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Characterization of the recombination enhancer, rec-1, in Caenorhabditis elegans Rattray, Bruce 1986

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CHARACTERIZATION OF THE RECOMBINATION ENHANCER, RECIN  CAENORHABDITIS ELEGANS by Bruce  B.Sc,  Rattray  Queens U n i v e s i t y , K i n g s t o n ,  O n t a r i o . 1981.  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE  in THE  FACULTY OF GRADUATE STUDIES GENETICS  We a c c e p t to  this  thesis  the required  as conforming standard  Dr.  A.M. Rqsfi^, co-^Ajzlvis^r  Dr.  C. F . Wehrhahn,  Dr.  R. Ward  Dr.  D.  Dr.  F. D i T l ,  THE  co-Advisor  Advisory  Committee  Juri.lof.fV E x a m i n e r Chairman  UNIVERSITY OF BRITISH  COLUMBIA  JANUARY 1986 BRUCE RATTRAY, 1986  7%  In p r e s e n t i n g  this  thesis  i n partial  f u l f i l m e n t of the  r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y of  B r i t i s h Columbia, I agree that  the Library  s h a l l make  it  freely  and study.  I further  available  agree that for  f o r reference  permission  f o rextensive copying o f t h i s  s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my  department o r by h i s o r h e r r e p r e s e n t a t i v e s . understood that for  financial  copying o r p u b l i c a t i o n  gain  of this  Department o f  r^XeA^c^  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3  /RI \ ,  T—,. 0  It is thesis  s h a l l n o t be a l l o w e d w i t h o u t my  permission.  Date  thesis  ~~2- °[  ( r e ^ t - ^ ^  Columbia  j *5 £  5  written  Abstract  The  hyper-rec  previously frequency further  mutation  rec-1,  b e e n shown t o i n c r e a s e i n t e r g e n i c by  a factor  of t h r e e  other meiotic processes  The  effect  of rec-1  conversion, mutation  on  has  Baillie,  1979a).  on  i n t r a g e n i c recombination,  rec-1  intragenic effects  thesis.  gene  nondisjunction  mutation  p r o d u c e d a two  recombination  are t y p i c a l  and  to t h r e e - f o l d  affected  gene c o n v e r s i o n .  of recombination  by  rec-1, exhibiting  (wild-type)  Pleiotropic  m u t a n t s , however, o n l y  determine  i f the  a premature  results  indicated The  by  rec-1 stop  mutation  of rec-1  as  Electrophoretic  banding  rec-1  altered  a c t i v a t e s Tc1  translation).  was to  amber  element, Tc1,  were compared  banding  mutation The was  This  mutation. e n h a n c e r on was  p a t t e r n s p r o d u c e d by  Tc1  to  phenotype  ( i e . sup-7).  a recombination  transposable  An  an amber, n o n s e n s e  suppressor an  m o b i l i t y of the  strains.  constructed  e x p r e s s i o n o f t h e Rec-1  i s not  fragments probed with  was  codon, p r e v e n t i n g  a tRNA amber  t h a t rec-1  affect  increase relative  above, a s t r a i n was  showed t h a t t h e  affected  a two-fold  the  strain.  In a d d i t i o n to the  (ie.  and  increase i n  o f X-chromosome n o n d i s j u n c t i o n a n d / o r chromosome l o s s  t h e N2  The  recombination  focus of t h i s  fecundity, competitive a b i l i t y ,  has  recombination  o f rec-1  been the  elegans,  r a t e were e x a m i n e d .  The  not  (Rose and  c h a r a c t e r i z a t i o n of the e f f e c t  and  rate  i n Caenorhabditis  p a t t e r n was  a l s o examined.  DNA  restriction  f o r t h e N2 found,  the  and  Rec-1  suggesting  that  m o b i l i t y . However, p r e d i c t e d d i f f e r e n c e s  between two other)  Rec-1  were n o t  DNA  samples  (one  found. Several  prepared  alternative  two  years  after  explanations  the  are  considered. High recombination r e v i e w e d and results  are  mutations  s e v e r a l hypotheses considered  as  reported  i n the  c o n s i s t e n t with  possible explanations  enhancer.  iii  literature the  above  of the  rec-1  are  TABLE OF  CONTENTS  Abstract  i i  List  of Tables  v i  List  of Figures  v i i  Ackowledgements  ix  I. I n t r o d u c t i o n : II.  Background  an o v e r v i e w Information  A) L i f e  History  8  B) S t r a i n s and C u l t u r e C) S t r a i n III.  IV.  V.  1  Conditions  Constructions  Recombination  9 10  Effects  Introduction  20  Methods  23  Results  31  Discussion  35  Pleiotropic  Effects  Introduction  38  Methods  41  Results  45  Discussion  51  The E f f e c t  o f an Amber S u p p r e s s o r on r e c - 1  Introduction  55  Methods  56  Results  57  Discussuion  60  iv  VI.  The  Effect  of  rec-1  on  Tc1  Introduction Materials  VII.  Mobility 63  and  Methods  65  Results  66  Discussion  68  Discussion:  Literature  an  overview  72  Cited  95  v  List  Table  1:  Strains  Table  2: M u t a t i o n s  Table  3: V e r i f i c a t i o n  Table  4:  The  used  effect  used  5:  P u t a t i v e Rec-1, bearing  Table  Table  6:  7:  of s t r a i n  and  Tables  study  in this  of rec-1  recombination Table  in this  of  on  11  study  12  construction  18  intragenic  gene c o n v e r s i o n  32  duplication  strains  34  The  effect  of rec-1  on f e c u n d i t y  and  competitive a b i l i t y  The  effect  of rec-1  46  on n o n d i s j u n c t i o n  a n d / o r chromosome l o s s Table  8:  The  effect  of rec-1  on  of r e c e s s i v e , l e t h a l Table  9:  Summary o f the  rec-1  48 the  frequency  mutations  the r e s u l t s  50  characterizing  enhancer local  73  Table  10:  A review  of  recombination  Table  11:  A review  of g e n e r a l enhancers of recombination  vi  enhancers  76  78  List  Figure  1: G e n e t i c  map  Figure  2: D e t e r m i n a t i o n  Figures  o f C. e l e g a n s  3: C o n s t r u c t i o n  13  o f p, t h e  recombination Figure  of  frequency  14  o f rec-1  homozygous s t r a i n s Figure  4: C o n s t r u c t i o n strain  16  of a heterozygous  unc-13  and i t s u s e i n m e a s u r i n g i n t r a g e n i c  recombination Figure  5: Sample  size  24 estimate  chromosomes s c r e e n e d Figure  f o r t h e number o f i n the i n t r a g e n i c expt  6: C a l c u l a t i o n o f i n t r a g e n i c  recombination  frequency Figure  27  7: C a l c u l a t i o n o f gene  conversion  frequency Figure  8  effect  9: A s s a y of  Figure  f o r determining  distances  30  the genotypes 43  10: S t r a i n c o n s t r u c t i o n and p r o c e d u r e t o d e t e r m i n e effect  o f r e c - 1 on m u t a t i o n  rate  11: S t r a i n c o n s t r u c t i o n and p r o c e d u r e the  Figure  o f r e c - 1 on l a r g e g e n e t i c  competitors  the Figure  28  S t r a i n c o n s t r u c t i o n p r o t o c o l to determine the  Figure  26  44  to determine  effect  o f sup-7 on t h e e x p r e s s i o n  o f r e c - 1 ....58  12: The e f f e c t  o f sup-7 on t h e e x p r e s s i o n  of the  Rec-1  phenotype  .....59  vii  Figure  13:  Tc1-hybridized from  Figure  14:  DNA  Screen alleles  restriction  samples  o f Rec-1  f o r the i s o l a t i o n of rec-1  fragment and  N2  patterns 67  of 89  viii  Acknowledgements  I would l i k e to thank Ree, unflagging and  support;  K e l l y and  T e r r y , L i n d a and  Linda  Kelly  for their  for their  patience  help i n the l a b ; Dr. Dave Holm f o r h i s c r i t i c a l review of  work and rec-1  my  story  s u p e r v i s o r , Dr. Ann  Rose, f o r her d e d i c a t i o n to  ...  II  I t was  a dark and  ix  stormy n i g h t  II  my  the  I.  Recombination generating insight  and  into  I n t r o d u c t i o n : An  i s central  Overview  to the process  characterizing  recombination  n o r m a l gene f u n c t i o n and  (1979a). in  as a s p o n t a n e o u s m u t a t i o n  which i s both  the recombination  same p r o p o r t i o n a l specific  established observed  interval that  effect  and  be  and Baillie  recombination  in i t s effect mutation  t o be  independent  l i n k a g e group examined. I t  a r e c e s s i v e mutation on r e c o m b i n a t i o n  was  the rec-1  of  mutation  that  provides  and  s e r v e as a framework f o r f u t u r e i n v e s t i g a t i o n nature  Hyper-rec recombination Escherichia  an  into  p o l A,  uncommon s u b c l a s s o f  uvrD, dut,  A r t h u r and  dam  L l o y d , 1980;  Marinus  and  frequency  t h e genome), b u t  different  pairs  the  recombination.  and  Tye  Konrad,  l i g (Konrad  e t a l , 1977;  1976;  Bale  ( i e . recombination  Konrad  et a l ,  the e x t e n t of the  of markers. A l l are 1  is affected  1979; on  through  increase varies for  mutators.  in  and  e t a l . , 1 9 7 3 ) . T h e s e m u t a n t s have a g e n e r a l e f f e c t  recombination out  form  of  in  of r e c - 1 ,  m u t a n t s . A number o f examples h a v e b e e n f o u n d  Lehman, 1975;  Konrad  the c h a r a c t e r i z a t i o n  evolutionary role  mutations  coli:  Lehman, 1974; and  and  an  recombinational processes  T h i s study d e a l s with  molecular  both  was  meiosis. will  The  r e s p o n s i b l e f o r the  frequencies. Given  opportunity to i n v e s t i g a t e  obtained.  three to f o u r - f o l d .  found  h y p e r - r e c mutants a r e u n u s u a l , unique  the rec-1  frequency  i n c r e a s e was  uniform  By  some  by Rose and  g e n e r a l and  1980). I n t h e homozygous s t a t e  increases  the  identified  I t i s t h e o n l y known m e i o t i c e n h a n c e r o f  eukaryotes  (Rose,  mutants,  mechanism may  R e c - 1 , i n C a e n o r h a b d i t i s e l e g a n s , was characterized  of m e i o s i s .  In y e a s t suggests  (Saccharomyces  cerevisiae),  t h a t many r e c o m b i n a t i o n  processes  a r e h e l d i n common between m i t o s i s and E s p o s i t o e t a l . , 1975; see  Haynes and  only mitotic intergenic  enhancers  and  Lasowski,  rev-2  and  i s i n c r e a s e d by  and  rad-4  (Lemontt,  results  frequency  of  decreases  meiotic recombination  mutants  inter-  and  crossing  Shanfield  mutation,  t o rem-1  (eg. Neurospora: and  uvs-3,  Kafer,  In Neurospora,  on  only  a t two  increase  but  but  rem-1  has  M a l o n e and  no  Schroeder,  Ustilago:  loci  1971;  Kowalski  for rev-1,  causes  chl a  high  and  increases  recombination affect  on  uvs-1,  and  meiosis  1984).  Aspergillus: Holliday  Mitotic  species uvs-B, et a l . ,  m e i o t i c exchange.  three meiotic hyper-rec  M u t a t i o n s . i n rec-1 recombination  mutations  have  produce and  a 10  t o 25  gene c o n v e r s i o n  ( C a t c h e s i d e e t a l . , 1964). M u t a t i o n s  gene c o n v e r s i o n and  intra2  and  H a b e r p e r s . comm.,  Hoekstra,  1970;  this,  deficient  have been r e p o r t e d i n o t h e r  1969;  i n intragenic  and  intra-  However, none o f them a r e g e n e r a l i n t h e i r  recombination.  increase  Cor,  1974;  mitotic  1 9 7 6 ) . None o f t h e s e m u t a n t s a l t e r  characterized.  the r e p a i r  over  (Haber,  intragenic  E s p o s i t o , 1977;  similar  UV-induced  o f chromosome t h r e e d u r i n g m e i o s i s  spontaneous m i t o t i c ( G o l i n and  spo-7:  r e c e s s i v e mutation  i n Baker e t a l . , 1976). F i n a l l y ,  spontaneous  (eg.  have b e e n f o u n d  1971 ). The  spontaneous m i t o t i c  cited  controls  E s p o s i t o , 1969;  ( H u n n a b l e and  increases  loss  their  meiosis  have b e e n i s o l a t e d .  1975). S i m i l a r  rev-3  evidence  f o r a r e v i e w ) . However, d e s p i t e  recombination rad-3  and  s p o - 8 : E s p o s i t o and  Kunz, 1981  mutants rad-1,  current  and  intergenic  been effect  fold but  i n rec-2  recombination  by  a factor  of t e n , but  1966).  Mutations  affect  intergenic  again, o n l y over  exchange  frequency  1971a,b; B r o o k s , enhancers The  (Smythe,  a variety Luccheci  frequency  and  Suzuki,  the d i s t a l  ends and  produces  i n an  1962).  Hinton  heterozygous T h i s mutant  can  be  (Stutervant,  found  on  the medial  results  with  a similar  is particularily  on  when homozygous  i n that  (Gowen and  of i n c r e a s e s i m i l a r  to the  the  X  frequency of  the  regions (Schultz  the e f f e c t  of  the  when  p a t t e r n of nonuniform  unusual  by  1919;  increase.  i t abolishes  Gowen, 1922;  C a r l s o n , 1 9 7 2 ) . I n Zea mays, t h e mutant as a l s o  pattern  induced  inversion  c(3)G which i n c r e a s e s recombination  and  recombination 1972;  Ramel,  (1966) compared t h e s e  mutation  increase i n  i n the r e g i o n of the centromere  and  1951;  t o 300%  effect.  increase i n recombination  effect  point  a 200  a uniform  on a l l chromosomes and  a u t o s o m e s , w h i l e h a v i n g no Redfield,  general, meiotic  1 9 6 8 ) . F o r example, an  result  also  (Chinnicci,  n e i t h e r has  o f chromosomal r e a r r a n g m e n t s  chromosome w i l l at  have b e e n s u c c e s s f u l  effect  but  increases i n  1 9 8 4 ) . However, o n l y two  interchromosomal  t o rec-1  (Smith,  1973).  for local  h a v e b e e n r e p o r t e d and  recombination  intervals  i n r e c - 3 behave s i m i l a r i l y  In D r o s o p h i l a , s e l e c t i o n recombination  two  interchromosomal  Hall, produces  effect  a  in  Drosophila. All either  o f t h e above e x a m p l e s d i f f e r they produce  frequency, increase  but  a uniform  increase i n  o n l y i n some l o c a l  i s quite variable  and  from  i n C.  elegans:  recombination  r e g i o n ; or the extent of  affects  3  rec-1  the e n t i r e  the  genome. I n  C.  elegans, and  the  general The  i n c r e a s e i n recombinatiom (Rose  specific  and  Baillie,  objectives  frequency  1979a; Rose,  of t h i s  i s both  1980).  work were  three-fold:  1) t o a d e q u a t e l y d e s c r i b e t h e e x t e n t o f t h e e f f e c t rec-1  mutation 2)  on  intragenic  to determine  indirect  effects  the g e n e t i c t o o l s first  t h e use  and  second  (1930) and  nominal  advantage  entered  the debate.  have a r g u e d  i n C.  (1932) f i r s t  of recombination, Nei  (1967) and selection  perhaps  eliminate) recombination  linkage  of  "well adapted"  investigated group  the  conditions  required  stringent.  T h e r e f o r e , group  under  special  evolution In  than  f o r group  order to account  of  such arguments n e c e s s i t a t e  (1972) has  shown t h a t  to operate are  i s a less level  only  quite operates in  individual.  f o r the p e r s i s t e n c e of r e c o m b i n a t i o n ,  to the i n d i v i d u a l  which y i e l d  (Maynard S m i t h , 4  a  the  general force  of the  number o f mechanisms h a v e b e e n p r o p o s e d advantage  the  alleles.  s e l e c t i o n most l i k e l y  a t the  (and  of r e c o m b i n a t i o n have been  selection  c i r c u m s t a n c e s and selection  (1971) amoung o t h e r s  should a c t to reduce  advantages  stance. Eshel  the  have  i n order to preserve  (eg. M u l l e r s r a t c h e t ) ,  selectionist  in evolution.  questioned  Lewontin  to  experiments  numerous a u t h o r s  combinations  l o n g term  using  importance  in population biology  Muller  by  elegans.  the r o l e of recombination  that natural  Although  (ie.i t s  mutation  p o i n t s are of c r i t i c a l  strain  to investigate  Since Fisher  the  gene c o n v e r s i o n ;  of rec-1  the nature of the rec-1  available  o f t h e Rec-1  intended  effects  of  on o t h e r m e i o t i c p r o c e s s e s ) ; and  3) t o i n v e s t i g a t e  The  r e c o m b i n a t i o n and  the p l e i o t r o p i c  uniform  1971;  a short  a  term  Williams,  1975;  Felsenstein  Strobeck  and  e t a l . , 1975;  Yokoyama, 1 9 7 6 ) . The  elegans  seems i d e a l l y  several  of these hypotheses.  genetically, Drosophila 1 9 7 4 ) . By designed  has  and  suited  rec-1  mutation  to provide empirical C.  elegans  1974;  i s well  in  C.  tests  for  characterized  a g e n e r a t i o n time which i s s h o r t e r than  is easily  cultured  using different  alleles  t o compare t h e e f f e c t  recombination  Felsenstein,  on  the  s h o r t and  i n the  laboratory  (Brenner,  of r e c - 1 , experiments  of d i f f e r e n t  could  be  amounts o f  even l o n g term  fitness  attempted,  full  of  an  individual. Before effects  such  of rec-1  d o c u m e n t e d and design. system  the  recombination taken  Gibson  into  and  intrinsic,  side  effects  fitness  must a t t e m p t  more f i t t h a n The  the  any  caused  by  the be  experimental  able to d i s c r i m i n a t e  c o m p e t i t i v e advantage or disadvantage  the p r i n c i p a l  due  i n c r e a s e d or  f u n c t i o n of the  t o answer t h e q u e s t i o n : " why  i s one  gene.  genotype  other?" of c r i t i c a l  of recombination  to  deduce the n a t u r e of p a r t i c u l a r In a d d i t i o n ,  that  pleiotropic  the  isolation  the  lower  for  s c r e e n i n g : i ) reduced  importance  a t the  a l . (1968) h a v e s u g g e s t e d  and  extent of  experimental  (1983) p o i n t o u t t h a t  et  meiosis.  i n the  o f t h e mutant v e r s u s an  above i s a l s o  understanding  the  on o t h e r p r o c e s s e s must  account  i n p o p u l a t i o n b i o l o g y must be  decreased One  on  then  Chao and  b e t w e e n an to  s t u d i e s c a n be  level  such  in aiding  of the c e l l .  side effects  one  effects  c a n be  are o f t e n used  of three c r i t e r i a fertility, 5  Sandler used  mechanisms a n d / o r s t a g e s  c h a r a c t e r i z a t i o n of recombination  eukaryotes,  our  i n w h i c h gamete  i n both  mutants.  are t y p i c a l l y  of  In  used  inviability  is  due  to a high  level  of aneuploidy  i n d u c e d by  the r e d u c t i o n or  a b s e n c e o f c r o s s - o v e r t e t r a d s ( e g . B a k e r e t a l . , 1976; 1974;  E s p o s i t o and  prototrophic in  E s p o s i t o , 1969);  p r o d u c t i o n , w h i c h s e r v e s as  t h e amount o f i n t r a g e n i c  al.,  i i ) enhanced or  1 9 6 4 ) ; and  an  recombination  i i i ) mitotic  mutagen s e n s i t i v i t y ,  frequencies  ( e g . G o l i n and  t h e above p r o c e d u r e s effect, 1985;  i f any,  In  and  mutants a l s o  o b s e r v a t i o n s . F o r example,  and  cause  rate  ( e g . mu:  well  as  al.,  1976; In  Hall,  and D5  mei-41  recombination  the p l e i o t r o p i c  result  mutants  rate,  and  chromosome l o s s . determine  1976,  their  effects  of genetic  reduced  fertility 1972;  Grell,  1962;  for a review). Also, repair  Setlow,  system  mutation 1973)  ( e g . m e i - 9 : Boyd  as et  1976).  pleiotropic  include:  and  reduced  effects  associated with  fertility  i n c r e a s e d spontaneous and/or  and  viability,  induced  c h a n g e s i n t h e r a t e o f n o n d i s j u n c t i o n and  The  which,  et a l . ,  i n a number o f t y p i c a l  mutagen s e n s i t i v i y  typical  by  i n D r o s o p h i l a a r e d u c t i o n i n the  : Boyd and  mutagen s e n s i t i v i t y ,  to  isolated  i n c r e a s e i n s p o n t a n e o u s and i n d u c e d A1 G o l d and G r e e n , 1974; mei-41 : Smith,  summary, t h e  mutation  recombination  an  radiation  the  mutation  (eg. Dowling  n o n d i s j u n c t i o n (eg. c ( 3 ) G : H a l l ,  see B a k e r and  defects  mitotic  examined t o determine  number o f c r o s s - o v e r t e t r a d s c a u s e s increased  induced  et  1984).  the h i g h e r eukaryotes,  recombination  altered  changes  (eg. C a t c h e s i d e  E s p o s i t o , 1977). Mutants  are then  Hoekstra,  of  d i s t u r b a n c e s , d e t e c t e d by  on m e i o t i c r e c o m b i n a t i o n  M a l o n e and  depressed  indicator  enhancement o r d e p r e s s i o n o f s p o n t a n e o u s and rates,  Perkins,  rec-1 i f any,  mutation of these 6  i n C.  e l e g a n s was  effects  examined  were p r e s e n t .  Finally,  elaborating  number o f g e n e t i c t o o l s allowed rec-1  several  on t h e t h i r d  objective  are available  specific  hypotheses  i n C. e l e g a n s w h i c h  on t h e m o l e c u l a r  C. e l e g a n s  i s the only higher eukaryote  nonsense  s u p p r e s s o r mutants have been i s o l a t e d  Brenner,  1978; W a t e r s t o n ,  the a p p r o p r i a t e s t r a i n , the presence ii) Baillie  to  study, a have  basis of  t o be t e s t e d :  i)  this  of t h i s  1981; H o d g k i n ,  the e f f e c t  assay  has b e e n d e v e l o p e d  determine  the mutation i f this  rate  ( W a t e r s t o n and  o f r e c - 1 on r e c o m b i n a t i o n i n  (1981) u s i n g t h e t r a n s l o c a t i o n  system,  tRNA  1 9 8 5 ) . By c o n s t r u c t i n g  o f a tRNA amber s u p p r e s s o r was  A mutation  i n which  investigated; by R o s e n b l u t h a n d  e T l as a b a l a n c e r .  