so t h a t the  one  other i n  c r o s s e s were  from  crosses.  of the d a t a :  Because  meiosis.  to those  Su(var)  Each  o r t h e O r e g o n R m a l e s was  Analysis  analogous  t  or S u ( v a r ) : 3 1 8 .  the 318  were  c h r o m o s o m e S_ b_ lt_ r_l j i w ^ P i n ^ / C y O and a  Su(var):325  the  procedures  were c r o s s e d t o f e m a l e s  carrying  half  suppressor  Su(var) mutations  c a s e T - 0 0 7 , OKI  chromosome  strain  by  Two  f o r the  a  single  mitotic  exchange of  may  progeny Most  in  one  premeiotically,  of  the  to  be  prior  to  r e p r e s e n t e d i n the  b e a r i n g one  often,  bears a s i n g l e  exchange  divisions be  i t is  a  single  or  other  product  offspring.  product  the  When  of exchange,  of a  i t is  22 clear  that  taken  one exchange e v e n t  place.  either  When  singly  product  or  both  has  been  more  number  one  study,  males  which  In  prior  from  of  t h e same  the number  done by c o n s i d e r i n g  In  at least  of a s i n g l e  i n groups  each  of s i n g l e each  estimated  given the t o t a l  This  a l l o w s some  each  interval.  falls event  that  there  the  data  t o know t h e  t h e number  of  directly  o f one P e l e m e n t .  Because  (high  experiments  levels  made  i t i s impossible of  recombinant  I t was events  cluster  mating to  tell  progeny  therefore necessary  which  which  of  occurred.  This  o c c u r r e d t o be t h e  event.  interval  single  single  one  T a b l e s 1-4, t h e number o f e s t i m a t e d s i n g l e  shown f o r each  a  of w h i c h ,  cluster  parent.  than  of t h r e e  to these  a  interest  individuals  impractical),  a l l members  estimate  product  of  analysing  i t was o f l e s s  place;  observed,  possibilities  that  exchange.  took  mated  observed  originated  was  were  individually  whether  are  groups  of t h e s e  i t i s clear  of r e c o m b i n a n t  events  F l males  sterility  is  large  o t h e r w i s e , r e p r e s e n t s the a c t i o n  the  to  than  i n this  recombinant or  products  p l a c e , but t h e r e i s a s m a l l p o s s i b i l i t y  generated total  or  (either  under t h e t e r m . " c l u s t e r " ) , taken  reciprocal  i n groups,  are observed  has  ( i n t h e r e g i o n s of i n t e r e s t ) has  on chromosome two as w e l l  events. number  On t h e l i n e  individuals  e s t i m a t i o n of the degree section,  as t h e t o t a l  below, i n parentheses,  of recombinant  In t h e r e s u l t s  events i s  observed.  of c l u s t e r i n g f o r  t h e number  and p e r c e n t  23  of  clusters  was  are  included  treatments exchange  The  of  (that  minor  of  experiment. indicate  had  any  would  single  identical  i s , from of  two  significance.  This  whether  effect  on  expected which events  the  information  the  butyrate  timing  t o show  up  separate  of  the  in  the  were c l u s t e r e d . is  of two  The  necessarily  p r o p o r t i o n of c a s e s , a  products  separate  parents),  but  an  cluster  exchange this  is a  error.  were a n a l y s e d  ( S o k a l and  be  events  since i n a small  source  data  G test  i t may  which  number  c o n t a i n the  very  each  of r e c o m b i n a n t  underestimate,  the  because  events,  estimated  events  for  or s u p p r e s s o r s  proportion  may  given  Rohlf,  for statistical 1969)  at  the  significance  standard  p=.05  using level  24 RESULTS  (a)  Standard  recombination  Meiotic  recombination  melanogaster general  males t h a t  absence  experiments  does  lack  for _b  the m u l t i p l y  and t h e i r  marked  virgin  recombinants  in  Drosophila  To d e m o n s t r a t e  this  o f m e i o t i c r e c o m b i n a t i o n , O r e g o n R m a l e s (M  chromosome  l._t r_ 1  occur  P elements.  s t r a i n ) were mated w i t h f e m a l e s second  not  male FI o f f s p r i n g ,  second  chromosome  females.  among  b e a r i n g the m u l t i p l y  As  t h e 7,919  F2  were  expected, offspring  marked  heterozygous backcrossed  there  examined  were  to no  ( d a t a not  shown). The  male  experiments substantial resulting  recombination are  presented  recombination  from  crosses  frequency  of r e c o m b i n a t i o n .  among  among  t h e OKI  and of  each  was  observed  produced  frequency  of  t h e chromosome  given i n Table  1.  both  cases,  among  male  hybrids  of  1.15%, whereas  that  f o r t h e T-007  The T-007 and  characteristic  only  and  0.74  OKI  p a t t e r n and  recombination the frequency  percent.  occurred OKI  a n d 0K_l  In  The f r e q u e n c y  m a l e s was  recombinants  1.  matings.  a  t h e T-007 m a l e s was strain  f o r t h e T-007_  i n Table  P male and M f e m a l e  untreated  events  data  The  i n each  experiments  number  interval are  also  25  Strains  1  T-007 n u m b e r  per cent  .216  number  per cent  3  1.  Interval 3  33 (67)  Total 34 (82)  0  .285  13 (49)  0  .178 (.292)  .165 (.621)  0  241  224  24 (34)  20 (63)  1.15 (2.18)  .207  1.000  11 (18)  58 (152)  .254 .140 (.786) (.228) .345  Flies Scored  116 10,087 (220)  .337 .238 (.813) (.337) .293  14 (23)  3  distri bution b  Table  25 (27)  .248 .327 (.367) (.664)  3  distribb tion OKI  3  Genetic 2  7,885  .736 (1.93) 1.000  . 190  R e c o m b i n a t i o n data, i n each g e n e t i c i n t e r v a l , f o r c o n t r o l d y s g e n i c c r o s s e s i n v o l v i n g P s t r a i n s T-007 and OKI. a  data f o r recombination events total recombinants given in " M a t e r i a l s and Methods").  b  data  based  on r e c o m b i n a t i o n  given f i r s t parentheses  events  only.  with (see  26  Sinclair strains  they  and  Grigliatti  (1985) found  e x a m i n e d , T-007 was  the  that  most p o t e n t  MR.  T h i s study c o n f i r m s t h i s o b s e r v a t i o n .  OKI  strain  pattern and  in  and  Grigliatti. the  thirds  that  In  study  was  Sinclair stock not  of  i n the  the f a c t  that  the  variations  induced not  events  by  may  have  variations.  other In  any  T-007 were s i m i l a r always  containing  would case,  f o r both  importantly,  in  as  the  euchromatin  f o r both s t u d i e s ,  my  of  clear  why  2  to  trends  The  as  4;  no  can this  used  by  i n the  culture  was  such  as  recombination one  P  these  strain slight  observed  the  for of  MR  intervals  heterochromatin.  