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Cloning fibrillarin and snoRNA genes from Sulfolobus acidocaldarius Wang, Xin 1996

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C L O N I N G FIBRILLARIN FROM  AND SNORNA  GENES  SULFOLOBUS ACIDOCALDARIUS  by  XIN  WANG  B . S c . N a n k a i University Tianjin, P . R . C h i n a ,  1994  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F THE REQUIREMENTS FOR THE DEGREE  OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE DEPARTMENT OF  STUDIES  BIOCHEMISTRY  AND MOLECULAR  BIOLOGY  W e a c c e p t this t h e s i s a s c o n f o r m i n g to t h e r e q u i r e d  standard  T H E UNIVERSITY O F BRITISH C O L U M B I A August  1996  © Xin W a n g ,  1996  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be granted her  for  It  is  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  11  Abstract  Sulfolobus acidocaldarius  Recently, a  R N A - p r o t e i n c o m p l e x w i t h r R N A p r e c u r s o r 5'  l e a d e r p r o c e s s i n g activity w a s purified a n d U 3 s n o R N A w a s f o u n d to b e a n e s s e n t i a l c o m p o n e n t ( P o t t e r et a l . , 1 9 9 5 ) . S i n c e fibrillarin is a u n i v e r s a l s n o R N A b i n d i n g p r o t e i n and  has  important  f u n c t i o n s in r i b o s o m e s y n t h e s i s , t h i s p r o c e s s i n g c o m p l e x  s u s p e c t e d to c o n t a i n fibrillarin. Fibrillarin g e n e of  Sulfolobus  p r o b e to a t t e m p t to isolate kb cross-hybridizing S. isolated.  Database  acidocaldarius  M. voltae  was  was u s e d a s a heterologous  fibrillarin g e n e . B y s o u t h e r n h y b r i d i z a t i o n s , a  D N A fragment w a s identified a n d s u b s e q u e n t l y  s e a r c h e s indicated  that  it w a s  not  the  fibrillarin  gene.  Some  h o m o l o g y w a s f o u n d b e t w e e n t h i s D N A f r a g m e n t a n d t h e m e t h a n o g e n fibrillarin p r o b e w h i c h m a y a c c o u n t for t h e s o u t h e r n s i g n a l . S i n c e this c o n t a i n s a 1.1  products  templates, Because  at  the  negative  the  kb D N A  fragment  p r i m e r s w e r e a l s o c o n d u c t e d to c l o n e this g e n e .  expected  lengths  were  results were obtained  degenerate  primer  obtained  using S.  using  t h i s g e n e . In o r d e r t o d e t e c t t h e  sequences were based on  protein  was  Sulfolobus  overexpressed  a n t i b o d i e s . In a w e s t e r n  in  M.  acidocaldarius  fibrillarin s e q u e n c e s , t h e n e g a t i v e P C R r e s u l t s u g g e s t e d that  fibrillarin  1.7  gene  k b o p e n r e a d i n g f r a m e , its s e q u e n c e w a s d e p o s i t e d i n t o g e n b a n k . P C R  reactions using degenerate PCR  1.7  voltae  and  as  DNA.  universally c o n s e r v e d  Sulfolobus  u s e d for  b l o t , t h i s a n t i b o d y c a n d e t e c t fibrillarin  l y s a t e s , b u t n o p r o t e i n t h e s i z e of fibrillarin c a n b e d e t e c t e d in  DNA  genomic  m a y not h a v e  fibrillarin p r o t e i n d i r e c t l y , t h e  E. coli  While  methanogen  raising  polyclonal  in m e t h a n o g e n  Sulfolobus  cell  extracts. T h i s  a n t i b o d y c a n a l s o c r o s s r e a c t with a p r o t e i n in a y e a s t c e l l l y s a t e w h o s e s i z e is s i m i l a r to that  anticipated  s u g g e s t that  for y e a s t  Sulfolobus  fibrilllarin  (NOP1).  Taken  together,  these  observations  l a c k s a c l o s e fibrillarin h o m o l o g u e . S i n c e fibrillarin  antibodies  c a n c o i m m u n o p r e c i p i t a t e t h e s n o R N A s a s s o c i a t e d with f i b r i l l a r i n , t h e y a r e a  powerful  tool  use  in s t u d y i n g e u k a r y o t i c  snoRNAs.  Experiments have  been  initiated  to  the  Ill  m e t h a n o g e n fibrillarin a n t i b o d y for i m m u n o p r e c i p i t a t i o n s of p o t e n t i a l s n o R N P s f r o m t h e m e t h a n o g e n cell extract.  R e v e r s e t r a n s c r i p t i o n c o u p l e d P C R w a s u s e d t o c l o n e o t h e r s n o R N A s in a d d i t i o n to  U3 t h a t obtained  a r e p o s s i b l y p r e s e n t in t h e  Sulfolobus  purified extract. F o u r c l o n e d c D N A s w e r e  but s e q u e n c e a n a l y s i s d i d not r e v e a l h o m o l o g y to a n y  s n o R N A . T h e y a r e p o s s i b l y R N A d e g r a d a t i o n p r o d u c t s . Interestingly, s h a r e s a n e i g h t n u c l e o t i d e s e q u e n c e that is i d e n t i c a l to t h e S . s e q u e n c e , a s e q u e n c e p r e s e n t in m o s t of t h e s n o R N A s .  known  eukaryotic  o n e of t h e c l o n e s  acidocaldarius  U3 b o x  C  TABLE OF CONTENTS  ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST O F T A B L E S  v  LIST O F F I G U R E S  vi  ABBREVIATIONS  vii  ACKNOWLEDGMENTS  ix  INTRODUCTION  1  1.1 A r c h a e a a n d Sulfolobus acidocaldarius  1  1.2 T h e r i b o s o m e a n d r R N A o p e r o n o r g a n i z a t i o n  4  1.3 T h e i m p l i c a t i o n s of R N A s in s o m e c a t a l y t i c p r o c e s s e s in t h e c e l l . .  6  1.4 r R N A p r o c e s s i n g a n d s n o R N P s 1.4.1  8  Eubacterial r R N A processing  8  1.4.2 A r c h a e a l r R N A p r o c e s s i n g  9  1.4.3  11  Eukaryotic r R N A processing a n d s n o R N P s  1.5 B a c k g r o u n d w o r k o n t h e U 3 - m e d i a t e d p r o c e s s i n g of pre-rRNA  5' E T S r e g i o n in  Sulfolobus acidocaldarius  1.6 T h e o b j e c t i v e of this project  21 22  MATERIALS AND METHODS  23  2.1 M a t e r i a l s  23  2 . 2 D e s c r i p t i o n of B a c t e r i a l s t r a i n s , p l a s m i d s a n d o l i g o n u c l e o t i d e s  24  2.3 M e d i a a n d Culture conditions  24  2.4 G e n e r a l Molecular Biology T e c h n i q u e s  27  2.5 S o u t h e r n hybridizations  31  2.6 D N A s e q u e n c i n g  32  2.7 P C R procedures  33  2 . 8 E x p r e s s i o n of t h e M e t h a n o g e n fibrillarin g e n e in  E. coli.  2.9 Protein electrophoresis a n d staining p r o c e d u r e s  35 35  2.10 Immunoanalysis procedures  36  2.11  39  Molecular s e q u e n c e s a n d alignments  RESULTS  40  (I) S E C T I O N O N E : E x p e r i m e n t s to c l o n e t h e S .  acidocaldarius  fibrillarin g e n e (i) D i r e c t s h o t g u n c l o n i n g f r o m t h e (ii) D e g e n e r a t e P C R a m p l i f i c a t i o n  40  Sulfolobus g e n o m e of t h e Sulfolobus  fibrillarin g e n e f r a g m e n t s (iii) R a i s i n g p o l y c l o n a l a n t i b o d i e s f o r m e t h a n o g e n  50 fibrillarin  to d e t e c t s i m i l a r s i z e d p r o t e i n in Sulfolobus e x t r a c t (II)  S E C T I O N T W O : R T - P C R cloning s n o R N A s from S .  43  53  acidocaldarius  e x t r a c t c o n t a i n i n g p r e - r R N A 5' E T S p r o c e s s i n g activity  61  DISCUSSION  65  SUMMARY AND PERSPECTIVES  69  BIBLIOGRAPHY  71  APPENDIX I  83  V  LIST O F T A B L E S T a b l e 1.1  M e t a z o a n small nucleolar R N A s  T a b l e 1.2  N u c l e o l a r proteins  T a b l e 2.1  E. coli  T a b l e 2.2  oligonucleotide s e q u e n c e s  26  T a b l e 3.1  C o m b i n a t i o n s of P C R p r i m e r s a n d t h e e x p e c t e d p r o d u c t s i z e s  51  s t r a i n s u s e d for c l o n i n g a n d p r o t e i n o v e r e x p r e s s i o n  14 20 25  LIST O F F I G U R E S F i g u r e 1.1  R o o t e d universal phylogenetic tree  3  F i g u r e 1.2  C o m p a r i s o n s of r R N A o p e r o n o r g a n i z a t i o n s  F i g u r e 1.3  A c o m p a r i s o n of e u b a c t e r i a l a n d  5  archaebacterial  r R N A p r o c e s s i n g recognition sites  10  F i g u r e 1.4  O r g a n i z a t i o n a n d p r o c e s s i n g s i t e s of e u k a r y o t i c p r e - r R N A  11  F i g u r e 3.1  M u l t i p l e a l i g n m e n t of t h e p r o t e i n s e q u e n c e s of all c l o n e d fibrillarin  F i g u r e 3.2  41  G e n o m i c s o u t h e r n h y b r i d i z a t i o n to  identify  S u l f o l o b u s fibrillarin g e n e  44  F i g u r e 3.3  S o u t h e r n h y b r i d i z a t i o n to v e r i f y t h e p o s i t i v e c l o n e s o b t a i n e d . . .  46  F i g u r e 3.4  S e q u e n c i n g s t r a t e g y of o n e s t r a n d of t h e 1.7 k b D N A f r a g m e n t . . 4 7  F i g u r e 3.5  S e q u e n c e of t h e 1.7 k b D N A c l o n e a n d t h e  translation  of its c o n t a i n e d orf  48  F i g u r e 3.6  A degenerate P C R Gel  52  F i g u r e 3.7  I n d u c t i o n of e x p r e s s i o n b y I P T G a n d t h e p r e s e n c e of r e c o m b i n a n t p r o t e i n in S - 3 0 f r a c t i o n  F i g u r e 3.8  W e s t e r n blot u s i n g a n t i b o d i e s r a i s e d  56 against  m e t h a n o g e n fibrillarin  57  F i g u r e 3.9  W e s t e r n blot to d e t e c t y e a s t fibrillarin  Figure 3.10  W e s t e r n d e t e c t i o n of p r o t e i n s from  M. voltae cell  lysate  60  immunoprecipitated 62  Abbreviations A  Adenosine  A  a b s o r b a n c e at 6 0 0 n m  bp  base  BSA  Bovine serum  C  cytosine  cDNA  DNA  ddNTP  dideoxyribonucleoside  DEPC  diethyl  DNA  Deoxyribonucleic  dNTP  deoxyribonucleoside  DTT  Dithiothreitol  EDTA  Ethylenediamine  ETS  external transcribed  G  Guanosine  IPTG  Isopropyl-p-D-thiogalactopyranoside  ITS  internal t r a n s c r i b e d  kb  kilobase  kDa  kilodalton  mRNA  messenger R N A  NTP  ribonucleoside  orf  open reading  PAGE  polyacrylamide  PBS  0 . 0 2 M p o t a s s i u m p h o s p h a t e , p H 7.4; 0 . 1 5 5  "600  pair albumin  reverse transcribed from R N A triphosphate  pyrocarbonate acid triphosphate  tetraacetic acid spacer  spacer  triphosphate  frame gel electrophoresis NaCl  PCR  polymerase chain  PMSF  phenylmethyl  RACE  r a p i d a m p l i f i c a t i o n of c D N A e n d s  RNA  ribonucleic acid  RNase  ribonuclease  rRNA  ribosomal  S  S v e d b e r g unit of s e d i m e n t a t i o n  SDS  sodium dodecyl sulphate  snoRNA  small nucleolar  snoRNP  small nucleolar ribonucleoprotein  T  reaction  sulfonyl fluoride  RNA coefficient  RNA  Thymidine  TBE  9 0 m M T r i s - b o r a t e , p H 8.3; 2 m M  TE  1 0 m M T r i s - H C I , p H 8.0; 1 m M  tris  tris ( h y d r o x y m e t h y l )  tRNA  transfer  U V  particle  Uridine volt(s)  RNA  EDTA  EDTA  aminomethane  ix  Acknowledgments  F i r s t , I t h a n k D r . P a t D e n n i s , m y first m e n t o r for s c i e n t i f i c r e s e a r c h , f o r g i v i n g m e t h e o p p o r t u n i t y to w o r k in h i s l a b , h i s g u i d a n c e a n d k i n d n e s s t h r o u g h o u t tediously  proof-reading  intelligence  my  of m y c o m m i t t e e  thesis.  I  also  members,  drew  heavily  Drs. G e o r g e  on  Mackie  the  and  m y study,  and  generosity  Caroline  and  A s t e l l , to  w h o m I a m v e r y grateful. I a l s o w o u l d like to a c k n o w l e d g e t h e h e l p a n d f r i e n d s h i p f r o m all m y f e l l o w in D e n n i s ' always  l a b : S i m o n for g u i d a n c e  just after I s t a r t e d  k e e p s p e o p l e a r o u n d him h a p p y ; Deidre  and  his British h u m o r  for t e a c h i n g  and  in  my  thesis  and  also  cakes; Jorg  and  Peter  for  which  later h e l p i n g  w i t h t e c h n i c a l stuff, t e a c h i n g m e E n g l i s h , " T h a n k y o u " in m a n y l a n g u a g e s , English  workers  sharing  me  correcting  with  me  their  e x p e r t i s e in m o l e c u l a r b i o l o g y ; M a r i o f o r critically r e a d i n g m y t h e s i s a n d c o n s u l t i n g in u s i n g c o m p u t e r p r o g r a m a n d molecular evolution; a n d fellow g r a d u a t e  students  Tony  a n d A r i n a for inspiring d i s c u s s i o n s . I f e e l l u c k y for m y s e l f for b e i n g a b l e to s t u d y in t h i s w o n d e r f u l the  help  r e c e i v e d from m a n y  people  department,  outside our lab, e s p e c i a l l y G l e n  and  I thank Ken  for  g i v i n g m e a h a n d in p r o t e i n e x p r e s s i o n a n d r a i s i n g a n t i b o d i e s . T h i s t h e s i s is d e d i c a t e d to m y p a r e n t s Mr. G o n g s h u W a n g  and  Mrs. Huijuan  T h e i r u n c o n d i t i o n e d l o v e is t o o m u c h to c o u n t a n d I c a n n e v e r c o m p e n s a t e .  Du.  1 I.  Introduction  R i b o s o m a l R N A p r o c e s s i n g is a v e r y c o m p l i c a t e d c e l l u l a r p r o c e s s . E u b a c t e r i a l a n d a r c h a e a l p r e - r R N A transcripts c a n form long helical structures bracketing small  and  l a r g e s u b u n i t r R N A s . E n d o n u c l e a s e c l e a v a g e s within t h e s e p r o c e s s i n g h e l i c e s r e l e a s e p r e - r R N A subunits. S u b s e q u e n t trimming g e n e r a t e s the m a t u r e r R N A e n d s . N o s u c h h e l i c a l s t r u c t u r e c a n b e f o r m e d in e u k a r y o t i c p r e - r R N A t r a n s c r i p t s . I n s t e a d , t h e processing  machinery  uses  unique  small  nucleolar  ribonucleoprotein  rRNA  particles  ( s n o R N P s ) , c o n s i s t i n g of s m a l l n u c l e o l a r R N A s ( s n o R N A ) a n d a s s o c i a t e d p r o t e i n s . Fibrillarin  is a m a j o r  protein  c o m p o n e n t of e u k a r y o t i c  s n o R N P s . It is e s s e n t i a l  r i b o s o m e b i o g e n e s i s in y e a s t a n d p r o b a b l y in o t h e r e u k a r y o t i c o r g a n i s m s a s  Sulfolobus acidocaldarius  is  a  hyperthermophilic  archaeon.  