Using  i n t h e Rec-1 s t r a i n was m e a s u r e d  hyper-rec  mutant a l s o  a c t s as a  mutator;  and iii) al.,  Tc1 i s a t r a n s p o s a b l e e l e m e n t  1 983).  Both  been i m p l i c a t e d Zupancic  The  i n transposition  the e f f e c t  mechanisms  that  that  these  t h e methods, r e s u l t s work. The f i n a l  results,  ( K l e c k n e r , 1981;  rec-1 i s a  recombination  work h a s b e e n o r g a n i z e d i n t o  c h a p t e r s . Each d e a l s w i t h a s p e c i f i c  to  have  o f t h e r e c - 1 on Tc1 m o b i l i t y was e x a m i n e d .  presentation of this  includes  (Emmons e t  nonhomologous and h o m o l o g o u s r e c o m b i n a t i o n  e t a l . , 1983). Given  enhancer,  i n C. e l e g a n s  reviews  s e t o f experiments  and some d i s c u s s i o n  chapter  summarizes  the proposed  mechanisms o f o t h e r  m u t a n t s a n d s p e c u l a t e s on t h e n a t u r e  mutation.  S e v e r a l experiments  are suggested  7  as i s r e l e v a n t  the s i g n i f i c a n c e of  hyper-rec  investigation.  and  of the rec-1  f o r future  II.  A)  Life  History  and  Information  General Reproductive  Caenorhabditis f e e d s on b a c t e r i a .  Background  elegans i s a free  nematode  which  I t i s h e r m a p h r o d i t i c ; however, m a l e s do  occur  s p o n t a n e o u s l y a t a f r e q u e n c y o f one 1974).  The  hermaphrodite  the  spontaneous  and  chromosome l o s s  1:1  sex r a t i o )  mating from  plates  i s XX  and  three  stages and  one  e a c h week. H e r m a p h r o d i t e s  (L1 half  t o L4) days;  is  on  indicater starvation  are passed  vulva.  This  of v i r g i n i t y conditions  growth  This  conditions  state  the dauer  Recombination  d e c r e a s e s w i t h age  B a i l l i e , 1979b).  larva  to drought,  returns  of to the per  1974).  increases with  Therefore, i n order to 8  Under  the r e t u r n  (Brenner,  and  with  t o form a dauer  larva  of  an  mean number o f p r o g e n y  i s a p p r o x i m a t e l y 300  Four  i s the  is resistant  e t c . . With  sperm  crescent  c a n be u s e d as  stage moults  hermaphrodite  (Rose and  This  new  use  increases  stage a t r a n s l u c e n t  pathway. The  (ie. a  up  1979).  f o r out-cross experiments.  starvation  developmental  setting  Carrel,  body w a l l .  1949);  nondisjunction  through over a p e r i o d  characteristic  t h e L2  (Nigon,  Male c u l t u r e s  speed of development  the hermaphrodite  high temperatures,  normal  from  (Brenner,  preferentially  (Ward and  I n t h e L4  (Daugherty e t a l , 1959).  favourable  t h e male i s XO  c a n be m a i n t a i n e d i n t h e l a b by  temperature.  undeveloped  soil  i n seven hundred  (Hodgkin e t a l , 1979).  increasing visible  living  p r o d u c t i o n of males a r i s e s  t h e male when o u t c r o s s e d  larval  Characteristics  temperature  accurately  measure t h e r e c o m b i n a t i o n individual  must be  f r e q u e n c y a l l progeny  s c o r e d and  2  o p t i m a l growth temperature is  further  c a n be  e n h a n c e d by  stored  The  guidelines  underlined, first  survive  lower  used  in this  thesis  letter  genotype  letters,  capitalized  follows  and  be u s e d  to r e f e r  like  that  o f N2  s t r a i n worms. L i n k a g e  line  underneath  t h e genes  the p u b l i s h e d  to  Genes a r e t y p e d i n have  to individuals i s denoted  a semicolon  their term  with a by  ( e g . dpy-5 dpy-14/+ +).  s e p a r a t e d by  of  the a b i l i t y  a r e n o t u n d e r l i n e d . The  shall  are  Strains  i s a group  w h i l e phenotypes  Wild-type  l i n k a g e groups  organism  freezing.  w h i c h has  o f t h e same g e n o t y p e .  case  The  Culture Conditions  o f some p a r t i c u l a r  individuals  constant.  study of t h i s  of H o r v i t z e t a l . (1979). A s t r a i n  individuals produce  to  held  an  n i t r o g e n ( H o r v i t z e t a l , 1979).  and  nomenclature  i s 2 0 C . The  its ability  in liquid  B) Nematode S t r a i n s  the temperature  from  a  phenotype continuous  Different  (eg. dpy-5/dpy-5;  unc-46/unc-46). The strain Baillie C.  Bristol  and  mutant  were o r i g i n a l l y  this  Vancouver,  Genetic Stock  strains  same e f f e c t  o f C.  most o f t h e mutant  (S.F.U.,  elegans  variety  are  identifies  Canada).  to that  f r o m N2.  in a l l strains.  s t u d y and  strains  C e n t e r . The  identical  isolated  e l e g a n s was  Table  The  studied.  were o b t a i n e d f r o m Others  N2 D.  were p r o v i d e d by  r e c o m b i n a t i o n maps o f o f N2  as a l l t h e s e  rec-1  1 lists  mutation  the  has  Table  the  these  strains the  s t r a i n s used  t h o s e c o n s t r u c t e d by me. 9  The  2  in lists  the  genes u s e d ,  illustrates  their alleles  and t h e i r p h e n o t y p e s . F i g u r e 1  the r e l a t i v e p o s i t i o n s  o f t h e s e genes on t h e g e n e t i c  map o f C. e l e g a n s . Nematodes were c u l t u r e d  i n 60 mm.  nematode g r o w t h m e d i a and s t r e a k e d 1 9 7 4 ) . S t o c k s were m a i n t a i n e d  petri  plates  with E. c o l i  filled  OP50  with  (Brenner,  a t 16 C, w h i l e a l l m a t i n g s  were  p e r f o r m e d a t 20 C. In a l l r e c o m b i n a t i o n  experiments crescent  h e r m a p h r o d i t e s were t r a n s f e r r e d allowed plates  to self every  (referred any  cross  twenty  Genetic distances configuration.  four  hours f o r three  made s c o r i n g  easier  were m e a s u r e d u s i n g  Recombination  and  culture transfers  number o f p r o g e n y on  and more  t h e markers  reliable. i n ac i s  f r e q u e n c y was c a l c u l a t e d recombinant  by p = 1 progeny  c h a r a c t e r i z a t i o n o f t h e r e c - 1 m u t a t i o n and i t s e f f e c t s processes  required  the a v a l i b i l i t y  s t r a i n s . The r e c - 1 homozygous  Baillie  (1979a) were u s e d  of several  strains described  i n t h i s s t u d y . However,  g e n o t y p e s had t o be c o n s t r u c t e d , rate  to fresh  Constructions  various  specific  plates,  1974) ( F i g u r e 2 ) .  C) S t r a i n  The  culture  days. These  2R, where R i s t h e f r a c t i o n o f v i s i b l y  (Brenner,  on  and t h e n t r a n s f e r r e d  t o as b r o o d i n g ) l i m i t e d t h e t o t a l  one p l a t e . T h i s  J l -  to separate  stage  by Rose  certain  e s p e c i a l l y f o r the mutation  experiment.  1 0  TABLE  Strain  1: S t r a i n s  Used i n t h i s  KR #  Study  Genotype  Rec-1 BC00196  36  dpy-5 dpy-14/dpy-5  dpy-14;  rec-1/rec-1  KR00177  177  dpy-5 unc-13/dpy-5 u n c - 1 3 ;  rec-1/rec-1  BC00313  25  KR00370  370  unc-46/unc-46; r e c - 1 / r e c - 1  KR00371  371  eT1/eTl;  KR00372  372  dpy-18/dpy-18;  rec-1/rec-1  KR00373  373  dpy-18/dpy-18;  unc-46/unc-46; r e c - 1 / r e c - 1 *  KR00377  377  dpy-18  BC00341  386  dpy-5 unc-13 u n c - 2 9 ;  rec-1/rec-1  rec-1/rec-1  *  *  unc-46/eT1; r e c - 1 / r e c - 1  *  rec-1  dpy-5 unc-13 unc-29 r e c - 1 BC00452  387  unc-13/unc-13;  rec-1/rec-1  N2 D e r i v e d BC00338  18  dpy-18/dpy-18  BC00356  50  unc-46/unc-46  BC00070  300  KR00353  353  BC00189  eT1/eT1 sDp2;  40  unc-13  l e t - 3 6 2 dpy-5  unc-13  unc-13/unc-13  BC00279 RW02070  l e t - 3 6 2 dpy-5  dpy-5 unc-13 unc-29/dpv-5 unc-13 653  s u p - 7 / s u p - 7 ; dpy-18/dpy-18  * C o n s t r u c t e d by B. R a t t r a y  11  unc-29  TABLE 2:  Mutations  used i n  this  ALLELE  study.  VISIBLE PHENOTYPE  GENE  LINKAGE GROUP  dpy- •5  I  (e61 )  dpy- •14  I  (e188)  dpy- 18  III  (e364)  s h o r t and f a t *; temperature s e n s i t i v e . s h o r t and f a t ; Unc-36; t r a n s l o c a t i o n ; u n c o o r d i n a t e d movement. lethal.  III,  eTl  V  l e t - 362  I  (h086)  rec- 1  ?  (s180)  unc- 1 3  I  (e51 ) (e450)  u n c - 29  I  (e403)  u n c - 46  V  (e177)  sup- 7  X  (st05)  short  and f a t ; moves wel!  increases recombination; o t h e r w i s e Wt p h e n o t y p e paralyzed; paralyzed. s l i g h t l y uncoordunated movement. very uncoordinated. amber s u p r e s s o r ; temperature s e n s i t i v e ; o t h e r w i s e Wt p h e n o t y p e .  * The Dpy a n d Unc p h e n o t y p e s a r e u n i q u e f o r e a c h gene ( i e . Dpy-5 c a n be d i s t i n g u i s h e d from D p y - 1 4 ) . W i t h t h e e x c e p t i o n o f Sup-7, a l l t h e o t h e r phenotypes a r e r e c e s s i v e .  LINKAGE GROUP  dpy-14  unc-13  dpy-5  let-362  _i u  I  unc-29  unc-54  i_  1  sDp2  0 %  U  eT1 breakpoint  III  i  h  dpy-18 i  LY  u n c - 4 6 e T 1 breakpoint • •  sup-7  FIGURE 1  '.  G e n e t i c M a p of C . elegans  a n d p o s i t i o n s of g e n e s s t u d i e d .  5  RECOMBINATION  The number o f v i s i b l y r e c o m b i n a n t p r o g e n y ( e g . Dpy-5 a n d Unc-13)  The f r a c t i o n o f v i s i b l y progeny,(R).  recombinant  2 [ § o f Dpy-5 ]  =  2 [ ff o f Dpy-5 ] T o t a l # Progeny  The  frequency of recombination: p = 1 - J l - 2R ; Where: 2R = t h e f r a c t i o n o f p r o g e n y c a r r y i n g 1 - 2R = f r a c t i o n o f p r o g e n y J l -  1 - J l -  not c a r r y i n g  a recombinant  chromosome;  a recombinant  chromosome;  2R ~ f r e q u e n c y o f a n o n - r e c o m b i n a n t e v e n t (oocyte o r spermatocyte); 2R = t h e f r e q u e n c y o f a r e c o m b i n a n t  i n e i t h e r gamete,  e v e n t i n e i t h e r gamete.  FIGURE 2: D e t e r m i n a t i o n o f p, t h e r e c o m b i n a t i o n f r e q u e n c y f o r s m a l l i n t e r g e n i c d i s t a n c e s . The c a l c u l a t i o n i s b a s e d on t h e number o f p r o g e n y t h a t v i s i b l y c a r r y a r e c o m b i n a n t chromosome. The p a r e n t a l g e n o t y p e was i n a c i s c o n f i g u r a t i o n ( e g . dpy-5 u n c - 1 3 ) . + + ( m o d i f i e d from Rose, 1980)  The  rec-1  l o c u s i s not unpubl.  gene has  linked  n o t b e e n mapped p r e c i s e l y .  t o any  o f t h e m a j o r gene c l u s t e r s  d a t a ) . H o w e v e r , R o s e has  by r e c - 1  t o u n c - 5 4 a t t h e end  Rec-1  recently detected  frequencies to i d e n t i f y  were t h e n  necessary  w e r e s e l f - c r o s s e d and One  to c a l c u l a t e segregants.  Wild-type  background  male progeny  d o u b l e m u t a n t . F2  individuals  t h e d e s i r e d p h e n o t y p e was  from a "high r e c o m b i n a t i o n "  p l a t e and  u s e d as  F i g u r e 3. The  s t r a i n Unc-46 Rec-1  dpy-5 t o dpy-14 i n t e r v a l on  h o m o z y g o t e s . Rec-1  h e r m a p h r o d i t e s and Dpy-14 Rec-1  of  was  c o n s t r u c t e d as  in  l i n k a g e group I  hermaphrodites.  f o r u n c - 4 6 . The The  t h e r e s u l t a n t males were c r o s s e d  ( F i g u r e 3 ) . One  The  counting  Dpy-5 and  recombination considered  segregating  t o Dpy-5  F2 w e r e h e t e r o z y g o u s f o r dpy-5 were e i t h e r r e c - 1 / r e c - 1  crossed  and  brooded.  f r e q u e n c i e s w e r e c a l c u l a t e d f r o m t h e F3 Dpy-14 worms. A d r a m a t i c  frequency  or  h a l f o f t h e F2 w e r e a l s o h e t e r o z y g o u s  F2 h e r m a p h r o d i t e s w e r e s e l f  recombination  was  m a l e s w e r e c r o s s e d w i t h Unc-46  dpy-14 i n a c i s c o n f i g u r a t i o n and  rec-1/+  picked  the p r o g e n i t o r  u s e d d u r i n g c o n s t r u c t i o n s as a means o f i d e n t i f i n g  and  then  strain.  For example, the  rec-1  recombination  t h e i r r e c o m b i n a n t progeny were c o u n t e d .  hermaphrodite having  t h e new  m a l e s . The  c r o s s e d w i t h a Rec-1  linkage  phenotype, t h e r e f o r e , d u r i n g  the rec-1/rec-1  w i t h Rec-1  (Rose,  loose  Hermaphrodites of the genotype r e q u i r e d i n a rec-1 were o u t - c r o s s e d  rec-1  of l i n k a g e group I .  does not have a v i s i b l e  s t r a i n c o n s t r u c t i o n s i t was  The  ( i e . approximately  t o i n d i c a t e a Rec-1  strain.  Ten  were p i c k e d from s e v e r a l h i g h r e c o m b i n a t i o n 15  by  increase i n threefold) Unc-46  was  individuals  l i n e s and  progeny  Po: rec-1 rec-1  [males]  a  [herm.]  F1 : ; rec-1 [males] +  X  dpy-5 dpy-14 ; r e c - 1 dpy-5 dpy-14 rec-1 [herm.]  F2: dpy-5 dpy-14 + +  ; rec-1 or rec-1 (self-cross)  rec-1; or + [herm.]  _+_  +  V  F3:  a) S c o r e r e c o m b i n a t i o n frequencies b) P i c k 10 a/a worms ( p h e n o t y p e = A) f r o m a p l a t e with a high recombination frequency. e  9*  a  i  r e c - 1 ; dpy-5 dpy-14 o r rec-1 (self-cross)  + + + +  V F4:  Progeny t e s t - p i c k the p l a t e which does not s e g r e g a t e Dpy-5 Dpy-14 worms.  FIGURE 3: C o n s t r u c t i o n o f r e c - 1 homozygous s t r a i n s . The procedure i n v o l v e d using the p r e v i o u s l y constructed double Dpy m u t a n t , Dpy-5 Dpy-14 R e c - 1 . " a " i s any m u t a t i o n w h i c h p r o d u c e s a d i s t i n g u i s h a b l e p h e n o t y p e when homozygous e g . Unc-46.  16  t e s t e d . Of t h o s e i n d i v i d u a l s w h i c h d i d n o t s e g r e g a t e Dpy-5 Dpy-14 p r o g e n y , (ie.  one was p i c k e d a s t h e p r o g e n i t o r o f t h e s t r a i n  unc-46/unc-46; r e c - 1 / r e c - 1 ) . The same m e t h o d was u s e d f o r t h e s u b s e q u e n t  the  strains.  I f t h e new  r e c - 1 , then a l l brooded  v e r i f i c a t i o n of  s t r a i n was a c t u a l l y homozygous f o r F2 h e r m a p h r o d i t e s w o u l d  have a h i g h  recombination frequency. Table 3 presents the r e s u l t s t e s t i n g t h e Unc-46 Rec-1 s t r a i n . R e c o m b i n a t i o n the  from  frequenciesf o r  d p y - 5 t o dpy-14 i n t e r v a l a r e p r o v i d e d f o r known Rec-1 a n d N2  s t r a i n s t o a l l o w comparison with the c o n s t r u c t i o n data. Despite the  e x p e c t e d l a r g e c o n f i d e n c e i n t e r v a l s on t h e e s t i m a t e s  o b t a i n e d f r o m i n d i v i d u a l h e r m a p h r o d i t e s , i t c a n be c o n c l u d e d that this  strain  i s homozygous f o r r e c - 1 .  The s t r a i n Dpy-18 Unc-46 Rec-1 was c o n s t r u c t e d f r o m t h e Dpy-18 Rec-1 and t h e Unc-46 Rec-1 s t r a i n s .  No r e c o m b i n a t i o n  m e a s u r e s w e r e r e q u i r e d a s b o t h s t r a i n s w e r e a l r e a d y homozygous for  r e c - 1 . Unc-46 Rec-1 h e r m a p h r o d i t e s w e r e c r o s s e d w i t h Rec-1  males.  The h e t e r o z y g o u s m a l e p r o g e n y  were t h e n c r o s s e d w i t h  Dpy-18 Rec-1 h e r m a p h r o d i t e s . Ten F2 h e r m a p h r o d i t e s w e r e c r o s s e d and brooded.  self  One h a l f o f t h e s e worms w e r e e x p e c t e d t o be  h e t e r o z y g o u s f o r b o t h u n c - 4 6 and d p y - 1 8 . F i n a l l y ,  a  single  s e g r e g a t i n g d o u b l e m u t a n t ( i e . Dpy-18 Unc-46) was p i c k e d f r o m the  F3 t o e s t a b l i s h t h e d e s i r e d s t r a i n  ( i e . Dpy-18  Unc-46  Rec-1). The r e c i p r o c a l t r a n s l o c a t i o n eT1 l i n k a g e group  ( i e . the r i g h t h a l f of  I I I a n d t h e l e f t h a l f o f V) p r o d u c e s  an  u n c o - o r d i n a t e d p h e n o t y p e ( U n c - 3 6 ) when i n t h e homozygous ( R o s e n b l u t h and B a i l l i e ,  1 9 8 1 ) . An e T l Rec-1 s t r a i n 17  was  state  Table  Strain  3: V e r i f i c a t i o n  of s t r a i n  N  # Proqeny  N2  9  2,907  40  Rec-1  10  3,104  1 65  1  331  1  Unc-36; * Rec-1  Unc-46; Rec-1  #  Recombinants  construction  **  Recomb .  Freq.  1 .4 %  _+  0.4  5.3  %  +_  0.6  17  5.3  %  +_  2.5  335  17  5.2  %  +_  2.4  1  307  17  5.7  %  +_  2.7  1  359  15  4.3  %  +_  2.2  1  267  17  6.6  %  +  3.2  1  353  19  5.5  %  1  1 96  14  7.4  %  +  3.8  1  245  13  5.5  %  +_  3.0  1 73  8  4.7  %  +  2.0  247  1 6  6.7  %  +  3.2  1  2.3  * Unc-36 i s t h e p h e n o t y p e o f eT1 h o m o z y g o t e s . * R e c o m b i n a t i o n f r e q u e n c y f o r t h e dpv-5 dpy-14  18  interval.  constructed  T h i s was t h e n u s e d  to construct the s t r a i n  dpy-18  r e c - 1 / r e c - 1 . T h i s was o b t a i n e d by c r o s s i n g Rec-1  hermaphrodites.  dpy-18 u n c - 4 6 / e T 1 ; r e c - 1 / r e c - 1  Rec-1 m a l e s w i t h e T l  The F2 W i l d t y p e  and dpy-18/+;  r e c - 1 / r e c - 1 . The e T l b a l a n c e d s t r a i n  eT1  produces  genotypes lethal).  pseudolinkage  of both Progeny  the phenotypic strain  unc-46/+; by t h e  o f brooded  F2 worms  Dpy-18 o r Unc-46 p h e n o t y p e s  (ie.  between dpy-18 a n d u n c - 4 6 ; t h e  from  ratio  dpy-18 unc-46/eT1 o f 4 Wt  : 1 Unc-36  by a l w a y s  individuals  occur  in  : 1 Dpy-18 Unc-46. The  transferring  Wild-type  ( i e . dpy-18 u n c - 4 6 / e T l ; r e c - 1 / r e c - 1 ) .  To d a t e ,  15 Rec-1 s t r a i n s  have b e e n c o n s t r u c t e d by e i t h e r  Dr. Rose o r m y s e l f . The a n a l y s i s rec-1  were  Dpy-18 and Unc-46 w o u l d be a n e u p l o i d a n d h e n c e  i s maintained  individuals  of either  progeny  was i d e n t i f i e d  s e g r e g a t i o n o f Unc-36 worms and t h e f a i l u r e segregate progeny  unc-46/eT1;  The F1 m a l e s were t h e n c r o s s e d w i t h  Dpy-18 Unc-46 Rec-1 h e r m a p h r o d i t e s .  to  (Table 3 ) .  a s d e s c r i b e d f o r t h e Unc-46 Rec-1 s t r a i n  mutation  and c h a r a c t e r i z a t i o n  i s b e c o m i n g more t r a c t a b l e  become a v a i l a b l e  i n a rec-1  background.  19  of the  as a d d i t i o n a l  markers  III.  Recombination  Effects  Introduction:  Isolated eukaryotes  i n C. e l e g a n s ,  which  recombination been working has  and  i s a g e n e r a l and u n i f o r m  (Rose and B a i l l i e ,  the l i n k a g e groups.  each  (1979a) t h a t  fairly  uniform  compared  extent  of the i n c r e a s e ranges  literature.  and  these  supports  to other hyper-rec from  that  two t o s i x - f o l d ,  m u t a n t s have b e e n r e p o r t e d i n  S e v e r a l have been i s o l a t e d repair  i n Escherichia  processes  1980; P u k k i l a e t a l . , 1 9 8 3 ) . Examples  recombination  upon m e i o s i s o t h e r t h a n  Kafer,  reducing f e r t i l i t y  M a l o n e and H o e k s t r a ,  recombination  1969; H o l l i d a y  increases the  b u t h a s no  uvs-3 i n  and uvs-1 i n U s t i l a g o  (Schroeder,  e t a l . , 1976).  effect  ( G o l i n and E s p o s i t o ,  1984). S i m i l a r i l y ,  uvs-B i n A s p e r g i l l u s  and  i n eukaryotes are  spontaneous m i t o t i c  frequency,  coli  (eg. Glickman  t o come by. I n y e a s t , rem-1  increase mitotic  with a  of three to f o u r - f o l d .  have had d e f e c t i v e  Neurospora,  the increase i s  mutants i e . t h e  much more d i f f i c u l t  1977;  on a l l  t h e c o n c l u s i o n o f Rose  i t demonstrates  few h y p e r - r e c o m b i n a t i o n  the  Radman,  o f t h e m a j o r gene c l u s t e r s  rec-1 i s a g e n e r a l enhancer o f  In a d d i t i o n ,  predominant e f f e c t  o f r e c - 1 on one t o two p e r c e n t  This data  recombination.  Very  ( u n p u b l . d a t a ) has  on mapping t h e r e c - 1 l o c u s . A t t h e same t i m e s h e  associated with  Baillie  enhancer o f m e i o t i c  1 9 7 9 a ) . Rose  o b t a i n e d d a t a on t h e e f f e c t  intervals of  rec-1 i s the only mutation i n  only  1970; S h a n f i e l d and  C(3)G,  a r e c e s s i v e mutation  suppresses  intra-  completely  when homozygous  produces  large,  frequencies by  for  Hinton,  effect  nonuniform  a variety  a review  1966;  1966;  1 9 6 8 ) . The  P r o c u n i e r and  Suzuki,  1972).  melanogaster recombination  effect  can  be  including (Ramel,  respectively  t h e as m u t a n t  i n a nonuniform  in  and Zea  pattern  1963). Baillie  (1979a) have shown t h a t  o f t h e above e x a m p l e s  recombination  i s uniform  i n that:  1)  throughout  increase i n recombination  regardless  of the  interval  i t s effect  frequency  mutations)  (unlike (Rose,  purpose  the m i t o t i c  over  effect  of the experiments of the  recombination,  frequencies  from  on  is  same  found  2)  i t acts  o f rem-1  and  other  such  1980).  the c h a r a c t e r i z a t i o n Intragenic  differs  or l i n k a g e group examined. T h i s  c o n t r a s t s w i t h t h e D r o s o p h i l a m u t a n t s ; and meiotically  rec-1  t h e genome. The  three-fold  The  Carlson,  1967,  see L u c c h e s i , 1 9 7 6 ) . F i n a l l y ,  Rose and each  but  compound chromosomes  mays i n c r e a s e s m e i o t i c r e c o m b i n a t i o n (Miller,  and  o f chromosomal r e a r r a n g e m e n t s and  almost  t h e genome when  r e p o r t e d i n D.  