t h e r e was  in two-  in  highest levels  and  and  Alternatively,  levels  general  and  used  the c u l t u r e  sensitive  this  this  conditions,  the  intervals  well  Perhaps  population.  studies.  in  consistently  to found  i t i s not be  frequency  be some v a r i a t i o n  laboratory  influenced  observed both  entire  both  of S i n c l a i r  study.  p r o p o r t i o n used  in  was  of  in  observed  t h e T-007 c u l t u r e  T h e r e may  the  The  interval  bottle  i n d u c e r of  identical  that  observed  a single  T-007, a l t h o u g h  the  from  P  data f o r the  events.  previous  that  Grigliatti.  temperature,  were  recombinant  r e p r e s e n t a t i v e of  small  and  every  d e r i v e d from  such  The  f o r t h e T-007 s t r a i n difference  observed  and  nearly  major  frequency  be e x p l a i n e d by  are  of r e c o m b i n a n t  events  s h o w e d one  total,  studies  frequency  p a t t e r n of  study  both  of the f o u r  Most  heterochromatic  27  recombination addition, events  a comparison  similarity  number  studies taken  practically  a s an i n d i c a t i o n  Despite characteristic  important  heterochromatic In both  total  recombination  2 and  4.  and  While  of  number  3;  Butyrate  a substantial  To t e s t  t h e p a t t e r n was  recombination  the e f f e c t  on the  may  be  60%)  a  there  The that  the only  most no  wholly  or the  OKI  p r o p o r t i o n of the  occurred  adjacent  somewhat  induce  i s  t h e T-007  was  strains.  this  each  above)  t h e m a j o r i t y of t h e r e c o m b i n a t i o n  two  Since  t h e two.  and  intervals,  based  recombination,  euchromatin  two  (b)  P strains  in either  a r e both  of both  much  frequency.  noted  (approximately  These i n t e r v a l s  are comprised  t h e two  in interval  strains,  on  the c o r r e c t e d data r e p r e s e n t the  (as  interval,  that  observed.  s i m i l a r i t i e s between  occurred  In  showed  i n procedure,  and f r e q u e n c y  observation  recombination  strains.  identical  that  pattern  some i n t e r e s t i n g  s t u d i e s than  of r e c o m b i n a t i o n  the f a c t  strains.  f r e q u e n c i e s based  individuals  that  the  for clustering)  the two  of r e c o m b i n a n t  were  of  of r e c o m b i n a n t  between  more a c c u r a t e e s t i m a t e  were  for either  (data c o r r e c t e d to account  greater the  observed  in  to the  intervals centromere  heterochromatin. observed  different  i n these  between t h e  experiments  of b u t y r a t e on male  recombination  the  28 same c r o s s e s as i n the u n t r e a t e d s e t were p e r f o r m e d hybrid been  males  were r a i s e d  added  butyrate had  no  to  a  i n the  effect  discovered crosses. does n o t  absence male  among  10,462  F2  the  In  recombination to  1.49%  importantly, (.048%) was  T - CJ CJ _7  (G was  a low,  observed  but  Rohlf,  in  this  1969).  interval  Recall in  that  the  absence  (intervals  4,  and  showed  relative  to  of  1 and  5)  comparable 2 (on t h e  treatment.  premeiotic  the  the  of  left  Finally,  exchange  a  events  the  the  male  and  OKI  overall from  pattern  of  of  Most  G-test,  i s never In  This Sokal  observed addition,  chromosome  two  significant On  the  other  w e r e u n a f f e c t e d by  small as  alone  interval.  levels.  arm)  R  recombination  a statistically control  Oregon  the  butyrate. arm  were  treatment.  (using  right  cross)  significantly  level  of  R  T-007  2),  recombination  on  butyrate  the  butyrate  significant  exchange  hand, i n t e r v a l s  in  f o r the h e t e r o c h r o m a t i c  T-007-induced 3,  altered  (Table  perceptible  had with  butyrate  Moreover,  by  the  FI  recombination.  increased  p=.05).  altered  is statistically  and  cross  of  male  occurred  Oregon  that  treatment  frequency  test,  of  acid  recombinants  progeny  levels  which  (event)  recombination  number  No  the  Treatment  ( i n the  recombination.  butyrate  crosses.  the  150mM.  P elements  perceptible  contrast,  increase  of  T h e r e f o r e , i t c a n be c o n c l u d e d  events  effect  of  the  recombination  1.15%  mediume t o w h i c h b u t y r i c  concentration  on  induce  In  on  and  increase  shown  by  a  in  the  slight  29  Genetic 2  Strains T-007 number  per cent  number  per cent  b  30 (45)  48 (77)  7 (7)  4  74 (221)  Total 56 (87)  .208 .332 .048 .512 .388 (.311) (.533) (.048) (1.53) (.602)  b  distribtion OKI  b  Interval 3  0  .140  223  13 (22)  34 (78)  .077 (.131)  distribution  033  4 (4)  .344  261  50 (106)  17 (44)  .202 .024 ,298 (.464) (.024) (.631)  110  .288  .034  .424  ,101 (.262) .133  215 (437)  Flies Scored 14,453  1.49 (3.02) 1.000  118 (254)  16,803  .702 (1.51) 1 .000  0  Table  2.  R e c o m b i n a t i o n data, i n each g e n e t i c i n t e r v a l , b u t y r a t e t r e a t e d dysgenic crosses i n v o l v i n g P s t r a i n s T-007 and OKI. 3  v  a  . 15M  b  data f o r recombination events given f i r s t with t o t a l recombinants given i n parentheses (see " M a t e r i a l s and Methods").  b  data  based  on r e c o m b i n a t i o n  events  only.  for  30 increase  i n the  untreated  (or  recombination 25%  experiment  78  corresponds  In of  of of  to  the  percent  recombination (about  the  events 0.8%  by b u t y r a t e t r e a t m e n t . pattern  of  Most  amount  recombination  heterochromatin significant whereas  notably,  of  events  the u n t r e a t e d c r o s s e s . percent  of  clusters  untreated,  or  recombination  exactly  clustered  of  One  of  the  118  events a  events  by  was  small  3.  which This  4  strain  remained  OKI  and  untreated  crosses)  t h e T-007 s t r a i n ,  altered  by  exposure  the to  within  the  centromeric  contrast,  there  frequency 5,  at about no  the the  for  was  a  region  1,  frequency same l e v e l  of  as i n  a p p r e c i a b l e change i n the  i n response  to  experiment,  20  same  frequency  significant  and  was  overall  but  were c l u s t e r e d the  butyrate  (0.24%),  In  exchange  There  the  This  observed.  the  the  occurred  2,  116  clusters. In  the  the  were c l u s t e r e d ,  treated  remained  standard, events  the  of  In  significant.  