In  the  for  well.  process  of  c h a r a c t e r i z i n g its p r e - r R N A p r o c e s s i n g within t h e 5' e x t e r n a l t r a n s c r i b e d s p a c e r ( E T S ) r e g i o n , a U 3 s n o R N A w a s i d e n t i f i e d a n d s h o w n to b e r e q u i r e d for p r o c e s s i n g . T h e w o r k presented  in t h i s  thesis  is m a i n l y  an  attempt  to  identify  a  fibrillarin  gene  in  S.  acidocaldarius.  1.1  Archaea and Sulfolobus acidocaldarius  1.1.1  Archaea as the third life lineage  Molecular  phylogenies  are  based  on  comparisons  of  gene  sequences  and  m o l e c u l a r p a t h w a y s a m o n g d i f f e r e n t o r g a n i s m s . O n e m a j o r c o n c l u s i o n of  molecular  phylogenetic  molecular  sequences eubacteria  studies  is t h e and  based  definition  eukaryotes.  on  small  subunit  of a r c h a e a  as  (Fox  1980;  et al.,  a  rRNA  as  well  monophyletic Woese  et al.,  as  other  group,  separate  1990). W h a t  are  from the  2 relationships  between  the  three  primary  domains?  At  the  cellular  and  genome  o r g a n i z a t i o n l e v e l , a r c h a e a r e s e m b l e e u b a c t e r i a m o r e t h a n e u k a r y o t e s ( K e e l i n g et 1994).  However,  at t h e  gene  sequence and  regulation  level, a r c h a e a  f e a t u r e s in c o m m o n with e u k a r y o t e s t h a n with e u b a c t e r i a ( D e n n i s , 1 9 8 6 ; et  al.,  1994). T h e s e features  sequences,  transcription  include similarities  mechanisms, and  in r R N A s a n d  certain  regulatory  have  more  and Keeling  ribosomal  pathways.  protein  Archaeal  p r o m o t e r s c o n t a i n a T A rich e l e m e n t r e s e m b l i n g t h e e u k a r y o t i c T A T A b o x ( R e i t e r 1988,  1 9 9 0 ) . A r c h a e a l R N A p o l y m e r a s e is i m m u n o l o g i c a l l y s i m i l a r to e u k a r y o t i c  p o l y m e r a s e (Puhler  et al.,  1989)  a n d e u k a r y o t i c t r a n s c r i p t i o n f a c t o r s T F IID  h a v e a l s o b e e n f o u n d in a r c h a e a ( C r e t i et  al.,  era/.,  1993; M a r s h  et al.,  era/., RNA  and  MB  1994; a n d R o w l a n d s  1994). S o m e characteristic eukaryotic regulatory p a t h w a y s  a r c h a e a , including ubiquitin-conjugated protein d e g r a d a t i o n (Wolf  al.,  a r e a l s o f o u n d in  era/.,  a n d B a u m e i s t e r , 1993) a n d s n o R N A m e d i a t e d r R N A p r o c e s s i n g (Potter  1991;  era/.,  Wenzel 1995).  M o l e c u l a r phylogenetic studies h a v e rooted the universal tree, a n d d e t e r m i n e d  the  evolutionary relationships a m o n g three d o m a i n s . A r c h a e a a p p e a r a s a n early branch o n t h e l i n e a g e l e a d i n g to e u k a r y o t e s . T h e root of t h e t r e e h a s b e e n p r e d i c t e d t h e e u b a c t e r i a a n d a r c h a e a - e u k a r y a a s d e p i c t e d in f i g u r e  1.1.  between  3 Bacteria  Eukarya  Archaea Green non-sulfur bacteria  Euryarchaeota Methanosarcina „„ . Entamoeb  Slime  Animals  Cyanobacteria Flavobacteria Flagellates Thermotogales  Trichomonads M i c r osp or i d i a Diplomonads  Figure 1.1  R o o t e d universal phylogenetic tree ( a d a p t e d from W o e s e a n d P a c e , 1993).  T h e b r a n c h o r d e r a n d b r a n c h length a r e b a s e d o n r R N A s e q u e n c e c o m p a r i s o n s . T h e p o s i t i o n of t h e root w a s d e t e r m i n e d b y c o m p a r i n g the s e q u e n c e s of p a i r s of p a r a l o g o u s g e n e s i n c l u d i n g the t R N A s y n t h e t a s e s (Brown a n d Doolittle, E F - T u a n d E F - G ( I w a b e et al., al.,  1993)  1995), elongation  factors  a n d t h e a a n d p s u b u n i t s of A T P a s e ( G o g a r t e n  et  1989).  1.1.2  Phylogeny within archaea  Archaea  are  divided  into  the  Crenarchaeota  and  the  Euryarchaeota.  E u r y a r c h a e o t a i n c l u d e t w o g r o u p s : h a l o p h i l e s t h a t g r o w o p t i m a l l y at 2 . 5 t o 5 M (nearly ten anaerobes  times and  the  require  salinity highly  generally hyperthermophilic  of s e a  water) a n d  r e d u c i n g c o n d i t i o n s for g r o w t h .  a r c h a e a and include  metabolizing,  hyperthermophilic  acidocaldarius  and  methanogens  t h e o r g a n i s m s s t u d i e d in t h i s t h e s i s .  voltae,  optimally  a representative  at  obligate  Crenarchaeota  Sulfolobus acidocaldarius,  organism growing  Methanococcus  that a r e  NaCl  pH 4  are  a sulfur-  and 80°C.  of the m e t h a n o g e n s ,  S. are  4  1.2  The  The ribosome and rRNA operon organization  ribosome  polymerizes  amino  is a  complexed  acids  into  ribonucleoprotein  proteins  along  an  structure  mRNA  that  template.  aligns  The  and  bacterial  r i b o s o m e contains 16S, 2 3 S a n d 5 S r R N A s a n d approximately 50 ribosomal proteins. The  eukaryotic  ribosome  contains  18S,  5.8S,  28S  and  5S  rRNAs  and  about  80  r i b o s o m a l proteins (Wool e r a / . , 1995).  Ribosomal  RNA  cotranscribed. T h e  (rRNA)  genes  are  organized  halophiles and methanogens  exhibit  into an  operons  and  r R N A operon  are  structure  s i m i l a r t o e u b a c t e r i a : t h e o r d e r is 1 6 S , 2 3 S a n d 5 S f r o m 5' t o 3' a n d t h e r e is u s u a l l y a t R N A g e n e b e t w e e n t h e 1 6 S a n d 2 3 S r R N A s a n d o c c a s i o n a l l y a n o t h e r t R N A g e n e 3' to the 5 S  gene  ( G a r r e t t et al.,  1 9 9 1 ) . In C r e n a r c h a e o t a , t h e  c o t r a n s c r i b e d without intervening operon  contains  tandomly  16S  and 23S  rRNAs  or distal t R N A s or 5 S r R N A . T h e e u k a r y o t i c  positioned  18S,  5.8S  and  28S  rRNAs.  are  rRNA  Because  the  eukaryotic 5 . 8 S a n d 2 8 S r R N A s e v o l v e d from a n ancient 2 3 S r R N A fragmentation,  the  o r g a n i z a t i o n of r R N A o p e r o n s in e u k a r y o t e s a n d C r e n a r c h a e o t a a r e s i m i l a r . F i g u r e  1.2  s h o w s s o m e e x a m p l e s of r R N A o p e r o n o r g a n i z a t i o n in t h e p r i m a r y l i n e a g e s .  T h e 5' a n d 3' s e q u e n c e s f l a n k i n g a r c h a e a l a n d e u b a c t e r i a l s m a l l a n d l a r g e s u b u n i t r R N A s c a n form helices  contain  maturation.  l o n g h e l i c a l s t r u c t u r e s with t h e r R N A s in t h e t e r m i n a l the  p r o c e s s i n g sites  Eukaryotic  a d d i t i o n to t h e s e two  pre-rRNAs  primary  do  used  in t h e  not  contain  helices, archaeal  first  step  these  of  rRNA  long stem  r R N A flanking  potential to f o r m s p e c i f i c additional s t e m s . ( K j e m s a n d Garrett,  loop. T h e s e excision  structures.  sequences have  1990).  and In the  5 16SrRNA  IRMA* '"  23SrRNA  2  5SrRMA  E.coH rrnB operon  16SrRNA  tRNA  23SrRNA  5S r R N A  tRNA  C y s  — H.cudrubmm  Ala  16SrRNA  tRNA  23SrRNA  5 S rRNA  M-thermoaitioiropfucitm.  16SrRNA  23SrRNA  intron  5SrRNA  Dmobi&s  16S rRNA  23S rRNA  5 S rRNA  S.acidoccddcirius  18SrRNA  5.8S  25S  5 S rRNA  S.cemvisiae  Figure 1.2 C o m p a r i s o n of r R N A o p e r o n o r g a n i z a t i o n in E. coli( a e u b a c t e r i a , B r o s i u s et al., 1 9 8 1 ) , Halobacterium cutirubrum ( a h a l o p h i l e , H u i a n d D e n n i s , 1 9 8 4 ), Methanobacterium thermoautotrophicum ( a m e t h a n o g e n , O s t e r g a a r d e r a / . , 1 9 8 7 ), Desulfurococcus. mobilis ( a h y p e r t h e r m o p h i l e , L a r s e n et al., 1 9 8 6 ), Sulfolobus acidocaldarius ( a t h e r m o p h i l e , D u r o v i c a n d D e n n i s , 1 9 9 5 ) a n d Saccharomyces cerevisiae ( a e u k a r y o t e , L a f o n t a i n e a n d T o l l e r v e y , 1 9 9 5 ) .  6  1.3  The implications of RNAs in some catalytic processes in  the cell  A n u m b e r of n a t u r a l a n d s y n t h e t i c R N A m o l e c u l e s p o s s e s s c a t a l y t i c  activities  a n d c a n a c t a s e n z y m e s ( R i b o z y m e s ) . C a t a l y t i c R N A s w e r e first d i s c o v e r e d in t h e s e l f s p l i c i n g introns of rRNA  Tetrahymena  c o d i n g r e g i o n of  sequence  r R N A a n d in t h e M 1 c o m p o n e n t o f R N a s e P . T h e 2 6 S  Tetrahymena  thermophila  contains  a 413 bp  intervening  (IVS). T r a n s c r i p t s c o n t a i n i n g t h e I V S a n d p o r t i o n s of t w o a d j a c e n t  s e q u e n c e s (exons)  rRNA  undergo self-excision a n d correct exon-ligation. T h e excision a n d  l i g a t i o n a c t i v i t y is i n t r i n s i c t o t h e s t r u c t u r e of t h e R N A ( K r u g e r et al., 1 9 8 2 ) . R N a s e P is the e n z y m e r e s p o n s i b l e for the maturation  of t h e 5' t e r m i n i o f t R N A t r a n s c r i p t s . It h a s  b e e n s h o w n t h a t t h e R N A m o i e t y of R N A s e P ( M 1 R N A o f  subtilis)  E. coli  in t h e p r e s e n c e o f a h i g h c o n c e n t r a t i o n o f m a g n e s i u m  a n d P - R N A of  B.  ions h a s catalytic  a c t i v i t y ( G u e r r i e r - T a k a d a et al., 1 9 8 3 ) . T h e r e a c t i o n r a t e is e n h a n c e d s e v e n f o l d b y t h e p r e s e n c e of t h e R N a s e P p r o t e i n s u b u n i t ( G u e r r i e r - T a k a d a a n d A l t m a n , 1 9 8 4 ) .  E u k a r y o t i c m R N A s usually contain intervening s e q u e n c e s (IVS) or introns. T h e y a r e p r o c e s s e d b y t h e s p l i c e o s o m e , a large ribonucleoprotein m a c h i n e containing U 1 , U 2 , U 4 , U 5 , a n d U 6 s m a l l n u c l e a r R N A s ( s n R N A s ) a n d their a s s o c i a t e d p r o t e i n s . T h e m R N A intron s p l i c i n g p r o c e e d s v i a t h e s a m e t w o s t e p t r a n s e s t e r i f i c a t i o n m e c h a n i s m a s t h e g r o u p II s e l f - s p l i c i n g i n t r o n s ( C e c h , 1 9 9 3 ) . R e c e n t s t u d i e s s u g g e s t t h a t t h r o u g h t h e c o m p l e x interactions involving U 2 , U 5 a n d U 6 s n R N A s , t h e intron, e x o n , a n d t h e introne x o n j u n c t i o n s of t h e m R N A , a c o m p l e x s t r u c t u r e r e s e m b l i n g t h e s e l f - s p l i c i n g g r o u p II intron is f o r m e d ( L e s s e r a n d G u t h r i e , 1 9 9 3 ; S o n t h e i m e r a n d Steitz, 1 9 9 3 ; W i s e , 1993). S n R N A s U 2 , U 5 a n d U 6 s n R N A s c o m p r i s e t h e catalytic c o r e of t h e s p l i c e o s o m e a n d mRNA  s p l i c i n g a p p e a r s to b e largely a R N A - c a t a l y z e d reaction ( U m e n  a n d Guthrie,  1 9 9 6 ) . A n u m b e r o f s p l i c e o s o m e p r o t e i n s , c h a r a c t e r i z e d in y e a s t , a r e r e q u i r e d f o r t h e  7 second  step  important Prp8  is  reaction  (Umen  and  Guthrie,  1995).  Prp16,  Slu7  and  Prp8  are  all  f o r 3' s p l i c e s i t e s e l e c t i o n . P r p 1 6 is a n A T P d e p e n d e n t R N A h e l i c a s e a n d required  reactions. T h e  for  spliceosome assembly and  both  the  first  and  second  p r e c i s e f u n c t i o n s of t h e s e s p l i c e o s o m e p r o t e i n s a r e still n o t  step  clearly  defined.  T h e r e is n o w a l s o b i o c h e m i c a l a n d g e n e t i c e v i d e n c e t o s u p p o r t t h e i d e a that translation  is a l s o a n  d i s c o v e r e d that w h e n  R N A - c a t a l y z e d reaction the isolated 5 0 S  (reviewed  by  Noller,  1991).  r i b o s o m e paricles w e r e partially  r i b o s o m a l p r o t e i n s , peptidyl t r a n s f e r a s e activity w a s  still r e t a i n e d  It  was  s t r i p p e d of  (Staehelin  er  al.,  1 9 6 9 ) . O n l y f i v e o u t of 3 4 5 0 S r i b o s o m a l p r o t e i n s a r e r e q u i r e d f o r p e p t i d y l t r a n s f e r a s e a c t i v i t y w h e r e a s 2 3 S a n d 5 S r R N A s a r e e s s e n t i a l ( H a m p l et al.,  1981).  Furthermore,  t h e p e p t i d y l - t r a n s f e r a s e a c t i v i t y of t h e r i b o s o m e is r e s i s t a n t to s e v e r a l r o u n d s of p h e n o l e x t r a c t i o n s o r p r o t e i n a s e K d i g e s t i o n ( N o l l e r e r a / . , 1 9 9 2 ) . In c o n t r a s t , c l e a v a g e of rRNAs  b y either  colicin E 3 or kethoxal  b i n d i n g activity ( B o w m a n  abolishes completely the  16S  ribosomes' t R N A  e r a / . , 1971; Noller a n d C h a i r e s , 1972). t R N A footprinting or  c r o s s l i n k i n g s t u d i e s h a v e s h o w n that s m a l l s u b u n i t r R N A is i m p o r t a n t f o r t R N A b i n d i n g to t h e  r i b o s o m e s . Affinity-labeling  studies using a  reactive  group  c o u p l e d to  the  a m i n o a c y l e n d of t R N A s h o w that t h e l a r g e s u b u n i t r R N A is in t h e i m m e d i a t e v i c i n i t y of peptidyl-transferase center (reviewed by Noller, 1993b). R e c e n t l y , a G - C b a s e pairing between  t h e 3 ' C C A t e r m i n u s of t R N A a n d t h e 2 3 S  r R N A w a s d e m o n s t r a t e d to  r e q u i r e d f o r t R N A b i n d i n g to t h e r i b o s o m e a n d p r o p e r p e p t i d y l t r a n s f e r a s e (Samaha  et  al.,  1995).  