Suzuki,  translocations,  Hinton,  Gowen, 1 9 2 2 ; ) ,  increases i n meiotic  ( L u c c h e s i and  inversions, 1962;  (Gowen and  (Gowen, 1933;  melanogaster,  meiotic recombination  i n c r e a s e throughout  interchromosomal  produces  induced  intergenic  a nonuniform  heterozygous The  and  in Drosophila  rec-1  r e p o r t e d here mutant  i n C.  gene c o n v e r s i o n and  was  of determining  frequency  over a broad  the e f f e c t  range  of rec-1  recombination  on  the  recombination  of g e n e t i c d i s t a n c e s . 21  extend  elegans.  l a r g e d i s t a n c e s were e x a m i n e d , w i t h  intention  to  The gene unc-13 was and an  gene c o n v e r s i o n . analysis  This  for several  (1979) have d e v e l o p e d elegans;  chosen t o study gene was  reasons:  particularily  first,  a procedure  (e450) a l l e l e s  and  Baillie,  unc-13 a l l e l e s  recombination over  Rec-1  large genetic  distances.  a homozygous  markers  (dpy-5 and unc-13) were  fifteen  map  units  to the l e f t  to balance  The  over  configuration  during  distance  control"  rec-1 s t r a i n  close  was  together  m u t a t i o n and  was  construction. scored  three  such t h a t  while  two  let-362  was  was  suppress the c i s  I n t h i s manner t h e l a r g e  a t t h e same t i m e as t h e interval  served  smaller  as an " i n t e r n a l  t o i n d i c a t e t h e p r e s e n c e o f a homozygous r e c - 1 during  Briefly,  homozygous  construction.  found  that  t h e Rec-1  approximately  constructed  the c o n s t r u c t i o n  of t h i s  three  exhibited  and gene  an  conversion  times g r e a t e r  than  f r o m t h e KR386 and KR387 s t r a i n s .  protocol  rec-1 d u p l i c a t i o n b e a r i n g  explanations  strain  intragenic recombination  to a l e v e l  heterozygotes  Secondly,  strain  i t was  i n both  frequencies the  both  containing  the marker i n t e r v a l s t o m a i n t a i n  Hence, t h e s m a l l e r  individual  increase  (Rose  protocol  o f d p y - 5 . The d u p l i c a t i o n  the l e t h a l  recombination  interval.  i n C.  reported  strains containing  m a r k e r s and t h e d u p l i c a t i o n sDp2. The d e s i g n  genetic  studies  t o examine t h e e f f e c t o f r e c - 1 on  constructing  required  Baillie  were a v a i l a b l e .  I a l s o attempted  involved  s u i t e d f o r such  f r e q u e n c y b e t w e e n t h e (e51)  o f unc-13 has b e e n p r e v i o u s l y  1 9 8 0 ) ; and t h i r d ,  recombination  Moerman and  for intragenic  second, the r e c o m b i n a t i o n  and  intragenic  followed strain.  r e s u l t a r e examined.  d i d not produce a Possible  Methods:  a ) I n t r a g e n i c Recombination of  rec-1  on  following  intragenic  and  e v e n t s was  Gene C o n v e r s i o n - The d e t e r m i n e d by u s i n g  2) u n c - 1 3 ( e 4 5 0 ); r e c - 1 unc-13(e450) rec-1 s c r e e n i n g method was Unc-13 males.  (e450)  Rec-1  Heterozygous  m o d i f i e d f r o m Rose and hermaphrodites  male p r o g e n y  individuals  both a l l e l e s  plates two  and  heterozygous  o f unc-13 p r e s e n t  hermaphrodites  Unc-13 i n d i v i d u a l s  (unc-13  Unc-13  (e450)  in this  Exceptional  from  performed  Rec-1  to loci,  with  4). Paralyzed individually  on  A l l p l a t e s were s c r e e n e d f o r  intervals  Dpy-5 Unc-13  to unc-29). to avoid  could  d i d not a f f e c t  and  Dpy-5 Unc-13  overcrowding.  n o t be  distinguished  the r e s u l t s  of  c r o s s - o v e r o r c o n v e r s i o n e v e n t s would  be  the  case. progeny  movement) and Unc-29 picked  flanking  were p l a c e d  i s epistatic  from the h e t e r o z y g o t e s ; t h i s  detected  (Figure  homozygous i n d i v i d u a l s  s c r e e n as n e i t h e r  hermaphrodites  segregants included  worms were removed a t r e g u l a r  (1980).  were c r o s s e d w i t h  f o r t h e two  allowed to s e l f - c r o s s . normal  Baillie  ( i e . unc-13/+; r e c - 1 / r e c - 1 )  (Unc-13 p h e n o t y p e )  g e n e r a t i o n s . The  and  .  were mated t o Dpy-5 Unc-13 Unc-29 Rec-1 produce  the  strains: 1) dpy-5 u n c - 1 3 ( e 5 1 ) u n c - 2 9 ; r e c - 1 ; dpy-5 u n c - 1 3 ( e 5 1 ) unc-29 rec-1  The  effect  consisting  (eg. s l i g h t l y  the background  to d i s t i n g u i s h  of Wild-type  (eg.  u n c o - o r d i n a t e d ) worms were  o f Unc-13 worms. P r o g e n y  between s i n g l e  23  normal  testing  c r o s s - o v e r and  was  apparent  CONSTRUCTION: rec-1 rec-1  [ m a l e s ] X unc-13 unc-13  (e450 ); r e c - 1 (e450) r e c - 1  [herm.]  u n c - 1 3 ; r e c - 1 X dpy-5 unc-13 (e51) u n c - 2 9 ; r e c - 1 + rec-1 dpy-5 unc-13 (e51 ) unc-29 r e c - 1 [males] [herm.]  dpy-5  unc-13 (e51)  + (e450) unc-13  unc-29; +  rec-1 rec-1  SCREEN: Po:  1 h e t e r o z y g o u s Unc-13 h e r m a p h r o d i t e p e r p l a t e ; [Self-cross];  F1 : 1/2 o f p r o g e n y ( a p p r o x . 65 p e r p l a t e ) h a v e t h e same g e n o t y p e a s t h e Po. T h e s e a r e s e l f - c r o s s e d ; Dpy Unc i n d i v i d u a l s a r e removed t o a v o i d o v e r c r o w d i n g a n d a s s i s t s c o r i n g i n t h e F2. F2: s c r e e n p l a t e s f o r e x c e p t i o n a l p r o g e n y e g . Wt ( n o r m a l movement) i n an Unc ( p a r a l y z e d ) b a c k g r o u n d . E x c e p t i o n a l Progeny from Wt  eg. S i n g l e + +  or  above a r e : Unc-29 Apparent e g . D o u b l e C r o s s -- o v e r  Cross-over  ++ unc-13  dpy-5 +  unc-29 ; +  or + dpy -5  ++ unc-13  ++ unc-29 + unc-1 3 or  + +  unc-29 . unc-29  ++ unc-13  + +  or + dpy-5  ++ unc-13  + unc-29  FIGURE 4: C o n s t r u c t i o n o f a h e t e r o z y g o u s Unc-13 Rec-1 s t r a i n and i t s u s e i n m e a s u r i n g i n t r a g e n i c r e c o m b i n a t i o n .  24  double cross-over the  events  exchange o f f l a n k i n g  e x c e p t i o n a l progeny intragenic either  genotype  1)  a chromosome  unc-13 +  + +  + unc-13 + dpy-5 + unc-29  the will  > 3 Wt  : 1 Unc-13;  > 3 Wt  : 1 Dpy-5 Unc-13  (Rose and B a i l l i e ,  be p r o v i d e d  be r e f e r r e d  conversion  later.  frequencies  : 2 Wt  s c r e e n e d was  o f t h e mean number o f p r o g e n y  remainder of the t e x t .  progeny  ratios:  > 1 Dpy-5 Unc-29  number o f chromosomes  cross-overs w i l l  unlike  on t h e  exceptional  phenotypic  > 1 Unc-13  4) dpy-5 unc-13 unc-29 dpy-5 + unc-29  generation  single  gene c o n v e r s i o n e v e n t s e g r e g a t e d  2) dpy-5 unc-13 unc-29 + + +  basis  progeny,  o r c o n v e r s i o n genotypes. Depending  i n one o f t h e f o l l o w i n g  The t o t a l  Unc-29  p r o d u c e d by a  e v e n t s e g r e g a t e d Unc-29  due t o a p u t a t i v e  + +  3)  W i l d - t y p e and  o f t h e h o m o l o g o u s chromosome,  recovered progeny  markers.  carrying  cross-over  parental  ( i e . gene c o n v e r s i o n ) by l o o k i n g f o r  1980)  ;  : 1 Dpy-5 Unc-29;  : 2 Wt  : 1 Dpy-5 , Unc-13  e s t i m a t e d on t h e  per hermaphrodite per (Figure  5 ) . Apparent  double  t o as gene c o n v e r s i o n s t h r o u g h o u t  Justification  Calculations  for this  interpretation  f o r r e c o m b i n a t i o n and  are presented i n Figures  6 and 7  respectively.  b) R e c o m b i n a t i o n examining follows: strain  the e f f e c t Rec-1  over l a r g e  distances  - The p r o t o c o l f o r  o f r e c - 1 on l a r g e g e n e t i c  d i s t a n c e s was  m a l e s were c r o s s e d w i t h a d u p l i c a t i o n - b e a r i n g  ( i e . sDp2/let-362  dpy-5 u n c - 1 3 / l e t - 3 6 2 dpy-5 25  unc-13).  as  GIVEN:  THEN:  1)  dpy-5 +  (e51)(+) unc-29 (+)(e450) + (self-crossed)  2)  Mean # p r o g e n y p e r X = 127 ;  3)  Total  ;  hermaphrodite:  # F1 = X [ § C u l t u r e P l a t e s ] ; = (127)(210) = 27,000 ;  4) # F1 Unc-13 H e t s =  27,000 ] ; 2 = 13,500 ;  5) T o t a l  § F2 = ( 1 2 7 ) [ 13,500 ] ; = 1.7 m i l l i o n ;  6) T o t a l  # screened  = # F1 + # F2 ; = 1.7 m i l l i o n .  THEREFORE: A p p r o x i m a t e l y 1.7 m i l l i o n i n d i v i d u a l s o r > 3 m i l l i o n chromosomes were s c r e e n e d . Sample c o u n t s were made and a s i m i l a r v a l u e was o b t a i n e d .  FIGURE 5: Sample s i z e e s t i m a t e f o r t h e number screened i n the intragenic experiment.  o f chromosomes  GIVEN: 1)  dpy-5 +  (e51) (+) unc-29 (+)(e450) + (self-cross)  V Recombinants a)  + +  ++ unc-13  unc-29; +  b)  + dpy-5  ++ unc-13  unc-29; unc-29  c ) dpy-5 +  (e51)(e450) + (e450)  d) dpy-5 dpy-5  2) R e c o m b i n a t i o n i s r e c i p r o c a l , i e . #(a)  + ; +  (e51)(e450) + . (e51) + unc-29 + #(b)  = #(c) + # ( d ) ;  3) R e c o m b i n a n t chromosomes i n (a) and (b) a r e a l w a y s " v i s i b l e " as Wt and Unc-29, r e s p e c t i v e l y ; however, ( c ) and (d) c a n n o t be d i s t i n g u i s h e d f r o m t h e s e g r e g a n t s (Unc-13, Dpy-5 Unc-13), THEN: 4)  Total  # Recombinant  Chromosomes = 2[ # Unc-29 + # Wt  ].  GIVEN: 5) Unc-29 i s u n d e r r e p r e s e n t e d due t o t h e d i f f i c u l t y o f s c o r i n g t h i s p h e n o t y p e on a b a c k g r o u n d o f p a r a l y z e d worms (eg. Unc-13); 6) B a s e d on t h e P u n n e t t S q u a r e : # Wt 7) Recomb. F r e q . : p = T o t a l  = #  Unc-29;  § R e c o m b i n a n t Chromosomes; T o t a l # Chromosomes  THEN: 9)  p = 2[  4[ # Wt ] Total # Individuals  ; ]  THEREFORE: p  = [ Total  2[ # Wt ] # Individuals  Screened ]  FIGURE 6: C a l c u l a t i o n o f i n t r a g e n i c r e c o m b i n a t i o n f r e q u e n c y . I n t r a g e n i c recombinants a r e d i s t i n g u i s h e d from c o n v e r t a n t s ( a p p a r e n t d o u b l e c r o s s - o v e r e v e n t s ) by e x a m i n i n g t h e i r p r o g e n y and l o o k i n g f o r t h e e x c h a n g e o f f l a n k i n g m a r k e r s i n t h e s e g r e g a n t s . N o t e t h a t unc-13 i s e p i s t a t i c t o u n c - 2 9 .  27  GIVEN:  1)  dpy-5 +  (e51)(+) unc-29 (+)(e450) + (self-cross)  i Convertants Genotype a) b) c) d) e) f) g) h)  Phenotype  dpy-5 (+)(+) unc-29/dpy-5 unc-13 u n c - 2 9 ; dpy-5 ( + )( + ) unc-29/+ unc-13 +; dpy-5 ( e 5 1 ) ( e 4 5 0 ) unc^29/dpy-5" unc-1 3 u n c - 2 9 ; dpy-5 ( e 5 1 ) ( e 4 5 Q ) unc-29/+ unc-13 + ; + (+)(+) +/dpy-5 unc-13 u n c - 2 9 ; + (+)(+) + / + unc-13 +; + ( e 5 1 ) ( e 4 5 0 ) +/dpy-5 unc-13 u n c - 2 9 ; + ( e 5 1 ) ( e 4 5 0 ) + / + unc-13 +;  Dpy-5 Unc-29; Wt; * Dpy-5 Unc-13; Unc-13; Wt; Wt; Unc-13; Unc-13;  2) H a l f o f t h e c o n v e r t a n t s c a n be d i s t i n g u i s h e d f r o m t h e s e g r e g a n t s : a) Dpy-5 Unc-29, b) Wt, e) Wt, and f ) Wt i n d i v i d u a l s c a n be p i c k e d f r o m a b a c k g r o u n d o f Dpy-5 Unc-13 and Unc-13 worms; i e . # C o n v e r t a n t Chromosomes = 2 ( # wt + # Dpy-5 Unc-29 ) ; 3) The Dpy-5 Unc-29 worms a r e u n d e r r e p r e s e n t e d due t o t h e d i f f i c u l t i e s i n s c o r i n g t h i s p h e n o t y p e i n t h e above b a c k g r o u n d ; THEN:  4) # C o n v e r t a n t  Chromosomes = 2 (4/3 [§ Wt  c o n v e r t a n t s ] );  GIVEN:  5) C o n v e r s i o n F r e q . =  # c o n v e r t a n t chromosomes T o t a l # chromosomes s c r e e n e d  THEN:  6) C o n v e r s i o n F r e q . =  4/3 [ # Wt ] [ # individuals ]  THEREFORE: Conversion Frequency  =  4/3 [ # Wt ] [ Total # Individuals  ]  FIGURE 7: C a l c u l a t i o n o f c o n v e r s i o n f r e q u e n c y . The c o n v e r t a n t s a r e d i s t i n g u i s h e d f r o m i n t r a g e n i c r e c o m b i n a n t s by e x a m i n i n g t h e i r p r o g e n y and l o o k i n g f o r t h e a b s e n c e o f f l a n k i n g m a r k e r exchange i n the s e g r e g a n t s . * unc-13 i s e p i s t a t i c t o u n c - 2 9 .  28  The  F1 h e t e r o z y g o t e s  individuals carrying (ie.  and t h o s e w i t h o u t  sDp2  (Figure  sDp2 were s e l e c t e d on t h e b a s i s  8 ) . Four i n d i v i d u a l s "  of slower  y o u n g e r a p p a r e n t age on b r o o d e d p l a t e s )  unco-ordinated and  consisted of duplication-bearing  ratio.  segregated  i n a 6 Wt  : 1 Unc-13  T h e o r e t i c a l l y , one h a l f o f t h e W i l d t y p e worms  were d u p l i c a t i o n b e a r e r s . (Figure  slightly  movement. T h e s e h e r m a p h r o d i t e s were s e l f - c r o s s e d  b r o o d e d . The p r o g e n y  phenotypic  and  developement  8 ) . One q u a r t e r  A l l o f t h e Unc-13 worms c a r r i e d sDp2  o f t h e Unc-13 worms were e x p e c t e d  homozygous f o r r e c - 1 ( b a s e d on i n d e p e n d e n t a s s o r t m e n t ) . Unc-13 h e r m a p h r o d i t e s were p i c k e d individually These  as p o s s i b l e  independent  strain  lines  h o m o z y g o t e . F o r any g i v e n  Unc-13 h e r m a p h r o d i t e s . T h i s without  interval and  threefold  scored  (Figure  (1979a) and was  increase.  frequencies  given  self  8 ) . The e f f e c t  with  progeny  crossed,  b r o o d e d and  o f r e c - 1 on t h e s m a l l  c a l c u l a t e d over  accordingly.  as a n i n t e r n a l  found  to e x h i b i t at least  i f t h e Unc-13  t e s t e d were r e c - 1 / r e c - 1 , the small  In e f f e c t ,  map  a strain  e x h i b i t i n g an i n c r e a s e  interval  let-362  t o dpy-5  unit distance. i n recombination  the e f f e c t 29  the recombination  a homozygous r e c - 1  unit interval  o v e r t h e dpy-5 unc-13 i n t e r v a l ,  strain  t h e dpy-5 unc-13  a t t h e same t i m e as t h e two map  a  i n t e r v a l were e x p e c t e d t o  control to identify  b a c k g r o u n d . The f i f t e e n scored  Rec-1 m a l e s were c r o s s e d  time the hermaphrodite  Therefore,  hermaphrodites being  increase  a rec-1  dpy-5 t o unc-13 h a s b e e n p r e v i o u s l y e x a m i n e d by Rose  Baillie  acted  progenitors.  t h e d u p l i c a t i o n were p i c k e d ,  recombinants  Five  p e r p l a t e and c u l t u r e d  were t e s t e d t o i d e n t i f y  line,  t o be  o f r e c - 1 on  was  Hence, frequency  CONSTRUCTION: Po: [males] rec-1 rec-1  F1 :  X  let-362 let-362  dpy-5 dpy-5  unc-13 unc-13  [herm]  V let-362 +  dpy-5 unc-13; rec-1 + + (self-cross)  and  let  dpy  unc; rec-1  V F2:  Pick, f i v e Unc-13 h e r m a p h r o d i t e s f r o m f o u r p l a t e s ; S e t up i n d i v i d u a l l y , a s p o s s i b l e s t r a i n p r o g e n i t o r s + + let-362 dpy-5 unc-13; r e c - 1 ; o r r e c - 1 ; o r + ; let-362 dpy-5 unc-13 rec-1 + +  eg.  STRAIN  TEST:  B a s e d on random s e g r e g a t i o n , 1/4 o f t h e s e p o s s i b l e p r o g e n i t o r s were e x p e c t e d t o be r e c - 1 h o m o z y g o t e s . T h i s was t e s t e d by c r o s s i n g them t o Rec-1 m a l e s eg. [males]  rec-1 rec-1  X  let-362 let-362  V let-362  py-s  unc-13; + + (self-cross)  d  dpy-5 unc-13; dpy-5 unc-13 [herm.]  (rec-1 ? ) ; ( r e c - 1 ?)  ( r e c - 1 ? ) ; [herm.] rec-1  SCORE: A recombination f r e q u e n c y o v e r t h e dpy-5 t o unc-13 i n t e r v a l o f a b o u t 6 % i n d i c a t e s t h a t t h e s t r a i n was homozygous r e c - 1 . * D u p l i c a t i o n b e a r i n g i n d i v i d u a l s i n t h e F1 were i d e n t i f i e d t h e i r s l o w e r d e v e l o p m e n t a l t i m e and s l i g h t l y uncoordinated movement.  by  FIGURE 8: S t r a i n c o n s t r u c t i o n p r o t o c o l t o d e t e r m i n e t h e e f f e c t o f r e c - 1 on l a r g e g e n e t i c d i s t a n c e s .  30  recombination  frequency over  the large  interval  c o u l d be  examined s i m u l t a n e o u s l y .  Results:  a) I n t r a g e n i c R e c o m b i n a t i o n determine  the e f f e c t  - The unc-13 gene was s t u d i e d t o  o f r e c - 1 on i n t r a g e n i c  gene c o n v e r s i o n . A s t r a i n  heterozygous  (e450) was a l l o w e d t o s e l f - c r o s s Exceptional Table were and  f o r the a l l e l e s  (e51)  and  f o r two g e n e r a t i o n s .  p r o g e n y were e x a m i n e d .  4 shows  performed;  the r e s u l t s the r e s u l t s  of these experiments. of the r e p l i c a t e s  were t h e r e f o r e c o m b i n e d . O n l y  reported  r e c o m b i n a t i o n and  i n Table  calculating  trials  consistent  recombinants are  4, and o n l y W i l d - t y p e worms were u s e d i n  the recombination  difficulties  the Wild-type  were  Two  scoring  f r e q u e n c i e s . T h i s was due t o  Unc-29 worms on a b a c k g r o u n d o f Unc-13  worms. The  f r e q u e n c i e s of both  conversion events strain.  c r o s s - o v e r and gene  i n c r e a s e d two t o t h r e e f o l d  Confidence  intragenic  single  intervals  recombination,  i n t h e Rec-1  do n o t o v e r l a p i n t h e c a s e o f  but j u s t  o v e r l a p f o r t h e gene  conversion frequencies. The  coefficient  o f c o i n c i d e n c e ( c . c . ) f o r N-2 was  calculated  as:  c.c.  =  observed frequency of apparent double c r o s s over events; expected frequency o f double c r o s s over events  31  TABLE 4:  Number o f Recombinants Rec-1 N2  Number o f Convertants  intragenic  # Chromosomes Screened  12  3.4  million  5  3  2.2  million  The  double  recombination,  Recomb. Freq. -6 26 x 10 -6 8.7 x 10  Conversion Freq. * -6 9.3 x 10 -6 3.5 x 10  crossovers.  p a r e n t a l g e n o t y p e and m a t i n g was: dpy-5 (e51 ) ( + ) unc-29; r e c - 1 + (+) (e450) + rec-1 (self-cross)  CONFIDENCE  N2  e f f e c t o f r e c - 1 on gene c o n v e r s i o n .  22  * Apparent  Rec-1  The and  o r +_ +  INTERVALS:  Recombination Lower l i m i t Upper l i m i t -6 -6 19 x 10 32 x 10 -6 -6 4.2 x 10 16 x 10  Confidence  intervals  Conversion Lower l i m i t Upper l i m i t -6 -6 7.0 x 10 12.2 x 10 -6 -6 1.5 x 10 7.0 x 10  are c a l c u l a t e d  as  i n Stevens  (1942)  where  the observed  the expected  f r e q u e n c y was  v a l u e was  unc-29  (e51) and  by t h e r e c o m b i n a t i o n  b) R e c o m b i n a t i o n  (e450) ( i e . 10  %)  f r e q u e n c y between unc-13 and  -6 3.5 X 10 (0.02)(10"*)  c.c. =  Over L a r g e  = 17.5;  D i s t a n c e s - I n o r d e r t o examine t h e  o f r e c - 1 on r e c o m b i n a t i o n o v e r  attempted  to construct a s t r a i n  that  large distances I w o u l d measure  f r e q u e n c y o v e r a two p e r c e n t and a f i f t e e n t h e same t i m e . As o u t l i n e d strain  frequency -5  ( i e . 2%). ie.  effect  as i n F i g u r e 7 and  equal to the recombination  between t h e unc-13 a l l e l e s multiplied  calculated  recombination  percent i n t e r v a l at  i n t h e Methods s e c t i o n  the intended  t o be c o n s t r u c t e d was:  s D p 2 / l e t - 3 6 2 dpy-5 u n c - 1 3 / l e t - 3 6 2 E x p r e s s i o n o f l e t - 3 6 2 was Unc-13 h e r m a p h r o d i t e s possibly  independent testing  lines.  During'subsequent  self  and h e n c e  crossed to establish  out c r o s s i n g  and p r o g e n y  one q u a r t e r o f t h e t w e n t y one l i n e s were e x p e c t e d  significant  distance  the c o n s t r u c t i o n  g e n o t y p e were  homozygous f o r r e c - 1 . However,  control  s u p p r e s s e d by t h e d u p l i c a t i o n .  o b t a i n e d from  of the d e s i r e d  dpy-5 u n c - 1 3 ; r e c - 1 / r e c - 1 .  none o f t h e s e  increase i n recombination  interval  dpy-5 t o unc-13  l e t - 3 6 2 t o dpy-5  lines  frequency  showed  for either  t o be a the  (2%) o r t h e l a r g e g e n e t i c  (15%) ( T a b l e 5 ) .  Discussion:  To c o n f i r m t h e o c c u r r e n c e o f gene c o n v e r s i o n one recover  must  b o t h p r o d u c t s o f a g i v e n e x c h a n g e and show t h e  nonreciprocal  nature of the event. 33  I n C. e l e g a n s  the g e n e t i c  T a b l e 5: P u t a t i v e Rec-1 d u p l i c a t i o n - b e a r i n g s t r a i n s . T h e s e were i s o l a t e d f r o m c o n s t r u c t i o n s i n t e n d e d t o d e t e r m i n e t h e e f f e c t o f r e c - 1 on l a r g e g e n e t i c d i s t a n c e s .  Parental Wt Dpy Unc 482 405 309 450 244 491 395 506 407 488 280 376 358 470 51 4 339 364 511 423 41 9 351  Recombinants Unc-13 Dpy-5  56 37 29 54 32 53 32 49 44 48 30 33 30 48 41 39 42 47 44 30 35  5 10 8 6 3 9 5 7 3 5 6 4 .3 5 6 3 7 7 8 4 6  0 1 1 0 0 0 1 0 1 1  Recombination unc-13 t o dpy-5 1 .4% 3.4% 3.5% 1 .8% 1 .6% 2.4% 1 .7% 1 .9% 1 .0% 1 .4% 2.8% 1 .4% 1.1% 1 .5% 1 .6% 1 .2% 2.5% 1 .9% 2.5% 1 .3% 2.3%  1 1 1 1 1 1 1 1 0 1  (0.7 t o + 1 .4 + 1 .5 (0.9 t o (0.6 t o + 1.0 (0.8 t o (1 .0 t o (0.4 t o (0.7 t o (1 .5 t o (0.6 t o (0.4 t o (0.7 t o (0.8 t o (0.1 t o (1.4 t o (1 .0 t o + 1 .2 (0.6 t o (1 .2 t o  Frequency dpy-5 t o l e t - 3 6 2  2.5%)*  3.1%) 3.5%) 3.2%) 3.1 %) 2.2%) 2.5%) 4.9%) 2.9%) 2.5%) 2.6%) 2.8%) 2.6%) 4.3%) 3.1 %) 2.6%) 4.0%)  To s c o r e t h e r e c o m b i n a t i o n f r e q u e n c y when one markers i s a l e t h a l mutation use: R = 4/3  2 [ Recombinant c l a s s ] [ Wt + Dpy Unc + Unc + 2 Dpy  then, the recombination as i n F i g u r e 2. * Confidence  interval  frequency  calculated  34  16.9% 13.1% 13.4% 17.4% 19.0% 14.4% 12.1% 14.1% 15.6% 14.3% 14.2% 13.0% 12.7% 14.8% 12.1% 16.0% 16.0% 13.4% 15.0% 11.0% 14.4%  of  the  ; ]  i s calculated  as  i n Stevens,  1942  + + +_ _+ +_ + +_ _+ +_ +  + + + + +_ + +  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  .0 .2 .5 .0 .2 .1 .2 .0 .1 .1 .4 .2 .2 .0 .0 .3 .2 .0 .1 .2 .3  tools necessary  to perform  However, a l i n e  o f argument c a n be d e v e l o p e d  suggests  that  conversion there  the experiments  i s reasonably high interference  in  males.  