both  produced  experiment, (40  in  intervals  recombination  of  region  decrease  for  as  However, as w i t h  butyrate. of  observed  induced  recombination  29  observed.  T-007 s t r a i n ,  for  observed.  appeared  events  events  with  was  experiment,  statistically  contrast  unaltered  clusters  observed  215 36  i s not  of  standard)  events  represents  increase  percent  (34%).  percent  butyrate. of  58  In the  In  the  observed butyrate  of  the  events  of  butyrate  was  events).  most  important  effects  is  the  31  occurrence  of r e c o m b i n a t i o n  i n the heterochromatin,  has  n o t p r e v i o u s l y been o b s e r v e d .  and  OKI,  a l l of t h e h e t e r o c h r o m a t i c  were s i n g l e , the  u n c l u s t e r e d events.  c r o s s e s , an a v e r a g e  degree  and  of t h e h e t e r o c h r o m a t i c  the euchromatin,  could have  clusters  euchromatic (c)  the t i m i n g  i s t h e same as t h o s e crossovers  the s m a l l sample difference  would  The a b s e n c e o f  size,  o r i t may  be an  i n h e t e r o c h r o m a t i c vs.  over.  Su(var)325 Preliminary  3 2 5 mutant Kroitsch, basic  may  which  important  dysgenic variegation may  experiments  suggest  meiotic crossing  1982, p e r s o n a l c o m m u n i c a t i o n ) .  determine  there  mapping influence  processes  probably to  that  of r e c o m b i n a n t s .  important  crossing  o f 3 2 % c a n be  t o be p r e m e i o t i c and a s s u c h  as a c l u s t e r  o f an  events  observed  f o r a l l of  4 o f t h e 11 h e t e r o c h r o m a t i c  may r e f l e c t  indication  on t h e d a t a  and i f i t i s assumed  have been e x p e c t e d appeared  o f b o t h T-007  events  of c l u s t e r i n g  Given  nature  this,  exchange  Based  obtained.  of  In the case  where i t  occur  crossing  the  over.  and b u t y r a t e have be some  i n meiotic  suppressor Also,  similarities  over  many  of the  over  are  i t was of i n t e r e s t  325  effect  i n their  ( T e j a n i and  crossing  since  t h e same  the Su(var)  Since  f o r male r e c o m b i n a t i o n ,  whether  that  could  influence  suppressors on  effects  of  variegation, on  chromatin  32 structure. can  Of  cause  the  key  interest  heterochromatic  mutant  has  recombination elements.  a  analogous  the  recombination  effect  marked  males  second  does  in  might expect  acts  similar  are c r o s s e d to M  (data  not  were o b s e r v e d shown).  and  recombination, exchange  Su(var)325  effects  backgrounds resulting  of  levels  dysgenic  different  in  total,  only  promote  bearing on  the  either  no  examined  _3 2__5  meiotic  the  third  cross),  progeny  effect  i n t r o d u c i n g .3 2J5 and  or  OKI  are  distribution  males  bearing  those  Furthermore,  has  or  on  somatic  altered  of  the  into  shown  the  in  dysgenic  Table  3.  p a t t e r n s of r e c o m b i n a t i o n 325  are  not  percent  (37  clustered  of  clusters  events  in  significantly  Xz.9_ _0 _7 c o n t r o l  the  The  of  (standard) observed 125  is  observed  30%).  Similarly, the  of  and  significantly  cross,  not  T-007  from  experiment. not  i t does  whatever  manner  crosses.  of t h i s  7,576 F2  P  i n males.  The  T-007  i n the  Thus,  a  of  alterations in  females  chromosome ( s e e F i g u r e 2(b) f o r a d i a g r a m recombinants  in  If male  absence  only i n dysgenic  chromosome  genes  butyrate.  the  i f 3^ _2_5  p a t t e r n or f r e q u e n c y  suppressor  recombination,  even  hand,  one  as  on  observed  other  When Oregon R multiply  be  to b u t y r a t e ,  i s whether  recombination  general  might  On  here  when 325  effects  significant  on  MR  i s introduced into are  only  difference  in  slight. regional  t h e OKI For  dysgenic  example,  the  recombination  33  P Strains  1  T-007 n u m b e r  per cent  3  distribtion  Interval 3  38 (52)  0  .312 .383 (.403) (.524)  0  3  31 (40)  Genetic 2  4  38 (58)  Total  18 (31)  .383 .181 (.585) (.313)  248  304  0  .304  144  35 (58)  68 (106)  0 0  85 (141)  24 (33)  Flies Scored  125 (181)  9,921  1.26 (1.82)  1.000  b  OKI  number  3  per cent®  .156 (.258)  .303 (.472)  . 165  321  distribution^  Table  3.  0  .379 .107 (.628) (.147) .401  .113  212 (338)  22,448  .944 (1.51) 1.000  Recombination data, i n each g e n e t i c i n t e r v a l , f o r d y s g e n i c c r o s s e s i n v o l v i n g P s t r a i n s T-007 and OKI, to w h i c h t h e mutant S u ( v a r ) 3 2 5 had been i n t r o d u c e d . a  data f o r recombination events given f i r s t , with t o t a l recombinants g i v e n i n parentheses (see " M a t e r i a l s and Methods").  b  data  based  on r e c o m b i n a t i o n  events  only.  34  frequency  that  was  males,  that  for interval  was  affected  by  the  events  observed  events.  Taken  the  _3 2_ 5_  presence  together,  mutation  heterochromatic  2.  of  3 2 5.  Seventy-one  control  of  results  clearly no  212  of  indicate  effect  significantly,  i s not  the  w h i c h r e p r e s e n t s 34%  virtually  Most  v_s.  Here a g a i n , c l u s t e r i n g  these  hsa  experimental  i t  on  the that  male  induces  no  recombination.  Su(var)318 Preliminary  mutant  325,  7,358 F2  shown). on  and  OKI  data  The  failed  progeny results  are  to  318  d i d not  (13,662  flies  were  observed represents  For  any  no  indication  meiotic crossing  the 3 2 5 m u t a t i o n . produce  of the  any  male  experiments  4.  significantly scored)  T-007  that  over.  As  was  the  Itthus true for  recombination  among  c r o s s ( d a t a not  to t e s t  the  effects  of  i n the d y s g e n i c b a c k g r o u n d s of T-007  i n Table  clustered  were  gave  of the Oregon R c o n t r o l  presented  experiments. events  with  male r e c o m b i n a t i o n  that  T-007  studies  influences  a contrast  Su(var)318  318  mapping  Su(var)318  provides  the  for  were c l u s t e r e d ,  recombination.  (d)  observed  48  of  or  clustered significant  (33%).  alter OKI  the  (31%).  I t can  50  Neither from  from  flies  observed  OKI,  difference  seen  recombination  (18,676  155  For  be  of  the  i n the scored)  recombination of  151  events  these  values  control  levels.  35  P Strains  Genetic 2  T-007 n u m b e r  per cent  3  distribtion  3  35 (49)  Interval 3  43 (86)  Total  49 (86)  .256 (.359)  .315 (.630)  .226  .277  0  29 (40)  .359 .212 (.630) (.293) .316  .187  Flies Scored  155 13,662 (261) 1.14 (1.91) 1.000  b  OKI  number  3  per cent  8  42 (58)  0 0  123 (.257)  .225 (.311)  0  152  278  distribution*  Table  4.  