r i b o s o m e ' s translation  All this  evidence  s u g g e s t s that  r R N A s are  be  function  important  for  activity.  T h e d i s c o v e r y of r i b o z y m e s l e d to t h e R N A w o r l d h y p o t h e s i s . In t h i s h y p o t h e s i s , c a t a l y t i c R N A s in m o d e r n c e l l s a r e t h e r e l i c s of a n c i e n t R N A e n z y m e s in t h e R N A w o r l d  8 w h e r e R N A s w e r e genetic information carriers a s well a s catalytic e n z y m e s 1984;  (Gilbert,  J o y c e a n d O r g e l , 1 9 9 3 ) . T o fulfill b o t h t h e s e r o l e s , R N A m u s t b e a b l e t o s e l f -  replicate. Indeed, complementary  s o m e modified R N A s can undergo template-dependent  o l i g o n u c l e o t i d e s r e s u l t i n g in s e l f - r e p l i c a t i o n  (Doudna  l i g a t i o n of  and  Szostak,  1989; D o u d n a e r a / . , 1991; a n d G r e e n a n d S z o s t a k , 1992).  1.4  Ribosomal RNA processing and small nucleolar ribonucleoprotein  particles  R i b o s o m a l R N A p r o c e s s i n g involves c l e a v a g e s a n d trimming  of a p r i m a r y  t r a n s c r i p t into i n d i v i d u a l m a t u r e r R N A s . T h e r R N A p r o c e s s i n g p a t h w a y s  rRNA  in t h e t h r e e  d o m a i n s a r e i n d i v i d u a l l y d i s c u s s e d in t h e f o l l o w i n g s e c t i o n s :  1.4.1  Eubacterial rRNA processing:  Ribosomal  RNA  processing  in  eubacteria  involves  primary  cleavages  e n d o n u c l e a s e s a n d s e c o n d a r y c l e a v a g e s b y e n d o - or e x o n u c l e a s e s ( r e v i e w e d  by by  S r i v a s t a v a a n d S c h l e s s i n g e r , 1 9 9 0 ; P a c e a n d B u r g i n , 1 9 9 0 ) . In t h e p r i m a r y c l e a v a g e s , R N a s e III  c u t s into b o t h s t r a n d s of t h e l o n g h e l i c a l s t r u c t u r e s b r a c k e t i n g t h e 1 6 S  and  2 3 S r R N A s in a s t a g g e r e d m a n n e r a n d e x c i s e s t h e i n d i v i d u a l r R N A p r e c u r s o r s f r o m t h e p o l y c i s t r o n i c r R N A t r a n s c r i p t . D e s p i t e i n t e n s i v e i n v e s t i g a t i o n , t h e s p e c i f i c i t y of R N a s e III,  w h i c h is c o d e d b y t h e r n c g e n e , is p o o r l y u n d e r s t o o d . Its R N A s u b s t r a t e s a l l a r e  a b l e to f o r m  a d o u b l e - s t r a n d e d r e g i o n in t h e  r a n g e of 2 0  to 4 0  b p s , o f t e n with  an  i n t e r n a l b u l g e c l o s e t o t h e R N A s e III c l e a v a g e s i t e ( G e g e n h e i m e r a n d A p i r i o n , 1 9 8 1 ) . T h e d o u b l e s t r a n d e d r e g i o n of t h e s u b s t r a t e R N A a l w a y s e x t e n d s 1 0 t o 1 4 n u c l e o t i d e s f r o m t h e c l e a v a g e s i t e to a 3 ' u n p a i r e d r e g i o n . T h i s s p a c i n g is i m p o r t a n t f o r c l e a v a g e site selection. A m o d e r a t e 26  nucleotide inverse d y a d c o n s e n s u s s e q u e n c e c a n  be  9 d r a w n f o r t h e R N A s e III c l e a v a g e s e q u e n c e s . T h e s u b s t r a t e R N A s a l w a y s h a v e 8 to nucleotides cleavages  identical can  nonetheless,  be  to  it ( K r i n k e a n d  detected  mature  16S  at  the  rRNA  Wulff,  RNase  can  form  1990).  III  In  an  E. coli  processing sites  (Gegeheimer  et  rnc' strain,  around  al.,  13 no  16S  rRNA;  23S  rRNA  1977).  m a t u r a t i o n f a i l s in t h e rnc" s t r a i n with s e v e r a l h u n d r e d a d d i t i o n a l n u c l e o t i d e s  remaining  u n c l e a v e d , b u t t h e m u t a n t 5 0 S r i b o s o m e s retain s o m e a c t i v i t y ( K i n g e r a / . , 1 9 8 4 ) . In t h e a b s e n c e of R N a s e III,  s o m e other e n d o n u l e a s e s s u c h a s R N a s e E a n d R N a s e P  l i k e l y t o c o n t r i b u t e to t h e p r i m a r y p r o c e s s i n g of p r e - r R N A . W h i l e t h e p r i m a r y c a t a l y z e d b y R N a s e III terminal maturases  are  cleavages  can occur on naked rRNA, subsequent processing by various  requires  the  rRNA  p r e c u r s o r to b e  a s s o c i a t e d with  ribosomal  proteins.  1.4.2  Archaeal rRNA processing:  A r c h a e a a l s o u s e a n e n d o n u c l e a s e t o c l e a v e in t h e h e l i c e s s u r r o u n d i n g 23S  rRNAs.  But  unlike  the  eubacterial  RNase  III,  the  archaeal  16S  and  endonuclease  r e c o g n i z e s a w e l l d e f i n e d s u b s t r a t e , t h e b u l g e - h e l i x - b u l g e m o t i f . T h i s m o t i f r e s i d e s in a double  stranded  region  and  opposite strands separated 1986;  G a r r e t t et  al.,  is c h a r a c t e r i z e d  by  two  three-nucleotide  by p r e c i s e l y four helical b a s e pairs ( C h a n t  1992). T h e  archaeal  endonuclease  bulges and  on  Dennis,  r e c o g n i z e s this motif  and  c l e a v e s w i t h i n t h e t w o b u l g e s . T h e b u l g e - h e l i x - b u l g e motif is a l s o p r e s e n t in t r a n s c r i p t s of  archaeal  introns  r e s p o n s i b l e for intron  containing removal  1 9 8 9 ) . A c o m p a r i s o n of t h e  rRNA  or  tRNA  a s well (Kjems  E. coli  R N a s e III  b u l g e - h e l i x - b u l g e motif is illustrated in F i g u r e  genes  and  a n d Garrett,  this 1988;  processing substrate  1.3.  endonuclease T h o m p s o n et a n d the  is al.,  archaeal  10 E. colirrnB  E. coli rrnB  (RNase III)  Hcu 16S  -16S-,  U-A G-C U-A U-A G-C | G-C  r  A-U  p  A-U C-G ^ ~ C-G \.AU«G C AG-C U-A G-C G«UU  V V.  \  G  _  II  X  G-C U -- A A U AC-G  G  A  Dmo 23S  G C A-U C-G C-G U-A A-U  -  h 7  C  C  G-C A  C  _  U A-UA"V G-C X  AV ^  G  G-C AG-C ^ A UG-C G-C G-C C-GA G  G  C-G U-A C-GA  U  _  G-C  G ^  C UC-G G-C C-G G«UU  A U-ACV U-A U-A G-C G-C  5' 3'  V  G-C  V  G-C C-G U-A U-A  HvOtRNA  „ „ ^  (RNase III)  i  Hcu 23S  frp  U-A  C-GAX  5rr ' i3i ' •  G»U /~i _ T T 5'3  5'3'  5'3'  Figure 1.3  A  recognition  sites  comparison (adapted  of  eubacterial  from  Dennis,  and 1991).  archaebacterial The  above  RNA  diagram  processing depicts  i n t r a m o l e c u l a r s e c o n d a r y s t r u c t u r e s a s s o c i a t e d with t h e e x c i s i o n of 1 6 S a n d 2 3 S  the  rRNA  E. coli a n d Halobacterium cutirubrum ( H c u ) , t h e t r p t R N A i n t r o n in Haloferax volcanii ( H v o ) a n d t h e 2 3 S r R N A intron in Desulfococcus mobilis ( D m o ) . T h e  precursors  in  arrows depict the c l e a v a g e sites.  B e s i d e t h e d i f f e r e n c e s in s u b s t r a t e s p e c i f i c i t y , t h e e n z y m o l o g y of t h e a r c h a e a l p r e rRNA  helix  processing enzyme  is a l s o d i f f e r e n t  from  eubacterial  RNase  a r c h a e a l e n d o n u c l e a s e c l e a v a g e p r o d u c e s a 5' h y d r o x y l a n d a 3 ' p h o s p h a t e eubacterial  RNase  III  cleavage  p r o d u c e s a 5' p h o s p h a t e  and  a 3'  III.  The  whereas  hydroxyl.  The  a r c h a e a l e n d o n u c l e a s e a n d e u b a c t e r i a l R N a s e III w e r e f o u n d t o b e s e n s i t i v e to p r o t e i n e x t r a c t i o n p r o c e d u r e s but r e s i s t a n t to R N a s e d i g e s t i o n , i m p l y i n g t h a t t h e a c t i v i t y is not R N A b a s e d (Kjems a n d Garrett,  1989).  11  1.4.3  Eukaryotic rRNA processing pathway and general mechanism  The  eukaryotic  rRNA  processing  pathway  is  different  from  counterparts; moreover, m a n y more r R N A processing intermediates (Gerbi  era/.,  1990,  Lafontaine  and Tollervey,  its  can be  1995). A s a eukaryotic  the  depicted  1.4.  Figure 1.4 et al.,  detected  example,  o r g a n i z a t i o n of y e a s t p r e - r R N A a n d t h e e n d o n u c l e o l y t i c p r o c e s s i n g s i t e s a r e in f i g u r e  bacterial  S t r u c t u r e of y e a s t p r e - r R N A a n d its p r o c e s s i n g s i t e s ( a c c o r d i n g t o v a n N u e s  1995;  Lafontaine a n d Tollervey, 1995). T r a n s c r i b e d s p a c e r s a r e r e p r e s e n t e d  by  lines a n d m a t u r e r R N A s a r e illustrated b y b a r s . E T S : e x t e r n a l t r a n s c r i b e d s p a c e r ; ITS: i n t e r n a l t r a n s c r i b e d s p a c e r , A - E : e n d o n u c l e o l y t i c p r o c e s s i n g s i t e s of p r e - r R N A .  5'ETS  18S  ITS1  5.8S  B 1 Eukaryotic eukaryotes,  rRNA  L  B 1  ITS2  25S  3'ETS  S  p r o c e s s i n g m e c h a n i s m s a r e just starting  to  be  r i b o s o m e s a r e s y n t h e s i z e d in t h e n u c l e o l u s , a c l u s t e r of  elucidated.  In  transcriptionally  a c t i v e r R N A g e n e s s u r r o u n d e d b y a c l o u d of d e n s e l y p a c k e d r R N A t r a n s c r i p t s in t h e f o r m of p r e r i b o s o m a l p a r t i c l e s at v a r i o u s s t a g e s of m a t u r a t i o n ( S c h e e r e r a / . , 1 9 9 3 ) . A g r o u p of s m a l l nucleolar  RNAs  nucleolar  ribonucleoprotein  (snoRNAs)  and  some  particles  nucleolar  p r o c e s s i n g a n d o t h e r s t e p s of r R N A m o d i f i c a t i o n  (snoRNPs),  proteins,  comprising  is i m p l i c a t e d  in  ( r e v i e w e d by B a s e r g a and  small rRNA Steitz,  1 9 9 3 ) . T h e r R N A p r o c e s s i n g m e c h a n i s m s a r e b e s t c h a r a c t e r i z e d in y e a s t ( r e v i e w e d b y Lafontaine  and Tollervey,  RNase  homologue,  III  SnoRNAs  U3, U14,  1995). After the transcription  RNT1,  c l e a v e s at  the  5'  of t h e  E T S site  p r e - r R N A , the  AO and  in t h e  3'  yeast ETS.  s n R 3 0 and s n o R N P proteins S o f l p , N o p l p , a n d G a r l p are  i n d i v i d u a l l y r e q u i r e d for c l e a v a g e s at t h e 5' E T S s i t e s A o , A i  a n d the ITS1  site  all A2,  12 c l e a v a g e s which e x c i s e the p r e - 1 8 S and  Fournier,  1995). T h e  nature  r R N A from the polycistronic transcript  of t h e i r  f u n c t i o n s in t h e s e  cleavages  (Maxwell  is still  not  u n d e r s t o o d . It h a s b e e n s u g g e s t e d that t h e s e s n o R N P s s i m u l t a n e o u s l y b i n d t h e  two  f l a n k i n g r e g i o n s of t h e r R N A s u b u n i t s , a n d b r i n g t h e t w o e n d s of r R N A in t h e p r e - r R N A t r a n s c r i p t t o g e t h e r to f o r m a s t e m - l i k e s t r u c t u r e t h a t is f u n c t i o n a l l y e q u i v a l e n t t o bacterial  helices  (Morrissey  and Tollervey,  1995;  Gerbi,  1995).  This  the  constrained  s t r u c t u r e m a y b e u n i v e r s a l l y r e q u i r e d f o r t h e p r o c e s s i n g of r R N A s u b u n i t s in t h e l o o p s . It h a s a l s o b e e n p r o p o s e d that s o m e s n o R N P s , like t h o s e i n v o l v e d in 1 8 S p r o c e s s i n g , can By  form a p r o c e s s i n g c o m p l e x called the p r o c e s s o m e (Fournier a n d M a x w e l l ,  1993).  e l e c t r o n m i c r o s c o p y , a 2 0 S r R N A p r o c e s s i n g c o m p l e x c o n t a i n i n g fibrillarin h a s b e e n  v i s u a l i z e d a s a t e r m i n a l b a l l - l i k e s t r u c t u r e b i n d i n g to t h e 5' E T S r e g i o n ( M o u g e y et 1993a).  It  is  p o s s i b l e that  after  cleavage  in  the  5'  E T S , this  complex  moves  d o w n s t r e a m to t h e 5' e n d of 1 8 S r R N A a n d p u l l s it into c l o s e p r o x i m i t y t o I T S 1 , forming a 1995).  small subunit r R N A  Molecular  l o o p a n d facilitating  d i s s e c t i o n of  the  yeast  requires  the  and  MRP  Potter  processome has  et al.,  1 9 9 5 b ) . T h e next  (Mitochondria  BNA  begun.  One  or m o r e u n k n o w n e n z y m e s  cleave  5'  ETS  Xenopus  cleavage  Erocessing)  p r o t e i n P o p 1 p ( S c h m i t t a n d C l a y t o n , 1 9 9 3 ; C h u et al.,  thus  subsequent processing (Gerbi,  c o m p l e x e s h a v e b e e n purified a n d partially c h a r a c t e r i z e d f r o m (Mougey era/., 1993b;  al.,  RNA  processing and archaea  at s i t e A 3 of I T S 1  and  in  its a s s o c i a t e d  1 9 9 4 ; a n d L y g e r o u et al.,  1994).  in t h e I T S 2 r e g i o n s i t e s C 1 a n d C 2 t o s e p a r a t e  5 . 8 S a n d 2 8 S r R N A . S u b s e q u e n t exonucleolytic trimming g e n e r a t e s the mature  termini  of i n d i v i d u a l  1p  r R N A s . S p e c i f i c a l l y in y e a s t , 5' e x o n u c l e a s e X r n  r e s p o n s i b l e for g e n e r a t i n g t h e 5' m a t u r e t e r m i n u s B1 the c l e a v e d s p a c e r R N A s (Henry  s  Rat  are  of 5 . 8 S r R N A a n d d e g r a d a t i o n of  et a / . , 1 9 9 4 ) . E x o n u c l e a s e R r p 4 p is n e c e s s a r y f o r  p r o c e s s i n g to t h e m a t u r e  3' e n d of 5 . 8 S  r R N A (Mitchell  u s e d for p r o c e s s i n g the  3' e n d s of 5 S  and 25S  uncharacterized  1p a n d  e n d o n u c l e a s e s c l e a v e at t h e  3'  et al.,  1996)  r R N A (Piper e n d s of  18S  et  al.,  while R n a 8 2 1983).  and 25S  is  Some  rRNAs  to  13 g e n e r a t e their final m a t u r e f o r m s .  SnoRNAs  c a n b e r e c o g n i z e d by s e v e r a l distinctive c h a r a c t e r i s t i c s (Maxwell  and  F o u r n i e r , 1 9 9 5 ) . T h e y a r e n u c l e o l a r l o c a t e d , s t a b l e R N A s p e c i e s w h i c h b y b i n d i n g to fibrillarin  and/or other  nucleolar proteins form  RNA-Protein  (RNP)  particles.  