was  double  than  in a coefficient This indicates  o c c u r r e d than expected.  Pritchard,  1960)  not been found  the  system  used  strongly  (Finnerty,  gene  melanogaster, The  b e e n e x a m i n e d on l i n k a g e group  my  the  that  The  cross-over  et a l . ,  1979).  the data  experiments  f a r more d o u b l e  classical  on with  explanation of a  ( i e . high negative  i s possible  but u n l i k e l y  i n C.  i s the d e t e c t i o n 1976).  C a r l s o n , 1971)  and  (Chovnick  and  B a l l a n t y n e and  Chovnick,  1970)  and  compound autosomes  the rudimentary  1971)  are used  the r o s y l o c u s  locus (Carlson,  completely accurate i n i d e n t i f y i n g  Rose and  Baillie  unc-13 a l l e l e s  (1980) c a l c u l a t e d  (e51 ) and  1970;  to provide a (Chovnick et a l ,  1971)  the absence was  of  found  gene c o n v e r s i o n e v e n t s .  t h e d i s t a n c e between  (e450) i n t h N2 35  1970;  et a l ,  o u t s i d e marker exchange i n r e c o v e r e d w i l d - t y p e f l i e s t o be  best  o f gene c o n v e r s i o n  and  In both  given  e l e g a n s . The  (Smith e t a l ,  analysis.  (see  cross-over  I n D r o s o p h i l a , a t t a c h e d X chromosomes  halftetrad  X  IV i n  o f c o i n c i d e n c e o f 17.5  previously  e x p l a n a t i o n of these r e s u l t s  available.  elegans.  (Hodgkin  from  i n c r e a s e i n exchange events  i t has  on  expected  c r o s s - o v e r events  section).  interference;  by  and  i n D.  d e s p i t e such h i g h i n t e r f e r e n c e ,  unc-13 r e s u l t e d Results  which  t h e number o f r e c o v e r e d d o u b l e  much l e s s  Secondly, apparent  o v e r has  i n hermaphrodites  In both cases  progeny  that  crossing  are not  r e p o r t e d here demonstrate as  l i n k a g e group  local  analysis  i n t h e unc-13 gene. F i r s t ,  frequency of double  events  a tetrad  strain  t o be  8.0  X  the  -6 10  map  units. This  controls map  f o r the  consistent  4). In the  frequency  with  the  i s less  slightly.  However, g i v e n  i t would  Rose and on  Since  that  distances  strains  rec-1  and  at  determine the The in  the  the  study  overlap gene of  l a c k of  o f one  the  same  statistical  rather  than  a  i t was over  was  not  21  separated  l a r g e r d i s t a n c e s . The  designed  t o answer t h i s  t h a t was  known t o be  of rec-1 the  on  the  by  a larger  36  to  attempts affected  work question  affected  by  by  interval.  d e s i r e d homozygous r e c - 1  tested  five  same c o n s t r u c t i o n ,  probability  lines  percent.  c l e a r whether rec-1  the  of  several  comm.). T h e s e c o n s t r u c t i o n  to recover  from the  effect  t o two  to construct  duplication construction i s puzzling  strain  seen and  products  limitations  order  same t i m e , w i t h i n  independent assortment, Rec-1  i n the  interval  effect  failure  end  is  conversion  e v e n t s and  c o n t a i n i n g markers  frequencies  examining a marker  on  intervals  t h a t the  attempted  (Rose p e r s .  in this  Rose  (1979a) have d e m o n s t r a t e d t h e  s u c c e s s f u l and  recombination  different  4). This  increases  of rec-1  cross-over  sample s i z e  t i m e , Rose has  units  performed  that  10  intragenic  3 and  confidence  the -6  rec-1.  Baillie  homozygous r e c - 1  were n o t  to  by  genetic  frequency  effect  of  X  t h r e e - f o l d (Table  seem most l i k e l y  i s due  lack of e f f i c a c y  rec-1  the  a r e b e l i e v e d t o be  significance  t e n map  c l e a r as  results  (p = 8.7  the  (compare T a b l e  4 ) . The  frequency  process,  strain  recombination  1979a t o T a b l e  conversion  Rec-1  increases  between i n t e r g e n i c m a r k e r s Baillie,  the  experiments performed here  u n i t s ; Table  recombination  i s i n agreement w i t h  of  (Table not  5).  strain Assuming  recovering  i s 0.24%. However,  a  Rose  (unpubl.  loose  l i n k a g e of  group I by  d a t a ) has  (p = 0.27  rec-1  possible earlier  rec-1  to the  recently obtained t o unc-54 a t t h e  explanation  interest  f o r the  Rose  (unpubl.  to the  unc-13 gene c l u s t e r  units  to the  left  place  rec-1  between 35  right  of unc-13. U s i n g  be  and  recovered  explanation  Rec-1  of  of  the An  of the  rec-1  strains  This using  the  map  a  linkage  (24  map  This  units to  strains  would  the  distances expected  to  c o n s t r u c t i o n i s b e t w e e n 3 and  5.  p o s s i b l e , but  I find  an  unsatisfactory. A  i s s u e r e q u i r e s a more p r e c i s e mapping  d u p l i c a t i o n , sDp2, h o m o z y g o s i t y f o r r e c - 1  is  m a r k e r s on  inability  c o u l d be  to  t o sDp2 a r e  circumstances.  strain  i n support  a sDp2 Rec-1  repeating  l i n k a g e group  chromosome). D u p l i c a t i o n a s s o c i a t e d i f a Rec-1  evidence  isolate  t e s t e d by  a different  comparable  only  i s that  failure the  The  strain  f o r the  in  i s the  by  is loosely  right.  speculative explanation  individual.  hypothesis  explanation  i f rec-1  were r e c o v e r e d ,  l i n k a g e t o be  linkage  locus.  to the  duplications  shown any  number o f Rec-1  c o n s t r u c t i o n t o p r o d u c e a Rec-1  possibility  of  However,  l i n k a g e group I  t h a n 50  of  linkage  range of p o s s i b l e l i n k a g e  attempted  a l t e r n a t i v e but  presence of lethal  greater  this  c o n c l u s i v e answer t o t h i s  5.  p o s i t i o n e d to the  unc-13 t h e  from the  G i v e n t h a t no  and  of  construction  not  of unc-54). Therefore,  t o unc-54 i t must be  between r e c - 1  on  end  i n Table  d a t a ) has  suggestive  possibility  i n the  results  rec-1  linked  right  _+ 0 . 1 6 ) . T h i s makes t h e  markers of  work by  data  the  (eg.  a v a i l a b l e on lethality  c a n n o t be  of  strain.  experiment several the  X  becomes a  isolated  this  under  likely these  IV.  Pleiotropic  Effects  Introduction:  It  i s important  pleiotropic provide  effects  some i n s i g h t  t o know t h e n a t u r e  and e x t e n t o f t h e  o f t h e rec-1 enhancer. into  Such i n f o r m a t i o n may  the basis of the mutation  and t h e  mechanism o f i t s i n f l u e n c e  on r e c o m b i n a t i o n .  Rec-1  i n p o p u l a t i o n experiments f o r  strain  studying  i s t o be u s e d  the e v o l u t i o n a r y importance  discussed  i n the f i r s t  m i g h t have on p r o c e s s e s  Moreover,  of recombination  chapter), the a d d i t i o n a l other than  i f the  (as  e f f e c t s rec-1  recombination  must be  documented. Several hyper-rec strains  types of " s i d e  and r e c o m b i n a t i o n  have a d r a m a t i c  increased meiosis.  effects"  aneuploidy  on f e r t i l i t y .  i n yeast,  Gowen,  Klapoltz  the s p o r u l a t i o n  ( E s p o s i t o and E s p o s i t o ,  e t a l . , 1985; H o l l i d a y  e t a l . , 1976; Gowen and  1922).  Radiation recombination of  include:  spo-1 t o s p o - 1 1 , uvs-1 i n U s t i l a g o  m a y d i s and c ( 3 ) G i n D. m e l a n o g a s t e r 1969;  T h i s i s due t o  by s e g r e g a t i o n d i s o r d e r s d u r i n g  Examples o f s u c h m u t a t i o n s  d e f e c t i v e mutants  both  d e f e c t i v e m u t a n t s . A number o f  affect  caused  are associated with  sensitivity  i s also  hyper-rec  o f a number o f  m u t a n t s . I n t h e Rad-3 and Rad-6 e p i s t a t i c  yeast, c e r t a i n mutations  recombination.  typical  Similarily,  increase radiation both  induced  of t h e spontaneous,  m u t a n t s , uvs-1 i n U. m a y d i s and rem-1 38  groups mitotic  mitotic  i n yeast, are  radiation sensitive  ( H a y n e s a n d K u n z , 1981). A s c o s p o r e  i s a l s o a f f e c t e d by t h e s e m u t a n t s , w i l d - t y p e gene p r o d u c t i n n o r m a l melanogaster, and  s u g g e s t i n g some r o l e f o r t h e  m e i o t i c development.  mei-9 d r a m a t i c a l l y reduces  i s defective i n excision repair  other r e c o m b i n a t i o n mutants  I n D.  recombination frequency  (Boyd e t a l . , 1976). S e v e r a l D5 (1)  ( e g . mei-41  defective i n postreplicative repair Recombination  viability  , mus  , etc.) are  (Boyd and S e t l o w ,  1976).  mutants a r e a l s o o f t e n found t o a f f e c t t h e  m u t a t i o n r a t e . The h y p e r - r e c m u t a n t s i n E. c o l i ,  f o r example,  produce  (eg. Marinus  a dramatic increase i n the mutation rate  and K o n r a d , All  1976; B a l e e t a l . ,  1979; A r t h u r a n d L l o y d ,  o f these mutants i n v o l v e d e f e c t s i n a r e p a i r  resulting  1980).  system,  i n n i c k s a n d b r e a k s i n t h e DNA. F o r e x a m p l e , t h e dam  m u t a n t s a r e due t o t h e l o s s o f m e t h y l d i r e c t e d m i s m a t c h producing a hyper-rec, hyper-mutable  phenotype  repair,  (Glickman and  Radman, 1980; P u k k i l a e t a l , 1983). Recombination  a s s o c i a t e d , spontaneous  mutation rate  changes  h a v e a l s o b e e n r e p o r t e d i n y e a s t . Spo-7, a r e c o m b i n a t i o n d e f e c t i v e mutant, has a reduced m i t o t i c m u t a t i o n r a t e et a l . ,  (Esposito  1975). C o n v e r s e l y , G o l i n a n d E s p o s i t o (1977) h a v e shown  t h a t rem-1  i n c r e a s e s b o t h m i t o t i c m u t a t i o n and r e c o m b i n a t i o n . I t  h a s b e e n s u g g e s t e d t h a t rem-1  causes  i t s mitotic  hyper-rec  p h e n o t y p e by p r o d u c i n g DNA l e s i o n s w h i c h a r e t h e n c o r r e c t e d by an e r r o r p r o n e  r e c o m b i n a t i o n - r e p a i r system  (Malone  and H o e k s t r a ,  1984). The  m e i o t i c m u t a t i o n r a t e , as w e l l as t h e r e c o m b i n a t i o n  f r e q u e n c y , a r e a f f e c t e d by mu i n D. m e l a n o g a s t e r Green,  (Green,  1976). The e f f e c t o n r e c o m b i n a t i o n i s n o n u n i f o r m ; 39  1970;  crossing all  over  on  t h e t i p o f t h e X chromosome i s d e c r e a s e d ,  other regions are unaffected. F i n a l l y ,  Moerman and  (1984) h a v e d e s c r i b e d t h e o n l y known m u t a t o r effect  on  evidence  recombination that  suggests  that  Finally, and  Beadle  have  the mutator phenotype i s caused  Tc1.  T h i s w o u l d make any  effect  segregation disorders resulting have b e e n f o u n d  mutants. S t u d y i n g  (1936) were t h e  tetrads.  recombination that  first  D.  t o be  associated with  melanogaster,  Sturtevant  to describe a dramatic  B a k e r and  events  properly with frequency, pairing,  i s necessary  i t s homologue. G i v e n  chromosomes t e n d  Grell,  1962;  pairing;  increases  (eg. f l i e s  decrease  and  i f recombination  may  nondisjunction (Hall, mei-1  and  i n higher plants,  a t metaphase I w i t h  the concluded  for regular segregation. a chromosome  size  pairs  recombination  (ie. distributive  decreases, nondisjunction  homozygous f o r c ( 3 ) G , B a k e r and  Hall,  n o t h o l d t r u e as b o t h when h e t e r o z y g o u s ,  but  alleles do  not  1972).  Mei-2 b o t h  i n c r e a s e n o n d i s j u n c t i o n (Smith,  mutants,  by  in  1976). T h i s i n c r e a s e s the chance of  increase recombination  In Neurospora,  have  a reduction i n  to p a i r  1 9 7 6 ) . However, t h e c o n v e r s e c(3)G  that  and  increase i n  (1976) h a v e r e v i e w e d  i n D r o s o p h i l a and  appear to ensure  incorrect  of  Hall  d e f e c t i v e mutants  normal recombination  Crossover  on  in nondisjunction  X chromosome n o n d i s j u n c t i o n a s s o c i a t e d w i t h a d e c r e a s e cross-over  by  unlikely.  chromosome l o s s  recombination  Waterston  elegans. I t s  i s n o t known. However, t h e y  the t r a n s p o s a b l e element, recombination  i n C.  while  40  recombination  1975); w h i l e a s y n d e t i c  have a h i g h  recombination  decrease  frequency  reduced  on  of u n i v a l e n t s  the b i v a l e n t s  (eg.  Beadle, al.,  1933;  Thomas and  Rajhathy,  S j o d i n , 1970;  Baker  et  1976). To  date,  elegans  investigations  have f o c u s e d on  s t u d y i n g the rec-1  i t s effect  p r e v i o u s work by  Hartman and  is  sensitive.  not  radiation  rec-1,  on  Herman To  In  viability,  complete  summary, i t was  fecundity, v i a b i l i t y  found and  that  mutation  C.  although  shown t h a t  of t h i s  mutation  rec-1  in  rec-1  the c h a r a c t e r i z a t i o n  effects  s e g r e g a t i o n and  enhancer  recombination,  (1982) has  I have examined the p o s s i b l e  fecundity,  on  1966;  had  rate.  no  mutation  detectable affect  However,  a n d / o r chromosome l o s s o f t h e X l i n k a g e g r o u p  appeared  i n c r e a s e d t w o - f o l d . Compared t o  recombination  mutants,  rec-1  has  on  rate.  nondisjunction t o be  of  surprisingly  other few  pleiotropic  effects.  Methods:  a) F e c u n d i t y genotypes culture  Crescent  (rec-1 /rec-1  plates,  maintained  and  +_/+)  self-crossed 2  a t 2 0 C and  stage hermaphrodites were t r a n s f e r r e d  and  brooded.  of to  both separate  C u l t u r e s were  t h e number o f p r o g e n y were c o u n t e d  when  mature.  b) of  Competitive A b i l i t y  both  petri  genotypes  plate  (rec-1 /rec-1  and  crescent stage  hermaphrodites  +_/+_) were p l a c e d on  a 100  c o n t a i n i n g nematode g r o w t h medium c o v e r e d w i t h  lawn o f w i l d - t y p e E. until  - Three  starvation  coli.  The  p o p u l a t i o n was  symptoms a p p e a r e d 41  allowed  a  t o grow  i n t h e most r e c e n t p r o g e n y  mm  (ie.  dauer  l a r v a e were p r o d u c e d ) . The p o p u l a t i o n  transferred  weekly  to fresh  a g a r cube c o n t a i n i n g culture  and p l a c e d  grew t h e p l a t e , stress.  about  culture  100 worms was c u t f r o m  by o u t - c r o s s i n g  exposed  of t e n hermaphrodites  The  were i n d i v i d u a l l y  o f males  p l a c e d on c u l t u r e  ( o r chromosome  ( i e . XO i n d i v i d u a l s )  d) M u t a t i o n R a t e  i n t h e Rec-1 S t r a i n  i n t h e Rec-1 s t r a i n  methods o u t l i n e d  using  were c o u n t e d  not c a r r y a spontaneous  lethal  t o ensure  The F2 was c o m p r i s e d  rec-1/rec-1 The and  i n the F1.  M u t a t i o n r a t e was  individually that  dpy-18 u n c - 4 6 / e T l ; (Figure 10).  p s e u d o - l i n k a g e b e t w e e n dpy-18  unc-46 and s u p p r e s s e s r e c o m b i n a t i o n w i t h i n 42  1  t h e P o s used  t o eT1/+; r e c - 1 / r e c - 1  o f two g e n o t y p e s :  eT1 p r o d u c e s  of the  (1981).  and dpy-18/+; unc-46/+; r e c - 1 / r e c - 1  translocation  brooded.  m u t a t i o n . A l l o f the progeny  o f an a c c e p t a b l e Po were t h e n c r o s s e d males.  were  was  l o s s ) was a s s a y e d by  a modification  Dpy-18 Unc-46 Rec-1 h e r m a p h r o d i t e s  did  -  plates f o r  20 C a n d 26 C;  found  i n R o s e n b l u t h and B a i l l i e ,  The p r o g e n y  determined  stage  r e c - 1 / r e c - 1 a n d +_/+) . E a c h h e r m a p h r o d i t e  t h e number  brooded.  crescent  (eg. temperature:  amount o f n o n d i s j u n c t i o n  estimated  were  i n t h e F2 g e n e r a t i o n ( F i g u r e 9 ) .  each e x p e r i m e n t a l c o n d i t i o n genotype:  starvation  +; r e c - 1 / r e c - 1 m a l e s a n d t h e n  c ) N o n d i s j u n c t i o n - Twenty t o t h i r t y hermaphrodites  to a  over  (approximately sixteen  t o dpy-5 dpy-14/+  the recombinants  the o l d  As t h e worms r a p i d l y  t h e y were r e g u l a r i l y  A t t h e e n d o f two months  scoring  p l a t e s . A two by two cm.  o n t o a new p l a t e .  g e n e r a t i o n s ) t h e genotypes  was t h e n  the translocated  1)  [males]  rec-1; rec-1  X  dpy-5 dpy-5  dpy-14; r e c - 1 ; dpy-14 r e c - 1  [herm.]  V 2)  [males]  dpy-5 +  dpy-14; r e c - 1 ; + rec-1  X  _+_', o r r e c - 1 ; [herm. ] + rec-1  V  [herm.] dpy-5 +  3)  dpy-14 +  or  + +  rec-1 +  or rec-1; rec-1  (self-cross)  SCORE:  V a) I f t h e r e c o m b i n a t i o n f r e q u e n c y i s h i g h t h e n t h e Po was homozygous r e c - 1 . b) I f t h e r e c o m b i n a t i o n f r e q u e n c y was ( e g . 1.5%) t h e n t h e Po was N2.  in  a t a normal  FIGURE 9: Assay f o r d e t e r m i n i n g the genotypes the p o p u l a t i o n experiment.  43  (eg about  5.0%)  level  of competitors  CONSTRUCTION:  Po:  1 hermaphrodite  (self-crossed) unc-46 unc-46  dpy-18 dpy-18  rec-1 rec-1  V F1  D e t e r m i n e t h a t t h e Po i s n o t c a r r y i n g a l e t h a l by c o u n t i n g t h e number o f p r o g e n y - a l e t h a l would r e d u c e t h e f e c u n d i t y . P i c k 100 F1 [herm.] (genotype as above)  X  eT1 +  ; rec-1 [males] rec-1  V F2:  S e t up 2,000 p l a t e s w i t h 1 Wt h e r m a p h r o d i t e e a c h and a l l o w t o s e l f - c r o s s i e .  dpy-18 unc-46; r e c - 1 eT1 +  and  dpy-18 +  ; unc-46 +  ;  rec-1 +  SCREEN:  V F3:  screen f o r : a b s e n c e o f Dpy worms; a b s e n c e o f Unc worms; reduced # of progeny.  s c r e e n f o r absence of Dpy Unc i n d i v i d u a l s  FIGURE 10: S t r a i n c o n s t r u c t i o n and p r o c e d u r e t o d e t e r m i n e t h e e f f e c t o f r e c - 1 on t h e s p o n t a n e o u s m u t a t i o n r a t e . The m u t a t i o n r a t e was m e a s u r e d by s c r e e n i n g f o r l i n k e d l e t h a l s i n t h e r e g i o n b a l a n c e d by e T l . Dpy-18 and unc-46 a r e p s e u d o - l i n k e d when b a l a n c e d by e T l .  44  regions lethal  ( a p p r o x i m a t e l y 40 map mutation,  units).  In t h e absence  t h e p r o p o r t i o n o f Dpy-18 Unc-46  t h e F3 w o u l d be 1 / 6 t h .  mutation i n  i n the absence  Unc-46 worms i n t h e F 3 . Any p l a t e w i t h two o r l e s s was p r o g e n y  tested  for a putative  lethal  unc-46 l e t h a l / e T l ;  self-crossed).  The p r o g e n y  carrying for  t h e w i l d - t y p e chromosome  the presence  reduced  number  of a l i n k e d  rather  lethal  o f Unc-46 worms; a n d t h e r e d u c e d  unc-46/eTl; was  stable  crossing  rec-1/rec-1)  i n the presence  over  hermaphrodites  t h a n eT1 were  screened  by n o t i n g : t h e a b s e n c e  number  f o r several  . T h i s was  ( i e . dpy-18  of  of  or reduced  or  numbers  progeny.  At the completion of the s c r e e n , w i l d - t y p e were a l l o w e d t o s e l f - c r o s s  o f Dpy-18  Dpy Unc worms  mutation  o f Dpy-18 worms; t h e a b s e n c e  linked  individuals i n  The o c c u r r e n c e o f a l e t h a l  t h e a r e a b a l a n c e d by eT1 w o u l d r e s u l t  of a  hermaphrodites  generations  to determine  ( i e . dpy-18  i f the s t r a i n  o f r e c - 1 ( i e . d o e s eT1  suppress  i n the rec-1 homozygote).  Results:  a) F e c u n d i t y - The mean number for  t h e N2 and Rec-1  individuals of  strains  of progeny  Table  6a shows t h e e f f e c t  number. No d i f f e r e n c e was  means o f t h e f e c u n d i t y  hermaphrodite  were compared by b r o o d i n g  and c o u n t i n g a l l p r o g e n y .  r e c - 1 on p r o g e n y  per  found  e s t i m a t e s o f Rec-1 a n d N2  45  between t h e  strains.  TABLE 6: The e f f e c t o f r e c - 1 on f e c u n d i t y and c o m p e t i t i v e a b i l i t y  a)  Fecundity: Mean # Progeny*  Standard Deviation  - Rec-1  281  39.9  4,774  N2  276  38.5  1,657  * Self  b)  Total Progeny  P >0.50  crossed.  Competition: Time (weeks) 0 8  Generation  0 16  Genotype Rec-1 N2 3 4  46  3 6  b) the  Competitve  Rec-1  Rec-1  strain  Ability  a population  hermaphrodites  environment.  The  results  Table  B o t h Rec-1  substantial  was  N2  difference  of  i n which limited  were d e t e r m i n e d  t e s t e d were  of the genotype and  performed  worms i n a f o o d  of ten i n d i v i d u a l s  hermaphrodites  selected.  