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An analysis of UNC-52/perlecan domain IV immunoglobulin repeats in myofilament assembly of Caenorhabditis… Bush, Jason Allan 1997

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AN ANALYSIS OF U N O 5 2 /PERLECAN DOMAIN I V IMMUNOGLOBULIN REPEATS I N MYOFILAMENT ASSEMBLY OF Caenorhahd.it is elegans. by JASON ALLAN BUSH B.Sc,  U n i v e r i s t y o f B r i t i s h C o l u m b i a , 1992  A THESIS SUBMITTED I N PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF MASTER OF SCIENCE in  THE FACULTY OF GRADUATE STUDIES (Department of Z o o l o g y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e required standard  THE UNIVERISTY OF BRITISH COLUMBIA O c t o b e r 1997 J a s o n A l l a n Bush  In presenting this thesis in partial fulfilment  of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely' available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by his or  her  representatives.  It  is understood that  copying or  publication of this thesis for financial gain shall not be allowed -without, my written permission.  Department of  CO \ O  The University of British Columbia Vancouver, Canada Date  DE-6 (2788)  ABSTRACT The unc-52(II) gene encodes the nematode homologue of p e r l e c a n , a mammalian basement membrane (BM) p r o t e o g l y c a n . UNC-52 i s e s s e n t i a l f o r p r o p e r m y o f i l a m e n t a s s e m b l y and m u s c l e a t t a c h m e n t i n C. elegans. The l o n g e s t p r e d i c t e d UNC-52 p r o t e i n h a s five s t r u c t u r a l domains, i n c l u d i n g a L D L - r e c e p t o r - l i k e domain (domain II), a l a m i n i n - l i k e domain (domain I I I ) , an N C A M - l i k e domain (domain IV) and a g l o b u l a r l a m i n i n - l i k e domain (domain V) . The unc-52 gene c o n s i s t s o f 37 exons w h i c h , t h r o u g h a l t e r n a t i v e s p l i c i n g , g e n e r a t e a number o f p r o t e i n i s o f o r m s . These i s o f o r m s show b o t h t i s s u e and t e m p o r a l s p e c i f i c i t y . The domain s t r u c t u r e of unc-52 l e n d s i t s e l f t o r e g u l a t i o n v i a a l t e r n a t i v e s p l i c i n g . In particular, domain IV contains multiple copies of an i m m u n o g l o b u l i n - l i k e r e p e a t ( I g - r e p e a t ) and many o f t h e r e p e a t s are encoded by i n d i v i d u a l exons. A l t e r n a t i v e s p l i c i n g o f exons 16, 17 a n d 18 a l t e r s t h e number o f I g - r e p e a t s w i t h i n t h i s domain. N u l l m u t a t i o n s i n unc-52 l e a d t o a l e t h a l P a t (paralyzed a r r e s t e d e l o n g a t i o n a t two-fold) phenotype ( e . g . st549), while m u t a t i o n s i n exons 16-18 l e a d t o a v i a b l e Unc p h e n o t y p e . Through the sequence a n a l y s i s o f two new unc-52 m u t a t i o n s , st560 and r a l l 2 , I e s t a b l i s h e d t h a t domain I V - c o n t a i n i n g i s o f o r m s o f unc-52 are c r i t i c a l f o r myofilament assembly d u r i n g e a r l y development. These two m u t a t i o n s b o t h l e a d t o a P a t p h e n o t y p e a n d r e s u l t i n p r e m a t u r e t r u n c a t i o n o f UNC-52 i s o f o r m s w i t h domain I V ; st560 i s a s t o p c o d o n i n exon 13; and r a l l 2 i s an d e l e t i o n removing s e v e r a l I g - e n c o d i n g exons w i t h i n domain I V . U s i n g a T e l e x c i s i o n and d o u b l e - s t r a n d b r e a k r e p a i r scheme t o v a r y t h e number o f I g r e p e a t s w i t h i n an i s o f o r m , I a d d r e s s e d t h e functional significance o f domain IV i n establishing proper muscle assembly. I s o u g h t t o d e t e r m i n e t h e m i n i m a l number o f I g r e p e a t s n e c e s s a r y w i t h i n domain I V t o a l l o w p r o p e r m y o f i l a m e n t assembly and t o determine the r o l e o f c o m b i n a t o r i a l I g r e p e a t s i n assembly. I characterized eight in-frame d e l e t i o n a l l e l e s created by T e l e x c i s i o n which e l i m i n a t e from 1 t o 4 Ig repeats. Animals homozygous f o r t h e s e d e l e t i o n s a p p e a r w i l d - t y p e i n movement and muscle s t r u c t u r e . The mec-8 gene encodes a p u t a t i v e R N A - b i n d i n g p r o t e i n t h a t i s r e q u i r e d f o r some o f t h e a l t e r n a t i v e s p l i c i n g o f unc-52 (Lundquist e t al., 1 9 9 6 ) . I n t h e a b s e n c e o f MEC-8 f u n c t i o n , o t h e r w i s e v i a b l e m u t a t i o n s i n t h e a l t e r n a t i v e l y - s p l i c e d r e g i o n o f unc-52 a r e l e t h a l b e c a u s e t h e a f f e c t e d exons a r e no l o n g e r s p l i c e d o u t o f e m b r y o n i c unc-52 pre-mRNA ( L u n d q u i s t e t al., 1 9 9 6 ) . I constructed several mec-8; unc-52(deletion) double mutants t o ask whether MEC-8 f u n c t i o n was s t i l l r e q u i r e d i n t h e a b s e n c e o f i t s s p l i c i n g t a r g e t . I f o u n d t h a t mec-8(null); unc-52(viable deletion) combinations are p h e n o t y p i c a l l y w i l d - t y p e , s u g g e s t i n g a complete independence from mec-8.  i i  TABLE OF CONTENTS Abstract  i i  Table of Contents  i i i  L i s t of Tables  v  L i s t of Figures  vi  L i s t of Abbreviations  v i i  Acknowledgement  viii  Introduction  1  I.  Extracellular Matrices  1  II.  Perlecan/UNC-52  2  III.  M u s c l e S t r u c t u r e i n C. e l e g a n s  4  IV.  Focal Contacts  4  V.  M u s c l e Development  7  VI.  Mutants and Isoform D i v e r s i t y  13  VII.  Model and S t r a t e g i e s  20  V I I I . G o a l s and Summary  24  M a t e r i a l s and Methods  25  I.  Nematode s t r a i n s , m a i n t e n a n c e , g e n e t i c c o n s t r u c t s . . .  25  II.  PCR m p l i f i c a t i o n o f genomic DNA  25  III.  PCR s e q u e n c i n g  26  IV.  I s o l a t i o n of d e l e t i o n revertants  28  V.  Lethal deletion revertants  29  VI.  C o n s t r u c t i o n o f mec-8;unc-52 d o u b l e m u t a n t s  30  VII.  D y e - f i l l i n g t o s t a i n amphid/phasmid  n e u r o n s , , 3 0  V I I I . I m m u n o f l u o r e s c e n c e t e c h n i q u e s and m i c r o s c o p y Results I.  30 32  Domain I V o f p e r l e c a n i s e s s e n t i a l f o r m y o f i l a m e n t a s s e m b l y i n m u s c l e and f o r nematode v i a b i l i t y iii  32  II. III.  A l t e r n a t i v e l y - s p l i c e d Domain I V i m m u n o g l o b u l i n e n c o d i n g exons a r e d i s p e n s a b l e  39  Domain I V d e l e t i o n s e l i m i n a t e t h e r e q u i r e m e n t f o r MEC-8-mediated s p l i c i n g o f unc-52 d u r i n g embryogenesis 48  Discussion  55  I.  Domain I V i s e s s e n t i a l f o r UNC-52 f u n c t i o n embryogenesis  II.  Effects unc-52  III.  Effects of mec-8;unc-52(deletion-revertant) on m u s c l e d e v e l o p m e n t  IV.  Summary  during  o f removing a l t e r n a t i v e l y s p l i c e d exon o f mutants  56 59 61 63  L i s t of References  66  Appendix  71  i v  LIST OF TABLES  Table 1  Table 2  Primer sets used i n a m p l i f i c a t i o n o f genomic DNA  27  The new domain I V - s p e c i f i c mutations  34  knockout  Table 3  R e v e r t a n t s o b t a i n e d from T e l e x c i s i o n  Table 4  C h a r a c t e r i z a t i o n of noteworthy a l o n e and i n d o u b l e homozygous w i t h a mec-8 n u l l  v  screen  revertants combination  41  45  LIST OF FIGURES  Figure 1  S t r u c t u r e o f t h e sarcomere and basement membrane...6  Figure 2  A n a t o m i c a l s t r u c t u r e o f t h e worm  Figure 3  Development  Figure 4  unc-52 gene s c h e m a t i c  Figure 5  Immunofluorescence s t a i n i n g o f w i l d - t y p e  o f C. elegans  and i t s m u s c l e  9 12 16  and mutant embryos  19  Figure 6  T e l e x c i s i o n scheme  23  Figure 7  D e l e t i o n s r e v e r t a n t s i n domain I V  38  Figure 8  Deletion breakpoints  43  Figure 9  Comparison o f n o r m a l and p o t e n t i a l l y h y b r i d I g s . . . 4 7  F i g u r e 10  Ribbon diagram o f C2-type immunoglobulin  50  F i g u r e 11  P o t e n t i a l s p l i c e choices occuring i n alternatively-spliced region  54  vi  LIST OF ABREVIATIONS  BM  basement membrane  bp  basepairs  dpy  dumpy  ECM  extracellular matrix  kb  kilobase  LDL  low-density  mec  mechanosensory  mL  millilitre  mM  millimolar  mut  mutator  pat  paralyzed, arrested elongation a t two-fold  PCR  polymerase c h a i n r e a c t i o n  pmol  picamole  U.L  microliter  uric  uncoordinated  lipoprotein  vii  ACKNOWLEDGEMENT  I w o u l d l i k e t o g r a c i o u s l y thank my s u p e r v i s o r , guidance,  h i sfinancial  stimulation. their  I t h a n k Greg M u l l e n ,  scientific  partaking  support,  and l i t e r a r y  and h i s constant  intellectual  Ken Norman a n d Poupak Rahmani f o r  assistance,  i n necessary ' d i s t r a c t i o n s ' .  f o r h e r guidance and i n v a l u a b l e  Don Moerman, f o r h i s  my f r i e n d s , a n d  I a l s o thank Teresa  suggestions.  viii  being  Rogalski  INTRODUCTION  I. E x t r a c e l l u l a r  Matrices  Basement membranes  (BM) a r e d e f i n e d a s s p e c i a l i z e d  the e x t r a c e l l u l a r matrix membranes times  a  types.  form zone  (ECM) (Yurchenco  1990) .  These  a barrier  and a t  f o r i n f o r m a t i o n t r a n s f e r between two d i s t i n c t  tissue  a dynamic network,  A l l extracellular  acting  m a t r i c e s have  & Schittny,  regions of  as both  t h e same b a s i c  components  i n c l u d i n g c o l l a g e n s (type I V ) , l a m i n i n s , n i d o g e n a n d h e p a r a n s u l f a t e p r o t e o g l y c a n s ( p e r l e c a n ) ( T i i n p l , 1993; T i m p l a n d Brown, 1996) . is  tremendous s t r u c t u r a l  which  of these  components  leads to a q u a l i t a t i v e l y diverse array of matrices  (Timpl and  Brown, 1996) .  diversity within  This d i v e r s i t y arises  f a m i l i e s f o r each  o f t h e s e components  H e i k k i l a a n d S o i n i n e n , 1996)  each  from t h e p r e s e n c e  and/or a l t e r n a t i v e  genes  al,  The BM i s i m p o r t a n t a s a s t r u c t u r a l  1996) .  attachment  (Noonan et al,  of cells  by  materials  acting  between  as  a  tissue  components o f t h e m a t r i x with  these  (reviewed  interactions  stabilizing interaction  the  I t appears molecular  types  substrate f o r the  receptors  (usually  e t al,  remain  and thus  static of  gradients this  latter  f o r the d i f f u s i o n  1994).  i n Calderwood  attachment  et  The  of  structural  a c t as l i g a n d s v i a i n t e r a c t i o n s  cells,  may be more dynamic t h u s  adjacent tissues.  1993; L u n d q u i s t  t o perform  sieve  (Inoue,  can also  v a r i o u s transmembrane  surfaces  1991; T i m p l ,  s p l i c i n g o f these  and a l s o as t h e mediator o f chemical  the migration of c e l l s .  function  of multigene  ( v a n d e n B r u l e e t al, 1995;  ECM e n c o d i n g  for  There  C e l l u l a r responses  1  integrins)  1997) . a c t as  but  I n some  on  cell  cases,  an  anchor  for  i n many  cases  the  leading to signaling  between  t o these e x t r a c e l l u l a r  signals  alters  gene e x p r e s s i o n w h i c h c a n have p r o f o u n d  behaviour  including  migratory  effects  p r o p e r t i e s , morphology,  on c e l l u l a r growth, and  differentiation.  Perlecan/UNC-52  II.  A t p r e s e n t we know a g r e a t d e a l about t h e s t r u c t u r a l of  t h e ECM b u t much  less  i s known  about  their  functional  This s i t u a t i o n i s e s p e c i a l l y acute  f o r proteoglycans.  are  have  glycosylated proteins  anionic  which  glycosaminoglycans  biological  function  physicochemical  (reviewed  of  proteoglycans  characteristics  of t h e molecule,  covalently  i n Timpl,  filtration  attached  1993).  from  o f the glycosaminoglycan  Other b i o l o g i c a l  (Timpl,  proteases  t h a t degrade t h e p r o t e i n core  component  pressure t o forces  functions include control of  1993).  inhibitors  the  compressional  t h r o u g h BMs a n d b i n d i n g o r s t o r a g e o f g r o w t h  protease  highly  The m a j o r  derives  the r e s p e c t i v e t i s s u e enabling i t t o withstand  roles.  Proteoglycans  which provides hydration and s w e l l i n g  ( Y a n a g i s h i t a , 1993).  components  The  concerted  and heparanases  f a c t o r s and action  of  t h a t remove  t h e h e p a r a n s u l f a t e may m o d u l a t e t h e b i o a v a i l a b i l i t y o f bound g r o w t h factors.  There a r e o t h e r p r o t e o g l y c a n s  found  t i s s u e s which a r e u s u a l l y d e f i n e d through sugar  side  chains  such  decorin  a g g r e c a n (Doege e t a l , 1990) Perlecan  i s recognized  proteoglycan i n vertebrates. and  sequenced  (Murdoch  et  i n mouse al,  1992;  i n various vertebrate  the binding of different  (Yamaguchi  1988),  and R u o s l a h t i ,  a n d bamacan (Couchman e t a l , 1 9 9 6 ) . a s t h e most  abundant  heparan  sulfate  The gene f o r p e r l e c a n was f i r s t  (Noonan  e t al,  Kallunki  Caenorhabditis  elegans  characterized.  A l l v e r s i o n s share  (Rogalski  2  & et  1991)  and l a t e r ,  Tryggvason, al,  1993)  an i d e n t i c a l  cloned  a  1992)  and  homologue  five-domain  human a  were  modular  structure with highest The f i r s t domain only  region  from  the other  receptor  homology w i t h of  two;  immunoglobulin  like  of  repeats  (Noonan  and  D.G.  the  second  laminin et  (domain  i n humans,  the neural  cell  regions  1991;  domain  to  the  I I I shares  I V i s composed  i n mouse  molecule  et  I n C.  locus.  