23 (48)  63 (137)  23 (27)  .335 .123 (.734) (.734) .417  152  151 (270)  18,676  .809 (1.45) 1 .000  R e c o m b i n a t i o n data, i n each g e n e t i c i n t e r v a l , f o r d y s g e n i c c r o s s e s i n v o l v i n g P s t r a i n s T-007 and O K I , t o w h i c h t h e mutant S u ( v a r ) 3 2 5 had been i n t r o d u c e d . a  data f o r recombination events given f i r s t , with t o t a l recombinants given i n parentheses (see " M a t e r i a l s and Methods").  b  data  based  on r e c o m b i n a t i o n  events  only  36 Figures second  chromosome  distribution crosses map in  3 a n d 4 ( T - 0 0 7 and OKI, derived  of r e c o m b i n a t i o n  from  data  events  female  figure,  on t h e  of  i n d i c a t e s any r e g i o n w h i c h shows a s i g n i f i c a n t the that and  control  dysgenic  there i s very either Thus,  little  suppressor these  cross.  difference  MR  figure,  between  A genetic  An  provided asterisk  difference  from  i t c a n be  seen  the c o n t r o l  cross  cross.  experiments  on h e t e r o c h r o m a t i c  In each  dysgenic  datais  comparison.  the  proportional  treatments.  meiotic recombination  f o r purposes  show  for control  and t h e b u t y r a t e and s u p p r e s s o r  d e r i v e d from each  maps  respectively)  that  have  revealed  are analogous  no  Su(var)  to those  effects  of b u t y r a t e .  37  FIGURE 3.  Second chromosome maps d e r i v e d from recombination d a t a of d y s g e n i c c r o s s e s i n v o l v i n g t h e P s t r a i n T007. The u p p e r l i n e i s d e r i v e d from f e m a l e m e i o t i c r e c o m b i n a t i o n d a t a , and i s i n c l u d e d f o r c o m p a r i s o n . An a s t e r i s k i n d i c a t e s i n t e r v a l s w h i c h e x p e r i e n c e l e v e l s of r e c o m b i n a t i o n s i g n i f i c a n t l y different from c o n t r o l s ( s e c o n d l i n e ) .  38  FIGURE 4 .  Second chromosome maps d e r i v e d f r o m recombination d a t a of d y s g e n i c c r o s s e s i n v o l v i n g the P s t r a i n , OKI. The u p p e r l i n e i s d e r i v e d f r o m f e m a l e meiotic r e c o m b i n a t i o n d a t a , and i s i n c l u d e d f o r comparison. An a s t e r i s k i n d i c a t e s i n t e r v a l s w h i c h experience l e v e l s of r e c o m b i n a t i o n s i g n i f i c a n t l y different from c o n t r o l s (second l i n e ) .  39  DISCUSSION  Sinclair  and G r i g l i a t t i  and  OKI  second  and  p a t t e r n of r e c o m b i n a t i o n .  specific for  chromosomes  distribution  these  strains.  confirm  their  results.  crosses  are very  It  that  has  Woodruff induced  and by  proximal that  report the  show  that  data  from  both  23,399 f o r OKI  both of  light  Sinclari  have  the l e f t  and  right  by and  most  OKI  in  chromosome this  report  the  control  Grigliatti,  and  around  assumed  the  (1985)  centric and  this  Most, i f not a l l , of Combining the f o r T-007  to date  no  with  entirely  d e l i n e a t e d by l i g h t  arms  w i t h i n the  chromosomes  the  are located  exchange  I t was  i s euchromatic.  examined  the  occurred  Grigliatti  15,639  within  of  chromosome.  and  intervlas  exchange  rolled  in  strain  identical.  i s not the case.  been  segment and  and  place  studies:  heterochromatic centromeric  T-007  took  exchange i n t h e s e  the second  presented  that  of t h e second  this  a non-random,  of S i n c l a i r  1977),  strains  heterochromatin.  frequency  p r e v i o u s l y ( H i r a i z u m i e_t a l . , 1973;  Thompson,  exchange  found  patterns generated  to those  t h e T-007  characteristic  which i s n e a r l y  shown  intervals  this  The  of OKI  the P  a  They  The^ d a t a  similar  been  induce  of exchange a l o n g  P  especially  (1985) r e p o r t e d t h a t  within  of chromosome  evidence  and of  heterochromatic  and  rolled.  Since  the  heterochromatin  2,  i t i s possible  40 that  recombination  heterochromatin  distal  formally  possible,  occur  this  in  within  the  crossing  over  in  the  i s that  P  into  1983;  into  identified  not  these  date  suggesting that  at  f l a n k e d by  the  lack  do  not  the  a  genome b u t  they  may  possibly  near  heterochromatin.  studies in  e_t a l . ,  1983;  P  and  elements do  they  insertions a  variegation into  and  or  near  H a z e l r i g g et  inserted  at the t i p  telomere. junction or  into  was  200  1983;  into  In  segments  i f ever,  have i n s e r t e d  i f i t inserted  r_l.  Spradling  exhibited  the  not  gene  These  over  euchromatin/heterochromatin to t e l l  of  (Goldberg  of t h e e l e m e n t s  but  insert  functioning  Rubin,  would  heterochromatic  a number  In  is  rare.  normally  rarely,  2 elements  it  l_t and  heterochromatic by  of  heterochromatic  of  ejt a l . , 1 9 8 4 ) .  ( S p r a d l i n g and  H o w e v e r , one  possible  from,  While  heterochromatin  S c h o l n i c k e t a l . , 1983;  only  segment  recombination  at a l l i s extremely  carrying  the  chromosome,  the  strains,  heterochromatin.  to  1984).  inserts  centric  the D r o s o p h i l a embryo  into  heterochromatin  the  why  This i s supported  Hazelrigg  insert  of  clear  P elements  excise  et^ a l . , 1983;  insert  al.,  of  element  readily  effect,  both  subsquently  transformed  Rubin,  the  in  of t h e s e m a r k e r s .  s i m p l e s t e x p l a n a t i o n f o r the  of a chromosome.  Richard  of  i f i t occurs  exchange e v e n t s  which  i t i s not  occurred  r e g i o n of h e t e r o c h r o m a t i n  then,  and/or  have  t o each  portion  summary  The  could  simply  The  other  and  i t is  near  the  41  Why There  would  are at least  may  lack  elements of  P elements  the bases  very  highly  recognition  the  recognition  sequence  possibility  (Foldback  small  P  Ruddell,  the h e t e r o c h r o m a t i c  those  hampered.  of the l a r g e r  that  of the t o t a l some  sequence  insertion  by t h e h i g h l y  condensed  communication;  much in  that  larger  than the  heterochromatin Truett  f o r review  P elements  of P e l e m e n t s  i s no a p r i o r i  of  to P element  f o r example;  elements  form  r e g i o n s of the D r o s o p h i l a  are found  that  that  i n heterochromatin.  