They  contain evolutionarily c o n s e r v e d s e q u e n c e elements (boxes) both a c r o s s s p e c i e s a n d a c r o s s different s n o R N A s . U 3 s n o R N A h a s b o x e s A , B , C , D a n d C ; m o s t of t h e  other  s n o R N A s h a v e b o x e s C a n d D w h i c h a r e t h e s e q u e n c e m o t i f s r e q u i r e d t o b i n d fibrillarin ( G e r b i et al.,  1 9 9 0 ) . A l s o , t h e s e c o n d a r y s t r u c t u r e s of s n o R N A s a r e c o n s e r v e d a m o n g  different o r g a n i s m s . S o m e s n o R N A s including U 3 , U 8 , a n d U 1 3 a r e t r a n s c r i b e d from their o w n p r o m o t e r s ( B a s e r g a a n d Steitz, 1993). After transcription, t h e y a r e  modified  at t h e 5' e n d t o a c q u i r e a 2 , 2 , 7 t r i m e t h y l g u a n o s i n e ( T M G ) c a p . O t h e r s n o R N A s a r e l o c a t e d in t h e  i n t r o n s o f c e r t a i n p r o t e i n - c o d i n g g e n e s a n d p o s s e s s a 5'  phosphate  g r o u p ( r e v i e w e d b y S o l l n e r - W e b b , 1993). T h e s e s n o R N A s * h o s t g e n e s u s u a l l y c o d e for nucleolar, ribosomal or other translation-related b y v i r t u e of t h e i r l o c a t i o n in t r a n s l a t i o n - r e l a t e d  proteins ( S o l l n e r - W e b b ,  1993). T h u s ,  p r o t e i n g e n e s , t h e p r o d u c t i o n of i n t r o n  s n o R N A s will b e c o o r d i n a t e d with r i b o s o m a l b i o g e n e s i s ( B a c h e l l e r i e e r a / . , 1 9 9 5 a ) . T h e c / s - a c t i n g s i g n a l s f o r s n o R N A s to b e p r o c e s s e d f r o m i n t r o n s a r e c o n t a i n e d w i t h i n t h e mature U24  s n o R N A s e q u e n c e . T h i s m a y explain how s n o R N A s s u c h a s U 2 0 ,  c h a n g e d h o s t g e n e s d u r i n g e v o l u t i o n ( B a c h e l l e r i e et al.,  1995b).  U21  and  Interestingly,  s i m i l a r t r a n - a c t i n g f a c t o r s a s t h e p r e - r R N A p r o c e s s i n g a r e i n v o l v e d in i n t r o n i c s n o R N A p r o c e s s i n g . F o r e x a m p l e , in y e a s t , X r n l p a n d R a t l p a r e r e q u i r e d f o r 5' m a t u r a t i o n  of  snR190  by  and  Lafontaine  U14,  and  Rna82  and Tollervey,  is r e q u i r e d f o r s n R 1 9 0  3'  processing (reviewed  1995). T h e s e intronic s n o R N A s e a c h p o s s e s s e s a  long  s e q u e n c e , u p to 21 n u c l e o t i d e s in t h e c a s e of U 2 0 , that is p e r f e c t l y c o m p l e m e n t a r y to a r e g i o n in t h e  mature  r R N A sequence (Sollner-Webb,  1 9 9 3 ) . A s u m m a r y of  m e t a z o a n s n o R N A s a n d s o m e of their c h a r a c t e r i s t i c s a r e l i s t e d in T a b l e  1.1.  known  c o  TJ  4—»  CD CD "D  00  CO © r— C LU O  CD  co >. CO E:  <  8f  Z  2§  DC  S  c o  Q. >*  CO  O  c  Q.  CD  sz  CD  Q  Q_  co  a  O .5  SZ  C c o r !? c  Q. Z5  \  DC i -  ~  CO  CD  2  CO CO CD CO  .Q  4—»  CD c ? £  4—»  c  ^ co  in CO Q. « O  sz -9 oLo c O  CO TJ  c C DO ^) O 0 > 0£0  C/)  "D  CO CO CD  o  22 '2 QJ  .y .Q  o o CO  O  Q..E I CD  rV) .—  Q-.!= =J C  « ^ TJ .=  o CO  CO ^ CD 0 5 CO  >  <  rr Z  9r  > CoJC03O> 00 \~o c •S O  ^  0 <  i-  T J |TJ T J |TJ  C  CO  Q . ^ C CO  2 co  03  CO  eO  r— LU  in  O  ..  CO 00  CO r—  si  w  CD C CD  oo  o sz CO  o2  O QJ CO ^ CO T J CO CD  C i  CO  If  C CO  g  CD «>  O  •t; co CD •—  Z DC  TJ D C CD >- CO  IO  CO oo  CO  lu  CD X  X ID  CD  rr < < co  CN  00  in  CD  00  CM  <  SZ\szz  co  Z DC  CO CO 00 oo  00  CVJ  TJ  c  c CO  lie  S3 LL  _Q LL  .Q LL  sz r—  CO  0) C  <  CD  lb b ccf o < E  CD CD X  C©  B  b  b  CO •4—»  CD N  CD  CO  00  CVJ CM I  CO  o  I  CO CO  CM  CO CD I  CO  CO  in CM  00  CO  CM •  m m co o  •  in  CO CM  CO  0_  co  Z DC  o .2 c  o oo  o  CO <  0)  lb b  o  E  N  b  cf  CO  CO  c (0 o  Q Q  rr  I  CO  3  00  3  CM CM  3  m  CM  3  CM  CM  Z3\LU  ~  15 Table 1.1 Metazoan small nucleolar RNAs 1  T h i s table w a s modified from three recent reviews ( S o l l n e r - W e b b , 1993;  1995b; Maxwell a n d Fournier, 2  TMG:  3  ETS,  Bachellerie,  1995)  trimethylguanosine external  transcribed spacer; ITS,  internal t r a n s c r i b e d s p a c e r ; x, c r o s s - l i n k i n g  d a t a ; p, p r o c e s s i n g d a t a , d e p l e t i o n of s n o R N A s i m p a i r s p r o c e s s i n g ; c , c o m p l e m e n t a r y to r R N A s  16 U 3 is t h e m o s t a b u n d a n t s n o R N A (2x 1 0  5  p e r c e l l ) a n d w a s t h e first t o b e  identified  a n d i m p l i c a t e d in r R N A p r o c e s s i n g b y R N A c r o s s - l i n k i n g s t u d i e s ( G e r b i , 1 9 9 0 ) .  Human  a n d rat U 3 b o x A e l e m e n t s c a n b e c h e m i c a l l y c r o s s - l i n k e d to t h e 5' E T S a f e w h u n d r e d n u c l e o t i d e s from the Stroke and Weiner,  U3  dependent  p r o c e s s i n g site ( M a s e r  and Calvet,  1989;  and  1 9 8 9 ) . In y e a s t , t w o r e g i o n s of U 3 h a v e b e e n c r o s s l i n k e d t o t h e  E T S . T h e b o x A e l e m e n t is c r o s s l i n k e d a r o u n d 5 0 n u c l e o t i d e s d o w n s t r e a m of t h e E T S p r o c e s s i n g s i t e A o a n d a 1 0 n u c l e o t i d e s i n g l e s t r a n d e d r e g i o n just d o w n s t r e a m of b o x A ( t h e h i n g e r e g i o n ) is p e r f e c t l y c o m p l e m e n t a r y to a n d c a n b e c r o s s - l i n k e d to t h e  -470  r e g i o n of p r e - r R N A ( B e l t r a m e a n d T o l l e r v e y , 1 9 9 2 ) . T h e s e c o n d a r y s t r u c t u r e of U 3 h a s b e e n m o d e l e d u s i n g its s u s c e p t i b i l i t y to R N a s e a n d c h e m i c a l m o d i f i c a t i o n s ( P a r k e r  and  Steitz, 1987; a n d Hartshrone a n d A g a b i a n , 1994). T h e U 3 b o x A a n d hinge region are s i n g l e s t r a n d e d a n d h a v e t h e p o t e n t i a l to b a s e p a i r with p r e - r R N A . the  U3  gene  in y e a s t  i n h i b i t s p r o c e s s i n g at t h e  In vivo  5" E T S s i t e A o a n d  d i s r u p t i o n of impairs  18S  a c c u m u l a t i o n . Little e f f e c t o n 5 . 8 S a n d 2 8 S r R N A p r o d u c t i o n is o b s e r v e d ( H u g h e s a n d A r e s , 1 9 9 1 ) . T h e y e a s t U 3 1 0 - n u c l e o t i d e c o m p l e m e n t a r y s e q u e n c e t o t h e p r e - r R N A 5' ETS  r e g i o n is r e q u i r e d f o r U 3 f u n c t i o n in 1 8 S p r o c e s s i n g ; m u t a t i o n s  in t h i s  m i m i c t h e e f f e c t s of U 3 d e p l e t i o n a n d c a n b e r e s c u e d b y c o m p e n s a t o r y  region  mutations  w i t h i n t h e E T S c o m p l e m e n t a r y r e g i o n of p r e - r R N A ( B e l t r a m e e r a / . , 1 9 9 4 ) . B y u s i n g a m o u s e or a n archaeal reproduce the U3  in vivo 5'  in vitro  r R N A leader processing system which c a n  faithfully  E T S p r o c e s s i n g e v e n t s , it h a s b e e n d e m o n s t r a t e d t h a t w h e n  is s p e c i f i c a l l y d e p l e t e d  by oligonucleotide-directed  RNase  p r o c e s s i n g a c t i v i t y at t h e 5' E T S s i t e is a b o l i s h e d ( K a s s et al.,  H  degradation,  1990;  Potter  et  the al.,  1 9 9 5 ) . A l l a v a i l a b l e d a t a s u g g e s t that U 3 is r e q u i r e d f o r 5' E T S a n d 1 8 S p r o c e s s i n g a n d its b a s e p a i r i n g with t h e 5' E T S is i m p o r t a n t .  R e c e n t l y , R N T 1 , a y e a s t p r o t e i n h o m o l o g u e of R N a s e III w a s s h o w n t o b e a b l e to c l e a v e in t h e a b s e n c e of a n y s m a l l R N A s at t h e 5' E T S U 3 - d e p e n d e n t AQ s i t e in a s h o r t  17 s y n t h e t i c s u b s t r a t e ( E l e l a e r a / . , 1 9 9 6 ) . T h u s U 3 is u n l i k e l y to p l a y a c a t a l y t i c r o l e in r R N A maturation. the  real  rRNA  processing  in vitro  H o w e v e r , b e c a u s e the  R N A s u b s t r a t e is m u c h s h o r t e r  p r e c u r s o r , this s t u d y c a n n o t d i s p r o v e  in vivo.  U3's  requirement  in 5'  than ETS  R e c e n t l y , t h r o u g h p h y l o g e n e t i c c o m p a r i s o n s of U 3 a n d p r e - r R N A  s e q u e n c e s a n d m u t a t i o n a l a n a l y s e s of U 3 , it w a s s u g g e s t e d t h a t U 3 h e l p s t h e 5' E T S l e a d e r f o l d i n t o a p s e u d o k n o t s t r u c t u r e t o f a c i l i t a t e t h e c l e a v a g e e v e n t s at t h e 5' E T S region ( H u g h e s , 1996).  U 1 4 w a s t h e first i n t r o n i c s n o R N A i d e n t i f i e d . T h e m o u s e U 1 4 g e n e h a s t h r e e c o p i e s a n d t h e y w e r e f o u n d to b e in intron 5, 6 a n d 8, o n t h e c o d i n g s t r a n d of t h e h s c 7 0 h e a t s h o c k g e n e (Liu a n d M a x w e l l , , rRNA  production and  required  for  sequence  to  its 1 3  its f u n c t i o n rRNA,  1990,  L e v e r e t t e et a l . , 1 9 9 2 ) . U 1 4 is e s s e n t i a l f o r  nts s e q u e n c e c o m p l e m e n t a r y  (Jarmolowski  some  et  s n o R N A s like  al., U3  to  18S  rRNA  1990). T h r o u g h their and  U14  might  is  18S  directly  complementary  function  as  rRNA  c h a p e r o n e s to h e l p p r e - r R N A f o l d into a f a v o r a b l e c o n f o r m a t i o n f o r n u c l e a s e c l e a v a g e . R e c e n t s t u d i e s of 21 snoRNA  led  to  the  h u m a n s n o R N A s a n d g e n e t i c d e p l e t i o n a n d r e s t o r a t i o n of conclution  that  these  antisense  snoRNAs  through  c o m p l e m e n t a r y s e q u e n c e s to p r e - r R N A function a s g u i d e R N A for s i t e - s p e c i f i c m e t h y l a t i o n of p r e - r R N A ( K i s s - L a s z l o et al., the methyl group, p r e - r R N A methylation f o l d i n g a n d f u n c t i o n ( L a n e et al.,  U24 their 2'-0-  1 9 9 6 ) . B e c a u s e of t h e h y d r o p h o b i c i t y of  is b e l i e v e d t o b e i m p o r t a n t f o r c o r r e c t r R N A  1 9 9 5 ) . In y e a s t , t h e  18S r R N A dimethylase  Dimlp  w a s f o u n d t o b e e s s e n t i a l f o r p r e - r R N A p r o c e s s i n g a n d p r e - r R N A m e t h y l a t i o n is likely to b e a n i m p o r t a n t r e g u l a t o r y s i g n a l in t h e r R N A p r o c e s s i n g p r o c e s s ( L a f o n t a i n e  era/.,  1 9 9 5 ) . T h u s , s n o R N A s might indirectly regulate r R N A p r o c e s s i n g b y controlling p r e r R N A methylations or other modifications.  In a d d i t i o n to r i b o s o m a l p r o t e i n s , t h e n u c l e o l u s a l s o c o n t a i n s a n u m b e r o f  other  18 p r o t e i n s w h i c h u s u a l l y a s s o c i a t e with s n o R N A s to b e c o m e s n o R N P p a r t i c l e s . A m o n g s n o R N P p r o t e i n s , fibrillarin, a p r o t e i n a p p r o x i m a t e l y 3 4 k D a in s i z e a n d l o c a t e d in t h e d e n s e fibrillar r e g i o n of the n u c l e o l u s , s e e m s to b e t h e m o s t i m p o r t a n t . associated  with  nearly  all s n o R N A s  and  it is a  very  F i b r i l l a r i n is  c o n s e r v e d protein.  Protein  a l i g n m e n t s s h o w 6 7 % of t h e h u m a n s e q u e n c e is i d e n t i c a l t o t h a t in y e a s t a n d 8 1 % of h u m a n s e q u e n c e is i d e n t i c a l to that in Xenopus ( S c h i m m a n g et al, al.,  1990;  Lapeyre era/.,  1990;  T o l l e r v e y et  al.,  1991;  1989; H e n r i q u e z  a n d Amiri, 1994).  et  Fibrillarin  s e q u e n c e a n a l y s i s r e v e a l s t w o c o m m o n R N A - b i n d i n g m o d u l e s ( D r a p e r , 1 9 9 5 ) . T h e first m o d u l e is t h e G A R r e g i o n , a g l y c i n e a n d a r g i n i n e r i c h r e g i o n t h a t c o n t a i n s m a n y A r g G l y - G l y ( R G G ) b o x e s i n t e r s p e r s e d with a r o m a t i c r e s i d u e s n e a r t h e N t e r m i n u s of t h e p r o t e i n . T h i s r e g i o n a c c o u n t s f o r m o s t of t h e p r o t e i n ' s s e q u e n c e d i v e r s i t y .  Fibrillarin  a l s o h a s t h e m o s t p r e v a l e n t R N A - b i n d i n g m o d u l e - - t h e R N P motif. It is c o m p o s e d of t w o consensus sequences, RNP1 a p a r t in a d o m a i n  of a b o u t  analysis, the  motif  RNP  and R N P 2 , which are  located about 30  amino  9 0 a m i n o a c i d s in s i z e . B y X - r a y d i f f r a c t i o n  structure w a s  demonstrated  and  to b e f o u r (3 s t r a n d s  acids NMR  forming  a n t i p a r a l l e l (3 s h e e t s p a c k e d a g a i n s t t w o a h e l i c e s . It h a s b e e n s u g g e s t e d that t h i s R N P d o m a i n f o r m s a n R N A b i n d i n g s u r f a c e i n s t e a d of a b i n d i n g p o c k e t ( D r e y f u s s et 1 9 9 3 ) . T h e s e t w o fibrillarin  R N A b i n d i n g m o d u l e s a p p e a r to a c c o u n t for  al.,  fibrillarin's  a s s o c i a t i o n w i t h s n o R N A s , a l t h o u g h a h i g h r e s o l u t i o n m o d e l of f i b r i l l a r i n ' s a s s o c i a t i o n with R N A is n o t y e t a v a i l a b l e .  Genetic  studies  in  yeast  orthologue N O P 1 (Nucleolar d e p l e t i o n of N O P 1 l e a d i n g to  18S  have  revealed  Protein 1).  