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Structure, expression and evolution of the 16 kilodalton heat shock protein gene family of C. elegans Russnak, Roland Hans 1986

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STRUCTURE, EXPRESSION AND EVOLUTION OF THE 16 KILODALTON HEAT SHOCK PROTEIN GENE FAMILY OF C. elegans  By  ROLAND HANS RUSSNAK  B.Sc,  The U n i v e r s i t y of C a l g a r y , 1979  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES Department of B i o c h e m i s t r y  We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d  THE UNIVERSITY OF BRITISH  COLUMBIA  October 1986  © Roland H. Russnak  In  presenting  degree  this  at the  thesis  in  partial  fulfilment  University of  British  Columbia,  freely available for reference and study. copying  of  department  this or  publication of  thesis by  this  for scholarly  his thesis  or  her  the  I agree  I further agree  purposes  may  representatives.  It  be is  requirements  for  an  Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  advanced  that the Library shall make it that permission  for extensive  granted  head  by the  understood  that  for financial gain shall not be allowed without  permission.  DE-6f3/81)  of  of  my  copying  or  my written  (i)  Abstract  Sequences coding f o r t h r e e  r e l a t e d 16 kd heat shock p r o t e i n s  (hsps) o f  the nematode C a e n o r h a b d i t i s elegans were i s o l a t e d and c h a r a c t e r i z e d . extensive  accumulation o f h s p l 6 mRNA d u r i n g  The  heat s t r e s s f a c i l i t a t e d the  i d e n t i f i c a t i o n o f two cDNAs, CEHS48 and CEHS41, which encoded hspl6  variants.  These plasmids were s e l e c t e d by t h e i r a b i l i t y t o h y b r i d i z e t o mRNA which d i r e c t e d the s y n t h e s i s  o f hspl6 i n v i t r o , and were f u r t h e r c h a r a c t e r i z e d by  sequence a n a l y s i s . Two-dimensional g e l e l e c t r o p h o r e s i s  o f hspl6 s y n t h e s i z e d  i n vitro  from  mRNA s e l e c t e d by h y b r i d i z a t i o n t o e i t h e r o f the cDNAs under c o n d i t i o n s stringency  revealed  the e x i s t e n c e  of a t l e a s t f i v e e l e c t r o p h o r e t i c  with s i g n i f i c a n t l y d i f f e r e n t i s o e l e c t r i c The  C. elegans genomic DNA.  recombinant  O v e r l a p p i n g phage  were used t o d e f i n e a r e g i o n o f approximately 30 k i l o b a s e s . f o r hspl6-48, p r e v i o u s l y  variants  points.  above cDNAs were used as s p e c i f i c probes t o i s o l a t e  bacteriophage containing  o f low  clones  The genes coding  i d e n t i f i e d by cDNA c l o n i n g , and f o r another 16 kd hsp  d e s i g n a t e d h s p l 6 - l were c h a r a c t e r i z e d  by DNA sequencing.  arranged i n a head-to-head o r i e n t a t i o n .  These two genes were  Both the coding and f l a n k i n g  regions  of these genes were l o c a t e d w i t h i n a 1.9 kb r e g i o n which was d u p l i c a t e d e x a c t l y t o form a p e r f e c t 3.8 kb i n v e r t e d repeat s t r u c t u r e . ended i n u n u s u a l G + C - r i c h sequences 24 bp i n l e n g t h . two  arms o f t h e i n v e r t e d r e p e a t a t the n u c l e o t i d e  the d u p l i c a t i o n event may have occurred A l t e r n a t i v e l y , gene c o n v e r s i o n  This  structure  The i d e n t i t y o f t h e  sequence l e v e l i m p l i e d  that  r e l a t i v e l y recently i n evolution.  between the two modules c o u l d have m a i n t a i n e d  homology between the two gene p a i r s .  (ii)  Comparison of the hsp!6-48 gene w i t h i t s c o r r e s p o n d i n g cDNA r e v e a l e d the presence of a s i n g l e , s h o r t i n t r o n .  An i n t r o n of comparable l e n g t h and i n an  analogous p o s i t i o n was a l s o found i n the h s p ! 6 - l gene.  The i n t r o n s  separated  v a r i a b l e and conserved r e g i o n s w i t h i n the amino a c i d sequences of the encoded heat shock p r o t e i n s .  A domain of approximatey 80 amino a c i d s i s c o n t a i n e d  w i t h i n the conserved second exon and i s homologous  to a s i m i l a r r e g i o n i n the  s m a l l hsps of D r o s o p h i l a , Xenopus,  as w e l l as the  soybean and man  a - c r y s t a l l i n p r o t e i n of the v e r t e b r a t e  lens.  Each hsp!6 gene c o n t a i n e d a TATA box upstream of the s t a r t of transcription.  Promoter sequences, which have been shown to be r e q u i r e d f o r  heat i n d u c i b i l i t y i n v a r i o u s systems, were l o c a t e d upstream of e i t h e r TATA box Northern b l o t a n a l y s i s showed t h a t the hsp!6-48 and h s p ! 6 - l genes a r e expressed a t l e v e l s a p p r o x i m a t e l y 20 - 40 f o l d lower than two c l o s e l y genes, hsp!6-41  and hsp!6-2, upon temperature e l e v a t i o n .  related  (iii)  TABLE OF CONTENTS  Page Abstract  i  L i s t of T a b l e s  viii  L i s t of F i g u r e s  ix  Abbreviations  xii  Acknowledgements  I.  xiv  INTRODUCTION  1  1.1  1  1.2  1.3  Response to Thermal S t r e s s 1.1.1  Locua 6 7B:  Puffing Activity  and P r o t e i n S y n t h e s i s  1.1.2  Locus 67B:  Gene O r g a n i z a t i o n  6  1.1.3  Locus 67B:  P r o t e i n Coding Regions  7  1.1.4  Locus 67B:  A c t i v i t y During  Development . .  ...  3  11  Conserved shsp F u n c t i o n i n Eucaryotes  14  1.2.1  Higher P l a n t s :  14  1.2.2  A v i a n and Mammalian Systems  16  1.2.3  Xenopus  23  1.2.4  Other Organisms  25  Soybean  T r a n s c r i p t i o n a l C o n t r o l and  I n d u c t i o n Mechanisms  26  1.3.1  Gene Promoter F u n c t i o n  27  1.3.2  Heat Shock Regulons  30  1.3.3  A c t i v a t i o n of T r a n s c r i p t i o n F a c t o r s  32  1.3.4  D i f f e r e n t i a l HSmRNA S t a b i l i t y  35  •  (iv)  Page 1.4  II.  Translational Control  36  1.4.1  HSmRNA S e l e c t i v i t y  37  1.4.2  High mRNA Turnover  41  1.5  Recovery and A u t o r e g u l a t i o n  42  1.6  Function  44  o f shsps  1.6.1  Intracellular Localization  45  1.6.2  Thermotolerance:  48  1.6.3  I n h i b i t i o n o f the Heat Shock Response  Role o f shsps  1.7  The B i o l o g y of C a e n o r h a b d i t i a  1.8  The P r e s e n t  51  elegans  52  Study  53  EXPERIMENTAL PROCEDURES  55  2.1  Maintenance o f Nematodes  55  2.2  A n a l y s i s o f Heat Shock P r o t e i n s  55  2.2.1  [ S ] - s u l f a t e L a b e l l i n g o f E. c o l i  55  2.2.2  I n v i v o L a b e l l i n g o f C. elegans ( B r i s t o l )  Proteins  and  . .  2.3  2.4  3 5  Induction  of.Heat Shock P o l y p e p t i d e s  55  2.2.3  A n a l y s i s of i n v i t r o L a b e l l e d Proteins  56  2.2.4  Polyacrylamide  56  Gel E l e c t r o p h o r e s i s of Proteins  RNA A n a l y s i s  57  2.3.1  I s o l a t i o n o f RNA from B r i s t o l N2 Nematodes  57  2.3.2  E l e c t r o p h o r e s i s o f RNA and Northern T r a n s f e r s  58  2.3.3  SI Nuclease Mapping  58  I d e n t i f i c a t i o n o f cDNAs 2.4.1  Screening  o f a cDNA L i b r a r y  59 . 59  (V)  Page 2.4.2 2.5  2.6  H y b r i d i z a t i o n S e l e c t i o n A n a l y s i s of cDNAs  60  General Methods f o r P l a s m i d A n a l y s i s  61  2.5.1  Bacterial Strains  61  2.5.2  Transformations  61  2.5.3  P u r i f i c a t i o n of P l a s m i d DNA  62  A n a l y s i s of B a c t e r i o p h a g e of C. elegans B r i s t o l Genomic DNA  -. . .  63  Libraries . .  63  2.6.1  Screening  2.6.2  E s t a b l i s h i n g High T i t e r Phage Stocks  64  2.6.3  I s o l a t i o n of Bacteriophage DNA  65  2.7  P u r i f i c a t i o n of C.  2.8  General DNA  elegans Genomic DNA  66  Techniques  67  2.8.1  R e s t r i c t i o n Endonuclease D i g e s t i o n of DNA  67  2.8.2  E l e c t r o p h o r e s i s of DNA  67  2.8.3  P u r i f i c a t i o n of S p e c i f i c DNA  2.8.4  End-Labelling  2.9  DNA  2.10  Preparation  of DNA  and  Southern T r a n s f e r s Fragments  68  Fragments  68  Sequencing  69  of H y b r i d i z a t i o n Probes  70  125 2.10.1 P r e p a r a t i o n  of  [  2.10.2 P u r i f i c a t i o n and  I ] - l a b e l l e d RNA  70  L a b e l l i n g of O l i g o d e o x y n u c l e o t i d e s  2.11  2.10.3 P r e p a r a t i o n Hybridization  of Double-Stranded DNA  2.12  Summary of B a c t e r i a l S t r a i n s Used  Probes  . .  ...  70 71 72 73  (vi)  I I I . RESULTS 3.1  Pase  The Heat Shock Response of C a e n o r h a b d i t i s elegans v a r . Bristol,  s t r a i n N2  74  3.2  Identification  of cDNAs Coding f o r Hspl6  3.3  Two-dimensional  3.4  Messages Coding f o r Hspl6 Are Not T r a n s c r i b e d i n  76  G e l E l e c t r o p h o r e s i s of Hspl6  C o n t r o l Nematodes  81  . . . .  83  3.5  Sequence A n a l y s i s o f the cDNAs CEHS48 and CEHS41  83  3.6  Isolation  89  3.7  Sequencing of the Hspl6 Genes and I d e n t i f i c a t i o n  of B r i s t o l Genomic DNA  Clones of a  P e r f e c t 1.9 kb I n v e r t e d Repeat  90  3.8  Identification  94  3.9  The shsp Genes of C. e l e s a n s C o n t a i n a S i n g l e I n t r o n  101  3.10  L o c a t i o n of the S t a r t s of T r a n s c r i p t i o n  102  of the Small Heat Shock Genes  3 .11 The 3 * F l a n k i n g Regions 3.12  O r g a n i z a t i o n of I n v e r t e d Repeats  104 i n the Region  C o n t a i n i n g Genes H s p l 6 - l / 4 8 3.13  D i f f e r e n t i a l E x p r e s s i o n of Hspl6 Genes i n C. elegans  3.14  Comparison  105 I l l  o f Locus Hspf6-l/48 i n C a e n o r h a b d i t i s elegans  B r i s t o l and Bergerac S t r a i n s  IV.  113  DISCUSSION 4.1  The Hspl6 Gene F a m i l y of C. elegans  4.2  E v o l u t i o n of the Hspl6 Gene Family and i t s R e l a t i o n s h i p to Other sHsps and t o V e r t e b r a t e a - C r y s t a l l i n s  118  . . .  122  (vii)  Page  V.  4.3  Gene Conversion W i t h i n Locus H s p l 6 - 4 8 / l  127  4.4  The Heat Shock Response o f C. elegans  133  REFERENCES  1  4  3  (viii)  LIST OF TABLES  Page II.  Hsp25 I n d u c t i o n i n CEF C e l l s  17  I I I . Hsp25 I n d u c t i o n i n Mammalian C e l l s  20  IV.  Genotypes o f B a c t e r i a l  73  V.  Amino A c i d Compositions o f Hspl6-1 and Hspl6-48  VI.  A s s o c i a t i o n o f G + C-Rich Sequences w i t h Gene C o n v e r s i o n Boundaries  Strains  97  132  (ix)  cy->-ft)V  LIST OF FIGURES  }  It,'", ft-  Page 1.  Gene o r g a n i z a t i o n a t l o c u s 67B o f D r o s o p h i l a  8  2.  A schematic diagram o f the v a r i o u s amino a c i d  homologies  between the shsps o f D r o s o p h i l a  10  3.  I n d u c t i o n o f h e a t shock p r o t e i n s i n C. elegans  75  4.  S t a b i l i t y o f the hsps o f C. elegans  77  5.  In v i t r o t r a n s l a t i o n p r o d u c t s o f mRNA from heat shocked  cells,  and enrichment o f messages coding f o r the shsps 6.  78  In v i t r o t r a n s l a t i o n o f RNA s e l e c t e d by h y b r i d i z a t i o n w i t h pCEHS41  7.  80  Two-dimensional  g e l e l e c t r o p h o r e s i s o f the p r o d u c t s from  t r a n s l a t i o n o f h y b r i d - s e l e c t e d polyA RNA  82  +  8.  N o r t h e r n b l o t and d o t - b l o t a n a l y s i s o f polyA RNA from c o n t r o l +  and heat shocked worms 9.  S t r a t e g y used t o determine the n u c l e o t i d e sequence and CEHS41 cDNA i n s e r t s  10.  84  Complete  .  85  n u c l e o t i d e sequences  and pCEHS41 w i t h the deduced 11.  Comparison  o f CEHS48  o f the deduced  o f the cDNA i n s e r t s from pCEHS48 amino a c i d sequence  86  amino a c i d sequences o f f o u r s m a l l heat  shock p r o t e i n s from D. melanogaster, a - c r y s t a l l i n and two 16,000 d a l t o n hsps from C. elegans 12.  13.  88  O r g a n i z a t i o n o f the hsp!6-48 and h s p ! 6 - l genes o f C. e l e g a n s , and the sequencing s t r a t e g y  91  A n a l y s i s o f C. elegans B r i s t o l genomic DNA  93  (X)  Page 14.  Complete sequence o f a s m a l l heat shock gene c l u s t e r o f C. elegans  95,96  15.  RNA d o t - b l o t a n a l y s i s u s i n g a probe s p e c i f i c f o r the h s p l 6 - l gene . . 99  16.  Comparison o f the deduced amino a c i d sequences f o t h r e e shsps of C. e l e g a n s . the mouse a A ^ - c r y s t a l l i n c h a i n , and t h e bovine a B ^ - c r y s t a l l i n chain  17.  100  SI n u c l e a s e p r o t e c t i o n a n a l y s i s o f t r a n s c r i p t i o n s t a r t  sites  of the hsp!6-48 and h s p ! 6 - l genes, and comparison o f the 5' flanking regions 18.  103  O r g a n i z a t i o n o f the i n v e r t e d repeats and G + C - r i c h boundary sequences i n l o c u s h3p!6-l/48  19.  106  A n a l y s i s o f G + C - r i c h boundary sequences i n v a r i o u s recombinant phage and B r i s t o l genomic DNA  20.  108  D e t e c t i o n o f boundary sequences i n 3.3R by d i s r u p t i o n o f t h e 370 bp i n v e r t e d r e p e a t  21.  D e t e r m i n a t i o n o f the s p e c i f i c i t y o f v a r i o u s probes used i n t h e mRNA e x p r e s s i o n s t u d i e s  22.  110  . .-  N o r t h e r n b l o t a n a l y s i s o f hsp!6 mRNA l e v e l s u s i n g  112 gene-  s p e c i f i c probes 23.  D e t a i l e d comparison o f the 1.9 kb i n v e r t e d r e p e a t  114 structure  i n the c l o s e l y r e l a t e d s t r a i n s Bergerac and B r i s t o l 24.  A comparison o f the p r o t e i n s encoded by the C. elegans 16 kd h e a t shock genes  25.  116  119  A model f o r the e v o l u t i o n a r y o r i g i n o f the hsp!6 gene l o c i o f C. e l e g a n s and the a - c r y s t a l l i n genes o f v e r t e b r a t e s  123  (xi)  Page 26.  The l e a d e r sequences of the hsp!6 genes of C. elegans  27.  A comparison o f the i n t e r g e n i c r e g i o n s o f h t e two hsp!6 gene l o c i of C. elegans  135  137  (xii)  ABBREVIATIONS  A  adenine  ATP  adenosine-5'-triphosphate  BUdR  bromouridineodeoxyribonucieoside  bp  base p a i r s  C  cytosine  cDNA  complementary DNA  cpm  counts p e r minute  DNA  deoxyribonucleic acid  dATP dCTP  deoxyadenosine-5'-triphosphate deoxycytidine-5 *-triphosphate  dGTP  deoxyguanosine-5'-triphosphate  dTTP  deoxythymidine-5'-triphosphate  ddTTP  dideoxythymidine-5 * - t r i p h o s p h a t e  EDTA  ethylenediamine t e t r a a c e t i c  G  guanine  Gu-HCl  guanidinium h y d r o c h l o r i d e  Hepes  N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid  hsg  heat shock granule  hsp  heat shock  hsmRNA  heat shock messenger RNA  HSTF  heat shock t r a n s c r i p t i o n  HSE, HIP  heat shcok promoter  kb  kilobases  kd  kilodaltons  acid  protein  factor  element  (xiii)  mRNA  messenger  N  adenine, c y t o s i n e , guanine o r thymine  PIPES  piperazine-N,N'-bis(2-ethanesulfonic acid)  Pi  isoelectric  polyA  +  RNA  point  polyadenylated  polyA  non-polyadenylated  RNA  ribonucleic  RNP  ribonucleoprotein  rpm  r e v o l u t i o n s p e r minute  SDS  sodium dodecyl  shsps  s m a l l heat shock p r o t e i n  T  thymine  tRNA  transfer  Tris  tris  ts  temperature  acid  sulfate  RNA  (hydroxymethyl) aminomethane sensitive  (xiv)  ACKNOWLEDGEMENTS  P e r s i s t e n c e i s e s s e n t i a l i n the s c i e n t i f i c process  and was encouraged  under t h e s p e c i a l working c o n d i t i o n s a f f o r d e d t o me d u r i n g my graduate studies.  C r e d i t f o r t h i s goes t o my s u p e r v i s o r , P e t e r Candido, who  the members o f h i s l a b o r a t o r y t o experiment. s p i r i t of cooperation Involved  allowed  This also contributed to a  i n which ideas were e a s i l y exchanged.  i n v a r i o u s aspects  o f t h i s p r o j e c t were T e r r y Snutch, Ann Rose,  E l i z a b e t h Burgess, Rashmi Kothary, P e t e r Candido and Marlys Koschinsky. S p e c i a l r e c o g n i t i o n goes to Don Jones who has been a s s o c i a t e d w i t h the c h a r a c t e r i z a t i o n o f the hsp!6 gene f a m i l y s i n c e i t s conception whose e l e g a n t m a n i p u l a t i o n  of the genes presented  and t o Rob Kay  i n t h i s t h e s i s together  with  h i s swim through t h e F a c u l t y Club pond made i t a l l worthwhile. In a d d i t i o n , I would l i k e t o acknowledge Susan Heming f o r h e r s k i l l and patience  i n t h e p r e p a r a t i o n o f t h i s document.  A s i n c e r e thanks t o E r i c Sommerman, Ev T r i p , P i e t e r C u l l i s and C r a i g Newton f o r a l l t h e d a n d e l i o n s .  1  I.  INTRODUCTION  1.1  Response t o Thermal S t r e s s Most c e l l s respond t o s e v e r e m e t a b o l i c s t r e s s by d r a m a t i c a l l y  t h e i r pattern of protein synthesis.  A l l organisms s t u d i e d ,  altering  including  b a c t e r i a , p l a n t s , f u n g i , p r o t o z o a and animals, both p o i k i l o t h e r m i c and homeothermic, r e a c t i n t h i s way t o abrupt temperature i n c r e a s e s , shock.  Altered patterns  o f p r o t e i n synthesis during  r e s u l t o f the r e g u l a t i o n o f mRNA s y n t h e s i s s i t u a t i o n being  a c o o r d i n a t i o n o f both.  heat shock p r o t e i n s  i . e . heat  thermal s t r e s s can be a  o r mRNA t r a n s l a t i o n , the  I n a l l cases,  usual  a well defined  set of  (hsps) can be i d e n t i f i e d .  H i s t o r i c a l l y , the heat shock response was f i r s t observed w i t h the  light  microscope a t the l e v e l o f p o l y t e n e  chromosome p u f f i n g i n l a r v a l s a l i v a r y  glands o f d i p t e r a n s  R i t o s s a , 1963).  ( R i t o s s a , 1962,  melanogaster. t h e r e are n i n e h e a t i n d u c i b l e p u f f s . a s e t o f p r o t e i n s upon heat shock was r e p o r t e d then i t has  In Drosophila I n 1974 the i n d u c t i o n o f  by T i s s i e r e s e t a l .  Since  become c l e a r t h a t the p u f f s are s i t e s o f RNA t r a n s c r i p t i o n and  t h a t most o f t h e s e new RNAs are t r a n s l a t e d i n t o heat shock p r o t e i n s . seven major heat induced p r o t e i n s o f D. melanogaster are designated hsp70, hsp68, hsp27, hsp26, hsp23 and hsp22 a c c o r d i n g m o l e c u l a r weights on SDS p o l y a c r y l a m i d e  The hsp83,  t o t h e i r apparent  gels.  Hsp83 i s encoded by a s i n g l e gene which i s l o c a t e d a t c y t o l o g i c a l l o c u s 63B  (Holmgren e t a l . , 1979).  1.2  kb i n t r o n i n the 5' noncoding r e g i o n  Lis,  1983).  The hsp83 t r a n s c r i p t i s c h a r a c t e r i z e d by a (Holmgren e t a l . , 1981;  Hackett and  Sequence a n a l y s i s o f a y e a s t hsp90 gene ( F a r r e l l y and  F i n k e l s t e i n , 1984)  and a c h i c k e n hsp90 gene ( C a t e l l i e t a l . , 1985a) has ~ ~  2  demonstrated t h a t the p r e d i c t e d p r o t e i n s are r e l a t e d to hsp83 of  Drosophila  (Hackett  chicken  and  L i s , 1983).  A p o l y c l o n a l a n t i b o d y prepared a g a i n s t  hsp90 r e a c t s w i t h p r o t e i n s of s i m i l a r m o b i l i t y i n heat shocked human, r o d e n t and  frog c e l l s  Drosophila  ( K e l l y and  Schlesinger,  1982).  Although the f u n c t i o n of  hsp83 i s unknown, hsp90 i s a s s o c i a t e d w i t h non-transformed  s t e r o i d receptors  i n chick oviduct  ( C a t e l l i e t a l . , 1985b) as w e l l  8S  as  src pp60  i n avian c e l l s  1981).  Drosophila  nonstress  i n f e c t e d w i t h Rous sarcoma v i r u s (Oppermann e t a l . ,  hsp83 and  i t s r e l a t e d p r o t e i n s are d e t e c t a b l e  under  c o n d i t i o n s , t h e i r l e v e l s i n c r e a s i n g s i g n i f i c a n t l y during  heat  shock. In D. melanoRaater, the hsp70 gene f a m i l y c o n s i s t s of both heat i n d u c i b l e and  c o n s t i t u t i v e l y expressed genes.  At l e a s t f i v e h i g h l y  homologous heat i n d u c i b l e genes are d i s t r i b u t e d over l o c i 87A (Schedl  e t a l . , 1978;  Livak e t a l . , 1978;  A r t a v a n i s - T s a k o n a s e t a l . , 1979; 1980;  I n g o l i a e t a l . , 1980).  homology to hsp70 and  Moran e t a l . ,  C r a i g e t a l . , 1979;  therefore  can be  considered  r e l a t e d p r o t e i n s are a l s o detectable  respectively 1986).  The  genes a r e  hsc4 which map  ( I n g o l i a and  Goldschmidt-Clermont, 75%  p a r t of the same f a m i l y .  i n normal c e l l s .  Hsp70  They are the p r o d u c t s  shock cognate genes) r e f e r r e d  c y t o l o g i c a l l y to 70C,  C r a i g , 1982a; C r a i g e t a l . , 1983;  h s c l and hsc2 genes c o n t a i n  shows  (Holmgren e t a l . , 1979).  c o n s t i t u t i v e l y expressed genes (heat  t o as h s c l . hsc2 and  87C  1979;  Hsp68 i s a l s o heat i n d u c i b l e , and  I t i s encoded by a s i n g l e gene at l o c u s 95D  of t h r e e  and  87D  and  88E,  P a l t e r et a l . ,  i n t r o n s w h i l e the heat  inducible  uninterrupted.  A. f a m i l y of i n d u c i b l e and -^••i^^S-a*?^'' those: i n D r o s o p h i l a  c o n s t i t u t i v e l y expressed genes r e l a t e d to  are found i n . yeast  ( I n g o l i a e t a l . , 1982;  Craig  and  3  Jacobsen, 1984; Caenorhabditis 1984;  C r a i g and  Jabobsen, 1985;  elegans (Snutch and  Lowe and Moran, 1986).  Baillie,  e t a l . , 1985;  1984)  and mouse (Lowe and  et a l . , 1984), Xenopus  (Rosen et a l . , 1985)  Hunt and Morimoto, 1985).  and man  which i s 48% homologous to D r o s o p h i l a  (Bienz,  (Voellmy e t a l . , 1985;  A r e l a t e d cognate gene has  c h a r a c t e r i z e d i n r a t (O'Malley et a l . , 1985).  i n c r e a s e s d u r i n g heat treatment  hsp70 (Bardwell  and  C r a i g , 1984).  inducible protein migrating been i d e n t i f i e d  and  i n mammalian c e l l s has  The  I t i s encoded  of the most h i g h l y  of the c o n s t i t u t i v e l y expressed hsp70 s p e c i e s  1985;  Chappell  enzyme w i t h  e t a l . , 1986).  d i s c u s s i o n which f o l l o w s w i l l be c o n f i n e d to the s t r u c t u r e  r e g u l a t i o n of the genes w i t h i n the heat shock p u f f at l o c u s 67B Drosophila  and  the e x p r e s s i o n  1.1.1  to r e l a t e d genes i n o t h e r organisms. of hsp27, hsp26, hsp23 and  Locus 67B:  P u f f i n g A c t i v i t y and  being  l a r v a e are s u b j e c t e d  T h i s l o c u s i s l i n k e d to  hsp22.  Protein  Synthesis  to a temperature of 37 C,  the normal c u l t u r e temperature, p u f f i n g at l o c u s 67B  minute.  Puffs continue  protein synthesis  to grow for- 30 - 40 minutes b e f o r e  i s blocked  by  and  of  o If Drosophila  by  weight of 70,000 has  been i d e n t i f i e d as a c l a t h r i n u n c o a t i n g  (Ungewickell,  i t s level  At the p r o t e i n l e v e l , a heat  w i t h an apparent molecular  One  been  C r a i g , 1984), i s  et §_1. , 1984).  i n every organism t e s t e d , making i t one  conserved p r o t e i n s known.  ATPase a c t i v i t y  (Neidhardt  Wu  In E. c o l i , p r o t e i n DnaK,  an extremely abundant p r o t e i n at normal growth temperature, but  a s i n g l e gene (Bardwell  Moran,  Heat i n d u c i b l e hsp70 sequences have been  c h a r a c t e r i z e d i n rainbow t r o u t (Kothary 1984a), D i c t y o s t e l i u m  C r a i g et a l . , 1985),  o 25  C  occurs w i t h i n regressing.  one If  i n c u b a t i n g e x c i s e d s a l i v a r y glands i n medium  4  c o n t a i n i n g c y c l o h e x i m i d e , the induced p u f f s f a i l to r e g r e s s u n l e s s the temperature i s r e t u r n e d t o normal.  A l s o , the s e v e r i t y of the temperature  i n c r e a s e i s r e f l e c t e d i n the maximum s i z e o f p u f f observed i n a g i v e n time. T h i s c y t o l o g i c a l a n a l y s i s l e d t o two o t h e r important o b s e r v a t i o n s . F i r s t l y , most o f the p u f f s e x i s t i n g p r i o r to heat shock r e g r e s s upon temperature e l e v a t i o n .  Upon prolonged exposure to h i g h temperature, they  r e t u r n to normal f o l l o w i n g , the r e g r e s s i o n of the heat shock p u f f s . Secondly, l o c u s 67B can be induced to p u f f by a v a r i e t y of o t h e r c o n d i t i o n s i n c l u d i n g exposure to d i n a c t i n 1963;  (Rensing, 1973), d i n i t r o p h e n o l  E l l g a r d and Rensing, 1972,  McCarthy,  1978)  1973), hydrogen p e r o x i d e (Compton and  and v a l i n o m y c i n (Rensing, 1973)  (Ashburner, 1970;  (Ritossa,  or by r e c o v e r y from a n o x i a  Zhimulev and Grafodatskaya, 1974).  Thus, p u f f i n g  activity  f i r s t d e f i n e d the parameters and k i n e t i c s of gene a c t i v i t y a t l o c u s 6 7B. Exposure to h i g h temperature a l s o r e s u l t s i n the r a p i d s y n t h e s i s of a p p r o x i m a t e l y seven hsps whereas the r a t e of s y n t h e s i s of most c e l l u l a r o ' p r o t e i n s n o r m a l l y made a t 25 C xs s t r o n g l y reduced ( T i s s i e r e s e t a l . , 1974).  T h i s phenomenon i s observed i n e x c i s e d s a l i v a r y glands as w e l l as  t i s s u e from b r a i n , m a l p i g h i a n t u b u l e s and wing i m a g i n a l d i s k s .  Various  D r o s o p h i l a m e l a n o s a s t e r t i s s u e c u l t u r e l i n e s show s i m i l a r p r o t e i n metabolism (McKenzie e t al.., 1975; et a l . ,  S p r a d l i n g e t a l . , 1975; Moran e t a l . , 1978;  Mirault  1978).  Upon h e a t shock, polysomes  from c u l t u r e d c e l l s d i s a g g r e g a t e and  newly  s y n t h e s i z e d heat shock mRNA (hsmRNA) sediments as two new polysomal peaks i n s u c r o s e d e n s i t y g r a d i e n t s (McKenzie e t a l . ,  1975).  The 12S RNA  fraction  c o n t a i n s hsmRNA t h a t has been shown to d i r e c t the s y n t h e s i s of 4 p r o t e i n s w i t h m o l e c u l a r weights o f 22,000, 23,000, 26,000 and 27,000 i n a r a b b i t  5  r e t i c u l o c y t e l y s a t e ( M i r a u l t e t a l . , 1978; Moran e t a l . , 1978) o r i n an a s c i t e s c e l l - f r e e e x t r a c t (McKenzie and Meselson, 1977). correspond t o 4 o f the 7 hsps i d e n t i f i e d molecular  These p r o t e i n s  i n vivo, the other three  weights of 70,000, 68,000 and 83,000.  Their  having  corresponding  t r a n s c r i p t s a r e found i n a 20S RNA polysomal f r a c t i o n . Pulse  l a b e l l e d RNA e x t r a c t e d from t h e 12S RNA peak h y b r i d i z e s i n s i t u  to chromosomal s i t e 67B ( S p r a d l i n g e t a l . , 1977; McKenzie and Meselson, 1977)  and thus a d i r e c t  l i n k can be made between p u f f i n g a c t i v i t y and hsmRNA  s y n t h e s i s a t l o c u s 67B and the appearance of a s p e c i f i c subset  of hsps upon  thermal s t r e s s . 35 Pulse  l a b e l l i n g with  [  S]methionine f o r 10 minutes a t t h e s t a r t o f a  heat ahock i s s u f f i c i e n t to d e t e c t t h e i n i t i a t i o n o f s y n t h e s i s of a l l seven hsps.  The maximum r a t e o f s y n t h e s i s  i s reached between 90 and 120 minutes o w i t h a subsequent d e c l i n e t o about 50% a f t e r 6 - 8 hours a t 37 C ( M i r a u l t e t a l . , 1978).  I n c o n t r a s t , when t h e c e l l s a r e r e t u r n e d  t o 25°C a f t e r a  o heat shock a t 37 C f o r 1 hour, and t h e r a t e o f s y n t h e s i s o f hsps i s examined by p u l s e l a b e l l i n g a t v a r i o u s times t h e r e a f t e r , t h e r e  i s a gradual  decrease o f t h e s e p r o t e i n s u n t i l , by 8 hours, no hsp s y n t h e s i s i s d e t e c t e d and normal p r o t e i n s y n t h e s i s has recovered (1978) a l s o demonstrated t h a t hsps, p u l s e  completely.  Mirault et a l .  l a b e l l e d during  shock, a r e s t a b l e f o r up t o one day a t 25°G.  a 1 hour heat  6  1.1.2  Locus 67B: The  RNA  coding  Organization  c l o n i n g of sequences coding  f a c i l i t a t e d by 12S  Gene  26 and  27 was  greatly  the f a c t t h a t t h e i r t r a n s c r i p t s were h i g h l y e n r i c h e d  p o l y s o m a l peak a f t e r heat shock. f o r hsp23 and and  experiments.  These cDNAs do not  h y b r i d i z a t i o n s e l e c t i o n (Wadsworth et a l . ,  do h y b r i d i z e t o l o c u s 67B melanoRaster genomic DNA regions  Thus, cDNAs made to 12S  using  and  (Voellmy  1980)  c r o s s - h y b r i d i z e under normal s t r i n g e n c y in situ hybridization.  Corces et a l . , 1980;  but  Drosophila  c l o n e s were subsequently i d e n t i f i e d t h a t  f o r . a l l 4 p r o t e i n s c l u s t e r e d w i t h i n 11 kb  McCarthy, 1980;  i n the  RNA,  hsp26, were i d e n t i f i e d by t r a n s l a t i o n a r r e s t  e t a l . , 1981)  coding  f o r hsp22, 23,  (Craig  Voellmy et a l . , 1981).  contained  and  The  o r i e n t a t i o n s of the genes were determined u s i n g R-loop mapping; the p o l y A ends of the mRNA were i d e n t i f i e d by t h e i r h y b r i d i z a t i o n to  poly(BUdR)-tailed  plasmid pBR345 (Voellmy e t a l . , 1981), or by the h y b r i d i z a t i o n of end-labelled  DNA  McCarthy, 1980;  fragments d e r i v e d  from the c l o n e s  Corces e t a l . , 1980).  The  to hsmRNA ( C r a i g  and  l a c k of i n t e r v e n i n g sequences  suggested a t t h i s p o i n t by R-loop mapping d a t a (Voellmy e t a l . , 1981) the f a c t t h a t the r e g i o n s  was  and  homologous to hsmRNA were mapped on the b a s i s  by  of  t h e i r r e s i s t a n c e to SI n u c l e a s e a f t e r h y b r i d i z a t i o n (Corces e t a l . , 1980). These genomic c l o n e s ( C r a i g and and  s e l e c t e d messages which s y n t h e s i z e d  McCarthy, 1980;  Corces e t a l . , 1980).  the 4 hsps i n v i t r o  Furthermore, the hsp27,  23 genes showed p a r t i a l homology under reduced s t r i n g e n c y  (Corces  e t a l . , 1980). The  f i r s t h i n t t h a t these genes were d e v e l o p m e n t a l l y r e g u l a t e d  came  from workers who  i d e n t i f i e d a genomic c l o n e which h y b r i d i z e d more s t r o n g l y  to p u p a - s p e c i f i c  cDNA than to e m b r y o - s p e c i f i c  cDNA ( S i r o t k i n and  Davidson,  26  7  1982).  This clone hybridized  i d e n t i f i e d by Corces e t a l .  i n s i t u to l o c u s 67B and overlapped the c l o n e  (1980).  F i v e r e g i o n s homologous  to pupa  RNA  were i d e n t i f i e d and the o r i e n t a t i o n s of f o u r of them were determined by R-loop mapping u s i n g poly(BUdR).  Two of these genes code f o r hsp26 and  hsp23 w h i l e the o t h e r t h r e e have been named genes 1, 2 and 3 (Southgate e t §_1., 1985).  Genes 1, 2 and 3 a r e heat i n d u c i b l e a l t h o u g h  their  c o r r e s p o n d i n g messages do not accumulate to the same h i g h l e v e l s as those of hsp23 and 26. (Ayme and T i s s i e r e s , 1985).  Gene 1 c r o s s - h y b r i d i z e s to the  hsp27 gene w h i l e genes 2 and 3 share a weak homology to the hsp22 gene (Ayme and T i s s i e r e s , 1985).  These seven genes, the o r g a n i z a t i o n of which i s shown  i n F i g u r e 1, w i l l be r e f e r r e d to c o l l e c t i v e l y as the s m a l l heat shock genes, and t h e i r products as the s m a l l hsps (shsps) of D r o s o p h i l a .  1.1.3  Locus 6 7B:  P r o t e i n Coding Regions  The p r o t e i n coding r e g i o n s of the hsp_22, 23, 26. and 27. genes have been sequenced by two groups ( I n g o l i a and C r a i g , 1982b; Southgate e t a l . ,  1983).  Both sequence d e t e r m i n a t i o n s i n d i c a t e the same s t a r t and stop codons f o r the f o u r u n i n t e r r u p t e d open r e a d i n g frames.  However, t h e r e a r e some d i f f e r e n c e s  (96% homology a t the amino a c i d l e v e l ) which are p r o b a b l y not due to sequencing e r r o r s s i n c e some changes demonstrably e i t h e r c r e a t e o r d e s t r o y 18 r e s t r i c t i o n s i t e s  (Southgate e t a l . ,  d e r i v e d from s t r a i n Oregon R DNA,  S i n c e b o t h c l o n e s were  the d i f f e r e n c e s must be due to  polymorphism i n the f l y p o p u l a t i o n . been sequenced a l s o  1983).  The p r o t e i n coding r e g i o n of gene 1 has  (Ayme and T i s s i e r e s , 1985).  The d e r i v e d m o l e c u l a r weights f o r the unmodified p r o t e i n s a r e 19,705, 20,603,  22,9976,  23,620 and 26,560 which correspond to hsp22, hsp23, hsp26,  8  gene2 hsp27  hsp23  •  F i g u r e 1.  gene!  hsp26  hsp22  - i  1 kb  Gene o r g a n i z a t i o n  a t locus 6 7B of D r o s o p h i l a .  The d i r e c t i o n of t r a n s c r i p t i o n  i s indicated  by the arrows except f o r  gene 2 f o r which the o r i e n t a t i o n has not y e t been determined. and  gene 3  3 are referred  Genes 1, 2  to as genes 1, A and 5 by S i r o t k i n and Davidson (1982)  9  hsp27 and t h e gene 1 product r e s p e c t i v e l y .  Although genes 1, 2 and 3 a r e  h e a t i n d u c i b l e as measured by t r a n s i e n t changes i n c o r r e s p o n d i n g t r a n s c r i p t l e v e l s , p r o t e i n p r o d u c t s have n o t been d e t e c t e d f o r these genes. i n two-dimensional et a l . ,  g e l e l e c t r o p h o r e t i c s e p a r a t i o n s of both i n v i v o  1978; B u z i n and P e t e r s o n , 1982) and i n v i t r o  (Mirault  ( B u z i n and P e t e r s o n ,  1982) t r a n s l a t e d p r o d u c t s , hsp27 c o n s i s t s of m u l t i p l e s p o t s . h e a t shocked  However,  Similarly, i n  s a l i v a r y glands from t h i r d i n s t a r l a r v a e , hsp22 runs as 2 s p o t s  on two-dimensional  g e l s ( B u z i n and P e t e r s o n , 1982).  A schematic i l l u s t r a t i o n of p o t e n t i a l f u n c t i o n a l domains w i t h i n the D r o s o p h i l a shsps i s shown i n F i g u r e 2.  Hsp22, 23, 26 and 27 are homologous  over a s t r e t c h of 108 amino a c i d r e s i d u e s i n which the same amino a c i d i s used i n a l l f o u r p r o t e i n s a t 35% of the p o s i t i o n s .  The same amino a c i d i s  used by t h r e e of the f o u r p r o t e i n s i n 71% of the p o s i t i o n s .  Among each  o t h e r , hsp 27, 26 and 23 have n e a r l y twice as many s i m i l a r i t i e s w i t h i n  this  r e g i o n as hsp22 does w i t h r e s p e c t to the o t h e r t h r e e , thus c o n f i r m i n g e a r l i e r h y b r i d i z a t i o n s e l e c t i o n experiments  (Corces e t a l . ,  1980).  The s i z e  d i f f e r e n c e s between these f o u r p r o t e i n s are accounted f o r by two h e t e r o l o g o u s r e g i o n s , v a r i a b l e i n l e n g t h , on e i t h e r s i d e of the conserved domain.  S u r p r i s i n g l y , the f i r s t 83 amino a c i d s of the conserved s t r e t c h of  108 amino a c i d s shows a h i g h degree of homology w i t h amino a c i d s 70 - 152 o f t h e b o v i n e a - c r y s t a l l i n B^ c h a i n (van der Ouderaa e t a l . ,  1973), the  a - c r y s t a l l i n s c o m p r i s i n g one of the most abundant p r o t e i n c l a s s e s i n the vertebrate lens.  I t i s t h i s 83 amino a c i d r e g i o n which l i n k s the gene 1  p r o d u c t t o t h e o t h e r shsps.  The gene 1 p r o d u c t shows the most s i m i l a r i t y to  hsp27 and, i n t e r e s t i n g l y , l a c k s the conserved 25 amino a c i d s t r e t c h on the c a r b o x y - p r o x i m a l s i d e which i s common to the o t h e r f o u r p r o t e i n s .  10  Gene!  122  213  Hsp23  186  66  ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ Hsp22  F i g u r e 2.  174  A schematic diagram o f the v a r i o u s amino a c i d homologies between  the shsps o f D r o s o p h i l a melanoRaster. The p r e d i c t e d amino a c i d sequences a r e based on the n u c l e o t i d e of t h e i r coding  regions  sequence  ( I n g o l i a and C r a i g , 1982b; Southgate e t a l . , 1983;  Ayme and T i s s i e r e s , 1985).  The 83 amino a c i d domain which i s common to a l l  shsps and to the v e r t e b r a t e a - c r y s t a l l i n s i s shown by hatched boxes. Other homologous regions a r e d e f i n e d by the amino-terminal 15 amino a c i d r e s i d u e s and the 25 amino a c i d s l o c a t e d carboxy-proximal to the major domain.  A l s o shown, f o r each p r o t e i n , i s the amino a c i d numbering  corresponds to t h e regions d i s c u s s e d  above.  which  11  A second r e g i o n of weaker homology e x i s t s w i t h i n the f i r s t 15 p r e d o m i n a n t l y hydrophobic amino a c i d r e s i d u e s of hsp23, 26, 27 and t h e gene 1 product.  The s i g n i f i c a n c e o f t h i s h y d r o p h o b i c i t y i n t h e N - t e r m i n a l  p o r t i o n o f 4 o f t h e 5 p r o t e i n s remains unknown; i t does n o t c o n s t i t u t e a s i g n a l p e p t i d e , s i n c e a comparison  o f i n v i v o and i n v i t r o  translation  p r o d u c t s suggests t h a t t h e r e i s no p o s t - t r a n s l a t i o n a l p r o c e s s i n g ( M i r a u l t et a l . ,  1978) .  The most s t r i k i n g f e a t u r e o f hydropathy p r o f i l e s i s a prominent h y d r o p h i l i c peak spread over 16 amino a c i d s i n t h e middle o f t h e 83 amino a c i d conserved domain (Southgate e t a l . ,  1.1.4  Locus 67B:  1983).  A c t i v i t y During Development  A l t h o u g h a l l seven genes a t l o c u s 67B respond t o an environmental s t i m u l u s such as heat shock,  i t i s now w e l l e s t a b l i s h e d t h a t they a r e under  complex and n o n - c o o r d i n a t e c o n t r o l d u r i n g f l y development. The hsp22, 23, 26_ and 2_7 genes a r e n o r m a l l y t r a n s c r i b e d from t h e t h i r d i n s t a r l a r v a l t o t h e mid-pupal  stages ( S i r o t k i n and Davidson,  and Shearn, 1983; Mason e t a l . , t h e i r l e v e l s of expression vary.  1982;  1984; Ayme and T i s s i e r e s , 1985);  Cheney  however,  The hsp23 t r a n s c r i p t i s t h e most abundant  by 2 - 5 f o l d as compared w i t h hsp26 and hsp27 mRNAs, w h i l e hsp22 mRNA i s b a r e l y d e t e c t a b l e (Ayme and T i s s i e r e s , 1985). 2, a r e a l s o expressed d u r i n g t h e same p e r i o d Ayme and T i s s i e r e s , 1985), respectively  Genes 1 and 3, but n o t gene ( S i r o t k i n and Davidson, 1982;  a t l e v e l s equal t o those o f hsp23 and hsp26/27,  (Ayme and T i s s i e r e s , 1985).  