segments f o r s t e r i c  on i n s e r t i o n  There  one f i f t h  elements  to p o s t u l a t e f u r t h e r  limitations of  imposed  elements,  I f one h y p o t h e s i z e s  sequence i s  an a c c e p t a b l e r e c o g n i t i o n  element  personal  i s known t o be  and c o n s i d e r i n g  to imagine  Transposable  and c o p i a  necessary  into  the r e c o g n i t i o n  inaccessible  constraints  of t h e DNA.  relatively  1983).  mostly  i n the heterochromatic  to s t e r i c  1981,  s e q u e n c e i s composed  i s not p r e s e n t  i s that  genome, b u t i s n o r m a l l y  state  the P  i n composition,  i tis difficult  Heterochromatin which  makes up a p p r o x i m a t e l y  in a cell,  due  sequence  This recognition  conserved  DNA  does e x i s t  heterochromatin?  explanations.  On t h e o t h e r hand,  heterochromatin  Another  from  G and C w h e r e a s h e t e r o c h r o m a t i n  A-T r i c h .  not  two p o s s i b l e  t h e 8 bp insert.  be a b s e n t  e_t  see Rubin,  are barred  reasons,  then  the c o n d i t i o n s must  which reason  differ  are not why  al.,  from  from i t is  and/or those  similarly  a l l eucharyotic  42 elements classes differ  should of  operate  quite radically was  to l e a r n  "steric  restraints"  is  P-induced butyrate  important  in their  used  attempt  that  the  w h e t h e r or not of h i g h l y  induced because  no  bromide  and  acridine  orange,  recombination melanogaster  on  the  males.  disrupt  the  DNA  occurs  which  recombination intercalating  agent,  the  fails  orange  somehow  broken  strands  induced  effect  than  that  unable  to suppress  to  that  suppress are  P  are  butyric  to  resolved ethoxy any  agents,  The acid  via  a  levels  male  of  agents breakage  homologous  caffeine,  another  recombination. and  acridine  mechanisms  so  that  incorrectly  alteration is clearly  of t h e a b o v e a g e n t s , normal r e p a i r  ethidium  chromosome  reannealed  male  JJ r_ <) j3 £P_ h_ JL 1^ a_  of  bromide  repair  cross  ( F e r r e s e_t a l . ,  intercalating  that  ethidium  R  induce  significant  produce  normal  observation  Oregon  known  elements  extent  chromosomes.  the  i n the  these  occasionally  by  The  chromosomes  However,  e_t a l . s u g g e s t  place.  cause  occasionally  Ferres  structure  take  intercalating  third  to  event.  recombinant  and  Presumably  helix is  condensed h e t e r o c h r o m a t i n  of  two  i n an the  absence  example,  structure  to reduce  recombination  For  various  occasionally  i t i s possible  to  1984).  the  elements  chromatin  some c h e m i c a l s  i n the  "rules";  behaviour.  to d i s r u p t  recombination  recombination  yield  same  prokaryotic transposable  Butyrate  allow  by  in  mechanisms.  chromatin  a less  or p e r h a p s  to  direct  butyrate i s In any  case,  A3  the  important  point  recombination In the  i s that  butyrate  i n a non-dysgenic  c o n t r a s t to the  previous  recombination  in  the  lack  of  butyrate  dysgenic  can  with  the  regular dysgenic  machinery.  that  can  be  butyrate  made  of  the  upon them by The  dysgenic  may  exchanges  event  to  this  reflects  of  the  exchange,  for  product  be  to  i s not 32%  The  on  influence  male  It  that  appears  in conjunction  simplest conclusion  facilitates the  insertion  constraints  is  that  for  both  hypothesis  elements. were a  lethal),  or  imposed  that  one  would  11  the  was  result  a  produced.  recognition are  normally  heterochromatic  of  were  expect  events  four events,  strains,  an  the  events.  the  to e u c h r o m a t i n  (if i t  merely  the  the  in timing  relative itself  but  butyrate-  (non-c1ustered)  difference  or Of  A l l of  single  P  exchange  in heterochromatin  example,  of them, o r  butyrate  recombination  reducing  the  exchange  clear.  of  o b s e r v a t i o n r e g a r d i n g the  in heterochromatin  nature  about  found  observed  effect does  of h e t e r o c h r o m a t i c  the  male  condensation.  favours be  inaccessible  Whether  by  crosses  level  strongly  sequences  size  elements  chromatin  significant  male  i s that  most i m p o r t a n t  treated  This  affect  induce  crosses.  butyrate  excision  only  to  cross.  above  crosses,  fails  exchange or  the  imprecise  "reciprocal"  of a s m a l l  which  in  were  sample  observed,  c o u l d have been e x p e c t e d  to  44 be  clusters  increase  (as noted  the  sample  i n the  size  may  results). indicate  of c l u s t e r i n g  of t h e h e t e r o c h r o m a t i c  Sinclair  and  Grigliatti  exchange  i n a cross involving  were  observed,  examined,  and  (1985)  among  each  of  Further  whether  the  P  was  strain  also  is  some  approximately these  or not  exchanges  observed  crosses the  to  lack  significant.  heterochromatic  hi2.  Seven  events  21,000  chromosomes  a single,  unclustered  event. It the  i s quite interesting  strain  would  h12  this  induced  strain  heterochromatic elements There  are  i s another  none  of  Sinclair  present  that  the  and  possible  decided  explanation.  1984;  the  copia-like  also  for  Ruddell,  possibility element  near  the  lt_ l o c u s ,  the  dysgenic  that  (perhaps and  crosses  that to  chromosome r e a r r a n g e m e n t s mutations easily  be  observed tested  locations  of t h i s  (which  e_t a l . , 1 9 8 2 ) .  heterochromatin  carries  was  some  m i g h t be  a  I  quiescent  heterochromatin, mobilized during  recombination  and  the b a s i s f o r the JLt  This suggestion  hybridization.  is  f a m i l y show  communication).  normally  strain).  strain.  (Rubin  personal  produce  P  dysgenesis  in  element  that  hybrid  gypsy) i n the this  for this  by jLn s i t u  hi2  why  display  suggest  o t h e r members of t h e c o p i a - l i k e  preferences  (Finnegan, suggest  and  tested  Grigliatti  i n the h e t e r o c h r o m a t i n  study  exchange.  others  known t o h a v e m o b i l i z e d c o p i a e l e m e n t s Copia elements  i n the above  some h e t e r o c h r o m a t i c  and  MR?  to note  could  45  It  is interesting  differently increased  that  to b u t y r a t e level  (including  the centromeric  of euchromatin  fact  active  conformation nuclease these  Steric  with  and  regions  of  defining  such  The  OKI  strain  treatment to  react  there e x i s t s  opened  that  as  of  by  butyrate  Although  by  excision  of P  regions.  