by  and  C.  family  of  short  a l , 1992;  1993; T.M.  results).  encodes p e r l e c a n i s t h e unc-52  separated  Murdoch  1992; R o g a l s k i e t al,  Moerman, unpub.  i s similar  14  adhesion  differs  ( R o g a l s k i e t a l , 1993) ; a n d domain V i s  globular al,  II)  o f l a m i n i n A; domain  (21 r e p e a t s  and i s t h e  significantly  c l a s s of receptors;  chains  (Ig) r e p e a t s  composed  homologue  elegans  o f t h e LDL  units  most  Tryggvason,  t h e C.  the short  contiguous  elegans)  (domain I ) i s u n i q u e t o t h i s p o l y p e p t i d e  where  domain  o v e r a l l homology t o w e l l - k n o w n p o l y p e p t i d e s .  Rogalski,  EGF-like  Kallunki G.P.  Mullen  t h e gene  elegans,  I t i s a l a r g e gene  &  that  spanning  o v e r 25 k b , a n d i s composed o f 37 exons t h a t p o t e n t i a l l y encodes a n open r e a d i n g al,  frame o f a l m o s t 3500  1993, 1995; T.M.  amino a c i d r e s i d u e s  R o g a l s k i , G.P.  Mullen  a n d D.G.  (Rogalski et  Moerman,  unpub.  results). To b e t t e r u n d e r s t a n d  the r o l e  have c h o s e n t h e nematode C. elegans simple  eukaryote,  cellular  and  we  nature  g r o u p s o f components,  i n this  more c o m p l e x o r g a n i s m s .  genetics  as a  components.  studying the molecular  i n BM  function,  a s o u r model s y s t e m .  can u t i l i z e  extracellular  of perlecan  tool  Results  By u s i n g  The g r e a t  s y s t e m may  a  t o manipulate gained  through  and f u n c t i o n a l r o l e s o f i n d i v i d u a l , simpler  we  be  or  applicable to  number o f m u t a n t s i n C.  elegans  and i t s we 1 1 - c h a r a c t e r i z e d genome make i t an i d e a l o r g a n i s m i n w h i c h to  s t u d y many b i o l o g i c a l p r o c e s s e s  1997) .  Research  interactions  i n our  between  (reviewed  laboratory  t h e BM  and  i n Wood, 1988,  i s aimed  the precursors 3  at  Riddle,  determining  o f t h e body  the wall  muscle  that  quadrant project  are necessary f o r the formation of a  i n C. has  elegans  been  to  (see  Figure  further  1  functional  for details).  characterize  the  My  muscle specific  functional  role  of  i n the adult  C.  nematode p e r l e c a n i n t h i s d e v e l o p m e n t a l p r o c e s s (see b e l o w ) .  I I I . Muscle  Structure  There elegans. are  Horvitz,  length  1977;  on  during  dorsal  and  postembryonic  cells  1983) .  two  the l o n g i t u d i n a l  spindle-shaped  cells  are present at b i r t h .  S u l s t o n e t al,  two  of  muscle  81 c e l l s  early  quadrants,  elegans  a r e 95 s t r i a t e d b o d y - w a l l m u s c l e Only  added  in C.  (Figure  (Sulston  Each  consists The  quadrant  7-10  sarcomeres  are  easily  of  basic  a  double  repeat  microscopy.  Myosin  through  The a  1997).  A-  or  I-bands  using  by  of  cell  polarized  M-lines while lattice  lateral  actin  i s anchored  i s anchored  attachments  to  (focal  the  these light  by  cell  dense surface  adhesions)  ( F r a n c i s and W a t e r s t o n , 1985,  which  as w e l l  1991).  Contacts general,  structural links  are  filaments.  as a d j a c e n t m u s c l e c e l l s  In  (Figure  and  t o t h e u n d e r l y i n g BM a n d h y p o d e r m i s  Focal  of  within  There  t i g h t l y adheres the c e l l  IV.  the  i s composed o f b u n d l e s o f m y o s i n - c o n t a i n i n g  myofilament  series  four  row  unit  interdigitated with actin-containing thin  i s anchored  bodies.  as  A sarcomere  thick-filaments  Moerman and F i r e ,  a c r o s s t h e w i d t h o f an a d u l t m u s c l e  visualized  and  runs  t h a t i s r e s p o n s i b l e f o r c o n t r a c t i o n i s t h e sarcomere  1; r e v i e w e d i n W a t e r s t o n , 1988;  cells  These a r e a r r a n g e d i n t o  and  2) .  remaining  development  ventral.  axis  The  any  disruptions  i n focal  l i n k between t h e c y t o s k e l e t o n , ECM  are c r u c i a l  since  adhesion  and s u b s t r a t e .  t h e y a r e r e g i o n s where s i g n a l 4  destroys  the  These  transduction  Figure  1.  S t r u c t u r e o f t h e sarcomere  Part  A.  A  schematic  contractile  unit  of  and basement membrane  representation C.  elegans  of  a sarcomere,  muscle.  The  the  major  structural  f e a t u r e s a r e i n d i c a t e d s u c h as t h e a c t i n - c o n t a i n i n g t h i n (blue)  which  vertebrate  are  Z  l i n e s ) and  orange)  whose  (purple)  . Some  The  unc-52  anchored  by  the  is  maintained  extracellular  gene p r o d u c t s  membranes u n d e r l y i n g  bodies  (light  muscle  green),  cells  and  of the f o r c e s generated w i t h i n the muscle  An  are  of  have been  the  focal  determined  analogs  M-line are  found  play  an  also  i n the  junction. adhesion  for lateral the b a s a l Many  complex  at  of  surface the  t h e membrane  1996)  but  particularly  obvious  demonstrates, could  be  in  occuring  in  the  v i t r o data between  the As  functional roles  show t h e BM  the  possible  components  of  itself of  the  This  is  transition  diagram  interactions but  the  (Burridge  well-characterized. BM.  the  in  structural  Chrzanowska-Wodnicka,  not  basement  transmission  i n t e r a c t i o n s documented  is  shown.  essential role  their  proteins  (light  components  and  interacting  of  sarcomere.  of a p o r t i o n of  cell-BM-hypodermal  components and  enlargement  by  filaments  filaments  components  a n c h o r i n g the dense b o d i e s t o the c u t i c l e P a r t B.  (mauve,  myosin-containing thick  alignment of  dense  basic  the  nature  that of  t h e i r assembly i s u n c l e a r . P a r t C.  Diagrammatic  showing  the  Yurchenco  representation  putative  and S c h i t t n y ,  layering 1990).  5  o f a p o s s i b l e BM  of  components  architecture  (Adapted  from  r e l a t e s t o t h e a s s e m b l y o f c e l l u l a r components. of i n t e g r i n receptors,  the constituents  s i d e o f t h e BM w h i c h i n c l u d e v i n c u l i n , adhesion kinase formation and  (FAK) s t i m u l a t e  aggregation  on t h e muscle c e l l talin,  membrane  oc-actinin, and f o c a l  s i g n a l cascades which i n f l u e n c e s t h e  of contractile proteins  Chrzanowska-Wodnicka,  Through  e t al,  (Jockusch  1995; B u r r i d g e  1996; Moerman a n d F i r e ,  1997).  The BM  network acts as a mediator o f adhesion t o t h e opposing s i d e as w e l l . Mechanical s t a b i l i t y  here  i s conferred  through m o l e c u l a r l y  distinct  j u n c t i o n s c a l l e d hemidesmosomes w h i c h a r e on t h e h y p o d e r m a l s i d e o f t h e BM (see F i g u r e  1) .  The e x o s k e l e t o n  o f C. elegans  therefore has  a d i r e c t l i n k a g e t o t h e m u s c l e , m e d i a t e d b y t h e ECM.  V.  Muscle  Development  The  development  characterized  of  (Sulston  1997) .  Muscle c e l l s  Several  of the early  specialized cell  C.  elegans  e t al,  body-wall  1983; r e v i e w e d  a r e n o t simply blastomeres  blastomere d e r i v a t i o n , a l l  give  i n Moerman  rise  t o muscle  a rather  ( S u l s t o n e t al,  body w a l l  o r i e n t e d w i t h i n the developing  i s well &  c l o n a l l y d e r i v e d i n most  types which i m p l i e s  commitment a n d d i f f e r e n t i a t i o n  muscle  muscle  embryo a l o n g  and  elaborate  (Figure 3).  This  1994; Moerman e t a l , 1996).  stage  and t h e muscle  within the c e l l hypodermis  1983).  Regardless o f  cells  are eventually  thel a t e r a l sides  ( E p s t e i n e t al,  Most c e l l s  components  a r e only  (Hresko e t a l , 1994) .  to their  final  other  of the first  i s t h e e a r l i e s t time a t which s t r u c t u r a l  p r o t e i n s o f muscle c a n be d e t e c t e d al,  cases.  pattern of  embryo a d j a c e n t t o t h e h y p o d e r m i s a t a r o u n d 290 m i n u t e s a f t e r cleavage  Fire,  dorsal  7  1993; H r e s k o e t  are postmitotic a t this diffusely  distributed  They t h e n m i g r a t e  or ventral  position  along t h e a n d form  F i g u r e 2. A n a t o m i c a l s t r u c t u r e o f t h e worm  Part  A.  Diagram  of a  posterior  cross-section  hermaphrodite showing major anatomical are  four muscle quadrants  (BM, green) j u x t a p o s e d P a r t B.  (red) w i t h  through  structures. an a d j a c e n t  Note  the adult that  there  basement membrane  t o t h e hypodermis.  L a t e r a l v i e w o f a c a n o n i c a l worm s h o w i n g o n l y two q u a d r a n t s  composed o f a d o u b l e row o f m u s c l e c e l l s . green t o represent  UNC-52/Perlecan  i n the adjacent  anal sphincters contain short isoforms muscles c o n t a i n long  isoforms.  8  Cells  are indicated i n BM.  o f UNC-52 w h i l e  Pharynx and t h e body w a l l  Figure 2. Anatomical s t r u c t u r e of the worm  9  quadrants al,  ( F i g u r e 3; H r e s k o , 1994;  1997) .  presumably  organellar  arrangement,  underlying  components  localized  to  minutes),  we  perlecan,  that i s  these see  Schnabel  through  signal  where of  they  the  the  form  BM  regions.  and  At  secretion  cascades focal  from  and  stage  muscle  encoded b y t h e unc-52  with  which  the  are  also  (approximately  350  of  locus  cell  subsequent  contacts  hypodermis  this  to the  for  the  analysis reveals  proper  assembly of  within  developing  Hresko  et  al,  body  1994;  that  the  the  proteoglycan,  ( F i g u r e 3; Moerman e t  wall  nematode p e r l e c a n  highly  muscle  Williams  and  ordered  cells  possibly  the  that  i t  integrin  may  myofilament et  1994) .  lattice  al,  1993;  After  (Moerman e t al,  interact  complex,  essential  being  U N C - 5 2 / p e r l e c a n f u n c t i o n s as  o f an a d h e s i o n complex t o a n c h o r m u s c l e hypothesized  is  (Rogalski  Waterston,  l o c a l i z e d o u t s i d e the muscle c e l l ,  on  with  the  1996) .  receptor  surface  of  muscle  1985,  1991;  genetic  G.P.  Mullen  hierarchy  double  Waterston,  of  G.P.  Mullen  in  RGD  sequences o f p e r l e c a n al,  1992;  extracellular contractions  D.G.  and  and  ( F r a n c i s and  Moerman, unpub.  established  integrin  the  et  vitro  and  (epistasis)  mutants 1994;  of muscle c e l l s  1)  perlecan  D.G.  genes  2)  (Williams  Moerman, unpub. r e s u l t s ) ;  Chakravarti attachment generated  by  et is the  specific al,  1995;  essential body-wall  10  integrin  subunits  Ruoslahti, to  transmit  muscles  to  over  the  construction  s t u d i e s done on mammalian homologs d e m o n s t r a t i n g bind  the  Waterston,  results;  through the  It  cells.  c o - l o c a l i z a t i o n o f i m m u n o s t a i n i n g o f b o t h i n t e g r i n and p e r l e c a n t h e dense b o d i e s and M - l i n e s  part  molecules,  S u p p o r t f o r t h i s model comes from s e v e r a l l i n e s o f e v i d e n c e :  of  et  1996) . Genetic  is  1996;  M y o f i l a m e n t components t h e n become l o c a l i z e d  membranes,  al,  Moerman e t al,  the  3) that  (Hayashi  1996). the  and  This muscular  hypodermis  F i g u r e 3. Development  P a r t A. in  C.  P a n e l s 1-4  summarize  depicting  elegans,  s t a g e s of development. in  Hresko  The  have begun  min.  after  sections  of  embryos  assembly  at  various  diagram i s adapted from d a t a p r e s e n t e d r e v i e w e d i n Moerman  first  to accumulate  and i t s m u s c l e  the process of myofilament  cross  e t a l . ( 1 9 9 4 ) and  embryo a t 290 cells  of C. elegans  cleavage.  myofilament  l i e adjacent to the l a t e r a l  (1997).  Muscle  cells  components  hypodermis  (1)  (circles)  (dots).  at this  An  Muscle  stage but are  beginning t h e i r m i g r a t i o n to f i n a l p o s i t i o n s beneath the d o r s a l ventral  hypodermis.  covering  most  of  hemidesmosomes hatched  to  in  350  (2)  adjacent  thin  layer  Components  the  embryo.  constituents to  other  Muscle (dots)  muscle  are  concentrated at  m u s c l e q u a d r a n t o f 420 m i n . become f l a t t e n e d  and m y o f i b r i l s  and hemidesmosome components Dorsal  muscle  organization  quadrant of  embryo.  the  of  Muscle c e l l s  (dots),  (hatched  450  min.  myofilament  regions.  the  components Dorsal have  oval)  basement membrane  (black)  are coextensive.  region)  embryo  lattice  are  and  (3) (now  shows  and  its  by  localized  cells  have  focal  for  cells  B o t h basement membrane and hemidesmosomal these  cells  (represented  hypodermis. become  of  destined  hypodermis  min.  myofibrillar  regions  is a  embryo.  present  .  because  membrane  hypodermis  the  are  regions)  asymmetric  The  or  the  (4)  complete  extracellular  anchorage. P a r t B.  Embryogenesis  g e n e r a t e d by reach fail  contractions  the 1.5-fold to s t a r t  i n w i l d - t y p e and P a t m u t a n t s  length  moving  and  of body-wall muscle (2)  only u n t i l 2 - f o l d stage then a r r e s t s . as m i s s h a p e n  o c c u r when  I n Pat mutants,  remain p a r a l y z e d .  larvae.  11  (1)  1.5-fold  Movement embryos embryos  Elongation continues  Embryos o f t e n h a t c h a n d d i e  Figure 3 . Development o f C. elegans and i t s muscle  A.  (1) 290 min  (3) 420 min  Dorsal  Dorsal  seam cell  Ventral  (4) 450 min  (2) 350 min  basement membrane hypodermis muscle quadrant  t  dense body • myofilament lattice hemidesmosome  B (1) Wild-type first cleavage  comma  1-1/2-fold  2-fold  3-fold  pharynx pumps  0  movement  (2) PAT mutant  0-0*0-0  first comma cleavage  1-1/2-fold  paralysis  12  2-fold  elongation arrest  2-fold  ^hatches  - M i l  P.