T h i s p e r i o d o f development i n  D r o s o p h i l a i s c h a r a c t e r i z e d by h i g h ecdysterone l e v e l s 1977)..  (Hodgetts e t a l . ,  12  The  e f f e c t s of ecdysterone on hsp s y n t h e s i s can be mimicked i n v i t r o .  I n c u b a t i o n o f D r o s o p h i l a t i s s u e c u l t u r e (S3) c e l l s w i t h ecdysterone the s y n t h e s i s o f hsp22, 23, 26 and 27 t o l e v e l s c o n s i s t e n t w i t h r e l a t i v e t r a n s c r i p t accumulation  induces  their  i n v i v o ( I r e l a n d and Berger, 1982).  In  i s o l a t e d i m a g i n a l d i s k s from l a t e t h i r d i n s t a r l a r v a e , hsp23, 26 and 2T_ mRNA l e v e l s a r e f u r t h e r induced when d i s k s a r e incubated i n ecdysterone et  al.,  1982).  (Ireland  T r a n s c r i p t s c o r r e s p o n d i n g t o genes 1 and 3 a r e o n l y weakly  induced i n e i t h e r hormone t r e a t e d S3 c e l l s o r i m a g i n a l d i s k s ( I r e l a n d et  al.,  1982).  I n primary c e l l  c u l t u r e s prepared from D r o s o p h i l a  g a s t r u l a t i o n stage embryos, ecdysterone and known t e r a t o g e n i c drugs coumarin  such as  and d i p h e n y l h y d a n t o i n i n h i b i t muscle and neuron development  in vitro.  Under these c o n d i t i o n s , o n l y hsp22 and hsp23 a r e induced  (Buzin  and B o u r n i a s - V a r d i a b a s i s , 1984). A subset o f t h e genes found a t l o c u s 67B a r e a l s o expressed n o r m a l l y d u r i n g embryogenesis.  Messages c o r r e s p o n d i n g t o hsp27 and hsp26'. as w e l l as  hsp23, can be d e t e c t e d i n t h e f i r s t 4 hours o f embryonic development (Zimmerman e t a l . ,  1983; Ayme and T i s s i e r e s , 1985) w h i l e gene 2 mRNA i s  p r e s e n t i n 3 - 24 hour embryos ( S i r o t k i n and Davidson, T i s s i e r e s , 1985).  1982; Ayme and  E a r l y hsp27 and hsp26 mRNAs accumulate  i n o v a r i a n nurse  c e l l s w i t h i n t h e egg chamber and a r e passed i n t o t h e oocyte et  al.,  1983).  (Zimmerman  Thus, these messages a r e a l s o d e t e c t e d i n 3 - 5 day o l d  females  (Zimmerman e t a l . ,  et  1984; Ayme and T i s s i e r e s , 1985) and gene 1 (Ayme and T i s s i e r e s ,  al.,  1985) flies.  1983; Ayme and T i s s i e r e s , 1985).  Hsp23 (Mason  t r a n s c r i p t s a r e d e t e c t a b l e i n f r e s h l y e c l o s e d male and female  adult  13  I f a h y p e r t h e r m i c shock i s a p p l i e d to animals from the l a r v a l t o a d u l t s t a g e s , t r a n s c r i p t i o n of the hsp22, 23, 26. and 27. genes  increases  s i g n i f i c a n t l y over t h e i r b a s a l , d e v e l o p m e n t a l l y c o n t r o l l e d l e v e l s , the t r a n s c r i p t s accumulating to a p p r o x i m a t e l y the same l e v e l a t a l l tested  (Ayme and T i s s i e r e s , 1985).  6 stages  The hsp23 gene, which g i v e s the h i g h e r  l e v e l of d e v e l o p m e n t a l l y r e g u l a t e d t r a n s c r i p t s , i s c o m p a r a t i v e l y l e s s h i g h l y induced d u r i n g h e a t shock than the hsp22, 26 and 27. genes. In c o n t r a s t , genes 1 and 3 a r e heat shock induced to 1 0 - f o l d h i g h e r l e v e l s i n white pre-pupae and middle pupae than i n t h i r d i n s t a r l a r v a e , pupae o r a d u l t s . all  Gene 2 becomes moderately t r a n s c r i b e d a f t e r heat shock i n  l a r v a l to adult  stages.  In p r e - b l a s t o d e r m embryos ( 0 - 3 n u c l e a r RNA  late  h o u r s ) , a p e r i o d i n which t h e r e i s no  s y n t h e s i s , the genes coding f o r hsp22, 23, 26 and 27 are  u n r e s p o n s i v e t o heat shock (Dura, 1981; Zimmerman e t a l . ,  1983).  These complex p a t t e r n s of t r a n s c r i p t i o n a l a c t i v i t y demonstrate t h a t the seven genes c l u s t e r e d w i t h i n 15 kb of DNA  can each be r e g u l a t e d  i n d e p e n d e n t l y of i t s neighbors under n o n - s t r e s s c o n d i t i o n s d u r i n g development.  Even under heat shock, t h e r e i s a v a r i a b l e  stage-specific  accumulation of t r a n s c r i p t s coding f o r the p u t a t i v e p r o d u c t s of genes 1, 2 and 3.  The s p e c i f i c i n d u c t i o n of c e r t a i n genes such as the e x p r e s s i o n o f  gene 2 o n l y i n 3 — 24 hour embryos, p r o d u c t s may  f o r example, suggests t h a t  s e r v e a v i t a l f u n c t i o n i n normal development.  their  14  1.2  Conserved shsp F u n c t i o n  1.2.1  Higher P l a n t s :  i n Eucaryotes  Soybean  o o o Upon temperature e l e v a t i o n to 35 C - 43 C from 28 C, max)  seedlings  (Key  tissue culture c e l l s  e t a l . , 1981; (Barnett  soybean  (Glycine  A l t s c h u l e r and Hascarenhas, 1982)  e t a l . , 1980)  and  respond by s h u t t i n g down  e x i s t i n g p r o t e i n s y n t h e s i s w i t h the concomitant i n d u c t i o n of heat shock protein synthesis synthesis  i n a s i t u a t i o n analogous to t h a t i n D r o s o p h i l a .  Hsp  i s c h a r a c t e r i z e d by major p r o t e i n products w i t h m o l e c u l a r weights  of 15,000 - 18,000 which are r e s o l v e d two-dimensional g e l s  (Key  e t a l . , 1981).  Is common to both root and in v i t r o translated  i n t o at l e a s t 10 components  hypocotyl  The  p a t t e r n of p r o t e i n  on synthesis  t i s s u e as w e l l as to both i n v i v o  and  products.  + Based on h y b r i d i z a t i o n s e l e c t i o n experiments, cDNAs made to p o l y A mRNA from heat shocked h y p o c o t y l Key,  1981).  tissue f a l l  into 2 classes  C l a s s I cDNAs h y b r i d i z e to mRNAs 800  - 900  ( S c h o f f l and  nucleotides  in  l e n g t h and which code f o r 20 hsps i n the 15 - 18 kd s i z e range when t r a n s l a t e d i n v i t r o u s i n g a wheat germ l y s a t e .  A c l a s s I I cDNA s e l e c t s a  message t h a t codes f o r a s i n g l e 18 kd p r o t e i n on two-dimensional g e l s . The coding  c h a r a c t e r i z a t i o n of s e v e r a l genomic DNA  f o r the shsp (hspl7)  groups. Table I.  These genes and  clones  containing  sequences  f a m i l y of soybean has been undertaken by  two  t h e i r c o r r e s p o n d i n g p r o t e i n products are l i s t e d  in  15  Table  I.  Soybean Hspl7 Gene  Family  Predicted Protein  Gene  cDNA C l a s s  Molecular Weight  Amino A c i d Residues  Reference  17.5- -M  I  17,544  153  Nagao e t a l . , 1985  17.6- -L  I  17,570  154  Nagao e t a l . , 1985  17.5- -E  II  17,533  154  Czarnecka ejb a l . , 1985  6871  I  17,345  153  S c h o f f l e t a l . , 1984  6834  -  -  S c h o f f l e t a l . , 1984  The  -  6834 Rene i s incomplete and i s l i n k e d to the 6871 gene,  by a p p r o x i m a t e l y 4 kb.  separated  The l i n k a g e of the other genes has n o t y e t been  determined. None o f t h e f o u r complete genes c o n t a i n i n t r o n s and a t the amino a c i d l e v e l they show an o v e r a l l homology o f 90%. i n t h e i r carboxy-terminal  The p r o t e i n s c o n t a i n a r e g i o n  h a l f which shows e x t e n s i v e homology t o t h e  conserved domain o f the p r o t e i n s encoded by l o c u s 67B i n D r o s o p h i l a . the apparent c o n s e r v a t i o n  Due t o  o f a f u n c t i o n a l domain i t i s not s u r p r i s i n g t h a t L  hydropathy p r o f i l e s of the soybean shsps d i s p l a y a prominent h y d r o p h i l i c peak i n an analogous p o s i t i o n t o t h a t found i n D r o s o p h i l a  (Czarnecka e t a l . ,  1985). The  shsps o f soybean can a l s o be induced  (Czarnecka e t a l . ,.. 1984)..  w i t h a r s e n i t e and cadmium .  .  .  .  16  Homologous shsp genes are found i n o t h e r h i g h e r p l a n t s s i n c e h s p l 7 c l a s s I cDNAs c r o s s - h y d r i d i z e to o t h e r p l a n t s p e c i e s such as pea, maize, and  sunflower  i n d u c i b l e products  (Key e t a l . , 1983).  field  grown c o t t o n  suspension  At the p r o t e i n l e v e l , major heat  with s i m i l a r molecular  ( N i c o t i a n a tobacum) t i s s u e c u l t u r e c e l l  millet,  weights occur  line  i n a tobacco  (Barnett e t §_1. , 1980), i n  (Gossyupium hirsutum) (Burke e t a l . , 1985), and  in  c u l t u r e s of tomato ( L y c o p e r s i c o n peruvianum) (Scharf and  Nover,  1982). In maize (Zea mays) s e e d l i n g s , 4 - 8 two-dimensional g e l s ( B a s c z c z y n s k i synthesized et  e t a l . , 1982).  on  These p r o t e i n s  are  i n a g r e a t v a r i e t y of maize t i s s u e s a f t e r heat shock (Cooper  a l . , 1984;  Baszczynski  the germinating stage  hsps of 18 kd are seen  e t a l . , 1985).  They are, however, not observed i n  p o l l e n g r a i n s of the maize p l a n t (Cooper e t a l . , 1984), a  i n which RNA  s y n t h e s i s takes p l a c e under n o n - s t r e s s  conditions.  This  non-responsiveness i s t h e r e f o r e analogous to t h a t seen w i t h hsp30 i n e a r l y Xenopus development ( s e c t i o n 1.2.3). germinating  p o l l e n g r a i n s has  The  absence of hsp  synthesis i n  a l s o been observed i n another p l a n t ,  T r a d e s c a n t i a p a l u d o s a (Xiao and Mascarenhas, 1985).  1.2.2  Avian  and Mammalian Systems  A 25 kd p r o t e i n i s i n d u c i b l e i n c h i c k embryo f i b r o b l a s t o  (CEF)  cells  o  d u r i n g a 41 C - 45 C i n c u b a t i o n ( K e l l y and s y n t h e s i s of t h i s hsp  can a l s o be  which are summarized i n Table I I .  induced  S c h l e s i n g e r , 1978).  The  w i t h a wide v a r i e t y of agents  17  Table I I .  Hsp25 I n d u c t i o n i n CEF C e l l s  Inducer  1.  Reference  Amino A c i d Analogues canavanine  K e l l y and S c h l e s i n g e r , 1978; Hightower , 1980  2.  3.  4.  5.  p-fluorophenylalanine  Hightower , 1980  hydroxynorva1ine  K e l l y and S c h l e s i n g e r , 1978  o-methyl t h r e o n i n e  K e l l y and S c h l e s i n g e r , 1978  Copper B i n d i n g Ligands kethoxal bis(thiosemicarbazone)  Levinson e t a l . ,  1978a  disulfiram  Levinson e t a l . ,  1978b  o-phenanthro1ine  Levinson e t a l . ,  1979  8-hydroxyqu i n o 1 i n e  Levinson e t a l . ,  1979  C h e l a t i n g Agents  Viral  Infection  Herpes simplex v i r u s  N o t a r i a n n i and P r e s t o n , 1982  Newcastle d i s e a s e v i r u s  Collins et a l . ,  1980  Metals copper,  cadmium, z i n c , mercury  Levinson et a l . ,  1980  18  Table I I .  Hsp25 I n d u c t i o n i n CEF C e l l s  Inducer 6.  (cont'd)  Reference  Others arsenite  Levinson e t a l . , 1980  puromycin  Hightower, 1980  Both i n v i v o and i n v i t r o s p e c i e s on 2-D g e l s (Johnston  s y n t h e s i z e d chicken hsp25 appears as two e t a l . , 1980; Wang e t a l . , 1981).  amounts of hsp25 have a l s o been observed et  (Johnston  a l . , 1980; Wang e t a l . , 1981). Using  was  i n uninduced c e l l s  Minor  found  c l o n e s coding f o r D r o s o p h i l a hsp27, 26 and 23, no h y b r i d i z a t i o n t o c h i c k e n mRNA (White and Hightower, 1984).  Similarly,  a n t i b o d i e s a g a i n s t hsp25 ( K e l l y e t a l . , 1980) f a i l t o r e a c t w i t h  cell  e x t r a c t s from o t h e r organisms i n c l u d i n g y e a s t , s l i m e mold ( D i c t y o s t e l i u m ) , maize s e e d l i n g r o o t s , C a e n o r h a b d i t i s Xenopus k i d n e y  elegans, D r o s o p h i l a melanogaster,  c e l l s , mouse L929 c e l l s and human WI38 c e l l s  S c h l e s i n g e r , 1982). e x t r a c t s prepared  However, anti-hsp25  ( K e l l y and  cross-reacts with a p r o t e i n i n  from 11 day o l d embryonic c h i c k e n l e n s and thus can be  c o n s i d e r e d analogous t o t h e shsps of D r o s o p h i l a , Xenopus and soybean ( S c h l e s i n g e r , 1985). Anti-hsp25 c r o s s - r e a c t s w i t h an a v i a n muscle p r o t e i n o f s u b u n i t molecular  weight 22,000 which i s u n u s u a l l y abundant i n c h i c k e n embryonic  h e a r t as well, as i n s k e l e t a l muscle ( S c h l e s i n g e r , 1985).  This antibody  also  19  shows t h a t s m a l l amounts of hsp25 are p r e s e n t confirming other  cells,  observations.  In d e v e l o p i n g  c h i c k e n embryos, hsp25 d i s p l a y s v a r i a b l e l e v e l s  i n d u c t i o n i n d i f f e r e n t t i s s u e s a f t e r a heat abundant i n h e a r t and Bromley, 1982).  i n unstressed  treatment.  l i v e r t i s s u e than i n b r a i n and  S i m i l a r l y , o t h e r workers f a i l e d  of  Hsp25 i s much more  lung  (Voellmy  and  to d e t e c t the i n d u c t i o n of  hsp25 s y n t h e s i s i n b r a i n i s o l a t e d from 9 day o l d embryos under c o n d i t i o n s i n which i t was  e a s i l y seen i n limb and b r e a s t t i s s u e ( A t k i n s o n e t a l . , 1985).  A 25 kd p r o t e i n i s a l s o one  of the major i n d u c i b l e p r o t e i n s i n primary  c u l t u r e s of Japanese q u a i l b r e a s t t i s s u e o s u b j e c t e d to a temperature of 43 I n t e r e s t i n g l y i t l o s e s i t s heat  (myoblasts)  when these  are  o - 46 C ( A t k i n s o n , 1981).  inducibility  by 100  development when the c e l l s have d i f f e r e n t i a t e d  - 120  hours of  i n t o myotubes, but i t s  ability  to be induced by a r s e n i t e , copper or z i n c remains ( A t k i n s o n e t a l . ,  1983).  A r s e n i t e a l s o induces hsp25 i n c h i c k e n embryonic myotubes (Wang  et  a l . , 1981).  Furthermore, q u a i l b l o o d c e l l s  (white and red) do  not  s y n t h e s i z e hsp25 when t r e a t e d w i t h copper o r z i n c ( A t k i n s o n e t a l . , 1983) but have the a b i l i t y to produce hsp25 under heat r e s u l t s suggest  shock c o n d i t i o n s .  t h a t d i f f e r e n t i n d u c e r s of the heat  These  shock response may  act  through d i f f e r e n t mechanisms. Q u a i l hsp25 runs as a s i n g l e band w i t h a p i of 5.4  ( A t k i n s o n , 1981)  appears t o be analogous to i t s c h i c k e n c o u n t e r p a r t s i n c e i t r e a c t s w i t h a n t i - c h i c k e n hsp25 ( A t k i n s o n e t a l . , 1983). The  i n d u c t i o n of a p r o t e i n w i t h an approximate m o l e c u l a r  25,000 has been observed reviewed i n T a b l e I I I .  weight of  i n a v a r i e t y of mammalian c e l l l i n e s which are  and  20  Table I I I .  Hsp25 I n d u c t i o n i n Mammalian C e l l s  C e l l Type  Inducer  Ref erence  human HeLa  heat  Hickey and Weber, 1982  human  a r s e n i te/ethano1  Rodenhiser e t a l . , 1985  r a t embryo f i b r o b l a s t s  heat  Welch and Feramisco, 1985  r a t embryo f i b r o b l a s t s  arsenite/proline  rat  heat/arsenite/arsenate  Kim e t a l . , 1983  arsenate  Kim e t a l . , 1983  r a t primary hepatoma  arsenate  Kim e t a l . , 1983  r a t p i t u i t a r y tumor  arsenate  Kim e t a l . , 1983  r a t hepatoma MH-7 7 77  heat  Lamarche e t a l . , 1985  arsenite  Bensaude and Morange, 1983  fibroblasts  arsenite  Bensaude and Morange, 1983  mouse  arsenite/ethano1  Rodenhiser e t a l . , 1985  mouse myeloma  heat  Hickey and Weber, 1982  mouse 3T3 f i b r o b l a s t s  heat  H i c k e y and Weber, 1982  lymphocytes  myoblasts  r a t primary  fibroblasts  analogue  Welch, 1985  mouse embryonal carcinoma mouse p r i m a r y  lymphocytes  21  Table I I I .  Hsp25 I n d u c t i o n i n Mammalian C e l l s  (cont'd)  C e l l Type  Inducer  Reference  Chinese hamster ovary  heat  Bouche e t a l . ,  heat  L i and Werb,  fibroblast  heat  A t k i n s o n and P o l l o c k ,  r a b b i t lymphocyte  arsenite/ethanol  Rodenhiser e t a l . ,  1979  Chinese hamster fibroblast  1982  hamster p r i m a r y  The human v a r i a n t does not i n c o r p o r a t e  [  1982  1985  S l m e t h i o n i n e but can be  3 labelled using [ Hjleucme  (Hickey and Weber, 1982).  Thus the i n d u c t i o n  of a shsp c o u l d n o t be observed i n human f o r e s k i n c e l l s 1980),  (Levinson et a l . ,  i n a human f i b r o s a r c o m a (HT1080) c e l l l i n e  ( S l a t e r e t a l . , 1981) o r 35 i n human lymphocytes ( A t k i n s o n and Dean, 1985) when [ S]methiomne was used i n the p r o t e i n l a b e l l i n g procedure. In human epidermoid carcinoma (KB) c e l l s , hsp25 i s n o t d e t e c t e d i n heat shocked, ( A t k i n s o n and P o l l o c k , 1982)  and may  14 [ C]leucine treated  cells  be an example of non-responsiveness i n  c e r t a i n transformed s t a t e s . Human hsp25 runs as two  components on two-dimensional g e l s , the more o  b a s i c one b e i n g p r e s e n t m  c e l l s grown a t 37 C (Hickey and Weber, 1982).  I n non-human mammalian c e l l s , hsp25 can be d e t e c t e d u s i n g methionine as the r a d i o a c t i v e amino a c i d .  The o n l y r e p o r t e d f a i l u r e to d e t e c t  an  22  [  S l m e t h i o n y l hsp25 under c o n d i t i o n s which r e a d i l y induced the o t h e r hsps  as w e l l as hsp25 i n CEF c e l l s , was cells  f o r mouse L c e l l s  ( K e l l y and S c h l e s i n g e r , 1978).  and baby hamster k i d n e y  T h i s cannot be a s p e c i e s s p e c i f i c  35 phenomenon s i n c e a [  S l m e t h i o n y l hsp25 i s induced i n Chinese hamster  ovary and Chinese hamster f i b r o b l a s t c e l l s  as w e l l as a mouse primary  f i b r o b l a s t c e l l c u l t u r e (see T a b l e I I I f o r r e f e r e n c e s ) . As i n HeLa c e l l s  and c h i c k e n c e l l s , o t h e r mammalian shsps appear  m u l t i p l e components on two-dimensional  gels.  as  Three v a r i a n t s of hsp25 have  been observed i n heat shocked primary c u l t u r e s of hamster f i b r o b l a s t s ( A t k i n s o n and P o l l o c k , 1982).  The d e t a i l e d a n a l y s i s of hsp25 isoforms i n  v a r i o u s r a t c e l l types (Kim e t a l . , 1983; complexity of shsp r e g u l a t i o n .  Welch, 1985)  In r a t myoblast  emphasizes the  c e l l s , 4 heat  p r o t e i n s i n the 25 kd - 30 kd range can be d i s t i n g u i s h e d on g e l s (Kim e t al^. , 1983).  inducible  two-dimensional  Of these, 2 are not l a b e l l e d w i t h methionine w h i l e  , a r s e n i t e o r a r s e n a t e treatment induces an a d d i t i o n a l 2 p r o t e i n s of comparable s i z e .  The same authors have a l s o shown t h a t a r s e n a t e induces a  30 kd p r o t e i n i n 13 day o l d c h i c k embryo muscle i n a d d i t i o n to hsp25. Furthermore,  the i n d u c t i o n o f these p r o t e i n s v a r i e d i n the d i f f e r e n t r a t  t i s s u e c e l l l i n e s t h a t were used. of  Welch (1985) has r e p o r t e d the e x i s t e n c e  f o u r hsp25 i s o f o r m s i n r a t embryo f i b r o b l a s t c e l l s , none of which are  l a b e l l e d w i t h methionine.  The i n d u c t i o n of these f o u r p r o t e i n s , which were  shown t o be r e l a t e d , d i f f e r e d depending heat, a r s e n i t e and amino a c i d In  r a t myoblast  cells,  r e s i d u e s (Kim e t a l . , 1984).  upon the agent used which i n c l u d e d  analogues.  two hsp25 p r o t e i n s are p h o s p h o r y l a t e d a t s e r y l S i m i l a r l y , i t has been shown t h a t t h r e e hsp25  i s o f o r m s i n r a t embryo f i b r o b l a s t c e l l s  are phosphoproteins  i n both  normal  23  and  stressed c e l i s  (Welch, 1985).  The p h o s p h o r y l a t i o n of two  of  these  p r o t e i n s i n c r e a s e s when the c e l l s are t r e a t e d w i t h a p h o r b o l d i e s t e r (phorbol-12-myristate-13  a c e t a t e ) , a c a l c i u m ionophore,  q u i e s c e n t c e l l s are given f r e s h serum. stress  A23187, o r i f  None of these treatments  induces  a  response.  The encoding  c h a r a c t e r i z a t i o n of two human hsp25 has  gene, d e s i g n a t e d HS11,  genes r e l a t e d to the 4 member gene f a m i l y  r e c e n t l y been r e p o r t e d  (Hickey e t a l . , 1986).  i s a member of a c l u s t e r of t h r e e genes l i n k e d w i t h i n  a 14 - 18 kb r e g i o n of the genome.  SI n u c l e a s e p r o t e c t i o n experiments  c o n f i r m that t h i s gene i s expressed  at low  induced  The  2 0 - f o l d d u r i n g heat  i n t e r r u p t e d by two  shock.  elements and  l e v e l s i n c o n t r o l c e l l s but i s  open r e a d i n g frame, which i s  i n t e r v e n i n g sequences of 723  p o l y p e p t i d e of 22,300 deduced m o l e c u l a r d e s i g n a t e d HS8,  One  weight.  appears to be a p r o c e s s e d  and 120 The  bp,  encodes a  second i s o l a t e d gene,  pseudogene l a c k i n g promoter  i s u n l i n k e d to the o t h e r members of the hsp25 gene f a m i l y .  The  deduced amino a c i d sequence of hsp25 shows homology to the v e r t e b r a t e a - c r y s t a l l i n s and is  to the shsps of D r o s o p h i l a , soybean and Xenopus and  i n d i c a t i v e of a c a r e f u l l y conserved  1.2.3  thus  essential physiological role.  Xenopus In Xenopus l a e v i s somatic  c e l l s o r c u l t u r e d f i b r o b l a s t s , a 30 o  p r o t e i n i s induced  kd  o  a t temperatures between 32 C and 37 C ( B i e n z , 1982).  In v i t r o t r a n s l a t i o n experiments demonstrate t h a t i t s c o r r e s p o n d i n g newly s y n t h e s i z e d d u r i n g heat  shock and  c o n t r o l , as are the genes a t l o c u s 67B  mRNA i s  thus i s under t r a n s c r i p t i o n a l i n Drosophila.  24  Sequences encoding hsp30 have been i s o l a t e d from cDNAs made t o heat shocked  Xenopus p o l y A RNA from an e p i t h e l i a l kidney f i b r o b l a s t c e l l  ( B i e n z , 1984a).  line  DNA sequence d a t a r e v e a l s a 45% homology t o the 83 amino  a c i d domain o f the D r o s o p h i l a shsps.  No homology i s d e t e c t a b l e i n the  p r e c e d i n g 80 amino a c i d r e s i d u e s o r i n the c a r b o x y - t e r m i n a l 40 amino residues.  Clones  acid  from a Xenopus l a e v i s b l o o d c e l l genomic DNA l i b r a r y have  been i s o l a t e d and two hsp30 c o d i n g r e g i o n s have been i d e n t i f i e d as hsp30A and hsp30B  ( B i e n z , 1984b).  sequenced cDNA. r e s u l t s , Bienz  Based on t h i s , along w i t h unpublished genomic Southern (1984b) suggests  10 members coding f o r hsp30. heat  N e i t h e r gene i s completely i d e n t i c a l t o the  t h a t t h e r e may be a multi-gene  blot  family of 5 -  T h i s i s supported by the o b s e r v a t i o n t h a t 5  i n d u c i b l e 30 kd p r o t e i n s can be separated on two-dimensional  gels  (Guedon e t a l . , 1985). The hsp30 cDNA has been used  t o q u a n t i f y corresponding mRNA l e v e l s  d u r i n g Xenopus development and i n v a r i o u s somatic t i s s u e s ( B i e n z , 1984a). Whereas hsp70 mRNA can be heat induced a t the b l a s t u l a stage, hsp30 i s n o t heat i n d u c i b l e u n t i l the swimming t a d p o l e stage.  T h i s i s i n agreement w i t h  the o b s e r v a t i o n t h a t hsp30 c o u l d - n o t be induced i n Xenopus oocytes  (Bienz  and Gurdon, 1982; B i e n z , 1982) b u t i n c o n f l i c t w i t h Guedon e t a l . (1985) who observed  that i t could.  Hsp30 a l s o shows some degree o f t i s s u e s p e c i f i c i t y , i t s messages accumulating  t o l e v e l s 1 0 - f o l d h i g h e r i n the kidney and gut as compared t o  lung o r l i v e r a f t e r heat shock. Although hsp30 genes may be r e p r e s s e d d u r i n g e a r l y development i n Xenopus, becoming r e s p o n s i v e t o heat shock i n l a t e r s t a g e s , t h e r e i s no r e p o r t o f these genes b e i n g t r a n s c r i b e d under n o n - s t r e s s c o n d i t i o n s .  25  1.2.4  Other Organisms Based on s i m i l a r i t i e s i n m o l e c u l a r weight, many hsps r e p o r t e d i n o t h e r  systems may be r e l a t e d t o the shsps a l r e a d y d i s c u s s e d .  A hsp  profile  analogous to D r o s o p h i l a melanogaster i s found i n another d i p t e r a n , Chironomus  t e n t a n s (Tanguay and V i n c e n t , 1981).  In the s k i p p e r b u t t e r f l y  (Calpodes e t h l i u s ) , a l e p i d o p t e r a n , shsps of 26 kd and 22 kd r e s o l v e  into  m u l t i p l e components on two-dimensional g e l s (Dean and A t k i n s o n , 1983). kd p r o t e i n i s induced w i t h cadmium and z i n c i n chinook salmon embryo ( H e i k k i l a et a l . ,  A 28  cells  1982) w h i l e a p a i r of p r o t e i n s a p p r o x i m a t e l y 30 kd i n s i z e  are induced w i t h heat and a r s e n i t e i n rainbow t r o u t f i b r o b l a s t (Kothary and Candido, 1982).  cells  H e i k k i l a e t a l . (1982) have observed t h a t the  m e t a l induced p r o t e i n i s not induced by a temperature i n c r e a s e to 24°C but t h i s may  have been below the t h r e s h o l d of i n d u c t i o n s i n c e the hsps of t r o u t  are induced a t 27°C (Kothary and Candido, 1982). Heat shock induces the s y n t h e s i s o f a p p r o x i m a t e l y 10 p r o t e i n s i n t h e m o l e c u l a r weight range of 23,000 t o 32,000 i n the s l i m e mold  Dictyostelium  discoideum (Loomis and Wheeler, 1982) and i n the p r o t o z o a n Tetrahymena pyriformis  (Guttman e t a l . ,  1980).  I n Tetrahymena,  these shsps a r e a l s o  induced by d e c i l i a t i o n o r by a r e l e a s e from anoxia. In V o l v o x c u l t u r e s , a p p r o x i m a t e l y 6 heat i n d u c i b l e p r o t e i n s between 18 kd and 26 kd have been observed ( K i r k and K i r k , 1985).  Interestingly,  t h e s e p r o t e i n s show d i f f e r e n t p a t t e r n s when s y n t h e s i z e d i n v i t r o o r i n v i v o , s u g g e s t i n g perhaps t h a t p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n i s i n v o l v e d . Fungal systems a l s o s y n t h e s i z e shsps under hyperthermic c o n d i t i o n s .  A  23 kd hsp which runs as a s i n g l e b a s i c band on two-dimensional g e l s has been identified  i n Neurospora c r a s s a (Kapoor, 1983).  In y e a s t  (Saccharomyces  26  c e r e v i s i a e ) . a 26 kd p r o t e i n i s s y n t h e s i z e d induced  Drosophila  of the developmental c o n t r o l of the genes at l o c u s 67B  The  expression  complexity  Induction  Mechanisms  of shsps a l t e r s d r a m a t i c a l l y upon heat shock.  The  of the response becomes e v i d e n t when the same p r o t e i n s are  induced  by a wide v a r i e t y of agents and  such as d i f f e r e n t i a l s y n t h e s i s d u r i n g non-responsiveness to s t r e s s d u r i n g s p e c i f i c r e g u l a t i o n and w i t h i n the same c e l l Despite  found  i s compounded by phenomena  c e r t a i n developmental  gametogenesis and  stages,  embryogenesis, t i s s u e  the d i f f e r e n t i a l response to d i f f e r e n t s t r e s s o r s  type.  t h i s complexity,  With the e x c e p t i o n due  in  (Kurtz e t a l . , 1986).  T r a n s c r i p t i o n a l C o n t r o l and  to be  It i s also  e a r l y i n s p o r u l a t i o n under normal growth c o n d i t i o n s , which i s  reminiscent  1.3  d u r i n g heat shock.  some important g e n e r a l i z a t i o n s can be made.  of e a r l y hsp27 and hsp26 e x p r e s s i o n  i n Drosophila  embryos  to s t o r e d mRNA i n the oocyte (Zimmerman e t a l . , 1983), the appearance of  shsps from p l a n t s to man  i s due  to new  transcriptional activity.  Although  i t has been suggested t h a t the i n d u c t i o n of hsp25 i n c a n a v a n i n e - t r e a t e d c e l l s i s due 1984)  to the t r a n s l a t i o n of e x i s t i n g message (White and  CEF  Hightower,  t h i s i s i n d i r e c t c o n f l i c t to i n h i b i t o r s t u d i e s i n which hsp25  s y n t h e s i s under the same c o n d i t i o n s i s blocked Schlesinger,  1978).  the o b s e r v a t i o n  A d d i t i o n a l support  t h a t i n CEF  by actinomycin  D (Kelly  f o r the above c o n c l u s i o n comes from  c e l l s , hsp25 s y n t h e s i s can occur  in virus  i n f e c t i o n s o n l y when the v i r u s i s d e f e c t i v e i n t e r m i n a t i n g h o s t synthesis  ( C o l l i n s e t a l . , 1980;  and  N o t a r i a n n i and  Preston,  1982).  RNA  27  1.3.1  Gene Promoter F u n c t i o n The o t h e r g e n e r a l i z a t i o n t h a t can be made comes from the a v a i l a b l e  sequence a n a l y s i s of gene promoter r e g i o n s .  Without e x c e p t i o n , a l l heat  i n d u c i b l e genes i n c l u d i n g gene 1 and the genes coding f o r hsp22, 23, 26 and 27 i n D r o s o p h i l a . the genes coding f o r f o u r hspl7 v a r i a n t s i n soybean, the human HS11  gene and the'gene coding f o r hsp30A  elements (HSEs) upstream from the TATA m o t i f .  i n Xenopus c o n t a i n heat shock These elements resemble the  14 bp p a l i n d r o m i c consensus sequence which has been shown to c o n f e r heat i n d u c i b i l i t y upon genes  (Bienz and Pelham,  B i e n z , 1982; M i r a u l t e t a l . ,  1982).  hsp30B gene of Xenopus, which may d i v e r g e d s i g n i f i c a n t l y compared  1982; Pelham,  1982; Pelham and  I t should be mentioned t h a t i n the  be a pseudogene,  the p r o x i m a l HSE has  to the hsp30A gene (Bienz,  1984b).  I t i s becoming more e v i d e n t that non-HSE promoter f u n c t i o n may  be  r e s p o n s i b l e f o r t r a n s c r i p t i o n a l a c t i v a t i o n i n the case of some i n d u c e r s o t h e r than heat shock.  An u n d e r s t a n d i n g of m u l t i p l e promoter f u n c t i o n has  come from the d e t a i l e d a n a l y s i s of promoter d e l e t i o n s p r e f e r a b l y , i n t o a homologous  reintroduced,  environment.  A s i z e v a r i a n t of hsp27 (hsp!8.5) has been i n t r o d u c e d i n t o the D r o s o p h i l a genome u s i n g P-element mediated t r a n s f o r m a t i o n  (Hoffman and  Corces, 1984) where i t d i s p l a y s heat i n d u c i b i l i t y and temporal r e g u l a t i o n through development i d e n t i c a l to endogenous  hsp27.  D e l e t i o n a n a l y s i s of  upstream sequences has demonstrated t h a t heat i n d u c i b i l i t y and e c d y s t e r o n e r e s p o n s i v e n e s s f u n c t i o n s are s e p a r a t e (Hoffman and Corces, 1986). t h a t remove 80% of i t s heat i n d u c i b i l i t y  (-2.1  on hsp!8.5 mRNA l e v e l s i n p r e - p u p a l s t a g e s .  Deletions  kb to -1.1 kb) have no  effect  A c o n s t r u c t which r e t a i n s o n l y  124 bp 5' t o the s t a r t of t r a n s c r i p t i o n completely a b o l i s h e s heat  28  a c t i v a t i o n y e t r e t a i n s 30% o f i t s hormone r e s p o n s i v e n e s s . c o n t a i n s homologies  t o sequences  shown t o be important f o r ecdysterone  induced e x p r e s s i o n o f the D r o s o p h i l a melanogaster Hogness, 1982;  McGinnis  This region  e t a l . , 1983;  g l u e genes (Muskavitch and  Bourouis and R i c h a r d s , 1985).  Full  heat i n d u c i b i l i t y o f the hsp27 gene may be due t o the c o n c e r t e d e f f e c t o f m u l t i p l e HSEs, 4 c l u s t e r e d w i t h i n 125 bp approximately 300 bp upstream o f the mRNA i n i t i a t i o n s i t e , and 2 l o c a t e d 30 bp a p a r t a t a d i s t a n c e o f g r e a t e r than 1 kb from the t r a n s c r i p t i o n s t a r t  and l o c a t e d , i n f a c t , w i t h i n the  a d j a c e n t hsp23 coding r e g i o n (see F i g u r e 1 ) . promoter  By a s s a y i n g v a r i o u s hsp27  c o n s t r u c t s i n D r o s o p h i l a t i s s u e c u l t u r e c e l l s , Riddlhough and  Pelham (1986) have a l s o found t h a t ecdysterone and heat  inducibility  elements  are d i s t i n c t .  elements  i n t o two c l u s t e r s , each c o v e r i n g approximately 100 bp, some 300 and  500 bp upstream  These authors, however, s e p a r a t e the f u n c t i o n a l  o f the cap s i t e .  n e c e s s a r y and s u f f i c i e n t heat i n d u c t i o n . at a p p r o x i m a t e l y  The r e g i o n between -579 and -455 i s  f o r ecdysterone i n d u c t i o n but i s n o t r e q u i r e d f o r  F u l l heat i n d u c i b i l i t y i s accomplished by the m u l t i p l e HSEs -300.  The heat i n d u c i b i l i t y and developmental been shown t o be d i s t i n c t and Meselson,  1985).  a t the promoter  r e g u l a t o r y f u n c t i o n s have a l s o  l e v e l f o r the hsp26 gene (Cohen  Upstream r e g i o n s o f the hsp26 gene were f u s e d t o  b a c t e r i o p h a g e lambda DNA and i n t r o d u c e d i n t o D r o s o p h i l a u s i n g the P-element system.  Sequences r e q u i r e d f o r the f u l l heat shock response are l o c a t e d  w i t h i n 341 bp upstream  o f the 5' end o f the mRNA and c o n t a i n 6 HSEs.  Sequences a p p r o x i m a t e l y 200 bp f u r t h e r upstream normal  o v a r i a n and p u p a l responses.  are r e s p o n s i b l e f o r the  29  U s i n g P-element mediated t r a n s f o r m a t i o n , Klemenz and Gehring  (1986)  have i n t r o d u c e d promoter d e l e t i o n s of the hsp22 gene i n t o a f l y s t r a i n which s y n t h e s i z e s an e l e c t r o p h o r e t i c v a r i a n t of hsp22.  The hsp22 gene c o n t a i n s  two c l o s e l y spaced HSEs which are j u s t upstream of the TATA box w h i l e a t h i r d d i s t a l element i s l o c a t e d a f u r t h e r 100 bp upstream.  Although  d e l e t i o n s r e t a i n i n g a l l t h r e e HSEs m a i n t a i n f u l l heat i n d u c i b l e f u n c t i o n and f u l l developmental e x p r e s s i o n , the authors suggest t h a t the sequence requirements f o r the two modes of gene a c t i v a t i o n might not be c o m p l e t e l y congruent.  A d e l e t i o n c o n t a i n i n g b o t h of the p r o x i m a l HSEs s t i l l  confers  a p p r o x i m a t e l y 20% of the heat i n d u c i b i l i t y but e x p r e s s i o n d u r i n g e a r l y p u p a l stages i s u n d e t e c t a b l e .  The use of d i f f e r e n t promoter sequences f o r hormone  and h e a t i n d u c t i o n has a l s o been shown f o r hsp23 u s i n g hsp23/betag a l a c t o s i d a s e h y b r i d genes i n D r o s o p h i l a t i s s u e c u l t u r e c e l l s et  al.,  (Mestril  1986).  The soybean 17.5-E gene has been i n t r o d u c e d i n t o p r i m a r y sunflower tumors w i t h the T-DNA r e g i o n of ARrobacterium tumefaciens ( G u r l e y e t a l . , 1986).  A c o n s t r u c t c o n t a i n i n g 3.2 kb of upstream sequences i s s t r o n g l y  induced by h e a t shock and a r s e n i t e and t o a l e s s e r degree by cadmium.  Once  a g a i n , a d d i t i o n a l a n a l y s i s w i l l be r e q u i r e d to a s c e r t a i n whether the a r s e n i t e and/or cadmium r e s p o n s i v e f u n c t i o n s can be s e p a r a t e d from the h e a t i n d u c i b l e ones. Cadmium and o t h e r heavy metal i o n s may  e x e r t t h e i r i n f l u e n c e on h e a t  shock gene i n d u c t i o n through sequences s i m i l a r to the metal i o n response element (MRE)  which has been shown by d e l e t i o n a n a l y s i s to be e s s e n t i a l f o r  i n d u c t i o n of t h e human m e t a l l o t h i o n e i n - I I A gene by cadmium ( K a r i n e t a l . , 1984).  S i g n i f i c a n t homologies t o t h i s r e g i o n have been r e p o r t e d i n the  30  soybean 17.5-E gene as two repeats between t h e TATA m o t i f and the transcription start The  (Czarnecka e t a l . ,  1985).  f a c t t h a t a r s e n i t e induces t h e e x p r e s s i o n o f p r o t e i n s which a r e n o t  heat i n d u c i b l e i n r a t myoblast muscle t i s s u e (Kim e t a l . ,  cells  (Kim e t al.., 1983), i n c h i c k embryo  1983) and i n rainbow t r o u t f i b r o b l a s t  cells  (Kothary and Candido, 1982) suggests t h a t a r s e n i t e , as w e l l , may be i n d u c i n g gene a c t i v i t y through sequences other than HSEs.  Furthermore,  t h e hsp25  gene i s induced by a r s e n i t e , copper and z i n c i n q u a i l myotubes which have l o s t t h e i r thermal s e n s i t i v i t y  1.3.2  (Atkinson e t a l . ,  1983).  Heat Shock Regulons The number and v a r i e t y o f ways i n which shsps can be induced s t r o n g l y  suggests  t h a t a common mechanism may be i n v o l v e d f o r many of them.  C o n s i s t e n t w i t h t h i s model would be t h e a c t i v a t i o n o f a f a c t o r d u r i n g c e l l u l a r s t r e s s o r damage which i n i t i a t e s t r a n s c r i p t i o n o f a b a t t e r y o f genes (regulon) through a common sequence element. A heat shock t r a n s c r i p t i o n f a c t o r (HSTF) has been p a r t i a l l y  purified  from n u c l e a r e x t r a c t s o f D r o s o p h i l a t i s s u e c u l t u r e c e l l s which i s s p e c i f i c a l l y r e q u i r e d f o r t r a n s c r i p t i o n o f a D r o s o p h i l a hsp70 gene i n v i t r o ( P a r k e r and T o p o l , 1984). has  DNAsel f o o t p r i n t i n g d a t a demonstrates t h a t HSTF  a h i g h a f f i n i t y f o r HSEs i n v i t r o  1985).  C o o p e r a t i v e b i n d i n g occurs a t a p a i r o f a d j a c e n t HSEs l o c a t e d i n t h e  D r o s o p h i l a hsp70 gene (Topol e t a l . , heat  (Parker and T o p o l , 1984; Topol e t a l . ,  1985).  Both a r e r e q u i r e d f o r maximal  i n d u c i b i l i t y i n v i v o (Dudler and T r a v e r s , 1984; Simon e t a l . ,  i n D r o s o p h i l a melanogaster  tissue culture c e l l s  (Amin e t a l . ,  1985) and  1985).  This  r e g i o n i s a l s o p r o t e c t e d by HSTF from exoIII n u c l e a s e d i g e s t i o n i n n u c l e i  31  p u r i f i e d from h e a t shocked D r o s o p h i l a c e l l s but not i n n u c l e i i s o l a t e d cells  grown a t normal temperature (Wu,  from  1984).  The p r o c a r y o t i c c o u n t e r p a r t to HSTF i s a 32 kd sigma f a c t o r (sigma 32) t h a t s t i m u l a t e s t r a n s c r i p t i o n i n i t i a t i o n from heat shock promoters et  a l . , 1984;  L a n d i c k e t a l . , 1984;  Yura e t a l . , 1984).  (Grossman  Interestingly, a  consensus sequence d e r i v e d from s i x o f the known heat shock genes of E s c h e r i c h i a c o l i c o n s i s t s of a -35 element which c o n t a i n s one h a l f of the e u c a r y o t i c HSE  concensus (Cowing e t a l . , 1985).  Mutations w i t h i n the gene coding f o r sigma 32 (rpoH gene) have shown t h a t the heat shock genes a r e under the p o s i t i v e c o n t r o l of sigma 32. nonsense m u t a t i o n i n a s t r a i n  (rpoH165)  A  c a r r y i n g a tRNA t h a t suppresses  o o . . amber mutations a t 28 C but not 42 C, e l i m i n a t e s the s y n t h e s i s of hsps a f t e r a s h i f t to the h i g h temperature and, i n f a c t , the c e l l s d i e a f t e r a o b r i e f p e r i o d a t 42 C (Neidhart and Van Bogelen, 1981; Yamamori and Yura, 1982). S i g n i f i c a n t amounts of HSTF and sigma 32 a r e , however, found i n n o r m a l l y growing c e l l s  (Beckman and Cooper, 1973; P a r k e r and T o p o l ,  1984).  Whether o r n o t they a r e r e q u i r e d under normal c o n d i t i o n s i s not y e t known. I f the rpoH gene c a r r y i n g a nonsense m u t a t i o n i s p l a c e d i n a n o n - s u p p r e s s i n g environment, the one hsp a n a l y z e d (groE) i s s t i l l s y n t h e s i z e d a t 30°C o a l t h o u g h no i n d u c t i o n i s observed a t 42 C (Yura e t a l . , 1984).  This  suggests t h a t sigma 32. i s d i s p e n s a b l e a t low temperatures. HSTF i s p r o b a b l y a c t i v a t e d d u r i n g heat shock s i n c e HSTF d e r i v e d from h e a t shocked c e l l s 1984).  i s more a c t i v e i n t r a n s c r i p t i o n assays (Parker and T o p o l , .  ,  32  An i n d i c a t i o n t h a t sigma 32 may  be found i n 2 d i f f e r e n t forms comes  from the o b s e r v a t i o n t h a t sigma 32 from c e l l two-dimensional  g e l s , but o n l y as 1 spot when c o p u r i f i e d w i t h RNA  (Grossman e t a l . ,  1.3.3  l y s a t e s migrates as 2 s p o t s on polymerase  1984).  A c t i v a t i o n of T r a n s c r i p t i o n F a c t o r s The evidence a l r e a d y p r e s e n t e d suggests t h a t an a c t i v a t e d  f a c t o r i n t e r a c t s w i t h genes w i t h i n the heat shock r e g u l o n to  transcription  initiate  coordinate expression. R e c e n t l y , s e v e r a l s m a l l molecules have been i d e n t i f i e d typhimurium  and E. c o l i  stress.  i n t r a c e l l u l a r c o n c e n t r a t i o n of f i v e adenylated n u c l e o t i d e s  The  t h a t accumulate  i n Salmonella  under heat shock o r e t h a n o l  (AppppA, AppppG, ApppG, ApppA, ApppGpp) i n c r e a s e s 5 - 1 0 minutes a f t e r i n s u l t nucleotides s t i l l  (Lee e t a l . , 1983).  accumulate  fold within 5  The o b s e r v a t i o n t h a t  these  i n the absence of hsp s y n t h e s i s i n the rpoH165  mutant l e d the authors t o suggest t h a t they were alarmones s i g n a l l i n g onset o f o x i d a t i v e s t r e s s and t r i g g e r i n g the heat shock response. accumulation of o n l y ApppA occurs i n heat shocked  yeast c e l l s  the  The  (Denisenko,  1984). A d e n y l a t e d n u c l e o t i d e s are b e l i e v e d to be s y n t h e s i z e d i n v i v o by a s i d e r e a c t i o n of aminoacyl-tRNA s y n t h e t a s e s (Rapaport e t a l . , 1975). one of the hsps i n E. c o l i  Curiously,  i s an i n d u c i b l e s p e c i e s of l y s y l - t R N A s y n t h e t a s e  encoded by the l y s U gene (Van Bogelen e t a l . ,  1983).  Many o b s e r v a t i o n s , however, c o n f l i c t w i t h t h i s model.  The k i n e t i c s of  the accumulation of these alarmones i s not c o n s i s t e n t w i t h the f a c t t h a t i n b a c t e r i a , t r a n s c r i p t i o n a f t e r heat shock i s observed w i t h i n one minute and  33  has  reached a maximum l e v e l and begun to decrease w i t h i n 5 minutes o f the  temperature  shift.  I n j e c t i o n o f Xenopus oocyte n u c l e i w i t h e i t h e r AppppA o r  ApppA does not induce hsp s y n t h e s i s (Guedon e t a l . , 1985).  A l s o , i n Xenopus  oocytes the accumulation of AppppA to 10 times i t s b a s a l l e v e l o c c u r s o n l y under severe heat c o n d i t i o n s such as 45°C whereas hsp s y n t h e s i s r e a d i l y o proceeds  a t 33 C (Guedon e t a l . ,  1985).  F i n a l l y , treatment of D r o s o p h i l a  t i s s u e c u l t u r e c e l l s w i t h cadmium c h l o r i d e under c o n d i t i o n s which i n d u c e the heat shock response  (Courgeon  e t §_1. , 1984)  does not s i g n i f i c a n t l y  the c e l l u l a r p o o l of AppppN and ApppN n u c l e o t i d e s (Brevet e t a l . , There  i s mounting evidence t h a t the heat shock response  a s s o c i a t e d w i t h p r o t e i n d e g r a d a t i o n systems.  affect 1985).  i s intimately  T h i s should not be  surprising  s i n c e most of the known i n d u c e r s must indeed l e a d to p r o t e i n damage. In some f l i g h t l e s s D r o s o p h i l a mutants, m o l e c u l a r l e s i o n s i n a c t i n I I I i s o f o r m s l e a d s to the d i s r u p t i o n of m y o f i b r i l s i n i n d i r e c t f l i g h t muscle, the o n l y t i s s u e i n which they a r e expressed.  This i s associated with  c o n s t i t u t i v e hsp s y n t h e s i s o n l y w i t h i n the same c e l l type ( K a r l i k e t a l . , 1984;  Hiromi and H o t t a , 1985).  P-element mediated  transformation with these  mutant a c t i n genes a l s o r e s u l t s i n c o n s t i t u t i v e e x p r e s s i o n of the heat genes w i t h i n the i n d i r e c t f l i g h t muscle (Hiromi et a l . , A mouse c e l l  shock  1986).  l i n e c a l l e d ts85 c o n t a i n s a t h e r m o l a b i l e u b i q u i t i n -  a c t i v a t i n g enzyme ( E - l ) ( F i n l e y e t a l . ,  1984).  At the  non-permissive  o temperature  of 39 C, s h o r t - l i v e d p r o t e i n s and abnormal p r o t e i n s are n o t  e f f i c i e n t l y degraded et a l . ,  1984).  t h r e s h o l d of hsp  s i n c e they do not become u b i q u i t i n a t e d  At t h i s temperature,  (Ciechanover  which i s w e l l below the normal  i n d u c t i o n , ts85 c e l l s s y n t h e s i z e hsps a t h i g h l e v e l s  (Ciechanover-et a l . ,  1984).  34  In E. c o l i , to canavanine  the s y n t h e s i s o f l a r g e amounts of a b e r r a n t p o l y p e p t i d e s due  i n c o r p o r a t i o n , the p r o d u c t i o n of t r u n c a t e d p r o t e i n s i n the  presence of puromycin,  the i n d u c t i o n of t r a n s l a t i o n a l e r r o r s w i t h  s t r e p t o m y c i n , o r the s y n t h e s i s o f a c l o n e d f o r e i g n p r o t e i n plasminogen  (human t i s s u e  a c t i v a t o r ) l e a d s to the i n d u c t i o n of the heat shock  (Goff and Goldberg, 1985).  response  T h i s phenomenon i s not seen i n the rpoH165  mutant i n which the sigma f a c t o r s p e c i f i c t o the heat shock r e g u l o n i s defective. The  i n d u c t i o n of the heat shock response by amino a c i d analogues i s ,  presumably,  a d i r e c t r e s u l t of t h e i r i n c o r p o r a t i o n Into newly s y n t h e s i z e d  p r o t e i n s , r e n d e r i n g them n o n f u n c t i o n a l .  Homoarginine,  i n c o r p o r a t e d i n t o p r o t e i n s ( N a z a r i o and Evans, shock response i n CEF c e l l s  (Hightower,  1974),  which i s not does not induce a heat  1980).  The most d i r e c t evidence f o r a l i n k between p r o t e i n d e g r a d a t i o n and heat shock response comes from the work of Ananthan e t a l . (1986).  the  A  D r o s o p h i l a h s p 7 0 / b e t a - R a l a c t o s i d a s e h y b r i d gene i s a c t i v a t e d i n Xenopus oocytes when c o - i n j e c t e d w i t h bovine b e t a - l a c t o g l o b u l i n or bovine serum albumin t h a t has been denatured by r e d u c t i v e c a r b o x y m e t h y l a t i o n . p r o t e i n s have no e f f e c t .  N a t i v e hemoglobin  a l s o has no e f f e c t , but  monomers which r e s u l t from e x t r a c t i o n of the heme groups do. the a n a l y s i s of promoter  The n a t i v e  d e l e t i o n s demonstrates  globin  Furthermore,  t h a t the p r o x i m a l HSE i s  r e q u i r e d f o r both heat shock i n d u c t i o n and f o r a c t i v a t i o n by  denatured  protein. A l l of these r e s u l t s suggest t h a t a d e p l e t i o n of a c t i v a t e d  ubiquitin  p o o l s i n eucaryotes and the overburdening of p r o t e i n d e g r a d a t i o n systems i n p r o c a r y o t e s l e a d s t o the a c t i v a t i o n of the heat shock  response.  35  I n t e r e s t i n g l y , E. c o l i p r o t e a s e La i s a hsp under the r e g u l a t i o n o f sigma 32 (Goff e t a l . ,  1984;  Phillips et al.,  1984;  Baker e t a l . , 1984).  Similarly,  i t has been shown t h a t u b i q u i t i n mRNA and u b i q u i t i n s y n t h e s i s i n c r e a s e 5 - f o l d a f t e r heat shock i n c h i c k e n embryo f i b r o b l a s t s  (Bond and S c h l e s i n g e r ,  1985). In e u c a r y o t e s , u b i q u i t i n a t i o n o f damaged p r o t e i n s c o u l d d e p l e t e a c t i v a t e d u b i q u i t i n p o o l s , mimicking  the l e s i o n found  i n ts85 c e l l s .  Under  these c o n d i t i o n s , a l l a v a i l a b l e u b i q u i t i n s t o r e s would be m o b i l i z e d t o d e g r a d a t i o n pathways.  T h i s i s c o n s i s t e n t with the o b s e r v a t i o n t h a t  u b i q u i t i n a t e d h i s t o n e H2A i s d e p l e t e d i n ts85 c e l l s a t the temperature  (Matsumoto e t a l . , 1983;  F i n l e y e t §_1., 1984)  c e l l s under heat shock ( G l o v e r , 1982a).  non-permissive  or i n Drosophila  Thus i t i 3 p o s s i b l e t h a t d u r i n g  heat shock o r m e t a b o l i c s t r e s s , HSTF may be a c t i v a t e d by the c o n v e r s i o n o f a u b i q u i t i n a t e d form t o a n o n - u b i q u i t i n a t e d form although t h e r e i s no d i r e c t evidence t o support t h i s as y e t .  A l t e r n a t i v e l y , HSTF and sigma 32 may have  a high turnover i n c o n t r o l c e l l s , being s u s c e p t i b l e to p r o t e i n pathways.  degradation  Under m e t a b o l i c s t r e s s , g e n e r a l c e l l u l a r p r o t e i n damage would  d i v e r t the pathway and HSTF would be s t a b i l i z e d .  Similarly, a defective  p r o t e i n d e g r a d a t i o n system such as i n ts85 c e l l s a t 39°C, would  stabilize  the t r a n s c r i p t i o n f a c t o r and l e a d t o hsp s y n t h e s i s .  1.3.4  D i f f e r e n t i a l HSmRNA S t a b i l i t y Although  the heat shock response  transcriptional activity, variability  i s c h a r a c t e r i z e d by new i n accumulated  mRNA l e v e l s due t o  d i f f e r e n c e s i n s t a b i l i t y may r e s u l t i n a l t e r e d p a t t e r n s o f hsp s y n t h e s i s . The  s y n t h e s i s o f shsps  i n heat shocked  versus ecdysterone t r e a t e d  36  D r o s o p h i l a melanogaster  t i s s u e c u l t u r e c e l l s i s one example o f t h i s .  The  r a t i o o f hsp23 t o hsp22 v a r i e s from 3 d u r i n g heat shock t o 20 d u r i n g ecdysterone treatment ( V i t e k and Burger, 1984).  Under heat  shock  c o n d i t i o n s , the mRNA l e v e l s f o r hsp23, 22, 26. and 27 i n c r e a s e r a p i d l y f o r 30 minutes  and p l a t e a u a t s i m i l a r l e v e l s .  With hormone treatment, however,  hsp23 mRNA i n c r e a s e s s t e a d i l y f o r 24 hours, r e a c h i n g a l e v e l 50% t h a t o f heat shock.  approximately  Hsp22 mRNA. on the o t h e r hand, i n c r e a s e s d u r i n g t h e  f i r s t 4 hours and reaches a p l a t e a u so t h a t by 24 hours, the r a t i o o f hsp23 mRNA t o hsp22 mRNA i s approximately 6.  Under the same c o n d i t i o n s hsp26 and  hsp27 t r a n s c r i p t s r i s e t o i n t e r m e d i a t e l e v e l s . f u r t h e r showed by pulse-chase experiments  V i t e k and Burger  (1984)  t h a t the v a r i a b i l i t y i n  s t e a d y - s t a t e mRNA l e v e l s i s due t o d i f f e r e n c e s i n mRNA s t a b i l i t y . o I n t e r e s t i n g l y , hsmRNAs are more s t a b l e d u r i n g a chase a t 35 C as compared to 25°C. 1.4  Translational Control The changing p a t t e r n s o f p r o t e i n s y n t h e s i s d u r i n g heat shock  usually  i n c l u d e the r e p r e s s i o n o f e x i s t i n g p r o t e i n s y n t h e s i s as w e l l as t h e appearance  o f heat shock p r o t e i n s .  The degree t o which t h i s o c c u r s depends  on the c e l l type i n q u e s t i o n and the n a t u r e o r s e v e r i t y o f the s t r e s s . F o r example, t h e r e i s no c e s s a t i o n o f t r a n s l a t i o n o f e x i s t i n g messages upon heat shock i n Xenopus somatic c e l l s as t h e r e i s i n Xenopus oocytes ( B i e n z , 1982).  Storage p r o t e i n s i n the soybean  s y n t h e s i z e d a t h i g h temperatures 1982).  embryo (seed) c o n t i n u e t o be  along the hsps  ( A l t s c h u l e r and Mascarenhas,  I n D r o s o p h i l a c e l l s . , heat: shock, a r s e n i t e , and canavanine  treatments  37  v a r y i n t h e i r a b i l i t y t o shut down normal p r o t e i n s y n t h e s i s even though hsps are  induced i n a l l t h r e e s i t u a t i o n s  (Olsen e t a l . , 1983).  These changes i n p r o t e i n s y n t h e s i s might o c c u r v i a changes i n t h e specificity  o f t h e t r a n s l a t i o n a l machinery so t h a t a subset o f mRNAs i s  t r a n s l a t e d more e f f i c i e n t l y than another, o r by changes i n the mRNA p o o l s t h a t a r e a v a i l a b l e t o t h e ribosomes. latter  s i t u a t i o n being u t i l i z e d  1.4.1  HSmRNA S e l e c t i v i t y  Both of these mechanisms a r e used, t h e  i n yeast.  In most organisms p r e - h e a t shock mRNAs (normal mRNAs) a r e s t a b l e under heat shock c o n d i t i o n s s i n c e they a r e e f f i c i e n t l y t r a n s l a t e d i n a v a r i e t y of in vitro  systems b u t n o t i n v i v o .  and HeLa c e l l s  In D r o s o p h i l a c e l l s  (Storti  e t a l . , 1980)  (McCormick and Penman, 1969; H i c k e y and Weber, 1982)  p r o t e i n s y n t h e s i s resumes  normal  i n the presence of a c t i n o m y c i n D upon r e t u r n t o  normal temperature s u g g e s t i n g t h a t t r a n s l a t i o n takes p l a c e on e x i s t i n g messages which have been i n a c t i v e d u r i n g s t r e s s . normal mRNAs i n v i t r o  Efficient  t r a n s l a t i o n of  f u r t h e r suggests t h a t they do n o t become m o d i f i e d i n  any way even though they a r e p r e f e r e n t i a l l y ignored by t h e t r a n s l a t i o n a l machinery i n v i v o . Polysome breakdown i s an immediate response t o heat shock.  T h i s has  been shown f o r D r o s o p h i l a (McKenzie e t a l . , 1975) and soybean (Key e t a l . , 1981).  I n D r o s o p h i l a , t h e p e r c e n t a g e o f ribosomes i n monosome form  to polysome  compared  form i n c r e a s e s from a p p r o x i m a t e l y 20% t o 50% d u r i n g h e a t shock  ( L i n d q u i s t , 1980; B a l l i n g e r and Pardue, 1983).  These changes a r e n o t due to  a f l o o d o f newly s y n t h e s i z e d hsmRNA s i n c e they o c c u r i n a c t i n o m y c i n D treated c e l l s  ( L i n d q u i s t , 1980).  As d i s c u s s e d e a r l i e r ,  a bimodal  38  d i s t r i b u t i o n o f polysomes i s found i n heat shocked D r o s o p h i l a c e l l s ,  which  r e f l e c t s t h e commitment o f t h e s e c e l l s t o t h e t r a n s l a t i o n of t h e two major s i z e c l a s s e s o f hsmRNA.  I n v i t r o t r a n s l a t i o n of polysomal mRNA from heat  shocked D r o s o p h i l a c e l l s  (Kruger and Benecke, 1981;  1983)  and a z e t i d i n e ( p r o l i n e analogue)  Mathews, 1982)  demonstrates,  normal messages a r e s t i l l being t r a n s l a t e d .  t r e a t e d HeLa c e l l s  (Thomas and  however, t h a t a s i g n i f i c a n t p r o p o r t i o n o f  a s s o c i a t e d w i t h polysomes even though they are n o t  Using s p e c i f i c h y b r i d i z a t i o n probes, i t was shown t h a t  a l p h a - t u b u l i n , b e t a - t u b u l i n and a c t i n f a l l Benecke,  B a l l i n g e r and Pardue,  i n t o t h i s category (Kruger and  1981).  B a l l i n g e r and Pardue (1983) have e s t i m a t e d that t h e r e i s a 15 - 30 f o l d decrease i n t h e i n i t i a t i o n / e l o n g a t i o n r a t e s of ribosomes heat shocked  Drosophila c e l l s .  on normal mRNAs i n  I n HeLa c e l l s , hsp t r a n s l a t i o n i s more  s e n s i t i v e t o c y c l o h e x i m i d e s u g g e s t i n g t h a t hsp mRNAs i n i t i a t e more e f f i c i e n t l y than most normal mRNAs (Hickey and Weber,  translation  1982).  The p r e f e r e n t i a l t r a n s l a t i o n o f hsmRNAs can be reproduced u s i n g c e l l l y s a t e s prepared from heat shocked cells  (Storti et a l . ,  an optimum temperature  Drosophila tissue  1980; Kruger and Benecke, 1981).  i n vitro culture  These l y s a t e s have  f o r p r o t e i n s y n t h e s i s o f 28°C s u g g e s t i n g t h a t a  o s t a b l e change takes p l a c e d u r i n g heat shock a t 36 C. o  A l y s a t e prepared  from c e l l s grown a t 25 C t r a n s l a t e s both normal mRNAs and hsmRNAs. F r a c t i o n a t i o n of l y s a t e s and subsequent demonstrated  that the ribosomal f r a c t i o n  supplementation  experiments  (222,600 x g p e l l e t ) of c o n t r o l  l y s a t e s c o u l d rescue normal mRNA t r a n s l a t i o n i n heat shock l y s a t e s and Pardue,  1981).  by a 0.5 M KC1 wash.  The a b i l i t y t o rescue normal  (Scott  t r a n s l a t i o n was d i m i n i s h e d  None o f t h e heat shock l y s a t e f r a c t i o n s c o u l d cause  39  the 25 C l y s a t e t o change i t s s p e c i f i c i t y negative  suggesting  t h a t there  isa  c o n t r o l o r i n a c t i v a t i o n o f a component i n t h e heat shock l y s a t e .  A c a n d i d a t e f o r t h i s type o f m o d i f i c a t i o n i s the d e p h o s p h o r y l a t i o n of r i b o s o m a l p r o t e i n S6 d u r i n g heat shock which has been shown t o occur i n Drosophila  (Glover,  1982b), i n suspension c u l t u r e s o f tomato (Scharf and  Nover, 1982), i n p r i m a r y c u l t u r e s d e r i v e d from human s k i n f i b r o b l a s t s and meningiomas ( R i c h t e r , 1983), as w e l l as i n human HeLa and baby hamster kidney c e l l s  (Kennedy e t a l . , 1984).  The o b s e r v a t i o n s  (1983) are i n c o n s i s t e n t w i t h t h i s simple c o r r e l a t i o n : rephouphorylation recovery  o f S6 i n D r o s o p h i l a  made by O l s e n - e t  al.  i n t h e i r s t u d i e s , the  c e l l s was found n o t to occur u n t i l  hod proceeded f o r 8 hours, w e l l a f t e r normal p r o t e i n s y n t h e s i s had  reuumed.  A l s o , a r s e n i t e and canavanine treatment o f D r o s o p h i l a  cells  induced hsp s y n t h e s i s but d e p h o s p h o r y l a t i o n of S6 was n o t observed. Recently, Drosophila  i t was r e p o r t e d  cell  t h a t the r i b o s o m a l supernatant from 25°C  l y s a t e s c o u l d rescue the t r a n s l a t i o n o f normal mRNAs i n heat  shock l y s a t e s (Sanders e t a l . , 1986). higher  The f a c t t h a t these authors used  i o n i c strength b u f f e r s i n t h e i r ribosomal p u r i f i c a t i o n s i s consistent  with the i n i t i a l  observation  made by S c o t t and Pardue (1981) t h a t rescue was  reduced w i t h s a l t washed ribosomes.  Sanders e t a l . (1986) a l s o showed t h a t  a r e c o n s t i t u t e d system c o n t a i n i n g heat shocked ribosomes and a c o n t r o l s u p e r n a t a n t had t h e a b i l i t y t o s y n t h e s i z e normal p r o t e i n s . The  change i n the s p e c i f i c i t y  o f t r a n s l a t i o n d u r i n g heat shock may  t h e r e f o r e depend on s o l u b l e f a c t o r s such as t r a n s l a t i o n i n i t i a t i o n f a c t o r s . D e t a i l e d immunoblot a n a l y s i s o f HeLa c e l l l y s a t e s demonstrates t h a t a v a r i e t y of modifications  occur i n v a r i o u s  i n i t i a t i o n f a c t o r s upon heat shock  40  (Duncan and Hershey, 1984).  Also, phosphorylation  o f eIF-2 alpha occurs i n  r e t i c u l o c y t e l y s a t e s heated t o 42°C ( E r n s t e t a l . , 1982). In D r o s o p h i l a c e l l s , some normal messages escape the t r a n s l a t i o n a l s e l e c t i v i t y d u r i n g heat shock. d u r i n g heat shock (Sanders,  H i s t o n e H2B s y n t h e s i s i n c r e a s e s 3 - 4  1981).  fold  Although t r a n s c r i p t i o n o f the h i s t o n e  gene i s r e l a t i v e l y u n a f f e c t e d , a g r e a t e r abundance o f h i s t o n e H2B s p e c i f i c mRNA i s found i n polysomes i s o l a t e d from heat shocked c e l l s as compared t o control c e l l s Schneider  ( F a r r e l l - T o w t and Sanders, 1984).  2 cells  Also, i n Drosophila  i n f e c t e d w i t h the double stranded  s p e c i f i c p r o t e i n s continue  DNA v i r u s HPS-1, HPS-1  to be s y n t h e s i z e d d u r i n g heat shock ( S c o t t  ot aJL. , 1980). The  sequences r e q u i r e d f o r h i g h temperature t r a n s l a t i o n a r e now under  investigation.  D r o s o p h i l a hsp70 genes w i t h d e l e t e d l e a d e r sequences o r  f u s i o n s between hsp70 promoter sequences and an a l c o h o l dehydrogenase (AdH) gene c o n t a i n i n g i t s own l e a d e r and coding Drosophila tissue culture c e l l s (Klemenz e t a l . , 1985).  r e g i o n s have been i n t r o d u c e d i n t o  (McGarry and L i n d q u i s t , 1985) and f l i e s  I n b o t h cases,  t r a n s c r i p t s accumulate t o h i g h  l e v e l s d u r i n g heat shock b u t they a r e n o t t r a n s l a t e d u n t i l the c e l l s a r e returned  t o normal temperature.  These r e s u l t s suggest t h a t the 5'  u n t r a n s l a t e d l e a d e r sequences o f hsp mRNAs a r e r e q u i r e d f o r p r e f e r e n t i a l t r a n s l a t i o n a t e l e v a t e d temperature.  I n i t i a l observations  (DiNocera and  Dawid, 1983; Lawson e t a l . , 1984) suggested t h a t o n l y the t e r m i n a l sequences of heat shock mRNA l e a d e r s f u l f i l l e d The  f u s i o n o f o n l y the f i r s t  this function.  95 bp o f the hsp70 250 bp l e a d e r sequence i  to t h e w i l d type AdH l e a d e r i s s u f f i c i e n t t o d i r e c t t r a n s l a t i o n a t h i g h temperature (Klemenz e t a l . , 1985).  D e l e t i o n s o f the same leader"between +3  41  and +26 o r between +14 and +114 have no e f f e c t on w i l d type t r a n s l a t i o n (McGarry and L i n d q u i s t ,  1985).  S u r p r i s i n g l y , a tandem d u p l i c a t i o n between  -29 and +2 which r e s u l t s i n an a d d i t i o n a l 39 bp a t t a c h e d t o the 5' end o f a p e r f e c t l y normal 250 bp l e a d e r ,  destroyed the t r a n s l a t i o n a l s e l e c t i v i t y of  t h a t mRNA upon heat shock (McGarry and L i n d q u i s t , S i m i l a r experiments demonstrate t h a t only of t h e hsp22 mRNA 250 bp l e a d e r during  stress  1.4.2  High mRNA Turnover In y e a s t  1985).  approximately the f i r s t 30 bp  sequence a r e r e q u i r e d  f o r i t s translation  (Hultmark e t a l . , 1986).  (Saccharomyces  cerevi3iae),  the p a t t e r n  of p r o t e i n  synthesis  a l t e r s r a p i d l y upon a o h i f t from 23°C t o 36°C ovon though both temperatures a r e c o n s i d e r e d t o be w i t h i n and F i n k e l s t e i n , 1980a; L i n d q u i s t , i n v i t r o translation pattern  proteins  1981).  during  Hybridization  1981).  (McAlister  (McAlister  In t h i s organism, however, t h e  f o r RNA i s o l a t e d from heat shocked  corresponds t o t h a t seen i n v i v o Lindquist,  i t s normal growth range  cells  and F i n k e l s t e i n , 1980a;  T h i s suggest t h a t t h e reduced s y n t h e s i s  of p a r t i c u l a r  heat shock c o r r e l a t e s w i t h the d e g r a d a t i o n o f t h e i r mRNAs.  o f s p e c i f i c gene probes t o mRNA supports t h i s s i n c e ura-3 mRNA  l e v e l s decrease 5 f o l d w i t h i n t h a t o f normal during  1 hour w h i l e lev-2 mRNA l e v e l s decrease t o 5%  t h e same time p e r i o d  (Lindquist,  1981).  Both t h e appearance o f hsmRNAs and t h e r a p i d disappearance o f normal mRNAs a r e dependent upon new t r a n s c r i p t i o n .  I f transcription i s inhibited  by t h e z i n c - c h e l a t i n g a n t i b i o t i c lomofungin or- i f t s r n a l s t r a i n s a r e used i n which RNA t r a n s p o r t / p r o c e s s i n g protein  synthesis  i s defective  a t 36°C, then r e p r e s s i o n o f  i s g r e a t l y reduced ( M i l l e r e t a l . , 1979; M c A l i s t e r and  42  F i n k e l s t e i n , 1980a).  T h i s phenomenon, however, does not r e q u i r e p r o t e i n  s y n t h e s i s s i n c e c y c l o h e x i m i d e treatment temperature  o r the use o f s t r a i n s which have a  sensitive defect i n translation i n i t i a t i o n  (ts!87) have no  e f f e c t on changing mRNA p o o l s d u r i n g heat shock as assayed by i n v i t r o translation  1.5  ( M c A l i s t e r and F i n k e l s t e i n ,  1980a).  Recovery and A u t o r e g u l a t i o n The  e f f e c t s o f heat shock and o t h e r s t r e s s c o n d i t i o n s are always  r e v e r s i b l e upon r e t u r n t o normal temperature, v a r i a b l e degree upon prolonged a d a p t i b i l i t y o f the c e l l  o r t o a l e s s e r and more  exposure t o s t r e s s depending on the  type.  In most organisms such as D r o s o p h i l a . mammals, and h i g h o r p l a n t s , r e t u r n t o normal p r o t e i n s y n t h e s i s i s p r o b a b l y simply a matter resumption  the  o f the  o f t r a n s l a t i o n o f e x i s t i n g messages, s i n c e r e c o v e r y i s  i n s e n s i t i v e to i n h i b i t o r s of t r a n s c r i p t i o n .  Yeast, on the other hand,  r e q u i r e s new mRNA s y n t h e s i s i n order t o r e c o v e r the p r e - e x i s t i n g  protein  synthesis pattern. The  s i t u a t i o n i n a v i a n systems, s p e c i f i c a l l y i n c h i c k embryo  f i b r o b l a s t s , p r e s e n t s an enigma.  Although  in vitro translation  i n d i c a t e t h a t normal mRNAs are p r e s e n t i n heat shocked 1980;  Johnston  e t a l . , 1980;  by a c t i n o m y c i n D (Hightower,  Voellmy 1980;  cells  results  (Kelly et a l . ,  and Bromley, 1982), r e c o v e r y i s b l o c k e d  Schlesinger e t a l . ,  1982). I f  c y c l o h e x i m i d e i s p r e s e n t d u r i n g an 8 hour r e c o v e r y p e r i o d and removed p r i o r to l a b e l l i n g o f newly s y n t h e s i z e d p r o t e i n , the heat shock p r o f i l e i s seen, s u g g e s t i n g t h a t t h e r e i s a l s o a need f o r new p r o t e i n s y n t h e s i s i n o r d e r f o r r e c o v e r y t o proceed  (Hightower,  1980;  Schlessinger et a l . ,  1982).  43  In D r o s o p h i l a , a l l normal mRNAs resume t r a n s l a t i o n a t the same r a t e i n r e c o v e r i n g c e l l s ; on the o t h e r hand, the r e p r e s s i o n of hsp s y n t h e s i s i s asynchronous and occurs i n a r e p r o d u c i b l e order (DiDomenico e t a_l., 1982a). A l t h o u g h r e c o v e r y times can v a r y depending on the s e v e r i t y of the shock, the r e p r e s s i o n of hsp70 always o c c u r s f i r s t , hsp83 l a s t and the shsps i n between.  The r e p r e s s i o n of hsp70 i s c o r r e l a t e d w i t h the r e c o v e r y of normal  protein synthesis.  R e p r e s s i o n of hsp  synthesis during recovery i s a r e s u l t  of hsmRNA d e g r a d a t i o n i n D r o s o p h i l a ( L i n d q u i s t , 1980; 1982a) and The  (Hickey and Weber, 1982).  a u t o r e g u l a t i o n of the heat shock response has been demonstrated i n  canavanine cells,  i n HeLa c e l l s  DiDomenico e t a l . ,  treated Drosophila c e l l s  (DiDomenico et a l . , 1982b).  In these  t h e r e i s no r e c o v e r y of normal p r o t e i n s y n t h e s i s at 25°G a f t e r a 1  hour heat shock and hsp s y n t h e s i s c o n t i n u e s unabated. cycloheximide  Also, i f  i s added to n o r m a l l y growing c e l l s immediately  b e f o r e heat  shock, s y n t h e s i s of hsmRNAs c o n t i n u e s f o r a t l e a s t 4 hours upon r e c o v e r y a t o 25 C where they remain s t a b l e  (DiDomenico e t a l . , 1982b).  authors a l s o demonstrated t h a t when the r a t e of hsp  The  same  synthesis i s l i m i t e d  d e c r e a s i n g the c o n c e n t r a t i o n of hsmRNA w i t h actinomycin D, both r e p r e s s i o n of hsp  s y n t h e s i s and  by  the  the r e s t o r a t i o n of normal s y n t h e s i s are  d e l a y e d , a p p a r e n t l y u n t i l a s p e c i f i c amount of f u n c t i o n a l hsp  has  accumulated. I f D r o s o p h i l a c e l l s are m a i n t a i n e d p e r i o d s , r e p r e s s i o n of hsp  a t h i g h temperature  s y n t h e s i s and  s y n t h e s i s i n i t i a t e s upon the accumulation r e p r e s s i o n of hsp  f o r prolonged  the onset of normal p r o t e i n of hsps as b e f o r e ; however, the  s y n t h e s i s i s p r o l o n g e d due to i n c r e a s e d hsmRNA s t a b i l i t i e s  a t h i g h temperature  (DiDomenico e t aj.. , 1982b) .  44  The  k i n e t i c s o f hsp70 r e p r e s s i o n and i t s c l o s e c o r r e l a t i o n w i t h t h e  recovery  o f normal p r o t e i n s y n t h e s i s  involved  i n the feedback c o n t r o l mechanism which seems t o be o p e r a b l e i n the  heat shock response.  I n E. c o l i ,  following observations.  i m p l i c a t e s i t as the p r o t e i n  t h i s hypothesis i s substantiated  The dnaK gene o f E. c o l i  by t h e  i s a heat shock gene whose  p r o d u c t i s approximately 50% homologous t o D r o s o p h i l a C r a i g , 1984).  directly  hsp70 (Bardwell and  Temperature s e n s i t i v e dnaK mutants such as dnaK756 f a i l t o  t u r n o f f the t r a n s i e n t heat shock response a t 43°C w h i l e b a c t e r i a t h a t overproduce dnaK p r o t e i n a t a l l temperatures undergo a d r a s t i c a l l y heat shock response ( T i l l y e t a l . , 1983). autoregulation, An  i f any,  The r o l e played  reduced  by the shsps i n  remains t o be e l u c i d a t e d .  a n a l y s i s o f hsmRNA sequences which are r e q u i r e d  f o r t h e i r own D2  d e s t a b i l i z a t i o n during contains truncated  recovery  has begun.  et  s t r a i n Df(3R) k a r  an X-ray induced 3' d e l e t i o n mutation o f hsp70 and s y n t h e s i z e s a p r o t e i n (hsp40) d u r i n g heat shock.  c u l t u r e c e l l s t r a n s f e c t e d w i t h the t r u n c a t e d during  Drosophila  recovery  a l . , 1985).  I n these f l i e s  or i n tissue  gene, hsp40 mRNA p e r s i s t s  whereas endogenous hsp70 mRNA decreases r a p i d l y (Simcox These data suggest t h a t the 3' sequences o f hsp70 mRNA are  i n v o l v e d i n the a c t i v e d e s t a b i l i z a t i o n o f the hsp70 mRNA a f t e r r e l e a s e  from  heat shock.  1.6  Function  o f shsps  Sequence i n f o r m a t i o n  f o r the shsp genes o f D r o s o p h i l a ,  and man i n d i c a t e s t h a t the p r o t e i n s acids with a - c r y s t a l l i n chains reacts with a p r o t e i n derived  soybean, Xenopus  share a domain o f approximately 80 amino  of vertebrates.  A l s o , a n t i - c h i c k e n hsp25  from 11 day o l d embryonic c h i c k e n  lens.  The  45  s i g n i f i c a n c e of t h i s homology i s not c l e a r .  Vertebrate a - c r y s t a l l i n s  the major p r o t e i n components of the v e r t e b r a t e eye l e n s , forming aggregates w i t h an average m o l e c u l a r 1971).  The  aggregation  are  large  weight of 800,000 (Bloemendal e t a l . ,  p r o p e r t i e s of a l p h a - c r y s t a l l i n s are i n t e r e s t i n g i n  l i g h t of the o b s e r v a t i o n s made f o r shsps d u r i n g c e l l u l a r  localization  s t u d i e s (see below). I n g o l i a and C r a i g (1982b) p o s t u l a t e d t h a t the r e g i o n of homology between m - c r y s t a l l i n and promoted a g g r e g a t i o n .  the shsps of D r o s o p h i l a r e p r e s e n t e d  Wistow (1985) has  suggested  a domain t h a t  t h a t the domain  r e p r e s e n t s a thermodynamically s t a b l e s t r u c t u r e which p r e - e x i s t e d the and  was  borrowed from a n c e s t r a l heat  of survLvLng  f o r years without  (Wannumacher and  1.6.1  Spector,  Intracellular  shock genes to b u i l d a p r o t e i n  turnover  i n the e n u c l e a t e d ,  avascular  e t a l . , 1980;  Velazquez e t a l . , 1980)  A r r i g o e t a l . , 1980)  and  tissue culture c e l l s  d u r i n g heat  shock.  Subsequent  27 were a s s o c i a t e d w i t h n u c l e a r f r a c t i o n s d u r i n g heat  t r a n s l o c a t e d to the cytoplasm  (Mitchell (Velazquez  showed t h a t newly s y n t h e s i z e d hsps were  f r a c t i o n a t i o n s t u d i e s w i t h D r o s o p h i l a Kc c e l l s confirmed  and V i n c e n t , 1982).  lens  Localization  r a p i d l y t r a n s p o r t e d to the nucleus  and  capable  1968).  E a r l y a u t o r a d i o g r a p h i c s t u d i e s on D r o s o p h i l a s a l i v a r y glands and L i p p s , 1975;  lens  during recovery  cellular  t h a t hsp22, 23,  26  shock and were  ( A r r i g o e t a l . , 1980;  Immunofluorescence s t u d i e s u s i n g an antibody  Tanguay specific  f o r hsp23 r e v e a l e d a s i m i l a r p a t t e r n ( A r r i g o and Ahmad-Zadeh, 1981).  The  a s s o c i a t i o n of the shsps w i t h the n u c l e a r p e l l e t i s r e s i s t a n t to e x t e n s i v e  46  nuclease  d i g e s t i o n and to 2.0 M s a l t treatment  ( S i n i b a l d i and M o r r i s , 1981;  L e v i n g e r and Varshavsky, 1981). A n u c l e a r f u n c t i o n f o r the shsps i s now i n doubt, however, due t o a b e t t e r understanding  of t h e changes which occur w i t h i n t h e c e l l u l a r  i n t e r m e d i a t e f i l a m e n t (10 nm) network d u r i n g heat shock. t h a t d u r i n g heat found  I t was  observed  shock i n D r o s o p h i l a , a 46 kd and a 40 kd p r o t e i n , n o r m a l l y  i n microsomal f r a c t i o n s , became e n r i c h e d i n n u c l e i  Biessman, 1980; Tanguay and V i n c e n t ,  1982).  ( F a u l k n e r and  An antibody made t o t h e 46 kd  p r o t e i n c r o s s - h y d r i d i z e d to t h e 40 kd p r o t e i n as w e l l as t o p r o t e i n s w i t h molecular  weights of 55,000 and 52,000 which have been i d e n t i f i e d as  v l m e n t i n and dosmin, r e s p e c t i v e l y , i n baby hamster kidney ot a l . , 1981) .  cells  (Faulkner  Thene p r o t o i n o a r e major components of the v e r t e b r a t e  i n t e r m e d i a t e f i l a m e n t c y t o a k e l e t a l system. Immunofluorescence s t u d i e s , u s i n g the antibody made a g a i n s t the D r o s o p h i l a 46 kd i n t e r m e d i a t e f i l a m e n t p r o t e i n , showed t h a t , upon heat shock, t h e p r o t e i n was l o c a l i z e d ( F a u l k n e r e t a l . , 1981).  i n the p e r i p h e r a l r e g i o n o f the n u c l e u s  Immunoelectron microscopy f u r t h e r c h a r a c t e r i z e d  the i n t e r m e d i a t e f i l a m e n t s t r u c t u r e o f D r o s o p h i l a and confirmed c o l l a p s e s upon t h e nucleus  a f t e r heat  that i t  shock (Walter and Biessman, 1984).  Furthermore, a n t i b o d i e s made a g a i n s t hsp23 and hsp26 behave l i k e a n t i b o d i e s made t o the 46 kd v i m e n t i n - l i k e p r o t e i n i n t h e i r s u b c e l l u l a r ( L e i c h t e t a l . , 1986), s u g g e s t i n g i n t e r m e d i a t e f i l a m e n t network. i n t o t h e n u c l e u s upon heat  t h a t the shsps a r e a s s o c i a t e d w i t h t h e  Thus the apparent t r a n s l o c a t i o n o f shsps  shock may be a r t i f a c t u a l due t o t h e c o l l a p s e o f  the c y t o s k e l e t a l s t r u c t u r e , throwing fractionation  results.  localization,  i n t o doubt the v a l i d i t y o f e a r l i e r  47  Support f o r t h i s e x p l a n a t i o n comes from the o b s e r v a t i o n t h a t the shsps are found  e n t i r e l y i n the c y t o p l a s m i c  f r a c t i o n of ecdysterone  D r o s o p h i l a l a r v a l i m a g i n a l d i s k s ( I r e l a n d at a l . , 1982).  treated  I n t e r e s t i n g l y , the  c r y s t a l l i n s show immunological c r o s s - r e a c t i v i t y w i t h k e r a t i n s which are a l s o members of the i n t e r m e d i a t e f i l a m e n t p r o t e i n f a m i l y (Kodama and  Eguchi,  1983). In D r o s o p h i l a , hsp22, 23, RNP  26 and  27 become a s s o c i a t e d w i t h  p a r t i c l e s which sediment at approximately  g r a d i e n t s ( A r r i g o e t a l . , 1985; detected  20S  i n sucrose  density  Shuldt and K l o e t z e l , 1985).  i n c o n t r o l c o l l s at l e v e l s l e s s than  accumulates a f t e r 6 hours of r e c o v e r y  cytoplasmic  The  shsps are  10% of the amount which  from heat  shock.  These RNP  particles  are c h a r a c t e r i z e d by the presence of 16 - 20 p r o t e i n s i n the 20 - 30 range on two-dimensional g e l s , c o n t a i n a s e t of s m a l l RNA  kd  s p e c i e s between  50  3 - 200  n u c l e o t i d e s and have a buoyant d e n s i t y of 1.365  C s C l a f t e r UV 1985).  