by  certain and  i n p a t t e r n of  3) a s a r e s u l t o f  Why  of  recombination  should  treatment?  the  r e g i o n s of t h e OKI there  a r e no  insertion  two  Possibly  between them s u c h  element that  f o r example,  change  to butyrate  i tis likely  "open"  interesting.  the c o n t r o l .  to permit  more  i n these  frequency  treatment  others;  butyrate relaxes  number  some s e q u e n c e p o l y m o r p h i s m  up  than  some  heterochromatin,  little  euchromatic  two  that  be i m p o s e d  quite  interval  so d i f f e r e n t l y  s e q u e n c e s n e w l y exposed regions.  prove  be  a  and/or  may  well  and t h e o v e r a l l  when t h e more c o n d e n s e d are  as  from  have  and p e r h a p s  words,  showed v e r y  (aside  to  recombination  i n other  chromosomal  condensed  insertion  r e g i o n s might  similar  strains  permits  euchromatin  recombination butyrate  studies),  shows an  of chromosome  (as determined,  concommittant  constraints,  highly  responded  strain  I t may  i s known  inactive  sensitivity  arm  region).  a r e more  strains  i n a l l three  the r i g h t  chromatin  than  regions  elements  was  on  P  The T-007  of r e c o m b i n a t i o n  examined  in  two  treatment.  intervals  regions  the  that  strain  recognition into  t h e two s t r a i n s  these  possess  46 some  sequence  temporally  differences  distinct),  unnecessary.  (as they  invoking  sequence  may  elements  i n t h e two s t r a i n s ;  simply  different  reflect  locations  differing  that  male  recombination  1983).  results  hybrid  dysgenesis  example, the element the  mobilization  crossover  mobilization  of  these  distinguish  whether  mobilization  of P elements  markers  centric Tejani  resides  observed  or o t h e r  at p o s i t i o n  a  recombination  possible  I t i s known  of o t h e r  frequency  are the r e s u l t  of Su(var)325  effect  of  transposons. 53.3 ( j ^ l . 4  map  u n i t s ) on  I t l i e s i n the i n t e r v a l  interval.  t h e gene, found  that  i n the m e i o t i c I chose  on male r e c o m b i n a t i o n  on m e i o t i c r e c o m b i n a t i o n .  between  includes the  of mapping  (50%)increase  i n this  with  i t i s i m p o s s i b l e to  personal communication)  significant  whether  i s associated  here  In the p r o c e s s  (1982,  that  elements ( f o r  e_t a 1., 1 9 8 4 ) ;  Gl_ ( g l u e d ) a n d S_b ( s t u b b l e ) w h i c h  and K r o i t s c h  the e f f e c t  present  In any c a s e  events  heterochromatin.  _3 2 5_ c a u s e s  which are a l s o  elements  t h e l e f t a r m o f c h r o m o s o m e 3. the  elements  md g4 ; Gerasimova  i s n o t known.  Su(var)325  of  i n t h e two P s t r a i n s .  causes  examined  possibility is  mobilization  o t h e r t h a n P, s u c h a s c o p i a e l e m e n t s , sites  a r e known t o r e s i d e  Another  from  be  of r e s i d e n c e of P  among t h e v a r i o u s P s t r a i n s  B r e g l i a n o and K i d w e l l ,  different  may  The d i f f e r e n c e i n  sites  the elements  (see  at  polymorphism  A simpler explanation exists.  response  in  a r e g e o g r a p h i c a l l y and  to study  because  of t h i s  In c o n t r a s t  t o 325,  47  the  other  such  effect  genetic are  Su(var)  mutation  in this  on m e i o t i c r e c o m b i n a t i o n .  interval  a s 325 a t p o s i t i o n  p a r t of a c l u s t e r  and/or  used  functionally  of S u ( v a r )  related  study, 318 l i e s  56.1.  marked  chromosome  and  recombination eliminates chromosome alters so  by  second M  chromosome  strain  observed  the  breakage  and m a l e  male r e c o m b i n a t i o n  structurally  R  that  b e a r i n g the  males, FI  there  is  progeny.  .3 2_ 5_ c a u s e s  recombination.  mechanisms  m e i o t i c or d y s g e n i c — a n d  of  no  This  localized  If  Su(var)325  f r e q u e n c i e s at a l l , i t appears  augmenting the normal  either  be  318  Su(var)325-bearing  i n the r e s u l t i n g  possibility  3 2 5 and  females  and a  Oregon  no  e_t a l . , 1983).  When a c r o s s i s made between M s t r a i n multiply  had  i n t h e same  Both  genes and may  (Sinclair  318,  t o do  recombination—  n o t v i a some o t h e r  independent  process. The  presence  original overall  male  cross In  nearly  325,  interval twice  comparable frequency  that  to the  i n the dysgenic  i s of  of male  events  lacking  this  parent  amount  recombinant  o f 325  was  OKI  to that  The found  increased distribution i n the  the of  dysgenic  t h e e x c e p t i o n of t h e _b t o l_t i n t e r v a l .  (number  2), the p e r c e n t  of the s t a n d a r d 1.5-fold  observed  strain  recombination. similar  with  the  c r o s s i n which the  increase  during  the  recombination  dysgenic  cross.  in meiotic mapping  of  was  This i s  recombination the  gene  on  48 chromosome recombination  frequency  Su(var)318 suggests and  that  had  not  cause  i n the T-007  cross.  no  i t may  did  effect  on  their  different  P  act quite d i f f e r e n t l y  some a s p e c t region,  so  meiotic  f u n c t i o n s are  responses of t h e  and o  does  meiotic  n  Previous  as  studies  apparently  take  affect  expected  as  have  male  apparently  an  affect  types  both  3 2 5,  shown  on  the  this  i n an  analagous  (meiotic  recombination  frequency.  hand  318  The  i n females),  to  cause  a c t upon transition  act  does  325  neither. that  not  _3 2_ 5_  is  i t seems  to  manner. left  hand  of chromosome two of  chromosome  increase  the  do  mutant  the  and  meiotic  alters  analogous  allow  between  of  1983)  and upon  not  alters  areas:  boundary  left  to  not  o b s e r v a t i o n as  and  of  of  dysgenic  effect  Engels,  boundary  right  cluster  interval  .3.2_5  euchromatin/heterochromatin the  318  meiotic recombination  of r e c o m b i n a t i o n acts  which  recombination. to  both does  while  (see  If  may  i n the  i n the  given  dysgenic,  exception  then,  manner  place  w h i c h a r e known t o a l t e r  to  right  recombination. well  318  in  which  325.  distinct  recombination,  in a  strain,  from  crosses.  