no  experiment  competed w i t h N2  Genotypes  16 g e n e r a t i o n s . The  6b.  - In order to assess the v i a b i l i t y  after  randomly  assay are presented i n  i n d i v i d u a l s were p r e s e n t . T h e r e  i n the c o m p e t i t i v e a b i l i t y  was  of the  two  strains. Moreover,  no d i f f e r e n c e was  t i m e s o f t h e Rec-1  and N2  n o t e d between t h e  strains  during  the maintenance  cultures.  F r e s h s t o c k p l a t e s were e s t a b l i s h e d  from both  strains  p l a t e s was  nondisjunction strain. in  - The  difference  indicator  chromosome l o s s  strain  was  cultured  temperature  and  the absence  sample  size,  temperature  abnormalities  t o N2  so does  of a s i g n i f i c a n t the experiment  worms  rate.  on  o f t h e amount o f  taking  was  no  place  i n the  significant  (Table  Rec-1  ( i e . N2)  phenocopies  predominated  increases  Baillie  with  the frequency of males. result  might  be a  Given  consequence  repeated at a higher be  hermaphrodites  with multiple  greater. d i d not  v u l v a and  t o s u c h an e x t e n t t h a t 47  statistical  7 ) . Rose and  t h e number o f m a l e s w o u l d  the c o n t r o l  develope properly;  C,  nondisjunction  therefore,  so t h a t  Unfortunetly,  a t 20  found r e l a t i v e  (1979b) have shown t h a t  of  and  f r e q u e n c y of males o c c u r r i n g  u s e d as an  and/or  of  D e s p i t e an a p p a r e n t d o u b l i n g i n t h e f r e q u e n c y o f m a l e s  t h e Rec-1  that  weekly  were f o u n d t o m a t u r e a t t h e same  c) Nondisj u n c t i o n brooded  developmental  other  t h e worms  TABLE 7: The e f f e c t o f r e c - 1 on n o n d i s j u n c t i o n a n d / o r chromosome l o s s .  Temperature  # Males  Sample  Size  Rec-1 N2  20°C 20°C  19 3  4,896 1,657  Rec-1 N2**  26°C 26°C  32 17  1,825 1,933  # Males per  * N o t s i g n i f i c a n t a t t h e 5% l e v e l **From Rose and B a i l l i e , 1979b.  48  1,000  3.9* 1.8 17.5 8.8  (P=0.15).  could  n o t be r e l i a b l y  experiment They may  were s l i g h t l y  The  when e x p o s e d  experiment  that  upper  was  limit  than t h e i r  c a n n o t be evident a  2 6 C Rec-1  used  2  tolerance  counterparts.  stage i n  increase.  too c l o s e  o f C.  - i t may to the  e l e g a n s . Note effect  i n C. e l e g a n s . A l t h o u g h i t  c o n s i d e r e d t o be a p r o p e r c o n t r o l ,  a two-fold increase  compares t h e f r e q u e n c y o f m a l e s f o u n d  experiment  in this  (1979b) h a v e e x a m i n e d t h e  on n o n d i s j u n c t i o n  i f one  used  unsuccessful  ( i e . 2 6 C ) was  Baillie  Rec-1  sensitive  t o the temperature  of the temperature  of temperature  hermaphrodites  r e p e a t e d , b u t was  the temperature  however, t h a t Rose and  is  younger  N2  have b e e n a t a t e m p e r a t u r e  development  be  s c o r e d . The  w i t h t h e N2  results  o f Rose and  i n the  Baillie  (1979b).  d) M u t a t i o n R a t e on t h e m u t a t i o n r a t e  i n t h e Rec-1 was  Strain  e x a m i n e d by  scoring  recessive,  lethal  by  s u c h m u t a t i o n s were i s o l a t e d  e T l . No  mutations  chromosomes o f t h e Rec-1 confidence -3  interval  as  - The  i n t h e 40 map  strain  (Table  i n Stevens  for  unit  from  spontaneous,  region  balanced  t h e 1,877  8). C a l c u l a t i n g  (1942), the upper  the  limit  per f o r t y  is  units.  T h i s o v e r l a p s e x t e n s i v e l y w i t h the c o n f i d e n c e i n t e r v a l  t h e N2  strain  by R o s e n b l u t h e t a l . ( 1 9 8 3 ) . The  not appear  to induce r e c e s s i v e  Wild-type h e t e r o z y g o t e s which several  mutations  screened  spontaneous,  reported  lethal  of rec-1  x 10  m u t a t i o n does  recessive,  effect  g e n e r a t i o n s p r o v e d t o be  dpy-18 u n c - 4 6 / e T l ; r e c - 1 / r e c - 1 ) . No  49  map of  rec-1  lethals.  were a l l o w e d t o s e l f stably  1.6  cross for  b a l a n c e d by eT1 ( i e .  Dpy-18 o r Unc-46  TABLE 8 : The e f f e c t o f r e c - 1 on t h e f r e q u e n c y of r e c e s s i v e , l e t h a l mutations.  # Lethals  Sample  Size  Rec-1  0  1,871  N2**  2  3,1 98  Frequency*  Conf. I n t e r v a l  0 6.2  x  0 10  -  to  4  to * P e r 4 0 map u n i t s . ** From R o s e n b l u t h e t a l . ,  1983.  1.6  1 .0 2 .3  x x  x 1 0 "  10 10  " J " 3  3  recombinants the  were r e c o v e r e d .  translocated region  Crossing  i n the  Rec-1  over d i d not  occur  within  strain.  Discussion:  Rec-1  appears t o have i n c r e a s e d  m a l e s by the  a factor  of  two  (Table  p r e l i m i n a r y work o f Rose  r e s u l t s were n o t sample s i z e event  obtained;  restrictions.  ( i e . XXX  however, t h i s Males are  either  chromosome l o s s  i n C.  are  ranging  from  2 to  There are r e c - 1 : The  two  24%  t h e N2  without  and  Herman, 1 9 8 2 ) . The  between t h e effect The  by  most l i k e l y putative  recovered  i n t h e Rec-1  due  in  strain.  increased  Him  this  In  and/or  other and  mutants: a s e r i e s  proportion  of  to  reciprocal  both n o n d i s j u n c t i o n  (eg. the  mutations  i s rad-4 which  chromosome by  recombination  an  the  significant  of  males,  35%).  only  first  value,  XO;  was  of  with  X-chromosome n o n d i s j u n c t i o n  elegans  increased  mutants which s e g r e g a t e  i s i n agreement  (1980). S t a t i s t i c a l l y  chromosome l o s s were i n c r e a s e d mutants  r a t e of p r o d u c t i o n  7). This  h e r m a p h r o d i t e s ) were n o t  experiment. Therefore,  segregation  the  elegans  increases  comparable  recombination  decreases X nondisjunction altering  authors  i s due  i n C.  t h a t the  affect  to d i f f e r e n c e s i n genetic  mutant and  control  strains,  rather  the  to one-tenth  autosome s e g r e g a t i o n  suggest  on  to  (Hartman  X of  and  on  background t h a n any  real  rad-4.  second  to one-eighth  i s him-8 of  the  N2  ( e l 489) value  which decreases X  and 51  increases  recombination  X-chromosome  nondisjunction autosomes a r e Goldstein complete  ( i e . segregating not  affected  (1 982 ) has  35%  males).  As  with  rad-4,  (Hodgkins e t a l , 1979). Moreover,  shown t h a t him-8  synaptonemal complexes;  ( e l 489 ) has  therefore,  structurally  i t appears  that  homologue p a i r i n g i s n o r m a l . Recently, allele  P r a t t and  i t s normal  nondisjunction.  The  nondisjunction  Drosophila  level  The  and  mutants  r e l a t i o n s h i p between by  f i t this  the  most o f t h e  may  affect  only  nondisjunction if  rec-1  small  Rec-1  strain Him  effect  recombination  1976;  Grell,  Esposito,  i t increases  s t r a i n were (Table  lethality  Similar results 1969). T h i s  X-chromosome s e g r e g a t i o n , effect  o f him-8  (Table on  6b),  of  1976). both  6a  not  and  b).  mutants i s o l a t e d  mutants  are  i s high,  found  suggests that similar  by  to  and in rec-1  the  ( d e s c r i b e d a b o v e ) . However,  caused autosomal n o n d i s j u n c t i o n  X l i n k a g e group a  taking place.  ( E s p o s i t o and  to  X  l a r g e amount o f a u t o s o m a l n o n d i s j u n c t i o n  hence a n e u p l o i d y yeast  Hall,  of the  Hodgkins et a l . (1979). In these reflecting  new  him-8 i s t y p i c a l  p a t t e r n as  t h a n t h e N2  i s i n c o n t r a s t with  a  nondisjunction.  viability  different  r a d - 4 and  ( B a k e r and  apparently  f e c u n d i t y and  significantly  dramatically increases  illustrated  meiotic  recombination  and  inverse  R e c - 1 , however, does n o t  This  ( p e r s . comm.) have i s o l a t e d  o f him-8 t h a t d e c r e a s e s X-chromosome r e c o m b i n a t i o n  1/80th o f  and  Herman  i t would be  f e c u n d i t y . A very  a t the  even  same r a t e as  difficult  to d e t e c t  l a r g e sample s i z e w o u l d  the such be  needed t o determine i f autosomal d i s j u n c t i o n i s a c t u a l l y altered.  Finally,  the  above r e s u l t s  findings  o f Hartman and  Herman  are  c o n s i s t e n t with  the  (1982) w h i c h showed t h a t Rec-1  not r a d i a t i o n 52  is  sensitive.  Most r a d i a t i o n  wild-type  and  B a k e r and  Hall,  o f t e n have r e d u c e d  Rosenbluth mutation  1976; and  unc-72  translocation.  this  the  Yeast  Baillie  (Rosenbluth  that  this  also  strain,  related  the  "captured" over  t h e b a l a n c e r eT1  strain  not  strain  radiation i s due  Hyper-rec increased  lethal  d i d not  (Table 8). This r e s u l t , is  in fact,  to develope  1985). In  effect  rec-1  sensitive,  to a r e p a i r  prepared  mutation  in  yeast,  increases mitotic  of  t e n and  i n c r e a s e s the c ( 3 ) G has  no  on  process  rate.  i n such  recessive,  To  a manner unc-46  be  lethal  the  fact  that  mutations that  the  rec-1  Rec-1  defect.  rate;  may  o r may  n o t have  however, a l l t h o s e  system  deficiency.  ( e g . A r t h u r and intergenic  with  on t h e  an  examined  I n E.  coli,  10  100-fold  to  L l o y d , 1 9 8 0 ) . Rem-1,  recombination  spontaneous mutation affect  might have  generation.  mutants a r e a l l hyper-mutable, rate  performing  would then  i n the next  mutants i n o t h e r organisms  i n mutation  an  i n a dpy-18;  makes i t u n l i k e l y  system  increases  a  available  increase i n mutation  i n conjunction with  have shown some s i g n o f r e p a i r  Drosophila,  elegans  to the recombination  induce  spontaneous mutation  the hyper-rec  1975).  t h e C.  i t was,  induce mutations  resultant  Rec-1  Laskowski,  that  s c r e e n was  could putatively  as  screen to e l i m i n a t e crossing  b a c k g r o u n d . The  The  any  e l i m i n a t e any  possibility  rec-1  and  e t a l . , 1983;  a b a l a n c e r i n the  over would then avoid  determined  r a t e m i g h t be  Using  - Kowalski  s c r e e n i n g technique p r e v i o u s l y not  s c r e e n u s i n g a Rec-1  itself.  as r o b u s t  f e c u n d i t y (eg. D r o s o p h i l a -  Renaming i t e T l t h e y p r o c e e d e d  species  the mutation  mutants a r e not  (1981) c h a r a c t e r i z e d  (e_873) and  elegant mutagenisis in  sensitive  rate  spontaneous  by  a  factor  five-fold. mutation  In  rate  i n meiosis  increase  implying  1 9 7 2 ) . The  mutation rate  Green,  some d e f e c t  effect  i n a repair process  lethal (Watson,  t h e above,  on  examined. i t i s possible  that  r e c - 1 may  on r e p a i r p r o c e s s e s e v e n t h o u g h i t had no e v i d e n t  on t h e s p o n t a n e o u s m u t a t i o n r a t e . X-ray  1 9 7 0 ) . However, i t d o e s  of the interchromosomal e f f e c t  has n o t b e e n  Considering effect  1971;  the frequency of X-ray induced, r e c e s s i v e ,  mutations, 1969,  (Hall,  induced mutation rate  To r e s o l v e  this  s h o u l d be measured  strain.  54  issue  i n the  have  an  effect the  Rec-1  V.  The  Effect  o f an Amber S u p p r e s s o r  on  rec-1  Introduction;  A suppressor mutation reverses (Smith,  the phenotypic  mutations  a t a new can  be  alteration  i n t h e DNA  e x p r e s s i o n o f an e a r l i e r  1979). R e v e r s i o n s  must o c c u r  i s a second  site.  are not Three  identified:  mutation  s u p p r e s s o r s ; t h e new  general classes  intragenic,  of  i n f o r m a t i o n a l and  (Sherman, 1 9 8 2 ) . I n f o r m a t i o n a l s u p p r e s s o r s  fidelity  of i n f o r m a t i o n f l o w from  convert (ie.  certain  the  amino a c i d  gene t o p r o t e i n  s t o p c o d o n s : amber - UAG,  o w i n g t o an  altered  anticodon  ochre  see: C e l i s  1982). In h i g h e r e u k a r y o t e s  - UAA,  insertion  (Laski  techniques,  this  and  the  Smith,  story  mammalian t i s s u e isolation in  C.  of  cultures  induced  elegans. Five  a r e amber  UGA).  stop  codon  1979,  has  and  mutation. coli  55  laevis  expressed  e t a l , 1 9 8 2 ) . However, has  see H o d g k i n ,  in  the  only occurred  s u c h m u t a n t s have been c h a r a c t e r i z e d ( f o r a review  Amber  microinjection  tRNA s u p p r e s s o r m u t a t i o n s  suppressors  and  Sherman,  u s i n g Xenopus  been s u c c e s s f u l l y  (Hudziak  that i t  i s quite different.  e t a l , 1 982)'. T h r o u g h suppressor  at the  e x t e n s i v e l y i n E.  s u p p r e s s o r s have b e e n s y n t h e s i z e d i n v i t r o DNA  may  opal -  b i n d s t o the nonsense  tRNA s u p p r e s s o r s have b e e n u s e d  cloned  ( e g . tRNA  i n t h e tRNA s p e c i e s s u c h  e r r o n e o u s l y r e c o g n i z e s and  ( f o r reviews  the  codons t o c h a i n t e r m i n a t i o n codons  o c c u r s by amino a c i d  yeast  alter  s u b s t i t u t i o n mutations  Suppression  now  mutation  suppressor  indirect  nonsense s u p r e s s o r s ) . Base p a i r  that  1985).  and a l l  Sup-7 i s t h e most p o w e r f u l and  has  a dominant e f f e c t  o f Unc-15 that  (Waterston,  that  Kimble  levels  e t a l . (1982) and  found  i n C.  to roughly  Wills  a tryptophan  40%  i n i t r e a d i n g UAG  In the experiment  which  c o u l d be  segregated  by  homozygous and  recombination  heterozygotes  were t h e n  These experiments rec-1  on  recombination  Bolten et a l . shown i t t o  at the  I determined  heterozygous  be  anticodon  i f the  compared  individuals.  two  cis-linked  homozygotes  i n a sup-7/+  background.  i s not  that  rec-1  constructed  of rec-1  demonstrated  frequency  rec-1  f o r sup-7 and  frequency  clearly  demonstrated  s u p - 7 . A s t r a i n was  p r o g e n y were a l s o h e t e r o z y g o u s  m a r k e r s . The  alteration  N2  codons.  reported here,  suppressed  ( s t 5 ) and  shown  of the  e t a l . (1983)  sup-7  tRNA gene c o n t a i n i n g an  which r e s u l t s  mutation  sequenced  analysis  d e f e c t i v e ) has  sup-7 i s an amber s u p p r e s s o r . More r e c e n t l y ,  (1984) h a v e c l o n e d and  elegans  1981). Chemical  (e1214) worms ( i e . p a r a m y o s i n  sup-7 r e s t o r e s p a r a m y o s i n  level.  The  suppressor  and  the e f f e c t  suppressed  by  of  sup-7.  Methods:  Sup-7 i s a d o m i n a n t , amber s u p p r e s s o r m u t a t i o n . to determine t o an  early  product.  i f the e f f e c t s stop s i g n a l  of rec-1  on  I t was  recombination  were  p r e v e n t i n g t h e p r o d u c t i o n o f a gene  Sup-7 i s X - l i n k e d .  56  used due  Rec-1 (Figure  m a l e s were c r o s s e d w i t h Sup-7 Dpy-18 1 1 ) . Dpy-18  constructions the presence transferred males,  was  p r e s e n t from  (Waterston, and a b s e n c e  hermaphrodites  the o r i g i n a l  strain  1981) and s e r v e d a s an i n d i c a t e r f o r o f s u p - 7 . The h e r m a p h r o d i t e s  to fresh plates  after  twenty  four hours.  a l l o f w h i c h were s u p - 7 / 0 , were c r o s s e d w i t h  Dpy-5  hermaphrodites.  heterozygous  f o r s u p - 7 , dpy-18, dpy-5 and u n c - 1 3 , and were  heterozygous  Crescent  s t a g e F2 W i l d - t y p e  C u l t u r e s were k e p t temperature  2  a t 22 C  hermaphrodites as t h i s  brooded  and r e c o m b i n a t i o n  were  f o r rec-1 (Figure 11).  was  were  brooded.  the optimal  f o r Sup-7 worms. The F3 was  recombinants each  o r homozygous  then  The F1  Unc-13 Rec-1  either  The F2 h e r m a p h r o d i t e s  were  growth  screened f o r  f r e q u e n c i e s were c a l c u l a t e d f o r  line.  Results:  Based  on random s e g r e g a t i o n , one h a l f  brooded  s h o u l d have been rec-1/+,  of the  hermaphrodites  while the other h a l f  have b e e n r e c - 1 / r e c - 1 ; b o t h were sup-7/+. I f sup-7 the e f f e c t should  suppresses  of rec-1, then a l l the recombination f r e q u e n c i e s  cluster  Alternatively, of  should  around  t h e w i l d t y p e v a l u e o f about  i f rec-1 i s not suppressed,  recombination  two p e r c e n t .  two d i f f e r e n t  f r e q u e n c i e s s h o u l d be d i s c e r n a b l e :  based  on t h o s e b r o o d e d  those  t h a t were  hermaphrodites  rec-1/+.  57  classes  calculations  t h a t were r e c - 1 / r e c - 1  and  CONSTRUCTION: Po: rec-1 rec-1  [males]  sup-7; sup-7  dpy-18 dpy-18  [herm.]  F1 : dpy-18; +  sup-7; 0  rec-1  X  dpy-5 unc-13 dpy-5 unc-13  F2:  SCORE  rec-1 rec-1  [herm.]  [males]  dpy-1 8 o r _+; + +  ;  V s u p - 7 ; dpy-5 + +  rec-1 unc-13; + rec-1 (self-cross)  or rec-1  [herm.]  RECOMBINANTS: F3:  V  a) I f sup-7 ( w h i c h i s d o m i n a n t ) s u p p r e s s e s r e c - 1 then a l l the scored recombination frequencies w i l l f a l l i n t h e r a n g e o f t y p i c a l N2 v a l u e s . b) H i g h r e c o m b i n a t i o n f r e q u e n c i e s t y p i c a l o f Rec-1 i n d i c a t e s an a b s e n c e o f s u p p r e s s i o n . G i v e n a l a r g e enough sample s i z e , a b i m o d a l d i s t r i b u t i o n w o u l d be e x p e c t e d .  FIGURE 11: S t r a i n c o n s t r u c t i o n and p r o c e d u r e t o d e t e r m i n e t h e e f f e c t o f sup-7 on t h e e x p r e s s i o n o f t h e Rec-1 p h e n o t y p e  58  Experimental: N U M B E R  7  *  6  *  (self-cross) dpy-5 unc-13; r e c - 1 o r r e c - 1 ; + + + rec-1 sup-7 +  5  *  *  4  *  *  *  *  *  *  *  *  *  *  0 F  3  JJ 2 E R " | M A O P H 5 R 0 4 D 1 3 T E 2 S 1 0  * 1  * 2  *  *  *  *  3  *  4  •  *  5  6  7  8  9  *  *  *  * 10  11  12  13  Control: (self-cross) dpy-5 unc-13; r e c - 1 + + rec-1 *  * 1  2  3  *  *  *  *  *  *  *  *  4 5 6 7 £3 9 RECOMBINATION FREQUENCY  * 10 (%)  *  T\  12  TT  FIGURE 12: The e f f e c t o f s u p - 7 , a n amber s u p p r e s s o r , on t h e e x p r e s s i o n o f t h e Rec-1 p h e n o t y p e . I f s u p p r e s s e d , the recombination f r e q u e n c i e s should c l u s t e r around the N2 v a l u e o f 2%. I f , o n t h e o t h e r h a n d , r e c - 1 h a s i t s t y p i c a l e f f e c t on r e c o m b i n a t i o n f r e q u e n c y , two c l u s t e r s s h o u l d be e v i d e n t : r e c - 1 / + ( a p p r o x i m a t e l y 2%) a n d r e c - 1 / r e c - 1 ( a p p r o x i m a t e l y 7%).  59  Figure frequency m.u.  12 c l e a r l y  shows t h a t  two c l a s s e s  are present. Confidence  f o r any p a r t i c u l a r  recombination  individual.  recombination  a r e a p p r o x i m a t e l y +_1  Although  the p a t t e r n of  f r e q u e n c i e s does n o t p r o v i d e a good f i t f o r a  bimodal  distribution,  limited  sample  with values  intervals  of  size.  this  i s most l i k e l y  Certainly,  i n excess  the c l u s t e r  o f 8% r e c o m b i n a t i o n  o f r e c - 1 . The r e c o m b i n a t i o n  frequency  the p r e v i o u s l y r e p o r t e d value  a consequence o f a of  hermaphrodites  i s due t o t h e e f f e c t  i s slightly  higher  than  (Figure 1). This i s probably  due  2  to the i n c r e a s e d i n c u b a t i o n temperature  of 22 C.  Discussion: Two First,  questions are of p a r t i c u l a r how  reasonable  m u t a n t ? The f r e q u e n c y C. e l e g a n s alleles  amber a t unc-54 tra-2,  i s i t to expect  (Hodgkin,  where one o f s e v e n  Certainly,  three out of f o u r s i x o f 22 a l l e l e s a r e  1 9 8 1 ) . T h i s c o n t r a s t s w i t h fem-1 a n d  (Doniach  and H o d g k i n ,  i n C. e l e g a n s  respectively,  1984; H o d g k i n , are not rare  f o r r e c - 1 t o be i n c l u d e d i n t h i s  mutants.  It i s interesting  mutation  i n E. c o l i  Secondly,  At t r a - 3 ,  and one o f 16 a l l e l e s ,  amber m u t a t i o n s  making i t p o s s i b l e  i n o t h e r mutant g e n e s i n  1985), w h i l e  (Waterston,  a r e amber m u t a t i o n s  r e c - 1 t o be an amber  o f amber a l l e l e s  i s quite variable.  a r e amber  relevance at this point.  t o note  that  i s an amber mutant  t h e uvrD  (for  with  comments  on t h i s  of nonsense mutations  topic  see: Waterston, 60  class of  ( A r t h u r and L l o y d ,  what i n f o r m a t i o n w o u l d be g a i n e d  the u t i l i t y  events,  (210) h y p e r - r e c 1980).  i f sup-7  s u p p r e s s i o n o f r e c - 1 was d e m o n s t r a t e d ? T h i s q u e s t i o n itself  1985).  concerns  as a g e n e t i c  1981; F e r g u s o n  tool and  Horvitz,  1985; H o d g k i n , 1 9 8 5 ) . I n t h e c a s e o f r e c - 1 ,  r e s u l t s w o u l d have i n d i c a t e d - a nontrivial gene p r o d u c t  that  t h e gene h a s a p r o t e i n  c o n c l u s i o n when t h e b i o c h e m i c a l  i s unknown. M o r e o v e r , t h e e x t e n t  any d o s a g e e f f e c t  ( i e . reduced  positive  suppression  product  function of the o f s u p p r e s s i o n and  o f t h e mutant  p h e n o t y p e when h e t e r o z y g o u s f o r sup-7) w o u l d d e m o n s t r a t e w h e t h e r t h e r e c - 1 gene p r o d u c t was r e q u i r e d stoichiometric enzymatic  quantities.  