^"* pumps  hatches  and  cuticle  in  produced by 1991;  order  facilitate  a l l the body-wall  Rogalski  functional  to  et  gene  al,  Mutants  and  Genetic mutant  Isoform  studies  alleles  al,  1980,  Williams  their  animals.  the  1974,  alternatively 1993,  et  al,  ( R o g a l s k i e t al, eliminate adult  two  broad  M a c k e n z i e e t al, 1992,  to  a the  One  while  other  becomes  1978;  Gilchrist  of  1978;  Waterston  et  completely  interval  exons  (exons  of  1993).  some o f t h e  these The  exons  in  18;  encodes  a  embryonic by  larval  1992).  As  for  some  c l a s s have  point  three  adjacent,  Rogalski single  e f f e c t of these molecular  l o n g UNC-52 i s o f o r m s  viable  later  except  containing 17,  1993,  characterized  Moerman,  paralyzed  16,  al,  contains  A l l mutants of t h i s  small  et  contains  apparent &  of  patterns  class  the  classes  and  Rogalski  which  are  a  Each  without  i s disorganized  v i a b l e c l a s s of mutants a r e  spliced  1995) .  is  Waterston,  1996),  phenotypes  1994).  animals  mutants  within  al,  defined  Moerman,  movement i n t h e head r e g i o n . mutations  have  their  phenotype  (Mackenzie  adults,  on  and  The  paralyzed  stages  unc-52  (Brenner,  (Unc)  ( F r a n c i s and  et  structure  UNC-52  occurs.  Waterston,  uncoordinated lethal  of  Gilchrist and  Since  Diversity  based  complementation  Moerman  muscle  e x t e n t t h a t embryonic d e a t h  VI.  muscle c e l l s  1993;  product,  movement.  et Ig  al,  repeat  l e s i o n s i s to  produced i n growing  and  animals. The  second  class  lethals,  a l l of  which  elongation at two-fold; results  described  in  of  unc-52  share  a  Pat  alleles  consists  phenotype  W i l l i a m s & Waterston, this  thesis, 13  only  two  of  (paralyzed 1994) . Pat  embryonic arrested  Prior alleles  to  the were  i d e n t i f i e d m o l e c u l a r l y , unc-52(st549)  and  a l t e r a t i o n r e s u l t s from t h e i n s e r t i o n  o f an  endogenous  element  st549  is a  ( c a l l e d Tel)  into  exon 2 and  unc-52 (ut111).  l e a d i n g t o a t e r m i n a t i o n codon i n exon 7 1995).  Since  these  these mutations product. either  are  lead to l i t t l e  I n utlll  t h e DNA  exons  transposable  nonsense  mutation  i n a l l UNC-52  expression of the  unc-52  gene  homozygous a n i m a l s , e x c i s i o n o f T e l c a n o c c u r  o r RNA  level  ( R o g a l s k i e t al,  defines the n u l l  al,  isoforms,  and  leads  1995).  phenotype  of  The  in  P a t phenotype o f st549  this  at  t o weak e x p r e s s i o n o f UNC-52  and a somewhat l a t e r l e t h a l phenotype t h a n what i s o b s e r v e d animals  utlll  ( F i g u r e 4; R o g a l s k i et  expressed o r no  The  locus  since  st549  st549  animals  mutants  lack  i m m u n o r e a c t i v i t y t o a n t i b o d i e s t h a t r e c o g n i z e a l l i s o f o r m s o f UNC-52 ( R o g a l s k i e t al, st549  and  isolated  1993;  utlll,  six  (rall2,  Identifying  G.P.  M u l l e n , unpub. r e s u l t s ) .  other  ra401,  st546,  the molecular  basis  f o r these  alleles  st560,  mutant p h e n o t y p e s has  1996).  t h e r e g i o n where t h e s e specific UNC-52  antisera. isoforms  locations  (G.P.  st572,  unc-52  One  been  splicing  unc-52  Mullen  and  Pat a l l e l e s  D.G.  has  thus the biochemical  c h a l l e n g i n g because that  gives  may  rise 1993,  to  of a  1995;  l i e i s t h e v a r i o u s UNC-52-  produced D.G.  revealed that  have  distinct  Moerman, p e r s .  the  ( R o g a l s k i e t a l , 1993;  Moerman, unpub. r e s u l t s ) .  T.R.  These  comm.).  14  are  1)  There  o r absence  R o g a l s k i , G.P.  isoforms  different  spatio-temporal  t h r e e g r o u p s o f unc-52 p r o d u c t s b a s e d on t h e p r e s e n c e domain IV/V  been  of the t o o l s a t our d i s p o s a l t o d e f i n e  alterations  are  have  Results).  ( R o g a l s k i e t al,  I m m u n o s t a i n i n g has  that  locus  st578—see  t h e l o c a t i o n w i t h i n unc-52 and  number o f p r o t e i n i s o f o r m s o f e t al,  this  thesis.  t h e complex p a t t e r n s o f a l t e r n a t i v e  Lundquist  of  l e s i o n s i n these  been a p r i m a r y g o a l o f t h i s Determining  Pat  In addition to  Mullen  are of and  a domain I ,  F i g u r e 4. unc-52 gene  Representation (boxes)  and  structure  of  t h e complete  intron  (line)  schematic  unc-52  structure  gene as  well  and 3 p o s s i b l e p r o t e i n products.  exons w h i c h  span  over  poly(A)-addition  sites  alternatively-spliced.  25 k b .  There  (colored To  are at least  only  st560  and r a l l 2  approximately  a r e shown.  3 500  residues  st549,  the text) .  (red)  The e p i t o p e s  are indicated  that  data  are  alternative exons  exists.  are that  Sequenced  and t h e domain I V k n o c k o u t s , into  5  t o sequences  The GMl e p i t o p e only  Representatives of both  indicated.  15  domains  ( p r o t e i n m o t i f s a r e as d e s c r i b e d  f o r a n t i b o d i e s GMl (green)  i s o f o r m s w h i l e MH2/3 r e c o g n i z e s Domain IV/V.  domain  demonstrating  c a n be d i v i d e d  and correspond  e x o n 19, r e s p e c t i v e l y .  the  exon  The l o n g e s t ORF e n c o d e s a p r o t e i n o f  (Domains I-V) b a s e d on h o m o l o g i e s in  4  Several  e x o n s 6, 16, 17 a n d 18 a r e i n v o l v e d i n s p l i c i n g Pat a l l e l e s i n c l u d i n g t h e n u l l  as  the  The gene c o n t a i n s 37  boxes).  date,  showing  a n d MH2/3  i n exon  10 a n d  i s common t o a l l UNC-52  the long  isoforms  containing  the short and long  isoforms  I I a n d I I I f o r m ; 2) a domain I , I I , I I I and I V f o r m ;  3) a  I,  V-containing  I I , I I I , IV  isoform  has o n l y  t h i s study. specific II,  and  Rabbit  epitopes  t o these  indicated,  form.  Because  the  r e c e n t l y been d i s c o v e r e d ,  domain  i t i s not included i n  p o l y c l o n a l a n t i s e r a was g e n e r a t e d t o  o f UNC-52 i n o r d e r  I I I isoforms  refer  V  from  t h e domain  as t h e s h o r t  the short  domain  recognize  t o d i s t i n g u i s h t h e domain I ,  I , I I , I I I , IV  and l o n g  isoforms,  isoforms.  We  respectively.  As  i s o f o r m l a c k s domain I V a n d i n c l u d e s o n l y  the  f i r s t t h r e e domains: i n embryos t h i s i s o f o r m i s l o c a l i z e d t o t h e BMs u n d e r l y i n g t h e pharyngeal and a n a l s p h i n c t e r / d e p r e s s o r Mullen  a n d D.G.  containing anal  Moerman, unpub. r e s u l t s ) .  long  muscles  isoforms  a t any  stage  variants are restricted cells  during  embryonic  unpub. r e s u l t s ; expressed  i n the pharyngeal  underlying  Instead,  short  the  of  UNC-52.  These  only  isoforms  include  muscles  in  W a t e r s t o n , 1991; G.P. M u l l e n , T.M.  later  Recently,  e v e n t s a t unc-52  insight  the  (i.e.  male  generating  co-  pharynx  and  has been  (Lundquist  et al,  the  importance  1996).  genes of  this  vulval  (Francis  and  Moerman, unpub.  shown  to  some o f t h e s e The g e n e t i c  encode  a  splicing  a n a l y s i s of  s u c h as mec-8 h a s  provided  alternative-splicing  isoform d i v e r s i t y w i t h i n a developmental  17  and  o f t h e gonad;  tail  R o g a l s k i a n d D.G.  t h e mec-8 gene  and i t s i n t e r a c t i o n w i t h into  Moerman,  a r e they  the uterine  p u t a t i v e RNA-binding p r o t e i n that r e g u l a t e s  unc-52  long  I n l a r v a e and a d u l t s , a d d i t i o n a l t i s s u e s a c c u m u l a t e  male-specific  results).  these  a n d D.G.  muscles i n the hermaphrodite; the m y o e p i t h e l i a l sheath and  or the  t h e body w a l l m u s c l e  (G.P. M u l l e n  5 for details), with  (G.P.  I n c o n t r a s t , domain I V -  embryogenesis.  development  i n t i s s u e s along  isoforms  of  t o t h e BMs  see F i g u r e  anal muscles). long  are not present  muscles  context.  for  F i g u r e 5. I m m u n o f l u o r e s c e n c e s t a i n i n g o f w i l d - t y p e a n d m u t a n t embryos w i t h a n t i b o d i e s t o UNC-52 a n d M y o s i n Heavy C h a i n A  Panel  A.  Wild-type  embryos  double-labeled  r e c o g n i z e s a r e g i o n common t o a l l unc-52 which recognizes is  basement the  panels  which  a n d DM5.6,  (MHC A) .  GMl  (FITC) a n d DM5.6 i s shown i n r e d (TRSC) .  The  l e f t p a n e l shows b o t h a n t i b o d i e s right  GM1,  gene p r o d u c t s ,  t h e minor body-wall muscle myosin  shown i n g r e e n  and  with  show  GMl  simultaneously,  a n d DM5.6  membranes u n d e r l y i n g  while  alone.  the middle  GMl  the body-wall muscles,  pharynx and a n a l s p h i n c t e r / d e p r e s s o r muscles.  stains  the  as w e l l  White  as  arrowhead  marks t h e p o s t e r i o r b u l b o f t h e p h a r y n x . Panel  B.  st549,  the putative  null  allele,  eliminates  a l l GMl  s t a i n i n g and has s e v e r e l y d i s o r g a n i z e d myosin. Panel  C.  premature  st560, stop  a  Domain  transition  IV-specific  (C -->  T) i n e x o n  marks t h e p o s t e r i o r b u l b o f t h e p h a r y n x . to  allele,  13. W h i t e  is a  arrowhead  Small white arrow  points  anal s p h i n c t e r muscles.  P a n e l D. IV.  lethal  White  rall2,  a lethal  arrowhead  marks  deletion  eliminating  the posterior  bulb  3283 bp o f Domain of  Small w h i t e arrow p o i n t s t o anal s p h i n c t e r muscles.  18  t h e pharynx.  Figure 5. Immunofluorescence s t a i n i n g of wild-type and mutants  G M l  ana  U M D . O  unc-52(st549i  fcj  it  *  \  »  }  J  GM1 and DM5.6  GM1  G M l and DM5.6 unc-52lra112)  GM1 and DM5.6  19  DM5.6  V I I . Model and We  Strategies  speculate  requirement  that  extensive  for particular  different  tissues.  As  demonstrate  that  and  Why t h i s  time.  isoforms  biomechanical  described  isoforms  above,  are differentially  Our p h e n o t y p i c  IV-containing  are  or i n  to  UNC-52  i n space  but the different stress.  adhering  Perhaps  properties or  characterization of the  essential  i n body w a l l m u s c l e s .  for  the loss  isoforms  Sequence a n a l y s i s o f  UNC-52  isoforms  while  (see R e s u l t s f o r f u r t h e r e x p l a n a t i o n  corroborates  t h e importance  development. exons.  of long  Domain  Analyses  of  these  long  splicing  shown t h a t much o f t h e d i v e r s i t y  the r o l e o f t h i s s p l i c i n g , Igs within  muscle formation,  this  isoforms  these  This  i n muscle over  16  messages h a s on a  limited  To a t t e m p t t o d e t e r m i n e  we e x p l o r e d t h e f o l l o w i n g q u e s t i o n s : A r e group  of particular  importance  o r do t h e I g s s i m p l y a c t a s s t r u c t u r a l  large repeat  the short  spread  centers  determine t h e l e n g t h o f t h e p r o t e i n , o r i s there for this  animals  o f domain V) .  o f unc-52  i n splicing  r e g i o n o f domain I V ( R o g a l s k i e t al, 1995).  individual  retaining  I V c o n s i s t s o f 14 I g r e p e a t s  of alternative  that  myofilament  two o f t h e m u t a n t s c o n f i r m s t h a t t h e p r i m a r y d e f e c t i n t h e s e is  a  times  (see R e s u l t s ) h a s l e d u s t o s p e c u a l t e isoforms  formation and s t a b i l i t y  reflect  distributed  d i f f e r e n t mechanical  o f UNC-52 have d i f f e r e n t  strength.  may  antibodies  i s required i s not clear,  l e t h a l c l a s s o f mutants domain  diveristy  i s o f o r m s needed a t d i f f e r e n t  m u s c l e s i n v o l v e d do e x p e r i e n c e different  isoform  domain?  during  spacers t o  some o t h e r  S t r a t e g i e s were d e v e l o p e d  reason  t o answer  questions. A k e y o b s e r v a t i o n that bears  repeats  on t h e r e l a t i v e  i n domain I V was made b y E r i n G i l c h r i s t  intragenic  revertants  of  unc-52  20  viable  importance o f I g i n her studies of  alleles  (Gilchrist  and  Moerman, 1992) . region  of  I t was  exons  15  found t h a t  through  mutations, w i t h i n t h i s region e t a l , 1995) . unc-52(ra38),  Also has  could  (Gilchrist  i n the  lead and  to  Moerman, 1992;  d e t e r m i n e d t h a t an i n t r a g e n i c  small  d e l e t i o n removing the  i s v i r t u a l l y wild-type  (Gilchrist  units  can  muscle.  We  be  removed w i t h o u t  extended these experiments to  of  revertant, of  & Moerman,  These r e s u l t s d e m o n s t r a t e t h a t  region  IV  Rogalski  equivalent  R o g a l s k i e t a l , 1995). from t h i s  domain  suppression  i t was  a  c o m p l e t e exon and  19  exon-skipping  one 1992;  i n d i v i d u a l Ig  adversely  affecting  determine whether i n t a c t  m u s c l e c o u l d be m a i n t a i n e d a f t e r t h e r e m o v a l o f m u l t i p l e I g u n i t s . A T e l e x c i s i o n s t r a t e g y was  u t i l i z e d to i s o l a t e  several  unc-52  d e l e t i o n r e v e r t a n t s and t h e n a s u b s e t o f t h e r e v e r t a n t s w i t h  altered  domain  IV  Ig  number  and  52 (stl96:  regions  were u s e d  specific  :Tel)  Ig  to  examine t h e  elements  a l l e l e has  for  importance  UNC-52  function.  a T e l i n s e r t i o n i n exon 18  ends c a n be or  a double-strand  i m m e d i a t e l y r e p a i r e d by t h e  t h e y may  1990;  Gloor  b r e a k t h a t must be  be  the  target  e t a l , 1991;  of  exonuclease  A  involved  i n strand  repair.  