crosslinking  ( A r r i g o e t a l . , 1985;  - 1.380  Shuldt and  g/cm  Kloetzel,  These p a r t i c l e s are v e r y s i m i l a r to the prosome which has  been  c h a r a c t e r i z e d i n duck e r y t h r o b l a s t s and mouse e r y t h r o p o i e t i c c e l l s e t a l . , 1984). p a r t i c l e s , 12 nm  Under the e l e c t r o n microscope they appear as r i n g i n diameter ( A r r i g o e t a l . , 1985;  in  Shuldt and  (Schmid shaped  Kloetzel,  1985). A 74 n u c l e o t i d e long RNA  a s s o c i a t i n g w i t h the D r o s o p h i l a prosome has  been sequenced ( A r r i g o e t a l . , 1985) U6  s m a l l n u c l e a r RNA  and  shown to be homologous to mammalian  (Ohshima e t a l . , 1981;  Harada e t a l . , 1980;  Epstein  et a l . , 1980). In suspension granules  c u l t u r e s of tomato, the shsps form c y t o p l a s m i c heat  (hsg) which are r e s i s t a n t to RNase, 0.5  M KC1,  EDTA, detergent  shock and  48  sonication  (Nover e t a l . , 1983).  prosomes i n many r e s p e c t s .  These are d i f f e r e n t from D r o s o p h i l a  F i r s t of a l l , c y t o p l a s m i c g r a n u l e s are not seen  i n the cytoplasm of D r o s o p h i l a c e l l s .  I n tomatoes, hsgs undergo  a massive  and r a p i d a c c u m u l a t i o n upon heat shock and d i s a p p e a r s l o w l y d u r i n g recovery.  I n c o n t r a s t , the prosome i s found i n equal amounts i n both  c o n t r o l and heat shocked c e l l s , the amount of a s s o c i a t e d shsps  increasing  s u b s t a n t i a l l y a f t e r heat shock. The f u n c t i o n of hsgs i n p l a n t s and of prosomes i n animal c e l l s  i s not  known.  1.6.2  Thermotolerance: Thermotolerance was  Role of shsp3 f i r s t used to d o a c r i b e the phenomenon i n which  i n i t i a l m i l d , n o n - l e t h a l heat treatment causes c e l l s  an  to be r e s i s t a n t to a  b r i e f s h i f t to h i g h e r temperatures which are n o r m a l l y l e t h a l .  In  o D r o s o p h i l a , a m i l d heat treatment a t 35 C f o r 50 minutes p r o t e c t s a l l developmental stages and t i s s u e c u l t u r e c e l l s for  20 minutes  ( M i t c h e l l e t a l . , 1979).  from c e l l death a t 40.5  In Chinese hamster  o  C  fibroblast  o c e l l s , a s h o r t 46 C treatment f o l l o w e d by a r e c o v e r y p e r i o d of 4 - 6 hours or  treatment a t 41°C f o r s e v e r a l hours p r o t e c t e d the c e l l s  o 45 C f o r 45 minutes  ( L i and Werb, 1982).  from death a t  o In y e a s t , a s h i f t t o 36 C f o r o  90 minutes r e s u l t s i n a t r a n s i e n t p r o t e c t i o n from death a t 52 C and F i n k e l s t e i n , 1980b). demonstrated  (McAlister  I n a l l of the p r e - t r e a t m e n t s d e s c r i b e d , i t was  t h a t hsp s y n t h e s i s o c c u r s .  Furthermore, the p e r s i s t e n c e of  t h e r m o t o l e r a n c e f o r up to 36 hours a f t e r heat shock c o r r e l a t e s w e l l w i t h the p e r s i s t e n c e o f heat shock p r o t e i n s i n hamster  cells  ( L i and Werb, 1982).  49  Many i n d u c e r s of the heat shock response a l s o induce thermotolerance. P r i o r exposure  of hamster c e l l s to a r s e n i t e and e t h a n o l o r a r e l e a s e  a n o x i a r e s u l t s i n an a c q u i r e d t o l e r a n c e to a subsequent  l e t h a l heat  c h a l l e n g e ( L i and Werb, 1982).  such as  I f amino a c i d analogues  canavanine  o r a z e t i d i n e are used to induce hsp s y n t h e s i s i n hamster c e l l s ,  then  t h e r m o t o l e r a n c e i s n o t a c q u i r e d ( L i and L a z l o , 1984), presumably hsps i n c o r p o r a t i n g analogues have a l t e r e d f u n c t i o n . cells,  y e a s t c e l l s to i o n i z i n g r a d i a t i o n resistance  because  Ethanol treated yeast  i n which hsp s y n t h e s i s i s l e s s than i n heat shocked  correspondingly l e s s thermotolerant (Plesset et a l . ,  from  1982).  cells,  are  Exposure  of  (gamma-rays) a l s o induces thermal  ( M i t c h e l and M o r r i s o n , 1982)  although i t i s not known whethor  t h e r e i s any heat shock p r o t e i n s y n t h e s i s under these c o n d i t i o n s . These f i n d i n g s show a s t r o n g c o r r e l a t i o n between the e x p r e s s i o n of the hsps and the development of thermotolerance but they do not prove t h a t events are f u n c t i o n a l l y r e l a t e d .  The requirement f o r heat shock  these  induced  t r a n s c r i p t i o n i n the a c q u i s i t i o n of thermotolerance has been demonstrated t s mutants o f y e a s t i n which a d e f e c t i n RNA  in  transport/processingresults i n  no i n c r e a s e of thermotolerance a f t e r heat shock.  Thermotolerance i s  a c q u i r e d , however, i f the c e l l s are allowed to r e c o v e r f o l l o w i n g a heat shock  ( M c A l i s t e r and F i n k e l s t e i n ,  1980b).  P r o t e i n s y n t h e s i s i n h i b i t o r s t u d i e s have a l s o been used to address question.  this  The e l i m i n a t i o n of thermotolerance w i t h c y c l o h e x i m i d e treatment  p r i o r t o a pre-treatment has been demonstrated  i n y e a s t by s e v e r a l workers  ( M c A l i s t e r and F i n k e l s t e i n , 1980b; M i t c h e l and M o r r i s o n , 1982; Jacobsen, 1984)  but not a l l . ( H a l l , 1983).  Hall  Craig  and  (1983) a l s o showed t h a t  y e a s t c e l l s t r e a t e d w i t h a p h e n y l a l a n i n e analog d i d not become  50  t h e r m o t o l e r a n t i n agreement w i t h experiments ( L i and L a z l o , 1984).  on analog t r e a t e d hamster c e l l s  However, i f these c e l l s were g i v e n a heat  treatment  o at  37 C, a c o n d i t i o n i n which hsps should s t i l l be n o n - f u n c t i o n a l ,  thermotolerance was  acquired.  Furthermore,  whereas c y c l o h e x i m i d e has been  shown to b l o c k the t h e r m o t o l e r a n t s t a t e i n D i c t y o s t e l i u m (Loomis  and  Wheeler, 1982),  treatment of r a t embryonic f i b r o b l a s t (Rat-1) c e l l s w i t h o o c y c l o h e x i m i d e f o r 6 hours a t 37 C a f t e r a 20 minute i n t e r v a l a t 45 C i n h i b i t s p r o t e i n s y n t h e s i s , i n c l u d i n g hsp s y n t h e s i s , but has no e f f e c t  on  o subsequent  s u r v i v a l a t 45 C ( W i d e l i t z e t a l . , 1986).  The reasons f o r  these d i s c r e p a n c i e s are not known. The s t r o n g e s t evidence f o r a f u n c t i o n of hsps found i n the rpoH165 mutant of E. c o l i  These c e l l s a l s o f a i l 42°C pre-treatment  i n which hsps are not s y n t h e s i z e d . o  to a c q u i r e r e s i s t a n c e to a 55 C c h a l l e n g e a f t e r a  (Yamamori and Yura,  1982).  Some o b s e r v a t i o n s suggest t h a t the shsps may thermotolerance. exposure  i n thermotolerance i s  play a role i n  In D r o s o p h i l a c e l l s , t o l e r a n c e i s a c q u i r e d by c o n t i n u e d  t o ecdysterone i n which o n l y the shsps are induced (Berger and  Woodward, 1983).  A l s o , D r o s o p h i l a pupae (a developmental  shsps are s p e c i f i c a l l y  s y n t h e s i z e d ) appear to be more r e s i s t a n t to heat  induced death ( M i t c h e l l e t a l . , 1979; A mutant c e l l  stage i n which the  Berger and Woodward, 1983).  l i n e of D i c t y o s t e l i u m discoideum c a l l e d HL122 has been  i d e n t i f i e d due t o i t s d e f e c t i n a c q u i r i n g thermotolerance (Loomis  and  Wheeler, 1982).  However,  These c e l l s do n o t s y n t h e s i z e any of the shsps.  even though hsp70 i s p r e s e n t i n heat shocked HL122 c e l l s , i t accumulates l e v e l s w e l l below those found i n w i l d type c e l l s and may of  these c e l l s to develop t h e r m o t o l e r a n c e .  to  a f f e c t the a b i l i t y  51  The  a c q u i s i t i o n of thermotolerance i n heat shocked Xenopus l a e v i s  embryos argues a g a i n s t a r o l e f o r hsp30 i n t h i s phenomenon ( H e i k k i l a et a l . , 1985).  Thermotolerance i s developed by the l a t e b l a s t u l a and  s t a g e s i n which the hsp30 genes are not 1984a).  Also,  i n yeast  strains containing  thermotolerance i s , nevertheless, phase c e l l s , (Petko and  1.6.3  (Bienz,  developed i n l o g phase or i n s t a t i o n a r y and  during  spore development  L i n d q u i s t , 1986).  I n h i b i t i o n of the Heat Shock Response  Induction  fibroblasts  of hsp  polyhydroxyl  a l c o h o l s such as g l y c e r o l b l o c k  s y n t h e s i s by heat or a r s e n i t e i n c h i c k embryo  (Hightower et a l . , 1985).  s t a b i l i z e macromolecules and may  to heat shock  an i n a c t i v e hsp26 gene,  i n mature or g e r m i n a t i n g spores,  Both deuterium oxide and the  responsive  early gastrula  Both of these compounds are known to  to p r o t e c t c e l l s from thermal k i l l i n g .  p r o t e c t s t r e s s - s e n s i t i v e p r o t e i n s from i r r e v e r s i b l e  denaturation,  p o s s i b l y by d i r e c t i n t e r a c t i o n but more l i k e l y by g e n e r a l i z e d effects.  I t has  denaturation  They  solvent  been suggested t h a t hsps, which are r e s i s t a n t to  by heat or e t h a n o l ,  can n o n - s p e c i f i c a l l y s t a b i l i z e  other  p r o t e i n s t h a t are h i g h l y s u s c e p t i b l e to i n a c t i v a t i o n (Minton e t al. , 1982). This  i s c o n s i s t e n t w i t h the i d e a t h a t hsps p l a y a r o l e i n p r o t e c t i n g  against  s t r e s s and  i n the a c q u i s i t i o n of thermotolerance.  the model whereby p r o t e i n damage may discussed  above.  cells  I t a l s o supports  t r i g g e r the heat shock response,  as  52  1.7  The B i o l o g y of C a e n o r h a b d i t i s elegans C a e n o r h a b d i t i s e l e g a n s , a member o f the f a m i l y R h a b d i t i d a e , i s a  m i c r o b i v o r o u s , f r e e - l i v i n g s o i l nematode.  A d u l t nematodes a r e found as  males o r s e l f - f e r t i l i z i n g hermaphrodites, the l a t t e r b e i n g s l i g h t l y and r e a c h i n g a l e n g t h of 1 mm.  larger  The l i f e c y c l e of C. elegans i s r a p i d  and  o takes about 3.5  days a t 20 C.  After fertilization,  the eggs b e g i n to  c l e a v e w i t h i n the hermaphrodite and a r e l a i d a t about the 3 0 - c e l l stage (mid-gastrula). embryogenesis,  Each hermaphrodite produces 200 - 300 progeny.  After  a j u v e n i l e c o n t a i n i n g about 550 c e l l s hatches from the egg  case and develops through f o u r l a r v a l stages, L1-L4, b e f o r e r e a c h i n g the a d u l t stage. somatic c e l l  Many c e l l d i v i s i o n s o c c u r d u r i n g the l a r v a l p e r i o d , the number I n c r e a s i n g to about 1000  i n mature a d u l t s .  The  first  l a r v a l stage c o n t a i n s o n l y two germ l i n e c e l l s w h i l e the a d u l t s c o n t a i n - 2000 germ l i n e n u c l e i .  L2 stage l a r v a e can t r a n s f o r m i n t o a r e s i s t a n t  s t a g e known as dauer l a r v a e under adverse environmental c o n d i t i o n s .  In t h i s  s t a t e , the nematode can t o l e r a t e s t a r v a t i o n f o r many months, resuming development  normal  when n u t r i e n t s become a v a i l a b l e .  S i n c e C. elegans i s t r a n s p a r e n t , development be observed w i t h a l i g h t microscope u s i n g Nomarski all  1000  of the l i v i n g organism can optics.  I n t h i s manner,  o f the c e l l d i v i s i o n s , deaths, and m i g r a t i o n s t h a t produce a mature  a d u l t from a s i n g l e egg have been determined ( S u l s t o n e t a l . , achievement  is a first  1983).  f o r an organism of t h i s degree of complexity, and has  been f a c i l i t a t e d a l s o by t h e f a c t t h a t nematode development  i s invariant.  T h i s means t h a t the f a t e and p o s i t i o n of a g i v e n p r e c u r s o r c e l l in a l l individuals.  This  i s the same  The anatomy and w i r i n g of the complete nervous  which i s composed o f o n l y 302 neurons, has a l s o been determined.  system,  53  C. elegans i s i d e a l l y s u i t e d to g e n e t i c  analysis.  mutations have been mapped t o about 500 genes. affect cell  fates during  development,  Thousands  of  Many i n c l u d e genes which  and o t h e r s which a f f e c t neuromuscular  function. C. elegans i s a d i p l o i d organism, c o n t a i n i n g  f i v e autosomal chromosomes  and one sex (x) chromosome.  Hermaphrodites c o n t a i n two sex chromosomes  w h i l e males c a r r y o n l y one.  The h a p l o i d DNA  content i s 8 x 1 0  bp p e r  7  genome ( S u l s t o n and Brenner, 1974), which i s o n l y 20 times t h a t of E. R e p e t i t i v e DNA makes up o n l y 17% of the t o t a l DNA overall  content which has an  low C/C base c o m p o s i t i o n of 367, ( S u l s t o n and Brenner, 1974).  s i m p l i c i t y of the nematode genome haa sparked a c o o p e r a t i v e the o n t i r o C. elegans genome. categorized greater  1.8  Prenontly,  i n t o about 900 segments,  coli.  e f f o r t to  The map  about 65% of the genome has been  many g r e a t e r  than 200 kb and  one  than 600 kb.  The P r e s e n t Study By the end of 1980,  i n Drosophila becoming  the heat shock response had been w e l l  characterized  (see r e v i e w by Ashburner and Bonner, 1979), but i t was  evident  t h a t the phenomenon was  organisms i n c l u d i n g E s c h e r i c h i a c o l i  conserved i n a d i v e r s e group o f  (Lemaux e t a l . , 1978; Yamamori e t a l . ,  1978), p r o t o z o a n s such as N a e g l e r i a  gruberi  pyriformis  Guttman e t a l . , 1980), s l i m e  ( F i n k and Zeuthen, 1980;  including Dictyostelium Polysphondylium pallidum  d i s c o i d e u m (Loomis and Wheeler, 1980) ( F r a n c i s and L i n , 1980), yeast  F i n k e l s t e i n , 1980a; 1980b), sea u r c h i n d i p t e r a n Chironomus  (Walsh, 1980) and  tentans (Vincent  Tetrahymena molds  and  ( M c A l i s t e r and  ( G u i d i c e e t a l . , 1980), another  and Tanguay,  1979), p l a n t s  including  54  tobacco and soybean  (Bamett  (Bouche e t a l . , 1979)  e t a l . , 1980),  Chinese hamster ovary  and c h i c k embryo f i b r o b l a s t s  cells  (see T a b l e I I , s e c t i o n  1.2.2) . The i n d u c i b i l i t y of the heat shock genes to h i g h l e v e l s ,  in a  r e v e r s i b l e f a s h i o n , made them a t t r a c t i v e models f o r the study of gene activation.  The l a t e s e v e n t i e s brought about  intense a c t i v i t y  directed  towards the c l o n i n g and c h a r a c t e r i z a t i o n of the major heat i n d u c i b l e genes of D r o s o p h i l a and a t the end of 1980, sequence of one complete 5' f l a n k i n g I t was  hsp70 gene along w i t h the comparison  DNA  of t h r e e hsp70  sequences. a t t h i s time t h a t the p r e s e n t i n v e s t i g a t i o n was  c h a r a c t e r i z e sequences elegans.  I n g o l i a e t a l . (1980) r e p o r t e d the  undertaken  to  coding f o r the shsps of the nematode C a e n o r h a b d i t i s  U n t i l then t h e r e had been no sequence i n f o r m a t i o n a v a i l a b l e  r e g a r d i n g the shsp genes of any organism  including Drosophila.  In the next  f i v e years i t would become c l e a r t h a t the shsps from a v a r i e t y of systems were r e l a t e d and h i g h l y conserved throughout  evolution.  During the same  p e r i o d , o t h e r workers were f o c u s i n g t h e i r a t t e n t i o n on the hsp70 gene f a m i l y of C a e n o r h a b d i t i s elegans (Snutch and B a i l i e , 1984).  1983;  Snutch and  Baillie,  55  II.  EXPERIMENTAL PROCEDURES  2.1  Maintenance of Nematodes C a e n o r h a b d i t i s elegans s t r a i n s B r i s t o l  m a i n t a i n e d on NG p l a t e s c h o l e s t e r o l , 1.0 mM  (N2) and Bergerac (BO) were  (0.3% NaCl, 0.25% b a c t o t r y p t o n e , 5.0  CaCl  2 >  1.0 mM  MgS0  4  and 25 mM  KH P0 2  c o n t a i n i n g E. c o l i 0P50 as d e s c r i b e d by Brenner (1974).  4  pH  yg/ml 6.0)  0P50 i s a u r a c i l  r e q u i r i n g mutant which p r e v e n t s the overgrowth of the b a c t e r i a l lawn.  The  o Bergerac s t r a i n was m a i n t a i n e d a t 17 C w h i l e the B r i s t o l s t r a i n was at  ambient  room temperature or a t 17°C.  Synchronous nematode p o p u l a t i o n s  were s t a r t e d from i s o l a t e d eggs by d i s s o l v i n g g r a v i d a d u l t s i n 2% h y p o c h l o r i t e , 0.05M NaOH f o r 10 minutes 2.2  kept  (Emmons ot a l . ,  sodium  1979).  A n a l y s i s of Heat Shock P r o t e i n s  35 2.2.1  [  S ] s u l f a t e L a b e l l i n g of E.  E. c o l i K12 was  coli  grown t o s t a t i o n a r y phase i n 100 ml of minimal medium 35  c o n t a i n i n g 5 mCi of [  S ] s u l f a t e (New  England N u c l e a r ) a c c o r d i n g to the  procedure o f B r e t s c h e r and Smith (1972). 2.2.2  In v i v o L a b e l l i n g of C. elegans ( B r i s t o l ) P r o t e i n s and I n d u c t i o n of  Heat Shock P o l y p e p t i d e s In at  a t y p i c a l experiment, a p l a t e of synchronous a d u l t nematodes growing  o room temperature (22 C) was  o t r a n s f e r r e d to a 35 C i n c u b a t o r .  After 1  o hour, the nematodes were washed o f f w i t h 35 C d i s t i l l e d water and a l l o w e d to  s e t t l e by g r a v i t y a t 3 5 ° C .  The nematodes were then washed t w i c e a t  56  35 C and yCi  of  t r a n s f e r r e d to prewarmed NG  35 [ S ] - l a b e l l e d E. c o l i K12  p l a t e s onto which a p p r o x i m a t e l y  p a s t e had  been spread.  Following  25 a  o l a b e l l i n g p e r i o d ranging  from 30 minutes to 5 hours at 35 C,  were washed o f f as b e f o r e  and  b a c t e r i a ( e i t h e r E. c o l i K12 elevated  temperature.  t r a n s f e r r e d to NG  plates containing  unlabelled  or E. c o l i OP50) f o r at l e a s t 30 minutes at  T h i s was  remaining i n the gut.  the nematodes  c a r r i e d out  to remove any  At t h i s time or a f t e r a recovery  labelled bacteria  p e r i o d a t room  temperature, the nematodes were washed thoroughly at room temperature resuspended i n 25 - 50  p i of t h r e e  b u f f e r (Laemmli, 1970).  times concentrated  by b o i l i n g f o r 5 minutes and  SDS-polyacrylamide g e l e l e c t r o p h o r e s i s and  2.2.3  A n a l y s i s of i n v i t r o L a b e l l e d T o t a l RNA,  RNA  was  polyadenylated  RNA  and  Laemmli sample  P r o t e i n s were then s o l u b i l i z e d by  freeze-thaw c y c l e s f o l l o w e d  the  2 - 3 rapid  were analyzed  using  autoradiography.  Proteins  (polyA RNA) or h y b r i d i z a t i o n s e l e c t e d +  t r a n s l a t e d i n a r a b b i t r e t i c u l o c y t e system (New  England N u c l e a r ) as  35 described  (Pelham and  Jackson, 1976)  using  [  S]methionine.  p r o d u c t s were f r a c t i o n a t e d on SDS-polyacrylamide g e l s and autoradiography.  The  p e l l e t was  washed w i t h e t h a n o l ,  slab gels  with, a 4.5%  (0.08  d r i e d and  resuspended i n  ( O ' F a r r e l l , 1975).  P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s of SDS  by  p r e c i p i t a t e d i n 10 volumes of acetone a t -20°C  O ' F a r r e l l loading buffer A  2.2.4  analyzed  For two-dimensional g e l e l e c t r o p h o r e s i s , the 25 y l  t r a n s l a t i o n r e a c t i o n was overnight.  Translation  cm X. 7.5  cm X. 10 cm)  Proteins containing  15%  s t a c k i n g g e l were p r e p a r e d u s i n g the d i s c o n t i n u o u s  polyacrylamide b u f f e r system  57  of Laemmli (1970).  Two-dimensional  polyacrylamide g e l electrophoresis  c a r r i e d out as d e s c r i b e d by O ' F a r r e l l  (1975).  A f t e r destaining, the gels  were d r i e d and a u t o r a d i o g r a p h y was c a r r i e d out u s i n g Kodak X-Omat AR  2.3  RNA  2.3.1  was  film.  Analysis  I s o l a t i o n of RNA from B r i s t o l N2 Nematodes C o n t r o l nematodes from which RNA was to be i s o l a t e d were washed o f f of  NG p l a t e s w i t h s t e r i l e 0.14 M NaCl a t 4°C.  The nematodes were then washed  twice w i t h c o l d 0.14 M NaCl a f t e r a l l o w i n g them to s e t t l e on i c e by g r a v i t y and removing  the supernatant by a s p i r a t i o n .  F i n a l l y they were c e n t r i f u g e d  o o a t 12,000 g f o r 5 minutes a t 4 C and the p e l l e t was s t o r e d a t -70 C. Nematodes were heat shocked by i n c u b a t i n g NG p l a t e s a t 35°C f o r hours b e f o r e c a r r y i n g out the washing  2-4  procedure d e s c r i b e d above.  One  hundred p l a t e s t y p i c a l l y y i e l d e d 1.5 - 2.0 gram of nematodes. The RNA  i s o l a t i o n procedure was based on t h a t of Chirgwin e t a l . (1979)  w i t h the f o l l o w i n g m o d i f i c a t i o n s .  Nematodes were passed t w i c e through a  p r e - c o o l e d F r e n c h p r e s s a t 8000 p s i i n t e n volumes of 6 M guanidinium h y d r o c h l o r i d e (Gu-HCl), 0.2 M sodium a c e t a t e pH 5.0, 0.1 M o a t 4 C.  A f t e r c e n t r i f u g a t i o n a t 12,000 g f o r 10 minutes  6-mercaptoethanol  o a t 4 C, 0.5  volume of 95% e t h a n o l was added to the supernatant and the RNA p r e c i p i t a t e d o v e r n i g h t a t -20°C. as d e s c r i b e d  (Chirgwin e t a l . ,  starting material.  was  The subsequent p u r i f i c a t i o n s t e p s were  1979).  T y p i c a l y i e l d s were 10 mg p e r gram of  C o n t r o l and heat shock polyA RNA were p r e p a r e d from +  t o t a l RNA by two passages through an o l i g o - d T c e l l u l o s e column ( C o l l a b o r a t i v e Research I n c . , Type 2) u s i n g the procedure of A v i v and Leder (1972) except t h a t the f i n a l polyA RNA f r a c t i o n s were e l u t e d w i t h +  58  s t e r i l e d i s t i l l e d water. diethylpyrocarbonate  A l l glassware and s o l u t i o n s were t r e a t e d w i t h 0.1%  (DEP) and baked o r autoclaved,  respectively,  before  use.  2.3.2  E l e c t r o p h o r e s i s o f RNA and Northern  Transfers  RNA was denatured w i t h g l y o x a l a c c o r d i n g and  t o t h e procedure o f McMaster  Carmichael (1977) except t h a t d i m e t h y l s u l f o x i d e  was o m i t t e d .  After  f r a c t i o n a t i o n on agarose g e l s i n 10 mM NaH^PO^ pH 7.0 w i t h b u f f e r recirculation,  the RNA was t r a n s f e r r e d d i r e c t l y t o n i t r o c e l l u l o s e  ( S c h l e i c h e r and S c h u e l l )  i n 20 X SSPE (Thomas, 1980).  EDTA, 10 mM Na^PO^, pH 7.0 and 0.18 M NaCl.  A l t e r n a t i v e l y , RNA was  denatured w i t h formaldehyde and e l e c t r o p h o r e s e d gels  (Maniatis  e t a l . , 1982).  e l e c t r o p h o r e s i s as d e s c r i b e d  1 X SSPE i s 0.1 mM  through formaldehyde agarose  Formaldehyde g e l s were p r o c e s s e d a f t e r ( M a n i a t i s e t a l . , 1982) and the RNA was  t r a n s f e r r e d t o n i t r o c e l l u l o s e as above.  A f t e r t r a n s f e r , the f i l t e r s were  o a i r d r i e d and baked a t 80 C f o r 2 hours.  2.3.3  SI Nuclease Mapping SI n u c l e a s e p r o t e c t i o n a n a l y s i s was c a r r i e d out e s s e n t i a l l y as  described  by Berk and Sharp (1977).  F o r mapping the hsp!6-48 mRNA, 10 t o 20  ng o f a TagI-RsaI fragment (map c o o r d i n a t e s with polynucleotide  kinase  +44 t o -191), 5'-end l a b e l l e d  32 and [y- P]ATP a t the TagI s i t e , was  +• o h y b r i d i z e d w i t h 200 ng o f heat shock polyA RNA f o r 20 hours a t 47 C m 10 y l o f h y b r i d i z a t i o n b u f f e r mM EDTA). to -85),  (50% formamide, 0.4 M NaCl, 40 mM PIPES, 1.0  F o r the h s p ! 6 - l mRNA, 10 t o 20 ng o f an A l u l - X b a l fragment (+28 5'-end l a b e l l e d as above a t the A l u l end, was h y b r i d i z e d t o 500 ng  59  of  the same RNA f o r 12 hours a t 50 C i n 10 y l o f h y b r i d i z a t i o n b u f f e r  without formamide.  I n each case, h y b r i d i z a t i o n was c a r r i e d out by f i r s t  o h e a t i n g t h e m i x t u r e a t 85 C f o r 15 min and then q u i c k l y submerging t h e tube i n a water b a t h a t the a p p r o p r i a t e temperature.  H y b r i d i z a t i o n was  terminated by the a d d i t i o n o f 300 p i of i c e - c o l d SI n u c l e a s e mixture c o n t a i n i n g 0.28 M NaCl, 50 mM sodium a c e t a t e pH 4.6, 4.5 mM Z n S O  l(  and 200  4 U o f SI n u c l e a s e (Boehringer-Mannheim).  SI n u c l e a s e d i g e s t i o n s were c a r r i e d  o out  a t 37 C f o r 30 min and t e r m i n a t e d w i t h 50 y l o f 4.0 M ammonium  acetate-100 mM EDTA.  The p r o t e c t e d fragments were p r e c i p i t a t e d w i t h  i s o p r o p a n o l w i t h 20 pg o f E. c o l i tRNA as c a r r i e r and a n a l y z e d on 8% a c r y l a m i d e g e l s c o n t a i n i n g 8 M u r e a (see DNA Sequencing). 2.4  I d e n t i f i c a t i o n o f cDNAs  2.4.1  S c r e e n i n g o f a cDNA L i b r a r y A cDNA l i b r a r y made w i t h polyA mRNA p u r i f i e d from heat +  B r i s t o l nematodes was k i n d l y p r o v i d e d by Don Jones.  shocked  The cDNA had been  i n s e r t e d i n t o t h e P s t I s i t e o f pBR322 u s i n g G-C t a i l i n g .  Annealed DNA was  used t o t r a n s f o r m E. c o l i RR1 t o c r e a t e the l i b r a r y which was used i n t h i s study.  The cDNA l i b r a r y was screened by. the c o l o n y h y b r i d i z a t i o n method o f 125  G r u n s t e i n and Hogness (1975), u s i n g as a probe mRNA which had been s i z e f r a c t i o n a t e d  [  I ] - l a b e l l e d heat  shock  (see s e c t i o n 2.10.1 below).  A m p i c i l l i n s e n s i t i v e , t e t r a c y c l i n e r e s i s t a n t c o l o n i e s were i n d i v i d u a l l y t r a n s f e r r e d from a master p l a t e t o a n i t r o c e l l u l o s e f i l t e r o  ( S c h l e i c h e r and  S c h u e l l ) and a l l o w e d t o grow o v e r n i g h t a t 37 C on LB (1.0% b a c t o t r y p t o n e , 0.5%  y e a s t e x t r a c t , 1.0% NaCl pH 7.5) p l a t e s c o n t a i n i n g  tetracycline  60  (15  yg/ml).  containing  I n some cases, t h e f i l t e r was t r a n s f e r r e d 170 yg/ml c h l o r a m p h e n i c o l a f t e r the c o l o n i e s  diameter o f a p p r o x i m a t e l y 1.0 mm for  12 hours a t 37°C.  This  s i g n i f i c a n t l y increase Cell  had grown t o a  and the plasmids were allowed to a m p l i f y  a l t e r n a t e procedure, however, d i d n o t  the h y b r i d i z a t i o n  s i g n a l of p o s i t i v e recombinants.  l y s i s was c a r r i e d out w i t h SDS and NaOH as d e s c r i b e d  (1982).  by M a n i a t i s e t a l .  The f i l t e r s were a i r d r i e d and baked a t 80°C f o r 2 hours p r i o r t o  hybridization.  Putative  p o s i t i v e s were p u r i f i e d from the master p l a t e and  the plasmids were analyzed  2.4.2  to an LB p l a t e  Hybridization  further.  Selection Analysis  of cDNAa  10 yg of plasmid DNA was l i n e a r i z e d with BamHI, p h e n o l - e x t r a c t e d and p r e c i p i t a t e d w i t h 2 volumes of 95% e t h a n o l . nitro-cellulose filters sintered  glass  Hybridization,  (Schleicher  The DNA was then a p p l i e d to  and S c h u e l l , B-6) u s i n g a M i l l i p o r e  f i l t r a t i o n u n i t as d e s c r i b e d  by Young e t a l . (1980).  washing and e l u t i o n were c a r r i e d out u s i n g the p r o t o c o l of  Tilghman e t a l . (1978).  T y p i c a l l y , the f i l t e r s c a r r y i n g plasmid DNA were  i n c u b a t e d w i t h 10 yg o f heat shock polyA RNA i n 400 y l of +  o hybridization buffer  f o r 20 hours a t 43 C.  N o n - h y b r i d i z e d RNA i n the  h y b r i d i z a t i o n b u f f e r was p r e c i p i t a t e d w i t h 2 volumes of 95% e t h a n o l and 1 yg was used f o r i n v i v o t r a n s l a t i o n .  H y b r i d i z e d RNA was e l u t e d o  i n 90%  formamide, 10 mM T r i s - H C l pH 7.5 and 1.0 mM EDTA a t 45 C f o r 1 hour, p r e c i p i t a t e d a f t e r the a d d i t i o n translated.  of 10 yg of c a r r i e r E. c o l i tRNA, and  61  2.5  General Methods f o r Plasmid A n a l y s i s  2.5.1  Bacterial E. c o l i RR1  recombinants  Strains was  used to propagate pBR322 and  i n c l u d i n g the cDNA l i b r a r y and was  s t r a i n s JM101  pBR325-derived  grown i n LB medium.  and JM103 were used f o r t r a n s f o r m a t i o n w i t h M13  E.  and  pUC-derived plasmids and were grown i n YT medium (0.8% b a c t o t r y p t o n e , y e a s t e x t r a c t , 0.5% NaCl pH  2.5.2  Transformations  C a C l ^ as d e s c r i b e d by Messing  (1983).  w i t h e i t h e r p u r i f i e d p l a s m i d DNA,  l i g a t i o n mixes or s i n g l e - s t r a n d e d M13  i n 20 y l of 50 mM  d i t h i o t h r e i t o l , 1.0 mM  serum albumin. u s i n g 1.0 DNA  mM  Competent c e l l s were transformed  L i g a t i o n s were u s u a l l y c a r r i e d out w i t h 10 ng of v e c t o r DNA  60 ng o f i n s e r t DNA mM  0.5%  7.0).  B a c t e r i a l c e l l s were made competent f o r t r a n s f o r m a t i o n u s i n g 50  DNA.  coli  T r i s - H C l pH 7.4,  spermidine, 1.0 mM  ATP  10 mM  MgCl  and 20 2 >  and 100 yg/ml bovine  L i g a t i o n s were done a t 4°C or 15°C f o r a t l e a s t 12  - 10 Weiss u n i t s of T4 DNA  10  hours  ligase.  i n s e r t e d i n t o the P s t I s i t e of pBR322 rendered the transformed  c o l o n i e s t e t r a c y c l i n e - r e s i s t a n t and a m p i c i l l i n - s e n s i t i v e .  pBR325  d e r i v a t i v e s c o n t a i n i n g i n s e r t s i n the EcoRI s i t e were screened by r e s i s t a n c e to a m p i c i l l i n and t h e i r s e n s i t i v i t y t o chloramphenicol.  their The  c o n c e n t r a t i o n s of a n t i b i o t i c s used are taken from M a n i a t i s e t a l . (1982). Plaques  i n f e c t e d w i t h recombinant  M13  phage were assayed f o r t h e i r  inability  to c l e a v e 5 - b r o m o 4 - c h l o r o 3 - i n d o l y l g a l a c t o s i d e (X-gal) as d e s c r i b e d by Messing  (1983).  c o n t a i n i n g pUC  The same c o l o r assay was plasmids.  used to screen b a c t e r i a l , c o l o n i e s  62  2.5.3  P u r i f i c a t i o n of Plasmid  DNA  F o r the l a r g e - s c a l e i s o l a t i o n of p l a s m i d DNA, amplified  500 ml c u l t u r e s were  i n the a p p r o p r i a t e medium w i t h chloramphenicol a c c o r d i n g to  M a n i a t i s e t a l . (1982).  pBR325 and  i t s d e r i v a t i v e s were a m p l i f i e d  s p e c t i n o m y c i n a t a c o n c e n t a t i o n of 300  yg/ml.  i s o l a t i o n of M13  5.0 ml of an e x p o n e n t i a l l y growing  JM101  r e p l i c a t i v e form DNA,  o r JM103 c u l t u r e and 100  supernatant  (see sequencing  - 200  For the  with  y l of a M13  large-scale  i n f e c t i o u s phage  s e c t i o n ) were added t o 500 ml of YT  and  o i n c u b a t e d a t 37 C f o r 5.-7  hours.  Plasmids were p u r i f i e d u s i n g the a l k a l i Doly was  (1979) as d e s c r i b e d by M a n i a t i s e t a l . (1982). dissolved  i n 8 ml of TE and was  phenol/chloroform WBB to  l y s i s procedure  cesium  The f i n a l DNA  e x t r a c t e d once w i t h an equal volume of  c o n c e n t r a t i o n of 25 yg/ml and d i g e s t i o n was  a t room temperature  f o r one hour.  The DNA  was  r o t o r a t 60,000 rpm  c o l l e c t i n g the p l a s m i d DNA  banded i n two  (12 - 16 hours, 20 C).  band from the g r a d i e n t , the e t h i d i u m bromide The  P r e c i p i t a t i o n was  allowed t o proceed  at -20  was  final  C  o v e r n i g h t and the DNA  was  minutes.  washed w i t h 95% e t h a n o l , d r i e d and resuspended  Y i e l d s ranged  1982)  d i l u t e d 3 - f o l d w i t h water p r i o r t o the a d d i t i o n of 2 o  volumes of e t h a n o l .  The DNA  4 ml  After  removed by s e v e r a l e x t r a c t i o n s w i t h w a t e r - s a t u r a t e d i - b u t a n o l . aqueous phase was  RNaseA  allowed  c h l o r i d e - e t h i d i u m bromide d e n s i t y g r a d i e n t s ( M a n i a t i s e t a l . , o  i n a Beckman VTi65  and  pellet  (1:1) and once w i t h the same volume of c h l o r o f o r m .  added to a f i n a l proceed  of B i r n b o i m  was  from 100  n a t u r e of the p l a s m i d .  p e l l e t e d by c e n t r i f u g a t i o n a t 12,000 g f o r 20  yg to 1.0  mg  per 500 ml c u l t u r e depending on  i n TE. the  63  Rapid  s m a l l - s c a l e plasmid  i s o l a t i o n s were c a r r i e d out on 1.5 ml of  o v e r n i g h t c u l t u r e s as d e s c r i b e d by M a n i a t i s e t a l . (1982) u s i n g the a l k a l i l y s i s procedure o f B i m b o i m and Doly purified  2.6  Bacteriophage  S c r e e n i n g o f C. elegans Two d i f f e r e n t B r i s t o l  study.  M13 r e p l i c a t i v e forms were  i n t h e same manner from c u l t u r e s i n f e c t e d w i t h M13 phage.  A n a l y s i s of  2.6.1  (1979).  B r i s t o l Genomic DNA L i b r a r i e s  (N2) genomic DNA l i b r a r i e s were used i n t h i s  One was a p a r t i a l EcoRI d i g e s t i n the lambda Charon4 v e c t o r .  I t was  c o n s t r u c t e d and k i n d l y p r o v i d e d by T e r r y Snutch o f Simon F r a s e r U n i v e r s i t y . T h i s phage was propagated i n E. c o l i DPSOsupF or LE392 hosts which were grown i n NZYT (1.0% NZ-amine, 0.5% yeast e x t r a c t , 0.2% casamino a c i d s , 10 mM MgCl^ and 40 v»g/ml thymidine  pH 7.0).  required i n a d d i t i o n , diaminopimelic yg/ml.  B a c t e r i a l s t r a i n DP50supF a c i d a t a c o n c e n t r a t i o n o f 0.1  The o t h e r l i b r a r y was a p a r t i a l Mbol d i g e s t cloned i n t o t h e BaraHI  s i t e o f t h e lambda d e r i v a t i v e EMBL4.  T h i s l i b r a r y was c o n s t r u c t e d by C h r i s  L i n k and k i n d l y p r o v i d e d by Mike Krause both a t the U n i v e r s i t y o f Colorado, Boulder.  T h i s l i b r a r y was used i n c o n j u n c t i o n w i t h E. c o l i Q358 o r Q359  s t r a i n s i n NZYC media (1.0% NZ-amine, 0.1% yeast e x t r a c t , 0.5% NaCl, 0.1% casamino a c i d s and 10 mM M g C l  2  pH 7.0).  S i n c e t h i s l i b r a r y had a low  p e r c e n t a g e o f w i l d type phage, t h e Q358 h o s t , i n which w i l d type phage a r e v i a b l e , was p r e f e r r e d s i n c e i t gave l a r g e r plaques hybridization  and s t r o n g e r  signals.  I n i t i a l s c r e e n i n g was c a r r i e d out on 50,000 - 100,000 plaques, r e p r e s e n t i n g approximately conservative estimate  5-10  genome e q u i v a l e n t s based on the  t h a t each recombinant clone contained 10 kb o f  64  C. elegans DNA.  Phage were p l a t e d a t a d e n s i t y of approximately 1,000  2,000 plaques per 10 cm p e t r i p l a t e . of 200 - 300 7.5,  0.01  A l i q u o t s of b a c t e r i o p h a g e i n a volume  y l of \ d i l u t i o n b u f f e r (0.1 M NaCl, 0.01  M MgCl^ and 0.02%  -  M Tris-HCl  pH  g e l a t i n ) were mixed w i t h an equal volume of an o  o v e r n i g h t c u l t u r e of the a p p r o p r i a t e b a c t e r i a and  incubated at 37 C f o r 20  minutes b e f o r e p l a t i n g i n the a p p r o p r i a t e medium c o n t a i n i n g 0.7%  agarose.  Both phage l i b r a r i e s were screened as d e s c r i b e d by M a n i a t i s e t a l . (1982), based  on the procedure of Benton and Davis  (1977).  Bacteriophage  agarose p l u g s c o n t a i n i n g phage c l o n e s were maintained  and  in \ dilution  buffer  o at 4 C i n the presence of c h l o r o f o r m . p u r i f i e d by subsequent  Recombinant c l o n e s of i n t e r e s t were  rounds of r e p l a t i n g and  d e n s i t i e s of phage u n t i l an i s o l a t e was 2.6.2  rescreening at decreasing  completely homogeneous.  E s t a b l i s h i n g High T i t e r Phage Stocks P u r i f i e d phage were p l a t e d as d e s c r i b e d above, a t a d e n s i t y of  a p p r o x i m a t e l y 10,000 plaques p e r p l a t e . media was  In t h i s case, however, 3 ml of  added to the phage-host mixture b e f o r e the a d d i t i o n of 3 ml of top  agarose p r i o r to p l a t i n g .  T h i s r e s u l t e d i n a more f l u i d  top l a y e r which  c o u l d be r e a d i l y scraped o f f a f t e r the plaques had grown a t 37°C. p l a t e s were r i n s e d w i t h an a d d i t i o n a l 2 ml of media. was  The  The phage suspension  c e n t r i f u g e d a t 12,000 g f o r 5 minutes to remove the agarose.  The  o supernatant chloroform.  (approximately 3 ml) was T h i s procedure  phage c o n c e n t r a t i o n .  s t o r e d a t 4 C i n the presence of  consistently resulted i n a 10-fold increase i n  65  2.6.3  I s o l a t i o n of Bacteriophage Phage DNA  was  DNA  r o u t i n e l y p u r i f i e d from 20 ml c u l t u r e s .  i n f e c t i o n s were o b t a i n e d w i t h the f o l l o w i n g c o n d i t i o n s . d i l u t i o n b u f f e r c o n t a i n i n g 0.5 for  20 minutes w i t h 100  LE392 o r Q358 or 200  - 1.0  x 10  6  phage was  Consistent  200  lytic  y l of \  o i n c u b a t e d a t 37 C  y l of a s t a t i o n a r y phase ( o v e r n i g h t ) c u l t u r e of  y l of DP50supF.  T h i s mixture was  the a p p r o p r i a t e growth medium i n a 125 ml Erlenmeyer  added t o 20 ml of  f l a s k and  incubated at  o 37 C w i t h v i g o r o u s shaking.  L y s i s u s u a l l y occurred within 5 - 7  which time a few ml of c h l o r o f o r m was shaking a f u r t h e r 5 - 1 0 glass  minutes.  The  hours,  added to the c u l t u r e which was  left  contents were t r a n s f e r r e d to a 30  (Corex) tube, being c a r e f u l to l e a v e most of the c h l o r o f o r m  and  the sample was  c e n t r i f u g e d a t 12,000 g f o r 10 minutes.  was  t r a n s f e r r e d to a c l e a n tube and c e n t r i f u g a t i o n was  remove a l l the b a c t e r i a l d e b r i s .  at  The  ml  behind,  supernatant  repeated i n o r d e r to  To the supernatant were then added 3 ml of  5.0  M NaCl and 3 g of p o l y e t h y l e n e g l y c o l (molecular weight 8,000 - 15,000). o The contents were mixed and l e f t a t 4 C f o r a t l e a s t 2 hours to  p r e c i p i t a t e the phage p a r t i c l e s . minutes,  the phage p e l l e t was  Hepes pH  7.5,  5.0  mM  MgCl  2  A f t e r c e n t r i f u g a t i o n a t 12,000 g f o r 10  resuspended  and 0.5  mM  i n 500  CaCl ) 2  y l of DNase b u f f e r (50  to which was  mM  added 10 y l  RNaseA (5 mg/ml) and 5 y l DNasel (1 mg/ml; Boehringer Mannheim, Grade I ) . RNaseA (Boehringer Mannheim; a n a l y t i c a l grade) was  i n a c t i v a t e contaminating minutes, was  DNase a c t i v i t y .  