function,  component.  and t h e p a r a l y z e d copy o f sup-7 mutations  were t r u e  increases of this  some a  structural  s u p p r e s s e d by one  g i v e n t h a t most occurs  no f u n c t i o n a l  as a r e p r e s s o r ;  study  early  suggest  that the  t h r o u g h o u t t h e genome.  clearly  show t h a t  there  partial  i s no  (Figure 15).  s u p p r e s s i o n o f t h e mutant  p h e n o t y p e w o u l d be d e t e c t a b l e when sup-7  i s heterozygous.  if  amounts,  t h e gene p r o d u c t  i s required  i n large  show some s u p p r e s s i o n when h e t e r o z y g o u s f o r sup-7 1 9 8 1 ) . F o r example,  (Epstein  1 9 7 7 ) . The w i l d - t y p e gene p r o d u c t  e l e m e n t and h e n c e  amber  Even  mutants  (Waterston,  Unc-54 worms have d e f e c t i v e m y o s i n h e a v y  c h a i n m o l e c u l e s and a r e p a r a l y z e d et a l . ,  i n the  i n i t s absence  o f r e c - 1 on r e c o m b i n a t i o n  at least  amber  gene p r o d u c t i s  f o r r e c - 1 i t would  uniformly  suppression of the e f f e c t I am a s s u m i n g t h a t  imply  partially  1981). F i n a l l y ,  such t h a t  gene p r o d u c t may f u n c t i o n  The r e s u l t s  would  t h e s t o p codon t y p i c a l l y  synthesis  recombination  suggest  Unc-15 worms a r e p a r a m y o s i n d e f e c t i v e  (Waterston,  produced. I f t h i s  the l a t t e r  phenotype i s o n l y  are n u l l ,  polypeptide  The f o r m e r w o u l d  whereas  F o r example,  i n c a t a l y t i c or  i s required  et a l . ,  i s a major  i n l a r g e amounts.  61  1974; MacLeod structural  Despite  this,  partial  s u p p r e s s i o n o c c u r s i n t h e sup-7 h e t e r o z y g o t e , r e s u l t i n g  i n an i n d i v i d u a l t h a t c a n move t o some e x t e n t . A n o t a b l e e x c e p t i o n h a s b e e n r e p o r t e d ; l i n - 2 4 i s an amber m u t a t i o n which a f f e c t s v u l v a development o f sup-7 t o show any  and r e q u i r e s two c o p i e s  s i g n s o f s u p p r e s s i o n ( F e r g u s o n and  Horvitz,  1985). However, l i n - 2 4 has s e v e r a l u n u s u a l t r a i t s w h i c h  may  explain this result.  exhibit  a mutant phenotype,  O n l y worms h e t e r o z y g o u s f o r l i n - 2 4 i m p l y i n g t h a t the phenotype  i s due  t o an  i n t e r a c t i o n o f t h e w i l d - t y p e and m u t a n t gene p r o d u c t s . A  single  c o p y o f sup-7 p r o v i d e s f o r a b o u t a 15% i n c r e a s e i n t h e w i l d - t y p e amount o f gene p r o d u c t ( W a t e r s t o n , 1 9 8 1 ) . I n t h e c a s e o f this apparently s t i l l result  i n any  lin-24  l e a v e s t o o much o f t h e m u t a n t p r o t e i n  improvement i n the phenotype.  to  O n l y when t h e amount  o f a b n o r m a l l y f u n c t i o n i n g gene p r o d u c t i s d r a s t i c a l l y  reduced  ( i e . homozygous f o r sup-7 and h e t e r o z y g o u s f o r l i n - 2 4 ) i s s u p p r e s s i o n of the mutant phenotype There  evident.  i s , h o w e v e r , no r e a s o n t o b e l i e v e t h a t t h e a b o v e  a p p l i e s t o r e c - 1 . The  Rec-1  strain  s e g r e g a t e s as an  r e c e s s i v e m u t a t i o n and d o e s n o t e x h i b i t any u n u s u a l (eg. semidominance  e t c . ) (Rose and B a i l l i e ,  62  mutant.  features  1979a). T h e r e f o r e ,  g i v e n t h e a b o v e and t h e r e s u l t o f t h i s e x p e r i m e n t c o n c l u d e t h a t r e c - 1 i s n o t a n amber  autosomal  (Figure 15), I  VI.  The  Effect  of rec-1  on  Tel  Mobility  Introduction:  Transposable position short  elements are mobile  i n the  genome c a n  p e r i o d s of time  prokaryotes;  Bristol A  1.6  DNA  extensive and  the  All  Bergerac  ( i e . Tc1)  has  t r a n s p o s i t i o n i n t h e BO  frames  1981,-  element  i n C.  fragment  variety  (BO)  Rubin,  elegans. length  and  the  Emmons e t a l . , 1 9 8 3 ) .  strain.  Tc1  to transposable  undergone has  been  elements  sequenced i n other  target site duplications  (Rosenweig e t a l . , 1983).  p r o p o s e d mechanisms o f t r a n s p o s i t i o n i n v o l v e  example,  the  i n one  form or another  a n a l y s i s of mutants of  indicates that a polypeptide  also necessary (Zupancic  f o r recombination  e t a l . , 1 9 8 3 ) . The  resemblence specific  relatively  S p r a d l i n g and  apparently  i t contains terminal repeats,  recombination  Tn5  - Yeast;  (Emmons e t a l . , 1979;  sequence  open r e a d i n g  Kleckner,  cause of r e s t r i c t i o n  structure i s similar  species: and  strain  t o change o v e r  i s a transposable  (RFLDs) between t h e  (N2)  kb  the  s e q u e n c e s whose  see:  Cameron e t a l . , 1979,  d i s c o v e r e d as  differences  observed  ( f o r reviews  1981,- D r o s o p h i l a ) . Tc1 I t was  be  DNA  (Kleckner,  The  required for transposition i s between IS50  mechanism b e a r s  authors  63  For  IS50 s e q u e n c e s t h a t f l a n k  t o homologous r e c o m b i n a t i o n  recombination.  1981).  sequences a  closer  r a t h e r than  suggest  that  the  site  IS50 e n c o d e d p o l y p e p t i d e f u n c t i o n s as b o t h in  the t r a n s p o s i t i o n The  rec-1  meiotic  p r o c e s s ) and  mutation  recombination  i n C. (Rose  s e e c h a p t e r I I I ) . The  and  dramatically, t h e Rec-1  that  i s a g e n e r a l enhancer  Baillie,  molecular basis  rec-1  1979a; R o s e , 1980;  of t h i s  effect  increases recombination  showed any  of and  i s not  nonhomologous r e c o m b i n a t i o n may  the q u e s t i o n addressed  strain  (required  recombinase.  elegans  known; b o t h homologous a n d / o r involved. Given  a  a resolvase  in this  be  frequency  work was  whether  signs of i n c r e a s e d t r a n s p o s i t i o n  by  Tc1 . The  approach  examining  the e l e c t r o p h o r e t i c  restriction different mobility  fragments  insertion i n N2  differences banding  t a k e n t o answer t h i s  site  i s very  separated  f o r over  Rec-1  N2  500  strains  two  derived strains:  rec-1  (unc-13  mobility banding  was  BC187  restriction  from  (e61)  no  fragment  had  been  years).  p a t t e r n s h o u l d be  The  of s i x  a heterozygote unc-15  significantly  then changes i n i n s e r t i o n  (e73)  banding  p a t t e r n was of t h i s  i n c r e a s e s the  sites  are suggested  64  found.  result  and  hence  However,  are considered.  for future  of  ) and  observed.  interpretations  experiments  that  (roughly eight  (dpy-5  DNA  e l e g a n s genome. Tc1  s t o c k s o f N2  isolated  by  band r e p r e s e n t s a  L i a o e t a l . (1983) f o u n d  (e51 ) ). I f r e c - 1  of Tc1,  alternative  i n t h e C.  generations  mutation  Such an a l t e r e d  Several  low.  Each  have been s e p a r a t e d f o r i n e x c e s s  The  CB51  by Tc1  involved  p a t t e r n s produced  with Tc1.  laboratory  years. N2  banding  amoung t h e T c 1 - h y b r i d i z i n g  p a t t e r n s of  and  probed  question  investigation.  the  Materials  and M e t h o d s :  1) DNA P r e p a r a t i o n - C. e l e g a n s methods m o d i f i e d f r o m pers.  DNA samples were p r e p a r e d by  Emmons e t a l . (1979)  (Baillie  and C u r r a n ,  comm.). Worms were washed o f f o f a p p r o x i m a t e l y  culture  plates with  pellet.  The p e l l e t  buffer  0.09 M N a C l  and t h e n  was r e s u s p e n d e d  c e n t r i f u g e d t o form a  i n 15 ml o f I x P r o t e i n a s e K  ( 1 % SDS, 0.05 M EDTA, 0.1 M T r i s  M NaCl);  12 l a r g e  buffer  (pH 8 . 5 ) ,  a n d 0.2  100 m i c r o g r a m s / m l o f p r o t e i n a s e K (EM B i o c h e m i c a l s )  were added and t h e s o l u t i o n was i n c u b a t e d a t 65 C f o r 30 minutes.  The s o l u t i o n was t h e n e x t r a c t e d t h r e e t i m e s  volumes o f w a t e r - s a t u r a t e d p h e n o l , each  time. A f i n a l  The in  resultant  phase  u s i n g an e q u a l  (24:1 by v o l . ) .  to stand a t l e a s t  C o l d 95%  one h o u r a t -20  was c e n t r i f u g e d a t 10,000 rpm f o r t e n m i n u t e s .  pellet  was a i r - d r i e d .  one m l . o f 1xTE (10 mM T r i s ,  c h l o r o f o r m was a d d e d t o p r e v e n t DNA p r e p a r e d  r e c o v e r i n g t h e aqueous  alcohol  e t h a n o l was a d d e d and a l l o w e d The s o l u t i o n  equal  e x t r a c t i o n was p e r f o r m e d  volume o f c h l o r o f o r m / i s o a m y l  C.  with  The DNA was t h e n  1 mM EDTA, pH 7 . 4 ) . bacterial  i n 1983 and N2 DNA p r e p a r e d  resuspended One d r o p o f  c o n t a m i n a t i o n . Rec-1 i n 1985 were  kindly  p r o v i d e d by N. M a w j i . 2) DNA H y b r i d i z a t i o n were s e p a r a t e d by s i z e electrophoretic filters  using horizontal  with a  genomic DNA d i g e s t s  0.7% a g a r o s e  The DNA was t r a n s f e r r e d  (unidirectional 32  hybridized Harris.  gels.  - Eco R l - r e s t r i c t e d  transfer:  P labelled  Smith  to nitrocellulose  a n d Summers,  1980)  and  Tc1 p r o b e p r o v i d e d by L-.  The Tc1 p r o b e c o n s i s t e d o f t h e c e n t r a l  65  1540 b a s e  pairs  of  t h e Tc1 e l e m e n t ; no f l a n k i n g s e q u e n c e was p r e s e n t .  f r a g m e n t u s e d as t h e p r o b e was plasmid  (pCeh14) u s i n g  electrophoresis Filters and  c u t o u t o f t h e pUC-19  E c o RV and i s o l a t e d  t h e n washed i n 2xSSPE, 0.2%  9  a t 62 C  a  XRP-1  by L .  Harris).  i n 5xSSPE, 0.3%  SDS f o r a t l e a s t  6 2 C . They were t h e n a u t o r a d i o g r a p h e d  derived  by a g a r o s e g e l  and e l e c t r o e l u t i o n ( p r e p a r e d  were h y b r i d i z e d o v e r n i g h t  The  f o r three  SDS  one h o u r a t days u s i n g  Kodak  film.  Results:  Genomic b l o t rec-1  h y b r i d i z a t i o n s were p e r f o r m e d t o d e t e r m i n e i f  has any e f f e c t  on Tc1 m o b i l i t y . T h i s was a c c o m p l i s h e d  e x a m i n i n g t h e T c 1 - h y b r i d i z i n g E c o RI b a n d i n g p a t t e r n Rec-1  strain  and c o m p a r i n g  DNA  using  i n the  i t t o t h a t o f N2. E x a m i n a t i o n o f N2  and  Rec-1  had  an a l t e r e d p a t t e r n o f Tc1 i n s e r t i o n s ( F i g u r e 1 3 ) . A t  Tc1 as a p r o b e r e v e a l e d  five  new  band  ( t o p a r r o w ) was m i s s i n g  includes had  bands  (lower  arrows i n F i g u r e  one band w h i c h was  double the i n t e n s i t y  by  13) were p r e s e n t  i n t h e Rec-1 present  t h a t t h e Rec-1  DNA.  This  i n b o t h Rec-1  DNA  least a n d one  estimate  and N2, b u t  o f h y b r i d i z a t i o n i n Rec-1  (center  arrow). Figure DNA:  13 a l s o shows a c o m p a r i s o n o f two s a m p l e s o f Rec-1  one was  August,  prepared  1985. The two p r e p a r a t i o n s  hundred g e n e r a t i o n s . patterns  i n 1983 w h i l e  were  the other  was p r e p a r e d  are separated  in  by a t l e a s t  No d i f f e r e n c e s i n t h e T c 1 - h y b r i d i z a t i o n  visible. 66  two  1  2  3  FIGURE 1 3 : T c l - h y b r i d i z e d r e s t r i c t i o n f r a g m e n t p a t t e r n s f r o m DNA s a m p l e s o f t h e 1) R e c - 1 ( 1 9 8 3 ) , 2) N2 a n d 3) R e c - 1 ( 1 9 8 5 ) s t r a i n s . Lambda s i z e m a r k e r s a r e shown on t h e r i g h t , w h i l e on t h e l e f t , a r r o w s i n d i c a t e t h e b a n d d i f f e r e n c e s b e t w e e n t h e N2 a n d R e c - 1 s t r a i n s .  67  Discussion:  The Tc1  N2  strain  whereas BO  i n C.  has  over  e l e g a n s has 300  ( L i a o e t a l . , 1983;  1983). T h i s  l a r g e d i f f e r e n c e has  postulating  extensive  mobility  i n N2.  This  inactive  strain  has  Molecular in  BO  suggestion received  this  1 9 8 4 ) . Tc1  o f Tc1  Snutch, stock  1984;  and BO N2. N2  500  studies  (1985a) e x a m i n e d  of  65  unc-54 gene and  18  but  This  the  independent found  that  contrasts  the  estimated  A l l evidence strain.  t o be  at  least  from g e o g r a p h i c a l l y  no  N2  the  three  68  a  and  laboratory  fragment  Eide  BO  to  of  these  and  insertions  mutation  stability C.  i n N2  at  i n w h i c h 10  times g r e a t e r  isolated  Anderson  Tc1  strain  same work, t h e  d i s t a n t regions  at  (Rose  spontaneous mutations  independently  and  ( L i a o e t a l . , 1983).  to date argues f o r the  Moreover,  but  separation  above r e s u l t .  with  excises  (Emmons  i n N2  restriction  none were due  1 9 8 5 b ) . From t h e  readily  i n N2,  not  Emmons, 1 9 8 4 ) . F i n a l l y ,  support  sources:  comm.). E x t r a c h r o m o s o m a l  i n BO,  generations  Tc1  relatively  somatic  s p o n t a n e o u s Unc-54 m u t a n t s a r o s e f r o m Tc1  Anderson, was  (Harris, pers.  of  by  limited  a c t i v e and  have been found, d e s p i t e  f o r roughly  transposition.  for  somatic e x c i s i o n a l s o occurs  Ruan and  copies  from a v a r i e t y of  differences in Tc1-hybridizing  Genetic  the  support  have b e e n i s o l a t e d  banding p a t t e r n stocks  o f an  and  excision is principally  much l o w e r r a t e t h a n BO copies  been a c c o u n t e d  t r a n s p o s i t i o n i n BO  30  Emmons e t a l . ,  i n v e s t i g a t i o n s have shown t h a t Tc1  and' t h a t  Yesner,  approximately  rate  out (Eide in  than that  o f Tc1  in  the  elegans s t r a i n s  have b e e n shown t o  have  in  few,  i f any,  d i f f e r e n c e s i n Tc1  Thus i t a p p e a r s t h a t strains  since  their  Tc1  has  patterns  (Liao et  been s t a b l e i n the  separation,  a l . , 1983).  genome o f  p o t e n t i a l l y hundreds of  most  years  ago. E x a m i n a t i o n o f N2 revealed  reported  the  N2  results.  banding p a t t e r n  Therefore,  (1983) w i t h activating  DNA Tc1  not  out  strain.  to  i n Tc1  as  the  w o u l d be c a s e as  identical  activity  as  increased  an the  i n Rec-1  one  cut  o f Rec-1  i n August,  or  test  prepared  1985.  I f rec-1  most l i k e l y  change c o u l d  result  i n t o the  gene c o n v e r s i o n  Tc1  gene c o n v e r s i o n  however, t h e  i n an  i n the  Eco  result  RI  possibility two  RI  69  a I ago  preparations. identical,  (Figure  i n the  cut  13). loss  may  Rec-1  site  in  the  precisely located,  site  pair  i s approximately  (Harris, pers. i n an  years  band a d d i t i o n and  t o be  cut  causing  was  were  mobility  Eco  Rec-1  Tc1-hybridized  occurring  an  and  progenitor  l o c a t i o n where a s i n g l e b a s e  element  could  this  N2  changes i n  was  DNA  Tc1-hybridized  yet  Any  rec-1  banding patterns  has  previously  its  e x p e c t e d between t h e s e  a c t i v a t o r of  site  to  l o s s were f o u n d  m o b i l i t y . To  v a r i a n t e x i s t s with  t r a n s p o s o n . The  probe  1 3 ) . As  i n N2.  suggested that  the  a  (Figure  m o b i l i t y , d i f f e r e n c e s i n the  rec-1  A Tc1  base p a i r s  and  Tc1  d i f f e r e n c e s between the  t o Tc1  result  prepared  Alternatively, due  due  the  banding p a t t e r n  banding p a t t e r n was  This  increase  compared t h e  be  using  o b s e r v e d was  F i v e band a d d i t i o n s  significant  ruling  DNA  must h a v e o c c u r r e d  banding p a t t e r n .  This  pattern  s t r a i n s were n o t  strain.  Rec-1  a number o f b a n d i n g d i f f e r e n c e s  expected,  Rec-1  and  increase  comm.). i n the  1,400  Increased number  of  copies this due  of  the  study, to  the  Eco  conversion  band w o u l d now  explanation  the  absence of  new  and  to  the  Tc1-hybridized  o l d Rec-1  DNA  the  expected  large  two  the  in  smaller  bands.  increase  i n gene  correction  of  v a r i a n t . Moreover, to account b a n d i n g d i f f e r e n c e s between  rates  h a v e r e a c h e d a new  as  that  preparations,  gene c o n v e r s i o n  were u s e d  band l o s s w o u l d be  favours  R1  digests  a single,  requires  Eco  RI  show up  disproportionately  h e t e r o d u p l e x DNA  variants  Eco  a b o v e ; what a p p e a r e d as  However, t h i s  the  v a r i a n t . As  b o t h band a d d i t i o n and  Tc1-hybridizing  that  RI  one  w o u l d have t o  between t h e  for  the  assume  d i f f e r e n t Tc1  equilibrium. Although p o s s i b l e ,  this  seems u n l i k e l y . A third Figure Rec-1  13, are  explanation, i s that  due  to  the  which  i s consistent  d i f f e r e n t Tc1  certain conditions  o f Tc1  activity.  This  strain  or  i t s progenitor  one  reproduce control  these conditions  of  Tc1  environmental evidence heat  could  may  s t r e s s might  s h o c k p r o m o t e r 5' copia  Dictyostelium  this to  element  discoideum,  sequence l o c a t e d  dramatically  as  i n the  pers.  comm.).  5'  putative  region  of  Tc1  heat  i n the  (1978) s u g g e s t e d  includes  the  the  discovery  of  the  McDonald,  RNA  t r a n s c r i p t s of a  1985).  Moreover, 2.5  kb  increase cells  (Zuker  shock promoters have elegans  to  Experimental  frame i n  the  burst  that  and  shocking  in  Rec-1  attempts  transposition.  i n C.  70  found  some i n s i g h t i n t o  i n a transposon  a r e s u l t of heat  1983). S e v e r a l  found  the  results in  produced a short  open r e a d i n g  (Strand  r e p e a t e d DNA  al.,  provide  proposal  the  that  s t r a i n s . I f so,  induce  the  insertion sites  have o c c u r r e d  mobility. McClintock  i n support of  Drosophila in  of  event  with  (Rose and  et  been Snutch,  a  Two e x p e r i m e n t s c a n be s u g g e s t e d 1)  the burst  earlier,  before  strains  o f Tc1 movement may have t a k e n p l a c e the i s o l a t i o n  are a v a i l a b l e , having  Hence, t h e T c 1 - h y b r i d i z i n g of  these 2)  s t r a i n s could  much  o f Rec-1. S t o c k s o f t h e p r o g e n i t o r been s t o r e d  restriction  i n liquid  nitrogen.  fragment banding  changes i n i n s e r t i o n s i t e s  itself, rather  bands i n t h e  may be a c o n s e q u e n c e o f t h e  t h a n a c a u s e . One o f t h e new  Rec-1 DNA may r e p r e s e n t t h e  insertion  o f a c o p y o f Tc1 i n t o t h e r e c - 1 gene,  implying  the  o f Tc1 m o b i l i t y o c c u r r e d  with the  burst  concurrently  a p p e a r a n c e o f t h e Rec-1 p h e n o t y p e . T h i s determining  then  could  outcross  that  be t e s t e d by  i f any o f t h e new bands s e g r e g a t e w i t h  phenotype d u r i n g  patterns  be compared t o t h o s e o f N2 and R e c - 1 ; and  t h e Rec-1 p h e n o t y p e ,  Tc1-hybridizing  f o r future investigation:  t h e Rec-1  experiments. P o s i t i v e r e s u l t s  could  l e a d t o t h e c l o n i n g o f t h e r e c - 1 g e n e . The i n s e r t e d  transposon could interest strain  be u s e d t o i d e n t i f y  i n a cloned  DNA  library  (Bingham e t a l . , 1 9 8 1 ) .  71  t h e gene s e q u e n c e s o f  constructed  f r o m t h e Rec-1  VII.  The  work p r e s e n t e d  recombination various  D i s c u s s i o n : Overview  in this  study  e n h a n c e r r e c - 1 , i n C.  experiments i n Table  three-fold  increase in intragenic  9.  The  c o n v e r s i o n . An  attempt  intervals,  problems  this  project The  from  effects  no  rec-1  was  being  rate,  crossing  strain  completed on  f e c u n d i t y and  by  nondisjunction  and/or  chromosome l o s s  increase  o f two.  Drosophila directly  and  A strain due  found  proportional  recombination  was  If real,  Hall  that  The  b e e n shown t o be  this  frequency  also showed  appeared  t o be  increased  i s very unusual.  with  an  For  m e i o t i c mutants i n was  in  IV).  c o n s t r u c t e d to determine  i f the  rec-1  enhancer  of a premature s t o p codon, p r e v e n t i n g  hyper-rec  mutant  i n E.  coli,  uvrD(210),  a c o n s e q u e n c e o f an amber, n o n s e n s e  showed t h a t  the e x p r e s s i o n of rec-1  mutation.  ability  t h e amount o f n o n d i s j u n c t i o n  L l o y d , 1 9 8 0 ) . The  amber  competitve  increase concomitant  ( A r t h u r and  amber s u p p r e s s o r  gene  to the extent of the decrease  to the presence  translation.  