Depending on w h e t h e r r e p a i r i s c o m p l e t e d o r  some p o i n t  the  rearrangement  i n v a s i o n which w i l l  site in  can  the  (Engels  e t a l , 1990;  process  i s diagrammed  unc-52  form Gloor  returned of  Ig repeats  development  a  i n Figure  c a n be  i n this  small  6) .  i t was  a  f r e e end  may  be  template  for  organism.  This  al,  interrupted  or  at  contain  a n a l y s i s of  this  several  as many as  four  adverse e f f e c t  suggests  a  deficiency  Moerman e t a l , 1991;  removed w i t h o u t any  21  et  DNA  determined that  free  (Engels  duplication  From an  and  machinery  t o w i l d t y p e o r may  e t a l , 1991;  deletion revertants,  consecutive muscle  be  provide  4)  These  repair  activity  Moerman e t a l , 1991) .  unc-  Upon e x c i s i o n ,  repaired.  c e l l ' s DNA  copy  The  (Figure  r e v e r t s a t a h i g h frequency i n a mutator background. T e l leaves  of  that  after  on a  Figure  6.  T e l e x c i s i o n can c r e a t e novel  insertion/deletion  m u t a t i o n s i n a gene  In a mutator background,  excise creating  from  the T e l element  i t s insertion  site,  d o u b l e - s t r a n d gaps.  or deletions  i n unc-52  at a r e l a t i v e l y  During  o f DNA c a n o c c u r .  22  (stl96::  high  the r e p a i r process,  Tel)  can  frequency, additions  Figure 6. T e l e x c i s i o n scheme  T d "hops" Out st196::Td homozygote  ' ' H  Double-strand Break  .llllllllllllllllllll® (gftrimsnr i m r ^ T  DNA ends Digested  Tc1  iiiiiimiimiMiufTeT  iiiiiiimiiii lllllllltlllDIIDIIIII  lllllllllllllll® / (gl iiiMliliUiiilllUl Tci PM T  tiuiiiiiiiiiii  Gap Repair gffijjjjjug Tci  HI  iiiiiiifflfti  ^  ^  i i i i i i i i n i i i i i i i i i i i i i i i i i i i  OR  3 Td pmnnnnnnnis  ^^^^^^^^^^^^^^^^^^^^^^  mnnnnnnnniq TCI  Additions  IIIIIIIIIIIIIIIIIIII  Deletions  23  c e r t a i n m i n i m a l number, I g copy number i s n o t o f p r i m a r y and  that  I g repeats  within  t h e exon  16-18  importance  interval  a r e non-  essential .  V I I I . Goals  and Summary  The g o a l o f t h i s role  o f domain  attachment.  IV of perlecan This  molecular  and  addressed  include,  development? and  within  1) I s domain  length  a r e addressed  t h e unc-52 two  importance  o f long  function  locus  have  structural  plasticity  redundancy  inherent  been  that  of  within  domain  gives  further  muscle  factor?  These  several  mutations  alterations. IV, t h e  domain  a n d may  ECM p r o t e i n s .  be  others?  illustrate  perlecan  to  of  than  f o r body  The v a r i o u s  characterized,  IV a l t e r a t i o n s  questions  IV a c r u c i a l  protein  combination  critical  specific  large  a  f o r body w a l l  have  o f nematode  in all  more  the analysis  demonstrated.  that  o f domain  o f domain  isoforms o f t h i s  h a s been  through Specific  Ig units  by  assembly and muscle  IV c r i t i c a l  Pat mutations  revertants  study  pursued  techniques.  2) A r e c e r t a i n  identifying  i n myofilament  h a s been  genetic  3) I s o v e r a l l  questions  t h e s i s h a s been t o examine i n more d e t a i l t h e  essential  wall IV  By  muscle deletion  the inherent reflect  the  I n summary,  this  insight  into  the  m o l e c u l a r f u n c t i o n o f UNC-52 a n d t e s t s t h e h y p o t h e s i s t h a t domain I V i s e s s e n t i a l d u r i n g embryonic m u s c l e d e v e l o p m e n t .  2 4  MATERIALS & METHODS  I . Nematode s t r a i n s , maintenance, We  have  followed al.,  genetic  standard  (Horvitz  et  1979).  employed  f o r the handling  Unless otherwise noted,  C.  elegans  Standard of  constructs genetic  laboratory  nematode  nomenclature  techniques  stocks  (Brenner,  g e n e t i c e x p e r i m e n t s were p e r f o r m e d  The s t o c k s u s e d were t h e w i l d - t y p e s t r a i n N2; RW6011, mnDp34H;  RW6013,  unc-52 (st560)/mnDp34H;  52 (st572) /mnDp34lI; 4 (st700)I/unc-52 4(st700)I;  (stl96::Tel)II;  TU74 ,  DM4413,  mec-8 (u74) I;  DM4411,  DM4416,  52 (ra517) II; DM4418, unc-52(ra518)II;  a t 20°C.  unc-52(st546)/  DM4414,  unc-  DM3102,  mut-  DM4301,  mut-  unc-52(ra511)II;  unc-52 (ra513)II;  unc-52(ra515)II;  1974).  RW6014,  unc-52 (st578)/mnDp34II;  unc-52 (stl96::Tel)II,  unc-52 (ra512)'II; DM4415,  DM5105,  were  DM4412,  unc-52(ra514)II;  unc-52(ra516)II;  DM4417,  DM4419, unc-52(ra519)II.  uncSome  s t r a i n s were p r o v i d e d c o u r t e s y o f t h e CGC.  II.  PCR a m p l i f i c a t i o n of genomic  DNA  S t a n d a r d PCR r e a c t i o n s were p e r f o r m e d e t al.  as d e s c r i b e d by B a r s t e a d  (1991) w i t h t h e f o l l o w i n g m o d i f i c a t i o n s .  E i t h e r 5-6 embryos  ( i n t h e c a s e o f l e t h a l s ) o r 2 a d u l t h e r m a p h r o d i t e s were p l a c e d i n 3 |IL o f l y s i s  buffer  0.45% Tween-20, 0.5 m l e p p e n d o r f briefly  spun  (50 mM KC1, 10 mM T r i s  0.45% NP-40,  (pH 8 . 0 ) , 2.5 mM  a  2  60 |lg/ml P r o t e i n a s e K) i n t h e l i d o f a  t u b e w i t h 15 (XL o f m i n e r a l o i l . in  MgCl ,  m i c r o c e n t r i f u g e and  then  These t u b e s incubated  were in  a  t h e r m o c y c l e r f o r 30 min. a t 37°C, t h e n 5 min. a t 95°C t o i n a c t i v a t e the  P r o t e i n a s e K.  These  lysates 25  were  .then  used  directly for  s t a n d a r d PCR (as d e s c r i b e d i n R o g a l s k i et al, 1993, 1995) o r l o n g r a n g e PCR w h i c h  differs  only s l i g h t l y  ( N i e l s o n e t al, 1994) .  Each  method i n c l u d e s p e r r e a c t i o n 10 mM dNTPs, 25 pmol f o r w a r d p r i m e r , 25 pmol r e v e r s e p r i m e r . (200 mM T r i s - H C l , X-100,  Long range PCR u s e d 2.5 (IL l o w - s a l t 10X b u f f e r  100 mM K C l , 100 mM (NH ) S0 , 20 mM MgS0 , 1% T r i t o n 4  2  4  4  1 mg/mL BSA, S t r a t a g e n e ) , a n d t h e c o r r e s p o n d i n g p r o p r i e t a r y  TaqPlus  Polymerase  (Stratagene)  i n t h e amount  3-5  units  on t h e s i z e o f p r o d u c t b e i n g a m p l i f i e d ) a n d dH 0 t o 22 | 1 L .  (depending  2  The s t a n d a r d PCR method i n c l u d e d 25 mM M g C l  2  a n d 2 .5 (IL s t a n d a r d 10X  r e a c t i o n b u f f e r , a n d -2.5 u n i t s T a q P o l y m e r a s e mixtures  of  were  amplified  in a  (Gibco-BRL) .  Perkin-Elmer-Cetus  480  A l l PCR  thermocycler  u s i n g t h e f o l l o w i n g c o n d i t i o n s o f 30 s e c a t 95°C, 30 s e c a t a n n e a l i n g temperature of  (53-57 °C) a n d 1 t o 5 m i n . a t p o l y m e r i z a t i o n  72 °C f o r 30 c y c l e s  temperature  f o l l o w e d b y 5 m i n . a t 72 °C.  s u c c e s s f u l i n g e n e r a t i n g genomic fragments  We  were  as l a r g e as 5 kb i n s i z e .  The f o l l o w i n g p r i m e r s e t s were u s e d ( T a b l e 1) :  III.  PCR  sequencing  The P C R - a m p l i f i e d genomic DNA f r a g m e n t s using  unc-52  described  p r i m e r s a n d t h e BRL dsDNA C y c l e  i n Rogalski  r e a c t i o n mixes reaction, MgCl , 2  e t al.  (1993,  mM  1995).  c o n s i s t e d o f 1-2 (XL from  4.5 (XL 10X T a q Sequencing  300  were d i r e c t l y  K C l , 0.5%  w/v  W-l  2 6  Sequencing  System a s  Briefly,  sequencing  PCR-amplif i e d  buffer  sequenced  genomic DNA  (300 mM T r i s - H C l ,  buffer,  B R L ) , 0.5  50 mM  |0,L T a q  VO  CN  in rH rH  C o X  rH  CN  co  C 0  C o X  <j\ rH rH  o  0  o  rH rH  IH  X  X  CO  H  a)  H  c 0 X  a)  rH  O  rH  u CJ  <! EH U CJ O EH EH EH EH  < < O EH EH CJ O CJ CJ  CN  rH  a o X  m  v o rH  EH  rH CJ tH  4J CO  •U 18  4J CO  CO rH  CO rH  CO rH  C 0  C cu o 4J • H cu CQ  o\  a CU 0 X 4J 0 •H 0)  a  •H  c  X d) C  •H  a  O -H 4J >H Q> CO C •H  0 -H 4J  rH  rH  <D cn  a  •H  X c  •iH  c  O •H 4J IH Q) CQ d •H  •^1  v o rH  CN rH  ro rH  rH rH  rH rH  CN rH  CN CO rH CN rH  CO  O  O  CN CO rH CN rH  < < V  ^1  CN  VO CN  m  tH  VO  VO  rH rH  rH rH  ^  ^  < U u < < u < o< < o U u < U u < < < < < cj u < u < U < < < U u < < < < o < < < < < < <  EH CJ  < CJ EH EH CJ CJ  O CJ CJ  CJ  EH  EH  O  EH  CJ  CJ EH  CJ  a  EH  EH  EH  CJ  EH  EH  CJ  CJ  CJ  CJ  O EH  U CJ  EH CJ  CJ  EH  O CJ EH U u CJ CJ U EH CJ CJ CJ  O  cu  m  trj  X  CI  rH  EH  a 0  a)  CN rH rH O rH  rH rQ  rH 0  rH  rH  o  rH U tH  CJ CJ  EH  < rtj  u  CJ  CJ EH  EH  CJ  EH  EH  EH  EH  EH  EH  EH  CJ  CJ  EH EH  o O  CJ  CJ  CJ  EH  EH  EH  CJ  CJ  O  CJ  CJ  CJ  CJ CJ  O  u  EH  o u CJ  < < CJ < CJ  EH  u  EH  <  CJ  CJ  (U W  3  Ui  u  CD  ft  u  CJ  EH  < u < < o < < u < < < u o  CJ  u  EH  EH  EH  U  EH  O  CJ EH  u  EH EH  CJ EH  CJ EH  CJ  ><  O CJ  U  U  CJ EH  CJ  CJ EH  CJ  EH EH  EH EH CJ EH  EH CJ EH CJ EH  O EH O CJ O  < < < EH  o CJ  CJ EH  CJ  EH  CJ EH  u u  O EH O  EH  CJ  CJ  CJ CJ  CJ CJ  EH  EH  CJ  CJ CJ  < < < o < o u < "S < o u < «5 2 < o u  CJ  CJ  EH  EH U  CJ  CJ  CJ EH  EH  CJ  CJ CJ CJ  EH CJ  CJ CJ  EH  CJ  EH  < CJ  < CJ EH CJ EH  < U  u  < EH  o < u EH EH CJ  o  CJ  CJ  EH  U  u u  <  CJ  EH  < EH  u  <  a o  EH  Polymerase  Four  JUL.  flL  of  the  5  (BRL) ,  0.5  the  mL  eppendorf  appropriate  of  (2  r e a c t i o n were  silicone  o i l .  DNA  30 s e c a t  95°C,  30 s e c a t  9 5 °C a n d 6 0  cycles  5  these  reactions  min.  before  sequencing  of  55 °C,  sec at  for  loaded  isolate  crossed  (N2)  males  mated  to  were  self  progeny.  and  Unc  dpy/mut-4; onto  new  segregate  Tel  G,  tube  8  or  with  following  7 0 °C f o r  10  was  (BRL)  a  then  with  standard  a  drop  the  to  each  6%  in  a  conditions:  At  placed  2.0  above  performed  added  and  and  the  20 c y c l e s  cycles.  36  containing  of  \ih  and o v e r l a i d  the  T  BRL)  were  5 min.  and  then  end of  the  tube  on  ice  and  for  5  polyacrylamide  allele  were  into  a  picked  onto  then  indicating  unc-52/+. and  Unc-52  Unc  new  progeny  to  (dpy-5/+;  Wild-type  hermaphrodite  scored  for  presence  that  the  progeny self.  2 8  Unc  the  allowed  parental were  expected  to  of  to  have  both  genotype  then  animals  were  unc-52/+)  and  were  Wild-type  unc-52(stl96)  plates  non-Dpy  allowed  background.  dpy-5(e61);  progeny  we  unc-52(stl96::Tcl),  mutator  hermaphrodites.  singly  P l a t e s were  from  to  male  mut-4(stlOQ)  plates  events  crossed  Outcross  progeny,  Dpy  revertants  excision  this  hermaphrodites.  progeny  and  to  2  gel.  f i r s t  then  C,  and H 0  reactions  for  onto  pmol)  mM e a c h ,  BRL)  60 s e c a t  7 0 °C  I V . I s o l a t i o n of deletion To  (2  each  solution  boiled  being  each,  thermocycler  stop  were  mM  (1  labeled A,  mix  sequencing  30 s e c a t  |IL  were  added to  The  Perkin-Elmer-Cetus  tubes  termination  dideoxynucleotides  sequencing  30  P-end-labeled primer  |lL  picked that  have  the  Dpy was  singly  failed  to  genotype  mut-4(stlOO),-unc-52(stl96::Tcl) maintained  and  spontaneous lines 3  ) .  Any  were  and  that  and  Revertants  6  were  the  exon  before  18  polymorphism  singly  were  of  at  a  to  the  new  allele  assayed by  unc-52.  with  plates  had  maintained  for  that  further  new  improved  Unc  siblings.  maintained  then  detectable  Candidates  to  their  their  We  10"  phenotype.  homozygous  PCR) .  x  discarded.  of  and  was  Unc  were  basis  (~1  animals  the  revertants  on  revertants  Unc  for  independent  frequency  of  were  screening  Several  high  reversion  isolates  by  single  compared  revertant  these  region  size  being  activity  picking for  independent  phenotype.  segregate  body  the  whether  by  identified  picked  until  generations  determine  were  larger  generations,  to  Unc  reverted  these  failed  Revertants movement  the  maintained  Several  mutator  that  monitoring  plates  for of  established  These  plates  tested  reversion  were  .  several  (usually used  4-  PCR  to  polymorphisms  appeared  to  analysis,  in  have  a  including  sequencing.  V.  Lethal  deletion  To were  isolate  crossed  lethal  Those  offspring  were of  excision  hermaphrodite  plates.  presence  Tel  unc-52(stl96::Tcl)  to  heterozygous  revertants  animals brooded  arrested  manner  were  mnDp34  (Herman  et  al.,  that  did these  embryos.  subsequently  wild-type  ; mut-4(st700)I  progeny  and  events,  were not  Lethal  balanced  1979).  29  singly  segregate were  alleles  with  the  males  homozygotes  picked  plates  (N2)  to  new  paralyzed  Unc  scored obtained free  and  for in  the this  duplication,  VI.  Construction To  of mec-8; unc-52 doubles  construct  were  isolated  male  stocks.  to  from  heterozygous  Strains  PCR  PCR f o r  carrying  wild-type  unc-52  product  VII.  were  were  a  plated  allele.  Strains  presumably  that  homozygous  for  Probes)  dye  to  that  by  bathed  a  i n  Herman solution  temperature to  remove  fluorescence  of  and then  retested  excess  microscopy  (using  i n  PCRf o r  a  a  wild-type  unc-52(revertant) these  to  a  animals  animals  for  excitation  Molecular  worms  as were  2-3 hours  plate  mounted  the  phenotype)  Briefly,  fresh  with  (DiO;  (Dyf  M9 b u f f e r  being  an  unc-52.  of  using  then  neurons  (1990).  dye before  were  was  allele  defects  transferred  plates  f o r the deletion.  dye-filling  10 m g / m l  double  From  hermaphrodite  stained  and Hedgecock  crossed  hermaphrodites  1  the  establish  then  d i d not produce  mec-8;  we  males  resulting  3,3 -dioctadecyloxacarbocyanine  evaluate  described  mec-8,  the  Mec  were  homozygous  putative  were  (revertant)  allele  to  individually.  