NOP1  the  functions  of  the  yeast  is e s s e n t i a l f o r y e a s t v i a b i l i t y ;  a f f e c t s all r R N A p r o c e s s i n g r e a c t i o n s , e s p e c i a l l y t h e  maturation  (Tollervey  era/.,  fibrillarin  1991). T h e  final  in vivo  cleavages  phenotype  shows  a  d r a m a t i c d e c r e a s e in r i b o s o m e n u m b e r s in t h e c y t o p l a s m . I n t e r e s t i n g l y , f u r t h e r s t u d i e s of t e m p e r a t u r e - s e n s i t i v e  point mutations  in t h e  NOP1  gene  indicate that  different  19 mutations NOP1.2 primary  have  and 35S  NOP1.4  and  1.5  different d e f e c t s mutations  transcript; NOP  1.7  in r i b o s o m e  inhibit t h e  NOP1.3 allow  maturation  mutations  proper  biogenesis  rRNA  inhibit  of  18S  (Tollervey and 25S  et  occur  1993):  r R N A from  r R N A methylation;  p r o c e s s i n g to  al.,  while  but  the  mutants  the  resultant  r i b o s o m e s m o v e a b n o r m a l l y in a s u c r o s e g r a d i e n t o w i n g to t h e i m p a i r m e n t  of a l a t e r  s t e p in r i b o s o m e a s s e m b l y . T h e s e o b s e r v a t i o n s s u g g e s t that fibrillarin is i n v o l v e d in a number  of d i f f e r e n t p o s t - t r a n s c r i p t i o n a l  s t e p s of r i b o s o m e f o r m a t i o n .  Presumably,  d i f f e r e n t fibrillarin m u t a t i o n s a f f e c t d i f f e r e n t s n o R N P s ' s t r u c t u r e a n d f u n c t i o n , u l t i m a t e l y i n t e r f e r i n g with t h e s e s n o R N P s ' f u n c t i o n s in v a r i o u s s t e p s of r i b o s o m e b i o g e n e s i s .  Another  n u c l e o l a r p r o t e i n that a p p e a r s to h a v e  important  functions  in  ribosome  b i o s y n t h e s i s is n u c l e o l i n . It is t h e m o s t a b u n d a n t n u c l e o l a r p r o t e i n a n d a l s o c o n t a i n s t h e G A R a n d R N P m o t i f s . N u c l e o l i n h a s b e e n s h o w n t o b e i n v o l v e d in t h e s t r u c t u r i n g of c h r o m a t i n in t h e v i c i n i t y of r i b o s o m a l g e n e s ( n u c l e o l o g e n e s i s ) , a n d in t h e a s s e m b l y a n d m a t u r a t i o n of p r e - r i b o s o m e s ( L p e y r e e r a / . , 1 9 8 7 ; C a i z e r g u e s - F e r r e r et al., al.,  1989; L e e  1 9 9 1 ) , b u t it h a s n o t b e e n r e p o r t e d to b e a s s o c i a t e d w i t h s n o R N A s . S o m e  nucleolar  proteins  s u m m a r i z e d in T a b l e  have 1.2.  been  discovered  and  characterized.  Their  features  et  other are  ai  .22  s  %  oo  0 cu 8  3  "o  •o  ^  §  2  3  01 o 0i O  s -s 0) o  XS C  0i o  2  5  00  ,7 s  CN  £  C  ai  00  0 1 00  I°o 00  s  43  o S  1,3  ai  d  S  a > -c3 so  a i <*-i  S  'O  oo o  .S -o  a) s fa _ *S § => «  00  I S -  1  I  ai  <  o S 5O  <n oo o ^  cx  oo  a I  00 w  ai g  a>  W  00 00  ai  O  3  O0  ai  OON©  §  Oi  ^ i c OO Oi o l | -i I Oi i l l o 3 -O  c  £ e O0 o i  2  :3  > 00 00 Oi >» •S 01 0i a ai o ti c is i ai •ti oo k-l s 13 & •ti  r-  §8.-g  •a a  •a .a  3  g § o I  2  s o •a o 3 TD  X I- 00 oo  00 in  oo  6  3  I i  OH  s  c  •ti 3  o a  00 — • I  e  5  c  3 °  o O0 o X5 •C  a | 01 00  ai ^ •i—* 3 T3 o  oo § s  e  &  oo p  Oi  ,o  C  oo o  oo  < -a  o s  Oi  .9  oo  •o  T3 Oi  Oi  .5  o s  Oi  s  OO T3  .3  s O  Oi T3 •*-»  •o  •4—*  c  Oi  & Oi  TD  o CO  o  o  c  3  oo  3  6 3 00  S  03  o  3 00  O  c  T3 Oi •S  o  •a  oo o o  00  01 w S3  Oi  •a o 3  oo  ai  •a  3  O "o oo  si:  5 3  6  3  O  OO  CN  00  oo2 *-< ^  C3  ° C o Ix: o Oi'  si o oo  oo cn oo  OO  00  a  8  0 1 3 00 Ui 01 *  oi  3 «u ^  o 00 3  CN ON  00  8 e  s  CN  CN  a>  •1.8s  in  o  8  3 00  ai  I  PQ <3  Oi O N Oi O N  O  .O  ai Oi  ilo .3  1° 1 ^  O  Oi  o  I-  3  oo  I .3  T3 g  00  .s  in  2  %  00  C3  01  00  01  PQ  oo oo  Oi  o  00  Oi  oi  00  g  >|  21  1.5  Background work on the U3-mediated processing of pre-rRNA 5' ETS  region in Sulfolobus acidocaldarius:  T h e r i b o s o m a l R N A o p e r o n of  S. acidocaldarius  has been cloned, sequenced and  t h e p r o c e s s i n g s i t e s h a v e b e e n m a p p e d ( D u r o v i c et al., of  the  Sulfolobus  hyperthermophilic  rRNA archaea,  operon with  has  16S  been  found  and 23S  1994). T h e g e n e to  be  the  same  organization as  r R N A cotranscribed and  in  other  5S  rRNA  s e p a r a t e l y t r a n s c r i b e d (figure 1.2). T h e f l a n k i n g s e q u e n c e s of t h e 1 6 S a n d 2 3 S  rRNAs  c a n i n d i v i d u a l l y f o r m l o n g h e l i c a l s t r u c t u r e s b r a c k e t i n g t h e m a t u r e r R N A s . In t h e h e l i x , p r o c e s s i n g s i t e s h a v e b e e n m a p p e d to t h e t w o b u l g e s o f t h e  23S  bulge-helix-bulge  motif~the typical a r c h a e a l h e l i x - p r o c e s s i n g e n z y m e recognition site. H o w e v e r , b e c a u s e t h e r e is a d e f e c t in t h e b u l g e - h e l i x - b u l g e motif in t h e 1 6 S - p r o c e s s i n g h e l i x ( a n u c l e o t i d e in t h e t h r e e - n u c l e o t i d e b u l g e of t h e a s c e n d i n g s t r a n d is m i s s i n g ) , o n l y w e a k p r o c e s s i n g e v e n t s h a v e b e e n d e t e c t e d at o n e b u l g e . I n s t e a d , t w o p r o c e s s i n g s i t e s at t h e 5' E T S r e g i o n a n d o n e p r o c e s s i n g s i t e in t h e 5' 1 6 S m a t u r e t e r m i n u s h a v e b e e n W h e n an  in vitro  detected.  t r a n s c r i p t c o n t a i n i n g t h e e n t i r e 1 4 4 n u c l e o t i d e 5' E T S s e q u e n c e p l u s  7 2 n u c l e o t i d e s of 5 ' 1 6 S s e q u e n c e w a s s u b j e c t e d to p r o c e s s i n g b y a  Sulfolobus  cell  f r e e e x t r a c t , p r o c e s s i n g at t h e s e t h r e e s i t e s o c c u r r e d ( D u r o v i c e r a / . , 1 9 9 4 ) . S i n c e t h i s artificial s u b s t r a t e c o n t a i n s o n l y t h e a s c e n d i n g s t r a n d of t h e 1 6 S p r o c e s s i n g h e l i x , t h e 1 6 S h e l i x a n d t h e b u l g e - h e l i x - b u l g e motif a r e n o t u s e d in 1 6 S 5' p r o c e s s i n g s . F u r t h e r r e s e a r c h h a s d e m o n s t r a t e d that t h e 5' E T S p r o c e s s i n g a c t i v i t y in t h e c e l l f r e e e x t r a c t is s e n s i t i v e to requires  either  essential  RNase RNA  A  or  micrococcal nuclease digestion,  components.  Since  in  p r o c e s s i n g h a s b e e n w e l l e s t a b l i s h e d ( K a s s et al., s u s p e c t e d that t h e p r o c e s s i n g of 1 6 S in  Sulfolobus  U 3 ' s a c t i v e i n v o l v e m e n t a s well. B y u s i n g the  eukaryotes,  U3's  indicating that role  in  5'  it  ETS  1 9 9 0 ; H u g h e s a n d A r e s , 1 9 9 1 ) , it is is s n o R N A m e d i a t e d , p r o b a b l y with  in vitro p r o c e s s i n g  o f t h e 5' E T S - 1 6 S  artificial s u b s t r a t e a s a n a s s a y , a p r o c e s s i n g a c t i v i t y w a s p u r i f i e d 1 0 0 - 2 0 0 f o l d . A  U3  22 h o m o l o g u e w a s c l o n e d f r o m it b y r e v e r s e t r a n s c r i p t i o n P C R ( R T - P C R )  amplification  a n d it h a s b e e n d e m o n s t r a t e d that U 3 R N A ' s s p e c i f i c d e g r a d a t i o n in t h e p u r i f i e d e x t r a c t a b o l i s h e s p r o c e s s i n g a c t i v i t y at all t h r e e  Sulfolobus  5'  E T S sites (Potter  et al.,  1995a).  This  U 3 p o s s e s s e s typical U 3 c o n s e r v e d s e q u e n c e e l e m e n t s - b o x e s A , C \ B, C  a n d D a n d a l s o p r e s e r v e s t h e e u k a r y o t i c U 3 - l i k e s e c o n d a r y s t r u c t u r e ( P o t t e r et 1 9 9 5 a ) . T h e p r e s e n c e of U 3 s n o R N A s u g g e s t s t h a t typical  archaeal  bulge-helix-bulge  rRNA  Sulfolobus,  processing pathway,  while also  retaining possesses  al., the a  eukaryotic s n o R N A - m e d i a t e d r R N A processing pathway.  1.6  The objective of this project  In a d d i t i o n t o U 3 , s o m e o t h e r R N A s a n d f i v e o r s i x p r o t e i n s p e c i e s a r e p r e s e n t in t h e p u r i f i e d p r o c e s s i n g a c t i v e f r a c t i o n a s j u d g e d b y 5' [ a  3 2  P ] p C p l a b e l i n g of t h e  RNAs  a n d s i l v e r s t a i n i n g t h e p r o t e i n s . T h i s p r o j e c t w a s initiated t o i d e n t i f y t h e e s s e n t i a l R N A and  protein  components  in t h i s  5'  E T S processing active  fraction.  Specifically,  e x p e r i m e n t s w e r e a i m e d at c l o n i n g t h e fibrillarin p r o t e i n g e n e a n d o t h e r s n o R N A g e n e s from S.  acidocaldarius.  A n e x t e n s i o n of t h i s p r o j e c t w a s to u s e m e t h a n o g e n fibrillarin a n t i b o d i e s to s t u d y methanogen r R N A processing. T h e methanogen antibodies may also be a  powerful  tool  used  in  the  cloning  immunoprecipitate  of  methanogen  snoRNAs  since  they  can  be  s n o R N A s from m e t h a n o g e n cell extracts, thus allowing  t r a n s c r i p t i o n P C R c l o n i n g to b e d o n e .  to  reverse-  23  II.  2.1  MATERIALS AND METHODS  MATERIALS  Yeast  extract,  Bacto  tryptone  and  Bacto  agar  were  purchased  L a b o r a t o r i e s . Ampicillin w a s bought from S i g m a C h e m i c a l C o . (Sigma), thiogalactopyranoside Technologies  (BRL),  (IPTG) and  from  Gibco  Bethesda  Research  from  Difco  isopropyl-p*-D-  Laboratories  5-bromo-4-chloro-3-indolyl-f3-D-galactopyranoside  Life  (X-gal)  from B R L or Biosyth A G .  Promega purchased  E. coli  supplied from  New  7zf(+/-) c a m e f r o m  strains J M 1 0 1  England  and  JM109.  Biolabs; pGEM-3zf(+/-),  The  vector  pBR322  pGEM-5zf(+/-)  and  was  pGEM-  Promega.  Restriction a n d modification e n z y m e s w e r e p u r c h a s e d from P h a r m a c i a Biotech (Pharmacia);  B R L ; N e w England Biolabs; Boehringer Mannheim; P r o m e g a ; and  Inc.  United  S t a t e s B i o c h e m i c a l C o . ( U S B ) / A m e r s h a m . T h e E r a s e - A - B a s e kit a n d p G E M - T v e c t o r kit  were  purchased  Sequencing  from  kits w e r e  Promega  bought  from  while  the  Sequenase  USB/Amersham.  version  Pharmacia  2  and  provided  s e q u e n c i n g kits, S e p h a g l a s - B a n d P r e p kits a n d P h a s t g e l a c c e s s o r i e s . T h e  Cycle the  T7  Enhanced  c h e m i l u m i n e s c e n t ( E C L ) w e s t e r n blot d e t e c t i o n kit w a s o b t a i n e d f r o m A m e r s h a m w h i l e B i o - R a d L a b o r a t o r i e s ( B R L ) s u p p l i e d t h e B i o r a d p r o t e i n a s s a y kit.  N T P s , d N T P s a n d d d N T P s w e r e obtained from either P h a r m a c i a or U S B . NEN  Research  Products  deoxyribonucleotides.  was  the  source  of  radioactive  Dupont  ribonucleotides  and  24 Acrylamide agarose  and  (genetic  N-N'-methylene  technology grade)  -bis-acrylamide were p u r c h a s e d from w a s from  ICN.  Bio-Rad;  All other c h e m i c a l s c a m e  from  S i g m a or F i s h e r Scientific.  The  Hybond-N  membrane XAR-5)  nylon  membrane  came  from  Amersham  while  the  w a s o b t a i n e d from Millipore. X - r a y films for a u t o r a d i o g r a p h y  and  films for t a k i n g  Immobilon-P (XRP-1  p i c t u r e s of s t a i n e d n u c l e i c a c i d o r p r o t e i n  gels  and were  p u r c h a s e d from E a s t m a n K o d a k a n d Polaroid, respectively.  2.2  DESCRIPTIONS OF THE BACTERIAL STRAINS, PLASMID CONSTRUCTIONS  AND OLIGONUCLEOTIDES USED IN THIS THESIS  T h e b a c t e r i a l s t r a i n s a n d p l a s m i d c o n s t r u c t i o n s u s e d a r e d e s c r i b e d in T a b l e  2.1,  w h i l e t h e o l i g o n u c l e o t i d e s a r e l i s t e d in T a b l e 2 . 2 .  2.3  MEDIA AND CULTURE CONDITIONS  V a r i o u s s t r a i n s of  E. coli  w e r e g r o w n in Y T m e d i a (8 g/l B a c t o t r y p t o n e , 5 g/l B a c t o  y e a s t e x t r a c t , 5 g/l N a C l , p H 7.5).  T h e c u l t u r e s w e r e s h a k e n in a r o t a r y b a t h o r a n air  s h a k e r at 3 7 ° C a n d g r o w t h w a s m o n i t o r e d b y m e a s u r i n g t h e a b s o r b a n c e at 6 0 0  nm.  W h e n r e q u i r e d , a n t i b i o t i c s w e r e a d d e d to t h e f o l l o w i n g c o n c e n t r a t i o n s : a m p i c i l l i n  (100  u.g/ml) a n d c h l o r a m p h e n i c o l (25 n g / m l ) .  25  Table 2.1 E. coli strains used for cloning and protein overexpression  Strain  Genotype  JM101  A ( l a c - p r o A B ) , s u p E , thi/F', l a c l Z A M 1 5 , t r a D 3 6 , p r o A B  JM109  r e c A 1 , s u p E 4 4 , e n d A 1 , h s d R 1 7 , g y r A 9 6 , r e l A 1 , thi, A ( l a c - p r o A B ) /  q  +  F'traD36, p r o A B , lacl , l a c Z A M 1 5 +  q  BL21(DE3)/  B L 2 1 s t r a i n ' s g e n o t y p e is F ' , o m p T , r B ' m B . D E 3 is a X  plysS  in  derivative  +  which  T7  RNA  promoter  was  Because  int g e n e  polymerase  inserted  into  under  the  int  is i n a c t i v a t e d ,  the  gene  control of  once D E 3  of  lacUV5  A, c h r o m o s o m e . was  inserted  into  B L 2 1 c h r o m o s o m e , it is s t a b l e . B L 2 1 ( D E 3 ) / p l y s S a l s o c a r r i e s t h e plasmid inhibit  l y s S w h i c h c o d e s for T 7 T7  RNA  polymerase  lysozyme. T7  when  it  E. coli c a n  r e c o m b i n a n t p r o t e i n t o x i c to the cell's inner m e m b r a n e  is  lysozyme  uninduced  can  so  be expressed. Also,  that when  was broken by freezing-thawing,  l y s o z y m e c a n i n d u c e r a p i d l y s i s of c e l l w a l l t o f a c i l i t a t e  T7  purifying  recombinant protein.  