and  to  c o n s t r u c t i o n prevented  (1976) s u r v e y e d  (see c h a p t e r was  a two  r e c - 1 ; however, X-chromosome  i n recombination  example, B a k e r and  the  are  o t h e r m e i o t i c p r o c e s s e s were  affect  a factor  over  of  (see c h a p t e r I I I ) .  substantial  by  results  I I I t o VI  mutant p r o d u c e d  the  made t o examine l a r g e g e n e t i c  i n the  of rec-1  studied. Mutation  e l e g a n s . The  reported i n chapters  summarized  but  characterized  ( i e . sup-7),  experiment was  indicating  performed not  rec-1  mutation  i n chapter  affected  that  has  by  a tRNA  i s not  an  V  TABLE 9: Summary  of r e s u l t s  Character I.  Recombination:  II.  Fecundity:  III.  IV. V.  Competitive Ability: Mutation  Rate:  Radiation Sensitivity:  VI.  Nondisjunction:  VII.  VIII.  Amber Suppression: Tc1  mobility:  characterizing  Effect  the rec-1 enhancer  Source  i n c r e a s e s gene c o n v e r s i o n , chapter I I I ; i n t r a - and i n t e r g e n i c Rose and B a i l l i e , exchange 3 t o 4 - f o l d 1 979a no  effect  chapter IV.  no  effect  chapter IV.  no  effect  chapter IV.  no  effect  Hartman a n d Herman, 1 982  2-fold  no  increase  effect  chapter IV. R o s e , 1980 chapter  V.  c h a p t e r VI. altered Tc1-hybridized r e s t r i c t i o n fragment banding p a t t e r n i n N2 v s R e c - 1 ; interpretation i s unclear  73  Finally,  the  effect  the m o b i l i t y of the chapter  Rec-1  rec-1  as a r e c o m b i n a t i o n  transposable  e l e m e n t Tc1  V I ) . E l e c t r o p h o r e t i c banding  restriction and  of rec-1  fragments probed with  strains.  An  a c t i v a t e s Tc1  two  explanations  years  found  of recombination  after  Tc1  DNA  were compared f o r t h e  p a t t e r n was  found,  can  be  Rec-1  suggesting  DNA  the o t h e r ) . S e v e r a l (chapter  N2 that  ( S i m c h e n and  Stamberg,  the  samples  (one  alternative  VI).  made between f i n e  c o n t r o l presumably a f f e c t necessary  (see  p a t t e r n s p r o d u c e d by  between two  were c o n s i d e r e d  A distinction  examined  on  m o b i l i t y . However, p r e d i c t e d p a t t e r n  d i f f e r e n c e s were n o t prepared  altered  was  enhancer  and  coarse c o n t r o l s  1 9 6 9 ) . Genes o f  essential,  coarse  s e q u e n t i a l steps  for meiosis  (eg. s y n a p s i s , breakage,  repair,  s e g r e g a t i o n e t c . ) and  h e n c e have a g e n e r a l and  u s u a l l y extreme  effect well  on  recombination.  characterized species  1969;  Drosophila;  Gowen, 1922;  Esposito,  1969;  Smith,  1975;  campestris: Genes o f  as3  Yeast:  Klapholz  Zea  control  ( S m i c h e n and  Lindsey  and  Sandler  recA  genetically  - Willets  Carpenter,  1972;  et a l . ,  c(3)G  - Esposito  1971;  -  and  N e u r o s p o r a : mei-1  - Palmer,  -  Brassica  1970). and  local  o f exchange i n s p e c i f i c  achieved  s i t e s which i d e n t i f y  enzymes  i n many  have a more l i m i t e d  Regulation  t h e genome i s a p p a r e n t l y  proposing  coli:  e t a l . , 1985;  - Stringham,  fine  found  s p o - 8 , spo-11  mays: a m e i o t i c  recombination.  recognition  (eg. E.  mei-9 - B a k e r and  Gowen and  on  Examples a r e  Stamberg,  by the  1969;  controlling location  Stamberg and  (1977) have e x t e n d e d t h i s  regions  access  to the  effect  to  recombinase  Koltin,  analogy  of  1973).  by  t h a t r e g i o n a l r e g u l a t i o n of exchange i n v o l v e s  two  components: 1) a c o n t r o l l i n g distribution  o f exchange;  system, which  and 2) a r e s p o n d i n g s y s t e m , w h i c h i s  r e c o g n i z e d by t h e c o n t r o l l i n g (interacting el  e l e m e n t s . Examples  (Angel e t a l . ,  Considering  1970; V a l e n t i n ,  h y p e r - r e c mutants  t h e same c l a s s i f i c a t i o n  of l o c a l  and g l o b a l  h o t s p o t s a r e DNA  recombination  immediate  sites  in their  1 9 8 3 ) . They  h a v e been  stimulate  recA-recBC-dependent effect  on e x c h a n g e  reduced  level  pombe  and t h e b u f f  stimulate meiotic Roeder,  changes  initiation  mutants  (Stahl et a l . ,  i n T a b l e 10. C h i  1974).  The maximum  but extends a t a 1980). I n  the cog mutation i n  t h e ade6 m u t a t i o n i n S c h i z o s a c c h a r o m y c e s (YS17) m u t a t i o n i n S o r d a r i a b r e v i c o l l i s a l l r e c o m b i n a t i o n i n a d j a c e n t sequences 1970; G u t z ,  ( K e i l and  1971; a n d M a c D o n a l d a n d  sequences  that  create  recognition  enzymes. D e t e c t i o n o f s u c h a s i t e or resolution  of a Holliday  produce  a local  sites for  results  i n the  junction.  h y p e r - r e c mutants  have a l e s s  on r e c o m b i n a t i o n and a r e n o t i n c l u d e d  intervals,  stimulate  ( f o r a review see  (McMilin et a l . ,  mutation i n yeast,  A number o f l o c a l , effect  that  1 9 7 9 ) . A l l o f t h e above a r e presumed t o i n v o l v e  i n DNA  recombinase  c a n be  r e c o m b i n a t i o n by t h e  pathway  1984; A n g e l e t a l . ,  Whitehouse,  vicinity  a r e summarized  up t o 10 kb away  crassa,  sequences  o c c u r s near the C h i s i t e ,  e u k a r y o t e s t h e HOT1 Neurospora  effects  f o u n d i n b o t h p r o k a r y o t e s and  t h e known m u t a n t s  i n E. c o l i  1973; N e l , 1 9 7 5 ) .  as a s u b c l a s s o f t h e a b o v e ,  a p p l i e d . Recombination  eukaryotes;  i n c l u d e cog  w i t h r e c - 2 ) i n N e u r o s p o r a , mei-1 i n D r o s o p h i l a and  i n Zea mays  Smith,  s p e c i f i e s the  i n c r e a s e over perhaps  regular  i n T a b l e 10. T h e s e one o r two  but decrease the recombination frequency elsewhere.  TABLE 10:  Species/gene E.  S.  S.  N.  Extent Mitotic  coli: Chi s i t e s cerevisiae: Hot 1  crassa: rec-1 rec-2/cog  * *  * No  local  recombination  enhancers  Increase Meiotic  Reference  Smith,  increase  7-fold  * *  S_. b r e v i c o l l u s : b u f f (YS17)  of  15-fold  pombe: ade 6 (M26)  rec-3/con  A review of  Keil  none  20-fold  intragenic  only;  10 t o 2 5 - f o l d i n t r a g e n i c ; 1 0 - f o l d i n t r a - and intergenically; 10 t o 2 5 - f o l d i n t r a and i n t e r g e n i c a l l y  10-fold  intragenic  only;  and  1983  Roeder,  Gutz,  1984  1971  Catcheside, 1964 Smith, 1966 A n g e l e t a l . , 1970 Catcheside, 1966  MacDonald Whitehouse,  and 1979  I n d i c a t e s t h a t t h e e f f e c t s on m i t o t i c r e c o m b i n a t i o n were not reported. p l e i o t r o p i c e f f e c t s were r e p o r t e d f o r any o f t h e l o c a l e n h a n c e r s .  Parry  (1973) and  mei-S282 and  decrease  on  by  (Baker  2.5-fold  local  Lycopersicum  e t a l . , 1 9 7 6 ) . Moens i n c r e a s e i n as(4)  esculentum  chiasma frequency metaphase I . The increase  and  an  recombination experiments  meiotic  General  enhancers  on  crosses  effects no  a two  and  in  fold  decreases chromosome 9  m o d i f i e r s of  through  selection  (Brooks,  1984;  Chinnicci,  recombination  stages  i n the  "landmark" e v e n t s  not  form  as  hypothesis  cohesive a  and  can  account  for  ( T a b l e 1 1 ) . B a s e d on  Simchen  and  likely  that  sequential control  a l . (1968) p r o v i d e d a s c h e m a t i c  o f chromosome b e h a v i o r  do  a r e more e x t e n s i v e  single  Stambergs' p r o p o s a l , i t appears different  be  homozygotes i n  t h e w i l d - t y p e v a l u e on  of r e c o m b i n a t i o n  suggesting that  effect  to  e t a l . , 1971).  Their pleiotropic  variable,  as(b)  the e x i s t e n c e of l o c a l  interstrain  1971a,b; S i m c h i n  believed  (1969) r e p o r t e d a 2  i n Zea mays c a u s e s  have been demonstrated  and  c h l mutant i n  1974). In h i g h e r  on chromosome 5, b u t  thirds  1975). F i n a l l y ,  but  i n c r e a s e d p r o p o r t i o n of u n i v a l e n t s at  e l mutation  t o two  and  and  associated with a general decrease  i n recombination  recombination  et  The  exchange, but d e c r e a s e s (Haber,  the X  f o r the g e n e r a l  both mutations.  chromosome t h r e e  that  increases i n  m e i o t i c d e f e c t s i n synapsis are t y p i c a l l y  involved  their  caused  increases mitotic  plants,  group.  (1974) have shown  i n the centromeric r e g i o n s of both  recombination  (Nel,  Sandler  chromosomes, a p p a r e n t l y c o m p e n s a t i n g  nonuniform yeast  and  mei-218 i n D r o s o p h i l a p r o d u c e  recombination second  Carpenter  t h e s e mutants of meiosis.  Sandler  r e p r e s e n t a t i o n of the  i n D r o s o p h i l a . They s u g g e s t e d  affect  control  that  ( i e . processes with g e n e t i c a l l y detectable  c o n s e q u e n c e s ) c a n be  used  to determine 77  t h e mode and  time  of  TABLE 1 1 ; A r e v i e w o f g e n e r a l  Soecies/cene E.  Extent of Increase Mitotic Meiotic  coli: uvr D  dut pol A  Aspergillus: uvs-3  Neurosoora: uvs-3  Drosochila: Ir.-erchrcmcsomaJ effect  Arthur  none  hyper-mutable rad. s e n s i t i v e  G o l i n and E s n c s i t c 1977."  10-fold i n t r a and i n t e r g e n i c  none  U.V. s e n s i t i v e reduced f e c u n d i t v  H c l l i d a y et a l . , 1976.  1-0-fold i n t r a and i n t e r g e n i c  none  reduced f e c u n d i t y  S h a n f i e l d and K a f e r , 1.969 .  2-fold intraand i n t e r g e n i c  none  reduced f e c u n d i t y  Schroeder,  3 t o 17  B o t h m i t o s i s and m e i o s i s ; 2 to 3 - f o l d ; nonuniform; intergenic oniv.  reduced fecundity  2 to 3-fold; low f e c u n d i t y i n t r a and i n t e r g e n i c ; nonuniform  C. e l e c a n s : rec-1  3 to 4 - f o l d ; i n t r a and i n t e r g e n i c ; g e n e r a l and u n i f o r m ;  mays: as  indicates  fold  hyper-rec (extent not reported) hyper-rec hyper-rec  c (3)G  Z.  Reference  hyper-mutable rad. s e n s i t i v e hyper-mutable rad. s e n s i t i v e hyper-mutable t.s. lethal hyper-mutable t.s. lethal  Saccharomvces ; rem-1 TO-fold i n t r a and i n t e r g e n i c Ustillagc: uvs-1  Pleiotroov  12-fold  dam lie  enhancers of r e c o m b i n a t i o n  1 to 10-fold; i n t e r,g, e= ni ii- cw ;, v a r i a b l e penetrance  that  and L l o y d , 1980. M a r i n u s and Konrad 1976. K o n r a d e t al.,1972 Tye e t a l . , 1977. K o n r a d and Lehman, 1974.  S t u r t e v a n t , 191 L u c c h e s i , 1976.  H i n t o n , V962 ; Watson, 1972.  increases Rose and B a i l l i e , nondisjunction 1979a; T h i s work. reduced fecundity  Miller,  t h e e f f e c t s on m i t o t i c r e c o m b i n a t i o n were n o t  78  1970,  1963  reported.  action  of  meiotic  m u t a n t s . The  h y p e r - r e c m u t a n t s may The  E.  coli  systems  (Table  12;  and  Hoekstra,  1984;  and not.  A l l of  a kind and  the  Hoekstra,  The  by  effect  1 9 8 4 ) . The  the  to  is typically  However, V a l e n t i n inversions  and  Lucchesi,  1 9 7 2 ) . The effects  pattern  occurring  i s not  of  breaks are  with  found  (eg.  1976  increase on  the  four  in flies  Suzuki  (Malone be  two  meiotic  heterozygous duplication  (1963) have  to  slightly  X-chromosome and  reported  e v e n when both  three-fold  affected  autosomes. Exchange i n  affected.  of  interchromosomal  i s nonuniform, with the  are  presumed t o  f o r a r e v i e w ) . However, only  rem-1  lesions  pathway  inversion,  recombination  distally the  and  mechanism.  (1972) and  a p p e a r s t o be  c e n t r a l l y on  regions  Rad-50 r e p a i r  interchromosomal e f f e c t increases  intergenic  see  (Malone  which enhance r e c o m b i n a t i o n  meiotic,  and  reduced  i n d u c e s DNA  i n t e r p r e t . The  a chromosomal r e a r r a n g e m e n t  recombination  and  by  rem-1  i n d u c e d DNA  more d i f f i c u l t  homozygous. The  i n yeast  a l . , 1976). Double mutants of  pleiotropic effects associated  exceptional  1964;  only)  repair  et a l . ,  however, rem-1, r a d - 5 0 i n d i v i d u a l s  corrected  i n Drosophila  etc.).  and  (rem-1  a recombination-repair  mutants are  in their  mutagen s e n s i t i v e , have  above s u g g e s t s t h a t  typically  repaired  for  viable,  et  are  Pukkila  e n h a n c e r s , rem-1  hyper-mutable  Holliday  defects  Radman, 1980;  mitotic  maydis are  are  rad-6 are  the  general,  landmarks.  implicating  G l i c k m a n and  in Ustilago  fertility  such  a l l h y p e r - m u t a b l e and  sensitive,  1983). S i m i l a r i l y , uvs-1  represent  mutants are  radiation/mutagen  p l e i o t r o p i c e f f e c t s of  mitotic (Ronen,  intragenic  (Carlson, the both  greatest distally  heterochromatic  Procunier  and  effect  decreases  single  cross-over  Suzuki  (1967) n o t e d t h a t  interference tetrads  ( i e . the  recombination  rather  than the  taking  as  latter  the  (Carpenter  and  Sandler,  challenges  the  assumption  constant  throughout  Finally,  Holm  i s increased  autosome, n o n d i s j u n c t i o n to the This the  control  implies  Two  with  Roberts  by  of  an  the  Structural  amount o f  i s required  heterozygosity  i n one  intervals.  As  required an  the  of  constraints the  imposed by  increased  she  exchange i s  compound  the  Hall  to e x p l a i n  bivalents  the  compete (eg.  reduces the of  the  to  the  time spent  the  inversion).  a  recombinase).  Suzuki  crossing  leave  80  larger  (1968) h a v e  time r e q u i r e d  also  a  competing  for  spatial  r e a r r a n g e m e n t . They p o s t u l a t e may  and  f o r some l i m i t e d  genome w o u l d  and  to  effect,  (1936)  amount o f  p h y s i c a l and  i n synapsis  due  e f f e c t of  enzyme f o r e x c h a n g e i n o t h e r  due  relative  (1976).  r e c o m b i n a t i o n . Mather  that  when  decreased  B a k e r and  a l t e r n a t i v e , Lucchesi  i s increased  1984;  interchromosomal  d e v e l o p e d a t i m e - d e l a y model i n w h i c h t h e synapsis  a  i s generally  f o r exchange  p a r t i c u l a r region  the  1978,  X-chromosome i s r e d u c e d  observations  r e a r r a n g e m e n t s on  which  Grell,  p r o b a b i l i t y of  r e c o m b i n a t i o n by  (1969) p r o p o s e d t h a t  material  exchange  interference  i n v e r s i o n on  nondisjunction  the  an  (1980) have shown t h a t  models have been s u g g e s t e d  structural  over  that  for  se o f  ( i e . a compound autosome w i t h o u t  enhancement o f  consistent  the  the  genome).  H a r g e r and  recombination  that  affect  see  to  suggests that  preconditions  does not but  interchromosomal  of m u l t i p l e  p r o b a b i l i t y per  1974;  the  ratio  increases). This  interchromosomal e f f e c t a l t e r s the  place,  the  increase  that the  probability  of exchange. T e s t i n g these hypotheses,  investigating  other p o s s i b i l i t i e s ,  difficult.  Recently, Szauter  nonuniform  increase induced  not  due  The  (1984) has by  the  rec-1  recessive;  mutant  an  i n C.  effect  elegans  shown t h a t  system  differs  increase i n recombination  interchromosomal recombination  effect,  and  effect  is  regulating  effect  on  f o r the  factor  of t h r e e or  penetrance;  any  recombination  rec-1  i n Zea  frequency  found  ( e g . up  induced  rec-1  the r a t e  enhanced r e c o m b i n a t i o n  exhibit  i n the  increase to unlike  the  has  same e x t e n t a  uniform  o f e x c h a n g e by  i n that  i t has  a  a dramatic  variable  increase in  to a 1 0 - f o l d i n c r e a s e ) or i n C.  observed  irregularities  elegans, which pairing  is  and  almost  100%  a t pachytene  i n s y n a p s i s . However,  the centromere  chromosomes. T h e s e were t h e  distal  i n as  pairing  ends o f  the  same r e g i o n s r e p o r t e d t o have  frequencies. Miller  c o m p e n s a t o r y mechanism s i m i l a r be  is  intragenic  mays i s u n u s u a l  (1963) has  normal around  D r o s o p h i l a may  rec-1  four.  none. T h i s c o n t r a s t s w i t h r e c - 1  and  increases  increasing  g i v e n p l a n t may  penetrant. M i l l e r  the  Secondly,  exchange. F i n a l l y ,  recombination,  a§_ mutant  from  a p p a r e n t l y gene c o n v e r s i o n t o t h e  i t does i n t e r g e n i c  appeared  the  i s o n l y found  a consequence of a s m a l l rearrangment.  plants  rather  interchromosomal  i n s e v e r a l ways. F i r s t ,  h o m o z y g o t e . T h i s makes i t u n l i k e l y  The  t o be  as  exchange.  interchromosmal  as  proven  to a d e f e c t i n the c o n t r o l l i n g  regional  be  has  as w e l l  to the  suggested  interchromosomal  r e s p o n s i b l e f o r the observed  81  that  a  effect  i n c r e a s e s and  in  predicted  that  recombination  i f medial frequency  chromosome l o s s if  r e g i o n s were e x a m i n e d , a w o u l d be f o u n d .  are also  decreased  N o n d i s j u n c t i o n and  i n c r e a s e d by t h e as m u t a n t , a s e x p e c t e d  s y n a p s i s i s d e f e c t i v e . As a c o n s e q u e n c e f e r t i l i t y  i s also  reduced. The  mutant c ( 3 ) G i n D r o s o p h i l a i s a r e c e s s i v e c r o s s - o v e r  suppressor  (Gowen and Gowen, 1 9 2 2 ) . R e m a r k a b l y ,  intergenic  recombination  heterozygous to  that  i n c r e a s e two t o t h r e e - f o l d  effect  1966). N o n d i s j u n c t i o n i s n o t d e c r e a s e d  In the  the increase i n recombination terms o f i t s p l e i o t r o p i c  spontaneous mutation  rate  i n the heterozygote, frequency  effects,  (Hall,  repair  lethals  from  c(3)Gf l i e s ,  (Hall,  1972).  c ( 3 ) G does n o t i n c r e a s e  1 9 7 1 ) . However, Watson of X-ray  induced,  s u g g e s t i n g some d e f e c t i n a  p r o c e s s . The homozygote d o e s n o t have a s y n a p t o n e m a l  complex, e x h i b i t s fecundity  e x t e n s i v e n o n d i s j u n c t i o n and h a s r e d u c e d  ( S m i t h and K i n g ,  Hinton deficiency  (1966),  found  1968; Gowen, 1933; H a l l ,  that  a f l y heterozygous  containing the c(3)G locus exhibited  frequency  i n t e r m e d i a t e between t h o s e  wild-type  flies.  i)  similar  ( C a r l s o n , 1972; H i n t o n ,  (1972) h a s shown a n i n c r e a s e i n t h e f r e q u e n c y recessive  i n females  f o r c ( 3 ) G . The p a t t e r n o f i n c r e a s e i s q u i t e  of the interchromosomal  despite  i n t r a - and  synapsis  i n c r e a s e s over  f o ra a  recombination  o f mutant homozygote and  T h i s prompted him t o suggest  c ( 3 ) G i s hypomorphic  1972).  that:  f o r some s y n a p t i c p r o c e s s  the genotypic  series  such  that  -  d e f i c i e n c y / c ( 3 )G, c ( 3 )G/c ( 3 )G, d e f i c i e n c y / - ! - , c ( 3 )G/+ , +/+_ a n d ;  82  ii)  the frequency  o f e x c h a n g e i s i n c r e a s e d by t h e amount o f  disturbance encountered and  Redfield,  1951; c i t e d  Presumably, and  i n Hinton,  s y n a p s i s i n c(3)G/+  t h e r e f o r e , causes  relative  by a b i v a l e n t  during synapsis (Schultz 1966).  flies  i s altered  an i n c r e a s e i n r e c o m b i n a t i o n  to the wild-type s t r a i n .  h y p o t h e s i s h a s some i n t e r e s t i n g model o f t h e i n t e r c h r o m o s o m a l  similarities  with  result  about  and h e n c e t h e f u n c t i o n  c a n be p r o p o s e d .  In chapter  In e i t h e r  case,  the nature of the rec-1  IV i t was  like  rate  I t f o l l o w s from  w o u l d a l s o be i n c r e a s e d (as f o u n d  assay  developed  by R o s e n b l u t h  rec-1  does n o t i n d u c e  mutations  radiation  sensitive  a repair  source that  system  o f DNA  coli  DNA  lesions.  i n t h e DNA  i n both  lethal  mutations  1,871  mutation  the E. c o l i  - a phenotype t h a t was e i t h e r  damage. T h i s e v i d e n c e  strain  or stressed  system  83  i s not  expected  by an e x t r a  does n o t c o n c l u s i v e l y  prove  related disorder  t h e example o f c ( 3 ) G as d i s c u s s e d i n c h a p t e r  d o e s make i t a n  Moreover,  w o u l d have b e e n  defective  that  lethal  chromosomes e x a m i n e d ) . t h e Rec-1  and  rate  I have shown  ( T a b l e 8: 0  (1982) r e p o r t e d t h a t  would  cross-over  the spontaneous  e t a l . (1983),  r e c - 1 i s n o t due t o a r e p a i r  (recall  r e c - 1 may  t h e b a l a n c e r e T l and t h e m u t a t i o n  r e c o v e r e d from  Hartman and Herman  product  the E.  for initiating  t h e above t h a t  Using  that  rem-1, i t m i g h t i n d u c e  an i n c r e a s e d number o f b r e a k s  yeast mutants).  if  suggested  process, l i k e  p r o v i d e an e l e v a t e d s u b s t r a t e s u p p l y events.  