single  deletion  and used  of  The parental  deletion  confirm  fluorescent  hour  were  spontaneous  males  Several  animals,  the  plates  D y e - f i l l i n g to s t a i n amphid/phasmid To  room  Mec  mutants,  revertant  unc-52  and brooded.  by  stock  hermaphrodites.  segregated  tested  revertant  hermaphrodites  transferred  double  unc-52  Homozygous  mec-8(u74)  that  mec-8;  mutants  on  at  f o r about slides  wavelength  of  1  for 488  nm) .  VIII.  Immunoflourescenee  techniques  Immunoflourescenee embryos  from  specimens  N 2 , CB444,  were  fixed  staining RW6013  and  microscopy  was performed  and adults  and stained  using  30  from  methods  on  populations  N2 a n d DM4415. previously  of The  described  (Rogalski  et  1993,  al.,  1995).  The  antibodies  polyclonal  antiserum  to  unc-52, G M l ( M o e r m a n e t  monoclonal  antibody  to  unc-52,  Rogalski myosin  et  heavy  antibodies, donkey  al. ,  1993,  chain  A,  1995); DM5.6  Laboratories,  F(ab') , 2  were  images  were  Optical  using  publication, and  arranged  computer  a  mouse et  purchased  al. , and  2  rabbit a  mouse  Waterston,  1983).  F(ab')  a  1996);  monoclonal  al. ,  anti-rabbit  collected  Rad Microsciences Division)  combined  and  were  antibody The  and  from Jackson  1991; to  secondary  TRSC-labelled ImmunoResearch  Inc.  Confocal  microscope.  (Francis  (Miller  F I T C - l a b e l e d donkey  anti-mouse  MH3  used  the  image and  images  attached were  PROJECT  ("maximum  were  annotated  For  polarized  following  procedures  to  sections  files  were  using  printed light  a  taken  on a  MRC 6 0 0  Nikon  at  0.2  Adobe  to  micron  a  described by Waterston  31  live et  3.0/4.0.  and For  computer The  printer.  worms al.  compound  function.  Photoshop  (Bio-  intervals  Macintosh  Codonics NP-1600  microscopy,  system  Optiphot-2  projection")  transferred  using  the  were  (1980).  viewed  RESULTS  I.  Domain  in muscle  protein.  been  encodes  These I,  form.  II,  Since  IV  of Unc  17  V-containing  only  to  i n  two o f  gene  I,  domain  IV.  a stop  codon  (Williams of  and  this  mutant  a l l  detectable  accumulation  both the  encoded  short  isoforms  remaining  to lead  st549  of  isoforms  alleles,  ra401  as  III,  has  only  study. were  this to  2)  IV  i n  a  and V  recently  of  the  to  I,  II,  and  long  complexity alleles,  A l l mutations  lethality  an that  exons  16,  locations  of  were  known.  The  exon  2 and  st549  7 ( R o g a l s k i e t a l , 1993).  UNC-52 UNC-52.  state  of the  a l ,  1993).  et  polyclonal  demonstrated  antibody  that  The GMl a n t i s e r a  exons UNC-52  9  a n d 10  (Moerman  has an identical  32  the null  Rogalski  GMl, a  of  the  generated  short  classes  study,  1994;  of  of  the domain  the  class.  complex  Polyclonal  and apparent  represents  with, of  II,  from  of a T e l insert  i n exon  by a portion  and long  lethal  Waterston,  recognizes  epitopes  these  a  form;  the alternatively-spliced  that  that  I,  UNC-52  two broad  Prior  i s the result  and III  this  the size  and a  l i e within  II  isoforms  to  has only  class  was determined  Staining  III  refer  the mutations  from  of  i s  isoforms  isoform  regions  II,  I,  domain  i n  Despite  i t  a  included  certain  locus,  utlll alteration  It  not  We  to Unc animals  results  i s  respectively.  a n d 18  a domain  domain  (uncoordinated)  lead  a r e 1)  the  t h e domain  t h e unc-52  different  form;  isoforms.  isoforms,  three  3)  C. elegans  of  least  and IV  i t  assembly  viability  III  specific  distinguish  at  isoforms  discovered,  antisera  III,  for myofilament  U N C - 5 2 / P e r l e c a n - e n c o d i n g gene  that  domain  is essential  and for nematode  The locus  IV of perlecan  there  i s  detects  al, 1996).  phenotype  no  recognizes  and thus et  that  Of  and staining  pattern  with  somewhat  UNC-52  similar  results).  the  stains  protein  remaining Pat  is  four  the  terminal  to  including  exhibit  a  the  pharynx  st546, do  st560,  not  UNC-52 four  have  mutant  IV This  antibody  four  strains  unpub.  in  results  staining  an  The  elsewhere  st578  alteration (i.e.  not were  Waterston,  detects contained .  st549 is  a l l  that  anal  the  3;  Table  wall but  any  indicates  region of  of  examined  1991;  embryos. antibodies  33  of  material is A  J .  material  in  muscles  of  wall  these  3  domains). a  by  exon  19  et  al,  UNC-52. to  consistent  and mutants  mutants  common  MH3,  possible  muscles  gene  with,  Rogalski  other  Rahmani,  that  f i r s t  encoded  isoforms  reactive  the  the  epitope  that  P.  the  body  MH3  with muscle  sphincter/retractor Mullen,  die  pattern  in  in  and  these  but  a  the  however,  immunoreactive  animals  share  This  body  embryos,  (G.P.  not  The  2).  staining  that  that  BM.  hatch  animals,  mutant  obseravtion  null  strains  of  long  any  This  these  a l l  presence  in  an  ral!2,  and  (Figure  pattern  also  recognizes and  and  ral!2  and  strains  results)  staining these  and  that  (Francis  pharynx  results).  isoforms  antibody  the  these  these  the  observed  in  for  to  immunoreactive  We  homozygous  from muscles  st549  to  unpub.  appears  from  identical  Bush  it  st578  is  J .  UNC-52  and  st572,  and  development  absent  wild-type  unpub.  st560,  progeny  UNC-52.  tissues  to  allele,  Norman  animals  antisera  different  was  K.  transported  of  is  a  in  st546,  stage  immunoreactivity  Bush,  be  homozygous  exhibit  antibodies  to  alleles,  phenotype  mutants  with  Another  st549.  Mullen,  muscle  weakly  two-fold  as  (G.P.  wall  unable  phenotype:  at  utlll  to  Body  mutations  antibodies  to  The  monoclonal in  domain  1993, None (G.  with  1995). of  P. the  these Mullen,  lack  interpretation allow  a l l  us  to  of of  Tj  TJ  0  0 M-l  o  o  JJ 4J (TJ  JJ  Tj  TJ CU 4J W CU  4-1 1  cu  4J IQ 0) (Tj  o  -H  4-> (Tj  4J (Tj PL.  1  (Tj  u u (Tj  4J (tj  3 4J  o u o u CU rH  Xi (Tj  -H MH •H U CU CQ I  > H  0.  o  4-)  'I 0  A 1  (Tj  CD  oo  o  CU  CN  (Ti CO  A  JJ (TJ  CN  CU EH  TJ CU >  u in  •H  Tj  4J CU rH CU TJ  0)  C  e o  o  -H  A I  I  n co oo  u o LO 4J W  CN rH rH (Tj  discern of  the  UNC-52  differential  within  the  the  pharynx  and other  are  present  in  In the  the  mAb,  myosin, DM5.6 wall  the  above  A  allows  into  the  ordered 5,  is  is  for  necessary body  wall  The short are  st578  panels  observations  in  st560,  long  if  for  we  st546,  st560,  of  these  mutants  led  us  lay  to  suspect  downstream  immunoreactive not.  Genomic  rall2  homozygous  and  upstream  While region  the  of  Pat exon  mutations  (see  Appendix  a  any  of  DNA w a s  The  is  with  muscle  pers.  comm.).  in  the  four  not  body lethal  organized  aggregates  (containing and/or  corollary  (see  of  these  domain  IV)  maintenance  within  The  10  these  alterations  because  in  19  Table  (see  st546  or  the 1  for  35  the  be  largely  IV  by in  one  nucleotide  should  10)  (exon  st560,  be  Mullen,  unc-52  downstream  not  any  G.P.  amplification  could  in  (exon  MH2  st546,  region  st578  details),  GMl but  isoforms  characterization  described  mutants  embryos  domain  of  long  would  phenotypic  as  sequence  distribution that  organization  mutation  exon in  for  wall  the  differential  PCR-amplified from  in  isoforms  implication  formation  antibodies,  material  of  large  UNC-52  a  ra!12.  or  (3')  in  elegans.  find  that  present  co-stained  body  myosin  important  myofilament  using  isoforms  long  Mullen,  forms  of  were  muscles  lattice  isoforms.  st578  are  myofilaments  rall2,  suggested  could  of  wall  form  C.  UNC-52  long  only  minor  the  An  long  model  but  G.P.  instead  D) .  of  the  of  and  myofilament  muscle  essential  confirmed  a  and  forms  embryos  1983;  body  but  and  that  above  and  C  short  muscle,  mutant  visualization the  short  muscle.  al,  Within  the  embryo;  recognizes et  A-bands  of  wall  experiments,  (Miller  st546,  Figure  non-body  which  muscles.  mutants  developing  body w a l l  DM5.6, MHC  expression  shows  19)  does  st578 of  exon  and 10  products) .  found  in  alterations  this in  st560  and  C  T  to  rall2 were transition  at  residue  (CAA)  glutamine Figure  4) .  products repeat the  At  are of  domain  overlying  body  low  result  cannot  the  is  is  that  (Figure  allele  than  that  approach  inserted  successfully  to  (Kiff  et  screen 52  al,  was  repair Moerman allele  mutation  et  in  al,  1991) .  the  alternatively  spliced  exons  using  in  the  muscles  in  this the  staining  product,  or  from  this  UNC-52, a l l  a the  possibility, at  BM  over  isoforms  isolated the  have  we time.  body  wall  myofilament  of  of  took  et  al,  36  domain  different  Pat  alleles. been  in  new  excision 1990; of  is 18  a  the  ideal which IV.  reversion  lethal  and  Gloor  used  elegans  C.  small-scale  isolating  strain  within  genome  A  advantage  exon  other  the  transposon  (Engels  using  previously  1991) .  intent  within  gene  unc-52  residual  of  2;  wild-type).  al,  This  (Table  observed  lacks  identify  after  We  is  isoform  a  present  former  and  genes  et  IV  is  possibilities  rearrangements  with  domain  stl96  long  to  to  unc—52(stl96::Tcl) in  two  changing  immunoglobulin  truncated  the  a  observations  UNC-52  i n i t i a l l y  Moerman  resulting  events  a  within  performed  mutations  favor  compare was  isolate  1988;  short  these  used  Transposons  the  disorganized  rall2  that  This a  is  (TAA)  second  is  C) .  detecting  without  5,  the  staining  panel  we  between  severely  organization The  is  5,  of  13  codon  reactivity  UNC-52  While  exon  means  at  mutation  sphincter/depressor  antibody  muscles.  clear  muscle  anal  expression  stop this  mutant  UNC-52  (Figure  distinguish  level,  in  immunofluorescence  that and  8989  ochre  this  residual  from  of  an  The  show  muscles  body-wall  in  IV.  only  to  unc-52(st560)  The  nucleotide  protein  pharynx  but  level  What  GMl  the  wall  could  the  truncated  antibody  mutant,  determined.  et  imprecise al,  paralyzed since  is From  one  unc-  1991; Tel  the  Tel  of  the  approximately  Figure  7.  D e l e t i o n s r e v e r t a n t s i n domain I V  Representation  o f t h e 10 d e l e t i o n r e v e r t a n t s i n unc-52 and  lethal  showing  allele  Molecularly, while  the  the  relative  the r e v e r t a n t s range  Pat  allele  i s an  i n size  out-of-frame  the l a r g e s t v i a b l e r e v e r t a n t , ra515, t h e r e g i o n , has deletions with  are  a virtually those  mec-8 w h i l e  the  used  extents  of  from 3283  the 311  bp  the  c o n s t r u c t i o n of  green-coloured  allele.  37  one  breakpoints. bp  to  1519  deletion.  which lacks 4 Ig repeats  w i l d - t y p e phenotype. in  the  The  bp Even from  red-coloured  double  d e l e t i o n represents  mutants the  Pat  1200  heterozygous  +/unc-52(stl96)  m u t a n t was i d e n t i f i e d results). and  insertion  embryos site  in a  5 kb  a l t e r a t i o n w i t h i n the locus.  observed  region  One  of  had  i n wild  type  animals  (data  across  the  region,  i t  alteration  is a  3283  bp  single  Mullen,  Pat  unpub.  unc-52 could  flanking detect  type  the T e l  a  molecular  of the a m p l i f i e d fragments  embryos  sequencing  a  fragments from both w i l d  i n e x o n 18 t o s e e i f we  homozygous  screened,  (P. Rahmani, J . B u s h a n d G.P.  We e x a m i n e d P C R - a m p l i f i e d  mutant  rall2  animals  a  smaller  fragment  not  was  out-of-frame  than  what  shown) ,  and  determined  deletion  from  (Table  we  after  that  this  2) .  This  l a r g e d e f i c i e n c y i s w h o l l y c o n t a i n e d w i t h i n t h e domain I V  encoding  ( F i g u r e s 4 and 7 ) .  results  portion  o f unc-52  demonstrate  that  the  deficiency  The  does  immunostaining  not  interfere  with  the  e x p r e s s i o n o f s h o r t i s o f o r m s o f UNC-52, and t o r e i t e r a t e ,  there i s  no  a  o r g a n i z e d myosin.  IV-containing myofilament  long  These isoform  assembly  to  results of  again  UNC-52  occur  at  confirm  is  this  that  required  time  to  domain permit  during  embryonic  IV immunoglobulin-encoding  exons  development.  II.  A l t e r n a t i v e l y - s p l i c e d Domain  are  dispensable P r e v i o u s s t u d i e s had suggested  the hypothesis  exons w i t h i n t h e r e g i o n o f a l t e r n a t i v e be  dispensable  1995) . this  and  Moerman,  1992;  individual  i n domain Rogalski  IV  r e g i o n by  screen  intragenic  that  again  utilizing  the  transposon  Instead of s e l e c t i n g yielded  revertants  unc-52(rall2)  with  viable  39  , we  insertion  f o r Pat animals, instead  phenotypes.  may  et a l ,  I n o r d e r t o t e s t t h i s , a s e r i e s o f e x o n s were removed  unc-52(stl96::Tel). the  (Gilchrist  splicing  that  from  allele, as i n  selected for We  identified  approximately  120 i n d e p e n d e n t  revertants which e x h i b i t e d  movement compared w i t h t h e p a r a l y z e d p a r e n t a l G.P.  Mullen,  unpub.  approximately  results) .  2.6 k b r e g i o n  Using  which  strain  PCR p r i m e r s  encompasses  (see Table  3).  