Sulfolobus acidocaldarious yeast  extract,  1 g/l  MgS04«7H 0, 2  and  w a s g r o w n in a m o d i f i e d D e R o s a m e d i u m  B a c t o tryptone, 250  mg/l  3.1  g/l  Krt.P0 , 4  CaCI «2H 0, 2  )  2  2.5  adjusted  to  s u p p l e m e n t e d with 1 m l / l t r a c e m e t a l m i x ( 1 8 0  mg/100ml  N a B O » 1 0 H O , 22  m g / 1 0 0 ml Z n S 0 « 7 H 0 ,  5 mg/100ml  Na MoB  mg/100ml  2  2  4  7  2  4  •2H 0, 2  3  4  V O S 0  2  4  » 2 H  2  0 and  g/l  (1 g/l B a c t o  (NH ) SO4,200 4  pH  2  4.0  with  M n C f e 4 H 0 , 450 #  2  CuCI »2H 0,  1 mg/100ml  2  2  3  HCI  c u l t u r e w a s g r o w n at 7 5 ° C with a d o u b l i n g t i m e of a p p r o x i m a t e l y f i v e h o u r s .  and  mg/100ml mg/100ml  CuS0 «7H 0). 4  mg/l  2  The  26  Table 2.2  Description of the Oligonucleotides used:  Designation Sequence 5 ' to 3' and corresponding amino acid of Methanogen fibrillarin protein  oXWl  (N62) L y s V a l L e u T y r L e u G l y A l a AAA GTT CTA TAT CTT GGA GCT  degeneracy  strand  1  oXW2  oXW3  length  21  Ser A r g A l a Lys l i e Ser Ile(Nl66) ACT TCT AGC TTT GAT TGA TAT  (N35)Val T y r G l y G l u L y s GTN TAY GGN GAR AA  oXW4  (N88)Val T y r A l a V a l G l u Phe GTN TAY GCN GTN GAR TT  oXW5  r e v e r s e o f oXW4  OXW6  (Nl37)Phe G l u A s p V a l A l a G i n P r o TTY GAA GAY GTN GCN CAR CC  0  sense  21  0  antisense  14  128  sense  17  128  sense  antisense 20  256  sense  OXW7  G i n D/N P r o G i n A l a V a l Asp(N139) 20 TG RTY NGG YTG NGC NAC RTC  1024  antisense  OXW8  H i s A s p K/R G l u F/Y Pro Glu TG RTC YYT YTC RA/TA NGG YTC  512  antisense  oXW9  oXWIO  GGGAATTCCATATGGCAAAAATT AAAGAAAAGTTCG (Nl-8)  20  36  sense  CGCCGGATCCTTATTGTCCATTC 0 CAAATTCC  oJCl OJC2 0SP14 1  (N222-228)  31  antisense  0 CGACGGATAGAAGAATTCTGTTCGTTGGAG  30  CTCCAACGAACAGAATTCTTCTATCCGTCG  30  CGAGCATGCGTCGACAGGCTTTTTTTTTTTTTTTTT  36  0 0  N followed  by a number  indicates the position  of t h e a m i n o  m e t h a n o g e n fibrillarin p r o t e i n c o u n t e d f r o m t h e N - t e r m i n a l e n d , e.g. m e t h i o n i n e in t h e fibrillarin will b e n u m b e r e d a s N 1 .  acid  in t h e  N-terminus  27  2.4  GENERAL MOLECULAR BIOLOGY TECHNIQUES  T h e g e n e r a l r e c o m b i n a n t D N A t e c h n i q u e s u s e d a r e d e s c r i b e d in M o l e c u l a r C l o n i n g ( S a m b r o o k e r a / . , 1989)  2.4.1  unless otherwise specified.  PREPARATION OF PLASMID DNA  Small and  large s c a l e plasmid preparations were d o n e  m e t h o d ( p 1 . 2 5 , 1.38,  S a m b r o o k et  al.,  1989)  using the  alkaline  lysis  with 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 in t h e  l a r g e s c a l e p r o c e d u r e . P l a s m i d s w e r e a m p l i f i e d o v e r n i g h t in t h e p r e s e n c e of 1 7 0 u.g/ml chloramphenicol.  O c c a s i o n a l l y , the  amplification  step was  omitted.  The  lysozyme  digestion s t e p w a s a l w a y s omitted. After the p o t a s s i u m a c e t a t e neutralization s t e p , the s u p e r n a t a n t w a s p r e c i p i t a t e d u s i n g 0 . 6 x t h e o r i g i n a l v o l u m e of i s o p r o p a n o l . F o l l o w i n g centrifugation,  the  pellet  was  r e s u s p e n d e d in 2  ml  c h l o r o f o r m , a n d p r e c i p i t a t e d with e t h a n o l u s i n g 2 . 5 x  T E , extracted  with  phenol-  the original v o l u m e . T h e  DNA  p e l l e t w a s r e s u s p e n d e d in 4 m l of T E a n d 1 m l 1 0 M L i C I w a s a d d e d t o p r e c i p i t a t e  the  high m o l e c u l a r weight R N A . After centrifugation, the supernatant w a s precipitated o n c e a g a i n b y e t h a n o l . T h e D N A p e l l e t w a s d i s s o l v e d in 0 . 9 7 x C s C I ( m a d e b y d i s s o l v i n g 9 7 g C s C I in 1 0 0 m l d H 0 ) a n d p u r i f i e d b y C s C I g r a d i e n t c e n t r i f u g a t i o n a s d e s c r i b e d o n 2  p 1 . 4 2 , S a m b r o o k et al.,  ( 1 9 8 9 ) . T h e d e n s i t y g r a d i e n t s w e r e c e n t r i f u g e d at 1 0 0 , 0 0 0  rpm  f o r t h r e e h o u r s in a T L V - 1 0 0 r o t o r in t h e B e c k m a n T L - 1 0 0 t a b l e t o p c e n t r i f u g e .  The  ethidium b r o m i d e w a s r e m o v e d by CsCI-saturated isopropanol extraction. After dialysis at 4 ° C w i t h t h r e e c h a n g e s of T E b u f f e r ( 1 0 m M T r i s 8 . 0 a n d 1 m M E D T A ) , t h e D N A w a s p r e c i p i t a t e d a n d r e s u s p e n d e d in T E .  28  2.4.2  RESTRICTION ENDONUCLEASE DIGESTION OF DNA  Restriction e n z y m e  d i g e s t s w e r e c a r r i e d out a c c o r d i n g to t h e  i n s t r u c t i o n s of  the  suppliers.  2.4.3  1%  G E L ELECTROPHORESIS  A g a r o s e s l a b g e l s w e r e r u n in 0 . 5 x  boric acid, 1 m M  T B E buffer  E D T A ) . T h e g e l s w e r e r u n in t h e  b r o m i d e or w e r e stained  (44.5  mM  Tris-HCI, 44.5  p r e s e n c e of 0.5  u.g/ml  mM  ethidium  afterwards.  A n a l y t i c a l (1 m m thick) o r p r e p a r a t i v e (3 m m thick) 5 % p o l y a c r y l a m i d e g e l s w e r e run in  0.5x  T B E  buffer.  The  gels  were  stained  with  0.5%  ethidium  bromide  after  electrophoresis.  2.4.4  DNA RESTRICTION FRAGMENT PREPARATION  D N A fragments  e l e c t r o p h o r e s e d on a g a r o s e gels w e r e e x c i s e d a n d purified using  t h e S e p h a g l a s B a n d P r e p Kit  DNA  fragments  (Pharmacia).  separated  by running on a 5 %  polyacrylamide  gel(PAGE)  e x c i s e d f r o m t h e g e l , p u t into s t e r i l e d i a l y s i s t u b i n g a n d e l e c t r o e l u t e d 2 0 0 V f o r o n e to t w o h o u r s ( d e p e n d i n g o n t h e s i z e of t h e f r a g m e n t ) . e x t r a c t e d with p h e n o l / c h l o r o f o r m a n d p r e c i p i t a t e d with e t h a n o l .  in 0 . 5 x  were  T B E at  T h e eluate  was  29  2.4.5  LIGATIONS  In a s t a n d a r d " s t i c k y e n d " l i g a t i o n , 5 0 n g d i g e s t e d v e c t o r D N A w a s i n c u b a t e d with a t w o t o t h r e e m o l a r e x c e s s of t a r g e t D N A in t h e p r e s e n c e of 1 unit T 4 D N A l i g a s e at 1 4 ° C o v e r n i g h t with t h e final b u f f e r c o n c e n t r a t i o n to b e 3 0 m M T r i s - H C I , p H 7 . 5 , 1 0 MgCI , 2  10 m M  mM  D T T a n d 1 m M A T P . T h e t o t a l l i g a t i o n v o l u m e w a s 2 0 uJ; h a l f of t h a t  v o l u m e w a s u s e d per transformation.  2.4.6  TRANSFORMATIONS  E. coli h o s t al.,  1989). T h e  cells w e r e m a d e c o m p e t e n t b y the C a C I  E. coli c e l l s  method (p1.82, S a m b r o o k  2  w e r e g r o w n in 1 0 0 m l Y T m e d i u m to a n  A26O  et  of 0 . 4 - 0 . 6 . T h e  c e l l s w e r e t h e n c h i l l e d b y s w i r l i n g o n i c e a n d c o l l e c t e d b y c e n t r i f u g a t i o n at 4 0 0 0 r p m ( 4 K ) f o r 1 0 m i n u t e s at 4 ° C . T h e c e l l p e l l e t w a s r e s u s p e n d e d in 4 0 m l of i c e c o l d mM  CaCl2 a n d  100  i n c u b a t e d o n i c e f o r 3 0 m i n u t e s with o c c a s i o n a l s w i r l i n g . T h e n t h e c e l l s  w e r e h a r v e s t e d b y c e n t r i f u g a t i o n at 4 K r p m f o r 1 0 m i n u t e s a n d r e s u s p e n d e d in 1 o r 2 m i s of i c e c o l d 1 0 0 m M C a C I w i t h 2  the competent  E. coli  1 5 % glycerol. After incubation o n ice for o n e hour,  c e l l s w e r e a l i q u o t t e d into 1 0 0 uJ p o r t i o n s a n d s t o r e d at  -80°C.  T r a n s f o r m a t i o n w a s c a r r i e d o u t b y i n c u b a t i n g h a l f of a l i g a t i o n r e a c t i o n ( a r o u n d 10uJ) with t h e c o m p e t e n t  E. coli  c e l l s o n i c e f o r 4 0 m i n u t e s , f o l l o w e d b y h e a t s h o c k at 3 7 ° C  for 2 m i n u t e s . T h e t r a n s f o r m e d  E. coli c e l l s  w e r e directly plated o n Y T p l a t e s c o n t a i n i n g  a m p i c i l l i n a n d i n c u b a t e d in a 3 7 ° C air i n c u b a t o r o v e r n i g h t .  2.4.7  5' end-labeling of oligonucleotides  1 0 p m o l e s of o l i g o n u c l e o t i d e s w e r e m i x e d with 2 \i\ of 1 0 x P N K b u f f e r ( 5 0 0 m M T r i s pH  7.5,  100mM  MgCI  2  and  1  mM  spermidine);  1  ul  100  mM  DTT,  1.2  jil  T4  30 P o l y n u c l e o t i d e K i n a s e a n d 4 LII [ y  3 2  P ] A T P , and made  u p to 2 0  LII w i t h d H 0 .  The  2  r e a c t i o n m i x t u r e w a s i n c u b a t e d at 3 7 ° C f o r o n e h o u r a n d t h e n t e r m i n a t e d b y a d d i n g 2 LII 0 . 5 M  E D T A , p H 8 . 0 a n d 1 1 8 ul d H 0 2  a n d i n c u b a t i n g at 6 5 ° C f o r 1 0 m i n u t e s .  labeled oligonucleotides w e r e precipitated by a d d i n g 50 yeast  RNA  (5  ugAil),  and  500  LII 9 5 %  ethanol.  LII 7 . 5  M  The  CH3COONH4,  Precipitates  were  10  collected  by  c e n t r i f u g a t i o n a n d t h e n r e s u s p e n d e d in 5 0 uJ T E b u f f e r ( 1 0 m M T r i s - H C I , p H 7 . 5 1mM  LII  and  EDTA).  2.4.8  Labeling DNA probes by the random priming method (Feinberg and  Vogelstein, 1983)  A p p r o x i m a t e l y 0.1  p m o l e of a D N A f r a g m e n t  (50  n g for a 7 0 0  bp fragment)  was  m a d e u p to 2 8 . 5 LII with d H 0 a n d b o i l e d with 2 . 5 LII r a n d o m h e x a n u c l e o t i d e s ( a b o u t 2  A  2 6 0  /ml)  for 5 m i n u t e s , t h e n  immediately  c h i l l e d o n i c e . 5 of 1 0 x  buffer  T r i s H C I , p H 6.6); 5 u l n u c l e o t i d e m i x ( 0.2 m M d G T P , d T T P , d C T P ) ; 5 u.l a  2LII  L i C i ) ; 2 |il 1 0 m g / m l B S A a n d  3 2  (0.5M  Bis-  P dATP  (50  K l e n o w e n z y m e (2 u n i t s ) w e r e a d d e d t o t h e  h e x a m e r - D N A m i x . T h e l a b e l i n g r e a c t i o n w a s a l l o w e d to p r o c e e d at r o o m  boiled  temperature  f o r m o r e t h a n t h r e e h o u r s o r o v e r n i g h t . 1 jxl y e a s t t R N A ( 1 0 m g / m l ) a n d 1 LII E D T A M , p H 8.0) terminate  (0.5  w e r e t h e n a d d e d a n d t h e m i x t u r e w a s h e a t e d at 6 5 ° C f o r 1 0 m i n u t e s the  reaction. T h e  labeled probes were precipitated  v o l u m e of 5 M a m m o n i u m a c e t a t e a n d 2 . 5 x  volume 9 5 %  50  by a d d i n g the  to  same  ethanol. After recovery,  the  pellet w a s r e s u s p e n d e d a n d b o i l e d for 5 m i n u t e s , t h e n c h i l l e d o n i c e b e f o r e b e i n g u s e d for hybridization  2.4.9  0.2-1  experiments.  Run-off transcription  i i g of l i n e a r i z e d t e m p l a t e D N A w a s m i x e d with 2 LII 5 X t r a n s c r i p t i o n b u f f e r  (200  31 m M T r i s - H C I , p H 7.5, mM  D T T , 0.5  ATP,  30 m M  MgC'2, 1 0 m M s p e r m i d i n e a n d 5 0 m M  uJ R N a s i n r i b o n u c l e a s e i n h i b i t o r ,  G T P and  UTP.),  1 ul o t  3 2  P  0.5  uJ n u c l e o t i d e m i x  C T P ( 1.0 u C i ), 0 . 5 .ul (3  p o l y m e r a s e ; m a d e u p to 2 0 |il w i t h D E P C t r e a t e d d H 2 0 , 1/2  hour. T h e labeled transcript w a s then precipitated  ammonium 95%  acetate  N a C I ) , 1 LII  units)  (2.5  SP6  100  mM  each  or T 7  RNA  a n d i n c u b a t e d at 3 7 ° C f o r 1  by adding  1/2x  v o l u m e 7.5  M  a n d 3 x v o l u m e s of 9 5 % e t h a n o l . T h e R N A p e l l e t w a s w a s h e d  by  e t h a n o l a n d d r i e d . T h e R N A w a s r e s u s p e n d e d in 1 0 u l D E P C t r e a t e d d H 2 0  and  w a s u s e d a s a p r o b e for s o u t h e r n hybridization.  2.5  Southern hybridization  T h e S o u t h e r n b l o t t i n g m e t h o d w a s initially d e s c r i b e d b y S o u t h e r n ( 1 9 7 5 ) . D N A  was  d i g e s t e d w i t h a v a r i e t y of r e s t r i c t i o n e n z y m e s a n d s e p a r a t e d b y s i z e o n a n a g a r o s e g e l at  low voltage  DNA  overnight.  (fragments  S o m e t i m e s for efficient  that a r e o v e r 4kb),  transfer  the a g a r o s e gel w a s  of l a r g e m o l e c u l a r  weight  s o a k e d in 0 . 2 5  HCI  in situ  partially d e p u r i n a t e the D N A . T h e D N A w a s then d e n a t u r e d for  30  minutes  equilibrating  the  in  denaturation  gel  in a l k a l i n e  solution transfer  (1.5 buffer  M  NaCI  (1.5M  and  NaCI,  M  b y s o a k i n g the  0.5M 0.25M  NaOH). NaOH)  to gel  After for  15  m i n u t e s , t h e D N A in t h e g e l w a s c a p i l l a r i l y t r a n s f e r r e d o v e r n i g h t t o a H y b o n d - N n y l o n membrane  in t h e s a m e b u f f e r . T h e m e m b r a n e w a s d r i e d b y i n c u b a t i n g at 8 0 ° C f o r t e n  m i n u t e s a n d t h e D N A w a s c r o s s l i n k e d to t h e m e m b r a n e b y e x p o s i n g t h e m e m b r a n e UV  light f o r t w o  minutes.  The  hybridization  protocol u s e d  in t h i s t h e s i s is w i t h o u t  f o r m a m i d e . T h e m e m b r a n e w a s p r e h y b r i d i z e d f o r half a n h o u r in h y b r i d i z a t i o n  solution  (5x S S P E ( 2 0 x S S P E : 3 . 6 M N a C I , 0 . 2 M s o d i u m p h o s p h a t e , 0 . 