50 y l of 10 x SET  b o i l e d f o r 5 minutes to o  A f t e r i n c u b a t i o n a t 37 C f o r 60  (0.1 M T r i s - H C l pH  7.5,  0.2  M EDTA and 5%  SDS)  added b e f o r e d i g e s t i o n w i t h 8 y l of p r o t e i n a s e K (25 mg/ml; B o e h r i n g e r o  Mannheim) f o r 60 minutes a t 68 C. e q u a l volume of p h e n o l / c h l o r o f o r m  T h i s mixture was  e x t r a c t e d once w i t h  (1:1) and once w i t h the same  an  66  volume of c h l o r o f o r m . g f o r 3 minutes.  The phases were s e p a r a t e d by c e n t r i f u g a t i o n a t 15,000  The DNA  was  p r e c i p i t a t e d from the aqueous phase w i t h 2  volumes of 95% e t h a n o l a t room temperature f o r 2 minutes, and c o l l e c t e d c e n t r i f u g a t i o n a t 15,000 g f o r 5 minutes. 1.0 ml of 70% e t h a n o l and r e c e n t r i f u g e d . resuspended i n 50 y l of TE (10 mM T y p i c a l l y , 2 - 5 yg of phage DNA  The DNA  p e l l e t was washed w i t h  The f i n a l p e l l e t was  T r i s - H C l pH 7.5, was  obtained.  10 mM  RNA  2.7  above f o r RNA  c o n t a m i n a t i o n was subsequent  DNA  (N2) and Bergerac (BO) nematodes were c o l l e c t e d as d e s c r i b e d isolation.  gram of nematodes. K buffer  and  (see below).  P u r i f i c a t i o n of C. elegans Genomic Bristol  dried  EDTA).  u s u a l l y h i g h , but c o u l d be removed by RNaseA d i g e s t i o n d u r i n g r e s t r i c t i o n endonuclease r e a c t i o n s  by  DNA  p r e p a r a t i o n s were t y p i c a l l y done on 0.5  Nematode p e l l e t s were resuspended  (0.1 M T r i s - H C l pH 8.5,  0.05  d e s c r i b e d by Emmons e t a l . (1979).  M EDTA, 0.2  c o n c e n t r a t i o n of 200 yg/ml and the s o l u t i o n was - 60 minutes a t which time the s o l u t i o n was  1.0  i n 10 ml of p r o t e i n a s e  M NaCl and 1% SDS)  P r o t e i n a s e K was  -  added  as  to a f i n a l  i n c u b a t e d a t 65°C f o r 30  clear.  This s o l u t i o n  was  e x t r a c t e d t h r e e times w i t h phenol and once w i t h c h l o r o f o r m i n a s e p a r a t o r y funnel with gentle mixing. centrifuge at f u l l  Phase s e p a r a t i o n was  speed f o r 3 minutes.  c a r r i e d out i n a desk top  The aqueous phase was  chilled  and 2  o volumes o f 957o e t h a n o l a t -20 C was  g e n t l y l a y e r e d over i t .  The DNA  was  p r e c i p i t a t e d e i t h e r by winding i t upon a g l a s s rod o r by r o t a t i n g the tube a t an angle to d i s t u r b the i n t e r f a c e .  The DNA  was  then washed w i t h c h i l l e d  70% e t h a n o l , d r i e d and resuspended i n an a p p r o p r i a t e volume of TE (1.0 ml).  This usually resulted  i n a DNA  s o l u t i o n of approximately 1.0 mg/ml.  2.0  67  RNA  was  d i g e s t e d w i t h RNaseA i n subsequent r e s t r i c t i o n enzyme r e a c t i o n s  below).  Banding of the DNA  i n C s C l d e n s i t y g r a d i e n t s was  found to  (see  be  unnecessary.  2.8  G e n e r a l DNA  2.8.1  Techniques  R e s t r i c t i o n Endonuclease D i g e s t i o n of DNA  using  (0.5  - 2.0  yg) was  DNA  usually digested  i n a t o t a l volume of 15 y l  the b u f f e r system d e s c r i b e d by M a n i a t i s  albumin ( u l t r a p u r e grade) was yg/ml.  In most cases,  each r e a c t i o n .  and  added to a f i n a l c o n c e n t r a t i o n  - 5.0  England B i o l a b s , Boehringer Mannheim and  C. elegans genomic DNA,  5.0  buffer  (0.25% bromophenol b l u e , 0.25%  2.8.2  E l e c t r o p h o r e s i s of DNA  used f o r  included.  volume of  25%  borate  and  2.0  mM  loading  ficoll).  and. Southern T r a n s f e r s  samples or r e s t r i c t i o n endonuclease r e a c t i o n mixtures were  89 mM  For  by e l e c t r o p h o r e s i s i n  x y l e n e c y a n o l and  on agarose g e l s which were poured and  DNA  100  Pharmacia.  yg of RNaseA was  agarose g e l s as d e s c r i b e d below a f t e r the a d d i t i o n of 0.1  8.3,  of  u n i t s of r e s t r i c t i o n enzyme was  R e s t r i c t i o n enzyme d i g e s t i o n mixtures were analyzed  DNA  Bovine serum  R e s t r i c t i o n enzymes were purchased from Bethesda Research  L a b o r a t o r i e s , New phage DNA  1.0  et a l . (1982).  run i n 1 x TBE  EDTA) c o n t a i n i n g 0.5  (89 mM  analyzed  Tris-borate  yg/ml ethidium  pH  bromide.  bands were v i s u a l i z e d under u l t r a v i o l e t l i g h t and photographs were taken  w i t h a P o l a r o i d camera u s i n g type 57  film.  The  DNA  was  t r a n s f e r r e d to  n i t r o c e l l u l o s e as d e s c r i b e d by Southern (1975) except t h a t the a c i d depurination  step was  u s u a l l y omitted.  T r a n s f e r was  c a r r i e d out i n e i t h e r  68  20 x SSPE o r i n 1.0 M ammonium a c e t a t e pH 7.0, the l a t t e r being efficient.  2.8.3  o The f i l t e r s were then a i r d r i e d and baked a t 80 C f o r 2 hours.  P u r i f i c a t i o n of S p e c i f i c DNA DNA  fragments were r e c o v e r e d  d i a l y s i s t u b i n g u s i n g 0.5 x TBE.  (BRL), o r DE-52 columns. c o n t a i n i n g 0.2 M NaCl.  Fragments  from agarose g e l s by e l e c t r o e l u t i o n  into  The DNA was then p u r i f i e d by  chromatography through RPC-5 analog  M NaCl.  more  (BRL) columns, NAGS PREPAC c a r t r i d g e s  In a l l cases,  the DNA was loaded and washed i n TE  The DNA was e l u t e d i n 300 y l of TE c o n t a i n i n g 2.0  A f t e r the a d d i t i o n of 0.1 volumes of 3.0 M sodium a c e t a t e pH 5.2,  the fragment was p r e c i p i t a t e d w i t h 2.0 volumes of 95% ethanol and resuspended i n a s m a l l volume of s t e r i l e d i s t i l l e d  2.8.4  E n d - L a b e l l i n g of DNA  water.  Fragments  Fragment ends c o n t a i n i n g 5' overhangs generated d i g e s t i o n s were l a b e l l e d i n one of two ways.  by r e s t r i c t i o n  enzyme  The 3' ends were e n d - l a b e l l e d  u s i n g the Klenow fragment of E. c o l i DNA polymerase I and the a p p r o p r i a t e 32 [a-  Pldeoxynucleoside  t r i p h o s p h a t e s , w h i l e the 5' ends were 32  e n d - l a b e l l e d w i t h T4 p o l y n u c l e o t i d e k i n a s e and [ytriphosphate  (ATP) a f t e r d e p h o s p h o r y l a t i o n  phosphatase (grade I ) . M a n i a t i s e t a l . (1982).  P]adenosine  with c a l f i n t e s t i n a l  These r e a c t i o n s were c a r r i e d out a c c o r d i n g to A l t e r n a t i v e l y , the 3' ends of 3' overhangs were 32  e n d - l a b e l l e d w i t h c a l f thymus t e r m i n a l t r a n s f e r a s e and [ a 5'-triphosphate  alkaline  P]cordycepm  as d e s c r i b e d by Tu and Cohen (1980).  S i n g l e - e n d - l a b e l l e d fragments were o b t a i n e d by cleavage w i t h a second r e s t r i c t i o n enzyme and were p u r i f i e d on 5% o r 8% p r e p a r a t i v e  polyacrylamide  69  s l a b g e l s (0.15 x 15 x 17 cm) c o n t a i n i n g a c r y l a m i d e : b i s a c r y l a m i d e a t a r a t i o of 29:1, 1 x TBE, 0.06% ammonium p e r s u l p h a t e and 0.03% TEMED (N.N.N*,N',t e t r a m e t h y l e t h y l e n e diamine). fragment o f i n t e r e s t .  Autoradiography  was used t o l o c a t e t h e  A f t e r e l e c t r o e l u t i o n i n t o d i a l y s i s t u b i n g as  d e s c r i b e d above, t h e fragment was p r e c i p i t a t e d w i t h 0.1 volumes o f 3.0 M sodium a c e t a t e pH 5.2 and e i t h e r 2 volumes o f 95% e t h a n o l o r 1 volume o f isopropanol.  I n a d d i t i o n , e i t h e r 20 yg o f E, c o l i tRNA o r 5 yg of  pBR322 was used as c a r r i e r d u r i n g t h e p r e c i p i t a t i o n  2.9  step.  DNA Sequencing The  cDNAs were sequenced u s i n g the base m o d i f i c a t i o n procedure  and G i l b e r t  (1980).  of Maxam  Maxam and G i l b e r t r e a c t i o n s were a l s o used t o determine  mRNA i n i t i a t i o n s i t e s  i n SI n u c l e a s e p r o t e c t i o n experiments.  of t h e genomic DNA c l o n e s was determined  The sequence  u s i n g the d i d e o x y n u c l e o t i d e  t e r m i n a t i o n technique o f Sanger e t a l . (1977).  chain  A d e t a i l e d d e s c r i p t i o n of  methods used, i n c l u d i n g t h e s u b c l o n i n g o f DNA fragments i n t o M13 v e c t o r s , the p r e p a r a t i o n o f s i n g l e stranded phage DNA and the d i d e o x y n u c l e o t i d e r e a c t i o n s u s i n g t h e Klenow fragment o f E. c o l i DNA polymerase I i s g i v e n i n Messing (1983).  Fragments were cloned i n t o M13mp8, M13mp9 o r M13mpll by  u s i n g e i t h e r cohesive-end  or blunt-end  c o h e s i v e ends had been f i l l e d (Klenow fragment).  l i g a t i o n s , t h e l a t t e r a f t e r the  i n w i t h E s c h e r i c h i a c o l i DNA polymerase I  I n most c a s e s , M13 c l o n e s were f i r s t  ddTTP r e a c t i o n s i n o r d e r t o a v o i d sequencing  screened u s i n g o n l y  redundant c l o n e s .  Fragments  c l o n e d i n t o t h e RF i n t h e o p p o s i t e o r i e n t a t i o n were sometimes screened by t h e i r a b i l i t y t o form a. f i g u r e e i g h t - l i k e s t r u c t u r e which migrates agarose  g e l s (Messing,  1983).  A l l sequencing  slower i n  r e a c t i o n s were analyzed on 6%.  70  polyacrylamide  s l a b g e l s (0.035 x 15 x 35 cm).  The g e l s c o n t a i n e d an  a c r y l a m i d e : b i s a c r y l a m i d e r a t i o o f 19:1 i n a d d i t i o n to 8.3 M u r e a , 1 x TBE, 0.06% ammonium p e r s u l p h a t e and 0.03% TEMED.  E l e c t r o p h o r e s i s was i n 1 x TBE  a t a c o n s t a n t c u r r e n t o f 17.5 m i l l i a m p e r e s .  The g e l s were d r i e d onto  Whatman f i l t e r paper and autoradiographed  u s i n g Kodak X-Omat RP  film.  2.10 P r e p a r a t i o n o f H y b r i d i z a t i o n Probes 125 2.10.1  Preparation of [  I]-labelled  RNA  F o r t y yg of heat shock polyA RNA was f r a c t i o n a t e d on a d e n a t u r i n g +  98%  formamide-polyacrylamide  M a n i a t i s e t a l . (1975). electroeluted  s l a b g e l (0.08 x 7.5 x 10 cm) as d e s c r i b e d by  The g e l was c u t i n t o 1.0 cm s l i c e s and the RNA  i n 20 mM T r i s - a c e t a t e pH 8.0, 0.4 mM  each such f r a c t i o n was assayed  EDTA.  was  Ten p e r c e n t of  i n a c e l l - f r e e t r a n s l a t i o n system a f t e r  c o n c e n t r a t i o n by e t h a n o l p r e c i p i t a t i o n .  The RNA f r a c t i o n which d i r e c t e d the 125  t r a n s l a t i o n o f hspl6 mRNA was l o d i n a t e d w i t h mCi/mg N a l ) a c c o r d i n g t o t h e procedure  [  I ] i o d i d e (Amersham, 17  of Commerford (1971) t o a s p e c i f i c  a c t i v i t y o f 2.5 x 10^ cpm p e r yg of RNA. 2.10.2  P u r i f i c a t i o n and L a b e l l i n g o f O l i g o d e o x y n u c l e o t i d e s  Two 18mer o l i g o d e o x y n u c l e o t i d e s were used as h y b r i d i z a t i o n  The  I.  CGGGGCCGCGCGCACGCA  II.  CAGGGCCGCGCGCACGCA  probes:  o l i g o d e o x y n u c l e o t i d e s were s y n t h e s i z e d by Tom A t k i n s o n i n the l a b o r a t o r y  of Dr. M. Smith, UBC, w i t h an A p p l i e d Biosystems 380A DNA s y n t h e s i z e r . O l i g o d e o x y n u c l e o t i d e s were p u r i f i e d through i s o l a t e d by C  SEP-PAK  20% sequencing  (urea) g e l s and  ( M i l l i p o r e ) chromatography as d e s c r i b e d by  71  A t k i n s o n and Smith  (1984).  To make h y b r i d i z a t i o n probes, 20 pmoles of 32  o l i g o d e o x y n u c l e o t i d e was  l a b e l l e d w i t h [y-  k i n a s e as d e s c r i b e d by Z o l l e r and Smith  2.10.3  P r e p a r a t i o n of Double-Stranded  P]ATP and T4 p o l y n u c l e o t i d e  (1983).  DNA  Probes 32  P u r i f i e d DNA  fragments were n i c k t r a n s l a t e d w i t h [ a -  P]dCTP and  [ a - p ] d G T P by the method of Rigby e t a l . (1977). 32  M13  templates c o n t a i n i n g C. elegans genomic DNA  fragments were a l s o  used to generate probes w i t h h i g h s p e c i f i c a c t i v i t i e s . c o n t a i n i n g 3.0  y l of template  (0.5 to 1.0  p r i m e r (P-L B i o c h e m i c a l s ; 0.03  yg), 2.0  An a n n e a l i n g mixture  y l of u n i v e r s a l  A„,„ u n i t s per m l ) , 2.0  y l of 10 x  260 annealing b u f f e r M g C ^ ) , was  (100 mM  T r i s - h y d r o c h l o r i d e , pH 7.5,  o i n c u b a t e d a t 65 C f o r 15 min  A f t e r c o o l i n g to room temperature dithiothreitol,  2.0  y l of 0.5 mM  each o f [ P ] d G T P and 32  E. c o l i DNA  polymerase  600 mM  NaCl, 70  i n a 1.5-ml microfuge tube.  (5 to 10 min), 1.0 dATP, 2.0  y l of 20  y l of 0.5 mM  mM  dTTP, 2.5 y l  [ P ] d C T P (25 y C i ; 3,000 Ci/mmol), and 0.5 32  I (Klenow fragment) were added.  allowed t o proceed f o r 10 min at-room chase a f t e r the a d d i t i o n of 2.0  temperature and was  y l of 0.5 mM  U of  The r e a c t i o n  dGTP and 2.0 o  was  then f o l l o w e d by a y l of 0.5  dCTP f o r 5 min b e f o r e t e r m i n a t i o n by h e a t i n g a t 70 C f o r 10 mm. primer-extended p r o d u c t was  mM  then d i g e s t e d w i t h H a e l l l f o r 30  mM  The  min.  For b o t h t e c h n i q u e s , f r e e t r i p h o s p h a t e s were separated from the labelled  s t r a n d s by chromatography  (Maniatis et a l . ,  1982).  on 1.0 ml spun columns of Sephadex  G50  72  2.11  Hybridization N i t r o c e l l u l o s e b l o t s o f DNA from b a c t e r i a l cDNA t r a n s f o r m a n t s were o i n 4 x SSPE, 50% formamide f o r 1 hour a t 37 C.  prehybridized  H y b r i d i z a t i o n was c a r r i e d out i n 4 x SSPE, 50% formamide c o n t a i n i n g 10  6  cpm p e r f i l t e r of [  hours.  The f i l t e r s  followed  1 2 5  1.0 x  I ] - l a b e l l e d RNA a t 37°C f o r a t l e a s t 12  were washed i n two changes of 2 x SSPE, 0.1% SDS  by two changes o f 0.1 x SSPE, 0.1% SDS a l l a t room temperature. o  F i n a l washes were c a r r i e d out a t 50 C i n 0.1 x SSPE, 0.1% SDS. In the case of double-stranded DNA probes, p r e h y b r i d i z a t i o n was done i n 5 x SSPE, 50% formamide, 5 x Denhardt's reagent, 0.1% SDS and 100 - 200 yg/ml of sheared, denatured E. c o l i , Prehybridization  c a l f thymus o r salmon sperm DNA.  was a t 42°C f o r a t l e a s t 1 hour.  Hybridizations  were  c a r r i e d out f o r a t l e a s t 12 hours a t 42°C i n the s o l u t i o n d e s c r i b e d f o r the p r e h y b r i d i z a t i o n except t h a t 1 x Denhardt's reagent was used and the 32 denatured  [  P ] - l a b e l l e d probe was i n c l u d e d .  i d e n t i c a l t o the c o n d i t i o n s  described  Washing of the f i l t e r s was  f o r the RNA probe above.  For 5 ' - l a b e l l e d o l i g o d e o x y n u c l e o t i d e s , o  p r e h y b r i d i z a t i o n and  h y b r i d i z a t i o n was done a t 37 C f-or a t l e a s t 1 hour and 12 hours, r e s p e c t i v e l y , i n 6 x SSPE, 2 x Denhardt's reagent and 0.2% SDS. were done i n 6 x SSPE.  for  A l l washes  Washing was u s u a l l y c a r r i e d out a t room temperature o  15 minutes, then 2 x 15 minutes a t 37 C f o l l o w e d  48°C f o r 15 minutes each.  by 2 f i n a l washes a t  1 x SSPE i s 0.1 mM EDTA, 10 mM NaH P0 2  4  pH  7.0 and 0.18 M NaCl. 1 x Denhardt's reagent i s 0.02% p o l y v i n y l p y r r o l i d o n e , 0.02% b o v i n e serum albumin and 0.02% f i c o l l . A f t e r washing, n i t r o c e l l u l o s e f i l t e r s from a l l of the h y b r i d i z a t i o n s o d e s c r i b e d were a i r d r i e d and f l u o r o g r a p h e d a t -70 C u s i n g e i t h e r Kodak  73  X-Omat AR o r X-Omat RP f i l m , the former b e i n g a p p r o x i m a t e l y 5 - f o l d more sensitive.  2.12  Summary o f B a c t e r i a l S t r a i n s Used. T a b l e IV.  of B a c t e r i a l  Strains  Genotype  Strain  DP50, supF  Genotypes  F  , tonA53,  dapD8, l a c Y l , glnV44  (supE44), A ( g a l - u v r B ) 4 7 ,  \~, t.yrT48(supF58), gyrA29, A(thyA57), hsdS3 b.  LE392  F~, hudK514(r~,m^), supE44, supF58,  l a c Y l or  A ( L a c l Z Y ) 6 , galK2, galT22, metBl, \~, RRI  F~, hsdS20(r~, m^),  ara-14, proA2,  trpR55  lacYl,  galK2,  rpsL20 (Sm ), x y l - 5 , m t l - 1 , supE44, X. r  d.  f.  -  Q358  — + + R hsdR^, hsdH^, su , 80  Q359  + + R hsdR^, hsdM^, s u ^ , 80 , P2  JM101  A l a c p r o , supE, t h i , F'traD36, proAB,  JM103  A l a c p r o , supE, t h i , F*traD36, s t r A , sbcB15, proAB,  laci ZAM15 q  endA, hspR4,  laci ZAM15 q  a,b,c - taken from M a n i a t i s e t a l . (1982) d,e, - t a k e n from Karn e t a l . (1980) f , g - taken from Messing  The h o s t s JM103 and JM101  (1983)  were s t r e a k e d out on p l a t e s c o n t a i n i n g  0.2%  g l u c o s e , 0.001% v i t a m i n B l and minimal s a l t s as d e s c r i b e d i n Messing  (1983).  74  III.  RESULTS  3.1  The Heat Shock Response of C a e n o r h a b d i t i s elegans v a r . B r i s t o l ,  strain  N2 C. elegans undergoes a t y p i c a l heat shock response  a t the e l e v a t e d  o temperature  of 35 C, the normal growth temperature  being  approximately  o 20 C.  As shown i n F i g u r e 3, t h e r e was  p r o t e i n s y n t h e s i s w h i l e a unique i n v i v o l a b e l l i n g experiments  a dramatic decrease  s e t of hsps was  i t appeared  are s y n t h e s i z e d i n c o n t r o l nematodes but  induced.  i n general From these  t h a t hsp81, hsp41, hsp38 and hsp29 thoy c o n t i n u e to be s y n t h e s i z e d a t  o 35 C.  These o b s e r v a t i o n s are i n agrooment with Snutch  except  f o r hsp29 which they r e p o r t e d to bo u y n t h e s i z e d o n l y d u r i n g heat  shock c o n d i t i o n s .  A p r o t e i n w i t h an apparent  accumulated  f o r the f i r s t  hours.  simplest explanation for this  has  The  (1983)  m o l e c u l a r weight of 36,000  3 hours of l a b e l l i n g but was  a h i g h t u r n o v e r r a t e and  and B a i l l i e  not d e t e c t a b l e by 5  i s that t h i s p a r t i c u l a r  is preferentially  protein  degraded d u r i n g prolonged  exposure of the nematodes to 35°C. Hsp70, h s p l 8 and hspl6 d i d not appear t o be s y n t h e s i z e d i n nematodes growing a t normal temperature h s p l 6 accumulating  but were induced d u r i n g heat shock, h s p l 8  to v e r y h i g h l e v e l s .  and  The nematode hsp70 i s p r o b a b l y  homologous t o hsp70 of D r o s o p h i l a and rainbow t r o u t s i n c e C. elegans genomic DNA  h y b r i d i z e s t o a D r o s o p h i l a hsp70 genomic DNA  1983)  c l o n e (Snutch and  and a t r o u t hsp70 cDNA (Kothary e t a l . , 1984).  appeared  as a d o u b l e t as shown i n F i g u r e 3, and  h s p l 9 d e s c r i b e d by Snutch and B a i l l i e  (1983).  Baillie,  Hspl8 sometimes  i s probably i d e n t i c a l  to  In a d d i t i o n , F i g u r e 3 shows  the i n d u c t i o n of a 50 kd p r o t e i n i n a. synchronous p o p u l a t i o n  75  Figure  3.  Induction  of  heat  shock  proteins  i n C.  elegans. o  Synchronously  growing  adult  worms  were  incubated  at  35  C  and  l a b e l l e d  lane  indicate  35 in the  vivo  with  duration  minute  chase  [  S ] - l a b e l l e d  of  exposure,  at  35°C.  E.  c o l i .  i n hours,  The  lane  on  The to  the  numbers  the  below  radioactive  r i g h t  (C)  each  bacteria  represents  before  t o t a l  protein  o synthesis  a t  22  C.  The  arrows;  the p o s i t i o n s  Numbers  r e f e r  to  of  p o s i t i o n s the  approximate  other  of  the  major  molecular  shsps hsps  are are  weights,  i n  indicated indicated  by by  k i l o d a l t o n s .  a  s o l i d lines.  30  76  of a d u l t nematodes.  T h i s p r o t e i n may be analogous t o the 50 kd p r o t e i n  i s s y n t h e s i z e d i n heat shocked dauer l a r v a e (Snutch and B a i l l i e ,  that  1983).  They, however, f a i l e d t o d e t e c t hsp50 i n n o r m a l l y growing nematodes which had been heat shocked. labelling protocols.  T h i s d i s c r e p a n c y may be due t o the d i f f e r e n c e i n Snutch and B a i l l i e  (1983) exposed  r a d i o a c t i v e b a c t e r i a p r i o r t o heat shock.  the nematodes t o  The h i g h e r background  o f normal  p r o t e i n s y n t h e s i s may have masked the appearance o f a heat i n d u c i b l e p r o t e i n i n the 50 kd range. Experiments were a l s o c a r r i e d out t o determine the s t a b i l i t y o f p u l s e o l a b e l l e d hsps.  I f the nematodes were l a b e l l e d f o r two hours a t 35 C and  allowed t o r e c o v e r a t room temperature on u n l a b e l l e d E. co 11, i t c o u l d be shown t h a t the hsps o f C. e l e g a n s , e s p e c i a l l y the c h a r a c t e r i s t i c 16 kd p r o t e i n s , were s t i l l  p r e s e n t a t 24 hours.  18 kd and  The r e s u l t s of t h i s  experiment are shown i n F i g u r e 4.  3.2  I d e n t i f i c a t i o n o f cDNAs Coding f o r Hspl6 In o r d e r t o c h a r a c t e r i z e the genes coding f o r h s p l 6 and h s p l 8 , a cDNA  l i b r a r y c o n s t r u c t e d from polyA RNA i s o l a t e d from heat shocked +  nematodes was s c r e e n e d .  Bristol  The probe used was a polyA RNA f r a c t i o n +  (Figure  5, l a n e D) which was h i g h l y e n r i c h e d f o r messages c o d i n g f o r h s p l 6 and hspl8.  T o t a l mRNA was f r a c t i o n a t e d by formamide p o l y a c r y l a m i d e g e l  e l e c t r o p h o r e s i s and monitored by i n v i t r o t r a n s l a t i o n i n a r a b b i t r e t i c u l o c y t e system as an assay f o r b i o l o g i c a l l y a c t i v e mRNA c o d i n g f o r the 125 shsps.  T h i s e n r i c h e d f r a c t i o n was then l o d m a t e d  s c r e e n f o r cDNAs s p e c i f i c t o t h i s  fraction.  with  I and used t o  77  Figure  4.  S t a b i l i t y  Synchronously l a b e l l e d the  f o r 2  presence  normal  the  growing  hours  of  of  at  35  unlabelled  temperature  f o r the  hsps  of  cultures o  C.  A f t e r  C.  elegans.  c o n s i s t i n g a  chase  b a c t e r i a ,  the  length  time  of  of  l a t e  f o r 30  worms  were  (hours)  l a r v a l  stages o  minutes  at  35  allowed  to  recover  shown  below  each  C  were i n at  lane.  o Lane in  C  vivo  represents l a b e l l e d  indicated.  t o t a l  p r o t e i n  proteins  of  E.  synthesis c o l i  K12.  at  22  The  C.  Lane  p o s i t i o n s  E of  i s a the  p r o f i l e shsps  of  are  78  AB C D  Figure and  5.  I n v i t r o  enrichment  t r a n s l a t i o n  of messages  Approximately  1.0  yg  products  coding of  RNA  o f mRNA  f o r the small was  from  heat  shocked  c e l l s ,  hsps.  t r a n s l a t e d  i n a  rabbit  r e t i c u l o c y t e  35 system  i n the presence  separated  on  a  heat  shock  formamide 16  kd  hsps  Lanes  polyA RNA; +  SJmethionine.  a r e A) D)  t o t a l  enriched  T r a n s l a t i o n  g e l and c o n t r o l f r a c t i o n  i s indicated  the c e l l - f r e e  by  the s o l i d  proteins  t r a n s l a t i o n  which  system  products  i d e n t i f i e d RNA; of  polyacrylamide g e l electrophoresis.  endogenously-labelled to  [  15% SDS-polyacrylamide  autoradiography. C)  of  B)  by  t o t a l  polyA  heat  RNA  Open  are also  present  (see Figure  6,  arrows  of  t h e 18  RNA;  by kd  and  indicate  when  lane  shock  p u r i f i e d  T r a n s l a t i o n  arrows.  were  C).  no  RNA  i s  added  79  Approximately 1000 t r a n s f o r m a n t s were screened by colony hybridization.  T h i s y i e l d e d 27 p u t a t i v e p o s i t i v e c o l o n i e s from which  p l a s m i d DNA was p r e p a r e d .  The DNA was r e s t r i c t e d w i t h P s t I and the  l i b e r a t e d cDNA i n s e r t s were analyzed  on a 1.5% agarose g e l .  The DNA was  t r a n s f e r r e d from the g e l t o n i t r o c e l l u l o s e and h y b r i d i z e d t o t h e 125 [  .. I ] - l a b e l l e d probe.  Of the o r i g i n a l 27 p l a s m i d s , 12 c o n t a i n e d  i n s e r t s which h y b r i d i z e d t o the r a d i o l a b e l l e d RNA ( r e s u l t s not i d e n t i f y h y b r i d plasmids coding was  c a r r i e d out.  6.  site.  To  or hspl8, h y b r i d i z a t i o n s e l e c t i o n  for hspl6.  A t y p i c a l p o s i t i v e s e l e c t i o n i s shown i n  R e s t r i c t i o n enzyme a n a l y s i s r e v e a l e d  inserts f e l l  shown).  Seven plasmids were i d e n t i f i e d which s e l e c t e d mRNA  s p e c i f i c a l l y coding Figure  f o r hspl6  PstI  t h a t the p o s i t i v e cDNA  i n t o two groups, based on the presence o r absence of an EcoRI  Two cDNA c l o n e s , pCEHS48 and pCEHS41, were used f o r sequence a n a l y s i s  s i n c e they c o n t a i n e d  the l o n g e s t  cDNA i n s e r t s r e p r e s e n t i n g  each group.  In the h y b r i d i z a t i o n s e l e c t i o n experiments, the o n l y mRNA s e l e c t e d by the cDNA h y b r i d plasmids t e s t e d were s p e c i e s hsp.  Those p l a s m i d s which d i d not  negative  results.  coding  f o r the 16,000 d a l t o n  s e l e c t hspl6 mRNA gave c o m p l e t e l y  T h i s suggests t h a t messages coding  f o r hspl6  are e i t h e r  v e r y abundant, o r t h a t t h e i r s t r u c t u r e i s p a r t i c u l a r l y w e l l s u i t e d t o cDNA s y n t h e s i s under the c o n d i t i o n s used.  I t i s perhaps s u r p r i s i n g t h a t no  p o s i t i v e recombinant plasmids s e l e c t e d mRNAs coding t o be induced t o the same h i g h degraded d u r i n g  l e v e l s as h s p l 6 .  f o r h s p l 8 , which appears  The h s p l 8 messages were not  the h y b r i d i z a t i o n s e l e c t i o n , s i n c e t o t a l polyA RNA removed  from the h y b r i d i z a t i o n mix contained  +  mRNA which was t r a n s l a t e d i n t o a 18,000  d a l t o n p r o t e i n as shown i n F i g u r e 6 ( l a n e T ) .  Again, i t i s p o s s i b l e  the h s p l 8 mRNA may possess some s t r u c t u r a l f e a t u r e which, i n t h i s  that  case,  30  S TC  16k>  Figure  6.  In  v i t r o  t r a n s l a t i o n  of  RNA  selected  by  h y b r i d i z a t i o n  w i t h  pCEHSAl. Plasmid polyA RNA +  selected  pCEHSAl  from by  the  heat  was  bound  shocked  recombinant  to n i t r o c e l l u l o s e  nematodes. cDNA  clone  After was  and  hybridized  h y b r i d i z a t i o n ,  eluted  and  to  the  translated  RNA i n  v i t r o •  35 [  S]-labelled  electrophoresis RNA; The  T)  products and  of  analyzed  by  autoradiography.  non-hybridized  p o s i t i o n  were  hspl6  or i s  t o t a l shown.  SDS-polyacrylamide  Lanes  polyA RNA +  are  and  S)  C)  control  selected  g e l with  no  added  polyA RNA. +  81  r e s u l t e d i n poor s y n t h e s i s  o f cDNA.  T h i s f e a t u r e i s n o t the l a c k o f a p o l y A  sequence, r e s u l t i n g i n inadequate p r i m i n g  w i t h o l i g o ( d T ) , s i n c e t h i s mRNA  binds t o o l i g o ( d T ) - c e l l u l o s e (see F i g u r e 5, lane C ) . coding  f o r Drosophila  I n t e r e s t i n g l y , cDNAs  hsp22 and hsp27 were n o t i d e n t i f i e d under  i n which cDNAs coding  conditions  f o r hsp23 and hsp26 were (Wadsworth e t a l . , 1980;  Voellmy e t a l . , 1981).  3.3  Two-dimensional G e l E l e c t r o p h o r e s i s o f Hspl6 The  f i n d i n g o f two d i s t i n c t l y d i f f e r e n t cDNAs coding  suggested the e x i s t e n c e possible existence  f o r hspl6  o f more than one gene f o r t h i s p r o t e i n .  of m u l t i p l e forms o f hspl6  The  was i n v e s t i g a t e d by a n a l y z i n g  the t r a n s l a t i o n p r o d u c t s o f mRNA s e l e c t e d by pCEHS48 o r pCEHS41 ( F i g u r e 6, lane S) on two-dimensional g e l s u s i n g  the system o f O ' F a r r e l l (1975).  g e l a n a l y s i s was k i n d l y c a r r i e d out by E l i z a b e t h Burgess. pCEHS41 a r e shown i n F i g u r e  7.  Five polypeptides  The  The r e s u l t s f o r  with d i s t i n c t l y d i f f e r e n t  i s o - e l e c t r i c p o i n t s , b u t i d e n t i c a l m o l e c u l a r weights were r e s o l v e d . I d e n t i c a l r e s u l t s were o b t a i n e d  when pCEHS48 was used i n t h e s e l e c t i o n .  T h i s i m p l i e s t h a t , under the c o n d i t i o n s used i n t h e h y b r i d i z a t i o n s e l e c t i o n experiments, b o t h pCEHS48 and pCEHS41 h y b r i d i z e d t o t h e same t r a n s c r i p t s . T h i s i s n o t s u r p r i s i n g s i n c e t h e cDNAs CEHS48 and CEHS41 c r o s s - h y b r i d i z e w i t h each o t h e r not  s t r o n g l y under normal h y b r i d i z a t i o n s t r i n g e n c i e s ( r e s u l t s  shown). These r e s u l t s a r e c o n s i s t e n t w i t h the e x i s t e n c e  d i s t i n c t b u t r e l a t e d hspl6 enzyme p a t t e r n s .  o f a t l e a s t two  sequences as suggested by t h e cDNA  restriction  32  IEF -  16k  5.0  Figure  7.  Two-dimensional  t r a n s l a t i o n  of  selected  +  v i t r o  (see  g e l  electrophoresis  hybrid-selected  PolyA RNA in  7.5  Figure  6,  by  the  products  from  polyA RNA. +  h y b r i d i z a t i o n  lane  of  S).  The  25  to  pCEHS41  was  t r a n s l a t e d  p i  t r a n s l a t i o n mixture  was  o p r e c i p i t a t e d washed  with  ( O ' F a r r e l l ,  i n 10  ethanol, 1975).  electrophoresis two  most  volumes  basic  are  dried The  acetone  and  by  of  arrows.  variants  at  -20  resuspended  d i r e c t i o n s  shown  protein  of  which  C  overnight.  i n O ' F a r r e l l  i s o - e l e c t r i c Arrows are  also  discussed  The loading  focusing show i n  the  and  p e l l e t buffer  A  SDS-gel  migration  section  was  4.1.  of  the  83  3.4  Messages Coding f o r Hspl6 Are Not T r a n s c r i b e d  i n C o n t r o l Nematodes  In v i t r o t r a n s l a t i o n o f c o n t r o l RNA sometimes showed the e x i s t e n c e o f what appeared t o be hspl6 might have been induced  (see F i g u r e 5, lanes A and B ) .  Hspl6 t r a n s c r i p t s  i n a d v e r t e n t l y d u r i n g c o l l e c t i o n and washing o f  c o n t r o l batches o f nematodes o r they may be p r e s e n t under normal conditions.  The a v a i l a b i l i t y o f s p e c i f i c DNA probes f o r hspl6 mRNA made i t  p o s s i b l e t o address t h i s q u e s t i o n .  As shown i n F i g u r e 8, no d e t e c t a b l e  h y b r i d i z a t i o n o f CEHS41 cDNA t o polyA RNA from c o n t r o l nematodes was +  seen.  Identical results  i n s e r t as probe. occurs  ( n o t shown) were obtained w i t h the CEHS48 cDNA  C l e a r l y , a very h i g h d i f f e r e n t i a l s y n t h e s i s o f hspl6 mRNA  d u r i n g heat uhock, demonstrating the s t r o n g i n d u c i b i l i t y o f these  genes.  Hspl6 t r a n s c r i p t s do n o t d i s p l a y any s i z e h e t e r o g e n e i t y  under  these  electrophoresis conditions.  3.5  Sequence A n a l y s i s o f t h e cDNAs CEHS48 and CEHS41 The  chemical  cDNAs were sequenced u s i n g Maxam and G i l b e r t base m o d i f i c a t i o n and cleavage  F i g u r e 9.  r e a c t i o n s on t h e e n d - l a b e l l e d DNA fragments shown i n  As seen i n F i g u r e 10, t h e n u c l e o t i d e sequences o f CEHS48 and  CEHS41 a r e v e r y s i m i l a r . a c i d sequences begins acid residues.  Numbering o f the n u c l e o t i d e and p r e d i c t e d amino  a t t h e 5' end o f CEHS48.  CEHS48 encodes 135 amino  CEHS41 i s a l i g n e d w i t h CEHS48, b e g i n n i n g  a t amino a c i d 32  s i n c e i t codes f o r 31 fewer amino a c i d r e s i d u e s a t the 5' end. most s t r i k i n g  One o f t h e  f e a t u r e s o f these sequences i s t h e c o n t r a s t i n degree o f  homology between d i f f e r e n t r e g i o n s .  From n u c l e o t i d e s 92 t o 112, t h e two  cDNAs d i f f e r a t a l l p o s i t i o n s except f o u r .  Sequence a n a l y s i s o f t h e  hsp!6-41 gene (Jones e t a l . , 1986) has r e v e a l e d t h a t t h i s s t r e t c h i n CEHS41  84  HS  origin  Figure and  8.  heat 1.0  and then  Northern shocked yg  t r a n s f e r r e d  and  dot-blot  analysis  was  denatured,  of polyA  RNA  from  control  worms.  of polyA RNA +  transferred applied  b l o t  t o n i t r o c e l l u l o s e .  as  a  RNA  spot  lanes.  1.5%  agarose  to the n i t r o c e l l u l o s e f i l t e r  a t  the top o f the  then  probed  RNA  g e l  t h e same  were  yg  a o f  b l o t s  0.5  on  sample  These  A  separated  f o r sequences  was  coding  32 f o r are  hspl6  with  indicated.  [  P ] - l a b e l l e d  CEHS41  cDNA.  Control  and heat  shock  lanes  85  cehs48 Hpall  PstI  Bgl II  EcoRI  Taq I  PstI Hpall  cehs41 Hpall Hhal  PstI  SstI  Pst I  Hpa I  200 bp  Figure  9.  CEHS41  cDNA  The  Strategy  are  inserts  a r e shown  clockwise  represented by thin  while  3' n o n - c o d i n g  d i r e c t i o n terminal  t o determine  t h e n u c l e o t i d e  sequence  o f CEHS48 a n d  r e l a t i v e  t o  inserts.  cDNA  conventional  used  numbering lines.  regions  o f sequencing  as they  o f t h e pBR322 Hspl6  a r es h o w n  from  t r a n s f e r a s e - l a b e l l e d  a r e oriented  coding  regions  b y shaded  Klenow-labelled fragments  sequence. a r e  boxes.  fragments  ( c i r c l e s ) .  pBR322 shown  Arrows  t h e sequences  b y open  boxe  r e p r e s e n tt h  (squares)  o r from  86a  F i g u r e 10.  Complete n u c l e o t i d e sequences of the cDNA i n s e r t s from pCEHS48  and pCEHS41 w i t h the deduced amino a c i d  sequences.  The c o d i n g s t r a n d s of each cDNA a r e a l i g n e d w i t h each o t h e r to demonstrate sequence homology.  A s t e r i s k s r e p r e s e n t d i f f e r e n c e s i n the  sequences a t both the n u c l e o t i d e and amino a c i d l e v e l s .  The  polyadenylation  s i g n a l AATAAA, found 12 base p a i r s b e f o r e the p o l y ( A ) s t r e t c h i n pCEHS48 i s a l s o shown.  86  E*23IS48  G  l g  TCT GAT TCA AAT GTT CTC GAT CAT T T C TTG GAT GAA ATC ACT GGA T C T GTT CAA TTT  pCEIIS-ll hspl6-48  S e r Asp S e r Asn V a l L e u A s p H i s Phe L e u A s p G l u H e  T h r G l y S e r V a l G i n Phe  hsplfi-41 1  10  60 80 100 120 CCA TAT TGG AGA AAT GCT GAT CAC AAC TCA T T C AAT T T T T^C GA(j AAT A T T GGA GAG A T T GTA AAT GAC G TA AAA TTG TGT T C T T T T f f f CAG ATT GTA AAT GAT Phe A s n Phe ie 8 L*** ys 20 30 40 w  140  160 160 TCT CAT T T C  180  TCT CAT T T C Glu  S e r L y s Phe S e r V a l G i n L e u A s p V a l S S eerr H H iiss P h e e L y s P r o G l u A s p L e u L y s H e G l u Leu Asp *** *** S e r H i s Phe : Pro G l u Asn Leu Lys H e Lys Leu Asp 50 60  GGA A G A ° G A A  CTA AAA A T T GAA  GGA  CTC AAA  AGA GAG  An  GAA  GGA ATT "CAA GAA AAA AAA TCA G A G ^ C A T G G A T A C GGG A T T CAA GAA ACA AAA K G  Gly  Arg Glu Leu Lys H e  Glu Gly H e  Gly  Arg G l u Leu Lys H e 70  Glu Gly H e  280  GAA  T £ G AAA CGA  TCA°TTT  CAT GGA TAC TTG AAA CGC TCA T T T  G i n G l u L y s L y s S e r G l u H i s G l y T y r S e r L y s A r g S e r Phe #** *** G i n G l u T h r L y s S e r G l u H i s G l y T y r Phe L y s A r g S e r Phe 80 ' 320  300  A T T T C G " "AAT  GAA GGA  A T T TCG AAT  GAA GGA  H e S e r Asn G l u Gly 90  H e S e r Asn G l u G l y 110  100  C T T CAA A T T GAG  GCT CCA AAG AAG^ACT AAC TCA T C T CGT T C T A T T ° C C C " ;  AAT T T T GTT  400 GCA AAA  CTC CAA A T T GAG  GCT CCA AAG AAG ACA AAC TCA TCA CGT T C T A T T CCG ,  AAT T T T GTT  GCA AAA  Leu G i n H e  G l u A l a Pro Lys Lys T h r Asn Ser S e r A r g S e r H e  Pro  A s n Phe V a l A l a L y s  Leu  G l u A l a Pro Lys L y s T h r Asn S e r S e r A r g S e r H e 120  Pro  A s n Phe V a l A l a L y s 130  Gin H e  460 CAT  TAA  CAT TAA H i s End H i s End 135  480  T,^TTTATT5TATJ^AAATAn$TTAATTTgAATAAAGTCATTAATTTAAAAAAAAAAAAAAAAAAAAAAAAC CACTTTT6TT6AAGAGAA6CTACTTATTATTTGTTCTTCTTTTT^  5  87  i s a c t u a l l y the 3' p o r t i o n of a 58 bp  i n t r o n , t h i s p a r t i c u l a r cDNA b e i n g  r e s u l t of r e v e r s e t r a n s c r i p t i o n of an u n s p l i c e d message.  the  CEHS48 corresponds  to a c o r r e c t l y s p l i c e d t r a n s c r i p t , the i n t r o n b e i n g found between amino a c i d r e s i d u e s 38 and  39  i n F i g u r e 10  From n u c l e o t i d e s 113 and  (see s e c t i o n  to the TAA  3.9).  t e r m i n a t i o n codons, the homology i s  r e s u l t s i n o n l y 6 amino a c i d d i f f e r e n c e s out of a t o t a l of  N o n - c o n s e r v a t i v e changes occur a t amino a c i d p o s i t i o n s 59 (Glu -» L y s ) , 77 regions  (Lys -> T h r ) , and  are h i g h l y d i v e r g e n t  homology of o n l y 30%.  101  (Thr •* P r o ) .  The  compared to the coding  CEHS48 c o n t a i n s a polyA  The  97.  (Asp •* Asn),  63  3' noncoding  r e g i o n , w i t h a sequence  s t r e t c h of 24 r e s i d u e s ,  n u c l e o t i d e s a f t e r the p o l y a d e n y l a t i o n s i g n a l AATAAA (Proudfoot 1976).  91%  and  12  Brownlee,  l e n g t h of the 3' noncoding r e g i o n s , e x c l u d i n g the polyA s t r e t c h ,  i s 49 n u c l e o t i d e s f o r CEHS48 compared to at l e a s t 92 n u c l e o t i d e s f o r CEHS41 which i s incomplete  at the 3'  end.  At the n u c l e o t i d e l e v e l , t h e c D N A s have A/T and  69%.  T h i s h i g h A/T  content  base compositions  F o r example, ATT  as codons f o r i s o l e u c i n e 23 times-* whereas the ATC S i m i l a r l y , l y s i n e i s coded f o r by AAA The  glutamic The  22 times w h i l e AAG  glycine also r e f l e c t s this  or ATA  and  are used  codon i s used o n l y once.  codon usage f o r v a l i n e , s e r i n e , asparagine,  a c i d and  67%  i s r e f l e c t e d i n the codon usage where A  T are p r e f e r r e d i n the wobble p o s i t i o n s .  times.  of  i s used o n l y 7 aspartic acid,  trend.  