Upon  ( J . Bush and that  f l a n k an  the alternatively-  s p l i c e d exons, f o u r t e e n w i l d - t y p e l o o k i n g s t r a i n s alterations  improved  sequencing,  showed c l e a r DNA  i t was  determined  t h a t f o u r o f t h e f o u r t e e n r e v e r t a n t s have s m a l l i n s e r t i o n s to  30 bp) ,  remaining  one h a s a s m a l l  9 revertants  hundred b a s e p a i r s  contain  (Table 3 ) .  i n t e r e s t because these  to  be  as  robust  between p l a t e s mechanical observed  deletion, while the  d e l e t i o n s removing  up t o s e v e r a l  These l a t t e r n i n e w e r e o f p a r t i c u l a r  strains a l l exhibit nearly wild-type  s t r u c t u r e a n d movement. be a m i n o r f r a g i l i t y  9 bp i n - f r a m e  (from 3  The o n l y  observable  o f the muscle.  as w i l d - t y p e  a n d o c c a s i o n a l l y seem  disruption  of  as i n d i v i d u a l  their  phenotype appears t o  Revertant  animals  worms b e i n g  worms do n o t a p p e a r  when, b e i n g  t o be more  body  muscle  wall  sluggish  transferred  susceptible to  muscles.  This  i s  o r uncoordinated  in  movement o n l y a f t e r b e i n g t r a n s f e r r e d . An  a n a l y s i s o f t h e DNA b r e a k p o i n t s  some i n t e r e s t i n g the  features.  rearrangements  endpoints ra.512,  The most p r o m i n e n t  ra515,  ra516,  site  ra517,  while  distribution.  the l e f t  impose  breakpoints  (Figure  8) .  The  a l l have  alleles a  right  Tel insertion dinucleotide  appear  more  random  i n their  T h i s may be a p r o p e r t y o f t r a n s p o s i t i o n o r t h e DNA  r e p a i r mechanism i t s e l f . also  characteristic of  a n d ra519  b r e a k p o i n t w i t h i n 12 bp o f t h e o r i g i n a l (TA) ,  shows  i s the close proximity of s i x of the deletion  to the T e l insertion  ra513,  i n the revertants  some  Our s e l e c t i o n  restraints  f o r v i a b l e r e v e r t a n t s may  on t h e d e l e t i o n b r e a k p o i n t s .  sequences e i t h e r s i d e o f t h e b r e a k p o i n t s  40  i n these  The  d e l e t i o n s do n o t  Table Revertants  3.  o b t a i n e d from T e l e x c i s i o n  Allele  Rearrangement  DM4305* DM4310* unc-52(ra507) DM4313* unc-52(ra506)* unc-52(ra511) unc-52(ra512) unc-52(ra513) unc-52(ra514) unc-52(ra515) unc-52(ra516) unc-52(ra517) unc-52(ra518) unc-52(ra519) unc-52(ra112)  3 0 bp insertion 2 4 bp insertion 12 bp insertion 3 b p insertion 9 bp deletion 5 6 6 bp deletion 1206 bp deletion 1293 bp deletion 1457 bp deletion 1519 bp deletion 9 8 2 bp deletion 1359 bp deletion 1205 bp deletion 1185 bp deletion 3 2 8 3 bp deletion  * denotes s t r a i n s  screen  t h a t were s u b s e q u e n t l y  41  lost.  Figure Deletion  (Top  line) Partial  (shown  in  breakpoints  e x o n 18 s e q u e n c e i n d i c a t i n g  outline)  insertion  site  surrounding  sequence.  breakpoints  d e n o t e d as a s l a s h ( / ) .  the  right-hand  ra519  endpoints  12 n u c l e o t i d e s  ' f o o t p r i n t ' remaining the  free  DNA  similarities ends  The a l l e l e s  (boxed n u c l e o t i d e s )  within  ends.  show t h e i r Notice  Italicized  and  some  of i t s  associated deletion  the close p r o x i m i t y of ra515,  to the T e l s i t e - - a l l  Ra516  to surrounding  i s clearly  for Tel  of ra512, ra513,  o f TA.  t h e TA d i n u c l e o t i d e  ra516,  ra517 and  s i x mutations  sequence  break  represents  the  a f t e r T e l e x c i s i o n f o l l o w e d b y gap r e p a i r o f  of T e l i t s e l f  Ra518  8.  a  20  nt  addition  s e q u e n c e so i t p r o b a b l y  acting a  has  as  template  d u p l i c a t i o n o f one  through  unequal  cross-over.  A l l other  occurred  through p r e c i s e e x c i s i o n .  42  during  with  arose  no  from t h e  strand  invasion.  end p o s s i b l y  resulting  mutations  have  likely  CJ O  cj CJ o CJ o EH CJ  CJ  o CJ CJ  o CJ EH EH EH EH  < CJ u %  (0  a •H • 00 ID  M  0  $  2 O H W to  id ID  Fi on  u o CJ < CJ u  3 EH  CJ  u  Q  CJ EH U EH U EH  < o; EH CJ EH EH  <:  H  o EH CJ  <:  u EH 00 rH  o x  cu  EH CJ EH EH CJ CJ CJ U  "S  CJ  < u % EH U < U U  CJ CJ  cj CJ u EH CJ < CJ CJ CJ CJ  CJ CJ  u CJ u EH CJ  CJ CJ U  u  CJ CJ EH  < CJ CJ CJ CJ CJ EH  9 CJ CJ CJ CJ  %  CJ CJ EH EH EH EH  CJ U  U  CJ  CJ CJ  %  %  < %  <  o  o  S CJ  El  CJ  3 3 u CJ  CJ  8  EH EH EH EH CJ \ EH U U U <C O EH  CJ CJ CJ  u o CJ EH CJ U CJ  < CJ %  < CJ  | EH EH EH EH EH CJ  cj EH CJ  a CJ u EH CJ  CJ EH CJ CJ EH  < EH EH CJ  ^ CJ  < cj u CJ  < u u EH  CJ CJ EH  3i CJ  %  u  EH EH CJ  CJ CJ  CN rH tn (d in  %  CJ CJ  CJ CJ EH EH EH EH  CJ CJ CJ CJ EH CJ CJ EH CJ  <  CJ CJ  CJ CJ EH EH EH EH  CJ CJ O CJ CJ EH CJ CJ EH <J  "S  •H •P ID H ID  ^ CJ CJ U U  CJ CJ CJ CJ CJ EH U CJ EH CJ  EH CJ CJ  &Au  CJ CJ O CJ O EH CJ  EH O  o EH < EH  a  ^ CJ CJ U U  <  EH U  |  «.  EH < CJ CJ O CJ O EH CJ  CJ CJ EH  EH CJ  <  W  u u  l£> rH in rd  rH in  (0  CJ CJ EH EH EH  < CJ  %  EH CJ < U CJ CJ CJ CJ EH CJ CJ EH CJ  EH CJ  < CJ  ui \  EH CJ CJ  %  < CJ <  I  CJ EH EH CJ CJ  EH CJ EH  % EH EH CJ  < CJ < CJ CJ CJ EH CJ O  < CJ CJ  < CJ EH EH O  % CJ CJ  in rH m <d  CJ CJ  cj u  < CJ EH  CJ CJ  o CJ CJ EH EH CJ CJ CJ EH CJ CJ EH CJ CJ  r> rH in td  § EH CJ CJ EH ^ EH EH CJ  < CJ  % CJ CJ EH EH  CJ  o\ \ EH CJ CJ CJ CJ EH CJ  CJ EH CJ CJ EH  CJ  %  9  % CJ EH CJ CJ CJ  EH EH EH EH CJ  CJ CJ CJ CJ CJ EH  EH CJ CJ CJ U EH CJ  CJ CJ CJ EH CJ  H  u u EH <  % CJ CJ CJ EH CJ EH EH  * S CJ  < CJ CJ CJ CJ  %  CJ  <c EH  <  CJ CJ CJ CJ CJ CJ CJ  CJ EH O EH CJ EH ^ CJ EH CJ EH EH  < EH <Cl CJ EH CJ  *S  CJ CJ  < CJ  < 0  u CJ  u EH U CJ CJ  1 9  CJ  U  o  CJ  u  CJ CJ EH EH CJ CJ EH CJ O EH ^ CJ EH EH  <  CJ EH CJ  < CJ  CJ CJ  *s  CJ CJ CJ CJ CJ  < u u EH  CJ CJ  u  < EH  CJ  *S CJ  EH CJ  CJ  < CJ  CJ  <  CJ CJ  EH  <:  < CJ CJ EH  CJ  % CJ CJ EH CJ EH EH CJ CJ CJ CJ  *s  9 CJ  EH CJ  < CJ  CJ EH CJ << U CJ  <  u u u CJ  < EH  % CJ -<  CJ CJ  CJ CJ  EH EH  < u:  CJ CJ CJ CJ  CJ CJ  CJ  ro rH in (d  <  CJ CJ EH  CJ  <  O EH CJ CJ CJ  %  < o  <  < CJ  3 CO rH in id  o o o 00 ro (d  U  CJ CJ CJ CJ CJ CJ CJ  CJ EH CJ EH CJ EH  < CJ EH CJ EH EH  < EH <J C EH <J C <J C CJ EH  rH rH in id  JH  U EH EH CJ CJ CJ  < < CJ U EH  CJ  < u CJ CJ EH CJ CJ CJ rtl  <]  ^  <: CJ CJ CJ CJ  <  CJ CJ  rH in cd in  s h a r e any o b v i o u s sequence s i m i l a r i t i e s the  result  of  intragenic the in  unequal  crossing  over,  r e v e r t a n t unc-52(ra38)  revertants  {ra516  so they as  correct  a n d ra518)  details).  reading  frame  do c o n t a i n  deletions  (refer  proposed  (see F i g u r e  to Figure  small  8  t o be  f o r the Two o f  rearrangements  alterations s t i l l  The o t h e r s e v e n members o f t h i s  in-frame  was  ( R o g a l s k i e t a l , 1995).  a d d i t i o n t o a d e l e t i o n , but these  the  are unlikely  legend  maintain  f o r further  set a l l contain  perfect  7 f o r a schematic view of the  genomic e x t e n t o f e a c h d e f i c i e n c y ) . T h e s e s e v e n domain I V d e l e t i o n s r a n g e f r o m 982 bp t o 1519 bp and  remove b e t w e e n two a n d f o u r I g r e p e a t s  7) .  I n some i n s t a n c e s  that  'chimeric'  These  have  ( f o r example  exons may r e s u l t  the  correct  of  folding  structure  o f an NGAM-like  encode (Figure couple an  only 9) .  (Table  and  Igs  (see F i g u r e Ig) .  Igs  and  these  The  10  are  al,  1983),  integrity.  of  conserved  exons  likely  ribbon  of the other appear t o  non-functional  removes  at least  a  a n d y e t t h i s does n o t a p p e a r t o h a v e much o f  strain,  n o r on m u s c l e  unc-52(ra515),  containing  d e l e t i o n was s t a i n e d w i t h GMl, an a n t i b o d y DM5.6, an a n t i b o d y  (Figure 9 ) .  for typical  strain  on nematode g r o w t h , m o t i l i t y ,  4).  Ig unit  spacing  The c h i m e r i c  deletion revertant  of I g repeats  effect  i n a hybrid  i t appears  s p l i c e d o u t o f t h e pre-mRNA a s t h e y  partial Each  a n d ra514)  amino a c i d s b e l i e v e d t o b e i m p o r t a n t f o r  tertiary  a l l e l e s are probably  ra512  arrangement  c y s t e i n e s and surrounding  (see Table 2 and F i g u r e  specific  t o determine  specific  of perlecan  T h i s mutant e x h i b i t s a c o m p l e t e l y  myosin s t a i n i n g p a t t e r n  longest  A  and  (Miller et t h e muscle  w i l d - t y p e UNC-52 a n d  ( d a t a n o t shown) d e s p i t e  four immunoglobulin repeats  the  t o UNC-52, a n d  t o a myosin heavy c h a i n  the state  development  lacking at least  f r o m domain I V (exons 15, 16, 17, a n d 44  Table 4. C h a r a c t e r i z a t i o n o f noteworthy r e v e r t a n t s alone and i n d o u b l e homozygous c o m b i n a t i o n w i t h a mec-8 n u l l  Strain  Muscle Structure  Movement  Amphid Staining  Phasmid Staining  ColdSensitivity  N2  wt  wt  12  4  no  unc-52(ra507)  wt  wt  12  4  no  unc-52(ra511)  wt  wt  12  4  no  unc-52(ra515)  wt  wt  12  4  no  unc-52(ra516)  wt  wt  12  4  no  mec-8(u74)  wt  variable  4-8  1-2  yes  mec-8(u74); ra507  mutant  pat  ?  ?  ?  mec-8(u74); ra511  wt  sluggish  4-8  1-2  yes  mec-8(u74); ra515  wt  sluggish  4-8  1-2  yes  mec-8(u74); ra516  wt  sluggish  4-8  1-2  yes  N o t e : N o t a l l d a t a f o r unc-52 (ra507) h a s b e e n  45  determined  Figure  9.  Comparison o f normal I g s and p o t e n t i a l h y b r i d s forced through chimeric  Amino a c i d and  sequence a l i g n m e n t  t h e most  cysteines  conserved  ( [C], boxed  of several  residues  Igs.  shown.  Those  revertants  that  may  present  resembling  conserved  The  two  as  they  e s s e n t i a l to proper f o l d i n g make  a functional  f r o m domain I V  boxes).  i n green) a r e a b s o l u t e l y  The o t h e r a l l e l e s do n o t have enough  make a n y t h i n g  Ig.  a  hybrid  Ig unit are  sequence a v a i l a b l e t o Note  the 3  cysteines  i n r a 3 8 a n d r a 5 0 7 w h i c h may a f f e c t c e r t a i n i s o f o r m s  MEC-8 i s removed motif  Ig units  (coloured  are r e q u i r e d f o r the d i s u l p h i d e bridge of  exons  (purple)  (see t e x t ) .  Also,  note  i n I g l 2 e n c o d e d b y e x o n 19.  46  when  t h e l o c a t i o n o f t h e RGD  18),  a n d r e d u c i n g t h e domain I V number o f I g r e p e a t s  to ten.  The o t h e r d e l e t i o n r e v e r t a n t a l l e l e s  polarized  light  n o t shown).  microscopy and they  These  combined  results  were  from  fourteen  examined  using  t o o had normal muscle  (data  indicate  t h a t many o f t h e I g  r e p e a t s i n t h i s r e g i o n o f domain I V a r e i n d e e d d i s p e n s a b l e . S i n c e a n i m a l s a p p e a r n o r m a l when homozygous f o r one o f t h e s e d e l e t i o n s we a t t e m p t e d t o s e e i f we c o u l d v a r y t h e gene d o s a g e a n d detect  a  phenotype.  unc-52(ra515)/unc-52(st560)  52 (ra.511) I unc-52 (st560) constructed  and  heteroallelic  examined.  These  p h e n o t y p e t o a n i m a l s homozygous a  single  copy  of  a  and  combinations  animals  were  f o r the deletions  revertant  unc-  chromosome  were  identical  in  indicating  that  i s sufficient  for  viability.  III.  Domain  mediated  IV  1995;  eliminate  s p l i c i n g of unc-52  Alternative unc-52  deletions  Lundquist  during  splicing within  transcripts  the  requirement  mec-8-  embryogenesis t h e domain  appears t o be r e g u l a t e d  et a l , 1996).  for  IV encoding region of (Rogalski  One component  e t al, 1 9 9 3 ,  of this  regulatory  s y s t e m i s a p u t a t i v e RNA b i n d i n g p r o t e i n encoded b y t h e gene mec-8 ( L u n d q u i s t e t al, 1 9 9 6 ) . i n t o a mec-8(null) a  synthetic  (Lundquist domain  lethal  When v i a b l e  background, t h e r e s u l t i n g phenotype  a n d Herman,  reminiscent  1994).  I V a r e mec-8 dependent:  unc-52(viable)  translational  stop  the s p l i c i n g 1996) .  embryos  mutations  double mutants  of Pat a l l e l e s  splicing  does  48  (Figure around  n o t occur  exhibit  o f unc-52  choices  o f exon  A l l other  t h r o u g h mec-8 i n d e p e n d e n t mechanisms  8(null);  mutations a r e crossed  Two o f t h e s p l i c e  exon 1 6 t o 1 9 ( L u n d q u i s t e t a l , occur  unc-52  within  15 t o 19 and  splice  decisions  11) .  I n mec-  exons  containing  and t r a n s l a t i o n i s  F i g u r e 10. Ribbon diagram of a C2-type  (3-sheet stranded cysteines residue  structure  of  tertiary a r e shown that  an  NCAM  subtype  folding. as  includes  well the  The as TA  immunoglobulin  of  I g showing  locations  the tyrosine dinucleotide  ( A d a p t e d f r o m Vaughn a n d B j o r k m a n ,  49  1996).  of (Y)  the  the  seven-  conserved  which  i s the  for Tel insertion.  Figure  10.  Ribbon diagram o f C 2 - t y p e  immunoglobulin  prematurely truncated  during  Therefore  t h e synthetic  52 (viable)  double  UNC-52  isoforms  lethal  mutants  with  that  I V , a s does  double 8(u74): the it  i f we c o u l d  To t e s t  constructed  alternatively-spliced the splice  8(u74);unc-52(ra516), e x o n s 17 a n d 18. synthetic 4) . with  this  hypothesis,  this  and a r e v i r t u a l l y  these  homozygous f o r mec-8,  that  i s also  these  & Methods).  of the  amphid a n d phasmid c e l l  This  i s simply  Mec p h e n o t y p e .  bodies  demonstrate  that  that  stained  confirmed  phenotype  (Table  o f mec-8  number o f  that  these  4) .  