structure  It so  to  T h i s i s as  recombination  seem  or  recombination  r_l.  factors  chromatin  unaffected.  heterochromatin, dysgenic  i n the dysgenic  changes  related  sufficiently  s p e c i f i c a l l y that  are  any  either  3 2 5 a r e r e a l l y p a r t of a f u n c t i o n a l l y  genes,  11  3 2 5_  three.  (MR) three  recombination  I t a l s o a c t s on t h e X c h r o m o s o m e ,to s u p p r e s s  the  49  inactivation the  of w  in w  +  , and  i t is likely  euchromatin/heterochromatin  that  i t acts  b o u n d a r y of t h i s  on  chromosome  also. The  euchromatin/heterochromatin  gradual  rather  than  appears  cytologically  heterochromatin, condensed 1974).  "A"  In t h e  interspersed  the  of  seems  mutant:  in  may  be  chromatin About  two,  the  sequences:  structure  or  structure  the  left  of  GJL  between  this  way  has  heterochromatin repeated  Jacob,  less  are  diffuse  B-'  involved in structure,  i n t h e makeup of to  be  affected  boundary  (because ^_b)  by  both  boundary  are of  zone of t h e X chromosome.  to  325  and  "B"  sequences  chromatin  right  It  heterochromatin.  the  genes  of  highly  ( L a k h o t i a and  appear  and/or  of  more  euchromatin/heterochromatin  upon w h i c h  short highly  1978).  euchromatic  regulating  the t r a n s i t i o n in  region  stretch  intercalary  suppressor  interval  possible  of  of  r e g i o n s which  the l e f t  the  80%  to  to look f o r s i m i l a r i t i e s  chromosome t h r e e , and It  the  likely,  i n those  chromosome  changing  blocks  maintaining  chromatin  included  diffuse  makes up  i t is logical  t h e 325  rather  a  zone t h e r e a r e  heterochromatin.  then  as  transition  type  establishing,  (Lifschytz,  heterochromatin  apparently  as  change  (centric)  between  is a  abrupt  gradually  This  If,  boundary  pinpoint  the  aspect  of  i t s effect.  i s composed  sequences  of  occupying  satellite distinct  50 regions  on  individual  satellite  sequences  distinguishable chloride obvious  in  aspect  A  chromosome  makeup  are  certain  with  proteins  certain  satellite  t o be  and V a r s h a v s k y , 1 9 8 2 ) .  observed  that  a_bo  two  (abnormal  partially  specific  relationship The  325  enhanced  of or  sequence  (Peacock  have  no  left  of  boundary  effect.  (1977)  could  be  be  of c e r t a i n with  X  one  a  satellite  sequence.  this  sequence  to  that  recombination i s  i s possible.  i s found  on  cause  the  X  There i s  heterochro-  b o u n d a r i e s of c h r o m o s o m e s two  1973).  may  Sandler  analogous  such  However,  of chromosome  This  1979;  of an  variegation  which  transition ejt a 1.,  with  structure  -1.697g/cc-  three  right  interact  associated  interacted  and  may  there  Brutlag,  copies  t h e 325  suppression  and  gene  mutant  an  interest.  (daughterless),  Each  is  of IX/ m e l a n o g a s t e r ,  the p o s s i b i l i t y  t h e DNA  matin  the  mutants  I envision  between  product  loosening  one  and  In a d d i t i o n ,  d_ a  and  sequences.  sequence.  each  easily  F o r example,  r e s c u e d i n t h e p r e s e n c e of e x t r a  heterochromatic  major  o r gene p r o d u c t s and  (Hsieh  maternal e f f e c t  oocyte)  be  preferentially  sequences  Levinger  can  i n the a r e a s of  a r e n o t unknown.  known  sequence  which  I n t e r a c t i o n s between e u c h r o m a t i c genes sequencs  six  3. a n o_£ a.^ t_^ J_»  m  satellite  f o r p r e s e n c e or absence  heterochromatic  are  of i t s s e d i m e n t a t i o n i n c e s i u m  gradients.  of  There  JJ j_ o_ js o_j> ti i_ 1^ a_  on t h e b a s i s  density  examined  chromosomes.  two,  explained  i t i s also where  and  found  on  325 appears  to  i n one  of  two  ways:  51  either  3 2 5 i s n o t i n any way  satellite interest  sequence,  appropriate Given it  of  the h i g h l y  left  hand  right  block  hand  effect  m a t i n boundary  a  dysgenic  areas  which  have  regions  need  as w e l l  as b l o c k s  two  form.  be  evidence,  3_ 2 _5  transition  paucity that  of  satellites,  also,  regions,  i n the  that  the  heterochro-  may  be  of r e l e a s i n g  The may  very  newly  sequences  of the t r a n s i t i o n  effect,  then  some  stem  or l a c k directly  recognition  exposed.  These  sequences,  sequences  will  and c a n t h u s  zone t o  o f o n e , on from  sites newly  as unique  the  i n the exposed  sequences,  o f e u c h r o m a t i n , a r e known t o r e s i d e These  i n the  present  than  of  region.  present  mutation  regions  n o t be s a t e l l i t e  zone.  t h e 1.697  a c t upon any h e t e r o c h r o m a t i n / e u c h r o -  of P-element  been  of  discussion).  compressed  or absence  transition  not a l s o  i s some  the  recombination  presence  would  with the net e f f e c t  condensed  with  P elements--in  sequences  There  of  regions  the  recognition  I t may  the t i g h t l y  less  to  structure  (see l a t e r  straightforward.  from  due  gene a c t s on e u c h r o m a t i c r a t h e r  sequences The  i n the  sequence  o f 1.697  block.  suppressor matic  that  two  sites—or  conserved  seems u n l i k e l y  particular  r e c o m b i n a t i o n i s o b s e r v e d on t h e  chromosome  recognition  this  or i t does i n t e r a c t  b u t no d y s g e n i c  boundary  with  i t s presence  i s coincidental;  sequence, right  and  involved  i nthe  n o t be t h e same f o r any explain  the d i f f e r e n c e s  52 between  the responses  presence  of  If  simplest  as does the e f f e c t possibility--then  ability  Rather, to  observations euchromatin crossing presence  that  than  may  the  butyrate.  Thus,  of chromosome  mutant  where  addition,  i t appears  the f a c t  indicates  sequences, chromatin, transition  that  that 325  to  zone.  rather  No  act  than 325  any  likely  infrequent  may  Two affect  c r o s s i n the of  on  variegation  does  a  probably  and  a c t s upon  zone ( t h e o n l y  effect  acts  as  different  butyrate,  at  meiotic recombination most  euchromatin  heterochromatic  the t r a n s i t i o n have  affect  the h e t e r o c h r o m a t i n  must  since meiotic recombination and  J3 2 5_  dysgenic  Therefore,  i n and a r o u n d  not  e x p e c t a t i o n s (see  suppressor  structure  more s p e c i f i c a l l y .  euchromatin  region  the  i s the  spreading.  