deduced amino a c i d sequences of the shsps of D. melanogaster a r e  v e r y s i m i l a r i n a r e g i o n of 83 amino a c i d s which shows homology to a - c r y s t a l l i n , the major w a t e r - s o l u b l e  p r o t e i n component of the  l e n s ( I n g o l i a and C r a i g , 1982b; Ayme and T i s s i e r e s , 1985). molecular  weight d i f f e r e n c e and  Due  vertebrate to  the  the f a c t t h a t no o t h e r 16,000 d a l t o n hsp  had  88  AA 27  87-  K D G  C M D V S Q  N E L T V K V V D N T V V V E  DMHSP 2 6  DMHSP  87-  KDG  CM  S E L N V K V V D D S  K D G  D V A Q  I  DMHSP 2 3  67-  DMHSP 2 2  61-  KDG  A-CRYS  72-  K D R F S V HjL II V[KJH F [ S J P E E L K J V K V L[G]D V I  B2  C M D V S H LT  G K H E E R G D G  L V E G K H E E R Q D D  S E L V V G V Q D N S V L V E  LD V KD  S E  V K V LD  L[K  G S V LV  CEHSP  16-48  43-  E S K F S V Q L D V S H F K P E  LK  L D G  CEHSP  16-41  43-  E S K F S V Q L D V S H F K P E  L K  L D G R E L K  RE  G N H E E R E D D  G G K S E Q Q F A E  E V H G, KH  LK  , E E R Q D[E  I E G I Q E K[KS^E I E G I Q E T K S -  E  AA H G fl I Q  V R K Y T L  H G H I M  FV  R RY  K'G L T P T K[V|V[S|T V f s l S D[G]V[L]T L - 1 5 9  LV  D G Y K A E Q V V S Q L S S D G V L T V  -159  H G F I T RH F V R R Y A L P  P G Y E A D K V A S T L S S D G V L T I  -139  Q G G Y S R H  L R R F V L P  EG  -133  H G F I S R  H RKY  H G Y S K R S f"S  11.  K V T S T L S S D G V L T I  K M I L L P E D V D L T S V KS A I S N EG  H G Y L K R S F S K M I L L  Figure  Y EAD  R I P [ A J D V D [ P L A | I I T [ S [S L [ S I S D | G | V | L | T V - 1 4 4  Comparison  P E D[A]D  L|P S V K S A  o f deduced  K LQTI-115  I S N E G K L Q  I -115  amino a c i d sequences o f four- shsps from  D. m e l a n o s a s t e r , a - c r y s t a l l i n and two 16,000 d a l t o n hsps from C. e l e g a n s . The r e g i o n  shown corresponds t o t h e 74 amino a c i d r e g i o n which was used  f o r comparison by I n g o l i a and C r a i g bovine a - c r y s t a l l i n i s that of the 1973)  (1982a, F i g u r e chain  w h i l e t h e D. melanogaster sequences  (1982b).  4).  The-sequence o f  (van d e r Ouderaa,  et a l . ,  a r e taken from I n g o l i a and C r a i g  The r e g i o n s of each p r o t e i n used f o r the comparison a r e i n d i c a t e d  by amino a c i d numbering. boxes and a r e d i s c u s s e d i s A, a l a n i n e ;  The d i f f e r e n t l e v e l s o f homology a r e shown by open i n Section  C, c y s t e i n e ;  3.5. The s i n g l e l e t t e r amino a c i d  code  D, a s p a r t i c a c i d ; E, g l u t a m i c a c i d ; F,  p h e n y l a l a n i n e ; G, g l y c i n e ; H, h i s t i d i n e ; I , i s o l e u c i n e ; K, l y s i n e ; L, l e u c i n e ; M, methionine; N, a s p a r a g i n e ; P, p r o l i n e ; Q, glutamine; R, arginine;  S, s e r i n e ; T, t h r e o n i n e ; V, v a l i n e ; W, tryptophan; Y, t y r o s i n e .  89  been i d e n t i f i e d  i n a e u c a r y o t i c system,  i t was s u r p r i s i n g to f i n d  that  C. elegans hspl6-41 and hspl6-48 c o n t a i n e d e x t e n s i v e homology to the shsps o f D r o s o p h i l a w i t h i n the conserved b l o c k .  The boxed r e g i o n s i n F i g u r e 11  show areas o f amino a c i d homology a t two d i f f e r e n t l e v e l s . a c i d s shared by bovine a - c r y s t a l l i n  amino  (B^ chain) and the two hspl6  p r o t e i n s o f C. elegans a r e i n d i c a t e d .  Amino a c i d numbering of the h s p l 6  p r o t e i n s corresponds to t h a t shown i n F i g u r e 10.  Over t h e r e g i o n s shown,  t h e same amino a c i d occurs a t 26 p o s i t i o n s out of 74. amino a c i d s 46 t o 61 (numbering  Firstly,  I n the s u b - r e g i o n of  r e f e r s to the C. elegans h s p s ) , the same  amino a c i d i s used a t 11 p o s i t i o n s out of 16.  A l s o i n d i c a t e d are the  p o s i t i o n s a t which the same amino a c i d i s used by a t l e a s t 6 of the 7 proteins.  T h i s occurs a t 19 p o s i t i o n s ,  7 f a l l i n g w i t h i n the s u b - r e g i o n  d i s c u s s e d above w h i l e another 5 a r e found over a r e g i o n of 11 amino a c i d s from p o s i t i o n s 103 to 113.  E x t e n s i v e homology among hsp22, 23, 26 and 2 7 o f  D r o s o p h i l a extends a p p r o x i m a t e l y 30 amino a c i d s i n a 3* d i r e c t i o n from t h e 74 amino a c i d domain (see F i g u r e 2 ) . h s p l 6 sequences  T h i s i s a l s o the case w i t h the two  where e x t e n s i v e homology i s maintained on the 3* s i d e o f the  f u n c t i o n a l domain and, i n f a c t , extends t o the c a r b o x y l t e r m i n i . no s i m i l a r i t i e s between the C. elegans and D. melanogaster  There a r e  sequences  i n this  r e g i o n a l t h o u g h a p r o l i n e r e s i d u e j u s t 3* t o the conserved domain shown i n F i g u r e 10 has a l s o been conserved  3.6  (see F i g u r e 16).  I s o l a t i o n of B r i s t o l Genomic DNA  Clones  The 0.5 kb cDNA coding f o r t h e hspl6-48 v a r i a n t was used to s c r e e n two s e p a r a t e phage l i b r a r i e s , one a. p a r t i a l EcoRI d i g e s t of B r i s t o l . DNA i n . Charon4 and the o t h e r a p a r t i a l Mbol d i g e s t i n EMBL4.  By- r e s t r i c t i o n  90  enzyme mapping of o v e r l a p p i n g  recombinant phage clones  d e f i n e a r e g i o n of 30 kb which i s shown i n F i g u r e r e s t r i c t i o n d i g e s t s was  i t was  12A.  p o s s i b l e to  Southern b l o t t i n g of  used to i d e n t i f y those EcoRI fragments which  h y b r i d i z e d to the hspl6-48 cDNA.  They i n c l u d e d two  fragments of 3.3  kb  which comigrated and which c o u l d be d i s t i n g u i s h e d by the presence or absence of a S a i l s i t e and ( F i g u r e 12A).  were p u r i f i e d  of s p e c i f i c DNA  fragments, the 3.3L  from the phage X.Charon4 A - l and  fragment was  A d e t a i l e d r e s t r i c t i o n map covering  7.6  kb,  another open r e a d i n g designated  kb  fragments  subcloned i n t o pBR325.  The  EcoRI 12A.  I d e n t i f i c a t i o n of a P e r f e c t 1.9  hsp!6-l.  The  an u n p a i r e d  loop,  two  sequenced u s i n g  genes are separated  kb  the  f o r another  by 347  bp  D e t a i l e d a n a l y s i s of the lambda  d u p l i c a t e d nearby as a p e r f e c t 1.9  T h i s unusual o r g a n i z a t i o n  i n v e r t e d repeats  kb was  frame which d e f i n e s a gene coding  t h a t t h i s gene p a i r was  inverted repeat. kb  1.0  for  Sequence a n a l y s i s i d e n t i f i e d the hsp!6-48  arranged i n d i f f e r e n t o r i e n t a t i o n s . revealed  also  Repeat  s t r a t e g y shown i n F i g u r e 12B.  16 kd hsp  and  i s a l s o shown i n F i g u r e  A contiguous r e g i o n of a p p r o x i m a t e l y 4.4  gene and  kb was  of these t h r e e adjacent  Sequencing of the Hspl6 Genes and  Inverted  respectively  subcloned i n t o pUC13 a f t e r i s o l a t i o n from phage  fragments, t o g e t h e r  3.7  3.3R,  For more d e t a i l e d r e s t r i c t i o n endonuclease a n a l y s i s and  bulk preparation  XEMBL4-2.  and  Another h y b r i d i z i n g EcoRI fragment of 1.0  identified.  3.3R  w i l l be r e f e r r e d to as 3.3L  were separated  by 416  i s shown i n F i g u r e  bp of unique DNA  i f the o t h e r w i s e p a l i n d r o m i c  12A.  and  are  clones kb The  1.9  which would form  s t r u c t u r e were d i s p l a y e d  in a  91a  F i g u r e 12. and  O r g a n i z a t i o n of the hsp!6-48 and h s p ! 6 - l  genes of C. elegans,  t h e DNA sequencing s t r a t e g y . (A) Map o f o v e r l a p p i n g  recombinant phage i n s e r t s and sequence  o r g a n i z a t i o n o f the s m a l l heat shock gene domain.  The complete EcoRI and  S a i l r e s t r i c t i o n maps of the 30 kb r e g i o n a r e shown. ends o f t h e \EMBL4 phage i n s e r t s r e p r e s e n t 1.6 kb EcoRI fragments a t the f a r l e f t \Charon4 B-3, b u t t h e i r  Solid  Sau3A s i t e s .  c i r c l e s a t the  The 0.9 kb and  ( i n p a r e n t h e s e s ) were i d e n t i f i e d i n  o r i e n t a t i o n s have n o t y e t been determined.  The  7.6 kb r e g i o n d e f i n e d by the 3.3L, 3.3R, and 1.9 kb EcoRI fragments has been enlarged  t o show t h e d e t a i l e d r e s t r i c t i o n map and gene o r g a n i z a t i o n .  arms of t h e p e r f e c t 1.9 kb i n v e r t e d repeat s m a l l heat shock genes a r e i n d i c a t e d . bars,  introns.  The  (IR) and the o r i e n t a t i o n of the  S o l i d b a r s , coding  sequences; open  The 0.9 kb BamHI-Xbal fragment which was used to d e t e c t  h s p l 6 - l s p e c i f i c mRNAs i s shown above the map.  (B) S t r a t e g y used to  sequence t h e 4.4 kb r e g i o n which i n c l u d e s both arms o f the i n v e r t e d  repeat,  the loop r e g i o n between them, and the d i s t a l boundary r e g i o n s . A l l sequencing was done w i t h M13 s i n g l e - s t r a n d e d phage. d i r e c t i o n and e x t e n t o f the sequence obtained shown.  Arrows i n d i c a t e the  from the r e s t r i c t i o n  The u n l a b e l l e d s i t e s a r e Sau3A r e s t r i c t i o n  sites.  sites  91 tn < < in  sz  JZ  r.  <  <  c  (J O O "J UJ (J  5 ]  92  stem-loop c o n f i g u r a t i o n . d e t a i l in section To G.  The  genes themselves w i l l be d i s c u s s e d  in greater  3.8.  show t h a t t h i s sequence arrangement i s a c t u a l l y p r e s e n t  elegans B r i s t o l genomic DNA,  extensive  out w i t h a v a r i e t y of probes ( F i g u r e 13). o r i g i n a l l y used to screen  Southern b l o t a n a l y s i s was Probe A,  the phage l i b r a r i e s ,  the cDNA t h a t  kb EcoRI fragment was  t h a t the p r e d i c t e d s e l f - c o m p l e m e n t a r i t y  carried  was  i d e n t i f i e d an i n t e n s e 3.3  EcoRI band which i s c o n s i s t e n t w i t h the phage map. e v i d e n c e of the expected 1.0  in  On  the other hand,  seen.  kb no  I t seemed p o s s i b l e  of t h i s fragment might  be  i n t e r f e r i n g w i t h h y b r i d i z a t i o n of the cDNA, which i s complementary to p a r t o f the i n v e r t e d repeat.  To circumvent t h i s problem, an attempt was  d i s r u p t the i n v e r t e d repeat  s t r u c t u r e by d i g e s t i n g B r i s t o l DNA  made to  with B e l l ,  r e s t r i c t i o n enzyme which c l e a v e s  i n the loop r e g i o n .  B,  bp B g l l l - B c l l i n s e r t , r e s u l t e d i n genomic  an M13  clone containing  B e l l fragments of 0.8  and  a 150 0.65  kb,  T h i s probe a l s o i d e n t i f i e d a 1.1 i t was  d e r i v e d from the two  The detect  arms of the i n v e r t e d  kb EcoRI fragment and  clone  c o n t a i n i n g a 500  to the loop r e g i o n , was  kb S s t l l fragment, a 4.0  since  repeat.  help  was  to i d e n t i f y other  also investigated.  used.  12A.  characteristic Probe C,  T h i s probe i d e n t i f i e d  kb B g l l l fragment, a 1.8  kb BamHI fragment, and  a l l of which were p r e d i c t e d from the i n v e r t e d repeat Figure  map.  more i n t e n s e  bp Ddel-EcoRI i n s e r t , of which 200  EcoRI fragment along w i t h a 1.1 3.75  c o n s i s t e n t w i t h the phage  p o s s i b i l i t y t h a t a probe which i n c l u d e d the loop r e g i o n might  the 1.0  specific  H y b r i d i z a t i o n to probe  kb B g l l l band which was  fragments from the i n v e r t e d repeat M13  again  a  bp  another  was  the 1.0  kb  kb P s t I fragment, a  a 4.1  kb Xhol fragment,  structure presented i n  93  F i g u r e 13.  A n a l y s i s of C. elegans B r i s t o l genomic  Approximately 2 yg of DNA  was  DNA.  digested with the indicated  enzyme, s e p a r a t e d by e l e c t r o p h o r e s i s on e i t h e r 1.5%  restriction  (probe B) o r  (probes A and C) agarose g e l s , and t r a n s f e r r e d t o n i t r o c e l l u l o s e . CEHS16-48 cDNA; probes B and C, i n s e r t s i n M13 shown i n k i l o b a s e s .  clones.  Fragment  0.7% Probe A, s i z e s are  Below the r e s u l t s f o r each probe i s shown the r e g i o n  t h a t i t r e p r e s e n t s , along w i t h the l e n g t h s of the expected h y b r i d i z i n g fragments f o r each r e s t r i c t i o n enzyme p r e d i c t e d from the map I, Regions from which probes B and C were d e r i v e d ; S,  i n F i g . 12A.  identical  sequences i n the o t h e r arm of the i n v e r t e d r e p e a t ( I R ) .  The markers  w i t h probes A and C were a H i n d l l l d i g e s t of lambda DNA;  those used w i t h  probe B were a mixture of A c c I , H i n d i ! , and H p a l l d i g e s t s of pBR322.  used  94  In F i g u r e 13, the hsp!6-48 cDNA (probe A) h y b r i d i z e d a l s o to a 2.2 EcoRI fragment.  T h i s i s a t t r i b u t e d to sequences coding f o r the hsp!6-41  v a r i a n t which have been c l o n e d and  c h a r a c t e r i z e d (Jones e t a l . , 1986).  a d d i t i o n a l f a s t e r m i g r a t i n g bands seen i n F i g u r e 12C palindromic  kb  c h a r a c t e r of t h i s r e g i o n and  are a r e s u l t of  can be e x p l a i n e d by the  The  the  formation  of f o l d b a c k s t r u c t u r e s d u r i n g the r e s t r i c t i o n enzyme d i g e s t i o n s done at h i g h e r temperatures.  The  a b i l i t y to form these s t r u c t u r e s i s d i r e c t l y  c o r r e l a t e d w i t h the degree of n i c k i n g o r s i n g l e - s t r a n d e d n e s s DNA,  which v a r i e d w i t h d i f f e r e n t p r e p a r a t i o n s .  s t r u c t u r e s are not found  i n high molecular  h i g h l y r e s i s t a n t to SI n u c l e a s e  3.8  was  The  i n the  foldback  weight genomic DNA  (data not shown).  s t r u c t u r e s can be seen i n F i g u r e 23 B r i s t o l DNA  Thus, these  found  which i s  absence of  foldback  i n which a d i f f e r e n t p r e p a r a t i o n of  used.  I d e n t i f i c a t i o n of the Small Heat Shock Genes As a l r e a d y mentioned, sequence a n a l y s i s i d e n t i f i e d f o u r open r e a d i n g  frames coding  f o r two  d i f f e r e n t hspl6s.  F i g u r e 14 shows 4.4.kb of  sequence which i n c l u d e s both arma of the 1.9 shsp genes. was  taken  to be the f i r s t ATG  i n the open r e a d i n g frame.  to CEHS48 coded f o r a p r o t e i n of 143  l o n g e r a t the NH^-terminal end  The  gene  amino a c i d s , 8 r e s i d u e s  than the p r o t e i n sequence determined from  The m o l e c u l a r weight of t h i s hsp was  a c i d composition molecular  kb i n v e r t e d repeat and a l l f o u r  In each case, the presumed i n i t i a t i o n s i t e of p r o t e i n s y n t h e s i s  corresponding  the cDNA.  DNA  c a l c u l a t e d from the amino  (Table V) to be 16,299, which i s i n good agreement w i t h  weight d e r i v e d from the r e s u l t s of SDS-polyacrylamide g e l  electrophoresis.  the  95a  F i g u r e 14. C.  Complete sequence of a s m a l l heat shock gene c l u s t e r of  elegans. The sequence begins 5* to t h e b o r d e r  of t h e l e f t arm of the i n v e r t e d  r e p e a t , which s t a r t s a t p o s i t i o n 114 and ends a t p o s i t i o n 2005.  In t h i s  arm  we have u n d e r l i n e d t h e noncoding s t r a n d o f the h s p ! 6 - l gene, and the coding s t r a n d o f the hsp!6-48 gene has been t r a n s l a t e d .  The converse  i s shown i n  the r i g h t arm, which begins a t p o s i t i o n 2422 and continues t o p o s i t i o n 4313.  The TATA sequences and the p o l y a d e n y l a t i o n s i g n a l s f o r a l l f o u r  r e a d i n g frames a r e boxed. u n d e r l i n e d and l a b e l l e d  The HIPs of the h s p ! 6 - l and hsp!6-48 genes are  (see t e x t f o r d i s c u s s i o n ) .  The s t a r t s of  t r a n s c r i p t i o n a r e shown with arrows over the coding strands of the hsp!6-48 ( p o s i t i o n 1344) and h s p ! 6 - l  ( p o s i t i o n 3339) genes.  r e g i o n s a r e shown s t r a d d l i n g R s a l s i t e s between Stretches  Potential  Z-DNA-forming  the two p a i r s of genes.  (12bp) of a l t e r n a t i n g p u r i n e - p y r i m i d i n e r e s i d u e s are a l s o shown  w i t h i n the G + C - r i c h boundary sequences. hexanucleotides change between  They a r e bordered  which form i n v e r t e d r e p e a t s as shown.  The  by one-nucleotide  the two i n v e r t e d r e p e a t s i s shown a t p o s i t i o n 4304 i n the  boundary sequence o f the r i g h t  arm.  95  DOE I 20  40  SO  I  tOO  80  120  IR XHOI Z 140. TCGGGGCCGCGCGCACGCAGCCC GC  Boundary  BAMHI  160 180 200 220 240 AATACGCAAAAATTAAGCTGTTGGCGAATCTCAACTCGAGACTGCCACACAGGTCAGCCCATCCAAAAGTGCAATGGATAGGCCTCACCATGOOGA  Sequerx*  SAC1I  260 280 SCO 320 340 360 TCCCGCCTTCATGATCGCGAGATAACCCCCAGCCAGOTAAQTTTAAGAAGTGGCGGTAAGAGAGGGTAAGGGTQTTGTATAGCATGACATCTGGCGGGTTCCGCGGACGAATGCAGAATG RSAI 380 400 420 440 460 480 TGTTAGGATGGGAGGGGCTGTGCAATACCCAAAAATAGGCATTATGCAAGAAGTACATTGCCGGCATTTACATATTTATTACTAACTTTCAAAAAATATCACAAATTCATATTATTTTTA  AAT|TTTATTJGAAACAGAATACTGGAATTTATAGTAATTACATGCATAGTTCAAAAAAATCATAAAATTACAATAAJTATTCAGAAGTTTTTTGTTCAACGGGCGCTTGCTGAATTGGAAT BGLII 620 640 660 680 70O 720 AGATCTTCCTTGAATCGCTTCCTTCTTTGGTGCTTCAATTGAAAGTTTTCCATCTTCTGAAAGATTTGAAGCAACTGCACCAACATCAACATCTTCGGGTAGAAGAATAACACGAGAAAA  740 760 780 800 820 840 TGATTTCTTTGAATATCCATGTTCAGTCTTTAATTCTTGTTCTCCTTGAATTGATAATGTATGTCCATCCAAATTAATTTTCAAATCTTCTGGCTTGAACTGCGAGACATTGAGATTTAT  BCLI  : t  OOEI  860 S80 9O0 920 940 960 GGCAAACTTTTGATCATTGTTAACAATCTGAAAGAAATCTTTTTTATACAAACTCTTGAAAAAAAAATGTGTTACTTACCTCAGAAGATTCAGATGGAGAGCCTCTGCAAACTGGAGTAA  DOE I 980  lOOO  ATTGACGTTCCATCTGAGCCATATCTCTCATGAGATCA  1020  1040  1060  1080  CCAAAAACAGAACGTTGAGCTGCACGGAAATAGTGOTAAAGTGACATGATTGTAGTTTGAAGATTTCACAATTAGAGTGAA  HSP16-1  XBAI  110O  1120  1140  1160  1180  120O  TGTTGTTTGGTTCGGTTTTGTCACTGTAITTTATAI^TCATTTCCACCTTTTTCTAGAACATTCGAGCTGCTTCTTGCAAAAGGAGGGCGACTCACATTCAGAACATTGAGAAATAGTGTGC RSAI  • H I P 1220  1240  X  1260  B  p  M  mr-w  128Q  )  3  0  S  T  Z  I  132Q  O  GTACTGAAGAAACCCAGATACTTTTTCAATCTGCGTCTCTTTGCACCTATGGGGTGTATTTTGAAATGAATG  t  HSP16~48 1360  ^  M  1380  L  M  L  R  S  P  1400  F  S  D  S  N  V  L  1420  D 1440  ATAA^CCAATCGTGTTCAGAGGAAACCAATACACTTTGTTCAAGTGCTTACTGTTCATTCTCTAAACTTCAAGAATGCTCATGCTCCGTTCTCCATTTTCTGATTCAAATGTTCTCGATC H  F  L  O  E  I  T  G  S  V  O  F  P  Y  W  R  N  A  O  H  N  S  F  N  F  S  D  N  I  G  E  BCLI 1460 1480 1SO0 1520 1540 1560 ATTTCTTGGATGAAATCACTGGATCTGTTCAATTTCCATATTGGAGAAATGCTGATCACAACTCATTCAATTTTTCCGACAATATTGGAGAGGTAAGAAAATAATCTCTTTTTCAATTGT I V N D E S K F S V Q L O V S H F K P E O L K I E L D G R E L  BGLI I  1580  16O0  1620  1640  1660  1680  TTATTTQTCAAATQTTTTATTTTTCAQATTQTAAATQACQAATCTAAATTCTCTGTTCAACTCGATGTTTCTCATTTCAAACCAGAAGATCTTAAAATTQAATTGGATGGAAGAGiAACTA K  I  E  G  I  O  E  K  K  S  E  H  G  V  S  K  R  S  F  S  K  M  I  L  L  P  E  O  V  O  L  T  S  V  K  S  A  I  S  N  ECORI 1700 1720 1740 1760 1780 180O AAAATTGAAGGAATTCAAGAAAAAAAATCAGAGCATGQATACTCGAAACGATCATTTTCAAAAATGATTCTTCTACCAGAAQATGTTGATTTAACTTCTOTCAAATCTGCAATTTCGAAT E  G  K  L  O  I  E  A  P  K  K  T  N  S  S  R  S  I  P  I  N  F  V  A  K  H  *  1820 1840 1860 1880 19O0 GAAGGAAAACTTCAAATTQAGGCTCCAAAQAAGACTAACTCATCTCQTTCTATTCCCATTAATT.TTGTTGCAAAACATTAATCTTTTATTGTATTCCAAATATTCTTAATTT(3AA  polyA •  1920 TAA~A)S  site 1940  1960  1980  2000  I  2020  2040  TCATTAATTTAATTTATTCATGTTCTCTAGCATAACAAAAACATCAAATCCGACTTTCCAATTCAAATATTTCAAAACAACATAAITTGAAGTTATTCCAGAAACTTTTTATGCAAAAAAT  206O  2080  2 tOO  IR » 2120  1 2140  2160  TTATTAGTCTCAATAAATGTTTTAOCTTGAATTTATGCTTAAAACAAAAAACATAAAAAATGTTTTAAAAAAATTACAGTGCQTGCAACTTCTACCAGGGCCCTCATAAAATAGGTTCTT DOE I  BCLI  2180 2200 2220 2240 2260 2280 CAAGAOAAAAATCAOAAAATATTTTAAOCTACCAOCTACTTCGGCTTCTC J0AAAACTCCTGCGAC7T0ATAAAAACACTTAAAAACTGTCAAAAGCTGTTA7AAAMTGCAOTTGATjCi J  96  23CO  3330  3340  3360  33SO  3400  ACACAGTTGTGAGCGAAATTAAGCTOAAAAACTQAATTTTGAAAAAQTTGCAAAAQTCGTTTGAAACGATGCAGGAATTTTTATAGCTAAACGTTTAACGGTTGGTAAAACGTAGCATGA  3430  I  3440  3460  34BO  2500  2520  QTATGCTQTAAAQTTGAQCCG|rTATGTTGTTTTGAAATATTTOAATTGGAAAGTCGGATTTGATGTTTTTGTTATGCTAGAGAACATGAATAAATTAAATTAATGAC|rTT ATT^SAAATTA  1  H R  3340  3560  3580  3600  3630  3640  AGAATATTTGGAATACAATAAAAGATTAATGTTTTGCAACAAAATTAATGGGAATAGAACGAGATGAGTTAGTCTTCTTTGGAGCCTCAATTTGAAGTTTTCCTTCATTCGAAATTGCAG ECORI 3660  3680  3700  3730  3740  3760  ATTTGACAGAAGTTAAATCAACATCTTCTGGTAGAAGAATCATTTTTGAAAATGATCGTTTCGAGTATCCATGCTCTGATTTTTTTTCTTGAATTCCTTCAATTTTTAGTTCTCTTCCAT BGLII 3780  3800  3830  3840  2860  2880  CCAATTCAATTTTAAGATCTTCTGGTTTGAAATGAGAAACATCGAGTTGAACAGAGAATTTAGATTCGTCATTTACAATCTGAAAAATAAAACATTTGACAAATAAACAATTGAAAAAGA BCLI 29O0  2930  2940  2960  2980  30O0  GATTATTTTCTTACCTCTCCAATATTGTCGGAAAAATTGAATGAGTTGTGATCAGCATTTCTCCAATATGGAAATTGAACAGATCCAGTGATTTCATCCAAGAAATGATCGAGAACATTT PSTI 3O30  3O40  3060  3080  3100  3130  QAATCAGAAAATGGAGAACGGAGCATGAGCAT.TCTTGAAGTTTAGAGAATGAACAGTAAGCACTTGAACAAAGTQTATTGGTTTCCTCTGAACACGATTGGC|TTATATA)CCCGTATCCTG  HSP16-48  X8A1 RSAI 3140 . 3160 3180 3200 3330 3240 CAGCCGTTTAGAATGTTO*T"AGAAGGTCCT'AGATGCATTCATTTCAAAATACACCCCATAGGTGCAAAGAGACGCAGATTGAAAAAGTATCTGGGTTTCTTCAGTACGCACACTATTTCTC  * XBAI * 3260 3280 3300 3320 33fO 3360 AATGTTCTGAATGTGAGTCGCCCTCCTTTTGCAAGAAGCAGCTCGAATGTTCTAGAAAAAGGTGGAAATGAG|rATAAA|rACAGTGACAAAACCGAACCAAACAACATTCACTCTAATTGT H  ,  P  Z  ^  H  HSP16 —1 " " " ^ ^ —  G  S  P  S  E  S  5  S  L  »  M  l  M  p  F « P * 0 » » V F Q 3380 340O 3430 GAAATCTTCAAACTACAATCATGTCACTTTACCACTATTTCCGTCCAGCTCAACGTTCTGTTTTTGG R  M  Y  V  R  O  L  M  A O DOEI  M  E  R  O  F  T  P  V  C  3440 3460 3480 TGATCTCATGAGAGATATGGCTCAGATGGAACGTCAATTTACTCCAGTTTGC  E  I V N N D Q K - F A I N L . N V ODEI BCLI 35O0 3520 3540 3560^, ^ _ 3580 3600 AGAGGCTCTCCATCTGAATCTTCTGAGGTAAGTAACACATTTTTTTTTCAAGAGTTTGTATAAAAAAGATTTCTTTCAGATTGTTAACAATGATCAAAAGTTTGCCATAAATCTCAATGT S  O  F  K  P  E  D  L  K  I  N  L  O  G  H  T  L  S  I  Q  G  E  Q  E  L  K  T  E  H  G  Y  S  K  K  S  F  S  R  V  I  3620 3640 3660 3680 3700 3720 CTCGCAGTTCAAGCCAGAAGATTTGAAAATTAATTTGGATGGACATACATTATCAATTCAAGGAGAACAAGAATTAAAGACTGAACATGGATATTCAAAGAAATCATTTTCTCGTGTTAT L  L  P  E  O  V O V G A V A S N L S E O G K L S I E A P K K E A I O G R S I P I O O BGLII 3740 3760 3780 3800 3820 3840 TCTTCTACCCGAAGATGTTGATGTTGGTGCAGTTGCTTCAAATCTTTCAGAAGATGGAAAACTTTCAATTGAAGCACCAAAGAAGGAAGCGATTCAAGGAAGATCTATTCCAATTCAGCA  AGCGCCCQTTGAACAAAAAACTTCTGAATAATTATTGTAATTTTATGATTTTTTTGAACTATGCATGTAATTACTATAAATTCCAGTATTCTGTTTCJAAT  A AAA|TTTAAAAATAAT ATGA  RSAI 3980  40O0  4020  4040  4060  4080  ATTTGTGATATTTTTTGAAAGTTAGTAATAAATATGTAAATGCCGGCAATGTACTTCTTGCATAATGCCTATTTTTGGGTATTGCACAGCCCCTCCCATCCTAACACATTCTGCATTCGT SAC 11  BAMHI 4100  4120  4140  4160  4180  4200  CCGCGGAACCCGCCAGATGTCATGCTATACAACACCCTTACCCTCTCTTACCGCCACTTCTTAAACTTACCTGGCTGGGGGTTATCTCGCGATCATGAAGGCGGGATCCCCATGGTGAGG XHOI 4220  4240  4260  4280  I  4300  4320  CCTATCCATTGCACTTTTGGATGGGCTGACCTGTGTGGCAGTCTCGAGTTGAGATTCGCCAACAGCTTAATTTTTGCGTATC?GGGGCTGCGTGCGCGCGGCCCTGAAAAATATA TTAATA RSAI 4335  4350  4365  GC 4380  4395  Boundary Sequence 4 4 10  CTGTTTTCAATAGTAATTGGCATAACGAATCAGTTTCAAGTAAAACGTACGTCA ATTTCAGTAGCAGAACGTTCAA ACAGTTTTCTATCTGATC  97  T a b l e V.  Amino A c i d Compositions o f Hspl6-1 and Hspl6-48  No. o f r e s i d u e s Amino A c i d  hspl6-l  hspl6-48  Ala  8  4  Arg  7  5  Asn  6  9  Asp  7  11  Cys  1  0  Gin  11  4  Glu  13  12  Gly  8  6  His  3  5  He  9  11  Leu  11  12  Lys  10  13  Met  4  3  Phe  - 6  9  Pro  8  6  Ser  16  19  Thr  4  3  Trp  0  1  Tyr  4  2  Val  9  8  98  The o t h e r gene codes f o r a p r o t e i n o f 145 amino a c i d r e s i d u e s with a c a l c u l a t e d molecular  weight o f 16,301.  To determine whether t h i s gene i s  t r a n s c r i b e d under heat shock c o n d i t i o n s , a 0.9 kb BamHI-Xbal  fragment  c o n t a i n i n g the gene f o r h s p l 6 - l ( F i g u r e 12A) was used t o probe dot b l o t s o f polyA RNA p u r i f i e d from c o n t r o l and heat shocked nematodes. +  of t h i s experiment a r e shown i n F i g u r e 15.  The r e s u l t s  Transcripts specific  t o the  h s p ! 6 - l gene were abundant i n mRNA i s o l a t e d from heat shocked nematodes b u t were n o t d e t e c t a b l e i n n o r m a l l y  growing nematodes.  This i s s i m i l a r to the  h i g h i n d u c i b i l i t y demonstrated f o r mRNA sequences complementary t o CEHS48 and  CEHS41 ( F i g u r e 8 ) .  identified inverted  Thus, two d i s t i n c t , heat i n d u c i b l e genn3 havo boon  i n t h i s r e g i o n , and t h i s gene p a i r hau been d u p l i c a t e d to form an  repeat.  Hspl6-1 shows e x t e n s i v e homology w i t h hsp!6-48 and hsp!6-41. and the t h r e e p r e d i c t e d amino a c i d sequences have been a l i g n e d i n F i g u r e 16. NH^-terminal r e g i o n s o f hspl6-48 and h s p l 6 - l are completely  The  different.  It  i s e v i d e n t t h a t t h i s d i v e r g e n t r e g i o n d e f i n e s an exon a t the l e v e l o f gene o r g a n i z a t i o n ( s e c t i o n 3.9).  The r e s t o f the p o l y p e p t i d e  c h a i n which d e f i n e s  the second exon, i s very s i m i l a r among the t h r e e C. elegans p o s i t i o n s being approximately  identical.  hsps, 55 o f 98  T h i s homologous r e g i o n c o n t a i n s t h e  80 amino a c i d domain t h a t shows homology t o the shsps o f  D r o s o p h i l a melanogaster and t o the a - c r y s t a l l i n p r o t e i n s .  I n t h i s domain  the same amino a c i d i s used by a t l e a s t f o u r o f the f i v e p r o t e i n s i n 32 o f 80 p o s i t i o n s .  A l s o shown a r e the 20 p o s i t i o n s t h a t are conserved between  the p r o t e i n s shown and the shsps o f D r o s o p h i l a .  99  F i g u r e 15.  RNA d o t - b l o t a n a l y s i s u s i n g a probe s p e c i f i c f o r the h s p ! 6 - l  gene. 0.5 yg o f polyA RNA p u r i f i e d  from c o n t r o l or h e a t shocked worms was  a p p l i e d t o n i t r o c e l l u l o s e b e f o r e b a k i n g and h y b r i d i z i n g .  A 0.9 kb  BamHI-Xbal fragment c o n t a i n i n g a h s p ! 6 - l gene was used as the probe.  100a Figure  16.  Comparison  o f t h e deduced  amino a c i d sequences  of t h r e e  shsps of  C. e l e g a n s , t h e mouse a A ^ - c r y s t a l l i n c h a i n , and t h e bovine aB^-crystallin  chain.  The p r o t e i n s have been a l i g n e d t o demonstrate emphasize t h e s i m i l a r l o c a t i o n s o f i n t r o n s .  sequence homology and t o  The l o c a t i o n o f t h e i n t r o n i n  the hsp!6-41 gene i s taken from Jones e t a l . (1986). a A ^ c r y s t a l l i n amino a c i d sequence  Most o f t h e mouse  i s taken from King e t a l . (1982), and  the N H - t e r m i n a l p o r t i o n and t h e l o c a t i o n o f t h e two i n t r o n s a r e taken 2  from K i n g and P i a t i g o r s k y (1983).  The p r o t e i n sequence  of bovine  a B ^ c r y s t a l l i n was i n c l u d e d t o i l l u s t r a t e the r e l a t i o n s h i p between t h e two a - c r y s t a l l i n c l a s s e s and i s taken from van d e r Ouderaa e t a l . (1973). I t i s n o t known a t t h i s time whether the bovine a B ~ c r y s t a l l i n gene 2  contains  i n t r o n s , a l t h o u g h the hamster a B ~ c r y s t a l l i n gene has 2  m a i n t a i n e d t h e e x o n / i n t r o n o r g a n i z a t i o n shown by t h e murine < * A - c r y s t a l l i n gene (see s e c t i o n 3.9). 2  B r a c k e t s show the b o r d e r s of the  74 amino a c i d r e g i o n which was used i n F i g u r e 11.  P o s i t i o n s o f homology i n  which t h e same amino a c i d i s shared by a t l e a s t f o u r o f t h e f i v e a r e boxed.  proteins  S o l i d c i r c l e s i n d i c a t e p o s i t i o n s t h a t have been conserved  between t h e s e f i v e p r o t e i n s and t h e shsps of D. melanogaster.  H $ P 1 6 - 4 1 H SP16  - 4 8  H 5 P1 a l p h a  • A  2  m o u s e )  a l p h a  • B  2  b o v i n e )  W  ©  ®  L  F  L  D  E  I  R  S  V  F  G  Y  W  F  K  R  A  L  G  P  F  -  Y  P  S  R  L  F  D  0  F  F  G  E  G  L  F  E  Y  0  i  L  P  F  L  S  S  P  U  I  R  R  P  F  F  P  F  H  S  P  S  R  L  F  0  0  F  F  G  E  H  L  L  E  S  D  F  L  P  -  A  s  T  I  V  N  0  E  S  K  I  V  II  0  E  s  1  • •  !  -  58  bp  I n t r o n  H  N  S  F  H  F  S  0  H  r  G  E  -  55  bp  I n t r o n  L  M  P.  0  M  A  0  M  E  R  0  F  T  P  V  c  R  G  S  P  S  E  s  S  E  -  52  bp  I n t r o n  T  I  s  p  Y  Y  -  P.  0  -  s  L  F  R  -  -  T  V  L  0  S  G  i  S  E  -  13 7 6  S  I  s  p  F  Y  L  R  P  P  s  F  L  R  A  p  S  W  I  0  T  G  L  S  E  -  P  E  M  L  K  I  K  L  0  G  R  E  L  K  I  E  G  I  Q  E  T  X  S  -  No  I n t r o n  P  E  D  L  It  I  E  L  D  G  R  E  L  K  I  E  G  I  0  E  K  K  s  -  No  I n t r o n  P  E  D  I  K  I  H  L  D  G  H  T  L  S  I  0  G  E  Q  E  L  K  I  -  Mo  I n t r o n  P  0  0  L  T  V  K  V  L  E  0  F  V  E  I  H  G  K  H  N  E  R  0  -  2 n d  P  E  E  L  K  V  K  V  L  _G_  D  V  I  E  _y_  K  H _E_  E  R  Q  -  I n t r o n 7  P  5  S  N  E  G  K  T  S  s  N  E  G  K  •  I  H  q  P  0  A  0  A  H  A  s  L  P  H  N  K  V  F  H  R  V  N  R  0  w  •  S  Y  I  Y  •  D  H  I  P  I  S  Y  T  F  A  F  L  A  0  s  P  S  I  V  K  S  V  S  V  R  0  G  • •  L  0  T  •  M  ®  6-1  •  •  H _G_  L  0  I  E  A  I  Q  i  E  A  •  •  bp  i  I n t r o n  -  D  V  0  T  K  F  s  V  0  ••  •  D  V  S  H  F  K  L  D  V  s  H  F  K  L  I  V  N  N  0  Q  K  F  A  I  N  H  V  5  0  F  K  V  R  S  0  R  D  K  F  V  I  V  K  H  F  s  M  R  L  E  K  0  R  F  S  V  K I D H -L H  V  K  H  F  5  H  G  Y  L  K  R  5  F  S  K  M  I  I  L  P  E  0  A  0  L  E  H  G  Y  S  K  R  S  F  S  K  M  I  L  I  P  E  D  V  0  L  E  H  G  Y  s  K  K  S  F  S  R  V  I  L  L  P  E  D  V  D  V  0  H  G  Y  I  S  R  E  F  H  R  R  Y  R  L  P  S  N  V  0  0  E  H  G  F  I  S  _R_  E  F  H  R  K  Y  R  I  P  A  D  V  D  P  I n t r o n  - - -- -0  S  -  I n t r o n ?  j  F  E  •  •  P  K  K  T  N  s  S  R  S  I  P  I  N  F  V  A  K  H  P  K  K  T  N  s  S  R  S  I  P  I  N  F  V  A  K  H  •  G  A  V  A  s  N  L  s  E  0  G  K  L  S  i  E  A  P  K  K  E  A  I  QG R  S  I  P  I  0  0  A  P  V  E  0  K  T  S  E  S  A  L  S  c  5  L  s  A  D  G  M  L  T  F  S  G  P  K  V  Q  S  G  L  D  A  G  H  S  E  R  A  I  P  V  S  R  E  E  K  P  -  -  L  A  I  T  s  S  L  s  S  0  G  V  L  T  V  0  G  P  R  K  Q  •-  -  -  -  A  S  G  P  E  R  T  I  P  I  T  R  E  E  K  P  A  V  S T  •  S  A  P  S  S  A  A  P  K  K  101  3.9  The shsp Genes of C. elegans C o n t a i n a S i n g l e I n t r o n Comparison of the hsp!6-48 gene w i t h the CEHS48 cDNA r e v e a l e d a s h o r t  i n t r o n of 55 bp which d i v i d e d the coding r e g i o n i n t o two exons. gene had a c o r r e s p o n d i n g i n t r o n of 52 bp.  The h s p l 6 - l  The e x o n - i n t r o n boundary  sequences match w e l l w i t h the consensus sequence d e s c r i b e d by Mount (1982). The l a c k of long p y r i m i d i n e t r a c t s p r e c e d i n g the 3' s p l i c e j u n c t i o n i s common to o t h e r C. elegans i n t r o n s ( S p i e t h e t a l . ,  1985) and may  s i m p l y be a  consequence o f s i z e r e s t r a i n t s , the average i n t r o n being a p p r o x i m a t e l y 50 bp. The homology between the shsps of C. elegans and v e r t e b r a t e a - c r y n t a l l i n was of the  also r e f l e c t e d  i n t h e i r gene o r g a n i z a t i o n .  The p o s i t i o n  the i n t r o n i n the nematode heat shock genes was p r e c i s e l y analogous to p o s i t i o n of the f i r s t  i n t r o n i n the a A ^ - c r y s t a l l i n genes of mouse  (King and P i a t i g o r s k y , 1983) and hamster (van den Heuvel e t a l . , the  hamster a B ^ - c r y s t a l l i n gene (Quax-Jeuken e t a l . ,  a A ^ - c r y s t a l l i n genes c o n t a i n w i t h i n t h e i r f i r s t  1985).  1985) or  The  i n t r o n a 23 amino a c i d  i n s e r t i o n sequence which i s expressed i n 10% of the messages due t o alternative splicing.  I n t e r e s t i n g l y , a l l of the c r y s t a l l i n genes mentioned  above c o n t a i n a second i n t r o n , not found i n the hsp genes, which i n t e r r u p t s the  conserved domain.  I n t r o n s have not been found i n the r e l a t e d shsp genes  from D r o s o p h i l a ( I n g o l i a and C r a i g , 1982b; Southgate e t a l . ,  1983; Ayme and  T i s s i e r e s , 1985), Xenopus ( B i e n z , 1984b) or soybean (see T a b l e I ) .  On t h e  o t h e r hand, t h e f u n c t i o n a l gene coding f o r human hsp25 c o n t a i n s two  introns  (Hickey e t a l . ,  1986).  A l t h o u g h human hsp25 shows more s i m i l a r i t y t o t h e  a - c r y s t a l l i n s than t o any o t h e r shsps, the l o c a t i o n s of the two have n o t been conserved.  introns  I n the C a e n o r h a b d i t i s hsp!6 genes and the  v e r t e b r a t e a - c r y s t a l l i n genes, the f i r s t  s p l i c e j u n c t i o n precedes the  102  r e g i o n which shows the g r e a t e s t homology between the shsps and the o-crystallins.  I n the human hsp gene, the second exon corresponds t o the  h i g h l y h y d r o p h i l i c r e g i o n o f 23 amino a c i d s which i s found w i t h i n t h e l a r g e r 80 amino a c i d conserved domain.  3.10  Location of the S t a r t s of T r a n s c r i p t i o n The h s p ! 6 - l and hsp!6-48 genes a r e c l u s t e r e d w i t h i n a r e l a t i v e l y  r e g i o n of DNA.  Furthermore, they are t r a n s c r i b e d i n o p p o s i t e d i r e c t i o n s ,  w i t h the r e s u l t t h a t o n l y 34 7 bp s e p a r a t e t h e i r r e s p e c t i v e ATG codons.  Therefore, i t i a l i k e l y  initiation  that a l l s i g n a l s r e q u i r e d f o r a c c u r a t e heat  i n d u c i b l e t r a n s c r i p t i o n w i l l be l o c a t e d i n t h i s SI  short  n u c l e a s e p r o t e c t i o n wxpariments  region.  ( F i g u r e 17) suggested t h a t the  t r a n s c r i p t s began w i t h adenine r e s i d u e s l o c a t e d a t p o s i t i o n s -42 and -51 f o r the  h s p ! 6 - l and hsp!6-48 genes, r e s p e c t i v e l y  initiation  codons).  ( r e l a t i v e to t h e i r  ATG  I n F i g u r e 17, the 5' f l a n k i n g sequences of the two  genes have been a l i g n e d t o compare the r e g i o n s upstream from the p u t a t i v e transcription starts. (M.L. Goldberg, Ph.D. first  I n b o t h genes t h e r e i s a Goldberg-Hogness o r TATA box t h e s i s , S t a n f o r d U n i v e r s i t y , S t a n f o r d , CA, 1979), t h e  thymine o f which f e l l e x a c t l y 26 bp from the t r a n s c r i p t i o n  start.  Approximately 30 bp f u r t h e r upstream were found e x c e l l e n t matches t o the consensus sequence CT-GAA—TTC-AG, which was d e r i v e d by comparing t h e 5' f l a n k i n g r e g i o n s o f the D. melanogaster h e a t shock genes (Pelham, 1982).  A  s y n t h e t i c promoter c o n t a i n i n g t h i s sequence has been shown t o c o n f e r h e a t i n d u c i b i l i t y on non-heat i n d u c i b l e genes such as the Herpes simplex v i r u s thymidine k i n a s e gene (Pelham and B i e n z , 1982).  The hsp!6-48 gene c o n t a i n e d  103a  F i g u r e 17.  SI nuclease p r o t e c t i o n a n a l y s i s of t r a n s c r i p t i o n s t a r t s i t e s of  the hsp!6-48 and h s p ! 6 - l genes, and comparison o f the 5' f l a n k i n g (A) S I n u c l e a s e mapping.  regions.  S p e c i f i c 5 ' - e n d - l a b e l l e d d o u b l e - s t r a n d e d DNA  fragments were allowed t o anneal t o h e a t shock poly(A) RNA under the +  h y b r i d i z a t i o n conditions described.  SI n u c l e a s e - r e s i s t a n t fragments were  s e p a r a t e d on d e n a t u r i n g p o l y a c r y l a m i d e g e l s along w i t h the Maxam and G i l b e r t c l e a v a g e r e a c t i o n s o f the same e n d - l a b e l l e d fragment.  RNA-, C o n t r o l  lanes  i n which the DNA fragment was taken through the same h y b r i d i z a t i o n and SI d i g e s t i o n r e a c t i o n s i n the absence o f mRNA.  (B) The 5' f l a n k i n g  sequences  of b o t h s m a l l heat shock genes were a l i g n e d t o show the r e l a t i o n s h i p between the t r a n s c r i p t i o n s t a r t s  (arrows), the TATA boxes, and the HIPs.  For each  gene, the A i n the f i r s t ATG a f t e r the mRNA s t a r t i s numbered +• 1. numbering  This  has a l s o been used t o d e s i g n a t e the r e s t r i c t i o n fragments which  were used i n the SI mapping experiments ( s e c t i o n 2.3.3).  The HIP elements  have been u n d e r l i n e d t o demonstrate t h e i r p a l i n d r o m i c n a t u r e , s o l i d l i n e s r e p r e s e n t i n g the dyad a x i s .  vertical  The hsp!6-48 gene has two o v e r l a p p i n g HIP  elements, t h e second i n d i c a t e d w i t h d o t s .  The dashed v e r t i c a l l i n e shows  the c e n t r a l a x i s o f a l a r g e 22 bp palindrome w i t h o n l y t h r e e  mismatches.  103  HSP 16-1  HSP16-48 GC  it  GA TC  \  \  B HSP16-1  G  HSP16-4B  A  C  C  T  C  C  T  T  T  T  G  C  A  A  G  A  A  A A -130  C  T  G  A  A  T  G  C  A  T  C  T  A  G  G  A  A  A  T  G  A  G  T  A  T  A  A  A  T  A  C  G  G  G  T  A  T  A  A  A  T  T  T  C  T A  G A  -20 G A  T G  T  -30  T  G  C  A  c|c  G  C  T  C  f T -110  C  T  A | G  G  A  A  A  G  T  T  C A  T  T  T  A  C  T  A  G  A  A  A  T  A  A  A  C  G  G  C  A C  1  T  A  G  G  T G - 90  C  T A  G G  G  A  G  A  -60 T A  C  A  G  T  G  A  C  A  A  A  A  C  C  G  A  A  C  C  A  -40 A A  C  A  A  C  A  T  T  C  A  C  T  C  A  A  G  C  C  A  A  T  C  G  T  G  T  T  C  A  G  A  6  G  A  A  C  C  A  A  T  A  C  A  C  T  T  T  C  A  A  A  C  T  A  C  A  A  C  A  C  T  A  T  T  T  G  T  T  C  A  T  T  C  T  C  A  -1 A G  A C  T T  C T  T A  C  T T  T T  C A  A A - 10  A  T C  G T  T T  C C  A A  C A  T G  T A  T  A A  - 1  C T  G  104  two  o f these heat i n d u c i b l e promoters (HIPs), which overlapped  each o t h e r t o  form a 22 bp i n v e r t e d r e p e a t w i t h o n l y t h r e e mismatches ( F i g u r e 1 7 ) . Only 128 bp separated genes.  t h e HIP sequences o f t h e h s p ! 