These  we c a n remove a n y d e p e n d e n c e o n  locus. associated  with  mec-8  mutants  embryonic and e a r l y l a r v a l c o l d - s e n s i t i v e l e t h a l i t y . mutants  i n mec-8  o f t h e unc-52  by elimination  (exons 15 t o 1 8 ) ,  MEC-8 f u n c t i o n a t t h i s Another  were  a diagnostic  The r e d u c e d  a n i m a l s were homozygous f o r t h e mec-8 m u t a t i o n  region  observed  t h e y were e x a m i n e d f o r t h e d y e - f i l l i n g ( D y f )  (see M a t e r i a l s  substrate  (Table  animals  mutants  observations  are not  i n phenotype  i n t h e amphid/phasmid neurons w h i c h a r e o b s e r v e d  indicative  since  d e l e t i o n removing  defects  tool  a l l of  combinations  wild-type  To c o n f i r m  mec-  18 a n d 19; a n d mec-  f o r exons  as i t i s the next smallest  homozygotes.  allele,  mec-8 (u74);unc-52(ra511),  region;  from  the following  eliminates  T h e s e worms a r e somewhat s l u g g i s h , b u t t h i s mec-8  region  become i n d e p e n d e n t o f a n y  because  junctions  the target  t h e mec-8 n u l l  with  I t was o b s e r v e d t h a t  lethal  o f unc-  thelethality  remove  mec-8 (u7'4);unc-52(ra515),  removes  of functional  homozygotes.  f o r MEC-8.  m u t a n t s were  1996).  mec-8(null);unc-  of  t h e absence  w i t h i n t h e unc-52 gene, p e r h a p s i t c o u l d requirement  e t al,  (Lundquist  phenotype  r e s u l t s from  domain  52 (st560) o r unc-52 (rall2) We r e a s o n e d  embryogenesis  exhibit  an approximately  51  3 0%  i s an  A t 15°C, n u l l mortality  rate  ( L u n d q u i s t a n d Herman, a  problem  the  i n muscle  double  phenotype. lethality, mutant  mutant  1994) .  attachment,  should  we  t o s e e i f unc-52  I f aborted then  Since t h i s  a no  unc-52  l e t h a l i t y may be due t o  were  interested  might  splicing  caused  longer  have  any  the c o l d  in  sensitive  attachment.  52  besides  this  sensitive double  cold-sensitive  s u g g e s t s t h a t MEC-8 has y e t a n o t h e r t a r g e t f o r muscle  implicated  mec-8(null);unc-52(deletion-revertant)  However, t h e d o u b l e m u t a n t s a r e a l l c o l d  important  be  i n examining  lethality.  ( T a b l e 4) unc-52  which  that i s  F i g u r e 11. Potential s p l i c e choices occuring i n the alternatively-spliced  The  mec-8-dependent  and  independent  region  splices  determined  through  cDNA a n d RT-PCR d a t a i n w i l d - t y p e worms ( R o g a l s k i et al 1993, 1995; Lundquist all  e t al, 1 9 9 6 ) .  r e v e r t a n t s e x c e p t ra38,  19 s p l i c e i s p r e s e n t is not  mec-8-dependent  only present  ra516,  splicing i s eliminated i n  ra518  a n d ra519.  i n a l l four of these  i n ra38.  Surprisingly,  The e x o n 15-  m u t a n t s b u t e x o n 16-19  ra516,  ra518  a n d ra519  l e t h a l when MEC-8 i s a b s e n t u n l i k e ra38 w h i c h i s l e t h a l .  53  are  Figure 11. Potential splice choices i n unc-52  54  DISCUSSION  The the  unc-52 gene e n c o d e s a l a r g e m u l t i - d o m a i n p r o t e i n  BMs  underlying  (Rogalski of  e t al,  myofilament  the muscle  1993)  and  et  al,  portion  of  isoforms single  the  of  regulation  of  gene  Much exons  and  f o r the proper  of  this  leads  the to  Ig unit.  dependent  on  Expression 1995;  G.  produced  the  of  P.  The  combinatorial  type  unc-52  of  varies  over  required  t i m e and  M u l l e n , unpub. r e s u l t s ) .  based  on  the presence  25 w h i c h  nature of  isoform  or  distinct  the  and  during  of  encode 19  may  isoforms are  wall  muscles  thesis  has  specifically  during  been  to  embryogenesis. determine  p e r l e c a n f u n c t i o n and m u s c l e that  have  body  wall  critical crucial within  been  addressed  muscle than  the  unc-52  the  To  include,  3)  address  locus  The  development.  (Rogalski of  et  al,  isoforms are  domain  IV.  that  1)  2) Is  Are  of  primary  goal  The  of  Short  IV  certain length  specific  body-  of  this  IV  for  questions  critical  for  units  more  Ig of  The  the  domain  specific  domain  questions,  possess  55  Is  overall  these  t o t h e BMs  importance  development.  development?  o t h e r s ? and  factor?  localized  a be  v a r i a n t s l a c k d o m a i n I V w h i l e l o n g forms c o n t a i n t h i s domain. long  a  which  Ig units  groups  absence  long  t o g e t h e r form  space  Two  IV  14  by  IV-encoding  domain  e x c e p t i o n s a r e exons  e n c o d e more t h a n one I g , and exons 24 and single  of  1995;  occurs  domain  several  undergoes  1993,  regulation  S e v e r a l exons  unit,  e t al,  elegans  initiation  locus that  (Rogalski  within  this  the p r o t e i n .  immunoglobulin  t h e nematode C.  T h i s i s a complex  1996).  alternative-splicing  of  i s required  assembly.  extensive transcriptional Lundquist  tissues  found i n  domain  several  IV  a  mutations  alterations  have  been a n a l y z e d .  A comparison o f the l o c a t i o n o f these  the phenotypes o f animals  homozygous f o r t h e s e  lesions  mutations  with  l e a d s us  t o a f u l l e r a p p r e c i a t i o n o f t h e c o m p l e x i t y o f unc-52  and a g r e a t e r  understanding  during  of the function of this  c o m p l e x gene  muscle  development.  I.  Domain  IV i s essential  The the  identification  unc-52  essential  gene  UNC-52  that  component f o r m y o f i l a m e n t  f o r muscle growth  al,  1978;  Waterston  Rogalski  e t al,  e t al,  1980;  1995;  UNC-52  isoforms  (Rogalski  e t al,  Antibodies  cellular  distributions  embryo.  Long  (G.P.  st549 (null) any st560  isoforms  body w a l l  Mullen  animals  Mackenzie e t 1992;  Williams  i sa null  1993).  during appear  muscle  D.G.  o f UNC-  lack only  What t h e s e t h r e e m u t a t i o n s  of  o f t h i s p r o t e i n have i n the  while  tissue  Moerman, types  short  during  unpub.  UNC-52  developing  other  from  are the  embryogenesis  results).  of isoforms  this  different  isoforms late  have  cause o f  t o be t h e p r e d o m i n a n t p r o d u c t cells  lack both  regions  and  a l l e l e and  The t r u e  morphogenesis  and cells  r e s u l t s when a n i m a l s  UNC-52 p r o t e i n ( R o g a l s k i e t al, 1 9 9 3 ) , and r a l l 2  1994;  e t al,  to different  from o t h e r muscle and  1974;  s h o r t and l o n g isoforms  r e v e a l t h a t l o n g and s h o r t isoforms  major product  important  i s more c o m p l i c a t e d a n d more s u b t l e t h a n  explanation.  developing  within  a n d Moerman,  The o b s e r v a t i o n t h a t st549  embryonic l e t h a l i t y simple  i s an  Gilchrist  52 l e d t o t h e s p e c u l a t i o n t h a t l e t h a l i t y no  of mutations  (Brenner,  Hresko  i t leads t o the loss of both  embryogenesis  assembly w i t h i n muscle  i n C. elegans  1993,  1994) .  Waterston,  during  and c h a r a c t e r i z a t i o n  demonstrate  and  that  for UNC-52 function  Although  a n d do n o t have Pat a l l e l e s  t h e body w a l l - s p e c i f i c  long  like  isoforms.  h a v e i n common i s t h a t t h e y a l l l e a d t o  56  disorganization  o f the myofilament array  within  body  wall  muscle  ( F i g u r e 5, p a n e l s B, C, D) a n d t h u s p a r a l y s i s a t a c r i t i c a l of  development  when  elongation  o f t h e embryo  should  stage  commence  (refer to Figure 3). C o m p l e t e a b s e n c e o f a l l unc-52 gene p r o d u c t s i s t h e r e f o r e n o t responsible complete  f o r the lethality.  domain  that  leads  lack  o f body  failure  t o embryonic wall  proper  base  1991) .  muscle-specific  The l e t h a l i t y  myofilament  assembly  and Waterston,  1994) .  o f dense  bodies  and M-lines  (Francis  the  results  i na  result  of a  (Francis and  o f t h e mutant  and Waterston,  i n vertebrates but  a l s o a c t a s t h e nematode homologue o f a v e r t e b r a t e We b e l i e v e  isoform  o b s e r v a t i o n t h a t UNC-52 c o n c e n t r a t e s a t  Dense b o d i e s a r e a n a l o g o u s t o Z - l i n e s  plaque.  of a  O u r model f o r  i s f o u n d e d on o u r c h a r a c t e r i z a t i o n  phenotypes and t h e e a r l i e r the  wall  i t i s the lack  function which i s a d i r e c t  1991; W i l l i a m s  function  body  lethality.  muscle  to initiate  Waterston, unc-52  IV-containing  Rather,  UNC-52  aids  focal  adhesion  i n the assembly and a n c h o r i n g o f  dense b o d i e s a s t h e y a r e formed d u r i n g morphogenesis.  Whether  UNC-52 p l a y s a n i n s t r u c t i v e / s i g n a l i n g r o l e o r s i m p l y a n a t t a c h m e n t role  i n t h e assembly  anchoring w i t h i n muscle functions stable act  primarily  of integrin is still  as an anchor,  sarcomere  organized  reviewed  i n Moerman a n d F i r e ,  described  by William  at later  (Burridge  within  muscle  and Waterston  Many  a  interface to c a n n o t be  et a l ,  1990;  o f t h e P a t mutants  affect  some a s p e c t o f  myofilament assembly o r anchoring a t t h i s c r i t i c a l  stage o f muscle  development and morphogenesis.  57  (1994)  Without  cells  e t a l , 1988; V o l k 1997) .  i tprobably  stages.  a t t h e membrane/BM  units  properly  and myofilament  notclear—although  i n attachment  f o c a l attachment s t r u c t u r e  complexes  At these  an  earlier  results  domain  was  IV of  stage  i n this  that  unc-52.  they One  project,  indicated  model  we  our  the  i n t e r p r e t a t i o n of  e s s e n t i a l nature  considered  was  domain IV i n t e r a c t s w i t h t h e i n t e g r i n complex. on  f u r t h e r sequencing  unlikely. we  had  previously We  spread  realized  over  about  region kb, pers.  comm.) .  (T.M.  This  and c o n t a i n s  repeats  (Noonan et a l ,  fold.  First,  probably rest  exons  several al,  et 1992).  The  i n i t s role  of the molecule.  Kallunki  Since  a  t h e gene and  a passive  sense o n l y  portion  of  laminin  contained  D.G.  capable  of  interacting  p o r t i o n o f domain V may surface between obtain  components long  and  between  this  may  isoforms.  mutations  domain  integrin  be t h e r e a l  and  and s h o r t  targeted  with  finding of  i n this  We  i s two-  V  and  V  be  the  present  Second,  i s thought This  important  to test  this  the  to  be  laminin  with  cell  difference  are c u r r e n t l y attempting  domain  IV  modular s t r u c t u r e of  (T. R o g a l s k i , C. W a r r e n , J . C u l o t t i and D. Moerman, p e r s .  58  1992;  domain  domain  complexes.  the  EGF-like  Tryggvason,  interactive partner be  10  Moerman,  and  p o s i t i o n domain V.  within  this  t o domain V o f mammalian  the p r e d i c t e d  to help  of  another  d o m a i n I V r e s e m b l e s a r o d (Noonan e t a l , 1991) , i t may in  26  genome  Analysis  importance  linker  see  and  the  l a m i n i n - l i k e segments  the  this i s  f u r t h e r s e q u e n c e 3' o f  Mullen  i s similar  that  based  1993;  recently  s i g n i f i c a n c e of t h i s  as  that  f o u r domains  extends  G.P.  results  al,  o f t h e gene. and  1991;  i t indicates  lies  More  Rogalski,  new r e g i o n  perlecan  Murdoch  11  et  us w i t h  the terminus  another  approximately  kb.  perhaps  gene i s e v e n l a r g e r t h a n  a gene w i t h  has p r o v i d e d  t h o u g h t was reveals  15  New  suggests  (Rogalski  had d e s c r i b e d  sequencing consortium what we  region  We now r e a l i z e t h a t t h e unc-52  Introduction) . exons  i n t h e unc-52  that  of  to  hypothesis comm.).  II.  E f f e c t s of removing Domain I V o f structure  proteins  with  proteins, the  i s typical  large  most  notably  twitchin/titin  1992).  I ti s large  family;  This  o f ECM a n d l a r g e  extracellular  1996) .  Bjorkman,  encodes 14 I g r e p e a t s .  unc-52  modular  and  a l t e r n a t i v e l y s p l i c e d exon of unc-52  components  also  found  in  structural  Benian  transmembrane  (reviewed  in  Vaugh  a few i n t r a c e l l u l a r  muscle  components  1989;  et al,  repetitive  Labeit  Long t r a c t s o f i m m u n o g l o b u l i n s w i t h i n p r o t e i n s  (e.g. etal,  have  been  p r o p o s e d t o a c t as r u l e r s t o e s t a b l i s h a p e r i o d i c i t y when b u i l d i n g larger molecular o n l y one the  bona  aggregates  fide  "ruler"  p r o t e i n has  lambda phage p r o t e i n , gpH,  a s c a f f o l d f o r the f o r m a t i o n results  showing  that  affecting  the  spacing  affecting  any other  supports The  the  skipping,  o r length  r e g i o n of can  o f muscle  that  domain I V does  combined  a n d Moerman, observations  alternative deletion and  of viable  o r b y an in-frame  splicing  revertants yet  conclusion o f the  we see that  can  unc-52  deletion  1992;  be d e r i v e d  alternatively  exon  occurs  s p l i c e d Igs  f u n c t i o n a l r o l e s i n muscle development.  via  two a d j a c e n t  exons  e t al, 1995).  about  the  these have  These  significance of  the largest alternatively  e f f e c t s on the  from  as a ruler.  mutants  a l l o f the  59  act  strongly  that  IV since  no d e l e t e r i o u s  without  s t u d i e s t h a t showed  Rogalski  o f domain  not  My  components, o r  development,  fusing  leave us puzzled  eliminate  t h i s phage.  o f myofilament  is  t o form  be e l i m i n a t e d  aspect  conclusion  suppression  (Gilchrist  none  of the t a i l Igs  This  1987) w h i c h a c t s  (Katsura,  up t o f o u r  To d a t e ,  been i d e n t i f i e d .  r e s u l t s a r e i n agreement w i t h e a r l i e r  intragenic  Igs  i n T r i n i c k , 1994).  (reviewed  the  spliced  animal.  observations unique  of  is  One that  oressential  Might  we  revertants  have  that  been  able  eliminate  to  obtain  e v e n more  even  Ig motifs?  