0 2  M E D T A , p H 7.4),  Denhardt's  2%  solution  (100x  polyvinylpyrollidone), 0.5% probe  was  added  and  Denhardt's  solution:  2%  BSA,  Ficoll  S D S and 20 ug/ml salmon s p e r m D N A ) . T h e hybridization  was  allowed  to  to  proceed  and  5x 2%  radiolabeled  overnight.  The  32 hybridization temperature  w a s d e t e r m i n e d e m p i r i c a l l y d e p e n d i n g o n t h e n a t u r e of t h e  probe used. T h e temperature hybridization  trial s t a r t s f r o m 6 5 ° C a n d is g r a d u a l l y l o w e r e d t o a l l o w  o f D N A s with m o r e  m i s m a t c h e s . D e p e n d i n g o n the  s t r e n g t h s of  s i g n a l s , t h e m e m b r a n e w a s s u b j e c t e d to e i t h e r l o w s t r i n g e n c y w a s h e s ( t w o m i n u t e w a s h e s at r o o m t e m p e r a t u r e s t r i n g e n c y w a s h e s (15  in s o l u t i o n # 1 ( 2 x  SSPE,  0.1%  m i n u t e w a s h at h y b r i d i z a t i o n t e m p e r a t u r e  S D S ) ) or  the  times  10  medium  in s o l u t i o n #2  (1x  S S P E , 0 . 1 % S D S ) ) . A f t e r e x p o s i n g to t h e X - r a y f i l m , t h e m e m b r a n e c a n b y s t r i p p e d b y p o u r i n g o n a b o i l i n g s o l u t i o n of 0 . 1 %  S D S f o l l o w e d b y i n c u b a t i o n w i t h light  agitation  until t h e s o l u t i o n c o o l s to t h e r o o m t e m p e r a t u r e . T h e m e m b r a n e c a n t h e n b e r e p r o b e d .  2.6  DNA sequencing  2.6.1 General sequencing strategy  All  DNA  sequencing was  done  using the  dideoxynucleotide  chain  termination  m e t h o d ( S a n g e r e r a / . , 1 9 7 7 ) . S p e c i f i c a l l y , t h e U S B S e q u e n a s e ( V e r s i o n 2) kits a n d USB  C y c l e S e q u e n c i n g kits w e r e u s e d . T h e D N A f r a g m e n t s o f i n t e r e s t w e r e c l o n e d  into p G E M SP6  vectors and s e q u e n c e d  u s i n g universal forward a n d r e v e r s e p r i m e r s or  a n d T 7 p r i m e r s . W h e n t h e i n s e r t w a s t o o l o n g to b e s e q u e n c e d in o n e r e a c t i o n ,  bidirectional strands complete  of  deletions  the  were generated  deletion  clones were  by  E x o n u c l e a s e III  sequenced and  (Henikoff,  organized  to  1984).  Both  represent  the  fragment.  2.6.2 Generation of unidirectional deletions of insert DNA by exonuclease III  T w o r e s t r i c t i o n e n z y m e s c u t in b e t w e e n t h e s e q u e n c i n g p r i m e r s i t e in t h e v e c t o r a n d t h e n e a r b y e n d o f t h e i n s e r t D N A . T h e e n d c l o s e to t h e s e q u e n c i n g p r i m e r h a s a  3'  33 o v e r h a n g t h a t is r e s i s t a n t to e x o n u c l e a s e III d i g e s t i o n , m e a n w h i l e t h e e n d c l o s e to t h e insert h a s a  r e c e s s i v e 3' e n d o r blunt e n d w h i c h  d i g e s t i o n . E x o n u c l e a s e III  is s u s c e p t i b l e to e x o n u c l e a s e  is a 3 ' t o 5' e x o n u c l e a s e t h a t d i g e s t s D N A at a  III  constant  s p e e d at a g i v e n t e m p e r a t u r e . T h e l i n e a r i z e d p l a s m i d w a s d i g e s t e d b y e x o n u c l e a s e a n d at f i x e d t i m e i n t e r v a l s , d i g e s t i o n s a m p l e s w e r e r e m o v e d a n d d i g e s t i o n  III  terminated.  W h e n all t h e t i m e p o i n t s a r e c o l l e c t e d , S 1 n u c l e a s e w a s u s e d t o d i g e s t a w a y t h e  5'  o v e r h a n g . A f t e r h e a t i n a c t i v a t i o n of S 1 n u c l e a s e , K l e n o w f r a g m e n t w a s u s e d a l o n g with f o u r d N T P s to flush the e n d s . T h e ligation a n d the  linear p l a s m i d w a s  resulting p l a s m i d s w e r e transformed  then  into  c l o s e d b y "blunt  E. coli  and  end"  subsequently  s c r e e n e d f o r d e l e t i o n c l o n e s in a p p r o p r i a t e l e n g t h s . T h e a d v a n t a g e of t h i s m e t h o d is to a l l o w s e q u e n c i n g into a n i n s e r t ' s i n t e r n a l s e q u e n c e b y u s i n g o n e p r i m e r , t h u s o b v i a t i n g t h e n e e d to s y n t h e s i z e m a n y p r i m e r s w h i c h c a n b e c o s t l y a n d of v a r i a b l e q u a l i t i e s , a n d a v o i d s t h e t e d i o u s w o r k to f i n d o u t e a c h p r i m e r ' s a p p r o p r i a t e a n n e a l i n g  temperature  a n d right s e q u e n c i n g c o n d i t i o n .  2.7  PCR procedures  T h e general P C R p r o c e d u r e s a n d reverse-transcription P C R ( R T - P C R ) p r o c e d u r e s are described below.  (1) G e n e r a l P C R p r o c e d u r e s w e r e d o n e a c c o r d i n g t o P C R P r o t o c o l s ( I n n i s et 1990).  For degenerate  Sulfolobus the  PCR  P C R a m p l i f i c a t i o n of t h e fibrillarin  D N A fragments  from  al., the  g e n o m e , all p o s s i b l e c o m b i n a t i o n s of s i x d e g e n e r a t e p r i m e r s w e r e u s e d in  amplifications.  The  amount  of  Sulfolobus  genomic  DNA,  primers,  the  a n n e a l i n g t e m p e r a t u r e a n d n u m b e r of c y c l e s w e r e v a r i e d in o r d e r t o g e t p r o d u c t s of t h e same  size  Touchdown  as  the  corresponding fragment  P C R was  also performed  in  the  in w h i c h 2 0  methanogen  to 2 5  fibrillarin  gene.  c o m p l e t e c y c l e s with  half  34 d e g r e e d e c r e a s i n g i n c r e m e n t s of a n n e a l i n g t e m p e r a t u r e  were  r u n , f o l l o w e d b y thirty  c y c l e s of f i x e d a n n e a l i n g t e m p e r a t u r e P C R a m p l i f i c a t i o n .  (2)  R e v e r s e - t r a n s c r i p t i o n P C R w a s p e r f o r m e d with m o d i f i c a t i o n s a c c o r d i n g to t h e  P r o m e g a P r o t o c o l a n d A p p l i c a t i o n G u i d e ( 1 9 9 1 ) . A p p r o x i m a t e l y 1 (j.g o f p u r i f i e d R N A w a s s u s p e n d e d in 2 5 ul D E P C t r e a t e d d H 0 . 2 5 ul 2 x p o l y A b u f f e r ( 8 0 m M T r i s - H C I , 2  2 0 m M M g C I , 5 m M M n C I , 5 0 0 m M N a C l , 0 . 5 m M A T P a n d 1 0 0 n g / m l B S A ) a n d 1 uJ 2  2  p o l y A p o l y m e r a s e w e r e a d d e d a n d t h e r e a c t i o n i n c u b a t e d at 3 7 ° C for 3 0 m i n u t e s . T h e r e a c t i o n w a s t e r m i n a t e d b y a d d i n g 2 u.l 0 . 5 M E D T A a n d p r e c i p i t a t e d w i t h e t h a n o l . T h e pellet  was  r e s u s p e n d e d in 4 0  \i\  DEPC  treated  dH 0. 2  12  ul  5x  AMV  reverse  t r a n s c r i p t i o n b u f f e r ( 2 5 0 m M T r i s - H C I , p H 8, 4 0 m M M g C I , 1 5 0 m M K C I , 5 0 m M 2  DTT  a n d 1 0 m M e a c h d N T P s ) a n d 7 uJ o S P 1 4 o l i g o n u c l e o t i d e ( s e q u e n c e d e s c r i b e d in t a b l e 2.2.)  (120  p m o l e s ) w e r e a d d e d a n d t h e m i x t u r e h e a t e d to 7 0 ° C f o r 3 m i n u t e s ,  a l l o w e d to c o o l to r o o m t e m p e r a t u r e .  then  1 n' A M V r e v e r s e t r a n s c r i p t a s e w a s a d d e d a n d  t h e r e a c t i o n w a s i n c u b a t e d at 4 2 ° C for 1 h o u r .  The  reaction  was  terminated  r e s u s p e n d e d in 4 0 u l d H 0 . 2  MgCI , 2  1 mM  D T T , 30 ng/ml  by precipitation  with e t h a n o l  a n d the  pellet  1 0 ul 5 x R N a s e H b u f f e r ( 4 0 m M T r i s - H C I , p H 7 . 5 , 4 B S A a n d 4 % g l y c e r o l ) , 1 u l R N a s e H (1.1  was mM  u) a n d 2  R N a s e A w e r e a d d e d to r e m o v e t h e R N A s t r a n d of t h e R N A - D N A h y b r i d . A f t e r  ul the  reaction mixture w a s extracted by phenol-chloroform a n d precipitated by ethanol, the first s t r a n d c D N A w a s r e s u s p e n d e d in 3 4 ul d H 0 2  buffer (250 ng/ml  m M T r i s - H C I , p H 7.5,  B S A ) , 5 n' 5'  50 m M M g C I ,  phosphorylated oJC1  2  (100  a n d m i x e d w i t h 1 0 uJ R N A l i g a s e 100 m M D T T , 5 m M A T P a n d  pmole)  and  1 n  1  R  N  A  ligase.  500 The  r e a c t i o n w a s a l l o w e d t o p r o c e e d at 3 7 ° C for 2 h o u r s . A f t e r p r e c i p i t a t i o n w i t h e t h a n o l , the  r e s u s p e n d e d D N A w a s s u b j e c t e d to P C R a m p l i f i c a t i o n with 1 0 0  o S P 1 4 o l i g o n u c l e o t i d e a n d t h e o J C 2 o l i g o n u c l e o t i d e (refer to t a b l e  2.2).  p m o l e s of  the  35 T h e P C R p r o d u c t s of i n t e r e s t w e r e c l o n e d into t h e p G E M T v e c t o r ( P r o m e g a )  and  sequenced.  2.8  Expression of Methanogen fibrillarin protein in E. coli  T h e s t r a i n P D # 1 1 9 4 w a s i n o c u l a t e d into 2 0 m l Y T m e d i u m w i t h 1 0 0 u.g/ml a m p i c i l l i n and  25  u.g/ml  chloramphenicol. The  i n o c u l a t e 1 liter of t h e s a m e m e d i u m  next  day,  10  ml of this c u l t u r e  was  used  to  ( d i v i d e d into 4 2-liter f l a s k s c o n t a i n i n g 2 5 0  e a c h ) . T h e s e w e r e s h a k e n at 3 7 ° C until t h e O D  6  0  0  ml  r e a c h e d 0.8. I P T G w a s then a d d e d  t o e a c h c u l t u r e t o a f i n a l c o n c e n t r a t i o n of 0 . 4 m M . T h e c u l t u r e s w e r e s h a k e n v i g o r o u s l y at 3 7 ° C f o r a n o t h e r t h r e e h o u r s a n d t h e c e l l s w e r e h a r v e s t e d at 4 ° C b y c e n t r i f u g a t i o n . T h e c e l l s w e r e w a s h e d with b u f f e r A ( 5 0 m M T r i s - H C I , p H 8 . 0 , 0 . 1 5 MgCI 8.0,  2  and 1 mM  1 mM  m M N a C I , 10  mM  E D T A ) a n d r e s u s p e n d e d in 3 0 m l l y s i s b u f f e r ( 5 0 m M T r i s - H C I ,  E D T A a n d 1% T w e e n 20). T h e cells w e r e l y s e d b y p a s s i n g two times  pH at  1 4 , 0 0 0 p . s . i . in a F r e n c h P r e s s . T h e l y s a t e w a s c e n t r i f u g e d at 3 0 , 0 0 0 g f o r o n e h o u r w i t h t h e o v e r e x p r e s s e d p r o t e i n p r e s e n t in t h e i n c l u s i o n b o d i e s . T h e pellet w a s w a s h e d b y buffer B ( 5 0  m M Tris-HCI, p H 8,1  inclusion body  m M E D T A a n d 1M Urea). T h e  p e l l e t w a s r e s u s p e n d e d in l y s i s b u f f e r , m i x e d with t w o t i m e s c o n c e n t r a t e d S D S l o a d i n g buffer (100  m M T r i s - H C I , p H 6.8, 2 0 0 m M dithiothreitdl, 4 % S D S , 0 . 2 %  blue a n d 2 0 %  glycerol)  and  b o i l e d for 5 m i n u t e s . T h e  a n a l y z e d or s e p a r a t e d on a 12.5% S D S - P A G E (Laemmli,  2.9  resulting  lysate w a s  either  1970).  Protein electrophoresis and staining procedures  SDS-polyacrylamide  gels  ( S D S - P A G E ) were  prepared  L a e m m l i ( 1 9 7 0 ) . A f t e r r u n n i n g , t h e g e l w a s s t a i n e d in 0 . 2 5 % 250,  bromophenol  45%  (v/v)  and (w/v)  used  according  to  C o o m a s s i e B l u e R-  m e t h a n o l a n d 1 0 % a c e t i c a c i d ( v / v ) f o r h a l f a n h o u r a n d d e s t a i n e d in  4 5 % methanol a n d 1 0 % acetic acid.  36 F o r s i l v e r s t a i n i n g , g r e a t e r s e n s i t i v i t y c a n b e o b t a i n e d if t h e g e l w a s p r e - s t a i n e d with C o o m a s s i e B l u e a n d d e s t a i n e d . After rinsing the g e l s with d H 0 2  for 10 m i n u t e s ,  the  g e l s w e r e s o a k e d in s i l v e r s t a i n s o l u t i o n (stain c o m p o n e n t A : s i l v e r n i t r a t e , 0 . 4 g in 2 m l dH 0; 2  s t a i n c o m p o n e n t B : 21  ammonium  ml 0.36%  (w/v)  sodium hydroxide and  1.4  ml 14.8  M  h y d r o x i d e ; s t a i n is f o r m e d b y a d d i n g A to B d r o p w i s e , s t i r r i n g c o n s t a n t l y  a n d m a k i n g t h e f i n a l s o l u t i o n u p t o 1 0 0 m l with d H 0 ) f o r 1 5 m i n u t e s . T h e s t a i n  was  r e m o v e d a n d t h e g e l s w a s h e d in d e v e l o p e r (2.5  38%  2  (v/v)  formaldehyde; made  u p to 5 0 0  m l 1 % (w/v)  m l with d H 0 ) 2  citric a c i d ; 0 . 2 5  f o r 5 to  15  minutes  ml  until  band  f o r m a t i o n o c c u r r e d . D e v e l o p m e n t w a s t h e n s t o p p e d b y t r a n s f e r r i n g t h e g e l s to 2 0 0 of s t o p s o l u t i o n ( 5 0 %  2.10  ml  (v/v) m e t h a n o l ; 1 0 % (v/v) a c e t i c a c i d ) .  Immunoanalysis procedures  Immunological  p r o c e d u r e s w e r e p e r f o r m e d a c c o r d i n g to A n t i b o d i e s : A  Laboratory  M a n u a l (Harlow a n d L a n e , 1988).  2.10.1  Immunization procedures  T w o white N e w Z e a l a n d f e m a l e rabbits w e r e u s e d for the i m m u n i z a t i o n . b o d i e s w e r e s o l u b i l i z e d in 6 M g u a n i d i n e h y d r o c h l o r i d e a n d t h e p r o t e i n quantified  against  BSA  standard  using  a  Bio-Rad  protein  Inclusion  concentration  quantitation  r e s u s p e n d e d i n c l u s i o n b o d i e s w e r e m i x e d with a n e q u a l v o l u m e of 2 x S D S  kit.  The  loading  buffer, b o i l e d for five m i n u t e s a n d fractionated o n a 1 2 . 5 % S D S - P A G E p r e p a r a t i v e g e l . T h e b a n d c o r r e s p o n d i n g to t h e o v e r e x p r e s s e d protein w a s e x c i s e d a n d  lyophilized.  T h e d e h y d r a t e d g e l w a s g r o u n d into a v e r y f i n e p o w d e r in a m o r t a r a n d p e s t l e  and  made  mM  into a s l u r r y u s i n g p h o s p h a t e - b u f f e r e d s a l i n e ( P B S - - 1 3 0  Na HP0 «2H 0, 2  4  2  p H 7.2).  