6 - l and hsp!6-48  W i t h i n t h i s r e g i o n i s a 10 bp s t r e t c h o f a l t e r n a t i n g  purine-pyrimidine  residues overlapping the Rsal s i t e  sequences can p o t e n t i a l l y adopt a l e f t - h a n d e d  ( F i g u r e 14).  Z-DNA conformation  Such  (Arnott  e t a l . . 1980), and have been i m p l i c a t e d i n t h e r e g u l a t i o n o f t r a n s c r i p t i o n of s i m i a n v i r u s 40 as w e l l as o t h e r DNA v i r u s e s and r e t r o v i r u s e s (Nordheim and  R i c h , 1983).  Adjacent  t o t h i s r e g i o n was a 35 bp p a l i n d r o m i c  w i t h t h e p o t e n t i a l t o form a stem-loop s t r u c t u r e .  structure  T h i s s t r u c t u r e would  c o n t a i n a 12 bp stem w i t h 2 mismatches and an 11 bp Loopnd-out r e g i o n . r e g i o n i s a l s o shown i n F i g u r e 27 and w i l l be d i s c u s s e d  This  i n more d e t a i l i n  s e c t i o n 4.4. The  l e a d e r s of t h e h s p ! 6 - l and hsp!6-48 t r a n s c r i p t s were 42 and 51  n u c l e o t i d e s r e s p e c t i v e l y , t h e d i f f e r e n c e s i n l e n g t h b e i n g due t o i n s e r t i o n s / d e l e t i o n s (see a l s o F i g u r e 26, s e c t i o n 4.4). When a l i g n e d t o compensate f o r these events,  t h e l e a d e r s show a s u b s t a n t i a l degree o f  s i m i l a r i t y , t h e sequences o f which may be conserved t o m a i n t a i n t r a n s l a t i o n a t higher  selective  temperature.  3.11 The 3* F l a n k i n g Regions The p o l y a d e n y l a t i o n s i g n a l AATAAA i s found i n t h e 3' noncoding of b o t h t h e h s p ! 6 - l and hsp!6-48 genes ( F i g u r e 14).  region  I n t h e hsp!6-48 gene i t  was found 33 bp downstream from the TAA t e r m i n a t i o n codon .  Sequence  a n a l y s i s of t h e cDNA p l a c e d t h e p o l y a d e n y l a t i o n s i t e 12 n u c l e o t i d e downstream o f t h e s i g n a l ( F i g u r e 10), and 74 bp b e f o r e t h e end o f t h e  105  inverted repeat.  The p r e d i c t e d l e n g t h of the hsp!6-48 t r a n s c r i p t was  n u c l e o t i d e s a f t e r s p l i c i n g and p r o c e s s i n g have been completed, not the p o l y ( A )  533  including  tail.  The h s p ! 6 - l gene c o n t a i n s two p o l y a d e n y l a t i o n s i g n a l s , one b e g i n n i n g p o s i t i o n 3937 and the second a t p o s i t i o n 3987 of F i g u r e 14. corresponding suggests  to h s p ! 6 - l was  Although  at  a cDNA  not i d e n t i f i e d , SI n u c l e a s e mapping data  t h a t the p r o x i m a l one  i s f u n c t i o n a l i n nematodes (Rob Kay,  communication), the s i t e of p o l y a d e n y l a t i o n o c c u r r i n g a t p o s i t i o n  personal 3961.  T h i s r e s u l t s i n a 3' u n t r a n s l a t e d r e g i o n of 90 n u c l e o t i d e s and a s p l i c e d t r a n s c r i p t w i t h a p r e d i c t e d l e n g t h of 570 n u c l e o t i d e s .  3.12  O r g a n i z a t i o n of I n v e r t e d Repeats i n the Region C o n t a i n i n g Genes  Hspl6-l/48 Unusual sequences were found a t the d i s t a l boundaries inverted repeat.  of the 1.9  kb  T h i s G + C - r i c h boundary sequence ( F i g u r e 14) i s  c h a r a c t e r i z e d by a s t r e t c h of 12 a l t e r n a t i n g p u r i n e - p y r i m i d i n e r e s i d u e s , f l a n k e d on e i t h e r s i d e by a G + C h e x a n u c l e o t i d e orientation. 1.9  The  i n an i n v e r t e d repeat  s i n g l e n u c l e o t i d e d i f f e r e n c e between the two  kb i n v e r t e d repeat was  found  i n t h i s boundary sequence.  arms of the  I t f a l l s on  one  of the f l a n k i n g h e x a n u c l e o t i d e s , where a c y t o s i n e i s changed to a thymine. F u r t h e r sequence a n a l y s i s of 3.3R  r e v e a l e d the presence  i n v e r t e d r e p e a t which o v e r l a p s the one o r g a n i z a t i o n i s shown i n F i g u r e 18. r e p e a t was  of another  370  bp  c o n t a i n i n g the heat shock genes.  U n l i k e the l a r g e r one,  not p e r f e c t , h a v i n g d i v e r g e d by approximately  4%.  Its  this inverted Its right  arm,  however, c o n t a i n e d a G + C - r i c h boundary sequence which shared p e r f e c t homology to i t s c o u n t e r p a r t .  In the s m a l l e r i n v e r t e d r e p e a t , the boundary  106a  Figure  18.  Organization  sequences i n locus A)  of i n v e r t e d r e p e a t s and G + C - r i c h boundary  hsp16-1/48.  R e s t r i c t i o n map  of the 7.6 kb r e g i o n  the 1.0 kb EcoRI fragment. containing are  c o n s i s t i n g of 3.3L, 3.3R,  The l o c a t i o n of the 1.9 kb i n v e r t e d  the heat shock genes and the o v e r l a p p i n g  i n d i c a t e d by open boxes.  and  repeat  370 bp i n v e r t e d  repeat  The two v a r i a n t forms of the G + C - r i c h  boundary sequences a r e shown a t t h e b o r d e r s o f the i n v e r t e d repeats by heavy lines.  Also  shown, above the r e s t r i c t i o n map,  a n a l y z e d f o r RNA  transcription.  B)  sequence of the 0.9 kb E c o R I - S a l l inverted  Strategy  fragment.  are the regions  which were  used to determine the DNA C)  D e t a i l e d map  repeat s t r u c t u r e showing the p a l i n d r o m i c  of the 370 bp  boundary sequences, the  s t r a t e g y used to sequence the r i g h t arm, and the l o c a t i o n s of the R s a l Hpall r e s t r i c t i o n the f o l d b a c k  s i t e s w i t h i n the loop r e g i o n which were used to d i s r u p t  structure.  single-stranded  B) and C)  A l l sequencing was done u s i n g  M13  phage, the d i r e c t i o n and e x t e n t of the sequence o b t a i n e d  from each r e s t r i c t i o n restriction  and  sites  s i t e shown w i t h arrows.  are Sau3A s i t e s .  A l l of the u n l a b e l l e d  1.0 l b Sol-Bom Sol  Sill  1.0  3.3 L  XholBam S n E  B a l l Bel  Xba Xba It'll Bel BglEEco  3.3R  Das Bel  H  | 1.9 kb loTt  ;  Xbo Eco BglOcI Pit I Xba  1.0 l b B a m - Eco  Bel B a l l S i l l Bom x h o l  XhoXBom S i l H  h 1.9kb,-'right  n. n. iaq  Taq  o OS  Hpall  rl 370  Sill  1 bp left  Bom X h o l  -+  1  Ria  HpoE  X h o l Bom  SnE  h370  bp  right'  Hpol  107  sequences were l o c a t e d on the p r o x i m a l b o r d e r s , b e i n g separated  by  approximately  were  500 bp of unique sequence.  c h a r a c t e r i z e d by a complete d i v e r g e n c e  The  d i s t a l boundaries  of sequence, the l e f t  one  i n t e r e s t i n g l y l o c a t e d p r e c i s e l y a t the f u n c t i o n a l p o l y a d e n y l a t i o n s i g n a l of the h s p ! 6 - l gene i n the r i g h t arm  of the 1.9  kb i n v e r t e d repeat.  Since the boundary sequences of the l a r g e r i n v e r t e d repeat d e f i n e d a l o c u s of t r a n s c r i p t i o n a l a c t i v i t y , an e f f o r t was any genes t h a t may Northern  precisely  made to l o c a t e  have been to the r i g h t of the s m a l l e r i n v e r t e d r e p e a t .  b l o t a n a l y s i s showed t h a t t h e r e were no d e t e c t a b l e messages i n  c o n t r o l polyA  , c o n t r o l polyA  probe the 3.3R-derived 1.0  t, or heat  + shock polyA RNA  . u s i n g as a  kb BamHI-KcoRI fragment (not shown).  n e g a t i v e r e s u l t s were o b t a i n e d u s i n g the 1.0  Similar  kb BamHI-Sall fragment of  which f l a n k s the l a r g e i n v e r t e d repeat on the other s i d e . The both of these probes are shown i n F i g u r e 18.  3.3L  l o c a t i o n s of  Thus i t would appear t h a t  t h e r e are no t r a n s c r i b e d r e g i o n s immediately  adjacent to the i n v e r t e d  r e p e a t s of l o c u s hsp!6-l/48 u n l e s s the t r a n s c r i p t s accumulate to l e v e l s below the l i m i t s of d e t e c t i o n . 0.9  Furthermore, the completed sequence of the  kb E c o R I - S a i l segment i n 3.3L  ( F i g u r e 18)  frames of s u b s t a n t i a l l e n g t h i n e i t h e r To examine the frequency  c o n t a i n e d no open r e a d i n g  direction.  of o c c u r r e n c e  of the boundary sequences i n the  C. elegans genome, o l i g o d e o x y n u c l e o t i d e s s p e c i f i c f o r the two were used as probes.  These o l i g o d e o x y n u c l e o t i d e s i n c l u d e d the a l t e r n a t i n g  p u r i n e / p y r i m i d i n e s t r e t c h but c o n t a i n e d o n l y one of the r e p e a t s to a v o i d s e l f - c o m p l e m e n t a r i t y . the two  v a r i a n t forms  The  hexanucleotide  s i n g l e base d i f f e r e n c e between  oligodeoxynucleotides i s located i n t h i s hexanucleotide.  r e s u l t s , u s i n g v a r i a n t I as probe, are shown i n F i g u r e 19.  The  Under c o n d i t i o n s  1 0 8  N  n  Figure phage  from  19. and  A n a l y s i s B r i s t o l  2.0  the i n d i c a t e d  phage  a f t e r  oligodeoxynucleotide Phage  inverted  clones  repeat  overlapping representing duplicated.  3 70  +  genomic  Approximately  n i t r o c e l l u l o s e  DNA.  of G  G  00  C - r i c h  M  1  S  CD  LU  boundary  sequences  yg  of B r i s t o l were  genomic  digested  with  DNA(G) EcoRI,  agarose g e l e l e c t r o p h o r e s i s v a r i a n t  B-3  i n various  recombinant  DNA.  and  s t r u c t u r e , bp  ^  1  inverted  the hsp!6-2/41  I .  B-7  The  markers  contain  r e s p e c t i v e l y . repeat locus  Clone  s t r u c t u r e . i n which  0.5  yg  of  transferred  to  and h y b r i d i z e d  are a  the l e f t  and  H i n d l l l r i g h t  B-7  includes  Phage  t h e hsp!6  arms  EMBL4-10 genes  to  digest  and  DNA  of  also  of  t h e 3.3 the  contains  are not  lambda  DNA  kb  109  i n which o n l y 3.3L from phage \Charon4 B-3  ( F i g u r e 12) h y b r i d i z e d to the  o l i g o d e o x y n u c l e o t i d e s p e c i f i c t o the boundary sequence i n t h a t fragment, genomic B r i s t o l DNA  d i g e s t e d w i t h EcoRI gave a h e t e r o d i s p e r s e s e r i e s of  bands which i n d i c a t e s t h a t such sequences a r e abundant i n the nematode genome.  The s i g n a l was not a b o l i s h e d a t washing temperatures as h i g h as  53°C i n 6 X SSPE. T h i s o l i g o d e o x y n u c l e o t i d e , s u r p r i s i n g l y , d i d not h y b r i d i z e to 3.3R  from  phage \Charon4 B-7 which c o n t a i n s a t l e a s t two c o p i e s of the v a r i a n t form.  To ensure t h a t the l a c k of h y b r i d i z a t i o n was not due to the s i n g l e  base p a i r mismatch,  an o l i g o d e o x y n u c l e o t i d e s p e c i f i c  sequences found i n 3.3R  was used as a probe ( v a r i a n t I I ) .  to those shown i n F i g u r e 19 were o b t a i n e d of 43°C.  f o r the boundary Identical  even a t low washing temperatures  C o n s i d e r i n g the d i f f i c u l t i e s encountered i n the genomic  a n a l y s i s of the 1.9 kb i n v e r t e d r e p e a t , h y b r i d i z a t i o n t o 3.3R  was  once a g a i n a r e s u l t of the f o r m a t i o n  t h e i n v e r t e d repeat of 3.3R  DNA  i t was p o s s i b l e t h a t the l a c k of  s t r u c t u r e s d e r i v e d from the s m a l l e r 370 bp i n v e r t e d r e p e a t . possibility,  results  was  disrupted with  of f o l d b a c k  To examine  this  various  r e s t r i c t i o n enzymes and probed a g a i n w i t h the o l i g o d e o x y n u c l e o t i d e .  As  shown i n F i g u r e 20, t h e r e i s r e l a t i v e l y l i t t l e h y b r i d i z a t i o n t o the  3.3R  EcoRI fragment o r the d e r i v e d 700 bp BamHI fragment i n which the i n v e r t e d repeat s t r u c t u r e i s maintained.  D i g e s t i o n of the same amount of DNA  with  e i t h e r H p a l l o r R s a l r e s u l t s i n s t r o n g h y b r i d i z a t i o n to two fragments o f 700 bp and 350 bp which c o n t a i n the boundary sequence. N e i t h e r o l i g o d e o x y n u c l e o t i d e h y b r i d i z e d t o any EcoRI fragments d e r i v e d from XEMBL4-10, the recombinant phage which c o n t a i n s a p a i r of r e l a t e d hsp16 genes (Jones e t a l . , 1986).  A l t h o u g h t h i s suggests t h a t sequences  110  Figure bp  20.  i n v e r t e d  Detection  A f t e r  EcoRI  0.5  fragment  ug  was  e l e c t r o p h o r e s i s  oligodeoxynucleotide DNA.  boundary  sequences  i n 3.3R  by  d i s r u p t i o n of  the  370  repeat.  Approximately 3.3R  of  of  a  recombinant  digested  and  with  Southern  v a r i a n t  I I .  the  pUC13  r e s t r i c t i o n  t r a n s f e r , The  plasmid  markers  the are  DNA a  containing  enzymes was  i n d i c a t e d .  h y b r i d i z e d  H i n d l l l  the  digest  to of  lambda  Ill  homologous to the boundary sequences a t l o c u s hsp!6-l/48  may  not  be  a s s o c i a t e d w i t h the other heat shock gene l o c u s , i t i s p o s s i b l e t h a t t h e i r a s s o c i a t i o n w i t h f o l d b a c k s t r u c t u r e s d i c t a t e s a more r i g o r o u s a n a l y s i s w i t h d i f f e r e n t r e s t r i c t i o n enzymes. In summary, G + C - r i c h sequences s i m i l a r or i d e n t i c a l to the boundary sequences d e s c r i b e d here are abundant i n the nematode genome.  Their  a s s o c i a t i o n w i t h i n v e r t e d r e p e a t s t r u c t u r e s i n the p r e s e n t i n s t a n c e i s i n t e r e s t i n g but whether they can be g e n e r a l l y c o r r e l a t e d w i t h o t h e r r e g i o n s of s i m i l a r s t r u c t u r e remains to be  3.13  seen.  D i f f e r e n t i a l E x p r e s s i o n of Hspl6  Genes i n C.  elegans  A p a i r of r e l a t e d hsp!6 genes i n C. elegans has been c h a r a c t e r i z e d by sequencing  (Jones e t a l . ,  1986).  The  two  genes are arranged  o r i e n t a t i o n s s i m i l a r to the hsp!6-48 and h s p ! 6 - l genes.  The  i n divergent two  genes are  d e s i g n a t e d hsp!6-2 and hsp!6-41, the l a t t e r c o r r e s p o n d i n g to the cDNA CEHS41, and both are h i g h l y expressed  d u r i n g heat shock.  The gene p a i r a t  l o c u s hsp!6-2/41 i s not d u p l i c a t e d t o form an i n v e r t e d repeat but i s f l a n k e d by r e p e t i t i v e elements.  To determine  from the f o u r hsp!6 genes, M13  the r e l a t i v e l e v e l s of t r a n s c r i p t i o n  probes were used t h a t showed  c r o s s - h y b r i d i z a t i o n between homologous genes. second  minimal  Probes corresponding to the  exons were avoided s i n c e the c a r b o x y - t e r m i n a l h a l v e s of the hsp!6-48  and hsp!6-41 genes, r e p r e s e n t e d by t h e i r r e s p e c t i v e cDNAs, c r o s s - h y b r i d i z e strongly.  Thus probes  (genes 1 and listed  c o r r e s p o n d i n g t o e i t h e r the 3' u n t r a n s l a t e d r e g i o n s  2) o r exon 1 (genes 48 and 41) were used.  i n the legend to F i g u r e 21.  The  v e r i f i e d by h y b r i d i z i n g them to Southern  These M13  c l o n e s are  s p e c i f i c i t i e s of these probes were t r a n s f e r s c o n t a i n i n g EcoRI d i g e s t s  112  ©  cn 2 i ! CO "J  1  •  mRNA  21.  1  Determination of  expression  DNA  was  h y b r i d i z e d probes  following B e l l  taken  Figure  3.3  EcoRI  2.2  counts  kb  were  from  M13  gels,  of various  templates  (1974) gene  -  by  TagI  (1910);  from  Jones  e t a l . (1986).  i n each  which  B g l H  and hsp!6-48  codes  genes  primer  (253) -  the numbering  which  t r a n s f e r r e d  t h e hsp!6  while  fragment  used  agarose  14  fragment  EcoRI  48  of EcoRI-digested \Charon4  16-1, Sau3A  16-2, TagI  i s taken  a  from  F o r the hsp!6-l  from  -3.3 -2.2  41  the s p e c i f i c i t y  derived  sequences:  probes kb  ng  separated on  derived  (1494);  (1169).  500  t o probes  were  1  probes  used  i n t h e  studies.  Approximately phage  © co «i i CO t  « »  t  2  Figure  o I I CO 1  tn y-  en "~ i CO  Phage  extension (601);  h y b r i d i z a t i o n .  TagI  2  and  B g l H  (933) -  and B-3  10^  t h e  (1647) TagI  hsp!6-41 contains  XEMBL4-10 x  The  include  the numbering  \Charon4 while  and  16-48,  probes,  f o r hspl6-2/41.  XEMBL4-10  a r e indicated.  f o r the hsp!6-2  f o r hspl6-l/48  codes  and  t o n i t r o c e l l u l o s e  which  16-41,  gene  B-3  i s gene a  contains  Cerenkov  -  113  of t h e phage \Charon4 B-3 and XEMBL4-10 which correspond t o the 1/48 and 2/41  loci,  respectively  ( F i g u r e 21).  T h i s experiment  probes were o f comparable s p e c i f i c a c t i v i t i e s .  also v e r i f i e d that the  Of the f o u r probes  used,  o n l y t h e one s e l e c t e d f o r the hsp!6-41 gene showed some degree o f c r o s s - h y b r i d i z a t i o n t o both phage c l o n e s b u t as the mRNA e x p r e s s i o n d a t a show, t h i s was o f no consequence. When these probes were a p p l i e d i n d i v i d u a l l y t o Northern t r a n s f e r s o f p o l y a d e n y l a t e d RNA from heat shocked the hsp!6-2/41 genes appeared  and c o n t r o l nematodes, mRNA l e v e l s  from  t o be 10 t o 20 times h i g h e r than those from  the genes hsp!6-48 and h s p ! 6 - l ( F i g u r e 22).  S i n c e t h e r e a r e presumably two  f u n c t i o n a l c o p i e s o f the l a t t e r gene p a i r , the r e l a t i v e t r a n s c r i p t i o n  level  per gene a t the hsp!6-2/41 l o c u s i s approximately 20 - 40 times t h a t o f t h e hsp!6-l/48  locus.  A l s o , these b l o t s i n d i c a t e d t h a t the p o l y a d e n y l a t e d hsp!6  t r a n s c r i p t s a r e between 600 and 700 n u c l e o t i d e s i n l e n g t h , as expected.  3.14 Comparison o f Locus H s p l 6 - l / 4 8 i n C a e n o r h a b d i t i s elegans B r i s t o l and Bergerac  Strains  The r e l a t e d s t r a i n s B r i s t o l and Bergerac can i n t e r b r e e d and thus any r e s t r i c t i o n fragment  length differences  markers t o determine l i n k a g e .  (RFLDs) can be used as g e n e t i c  T h i s can be done by making use o f  B r i s t o l - B e r g e r a c h y b r i d p o p u l a t i o n s homozygous f o r a s i n g l e p a r t i c u l a r B r i s t o l o r Bergerac chromosome, t h e remaining chromosomes b e i n g heterozygous (Rose e t a l . ,  1982).  The two s t r a i n s d i f f e r a t t h e i r n u c l e o t i d e l e v e l by  a p p r o x i m a t e l y 1% (Emmons e t a l . ,  1979; Rose e t a l . ,  1982).  The observed  f r e q u e n c y o f RFLDs (Rose e t a l . ,  1982) i s c o n s i s t e n t w i t h t h e occurrence o f  114  H m H m  Figure  22.  Northern  b l o t  HCm  analysis  o f hsp!6  HCm  mRNA  l e v e l s  using  gene-specific  probes. Approximately shocked gel, The  (H) worms  t r a n s f e r r e d markers  end-labelled t r a n s f e r r e d  1.0  yg o f polyA RNA +  was separated  e i t h e r  by t h e f i l l i n g - i n with  t h e RNA  digest  control  (C) o r  1.2% agarose-formaldehyde  t o n i t r o c e l l u l o s e and hybridized  (m) a r e a H i n d l l l  along  on a  from  o f lambda  reaction  using  t o t h e probes  DNA. Klenow  t o n i t r o c e l l u l o s e .  Fragments polymerase  heat  denaturing indicated. were and  115  a base p a i r change per 120 bp o r one i n s e r t i o n / d e l e t i o n rearrangement per 52,000 bp. In an attempt t o determine the l i n k a g e of l o c u s h s p ! 6 - 4 8 / l. s e v e r a l p u r i f i e d EcoRI fragments d e r i v e d from \Charon4 A - l o r A-4 were used to probe f i l t e r s c o n t a i n i n g b o t h B r i s t o l and Bergerac genomic DNA EcoRI.  digested with  No d i f f e r e n c e s were seen i n the m i g r a t i o n of h y b r i d i z i n g bands  between the two s t r a i n s  (Ann Rose, p e r s o n a l communication).  t o t a l o f a p p r o x i m a t e l y 16,000 bp was  However, o n l y a  a n a l y z e d , which i s w e l l below the  average d i s t a n c e r e q u i r e d to d e t e c t an i n s e r t i o n / d e l e t i o n event (Rose et a l . ,  1982).  Snutch and B a i l l i e  (1984) have proposed t h a t the h i g h  mutation r a t e (10% sequence d i v e r g e n c e ) a s s o c i a t e d w i t h a C. elegans hsp70 gene i s due t o i t s s t a t u s as a h i g h l y i n d u c i b l e gene.  T h i s does not appear  to be the case w i t h the hsp!6 genes which a r e a l s o h i g h l y  inducible.  A d e t a i l e d comparison of the 1.9 kb i n v e r t e d r e p e a t was undertaken.  B r i s t o l and B e r g e r a c DNA  was  d i g e s t e d w i t h 12 d i f f e r e n t  r e s t r i c t i o n enzymes which c u t w i t h i n each arm of the p e r f e c t repeat.  As a r e s u l t 136 bp of DNA  was  also  inverted  a n a l y z e d f o r mutations.  F i g u r e 23, no r e s t r i c t i o n fragment polymorphisms  As seen i n  c o u l d be d e t e c t e d ,  emphasizing the sequence c o n s e r v a t i o n between the two s t r a i n s w i t h i n p a r t i c u l a r locus.  The probe used i n t h i s s e t of experiments was  t h a t used i n F i g u r e 13C. p a r t i c u l a r DNA  this  the same as  Foldback s t r u c t u r e s were not seen i n these  p r e p a r a t i o n s i n which i t c o u l d be shown t h a t there was  a much  lower degree o f n i c k i n g . S i n c e t h e s e two s t r a i n s cannot be d i s t i n g u i s h e d m o r p h o l o g i c a l l y , the s p e c i f i c i t y o f the B r i s t o l and Bergerac DNA  was v e r i f i e d by p r o b i n g  n i t r o c e l l u l o s e f i l t e r s c o n t a i n i n g genomic DNA  w i t h the p l a s m i d pCeh2 which  116  Bell N2 BO  Eco N2 BO  BglD N2 BO  Pst N2 BO  Sstn N2 BO  Bam N2 BO  Xhol N2 BO  -4.0  '375  1.0-  Hpctl N2 BO  3  Nrul N2 BO  Ncol N2 BO  Ddel N2 BO  N2  Rsa  BO  ,r  2.7-  Figure the  23.  Detailed  c l o s e l y  r e l a t e d  Approximately digested  with  the  electrophoresis probe 200  used  bp  were  Figure  13C).  kilobase  was  on an  comparison s t r a i n s  2  jig o f  M13  s p e c i f i c to The  p a i r s .  s i z e  the of  1.9  (N2)  gels  the  region  inverted  repeat  structure  i n  B r i s t o l . and  Bergerac  enzymes,  and  containing loop  kb  and  r e s t r i c t i o n  agarose clone  the  Bergerac B r i s t o l  indicated 1.0%  of  500 of  h y b r i d i z i n g  bp the  band  genomic  separated  t r a n s f e r r e d  a  (BO)  to  i n  each  was  by  n i t r o c e l l u l o s e .  Ddel-EcoRI inverted  DNA  i n s e r t  repeat case  i s  of  (Probe shown  The  which C, in  117  c o n t a i n s a copy of T e l , a t r a n s p o s a b l e element of C. elegans.  T h i s element  was i s o l a t e d from B r i s t o l DNA and was k i n d l y p r o v i d e d by L i n d a H a r r i s , U n i v e r s i t y o f B r i t i s h Columbia. bands c o u l d be observed found w i t h Bergerac  When h y b r i d i z e d t o genomic DNA, 20 - 30  w i t h B r i s t o l DNA but a more complicated p a t t e r n was  DNA s i n c e t h e l a t t e r c o n t a i n s approximately  10 times as  many c o p i e s o f t h e t r a n s p o s a b l e element (Emmons e t a l . , 1983; L i a o e t a l . , 1983).  These r e s u l t s a r e n o t shown.  118  IV.  DISCUSSION  4.1  The Hspl6 Gene Family of C. The sequences p r e s e n t e d  elegans  i n t h i s t h e s i s along w i t h those of Jones e t a l .  (1986) d e f i n e a f a m i l y of f o u r r e l a t e d hsp16 genes.  They are arranged i n  d i v e r g e n t l y t r a n s c r i b e d p a i r s a t two s e p a r a t e  The hsp!6-48/41  loci.  genes  code f o r one c l a s s of h s p l 6 , 143 amino a c i d r e s i d u e s long w h i l e the hsp!6-l/2 genes encode the o t h e r c l a s s , which i s two amino a c i d longer. hspl6.  residues  Thus each l o c u s and each gene p a i r codes f o r the two major types of The complete amino a c i d sequences of a l l 4 p r o t e i n s are a l i g n e d i n  F i g u r e 24 to show the r e l a t e d n e s s of hspl6-41 to hspl6-48 and of hspl6-2 to hspl6-l.  The b a s i s f o r t h e i r c l a s s i f i c a t i o n i n t o two d i s t i n c t groups i s  most e v i d e n t i n the comparison of t h e i r f i r s t  exons.  F o r example,  t h r e e amino a c i d changes occur between the r e l a t e d p r o t e i n s i n t h i s  only region.  Between the two c l a s s e s o f p r o t e i n s , the r e g i o n s encoded by exon 1 show no homology. In t h e r e g i o n encoded by the second exon, 61 of the p o s s i b l e 98 amino a c i d p o s i t i o n s which can be compared a r e i d e n t i c a l i n a t l e a s t t h r e e of the f o u r p r o t e i n s as shown i n F i g u r e 24.  The second exons of hsp!6-41 and  hsp!6-48 c o n t a i n o n l y 6 codon changes w h i l e the homology i n the same r e g i o n of  h s p ! 6 - l and hsp!6-2 i s comparable, w i t h o n l y 7 codon d i f f e r e n c e s .  s i m i l a r i t y i s seen between genes of the same l o c u s .  Less  F o r example, a  comparison of the second exons of hsp!6-48 and h s p ! 6 - l shows t h a t there are 37 codon d i f f e r e n c e s w i t h i n 98 amino a c i d s . p r o t e i n domain r e p r e s e n t e d by the f i r s t p r o t e i n c o n t a i n s a conserved  Thus t h e r e i s a h i g h l y v a r i a b l e  exon w h i l e the remainder of the  domain which shows homology to the shsps o f  119  MLMLRJSIPJYSDSNALDHFLDELTGSVQFPYV&ADHNSFNFSD MLMLRSPFSDSNVLDHFLDEITGSVQFPYWRNADHNSFNFSD MSLYHYFRPAQRSVFGDLMRDMAQMERQFTPVCRGSPS  HSP16-41 HSP16-48 HSP16-1 H S P 1 6-2  I^LTYJHYFRPAQRSVFGDLMRDMALMEYQFAPVCRI SPS  HSP16-41 HSP16-48 HSP16-1 HSP16-2  NIGENIGEESSEESS[E]-  HSP 1 6-•41 HSP1 6- •48 HSP1 6- •1 HSP 1 6-•2.  heat  shock In  A  KSAI SNEGKLQ I E A P K K T N S S - R S I P I NFVAKH KSAI SNEGKLQ I E A P K K T N S S - R S I P I NFVAKH ASNLSEDGKLSIEAPKKEAIQGRSIPI QQAPVEQKTSE A S N L S E D G K L S I E A P K K EAVQGRSIPI Q Q A I V E E K S A E  comparison  o f t h e proteins  encoded  by  t h e C.  elegans  16 k d  genes.  t h e boxed  i d e n t i c a l  amino  conserved  domains  acid  IVNDESKFSVQL DVSHFKPENLK IVNDESKFSVqi DVSHFKPEDLK IVNNDQKFAl NpLjNjV'SQFKPEDLK IVN|NDQ|KFAIN1L{N1V" SQFKPEDLK  KI E G I Q E T K S E H G Y L K R S F S K M I L L P E D A D L P S V KlI E G I Q E K K S E H G Y S K R S F S K M I L L P E D V D L T S V NLDGHTLS IC G E Q E L K T E H G Y S K K S F S R V I L L P E D V D V G A V NLDGRTLS IQGEQELKTDHGYSKKSFSRVIILLPEDVDIVGAIV  1 6'4 1 1 6-48 HSP1 6- •1 H S P 1 6-2  24.  INTRON INTRON INTRON INTRON  KLDGREL ELDGREL  HSP HSP  Figure  58bp 55bp 52bp 46bp  sequences  regions,  acids. which  a t least  Also  shown  three  o f t h e four  a r e t h e h i g h l y  a r e p r e c i s e l y defined  o f hspl6-2  and hspl6-41  by  a r e taken  proteins  v a r i a b l e a  short  from  and  have h i g h l y  intron.  Jones  The  amino  e t a l . (1986).  120  Drosophila,  Xenopus, soybean and man.  m a i n t a i n e d i n the v e r t e b r a t e  eye  The  s t r u c t u r e of t h i s domain has  been  lens p r o t e i n a - c r y s t a l l i n .  Two-dimensional g e l e l e c t r o p h o r e s i s of the i n v i t r o t r a n s l a t i o n p r o d u c t s of RNA  h y b r i d - s e l e c t e d by e i t h e r CEHS48 or CEHS41 r e v e a l e d  f i v e e l e c t r o p h o r e t i c v a r i a n t s of hspl6 i s o e l e c t r i c points. be  The  which had  significantly different  p o s s i b l i l i t y t h a t another hspl6  gene e x i s t s cannot  excluded although i t i s a l s o p o s s i b l e t h a t a given gene product may  i n more than one  e l e c t r o p h o r e t i c form s i n c e a c e t y l a t i o n of p r o t e i n s has  shown to occur i n a r a b b i t r e t i c u l o c y t e system ( P a l m i t e r , 1977; Hunter, 1979; The  Rubenstein and  distinct  occurring  i n q u a i l (Atkinson  isoforms  are c h a r a c t e r i z e d by two tyP  e s  ( b a s i c ) and  (Schoenmakers and by  Xenopus, p l a n t s and  At the p r o t e i n l e v e l ,  l e a s t two  Garrels  man  are a l s o encoded  shsps are always p r e s e n t  on two-dimensional g e l s , the only  and  e t a l . , 1981).  The  by  i n at  exception  vertebrate a - c r y s t a l l i n s  p r i m a r y gene p r o d u c t s which have been designated  A^ types ( a c i d i c ) due  Bloemendal, 1968).  to t h e i r i s o e l e c t r i c  as  points  In hamster, these p r o t e i n s are encoded  In the v e r t e b r a t e  l e n s , the A^  and  crystallins  l a r g e aggregates w i t h t h e i r deamidation p r o d u c t s which have been and  B^.  C-terminal  With age,  a l l of the a - c r y s t a l l i n chains  can be  grouped a c c o r d i n g  hsp26 are b a s i c whereas hsp23 and  1978;  S t o r t i e t a l . , 1980).  form designated  undergo  d e g r a d a t i o n ( f o r a review see Bloemendal 1982).  shsps of D r o s o p h i l a and  been  s i n g l e copy genes which are l o c a t e d on d i f f e r e n t chromosomes (Quaz-Jeuken  e t a l . , 1985).  A^  exist  Deuchler, 1979).  shsps of D r o s o p h i l a ,  multigene f a m i l i e s .  ^2  at l e a s t  to t h e i r net  hsp22 are a c i d i c  S i m i l a r l y , the charges:  hsp27  ( M i r a u l t et a l . ,  121  It to  i s p o s s i b l e t h a t the two c l a s s e s of h s p l 6 i n C. elegans correspond  a c i d i c and b a s i c t y p e s .  S i n c e each heat shock l o c u s codes f o r both types  and s i n c e t r a n s c r i p t s from one l o c u s accumulate t o l e v e l s much h i g h e r than those c o r r e s p o n d i n g to the o t h e r , one c o u l d expect to see a p o o r l y expressed a c i d i c v a r i a n t and a p o o r l y expressed b a s i c v a r i a n t . be a more abundant  a c i d i c and a more abundant  S i m i l a r l y t h e r e would  basic isoform.  i s observed on the two-dimensional g e l shown i n F i g u r e 7. r e l a t i v e abundance of the two most b a s i c v a r i a n t s  T h i s i s what  For example, the  (shown by arrows) i s  c o n s i s t e n t w i t h the r e l a t i v e accumulation of t r a n s c r i p t s from the two  loci,  one b e i n g 20 to 40 times h i g h e r than the o t h e r . Based on the o v e r a l l p r o p o r t i o n of a c i d i c amino a c i d  residues  (glutamate and a s p a r t a t e ) t o b a s i c amino a c i d r e s i d u e s ( l y s i n e and as p r e d i c t e d by the d e r i v e d amino a c i d sequences, a s s i g n the two c l a s s e s of h s p l 6 i n t o d i s t i n c t  arginine)  i t i s not p o s s i b l e to  i s o e l e c t r i c forms. However,  t h e r e are some i n t e r e s t i n g charge d i f f e r e n c e s w i t h i n l o c a l i z e d r e g i o n s o r domains.  The f i r s t  exon of both hspl6-41 and hspl6-48 has a p r e d i c t e d n e t  charge of -3 w h i l e the c o r r e s p o n d i n g r e g i o n of hspl6-2 and h s p l 6 - l has a n e t charge of +1. of  The second exons of hspl6-41 and hspl6-48 have a net charge  +3 and 0, r e s p e c t i v e l y , w h i l e the second exons of both hspl6-2 and  h s p l 6 - l have a n e t charge of -4. c o r r e s p o n d i n g domains may s p a t i a l arrangement  These d i f f e r e n c e s i n net charge between  be important depending on the d i s t r i b u t i o n  of charged groups as a r e s u l t of p r o t e i n f o l d i n g .  and The  a v a i l a b i l i t y of s p e c i f i c c l o n e d sequences now makes i t p o s s i b l e to a s s i g n a s p e c i f i c i s o e l e c t r i c v a r i a n t to a p a r t i c u l a r gene u s i n g h y b r i d i z a t i o n s e l e c t i o n under s t r i n g e n t c o n d i t i o n s and subsequent i n v i t r o  translation.  122  4.2  E v o l u t i o n o f the Hspl6 Gene F a m i l y and I t s R e l a t i o n s h i p t o Other shsps  and t o V e r t e b r a t e a - C r y s t a l l i n s The presence o f homologous multigene f a m i l i e s coding f o r shsps of many organisms suggests t h a t an a n c e s t r a l heat shock gene has undergone a number of d u p l i c a t i o n events. genes i n v e r t e b r a t e s .  The same i s t r u e f o r the homologous a - c r y s t a l l i n I t has been p o s t u l a t e d t h a t the p r o g e n i t o r heat  shock  gene and the p r o g e n i t o r a - c r y s t a l l i n gene arose from the d u p l i c a t i o n o f a . s i n g l e a n c e s t r a l gene (van den Heuvel e t a l . ,  1985).  I t i s proposed  that  t h i s p r o g e n i t o r a - c r y s t a l l i n gene then e v o l v e d through an i n t e r n a l d u p l i c a t i o n w i t h i n the sequences  r e p r e s e n t e d by the f i r s t exon, f o l l o w e d by  a d u p l i c a t i o n of the e n t i r e gene which gave r i s e to the aA and aB type genes.  An a l t e r n a t i v e p r o p o s a l can be made i n which a s i n g l e a n c e s t r a l gene  d u p l i c a t e d to form a p a i r of genes r e p r e s e n t i n g A and B types.  T h i s p a i r of  genes gave r i s e t o the a - c r y s t a l l i n genes and the h s p l 6 gene f a m i l y . scheme i s i l l u s t r a t e d i n F i g u r e 24.  This  The argument i n f a v o r o f t h i s scheme  comes from the comparison o f the C. elegans h s p l 6 amino a c i d sequences w i t h the a - c r y s t a l l i n s o f the d o g f i s h , Squalus a c a n t h i a s .  The a - c r y s t a l l i n s  from t h i s s p e c i e s a r e c o n s i d e r e d t o be p r i m i t i v e r e l a t i v e t o those of o t h e r v e r t e b r a t e s based on immunological comparisons De Jong, 1982). basic  (B) forms.  (Manski and Malinowsky, 1978;  This p r i m i t i v e a - c r y s t a l l i n also exists i n acidic Computer a n a l y s i s  d e s c r i b e d by Delaney  (Jones e t a l . ,  (A) and  1986) u s i n g the program  (1982) w i t h a MINBLOCK parameter s e t of 2 r e v e a l e d  that  hspl6-2 resembled the A form o f d o g f i s h c r y s t a l l i n more than the B form. C o n v e r s e l y , hspl6-41 showed g r e a t e r s i m i l a r i t y t o the B type c r y s t a l l i n o f Squalus.  123  ANCESTRAL GENE  DUPLICATION / INVERSION A  |  IE  B  IE  DIVERGENCE A N D / O R |  EXON SHUFFLING  •  olpha-cryitaltm genes  IE HSP 16-48  DUPLICATION & MINOR DIVERGENCE  HSP16-1 • ™  DUPLICATION/INVERSION HSP 16-1 HSP 16-1  HSP 16-48  H  HSP 16-48  HSP 16-41  LOCUS 1 (CONVERSION UNIT )  F i g u r e 25. C. elegans  LOCUS 2  A model f o r the e v o l u t i o n a r y o r i g i n o f the hspl6  gene l o c i of  and t h e a - c r y s t a l l i n genes of v e r t e b r a t e s .  Open boxes show the s e p a r a t e divergence  HSP 16-2  i n the f i r s t  exons, and h a t c h i n g  exon sequence.  i n d i c a t e s a major  124  The followed  e v o l u t i o n o f an a n c e s t r a l gene i n t o A and B types must have been by a major d i v e r g e n c e i n the sequence of the f i r s t  C. e l e g a n s , genes w i t h i n the same p a i r share l i t t l e region.  exon s i n c e i n  o r no homology i n t h i s  T h i s d i v e r g e n c e may a l s o have been brought about by exon s h u f f l i n g  (van den Heuvel e t a l . , 1985) s i n c e the d i v e r g e d i n t r o n i n the C. elegans genes.  r e g i o n i s demarcated by an  Furthermore, t h i s event p r o b a b l y took p l a c e  a f t e r the d i v e r g e n c e o f p l a n t s and animals s i n c e the shsps of soybean a r e h i g h l y homologous t o each o t h e r amino-terminal p o r t i o n  i n a l l regions  of the p r o t e i n s  (Nagao, 1985); however, the amino a c i d sequences o f  o n l y 4 out o f a t l e a s t 10 shsps have been determined. Drosophila. regions  i n c l u d i n g the  on the other hand, show l i t t l e  The shsps of  homology i n t h e i r amino-terminal  (see F i g u r e 2 ) .  In C. e l e g a n s . t h i s p e r i o d of d i v e r g e n c e must have been f o l l o w e d by a second d u p l i c a t i o n event which i s represented found a t d i f f e r e n t chromosomal l o c a t i o n s .  Following  d u p l i c a t i o n , o n l y minor changes have o c c u r r e d genes.  by t h e homologous gene p a i r s the proposed second  i n the coding  regions  A l s o , over the l a t t e r p e r i o d the sequence o f the f i r s t  e v i d e n t l y changed a t the same r a t e as t h a t o f the second.  of the  exon  Perhaps the  a c q u i s i t i o n o f f u n c t i o n a t t h i s stage f i x e d the r a t e o f mutation w i t h i n the first  exon.  A t h i r d d u p l i c a t i o n and i n v e r s i o n event has occurred  hsp!6-48/l l o c u s , r e s u l t i n g i n a 1.9 kb i n v e r t e d r e p e a t . i d e n t i t y o f t h e two "arms" o f t h i s repeat  has  been m a i n t a i n e d by f r e q u e n t  argument f o r a gene c o n v e r s i o n  The p e r f e c t  a t the n u c l e o t i d e  i m p l i e s t h a t t h e d u p l i c a t i o n event was v e r y r e c e n t  w i t h i n the  sequence l e v e l  o r t h a t sequence i d e n t i t y  i n t r a l o c u s gene c o n v e r s i o n  events.  The  mechanism w i l l be p r e s e n t e d i n s e c t i o n 4.3.  125  Thus, the e v o l u t i o n of the hspl6 genes o f C. elegans has been characterized  by a s e r i e s of d u p l i c a t i o n s , a t l e a s t two of which were  accompanied by i n v e r s i o n s .  Leigh-Brown and Ish-Horowicz (1981) have  suggested t h a t the a n c e s t r a l o r g a n i z a t i o n as  of the D r o s o p h i l a  i n v e r t e d repeats f o r both the 87A and 87C l o c i .  87A a r e a p p r o x i m a t e l y 1.7 kb a p a r t (Goldschmidt-Clermont, 1980).  i n opposite  The c o p i e s  hsp70 genes was  The two hsp70 genes a t  orientations  a t 87C a r e p r e s e n t i n two domains,  a s i n g l e p r o x i m a l sequence separated from tandem d i s t a l sequences by about 38 kb (Ish-Horowicz and P i n c h i n , and  D. m a u r i t i a n a ,  1980).  In the r e l a t e d s p e c i e s ,  t h i s 38 kb i n s e r t i s m i s s i n g ,  genes which a r e i n a head t o head c o n f i g u r a t i o n S i m i l a r l y , a t l o c u s 6 7B, were o p p o s i t e  r e s u l t i n g i n two hsp70 l i k e those a t locus 87A.  i f the o r i e n t a t i o n of gene 2 (which i s not known)  t o t h a t o f gene 3, the shsp genes of D r o s o p h i l a  t h r e e gene p a i r s , t h e o r i g i n s of which c o u l d be e x p l a i n e d duplications during  (see F i g u r e  1).  Inverted  would e x i s t i n  by i n v e r t e d  d u p l i c a t i o n has been shown to o c c u r  t h e a m p l i f i c a t i o n o f a c e l l u l a r oncogene (c-myc) i n a v a r i e t y o f  tumor c e l l  l i n e s and d u r i n g  the a m p l i f i c a t i o n of the CAD gene i n  N-(phosphonacetyl)-L-aspartate-reeistant  cell  l i n e s (Ford and F r i e d , 1986).  