how s m a l l c o u l d one make domain I V a n d s t i l l This The  i s an i n t e r e s t i n g q u e s t i o n T e l e x c i s i o n screen  has  several  restrictions  most s e r i o u s genetic  that  with  a high  occurred analyze  probably  produced  difficult  better 2  (Table  2).  not s p e c i f i c a l l y to precise  homologues  to pair,  machinery  and i n these  phenotype  (see Engels  animals. were  proportion  The m a j o r i t y  address  of  This able  to  of these revertants  ratio  of  r e p a i r i s determined by the a b i l i t y  of  this  the s p e c i f i c experiments,  e t al  of  our a b i l i t y  The ra515 that  the repair  to detect  e t al,  i s possible to obtain  in  t h i s i n s t a n c e we d i d n o t demand v i a b i l i t y .  m u t a t i o n a t o v e r 1.5 still  segregated w i l d  i n the d e l e t i o n revertants  preferentially  extend  5' r a t h e r  insertion  site  (Figure  7) .  speculate  that  this  implies  than  While  UNC-52 of  may  3'  the  on m a t e r i a l  and s t i l l  appear t o transposon  i s small, that  maintain  interact with  the muscle c e l l s .  60  from  o u r sample s i z e  a restraint  removed 3' o f t h e T e l i n s e r t i o n s i t e  transmembrane s u r f a c e  that  larger deletions a f t e r T e l excision, but  d e l e t i o n breakpoints  earlier  the  1991 f o r d a t a  The 3.2 k b ra!12 d e l e t i o n m u t a n t d e m o n s t r a t e s  it  mentioned  The  properties  kb was t h e l a r g e s t d e l e t i o n we i s o l a t e d type animals.  point.  1990; G l o o r  pertaining to the repair process).  As  for' those  arose as a r e s u l t o f p r e c i s e e x c i s i o n events although our  does  The  Perhaps t h e  a n d we  3  survive?  rearrangements  was b i a s e d  moving  (~10~ -10~ )  words,  one t o answer.  limitations.  the screen  only  frequency  had d e l e t i o n s  rearrangements the  was t h a t  In other  t o obtain these  and p o s s i b l e  deletion  have a n i m a l s  o v e r 120 r e v e r t a n t s , b u t o n l y a s m a l l  actually  data  limitation  events  but a  we u t i l i z e d  larger  we  c a n be  viability.  i n t e g r i n s on t h e  I n t e r e s t i n g l y , 600 bp  3'  of the T e l insertion site  within for  e x o n 19 a n RGD  integrin  in  (arg-gln-asp)  receptors  R u o s l a h t i , 1996) .  within  (Lawler  i s over  motif,  and  I f one c o n s i d e r s  the revertants  exon  site  should  viable  b e removed.  deletion  Since  revertants,  we  ligand  1989; r e v i e w e d  and  presuming  an  suggest  that  this  motif  that  this  o r some  f o r domain IV  i s a difficult  t o s t r e s s b e c a u s e o u r sample s i z e i s s m a l l .  This  excision  direction  and t h e f a c t  d i d not recover  deletion  revertants  that  region  further  compatible with  III.  Effects  suggests  that  equal  i t i s n o t removed b y a n y o f t h e  We r e c o g n i z e  extend  in  t h e n b y i n s p e c t i o n , t h e RGD  o t h e r a s p e c t o f t h e r e g i o n 3' t o e x o n 18 i s c r i t i c a l f u n c t i o n i n UNC-52.  i s encoded  t h e average d e l e t i o n s i z e  bp  d i s t r i b u t i o n around t h e e x c i s i o n s i t e ,  there  a demonstrated  Hynes,  that  1200  18  that  we  beyond  perhaps  point  possible bias i n any  the a l t e r n a t i v e l y s p l i c e d  a smaller  domain  IV i s  not  viability.  of mec-8;unc-52(deletion-revertant)  mutants  on  muscle  development The has  mec-8 l o c u s p r o d u c e s a p u t a t i v e R N A - b i n d i n g p r o t e i n  b e e n shown t o p r o m o t e a l t e r n a t i v e s p l i c i n g  which  o f two unc-52 p r e -  mRNA t r a n s c r i p t s , exon 15 - 19 a n d e x o n 16 -19 ( L u n d q u i s t ,  1994;  1996).  unc-52  Accumulation  transcripts  o f normal  unc-52 (paralyzed) phenotype  alternatively-spliced The  i n unc-52(+) muscle double  animals  development. homozygotes  s i m i l a r to the Pat a l l e l e s  Herman, 1 9 9 4 ) .  MEC-8-dependent  (15 - 19 a n d 16 - 19 ) a r e n o t a b s o l u t e l y  embryogenesis  capable  other  i s mec-8-independent.  s p l i c e products for  of  Like true Pat a l l e l e s ,  61  since  mec-8  However, exhibit  a  o f unc-52  unc-52 required  mutants a r e  mec-8(null) synthetic  and lethal  (Lundquist  and  these double mutants produce  only  truncated  UNC-52 i s o f o r m s  (Lundquist  e t al, 1996) .  b e c a u s e t h e m u t a t e d exon t h a t w o u l d n o r m a l l y embryogenesis embryos.  h a s become  We a t t e m p t e d ,  be s p l i c e d o u t d u r i n g  the default expression  o f unc-52,  eliminate  MEC-8  transcripts. if  we  necessity  for  help  i n these  define  the  between  t o s e e i f we  processing  A secondary reason f o r these  could  pattern  through t h e use o f double mutants  mec-8 a n d t h e d e l e t i o n r e v e r t a n t s the  This i s  could  of  unc-52  e x p e r i m e n t s was t o s e e  target  region  within  unc-52  t r a n s c r i p t s t h a t MEC-8 a c t s upon. Our  reasoning  interacts  with  removing  this  requirement  i n these  t h e e x o n 15 t h r o u g h region  structure  indeed  the processing  independent that  a n d movement  low  includes  this  This  region  phenotype  (Table  all  three  target  o f unc-52, unc-52 three  4) .  (Figure  This  11) .  the only  only  then by from  any  mec-8;  unc-  as w e l l  I t also  e t al, 1986; L u n d q u i s t has been  a n d Herman,  1994).  Mec p h e n o t y p e s  indicating  t r a n s c r i p t s besides  double mutants a r e s t i l l  unc-52.  weakly  that  defects  and  (Chalfie  and  hypodermis mutants  1994). i n the  and c u t i c l e  still  MEC-8 must  exhibit  have  other  since  these  f o r the cold-sensitive  l e t h a l p h e n o t y p e , MEC-8 must i n t e r a c t w i t h o t h e r 62  i s complex  defects  In particular,  penetrant  unc-52  a n d Herman,  correlated with  The d o u b l e  further  portion of  arrest  that  examples i s  as chemosensory  embryonic  type  indicates  The p h e n o t y p e o f mec-8 m u t a n t s  attachment o f body w a l l muscle t o a d j a c e n t (Lundquist  i f MEC-8  t r a n s c r i p t s i n these  i s probably  cold-sensitive  1981; P e r k i n s  latter  that  liberate  constructed  function  mechanosensory  penetrance  Sulston,  o f unc-52  o f mec-8  t h a t MEC-8 r e g u l a t e s . and  19 r e g i o n  should We  was  mutants t h a t a r e v i a b l e and have w i l d  muscle  confirms  we  f o r MEC-8.  52 (deletion-revertant)  now  experiments  t a r g e t genes  that  affect of  muscle attachment.  t h e Mec-8;  revertants  Unc-52  Suppressor synthetic  unc-52 p a r a l y z e d  Not  a l l double  viable;  lethal 4) .  mutants  While  synthetic (ra507)  we  I g formed  we  accumulate release  high  (Figure  Each  why  lead  trafficking  Synthetic  lethal  protein  to their  mec-8 b u t  comm.). revertants  i sa  (ra38)  synthetic  mec-8;  within  altered  or  form  altered  unc-52(ra38)  the c e l l ,  with  accumulation  within  These  b u t do n o t  three  proper  with  embryos  (G. M u l l e n  Igs contains  unpub.  cysteines  I g f o l d i n g and  the  intracellular  observations  imply  t o o l t o determine whether chimeric  are f u n c t i o n a l a t t h e p r o t e i n  (Table  may n o t be c o m p a t i b l e  t o t h e ECM  of the c e l l .  mec-8 may b e a u s e f u l  with  has been  two c o n s t r u c t s  the chimeric  o f UNC-52  o f these  pathway  lethal  these  9) w h i c h may b e i n c o m p a t i b l e  therefore  IV.  suspect  levels  found  comm.) a s i s mec-8; unc-52(ra507)  any d e t e c t a b l e  results).  unc-52  a new l o c u s  double mutant  i n these revertants  function.  have  b e t w e e n mec-8 a n d unc-52  are not certain  lethals,  f o r rescue  ( s e e Smu m u t a n t s i n  (Bob Herman, p e r s .  t h e mec-8; unc-52(ra38)  (B. Herman, p e r s .  unc-52  loci  More r e c e n t l y ,  mutants  looking  phenotype  t h a t when m u t a t e d forms a s y n t h e t i c  not w i t h  are  lethal  w h i c h map t o two i n d e p e n d e n t  L u n d q u i s t a n d Herman, 1994) . defined  screens  that  exons o f  level.  Summary A common theme among many e x t r a c e l l u l a r p r o t e i n s ,  perlecan, finite  laminins, number  polypeptides.  and f i b r o n e c t i n  of  modular  Over h a l f  extracellular proteins (Bairoch,  1995).  A  domains  including  i s the incorporation into  large  of  a  multi-domain  o f t h e 40 modules w h i c h a r e t y p i c a l f o r  have so f a r been d e t e c t e d series  of repeating  63  units  i n mammalian BMs based  on t h e  Ig-  superfamily motif molecules.  i s one s u c h module p o p u l a r w i t h i n  I t has been s u g g e s t e d t h a t s t r i n g s  important f o r homophilic heterophilic spacing  binding  of globular  extracellular  of multiple  (Ranheim e t al,  interactions (Brummerdorf  and  domains a l t h o u g h  Rathjen,  Igs are  1996) a n d / o r 1996)  and/or  t h e r e does n o t a p p e a r t o be  any d i r e c t e v i d e n c e i n t h e l i t e r a t u r e s u p p o r t i n g t h i s c o n c e p t . h a s b e e n shown t h a t are  the a l t e r n a t i v e l y  n o t important f o r any o f these Alternative-splicing  different  isoforms  splicing  stress  diversity  is  reasonable  Agrin of  that to  key  Most  functional  each  example  mechanism  isoform  should  comes  from  i s a heparan s u l f a t e receptors  a  work  of  alternative  this  structural  It  proteoglycan  t h e ECM that  muscle  and n e u r a l - s p e c i f i c  1991) .  Studies  have  1995) .  We have  (Stone  short  but  t h i s does n o t a p p e a r t o be t r u e f o r t h e v a r i a n t s At this point  o f UNC-52 does  we a r e l e f t  have  with  for the alternative-splicing within  unc-52  a complex r e g u l a t o r y  i s clearly  limited  et  isoforms  al, have  and N i k o l i c s ,  functional  implications, within  no s a t i s f a c t o r y  interactions  interaction.  explanation  have  further  MEC-8 r e g u l a t i o n  t o t h e e x o n 15 t o 19 i n t e r v a l  64  domain  domain I V .  My e x p e r i m e n t s on mec-8 a n d unc-52 elucidated  s p l i c i n g and  the d i f f e r e n t i a l d i s t r i b u t i o n of long  and  IV.  agrin.  aggregation  (Hamshere  different  s p e c i f i c i t i e s and t a r g e t s  isoforms  An  protein,  induces  isoforms  demonstrated that  shown t h a t  regulated  a t t h e neuromuscular synapse.  includes  binding  seem  function.  This aggregating a c t i v i t y i s modulated by a l t e r n a t i v e  different  does  through  distinct  on  (AChRs)  generating  diversity.  generated  have  for  reviews  the purpose f o r generating  yield  that  acetylcholine  a  exons o f domain I V  functions.  of a protein.  alternative-splicing excellent  is  spliced  It  of  a n d one  could lies  i n t e r p r e t my r e s u l t s between  earlier  exon  18 a n d 19.  speculations  be n e c e s s a r y  t o a r g u e t h a t t h e MEC-8 b i n d i n g  (Lundqist  While  this  i s i n agreement  e t al, 1996) o t h e r  to confirm this idea.  u s e f u l reagents This  interactions  has  UNC-52  function. 'glue'  validity,  then  important  f o r this  molecular  helped  to  further  using  c o u l d be  the  Particularly  molecular u s e f u l has  o f t h e e s s e n t i a l i m p o r t a n c e o f domain I V t o  adhering my  will  (A. D a v i e s a n d B.  resolve  locus.  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F o r completeness sake, t h e f o l l o w i n g p r i m e r s f r o m t h e Moerman L a b p r i m e r s t o c k s ( s e q u e n c e a v a i l a b l e ) should be t r i e d a g a i n o n a m p l i f i e d DNA f r o m t h e r e s p e c t i v e strains: s t 5 4 6 - p27 a n d p28 o n l y a s a l l o t h e r r e g i o n s h a v e b e e n s a t u r a t e d , s t 5 7 8 - e x o n 14 s h o u l d b e r e - s e q u e n c e d . If n o t i n these regions, the mutations most likely occur d o w n s t r e a m o f t h e a l t e r n a t i v e l y - s p l i c e d e x o n s , p o s s i b l y e x o n 19 o r e v e n more 3' i n domain V. I n a d d i t i o n , t h e m u t a t i o n f o r unc-52(st572) was n o t d e f i n e d . I t e x h i b i t s a u n i q u e i m m u n o s t a i n i n g p a t t e r n compared t o t h e above m e n t i o n e d m u t a n t s . U s i n g GMl, t h e r e i s o n l y f a i n t s t a i n i n g o f t h e BM o v e r t h e p h a r y n x s u g g e s t i n g t h a t t h e m u t a t i o n may b e i n e x o n 10 s i n c e t h i s e x o n i s common t o a l l UNC-52 i s o f o r m s . No a l t e r a t i o n s w e r e f o u n d i n e x o n 10 o r t h e 3' e n d o f e x o n 9. Because t h i s m u t a n t may b e a r e p r e s e n t a t i o n o f a missense mutation and t h e r e f o r e o c c u r r i n g anywhere i n t h e amino t e r m i n a l r e g i o n , f u r t h e r s e q u e n c i n g 5' o f e x o n 9/10 c o u l d i s o l a t e i t . A l s o , t h e o t h e r p o t e n t i a l n u l l m u t a t i o n , unc-52(ra401), was p r e d i c t e d t o b e i n t h e i n t e r v a l b e t w e e n exons 6-9 ( s i n c e t h e r e i s no UNC-52 i m m u n o r e a c t i v i t y i n t h i s mutant c o n s i s t e n t w i t h s t 5 4 9 s t a i n i n g w h i c h i s i n e x o n 7) . The f o l l o w i n g n u c l e o t i d e s e q u e n c e s a r o u n d e x o n 6 a n d 7 do n o t c o n t a i n a l t e r a t i o n s 4570-4692, 4 9 1 0 5000, 5040-5178, 5570-5725.  71  

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