mutant  in either  i s a stronger  may  c o n f e r upon  heterochromatin.  observed 325  to i n i t i a l  i s b u t y r a t e , yet i t d i d not a f f e c t  probably  325  was  gene  heterochromatic  suggest  o f 325.  component  the  the s u p p r e s s o r  t h e mutant  resist  rather  over  on r e c o m b i n a t i o n — w h i c h  at a l l , c o n t r a r y  Introduction).  did  i n the  325.  heterochromatin  than  regions  t h e s u p p r e s s i o n a c t i v i t y o f 3 2 5 o p e r a t e s by t h e same  mechanism  the  of v a r i o u s t r a n s i t i o n  upon  all).  In  i s altered  by  euchromatic  i s very r a r e i n h e t e r o i n the  region  of  the  53  CONCLUSIONS  In OKI,  dysgenic  rarely,  chromosome reflected  crosses  a  two.  I t was  steric  strains,  constraints  butyric  recombination  is  response  the  differences  strains  insertion  and/or  male  butyrate i n the  by  sites  of  t o .15M  significant  that  l_t t o  excision  two  by  P  of  exposing  In  levels  acid,  both of  P  male  r_l i n t e r v a l  of the  on  exchange  butyric  chromatin  recombination. the  of  structure.  heterochromatic  and  packaging  tested  strain  induced  lack  tight  was  T-007  exchange occur  this  the  chromatin  acid  in  to  either  Thus i t a p p e a r s  reflected to  due  hypothesis  of  i n the  chromosome two.  this  This  modifier  the  influence  P  postulated that  h y b r i d males from  presumed  the  i f e v e r , does h e t e r o c h r o m a t i c  heterochromatin. dysgenic  involving  of  structure  can  elements  and  Differences in  strains  may  r e s i d e n c e of P e l e m e n t s  reflect in  each  strain. The with  two  same  dysgenic  Su(var)  heterochromatic of a c t i o n the  genes:  recombination,  an  318.  from,  in  Neither  and  combination  produced  any  their  modes  more s p e c i f i c  than,  of b u t y r a t e .  i n c r e a s e i n the  containing  performed  which suggests that  be q u i t e d i f f e r e n t  caused  interval  were  3 2 5 and  rather general e f f e c t s 325  the  may  crosses  the  left  recombination  frequency  in  heterochromatin/euchromatin  54 boundary of chromosome two that  the  325  (although matin  less  mutant most l i k e l y  the  possibility  is also  release  some  of  OKI  sequences  from  form.  either  In  strain.  an  interaction  areas  the  euchromatin  with  heterochro-  condensed  where n e w l y  sequences  The  fact that  the  r i g h t boundary  act  similarly  and  that  increase  in  sites  residence  of  locations, The  and  of  between  does  thus  (by  not  introduced  into  usually "rescue" The one  possible  observation  by  line  direct  for  the  insertion site;  i s apparently  place. not  similar  differing chromosome  another.  a l t e r i n g chromatin  dysgenic of  the  which  P  the  or  functional  of  of  the  genes  copies  of  At  may but  be i t is  elements, which the  can  gene.  a r e c o g n i t i o n - l i k e sequence  direction the  P elements.  embryos,  insertion copies  recombination  elements  of D r o s o p h i l a  functioning  sequence,  prerequisite  by  different  i s that  mobility  individuals lacking  recognition  in  influence  the  germ to  to  exist  c a r r i e r s for  particular site  the  elements  a  the  does  e x p e r i e n c e s no explained  or  take  c h r o m o s o m e two  be  to  strains.  inference)  techniques  yet  P  appear  present,  not  the  may  may  to  sequences  for P elements,  T-007 s t r a i n  recombination  most i m p o r t a n t  structure and  male  of  acts  state  exposed  elements themselves, dysgenic recombination  the  suggested  t r a n s i t i o n z o n e , and  their highly  recognition  It i s  i n t e r a c t s with  d i s c u s s e d ) i n the  compressed  contain  of t h e  state  of  of the  a  P  element  chromatin  at  to  is a  that  55  LITERATURE  CITED  BERG, R., W.R. 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(b) S u p p r e s s i o n of male r e c o m b i n a t i o n i n r e c i p r o c a l c r o s s e s . Genet. Res. Camb. 29: 231-238. Y A N N O P O U L O S , G., N. S T A M A T I S , A. Z A C H A R 0 - P 0 U L 0 U a n d M. PELECANOS. 1983. S i t e - s p e c i f i c b r e a k s i n d u c e d by t h e male r e c o m b i n a t i o n f a c t o r 23.5 MRF i n D r_ o_ s^ .ojpji _i _1 _a melanogaster. M u t a t i o n Res. 108: 185-202.  64  APPENDIX I  I performed OKI. of  an a d d i t i o n a l  A reciprocal the  P  c r o s s (one  strain,  hearing  the  treated  with  and  multiply 0.15M  act  a  P  cytotype).  (7  A  in  control  (the  for review).  These  cross  should  male  lower  reciprocal  than  cross  frequencies f i f t e e n would  have  progeny  4.  been  no  were  and  MATERIALS  AND  butyrate did  mechanisms  was  no  and  13  individuals),  interval  There  (to  being i n  scored,  (36 r e c o m b i n a n t in  strain  s e t up  that  dysgenic  reciprocal  numerous  yield  i n the  M  was  2  and  8  (29  recombination  interval.  1983,  ten-fold  F2  not performed,  performed  the  f o r example, d e s p i t e t h e i r  interval  other  b u t y r a t e ) was been  regular  individuals)  i n any  was  strain  parent  chromosome) was  outlined  were o b s e r v e d  recombinant  observed  the  10,183  events  individuals)  second  the P  maternal  done t o a s c e r t a i n  the P elements,  recombinant 5  marked  of  the  with  paternal parent  T h i s was  outside  derepress  i n which  b u t y r a t e , as  METHODS s e c t i o n . not  the  experiment  times  times  suggest  the  with  experiments  that  the  dysgenic  performed  reciprocal  lower  than the (G t e s t ,  cross.  and  had  dysgenic p = .05)  have  Kidwell,  at f r e q u e n c i e s  been  significant  treated  B r e g l i a n o and  recombination of  not  analogous (see  studies  those had  but  cross,  about  If  the  yielded  cross,  there  difference  65 between butyrate. the  this  cross  heterochromatic  not an  the  Thus, b u t y r a t e  dysgenic  However,  and  because  system.  appears Most  recombination the  control  a c t u a l l y performed, appendix,  r e c i p r o c a l cross  rather  than  not  operate  important,  outside (untreated  I decided i n the  to  of  the  i t  with  outside  of  cause  no  dysgenic  system.  reciprocal) cross  to p l a c e  text.  treated  this  observation  was in  

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