CAD i s an acronym f o r the m u l t i f u n c t i o n a l enzyme which c a t a l y z e s three  D. simulans  steps i n u r i d i n e b i o s y n t h e s i s .  the f i r s t  These authors suggest t h a t the wide  o c c u r r e n c e o f i n v e r t e d d u p l i c a t i o n s i s r e l a t e d t o the mechanism o f gene amplification. The  shsps of.C.  elegans resemble the a - c r y s t a l l i n s o f v e r t e b r a t e s  more than they do the shsps of D. melanogaster.  This i s c l e a r l y  demonstrated by the amino a c i d comparisons shown i n F i g u r e s example, i n F i g u r e  16, t h e r e  11 and 16.  For  a r e 32 p o s i t i o n s conserved between the shsps of  126  C. elegans and the a - c r y s t a l l i n s w h i l e t h e r e are o n l y 20  positions  conserved between the shsps of C. elegans and D r o s o p h i l a .  Furthermore,  the  30 amino a c i d r e s i d u e s a t the c a r b o x y - t e r m i n a l of the a - c r y s t a l l i n s show no obvious homology to c o r r e s p o n d i n g r e g i o n s i n the D r o s o p h i l a shsps sequences a l t h o u g h there i s a s l i g h t s i m i l a r i t y w i t h the C - t e r m i n a l 20 amino a c i d r e s i d u e s of nematode h s p l 6  (Wistow, 1985).  A l s o , each of the hspl6  genes c o n t a i n s a s m a l l i n t r o n i n a p o s i t i o n analogous i n t r o n of the a - c r y s t a l l i n genes. of  T h i s suggests  to t h a t of the  first  t h a t some common p r o p e r t y  shsps and a - c r y s t a l l i n s has been more r i g o r o u s l y conserved  in  C. elegans than i n D r o s o p h i l a . Human hsp25 i s even more homologous to a - c r y s t a l l i n than any of the shsps  i n c l u d i n g those of C. elegans  (Hickey e t a l . , 1986).  A l s o , r e g i o n s of  amino a c i d i d e n t i t y between hsp25 and a - c r y s t a l l i n are found along the e n t i r e l e n g t h of a - c r y s t a l l i n .  The  l a c k of a d i v e r g e n t amino t e r m i n a l  r e g i o n , as demarcated by an i n t r o n i n the hsp!6 genes of C. suggests  t h a t the human hsp  elegans,  genes evolved from an a n c e s t r a l p a i r of genes  which d i d not experience major divergence and/or exon s h u f f l i n g . supported by the f a c t t h a t n e i t h e r of the two gene i s i n a p o s i t i o n analogous hsp!6 genes o r to the f i r s t The  almost  This i s  i n t r o n s of the human hsp25  to the s i n g l e i n t r o n s of the C.  elegans  i n t r o n s of the a - c r y s t a l l i n genes.  shsps of D r o s o p h i l a (except hsp22) c o n t a i n a hydrophobic  r e g i o n of 14 amino a c i d r e s i d u e s .  N-terminal  I f t h i s domain has a p r o p e r t y which i s  r e q u i r e d f o r the f u n c t i o n of these p r o t e i n s , then i t must have e v o l v e d the d i v e r g e n c e of nematodes and  after  i n s e c t s s i n c e the N - t e r m i n i of the shsps  C. elegans do not show t h i s degree of h y d r o p h o b i c i t y . shsps of soybean c o n t a i n an N - t e r m i n a l hydrophobic  Interestingly,  r e g i o n of  of  the  approximately  127  10 amino a c i d r e s i d u e s (Nagao e t a l . , 1985).  Human hsp25 shows no degree of  h y d r o p h o b i c i t y w i t h i n i t s amino t e r m i n a l p o r t i o n (Hickey e t a l . , 1986).  4.3  Gene C o n v e r s i o n  W i t h i n Locus  Hspl6-48/l  The h s p ! 6 - l and hsp!6-48 genes, i n c l u d i n g t h e i r coding and f l a n k i n g sequences, are l o c a t e d w i t h i n a 1.9 compactness, f u n c t i o n a l u n i t y , and module.  Two  kb r e g i o n which, because of i t s r e p e t i t i o n , might be r e f e r r e d to as a  such modules, d u p l i c a t e d p e r f e c t l y w i t h o n l y a s i n g l e base p a i r  change, e x i s t i n the genome i n an i n v e r t e d o r i e n t a t i o n r e l a t i v e to each o t h e r and  are separated by 416  o r g a n i z a t i o n was genomic DNA  confirmed  bp of u n r e l a t e d sequence.  by t h r e e l i n e s of evidence.  Firstly,  a n a l y s i s has been c a r r i e d out u s i n g 12 d i f f e r e n t  enzymes which cut i n the r e g i o n of i n t e r e s t .  The  e n t i r e l y c o n s i s t e n t w i t h the p r e d i c t e d s t r u c t u r e . phage c l o n e s were i s o l a t e d from two which c o n t a i n e d both the 3.3L r i g h t and  T h i s n o v e l gene  and  independently  3.3R  d i f f e r e n c e was  identified.  restriction  fragments obtained were Secondly,  overlapping  constructed  libraries  EcoRI fragments.  l e f t modules were sequenced completely,  T h i r d l y , both  The p a l i n d r o m i c r e g i o n has  approximately otherwise  stem p o r t i o n w i t h a looped-out  400  nucleotides.  denaturing  c o n f i g u r a t i o n migrate analysis.  the  and a s i n g l e base p a i r the p o t e n t i a l to  adopt a s t r u c t u r e i n which i n t r a - s t r a n d p a i r i n g r e s u l t s i n a 1.9 double-stranded  extensive  kb  s i n g l e - s t r a n d e d r e g i o n of  T h i s f o l d b a c k form remains s t a b l e under  conditions.  DNA  fragments c o n t a i n i n g t h i s a l t e r n a t e  f a s t e r i n agarose g e l s as seen i n the genomic  More d i r e c t p r o o f of the e x i s t e n c e of f o l d b a c k DNA  comes from the o b s e r v a t i o n t h a t the p r e d i c t e d 1.9  DNA  at t h i s  kb stem s t r u c t u r e i s  locus  128  r e s i s t a n t t o SI n u c l e a s e d i g e s t i o n a f t e r samples of genomic DNA have been denatured (Don Jones, p e r s o n a l communication). Evidence a t t h e m o l e c u l a r l e v e l f o r gene c o n v e r s i o n has a r i s e n from the sequencing o f d u p l i c a t e d n o n a l l e l i c genes such as the tRNA (Amstutz e t a l . , 1985) and cytochrome C ( E r n s t e t a l . , genes o f goats (Schon e t a l . ,  1981) genes of y e a s t , t h e a - g l o b i n  1982) and humans (Liebhaber e t a l . , 1980,  1981), t h e human y - g l o b u l i n genes ( S l i g h t o m e t a l . ,  1980; Shen e t a l . ,  1981), t h e human immunoglobin \2 c h a i n genes ( O l i o and Rougeon, 1983), and the h i g h - c y s t e i n e c h o r i o n genes o f the s i l k moth, Bombyx mori ( I a t r o u et a l . ,  1984).  In each o f these cases, t h e r e a r e r e g i o n s o f g r e a t e r  homology between genes than would be expected from normal r a t e s of sequence d i v e r g e n c e s i n c e the time o f t h e o r i g i n a l d u p l i c a t i o n event. words, these r e g i o n s have n o t e v o l v e d  In other  independently.  Gene c o n v e r s i o n may be i n v o l v e d i n m a i n t a i n i n g m i c r o d i v e r s i t y among the immunoglobulin v a r i a b l e - r e g i o n gene f a m i l i e s (Cohen e t a l . , et a l . ,  1982) and among t h e c l a s s I (Lalanne e t a l . ,  1983; Schulze e t a l . ,  1982; D i l d r o p  1982; M e l l o r e t a l . ,  1983) and c l a s s I I (Widera and F l a v e l l , 1984; Denaro  e t a l . 1984) major h i s t o c o m p a t a b i l i t y complex a n t i g e n s .  As an example, a  c o n v e r t e d r e g i o n o f o n l y 39 bp has been d i s c o v e r e d i n the gene f a m i l y encoding the v a r i a b l e r e g i o n s o f human immunoglobulin kappa l i g h t ( B e n t l e y and R a b b i t s , 1983).  chains  T h i s s t r e t c h accounts f o r 7 of 10 base  s u b s t i t u t i o n s over a l e n g t h o f 940 bp and i s evidence t h a t gene c o n v e r s i o n may l e a d t o t h e s h u f f l i n g o f gene segments. The b o v i n e prepro-AVP-NPII ( v a s o p r e s s i n ) and prepro-OT-NPI genes a r e r e l a t e d and p r o b a b l y arose from a d u p l i c a t i o n event.  (oxytocin) In t h i s  case  t h e r e i s a s t r e t c h o f 332 bp which has p e r f e c t sequence i d e n t i t y between t h e  129  two  genes.  The s i m i l a r i t y drops t o 81% i n the preceding  60 bp, and the  o t h e r s i d e can be o n l y p o o r l y a l i g n e d (Ruppert e t a l . , 1984). have suggested the occurrence the p r e s e n t  o f two s u c c e s s i v e c o n v e r s i o n  These  authors  events l e a d i n g t o  sequence o r g a n i z a t i o n , t h e second one being very  recent.  The p e r f e c t homology between the two 1.9 kb modules c o n t a i n i n g the h s p ! 6 - l and hsp!6-48 genes c o u l d be e x p l a i n e d f o r t u i t o u s l y very r e c e n t . c o n t i n u o u s l y maintained conversion reasons,  events.  i f the d u p l i c a t i o n event were  A l t e r n a t i v e l y , the homology c o u l d have been  a f t e r t h e second d u p l i c a t i o n by repeated  The l a t t e r e x p l a n a t i o n  gene  i s favored f o r p r o b a b i l i s t i c  because the sequence i d e n t i t y would be i n a steady  s t a t e and  t h e r e f o r e d e t e c t a b l e over a much longer p e r i o d o f e v o l u t i o n .  This  c o n v e r s i o n process might be f a c i l i t a t e d by the p r o x i m i t y of the two modules and by t h e unusual sequences which have been found a t e i t h e r end of the i n v e r t e d repeat s t r u c t u r e . borders  T h i s mechanism must a l s o be p r e c i s e s i n c e the  o f the " c o n v e r s i o n u n i t " a r e v e r y s h a r p l y d e f i n e d i n the p r e s e n t  example, c o n t r a s t i n g 100% sequence homology w i t h i n the converted  1.9 kb  r e g i o n w i t h complete d i v e r g e n c e  on e i t h e r s i d e of i t . I t i s i n t e r e s t i n g  that a procaryotic transposable  element such as transposon  two  Tn5, which has  IS50 modules o f 1,530 bp i n an i n v e r t e d repeat, can m a i n t a i n the  sequence i d e n t i t y o f these modules.  Perhaps i n the hsp gene c l u s t e r t h e  elements occupy s t a b l e p o s i t i o n s i n t h e genome ( u n l i k e  transposable  elements) b u t undergo s t r a n d exchange and mismatch r e p a i r f r e q u e n t l y .  It  has been demonstrated t h a t intrachromosomal, n o n r e c i p r o c a l t r a n s f e r o r gene conversion  can occur a t a h i g h frequency  Stachelek,  1983) and i n y e a s t  i n c u l t u r e d mouse c e l l s  ( K l a r and S t r a t h e r n , 1984; K l e i n ,  ( L i s k a y and 1984).  130  Given the p o s t u l a t e t h a t the hsp!6-l/48 gene c l u s t e r i s a hot spot f o r gene c o n v e r s i o n , t h e r e should be sequences which a r e i n v o l v e d i n f a c i l i t a t i n g and d e l i m i t i n g t h i s phenomenon. d i s c u s s e d i n S e c t i o n 3.12 the i n v e r t e d r e p e a t .  The G + C - r i c h sequences  a r e i n t r i g u i n g l y s i t u a t e d a t the d i s t a l borders of  T h i s sequence i s 24 bp i n l e n g t h and i s c h a r a c t e r i z e d  by a s t r e t c h o f 12 a l t e r n a t i n g p u r i n e - p y r i m i d i n e r e s i d u e s , f l a n k e d on e i t h e r s i d e by a G + C h e x a n u c l e o t i d e  i n an i n v e r t e d repeat o r i e n t a t i o n .  In f a c t ,  the e n t i r e 24 bp s t r e t c h has the p o t e n t i a l t o adopt a p a l i n d r o m i c s t r u c t u r e , the l e f t v a r i a n t c o n t a i n i n g 2 mismatches c o n t a i n i n g 3 mismatches.  i n a 12 bp stem, the r i g h t v a r i a n t  A l l of the base p a i r s i n these s t r u c t u r e s would be  G - C p a i r s , t h e o r e t i c a l l y maximizing t h e i r  stability.  S i m i l a r G 4- C - r i c h sequences are found a t the 3' border of a c o n v e r s i o n event between the human immunoglobulin a l and a2 constant r e g i o n genes (Flanagan e t a l . , 1984)  (Table V I ) .  S t r i k i n g l y , G + C hexanucleotides  i d e n t i c a l t o t h e ones i n the heat shock l o c u s f l a n k a 40 bp s t r e t c h of a l t e r n a t i n g p u r i n e - p y r i m i d i n e r e s i d u e s and are o r i e n t e d i n an i n v e r t e d repeat.  These sequences a r e shown i n upper case l e t t e r s .  The  similarity  even extends t o a c y t o s i n e t o thymine change which breaks up the i n v e r t e d r e p e a t and which i s c h a r a c t e r i s t i c of the boundary sequence v a r i a n t i n the r i g h t module of the hsp!6-l/48 l o c u s . A 2.1 kb r e g i o n of DNA  c o n t a i n i n g a p a i r of c h i c k e n h i s t o n e genes  and H2A) has been d u p l i c a t e d and i n v e r t e d , the two arms b e i n g 97% and separated by approximately  (H4  homologous  2.0 kb of u n r e l a t e d sequence which c o n t a i n s a  s i n g l e h i s t o n e H3 gene (Wang e t a l . , 1985).  Once again the borders are  d e f i n e d by 10 bp G + C - r i c h sequences, the core of which i s i d e n t i c a l t o the hexanucleotides  found  i n l o c u s hsp!6-48/l (Table V I ) .  In t h i s  case,  131  however, t h e sequences a r e found  a t b o t h t h e p r o x i m a l and d i s t a l  borders  where they o c c u r as i n v e r t e d r e p e a t s and d i r e c t r e p e a t s , r e s p e c t i v e l y .  They  a r e s e p a r a t e d by DNA s t r e t c h e s which do n o t c o n t a i n a l t e r n a t i n g p u r i n e / p y r i m i d i n e r e s i d u e s o r , i n t h e case o f the p r o x i m a l b o r d e r s , do n o t contribute to a large palindromic structure.  A C/G t o A/T t r a n s i t i o n has  a l s o o c c u r r e d i n an analogous p o s i t i o n i n one o f t h e d i r e c t repeats a t t h e right distal  border.  A computer s e a r c h o f DNA sequences i n p r o x i m i t y t o 13 r e c o m b i n a t o r i a l b r e a k p o i n t s presumed t o be a r e s u l t o f gene c o n v e r s i o n has l o c a t e d i n each case a p a l i n d r o m i c sequence (Krawinkel e t a l . , 1986).  These sequences have  stem s t r u c t u r e s ranging i n l e n g t h from 9 t o 16 bps w i t h loop s i z e s from 0 to 28 n u c l e o t i d e s .  A c o r r e l a t i o n o f c o n v e r s i o n boundaries  d i r e c t r e p e a t s was n o t observed. d i s c u s s e d above suggest  ranging with  These r e s u l t s , i n c o n j u n c t i o n w i t h  those  t h a t p a l i n d r o m i c sequences may promote gene  c o n v e r s i o n and t h a t they may serve as r e c o g n i t i o n s i t e s f o r one o r more enzymes i n v o l v e d i n g e n e t i c recombination.  The c h a r a c t e r i z a t i o n o f o t h e r  s i m i l a r G + C - r i c h sequences, which appear t o be abundant w i t h i n the genome of C. e l e g a n s , should h e l p i n u n d e r s t a n d i n g and  their function.  t h e 3/0 bp i n v e r t e d r e p e a t s t r u c t u r e s a r e a s s o c i a t e d w i t h  sequences.  Both the 1.9 kb these  The a v a i l a b i l i t y o f s p e c i f i c o l i g o d e o x y n u c l e o t i d e probes should  f a c i l i t a t e t h e i d e n t i f i c a t i o n o f o t h e r cloned f o l d b a c k s t r u c t u r e s t o determine whether they a r e a s s o c i a t e d w i t h f u n c t i o n a l genes which have a l s o undergone gene c o n v e r s i o n p r o c e s s e s .  '  T a b l e  V I .  A s s o c i a t i o n  S t r u c t u r e s  L o c a t e d  P a l i n d r o m i c  W a t s o n - C r i c k S t e m  C G G G G C c g c g c g  o f  p a i r /  G  +  a t  C - R i c h G e n e  S e q u e n c e s  C o n v e r s i o n  Location  L o o p  S i z e  h s p l 6 - l / 4 8  1 0 / 1 2  G C C C C G  l e f t  d i s t a l  l o c u s  9 / 1 2  0  h s  P  r i g h t  1 6 - l / 4 8  d i s t a l  l o c u s  1 0 / 1 0  2 1  c h i c k e n g e n e  c c G C C C C G c c  C A G G G C  B o u n d a r y  G e n e  L e n g t h  G C C C C G a c g c a c  g g C G G G G C g g  P a l i n d r o m i c  B o u n d a r i e s  S t r u c t u r e  G C C C C G a c g c a c  C A G G G C c g c g c g  w i t h  5/6  4 0  m o u s e a l  a n d  h i s t o n e l o c u s  I g - c o n s t a n t c<2  g e n e s  l e f t  a n d  r i g h t  p r o x i m a l  3 " - b o u n d a r y  133  4.4  The Heat Shock Response of C. elegans C. e l e g a n s undergoes a t y p i c a l response when exposed to h y p e r t h e r m i c  conditions.  In v i v o l a b e l l i n g of nematodes a t 35°C shows t h a t most of the  p r e - e x i s t i n g p r o t e i n s y n t h e s i s d e c l i n e s w h i l e a p p r o x i m a t e l y 10 p r o t e i n s a r e abundantly s y n t h e s i z e d .  These experiments cannot d i s t i n g u i s h between those  p r o t e i n s whose s y n t h e s i s i s induced a t the h i g h e r temperature and those whose p r e v i o u s s y n t h e s i s under normal temperatures i s u n a f f e c t e d by heat shock.  F o r example, hsp70 c o n s i s t s of a t l e a s t two p r i m a r y gene p r o d u c t s ,  one which i s s y n t h e s i z e d o n l y under heat shock c o n d i t i o n s w h i l e the o t h e r i s a p r o d u c t of an hsp70 cognate gene which i s a c t i v e a t normal temperatures (Snutch and D a i l l i e ,  1984).  Hspl6, however, appears to be newly  synthesized  at the e l e v a t e d temperature and t h i s has been v e r i f i e d by the h y b r i d i z a t i o n of s p e c i f i c c l o n e d DNA  sequences to polyA RNA i s o l a t e d from heat shocked +  nematodes b u t n o t to a s i m i l a r RNA nematodes.  fraction purified  from n o r m a l l y growing  Thus the hsp!6 genes, of which t h e r e are a t l e a s t f o u r , are  under a t i g h t t r a n s c r i p t i o n a l c o n t r o l mechanism.  A l t h o u g h the t r a n s i e n t  n a t u r e of the h e a t shock response i n C. e l e g a n s has n o t been it  investigated,  appears t h a t the resumption of normal p r o t e i n s y n t h e s i s does not o c c u r o  within 5 - 6  hours i f the nematodes a r e m a i n t a i n e d a t 35 C ( F i g u r e 3 ) .  T h i s i s s i m i l a r t o the s i t u a t i o n seen i n HeLa c e l l s i n which normal p r o t e i n s y n t h e s i s does n o t resume f o r a t l e a s t 10 hours i f the c e l l s are m a i n t a i n e d a t 42°C (Hickey and Weber, 1982).  I n c o n t r a s t , resumption of e x i s t i n g o p r o t e i n s y n t h e s i s can o c c u r w i t h i n 10 - 20 minutes a t 36 C i n y e a s t o ( M c A l i s t e r and F i n k e l s t e i n , 1980) o r w i t h i n 2 - 3 D r o s o p h i l a (DiDomenico e t a l . ,  1982b).  hours a t 37 C i n  Assuming t h a t the heat shock  p r o t e i n s s e r v e a p r o t e c t i v e r o l e d u r i n g c e l l u l a r s t r e s s , then i t would be b e n e f i c i a l f o r them to p o s s e s s  134  r e l a t i v e l y extended h a l f l i v e s . emphasized  by the presumably  The  importance  of t h i s p r o p e r t y i s f u r t h e r  t r a n s i e n t n a t u r e of the heat shock  i t has been found t h a t i n D r o s o p h i l a . hsp70 r e p r e s s e s i t s own continued s t r e s s .  response:  s y n t h e s i s upon  As shown i n F i g u r e 4, the hsps of C. e l e g a n s . which were  p u l s e - l a b e l l e d a t 35°C, are d e t e c t a b l e i n c e l l s 24 hours f o l l o w i n g heat shock, a t l e v e l s comparable t o those observed d u r i n g o r immediately the  after  shock. In v i t r o t r a n s l a t i o n of polyA RNA from heat shocked nematode c u l t u r e s +  demonstrated temperatures.  t h a t a t r a n s l a t i o n a l c o n t r o l a l s o operates i n nematodes at h i g h Although p r e - e x i s t i n g messages are s t i l l p r e s e n t i n heat  shocked  c e l l s as d e t e c t e d by t r a n s l a t i o n i n v i t r o , they are not  in vivo  (compare F i g u r e 3 and F i g u r e 5, lane C).  sequences  of hsp70 (McGarry  and L i n d q u i s t , 1985;  hsp22 (Hultmark e t a l . . 1986)  In D r o s o p h i l a . the l e a d e r Klemenz et a l . ,  mRNA are r e s p o n s i b l e f o r t h e i r  t r a n s l a t i o n , a l t h o u g h i t i s not y e t known how  translated  1985)  selective  t h i s i s accomplished.  The  mechanism i n v o l v e d does not appear to have s p e c i f i c sequence (McGarry L i n d q u i s t , 1985) 1985;  o r l e n g t h (McGarry  Hultmark e t a l . , 1986)  sequenced  and L i n d q u i s t , 1985;  requirements.  and  Klemenz e t a l . .  The l e a d e r sequences  hsp!6 genes are shown i n F i g u r e 26.  and  of a l l 4  When a l i g n e d to compensate  f o r d e l e t i o n s / i n s e r t i o n s , the l e a d e r s of the C. elegans hsp!6 genes d i s p l a y a g r e a t d e a l of s i m i l a r i t y w i t h an average nucleotides.  l e n g t h of approximately 45  The involvement of these l e a d e r s i n the a b i l i t y of hspl6  t r a n s c r i p t s to be r e c o g n i z e d by heat shocked  ribosomes  which mutated l e a d e r r e g i o n s a r e assayed f o r s e l e c t i v e Due  awaits experiments i n translation.  to the d i v e r g e n t d i r e c t i o n s of t r a n s c r i p t i o n , the 5'  r e g i o n s of the hsp!6 gene p a i r s are p r e c i s e l y d e f i n e d .  The  noncoding  intergenic  135  TRANSCRIPTION STARTS  TATA BOX "  »  MET  "I  GAG|GAA AjqqAATA  CAC T T T G T T C A A  HSP16-48  GGjGTATATA  AGCCAAlCGTGTTCA  HSP16-41  GG3TATATA  1 3 G A A A C C 3GGCTC * - G A G 2AA G ZZ&ACA - C G O T T T G M T C T A W v  CGTTGlAhAATAA  HSP16-2  TA J T A T A A A r  HSP16-1  GAJGTATAAAfrlAbAGTGACHAAA  F i g u r e 26.  TACC  V  •ACAlAfcA  CCfeAAjCttl! AACAlAlCA  GTG C T T A C T G T T C A T T C T C T A A A C T 1 C A A GAATG k3TG  A T C T A A p A A A C T T CGA 4 A I T G  TTTG  rCTApTTpTGfVAATTAGAA- \TCTTC  TTCAi  rCTAlATTlGTGjAA  J A A C T 1 rAA TC i T G  I A T C T T C i &AACTA :AAh~CiATG|  The l e a d e r sequences of the hsp_16_ genes of C. elegans  The TATA m o t i f s and the t r a n s c r i p t i o n s t a r t s deduced from SI n u c l e a s e p r o t e c t i o n a n a l y s i s are indicated.  P o s i t i o n s where a t l e a s t three out o f  f o u r n u c l e o t i d e s a r e i d e n t i c a l are boxed. b e s t alignment.  Gaps were i n t r o d u c e d to g i v e the  The hsp!6-2 and hsp!6-41 sequences and s t a r t s i t e s a r e  taken from Jones et. a l .  (1986).  136  r e g i o n s of the hsp!6-41/2 and 27.  This  region  nucleotide  hsp!6-48/l gene p a i r s are a l i g n e d  shows an o v e r a l l homology of 85%  differences.  The  sequences; two  hsp!6-48 and  of the  hsp!6-41 genes.  26  sequences of  These i n c l u d e the TATA sequences  l a t t e r overlap The  Figure  c o n t a i n s only  known f u n c t i o n a l l y important 5'  the hsp!6 genes have been conserved. the HIP  and  in  each other upstream of  conserved r e g i o n s i n c l u d e  and  the  sequence  elements the s i g n i f i c a n c e of which i s unknown, such as the s t r e t c h of a l t e r n a t i n g purine-pyrimidine residues region  of dyad symmetry.  s i t u a t e d adjacent to a prominent  T r a n s c r i p t i o n a l s t u d i e s of the hsp!6-48  and  h s p ! 6 - l gene p a i r i n mouse f i b r o b l a s t c e l l s have demonstrated that heat i n d u c i b i l i t y as w e l l as a r s e n i t e sequences (Kay  e t a l . , 1986).  bidirectionally,  inducing  between the TATA boxes.  i n d u c i b i l i t y i s dependent on the  A s i n g l e HIP  sequence can  HIP  function  the t r a n s c r i p t i o n of both genes when p l a c e d In t h i s case, however, the e f f i c i e n c y i s reduced  f o l d r e l a t i v e t o the w i l d type gene p a i r . elements between the genes r e s u l t e d  Placing  four overlapping  10  promoter  in inducible bidirectional transcription  a t l e v e l s h i g h e r than those found w i t h the w i l d type gene p a i r . The  h i g h degree of s i m i l a r i t y between the  heat shock l o c i  implies  the e x i s t e n c e  a d d i t i o n t o the heat shock promoters. yet,  i n t e r g e n i c r e g i o n s of the  of f u n c t i o n a l l y important sequences i n Although none have been i d e n t i f i e d as  i t i s p o s s i b l e t h a t o t h e r m e t a b o l i c s t r e s s o r s or p h y s i o l o g i c a l  a c t i v a t e the hsp!6 genes through a l t e r n a t i v e c i s - a c t i n g elements. o b s e r v a t i o n s made i n D r o s o p h i l a . s e r v e some f u n c t i o n during subset t h e r e o f  may  two  states Based  t h e r e e x i s t s the p o s s i b i l i t y t h a t h s p l 6  on may  nematode development, i . e . the hsp!6 genes or a  be a c t i v a t e d  i n a s t a g e - s p e c i f i c manner.  a v a i l a b i l i t y o f s p e c i f i c gene probes now  The  a l l o w s a d e t a i l e d a n a l y s i s of  RNA  13 7  MET  V  W  HSP 1 S - 4 1 - C A T T T T C G A A G T T T T T T A GATQCACTAGA ACAAAGCGTGTTGGCTTCCTCTGAGCCCGCTT H S P 16 - 4 8 - C A T T C T T G A A G T T T A G A G A A T G A A C A G T A A G C A C T T G A A C A A A G T G T A T T G G T T T C C T C T G A A C A C G A T T GTA  — H I P  A  TCCTT ATATAJCCCGCATTCTGCAGCCTT ACAGAATGTTCTAGAAGGTCCTAGATGCATTCGTTTGAAA'ATACTCCCGGT GGCTT A T A T A C C C G T A T C C T G C A G C C G T T T A G A A T G T T C T A G A A G G T C C T A G A T G C A T T C A T T T C A A A A T A C A C C C C A T CRUCIFORM  FIGURE  GGGTGCJAA A G A G A C G C A G A C G G A A A ATGT ATCTGGGTCTCTTTJATJTGTGT AC ACfT A C T T T T C C A T G T A C C G A A T G T G A G P ltG T A C G C A C A C [ T A f T T C T C A A.T-r^-r-r^-rs* A G G T G C ^ A A G A G A C G C A G A T T G A A A A A G T A T C T G G G T f T C TITlp A G T T C T G AA A T G T G A G HIP — * •  VW  TCGCCCTCCTTTTGCAACAAGCAGCTCGAATGTTCTAGAA  AAAGGTGGAAAATAGrATAAATACCGTTGAAAATAAATA  TCGCCCTCCTTTTGCAAGAAGCAGCTCGAATGTTCTAGAAAAAGGTGGAAAT GAG[TATAAATA|CAGTGACAAA--W MET CCGAAC -AACATTTGCTCTAATTGTGAAATTAGAAATCTTCAAACTATAATCATG-HSP16-2 CCGAACCAAACAACATTCACTCTAATTGTGAAA TCTTCAAACTACAATCATG-HSP16-7  Figure l o c i  27.  A  o f C. Dots  TATA  f i g u r e  o f t h e i n t e r g e n i c  regions  o f t h e two hsp!6  gene  elegans. i n d i c a t e  introduced and  comparison  p o s i t i o n s  t o obtain boxes  with  t h e best  a r e i n d i c a t e d ,  t h e p o t e n t i a l  purine-pyrimidine  sequence  t r i a n g l e s ,  with  t h e major  i n t e r g e n i c  region  i s taken  o f sequence alignment.  d i f f e r e n c e s The heat  as i s t h e p o s i t i o n  t o adopt ( Z ) .  a  stem-loop  T r a n s c r i p t i o n  and gaps  inducible  promoters  o f t h e large  s t r u c t u r e s t a r t  s i t e s  shown  as f i l l e d  from  Jones  e t a l . (1986).  were (HIPs)  cruciform  and i t s a d j o i n i n g  s i t e s  t r i a n g l e s .  a r e i n d i c a t e d  by  The hsp!6-2/41  A  138  purified four  from d i f f e r e n t developmental stages i n c l u d i n g  i n t e r m e d i a t e l a r v a l forms.  t h e s i s was  Although the  prepared from mixed c u l t u r e s ,  of a p a r t i c u l a r hsp!6 gene d u r i n g one  c o n t r o l RNA  s p e c i f i c stage out  In v i v o  h s p l 6 i s heat i n d u c i b l e (Snutch and  Baillie,  protein  i n a l l of the  two  i n t h e i r rates  but  not  The  identical.  a f f e c t the  intergenic  r e g i o n s of the  It i s possible  that  s i x major  limited s e n s i t i v i t y  elegans have r a d i c a l l y T h i s may  reflect a  two  gene p a i r s are v e r y  o u t s i d e the  the  transcription.  c e n t r a l l y located  For  Alternatively,  r e g i o n s d i s t i n g u i s h the  example, a  palindromic 12  bp  two  to  t h r e e major f e a t u r e s d i f f e r e n t gene p a i r s .  nonhomologous 3' noncoding sequences, the presence of  r e p e t i t i v e elements f l a n k i n g duplication  could  between the hsp!6-l/48 gene p a i r , which would be expected  intergenic  These i n c l u d e  similar  some minor sequence v a r i a t i o n  i n t r o d u c e s a second base p a i r mismatch i n t o the p r e d i c t e d  significantly affect i t s stability.  are  of the  of t r a n s c r i p t i o n or i n t h e i r r e l a t i v e message  r e l a t i v e l e v e l s of heat i n d u c i b l e  stem s t r u c t u r e  The  transcription  above-mentioned developmental stages  hsp!6 gene p a i r s o f C.  c y t o s i n e t o thymine t r a n s i t i o n w i t h i n structure  the  l a b e l l i n g experiments have shown t h a t  d i f f e r e n t e x p r e s s i o n l e v e l s d u r i n g heat shock.  stabilities.  the  1983).  Remarkably, the  difference  t o the  and  used i n t h i s  i t i s l i k e l y that  i d e n t i f i a b l e ones would have gone undetected due of N o r t h e r n a n a l y s i s .  eggs, a d u l t s  the hsp!6-2/41 gene p a i r and  the  inverted  of the hsp!6-l/48 gene p a i r .  3' noncoding r e g i o n s of the  two  r a d i c a l l y d i f f e r e n t i n l e n g t h (38  sequence s i m i l a r i t y .  The  o t h e r two  r e l a t e d genes hsp!6-48 and  v e r s u s 94 n u c l e o t i d e s ) and  r e l a t e d genes, h s p ! 6 - l and  homology e x t e n d i n g 45 bp p a s t the p o l y a d e n y l a t i o n s i g n a l .  hsp!6-41  show no  hsp!6-2. show  Perhaps more  139  s i g n i f i c a n t i s the presence of an A + T - r i c h sequence i n the 3* noncoding r e g i o n o f t h e hsp!6-2 gene which c o u l d form a p e r f e c t 7 bp h a i r p i n . occurs  i n a r e g i o n which has been d e l e t e d i n t h e corresponding  expressed h s p ! 6 - l  gene.  less actively  The o t h e r h i g h l y expressed gene, hsp!6-41. has a  p e r f e c t 11 bp A + T - r i c h h a i r p i n l o c a t e d adjacent signal.  It  I t i s conceivable  to the p o l y a d e n y l a t i o n  t h a t these secondary s t r u c t u r e s may  i n t r a n s c r i p t processing, polyadenylation  be i n v o l v e d  or s t a b i l i t y .  Three c o p i e s of the 200 bp r e p e t i t i v e element f a m i l y CeRep-16 f l a n k t h e more a c t i v e hsp!6  locus  (Jones e t a l . , 1986).  c o n t a i n i n g the proximal of the hsp!6-41  An EcoRI-EcoRV  fragment  h a l f of the s i n g l e r e p e t i t i v e element at the 3' end  gene was h y b r i d i z e d to phage c l o n e s \Charon4  \Charon4 B-7, which r e p r e s e n t  B-3  and  the e n t i r e 30 kb r e g i o n shown i n F i g u r e  12.  Under c o n d i t i o n s i n which the r e l a t e d r e p e t i t i v e elements from the 3' end of the hsp!6-2 gene could be d e t e c t e d ,  t h e r e was no h y b r i d i z a t i o n to any of the  EcoRI fragments d e r i v e d from the above-mentioned phage. shown) i n d i c a t e t h a t homologous  These r e s u l t s (not  sequences a r e not p r e s e n t  within at l e a s t  kb on e i t h e r s i d e of the l e s s a c t i v e l o c u s c o n t a i n i n g the hsp!6-l/48 gene pairs.  Members o f the CeRep-16 element c o n t a i n m u l t i p l e repeats  o f the  sequence of G TTTGC, which i s very s i m i l a r t o p a r t of the "enhancer G c o r e " sequence GGTTTG found i n a v a r i e t y o f v i r a l and c e l l u l a r enhancers ( S e r f l i n g e t a l . , 1985).  Thus i t i s p o s s i b l e t h a t an  mechanism may be r e g u l a t i n g t h e e x p r e s s i o n locus.  Unfortunately,  of t h e heat shock genes a t t h i s  the d i f f e r e n t i a l e x p r e s s i o n  of these gene p a i r s  cannot be reproduced when t r a n s f e c t e d i n t o mouse c e l l s communication) suggesting  enhancer-like  (Rob Kay,  t h a t the f a c t o r s t h a t mediate 20 f o l d  personal higher  t r a n s c r i p t l e v e l s of the hsp!6-2/41 gene p a i r r e l a t i v e to the hsp!6-l/48  10  140  gene p a i r i n C. elegans a r e n o t conserved i n mouse c e l l s . homologous  The use o f a  t r a n s f o r m a t i o n system, which has r e c e n t l y been developed f o r  C. e l e g a n s (Stinchcomb e t a l . , 1985), may prove t o be h e l p f u l i n demonstrating the e x i s t e n c e and n a t u r e o f such f a c t o r s . Another reason f o r the observed d i f f e r e n t i a l e x p r e s s i o n may simply l i e i n t h e p o s s i b i l i t y t h a t the two heat shock l o c i a r e s i t u a t e d i n d i f f e r e n t chromosomal  environments o r domains. The l o c i have n o t y e t been  genetically  mapped b u t even i f they were l i n k e d , they would be separated by a t l e a s t 10 kb o f DNA. In of  a d d i t i o n to t r a n s c r i p t i o n a l and t r a n s l a t i o n a l c o n t r o l ,  the presence  i n t r o n s i n heat i n d u c i b l e genes i n t r o d u c e s the p o s s i b i l i t y of r e g u l a t i n g  gene e x p r e s s i o n a t the l e v e l o f s p l i c i n g .  I t i n i t i a l l y appeared t h a t the  presence of i n t e r v e n i n g sequences was c o n f i n e d to genes which were t r a n s c r i b e d a t normal growth temperatures.  These i n c l u d e the c o n s t i t u t i v e l y  expressed cognate genes o f t h e D r o s o p h i l a hsp70 gene f a m i l y as w e l l as t h e D r o s o p h i l a hsp83 and human hsp25 genes which a r e expressed under normal c o n d i t i o n s b u t a r e induced t o much h i g h e r l e v e l s d u r i n g heat shock. hsp16  The  genes o f C a e n o r h a b d i t i s comprise a t h i r d group o f i n t r o n - c o n t a i n i n g  genes, b e i n g i n a c t i v e a t normal temperatures. Hsp83 has a p a t t e r n o f e x p r e s s i o n which i s d i f f e r e n t from any o f the o t h e r hsps i n D r o s o p h i l a (Yost and L i n d q u i s t , 1986). it  o i s produced a t s u b s t a n t i a l l e v e l s a t 25 C  observed between o  by 38 C.  As a l r e a d y mentioned,  Maximum e x p r e s s i o n i s  o o 33 C and 35 C, d e c r e a s i n g t o b a r e l y d e t e c t a b l e  o I n c o n t r a s t hsp70 i s h i g h l y induced a t 38 C.  levels  A l t h o u g h the 6  amount o f hsp83 mRNA remains c o n s t a n t a t temperatures above 35 C, t h e decreased e x p r e s s i o n of hsp83 i s due t o the r e v e r s i b l e thermal s e n s i t i v i t y  141  of the s p l i c i n g apparatus, v i r t u a l l y a l l o f the hsp83 mRNA b e i n g found as o u n s p l i c e d p r e c u r s o r a t 38 C (Yost and L i n d q u i s t , 1986). o b s e r v a t i o n s were made by S t e l l a r and P i r r o t t a  Similar  (1985) i n a transformed  D r o s o p h i l a s t r a i n c a r r y i n g the white gene which had been l i n k e d t o an hsp70 promoter.  A t 37°C, the white gene t r a n s c r i p t s , which c o n t a i n e d a l a r g e  i n t r o n , were i n e f f i c i e n t l y s p l i c e d .  Proper s p l i c i n g resumes upon r e c o v e r y  a t 25°C ( S t e l l e r and P i r r o t t a , 1985;  Yost and L i n d q u i s t , 1986).  I f a mild  35°C heat shock, which induces the s y n t h e s i s o f hsps, i s a d m i n i s t e r e d p r i o r t o a severe 38°C heat shock, hsp83 t r a n s c r i p t p r o c e s s i n g occurs under o t h e r w i s e r e s t r i c t i v e c o n d i t i o n s (Yost and L i n d q u i s t , 1986). suggested by these authors t h a t one o f the f u n c t i o n s o f the heat response i s t o p r o t e c t the s p l i c i n g I t remains  apparatus.  o t h e r than D r o s o p h i l a .  s i m i l a r phenomenon occurs i n C. elegans.  I t i s p o s s i b l e that a  Snutch and B a i l l i e  (1983) have  observed t h a t hspl6 i s not expressed a t h i g h e r temperatures. pCEHS41 corresponds to an u n s p l i c e d t r a n s c r i p t .  A l s o , the cDNA  Although SI mapping  i n d i c a t e d t h a t hspl6-41 t r a n s c r i p t s were completely s p l i c e d  RNA  shock  t o be seen whether RNA p r o c e s s i n g i s s i m i l a r l y more  t h e r m o - l a b i l e i n organisms  1986),  It i s  (Jones e t a l . ,  the mRNA used i n these s t u d i e s was i s o l a t e d independently from the  used i n the c o n s t r u c t i o n o f the cDNA l i b r a r y and which was k i n d l y  p r o v i d e d by T e r r y Snutch, Simon F r a s e r U n i v e r s i t y .  Thus the nematodes from  which RNA was i s o l a t e d were s u b j e c t e d t o d i f f e r e n t heat shock  conditions.  C. e l e g a n s hsp70 i s expressed under severe heat shock c o n d i t i o n s i n which h s p l 6 i s not (Snutch and B a i l l i e ,  1983).  Although one o f the members o f the  hsp70 gene f a m i l y c o n t a i n s an i n t r o n (Mark H e s c h l , p e r s o n a l the presence o f i n t e r v e n i n g sequences  communication),  i n the o t h e r r e l a t e d genes has n o t  142  been determined.  Thus the s y n t h e s i s of hsp70 at h i g h e r temperatures  may  take p l a c e on t r a n s c r i p t s which have bypassed the s p l i c i n g mechanism. hsp25 mRNA i s completely 1986)  s p l i c e d a t 42°C i n HeLa c e l l s  but t h i s temperature may  i n a c t i v a t i o n f o r a mammalian The  Human  (Hickey e t a l . ,  be below the t h r e s h o l d of s p l i c i n g cell.  i n c r e a s e d temperature s e n s i t i v i t y of s p l i c i n g r e l a t i v e to  t r a n s c r i p t i o n and  t r a n s l a t i o n may  r e f l e c t a physiologically relevant  control  mechanism i n which the e x p r e s s i o n of t r a n s c r i p t s c o n t a i n i n g i n t e r v e n i n g sequences i s i n h i b i t e d d u r i n g severe unspliced transcripts  stress.  A l s o , the accumulation  d u r i n g heat shock r e s u l t s i n the delayed  these RNAs i n t o p r o t e i n  products  can o n l y occur d u r i n g r e c o v e r y  s i n c e s p l i c i n g and  subsequent  of  expression translation  at lower temperatures or upon a d a p t a t i o n  the s p l i c i n g mechanism d u r i n g maintained  stress.  of  of  143  V.  REFERENCES 1. A l t s c h u l e r , M. and J.P. Mascarenhas. 103-115.  1982.  P l a n t Mol. B i o l .  1:  2. Amin, J . , R. M e s t r i l , R. Lawson, H. K l a p p e r , and R. Voellmy. Mol. C e l l . B i o l . 5: 197-203.  1985.  3. Amstutz, J . , P. Munz, W.-D. C e l l 40: 879-886.  1985.  Heyer, U. Leupold, and J . K o h l i .  4. Ananthan, J . , A.L. Goldberg, and R. Voellmy. 522-524.  1986.  Science 232:  5. A m o t t , S., R. Chandrasekaran, D.L. B i r d s a l l , A.G.W. L e s l i e and R.L. Rattliff. 1980. Nature 283: 743-745. 6. A r r i g o , A.P., S. Fakan, and A. T i s s i e r e s .  1980.  7. 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