obtained i n this  o f t h e w i l d - t y p e gene  i n a d e f e c t i n some r e p a i r  mutants. A l t e r n a t i v e l y ,  the time-delay  effect.  a number o f h y p o t h e s e s  mutation  frequency  This synaptic disturbance  B a s e d on t h e a b o v e e x a m p l e s and t h e r e s u l t s study  slightly  I V ) , but i t  unlikely explanation. Alternatively, recombinase  the rec-1  w i l d - t y p e gene p r o d u c t may  enzyme. T h i s enzyme c o u l d be  f o r m a t i o n of the H o l l i d a y  junction,  rec-1  and  p r o t e i n s i n E. c o l i  (Tsang 1982,  e t a l . , 1985; 1 9 8 4 ) . On  assist  i s the case w i t h  U s t i l a g o maydis,  of the H o l l i d a y purified  junctions.  amount o f enzyme  ( i e . a h y p e r m o r p h ) o r an  o f t h e enzyme c o u l d r e s u l t  recombination  frequency.  dominant e f f e c t mutation rec-1  (Rose and  Thirdly,  effect  Baillie,  rec-1  an  may  recombination.  Neurospora  be  frequency  result  The  1966;  that modify increases 1970;  Smith,  i n c r e a s e i n the  increase i n the  i n an  increase i n would have  that  been r e p o r t e d i n  rec-1, rec-2 specific  and  i n the r e c  Angel,  local  genes  regions. Mutations  increase in  1 9 6 6 ) . Two  rec-3  at  recombination  ( C a t c h e s i d e e t a l . , 1964;  elements  have been  the e x p r e s s i o n o f the r e c mutants: the cog  recombination  a  b e h a v e s as a r e c e s s i v e  t h e genes i n v o l v e d have o n l y a  i n a dramatic  C a t c h e s i d e and  modifies  has  i n the a s s o c i a t e d i n t e r v a l s  Catcheside  an  and  which  1 9 7 9 a ) . T h i s makes i t u n l i k e l y  A precedent  recombination.  loci  Symington  a repressor involved i n regulating  r e g u l a t e exchange i n s e v e r a l these  case,  rec-1  could  enzyme.  crassa, although  on  Holloman,  enzyme i n y e a s t  However, s u c h m u t a t i o n s  on r e c o m b i n a t i o n ;  codes f o r such  meiotic  In e i t h e r  junction.  an  and  respectively  Kmiec and  cleaves Holliday  efficiency  recA  t h e o t h e r hand, t h e p u t a t i v e r e c o m b i n a s e  (1985) have r e c e n t l y  a  i n v o l v e d i n the  T s a n g e t a l . , 1985;  i n the r e s o l u t i o n  Kolodner  as  be  mutants  identified + factor  (Angel e t a l . ,  1 9 7 4 ) , whereas t h e c o n  the extent of s u p p r e s s i o n of recombination 84  element by  the  rec  alleles  Catcheside bind with blocking  ( C a t c h e s i d e , 1975). Angel (1975) have s u g g e s t e d  an o p e r a t o r - l i k e s i t e translation  enzymes n e c e s s a r y rec  ) these  factor, site  just  general  gene  element),  "recombinase"  limit  products thereby  coding  the p r o d u c t i o n of  f o r exchange. In the d e r e p r e s s e d to i n t e r a c t  with  one  the rec-1  locality.  Therefore,  r e c o m b i n a s e w i t h no  mutation  sites.  state ( i e . the  of  O c h r e and  Other  mutation  in a critical  mutation  mutation producing  This hypothesis pleiotropic  genome, product  i t s c o n t r o l must be  for  rec-1  noted  homozygote. A  earlier  have an  the  affect  amber on  effects,  such  are  which  tRNA  but  strain. cannot  isolated  as a s m a l l d e l e t i o n ,  r e g i o n of the p o l y p e p t i d e , or  can as  the p r o b a b i l i t y  account  gene  in  f o r the g e n e r a l absence of m e i o s i s  mutant  of  affected  o f e x c h a n g e . However, n o n d i s j u n c t i o n  85  C.  a  product.  the only aspect  be  a point  a r e p l a u s a b l e means o f p r e v e n t i n g t h e a functional  of  nonsense  t h e Rec-1  r e m a i n as p o s s i b i l i t i e s ,  events  a  particular  t h e a p p r o p r i a t e tRNA s u p p r e s s o r s  elegans.  gene f r o m  As  opal mutations  frameshift  the e n t i r e  i f t h e w i l d - t y p e gene  specificity  i n the  s u p - 7 , does n o t  until  f o r the  s e v e r a l p o s s i b l e c l a s s e s of mutations  requirement.  suppressor,  cog  T h i s h y p o t h e s i s would r e q u i r e the absence  gene p r o d u c t  i s one  meet t h i s  w o u l d be  enhancer a f f e c t s  i s a r e p r e s s o r , t h e enzyme u n d e r  functional  the  exchange.  elegans,  recognition  tested  or  enzymes a r e a v a i l a b l e  of enhanced  of rec-1  a  (the con  and  a r e c o g n i t i o n sequence p r o v i d i n g s p e c i f i c i t y  I n C. not  t h a t the r e c  o f an a d j a c e n t  sequence. T h i s would p r e v e n t  e t a l . (1970)  a n d / o r chromosome l o s s a p p e a r  t o be  no  model t h a t  mechanism  i n the  repressor  increased  i n Rec-1. There  could  produce  such  is  an  increase. Finally, proposed  the  f o r c(3)G  considered.  see  synapsis  i n the  discrete  heterozygote  Instead,  i n synapsis  This  suggest  gene p r o d u c t . the  and  some r e g u l a t o r y  rec-1  (pers.  synaptonemal complexes discriminate  At  have been p r o p o s e d  locus  order  must be  genetically  rec-1  are  end  of  mapped. Any  characterize  as  f o r the the is a  wild-type  only  side  segregational control  process  of  of  examining  strain. fashion  between  these  f u r t h e r c h a r a c t e r i z a t i o n of  experiments designed could  86  be  accomplished  the  experiments  i n v e s t i g a t o r s . I would a s s i g n  other  the  exchange.  to these p r o j e c t s . F i r s t ,  rec-1  (ie. a  some  e a c h c h a p t e r a number o f  for future  or p r i o r i t y  such  involve  a l t e r a t i o n i n the  conclusive  require  only  affects  p r o b a b i l i t y of  function  Rec-1  1976;  consistently increases  comm.) i s i n t h e  i n any  the  an  i n the  hypotheses w i l l  following  to  mutation appears to pocess  Goldstein  strain.  Lucchesi,  i f rec-1  I t i s i n t e r e s t i n g to note that  synapsis.  Rec-1  and  hence the  perhaps r e f l e c t i n g  various  1966;  e f f e c t s of  i t i s more l i k e l y  disorder,  To  (Hinton,  increase  some hypomorph i n d u c e d d i s t u r b a n c e  f u n c t i o n which u n i f o r m l y  would  mutants  as  be  a consequence of a p h y s i c a l c o n s t r a i n t  time spent  effect  i n t e r c h r o m o s o m a l e f f e c t must  homozygote. T h e r e f o r e ,  i t i s not  i n c(3)G.  a synaptonemal d i s t u r b a n c e ,  1 9 7 2 ) , whereas t h e  rearrangement) or that  the  of  these Drosophila  i n the  Valentin,  expressed  and  Both of  recombination but  possibility  the to more  the rec-1  efficiently  i f the l o c a t i o n of the recombination  known. N e x t , region  I would  isolate  d e f i c i e n c i e s covering  about t h e rec-1 l o c u s .  deficiencies  would a l l o w  of p r o d u c t i o n  enhancer  By v a r y i n g  was  t h e immediate  t h e gene d o s a g e  such  the i n v e s t i g a t o r to determine the l e v e l  o f t h e gene p r o d u c t  i n t h e mutant. F o r example, a  hypomorph w o u l d e x h i b i t a more e x t r e m e p h e n o t y p e as a h e m i z y g o t e (ie.  d e f i c i e n c y / r e c - 1 ) t h a n as a homozygote  (Muller,  1 933).  However, an amorph w o u l d n o t p r o d u c e a f u n c t i o n a l gene and,  therefore,  product  i t s mutant p h e n o t y p e w o u l d be i n d e p e n d e n t o f  gene d o s a g e . The alleles  d e f i c i e n c i e s c a n a l s o be u s e f u l of rec-1.  way o f s c r e e n i n g especially effect  when t h e mutant  In yeast,  the basis  isolating  recover region  process  i n higher  eukaryotes, of i t s  used  one r u n s t h e r i s k o f  i s f u n c t i o n a l l y i n c i d e n t a l to the  (Esposito  and E s p o s i t o ,  1969).  (1984) h a s made u s e o f a d e f i c i e n c y t o p r e f e r e n t i a l l y  to i d e n t i f y  alleles  d e f e c t i v e mutants from a  The p r o c e d u r e  only  of several  a comparison of the extent and o t h e r  i s outlined  homozygous f o r some v i s i b l e  processes i n Figure  (eg.  of t h e i r  pleiotropic  14. Rec-1 m a l e s  m a r k e r , B, a r e m u t a g e n i z e d .  87  specific  three  t h e m u t a n t . The i s o l a t i o n  would a l l o w  on r e c o m b i n a t i o n  effects).  i s no e a s y  side effects are often  o f t h e genome. Her a p p r o a c h r e q u i r e s  effects  there  other  t h a n some a s s o c i a t e d p l e i o t r o p i c  procedure,  induced recombination  generations rec-1  rather  i n which m e i o t i c  a mutant t h a t  Grell  that  i s s e l e c t e d on t h e b a s i s  of a screening  recombination  be e m p h a s i z e d  f o r r e c o m b i n a t i o n mutants  on r e c o m b i n a t i o n  effect. as  I t should  in isolating  These  a r e mated t o h e r m a p h r o d i t e s deficiency  (Def.) c o v e r i n g  m a r k e r , A,  on t h e homologue  males c a r r y i n g  the t r e a t e d  deficiency/rec-1)  carrying the rec-1 (Figure  are selected  be  exposed  by  l o c u s and  and  scoring  and  induced, r e c e s s i v e mutation i n (eg. the rec-1  i n t h e F 2 . Any  worms) s h o u l d be The  readily  s c r e e n c a n be  locus) w i l l  i n the F1. T h e r e f o r e ,  p s e u d o r e v e r t a n t s of rec-1 w i l l  the frequency of the recombinant  C;  c r o s s e d w i t h homozygous  the d e f i c i e n c y  the recombinants  dominant  ( i e . B +/+  e x p r e s s e d i n the hemizygous c o n d i t i o n  revertants  a  a  14). In the F1, w i l d - t y p e  chromosome  d o u b l e mutant h e r m a p h r o d i t e s . Any the r e g i o n  a s e c o n d m a r k e r , C,  be d e t e c t e d by  significant  phenotypes  reduction i n  ( i e . Wild-type  observed.  adapted  to the i s o l a t i o n  of h y p e r - r e c  alleles  o f r e c - 1 by d e c r e a s i n g t h e d i s t a n c e between t h e  trans  markers  B and  become  C such t h a t  rare  would  not have a r e c o m b i n a n t ) . A t h r e e - f o l d  one  map  units  i n t h e N2  quite  in  ( e g . 0.3  recombinants  - most b r o o d s  r e c o m b i n a t i o n f r e q u e n c y would Wt  recombinant  identify  mating  100  or g r e a t e r  progeny  increase least  p e r b r o o d a much more common e v e n t , and only  constraint  t h e m a r k e r , B, must n o t s i g n i f i c a n t l y  efficiency  reported  of about  make t h e p r e s e n c e o f a t  t h e r e c o m b i n a t i o n m u t a n t . The  above i s t h a t  strain  of the mutagenized  on t h e m a t i n g  elegans. His r e s u l t s  efficiency  males.  Hodgkin  on  to select  an  the  reduce  the  (1983)  has  o f a number o f m u t a n t s  c o u l d be u s e d  hence  i n C.  appropriate  marker. Finally, extent  given a set of a l l e l e s  of t h e i r  experiments  effect  could  on c r o s s i n g  be p e r f o r m e d  w i t h some v a r i a t i o n over, long  to investigate 88  term the  i n the  population evolutionary  Po:  EMS  [males]  rec-1; rec-1  B B  X  Def.  _C_ C  [herm.]  V Fi:  P i c k Wt m a l e s d o u b l e mutant Eg.  [1  male] (Wt)  ( r e c a l l A i s d o m i n a n t ) and hermaphrodites. Def . rec-1  B  X  cross  to  [ 1 herm.] B  V F_2:  S c o r e f o r t h e absence o f r e c o m b i n a n t s . F o r example, few o r no Wt p r o g e n y i n t h e F2 i d e n t i f i e s a r e v e r t a n t or p u t a t i v e recombination d e f i c i e n t a l l e l e of r e c - 1 .  F i g u r e 14: S c r e e n f o r t h e i s o l a t i o n o f a l l e l e s o f r e c - 1 . M o d i f i e d f r o m G r e l l ( 1 9 8 4 ) . The u s e o f a d e f i c i e n c y t a r g e t s t h e r e c - 1 l o c u s f o r mutant i n d u c t i o n .  89  role  of r e c o m b i n a t i o n . Given the i n t u i t i v e  linkage of w e l l expect 1967; for  against  five  offspring  varies  the a v a i l a b l e  should,  categories.  produced)  environment  either  Smith,  First, may  would  ( N e i , 1967;  genetically  be  Williams,  1975;  be  when t h e The  diversity  the o p t i m a l genotype  o u t compete o t h e r s i n t h e  1971;  can  variable (ie.  or temporally.  l a r g e and  Turner,  to account  hypotheses  be a d v a n t a g e o u s  spatially  niches w i l l  therefore,  (Maynard  one  1 9 7 2 ) . A number o f m o d e l s have a t t e m p t e d  into  sexually  of  selected  the p e r s i s t e n c e of r e c o m b i n a t i o n . These  divided  to p r e s e r v e the  adapted combinations of a l l e l e s ,  r e c o m b i n a t i o n t o be Eshel,  need  microhabitat  H a m i l t o n , 1975;  Bulmer,  1980). Secondly,  f r e q u e n c y dependent  favour of genetic v a r i a t i o n  action their  environment.  p o s s e s s e d by t h e m i n o r i t y  of pathogens own  strategy  and  predators,  to e x p l o i t  by  (ie.  introducing  d e f e n c e s ) between t h e progeny Thirdly,  advantage  hence,  dependent  for sexually  (1978) and variable  density  Price  siblings  when c o m p e t i n g  biotic  i s due  factors  disimilarities and  that  are reduced  in  phenotype  a l s o p r o v i d e an Maynard  Smith  genetically  r e s o u r c e r e q u i r e m e n t s and  than a s e x u a l l y  i n a uniform, resource l i m i t e d 90  adjust  their parents.  (1982) p r o p o s e d t h a t  fitness  that  on t h e o f f s p r i n g  s e l e c t i o n may  have d i f f e r e n t  to the  available  reproducing individuals.  and Waser  have a g r e a t e r  genotype  selective  (1983a,b) h a v e s u g g e s t e d  o f p e s t s and p a t h o g e n s  sexual reproduction  The  t h e most r e a d i l y  r e s o u r c e . J a e n i k e (1978) and R i c e the e f f e c t s  operate i n  p r o d u c e d by r e c o m b i n a t i o n . T h i s  model i s b a s e d on t h e b i o t i c advantage  s e l e c t i o n may  produced  progeny  environment.  Fourth, the p e r s i s t e n c e of recombination may  be determined  mutations.  The  by t h e i r a s s o c i a t i o n with  recombination  gene may  promoting  alleles  favourable  h i t c h - h i k e with  favourable  recombinants t h a t i t c r e a t e s ( F e l s e n s t e i n and Yokoyama, Strobeck  1976;  e t a l . , 1975).  F i n a l l y , a l l of the above hypotheses are c o n c e p t u a l l y s i m i l a r i n t h a t they assume t h a t the s e l e c t i v e advantage recombination  provides to an i n d i v i d u a l stems from i t s a b i l i t y  to i n c r e a s e the genotypic v a r i a t i o n of the progeny. B e r n s t e i n e t al.  (1985) have proposed a d i f f e r e n t b a s i s f o r t h i s nominal  s e l e c t i v e advantage - the r e c o m b i n a t i o n a l r e p a i r of damaged Drawing from n a t u r a l h i s t o r y data, they suggest b a s i c r o l e f o r recombination  e x p l a i n s why  DNA.  t h a t t h i s more  many s p e c i e s have  abandoned o u t - c r o s s i n g but have r e t a i n e d recombination.  In t h e i r  view, any advantage produced by i n c r e a s e d g e n e t i c v a r i a t i o n i s l i m i t e d to s p e c i a l i z e d  situations.  As an a s i d e , S h i e l d s (1982) has  taken a novel approach by  suggesting t h a t the e f f e c t s of recombination advantages or disadvantages  these might e n t a i l ) may  by b e h a v i o u r a l m o d i f i c a t i o n . The the reduced  (whatever be r e g u l a t e d  amount of i n b r e e d i n g , and  e f f i c a c y of recombination  hence  i n producing v a r i a t i o n i n  the progeny, i s c o n t r o l l e d by the organism's d i s p e r s a l p a t t e r n s . S e l e c t i o n may breeding  a c t on the d i s t a n c e t r a v e l l e d b e f o r e choosing  a  s i t e and a mate, such t h a t an optimal l e v e l of g e n e t i c  variability  i s maintained  - not too much and not too  little.  A major l i m i t a t i o n of these v a r i o u s models has been the absence of an a p p r o p r i a t e system to t e s t t h e i r p r e d i c t i o n s . Very few attempts have been made to e x p e r i m e n t a l l y i n v e s t i g a t e the 91  role  of recombination  fitness.  Malmberg  recombination trait  i n the d e t e r m i n a t i o n of  (1977) u s e d  T4  r e c o m b i n a t i o n were n o t w e l l results  that  bacteriophage t o determine i f  i n c r e a s e s the r a t e  under p o l y g e n i c c o n t r o l .  of response  Although  quantified,  the h i g h recombination  that  r e c o m b i n a t i o n had  chromosomal r e a r r a n g e m e n t s crossing  over  of  response  in  a wild-type  i t appears  line  any  affect.  available  to s e l e c t i o n  Thompson made u s e  pair  c o n c e r n i n g the  and  Antonovics  Anthoxanthum odoratum  flies  sexually  these  Ellstrand  solution  hypothesis  the  rate  found  reproduced  plants  No  studies  cannot  make any  forces  i n the  and  grass  have a  fitness  subject to  such advantage  t o d e n s i t y dependent  to  was  observed  selection. c o n c l u s i o n s about which  optimum. l o c u s i n C.  to this  gene c a n be  suppress  (1984)  t h e o p t i m a l amount o f r e c o m b i n a t i o n , o r t h e f a c t o r s  rec-1  complex,  to that  selective  i n environments  selection.  when p l a n t s were e x p o s e d  The  of  have attempted  (1985) have shown t h a t  asexual plants  frequency dependent  that  (1977)  t h e n compared  of i n v e s t i g a t o r s  Ellstrand  determine  rapidly  strain.  r e c o m b i n a t i o n . A n t o n o v i c s and  Unfortunately,  He  He  favouring  over  for a  his  " e v o l v e d " more  i n the suppressed  of the hypotheses  advantage  from  i n Drosophila to  in a constructed strain.  To d a t e , o n l y one test  no  to selection  the f r e q u e n c i e s of  than the o t h e r s . Contrary to these r e s u l t s , found  individual  problem,  isolated. of Strobeck  As  e l e g a n s may especially  provide at least i f several  an example c o n s i d e r t h e  alleles  a  partial of  this  "hitch-hiking"  e t a l . ( 1 9 7 5 ) . They s u g g e s t e d  which c o u l d i n c r e a s e the frequency of a recombination  a mechanism enhancing  allele.  Just  as a mutator  hitch-hiking  gene c a n i n c r e a s e i n f r e q u e n c y by  with a favourable mutation  i t has induced  Gibson,  1974; Chao a n d Cox, 1983), computer  authors  demonstrated  in  frequency  They made t h e f o l l o w i n g  two a l l e l e s  population  enhancer  assumptions  A and a a r e m a i n t a i n e d  i s fixed  f o r the a l l e l e  dominant  suppressor  increase  recombination  of the experiment is  a recombination  s i m u l a t i o n s by t h e can i n c r e a s e  because o f i t s a s s o c i a t i o n with a f a v o u r a b l e  recombinant. i)  that  by h e t e r o s i s ;  b; i i i )  of recombination,  i n their  By  the a l l e l e ,  D, i s a  where a s d u n i f o r m l y  t o some s e t l e v e l ;  a favourable mutation,  and i v ) a t t h e s t a r t B  (codominant  with b ) , o f one i n  individuals.  choosing  t h e a p p r o p r i a t e g e n e t i c markers each  assumptions  c o u l d be s a t i s f i e d  C. e l e g a n s .  Initial  allele  i n an e x p e r i m e n t a l  c o u l d be e s t i m a t e d  from  c o u l d be d e t e r m i n e d  rec-1  in different  experimental  of Strobeck  generations  are required f o r a l l e l e  equilibrium  i n such  Such a l o n g t e r m  Selection  study  trials.  e t a l . (1975) i n d i c a t e  differences  100 g e n e r a t i o n s  The computer that  C. e l e g a n s '  i n a year  d o e s n o t seem i m p r a c t i c a l  93  pairs of  roughly  70  frequencies to reach  a p o p u l a t i o n . Given  over  coefficients  by u s i n g d i f f e r e n t  simulations  time,  using  o f v a r y i n g t h e amount o f  recombination alleles  system  f e c u n d i t y and d e v e l o p m e n t a l  between m u t a n t s . The a f f e c t  of these  f r e q u e n c i e s and g e n e t i c d i s t a n c e s  between t h e m a r k e r s c o u l d be v a r i e d .  generation  model:  i i )the  i n t r o d u c e d i n c o u p l i n g phase w i t h d a t a f r e q u e n c y  a thousand  (Cox and  rapid  i s possible. when  compared  to  the  Endler  one  year  Drosophila  population  experiment performed  by  (1973).  Taking  the  investigation important, theoretical  a b o v e as c o u l d be  albiet  small  population  a starting developed.  p o i n t , a number o f  E a c h e x p e r i m e n t w o u l d be  s t e p towards narrowing b i o l o g y and  models.  94  lines  the  empirical tests  gap  of an  between  of i t s  Literature  cited  A n g e l , T., B. A u s t i n and D. G. C a t c h e s i d e , 1970. R e g u l a t i o n o f recombination a t the h i s - 3 locus i n Neurospora c r a s s a . Aust. J . B i o l . S c i . 23: 1229.  A n t o n o v i c s , J . and N. C. E l l s t r a n d , 1984. 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