mM  NaCl and 8  T h i s s l u r r y w a s e m u l s i f i e d in a n e q u a l v o l u m e of F r e u n d ' s  37 A d j u v a n t ( S i g m a ) a n d u s e d f o r t h e i n j e c t i o n . F o r t h e p r i m a r y i n j e c t i o n , 3 0 0 Lig of p r o t e i n w a s e m u l s i f i e d in F r e u n d ' s C o m p l e t e A d j u v a n t a n d i n j e c t e d s u b c u t a n e o u s l y into e a c h r a b b i t . F o r s u b s e q u e n t b o o s t i n j e c t i o n s , 1 0 0 Lig of t h e p r o t e i n , e m u l s i f i e d in F r e u n d ' s I n c o m p l e t e A d j u v a n t ( S i g m a ) , w a s i n j e c t e d i n t r a m u s c u l a r l y into e a c h r a b b i t . T h e s e r a w e r e c o l l e c t e d ten d a y s after the b o o s t injection.  2.10.2  Western blot procedures  F o r u s e in w e s t e r n b l o t s , p r o t e i n s a m p l e s w e r e s e p a r a t e d o n a 1 2 . 5 %  SDS-PAGE  a n d t h e n e l e c t r o b l o t t e d o n t o a n i m m o b i l o n - P m e m b r a n e ( M i l l i p o r e ) b y e i t h e r w e t (tank) transfer or s e m i - d r y transfer. T h e m e m b r a n e w a s then b l o c k e d with 5 %  skim  milk  p o w d e r d i s s o l v e d in P B S - T ( P B S with 0 . 1 % T w e e n - 2 0 ) f o r o n e h o u r . A f t e r r e p e a t i n g a five m i n u t e s ' w a s h with P B S - T buffer t h r e e t i m e s , t h e m e m b r a n e w a s  p r o b e d with  p r i m a r y a n t i b o d y ( 1 : 5 0 0 0 d i l u t i o n in P B S - T b u f f e r with 1 % s k i m m i l k p o w d e r ) f o r o n e h o u r . T h e m e m b r a n e w a s w a s h e d with P B S - T b u f f e r f o u r t i m e s f o r 1 0 m i n u t e s e a c h , a n d t h e n w a s p r o b e d with a s e c o n d a r y a n t i b o d y ( 1 : 7 0 0 0 d i l u t i o n in P B S - T b u f f e r with 1 % s k i m p o w d e r ) . T h e s e c o n d a r y a n t i b o d y is d o n k e y s e r u m r a i s e d a g a i n s t r a b b i t a n d is c o n j u g a t e d to h o r s e r a d i s h p e r o x i d a s e . A f t e r f o u r a d d i t i o n a l w a s h e s ( 1 0  IgG  minutes  e a c h ) , t h e m e m b r a n e w a s s u b j e c t e d to e n h a n c e d c h e m i l u m i n e s c e n c e ( E C L ) d e t e c t i o n u s i n g a n A m e r s h a m E C L w e s t e r n blot d e t e c t i o n kit.  2.10.3  Immunoprecipitation procedures  A p p r o x i m a t e l y 0.6  g of b a c t e r i a l c e l l s w e r e u s e d f o r e a c h  immunoprecipitation.  0 . 6 g of b a c t e r i a c e l l s w e r e r e s u s p e n d e d in 2 m l N E T - 2 0 0 b u f f e r ( 5 0 m M T r i s p H  7.4,  150  was  mM  NaCI and 0.05%  NP-40) a n d lysed using a F r e n c h P r e s s . T h e lysate  c e n t r i f u g e d at 9 K in t h e S o r v a l l S S - 3 4 rotor f o r 1 0 m i n u t e s . T o l o w e r t h e b a c k g r o u n d ,  38 p r e - i m m u n e s e r a w e r e u s e d for pre-clearing of the cell lysate. 100 ul of p r e - i m m u n e rabbit s e r u m w a s a d d e d into the lysate (from 0.6 g of bacterial cells) a n d the mixture w a s i n c u b a t e d o n ice for o n e hour. T h e antibody precipitated c o m p l e x w a s r e m o v e d by two s u c c e s s i v e i n c u b a t i o n s with p r o t e i n - A s e p h a r o s e b e a d s ( P h a r m a c i a ) for 3 0 m i n u t e s (on a n automatic mixer in a c o l d room) a n d centrifugation to r e m o v e the resin. In the immunoprecipitation using the fibrillarin antibody, 5 0 ul of s e r u m w a s i n c u b a t e d with a p r e - c l e a r e d c e l l lysate on ice for o n e hour. 100 ul p r o t e i n - A s e p h a r o s e (1mg P r o t e i n A/ml) w a s a d d e d a n d m i x e d with a n automatic m i x e r in the c o l d room for o n e hour. A f t e r c e n t r i f u g a t i o n , the resin w a s w a s h e d with four s u c c e s s i v e r o u n d s of suspension  in  1 ml N E T - 2  buffer  and reprecipitation.  After  a final  round  of  centrifugation, 3 0 0 ul N E T - 2 buffer a n d 3 0 ul 1 0 % S D S w e r e a d d e d to r e s u s p e n d the resin. T h e s u s p e n s i o n w a s incubated at 37°C for 15 minutes with o c c a s i o n a l vortexing. Half of the v o l u m e w a s s a v e d a s a protein s a m p l e ; the other half w a s extracted with a n e q u a l v o l u m e of phenol-chloroform a n d precipitated with ethanol.  T o a n a l y z e the protein precipitates, the protein s a m p l e w a s s e p a r a t e d o n a 1 2 . 5 % p o l y a c r y l a m i d e gel a n d silver-stained. T h e precipitated R N A w a s a n a l y z e d by 3' e n d labeling with 5' [y  3 2  P ] p C p a n d separation a 8 % p o l y a c r y l a m i d e - u r e a g e l . T h e 3' p C p  labeling protocol w a s a s follows: approximately 1 u g of R N A w a s m i x e d with 1 ul 0.15 m M A T P , 2 u l 0.75 M H E P E S - N a O H (pH 8.3), 3 ul 0.1 M M g C I , 1 ul 0.1 M D T T , 3ul 2  D M S O , 3ul 100 ug/ml B S A , 2 0 u C i [ y ^ P ] p C p , 1 ul (3 units) T 4 R N A l i g a s e a n d m a d e up to 3 0 u l with D E P C treated d H 2 0 . T h e reaction w a s i n c u b a t e d at 4 °C overnight. A f t e r p r e c i p i t a t i o n with e t h a n o l a n d w a s h i n g with 9 5 % e t h a n o l , the pellet  was  r e s u s p e n d e d in 2 0 ul D E P C treated d H 2 0 . A n aliquot w a s m i x e d with l o a d i n g d y e , h e a t - d e n a t u r e d a n d run on a p o l y a c r y l a m i d e - u r e a denaturing g e l . A u t o r a d i o g r a p h y w a s u s e d to v i s u a l i z e the b a n d s .  39  2.11  Molecular sequences and alignments  T h e m o l e c u l a r s e q u e n c e s w e r e o b t a i n e d f r o m G e n b a n k at N C B I , N I H  b y u s i n g the  p r o g r a m E n t r e z . T h e B a s i c l o c a l a l i g n m e n t s e a r c h t o o l ( B L A S T ) , ( A l t s c h u l et al., was  u s e d t o s e a r c h in G e n b a n k  for h o m o l o g o u s s e q u e n c e s to s e q u e n c e s  1990), under  inquiry. T h e A u t h o r i n p r o g r a m (Intelligenetics) w a s u s e d to s u b m i t the s e q u e n c e d a t a to  Genbank.  unpublished,  The 1991)  program was  CLUSTALV  u s e d to m a k e  (Higgins  era/., 1 9 8 9 ;  and  Higgins  protein s e q u e n c e a l i g n m e n t s . T h e  program  F A S T A w a s u s e d to m a k e t w o n u c l e o t i d e s e q u e n c e c o m p a r i s o n s . T h e d e f a u l t w e r e u s e d f o r initial a l i g n m e n t s , a n d later t h e a l i g n m e n t s w e r e a d j u s t e d b y e y e .  er al., values  40  Ill  Results  SECTION ONE: Experiments to clone the S. acidocaldarius  (I)  fibrillarin gene: F i b r i l l a r i n , a p r o t e i n w i t h i m p o r t a n t f u n c t i o n s in e u k a r y o t i c r R N A p r o c e s s i n g , s u s p e c t e d to b e a f u n c t i o n a l c o m p o n e n t of a p u r i f i e d S .  acidocaldarius  was  fraction which  h a s 5' r R N A p r o c e s s i n g a c t i v i t y . E x p e r i m e n t s d e s c r i b e d in t h i s t h e s i s a r e m a i n l y e f f o r t t o c l o n e fibrillarin g e n e f r o m S . g e n e s from two a r c h a e a l s p e c i e s ,  acidocaldarius.  Methanococcus  R e c e n t l y , fibrillarin-like  voltae  and  Methanococcus  protein  vannielii,  h a v e b e e n i d e n t i f i e d a n d c l o n e d ( A m i r i , 1 9 9 4 ) . T h e a r c h a e a l fibrillarin p r o t e i n s  retain  t h e R N A - b i n d i n g R N P motif a n d s h a r e 3 5 % o v e r a l l i d e n t i t y with h u m a n , Xenopus, y e a s t f i b r i l l a r i n s . M u l t i p l e a l i g n m e n t s of fibrillarin p r o t e i n s f r o m d i f f e r e n t  an  and  s p e c i e s are  g i v e n in f i g u r e 3 . 1 .  As  i l l u s t r a t e d in t h e a l i g n m e n t ,  s e q u e n c e identities a n d similarities b e t w e e n  m e t h a n o g e n i c a n d e u k a r y o t i c fibrillarins a r e suggesting  high  conservation  of  this  distributed throughout  protein.  Since  archaea  the  are  the  alignment, a  coherent  p h y l o g e n e t i c g r o u p , t h e i r fibrillarin p r o t e i n s s h o u l d s h a r e a h i g h e r d e g r e e of s i m i l a r i t y with e a c h o t h e r t h a n with e u k a r y o t e s . T h e r e f o r e , t h e fibrillarin g e n e f r o m a m e t h a n o g e n w a s u s e d a s a h e t e r o l o g o u s p r o b e for identifying a n d c l o n i n g t h e S .  acidocaldarius  fibrillarin g e n e . T h r e e a p p r o a c h e s w e r e u n d e r t a k e n : (i) S o u t h e r n h y b r i d i z a t i o n u s i n g t h e m e t h a n o g e n g e n e a s p r o b e ; (ii) P C R u s i n g d e g e n e r a t e o l i g o n u c l e o t i d e s t a r g e t e d  to  a m i n o a c i d s e q u e n c e s h i g h l y c o n s e r v e d in fibrillarin a n d (iii) i m m u n o d e t e c t i o n u s i n g a n a n t i b o d y p r e p a r e d a g a i n s t t h e e x p r e s s e d m e t h a n o g e n fibrillarin.  41 Figure 3.1  Multiple alignment of the protein sequences of all cloned fibrillarins  Fibrillarin s e q u e n c e s from ten s p e c i e s - eight e u k a r y o t e s  a n d two a r c h a e a , are  a l i g n e d u s i n g the clustalV p r o g r a m (Higgins e r a / . , 1989). T h e eight e u k a r y o t e s include  Homo sapiens ( J a n s e n  Saccharomyces cerevisiae ( S c h i m m a n g et al, Xenopus laevis ( L a p e y r e et al., 1 9 9 0 ) ; m o u s e ( T u r l e y et al., 1 9 9 3 ) ; S . pombe ( G i r a r d et al, 1993); Leishmania ( C a p p a i et al., 1 9 9 4 ) ; Tetrahymena thermophila ( D a v i d a n d P e a r l m a n , 1 9 9 4 , u n p u b l i s h e d s e q u e n c e , G e n b a n k a c c e s s i o n n u m b e r # X 7 7 9 6 2 ) ; Giardia lamblia ( N s a r c i s i et al., 1 9 9 4 , 1989;  a n d Henriquez  era/.,1991);  era/.,  1990);  unpublished sequence, Genbank accession number #L28115). T w o archaeal species are:  M. voltae a n d M. vannielii ( A m i r i ,  1 9 9 4 ) . T h e a m i n o a c i d s t h a t a r e c o n s e r v e d in all  ten s p e c i e s a r e d e n o t e d by stars under the alignment; highly c o n s e r v e d a m i n o a c i d s are denoted by dots.  Hs f i b Yeast Xenopus Mouse SacPomb Leishman Tetrahy Giardia Mvannielii M.voltae  MK PGF S PRG - GGFGGRGGFGDRGGRGG RGGFGGGRGRGGGFRGRGR MSFRPGSRG-GSRGGS RGGFGGRGGSRGGARGGSRGGFGGRGGSR MRPGFSPRG-G RGGFGDRGGFGGRGGFGDRGGFRGGSRGGFGGRGR MKPGFRPRG-GGFGGRGGFGDRGGRGGGRGGRGGFGGGRGGFGGGGRGRG MAYTPGSRG-GR GGSRGGRGGFNGGRGGFGGGRGGAR MRGGFGRGG-GGRGGSRG MGKDFKSGG-GNAGGKPFNKGPGGPGGRPFNKGPGGPGGP MGTDYRNSGRGGRDGPGGRGPGNDRRDSGRSFGDRRPERPDFKRGDGGRG M M  Hs f i b Yeast Xenopu s Mouse S a c Pomb L e i shman Tetrahy Giardia Mvannielii M.voltae  GGGGGGGGGGGGGRGGGGFHS-GGNRGRG--RGGKRGNQSGKNVMVE PHGGARGG S RGGFGG-RGGSRGG-ARGGSRGGRGGAAGGARGGAKWIEPHGGDRGGRGGFRGGFS S PGRGG-PRGGGRGGFGGGRGGFGAGRKVIVEPHGGGGGFRGRGGGGVRGGGFQS-GGNRGRGGGRGGKRGNQSGKNVMVEPHGGGRGGARGGRGG-RGGARGG RGGSSGGRGGAKGGAKV11EPHGRGGFGRGGGRGGGRGGGRGG-GRGGGRGGGRGGGRGGAGAKVIVE PHM GGKFGG-GRPGGPGGKFGAKG- - P R G P K T I I V K H FGDRRGSGPPGGPDRGDRRGPRDGPGGRGGPGGPGGGFKGGAKTMVKPHP EKIKVKEIF - -AKIKEK- -F  Hsf i b Yeast Xenopus Mouse SacPomb L e i s hman Te t r ahy Giardia Mvannielii M. v o l t a e  RHEGWICRGKEDAL VTKNLVPGESVYGEKRVSISEGDD KIEY RHAGVYIARGKEDLL VTKNMAPGESVYGEKRISVEEPSKEDGVPPTKVEY RHEGIFICRGKEDALVTKNLVPGESVYGEKRISVEDGEV KTEY RHEGVFICRGKEDALFTKNLVPGESVYGEKRVSI SEGDD TIEY RHAGVFIARGKEDLLVTRNLVPGESVYNEKRISVDS PD GTKVEY LH PGVFISKAKTDSLCTLNMVPGIS VYGEKR IELGATQGGDD KKEY RLEGVFICKGQQE AL VTKNFF PGES VYNEKRMS VEENGE KIEY KYDGIFISHGRGDVLVTKSL APGVAVYGEKRISVEGTE SKIEY SNVYGVDLGDGLKRIATKS L APGKRVYGEKLIY S EN KEY DNVFELDLGDGIKRIGTKSLVPNKRVYGEKLVNVKN TEY  *  42 Hsfib  RAWNPFRSKLAAAILGGVDQIHIKPGAKVLYLGAASGTTVSHVSDIVGPD  Yeast  RVWNPFRSKLAAGIMGGLDELFIAPGKKVLYLGAASGTSVSHVSDWGPE  Xenopus  RAWNPFRSKIAAAILGGVDQIHIKPGVKVLYLGAASGTTVSHVSDWGPE  Mouse  RAWNPFRSKLAAAILGGVDQIHIKPGAKVLYLGAASGTTVSHVSDIVGPD  SacPomb L e i shman Tetrahy Giardia Mvannieli i M.VOltae  RVWNPFRSKLAAGILGGLDNIYIKPGARVLYLGAANGTSVSHVADWGPE RLWNPYRSKLASAIYAGVSSIHMGPGSKVLYLGGATGTTVSHVSDLVGPE RVWNPYRSKIAAAWGGISDIHIKPGSKVLYLGGASGTTVSHVADIVGPT REWNPFRSKLGAAVRLNVLDMPIKPGAKVLYLGAASGTTVSHVSDIVGPT RIWNPNKSKLGAAIINGLKKMPIKKGTKVLYLGASAGTTPSHVSDI-SED RVWNPNKSKLGASIINGLKEMPIKKGSKVLYLGASAGTTPSHVADV-AED  * *** Hsfib Yeast Xenopus Mouse S a c Pomb Leishman Tetrahy Giardia Mvannielii M.voltae  GLVYAVEFSHRSGRDLINLAKKRTNIIPVIEDARHPHKYRMLIAMVDVIF GWYAVEFSHRPGRELISMAKKRPNIIPIIEDARHPQKYRMLIGMVDCVF GLVYAVEFSHRSGRDLINVAKKRTNIIPVIEDARHPHKYRILVGMVDWF GLVYAVEFSHRSGRDLINLAKKRTNIIPVIEDARHPHKYRMLIAMVDVIF GLVYAVEFSHRSGRDLLNMAKKRTNVIPIVEDARHVQKYRMLVGMVDWF GMVYAVEFSHRVGRDLVEMSKRRP-IVPIVEDARYPMKYRMLVPMVDCIF GVVYAVEFSHRSGRDLVNMAKKRTNVVPIIGDARKPQEYRFLVGMVDWF GAVYAVEFSQRSGRDLLEVAKARTNVYPIIADARHPYKYRMIVPEVDCIF TIVYAVEFAPRIMREFIDSCNERINLIPILGDANRPFEYSNIVGKVDVIF SPVYAVEFAPRIMREFIESCEGRKNLFPILGDANKPEEYANIVEKVDVIF  Hsfib Yeast Xenopus Mouse SacPomb Leishman Tetrahy Giardia Mvannielii M.voltae  ADVAQPDQTRIVALNAHTFLRNGGHFVISIKANCIDSTASAEAVFASEVK ADVAQPDQARIIALNSHMFLKDQGGWISIKANCIDSTVDAETVFAREVQ ADVAQPDQTRIVALNAHNFLKNGGHFVISIKANCIDSTAAPEAVFAAEVK ADVAQPDQTRIVALNAHTFLRNGGHFVISIKANCIDSTASAEAVFASEVK ADVAQPDQARIVALNAAAFLKNEGGWISVKASCIDSTADAAWFAREVK MDVAQPDQARILALNAQAFLQNGGHYVISIKANCIDSTLEAPWIASELN ADVAQ PDQ AR IMGMNCQYFLKNGGHFLISIKACCIDSTNE P A W F AAEVQ SDVAQ PDQARIVAENARYYLKANGGMLISIKASSVDSTLKPEAVFAREIE EDVAQ PNQAEILVKNAKWFLNKDGYAMISIKARSIDVTKNPKEIFLEQKK EDVAQPNQAEILIKNAKWFLKKGGYGMISIKARSVDVTENPRVIFEAQKE *****_*_•_ * * * _**_** .**  Hsfib Yeast Xenopus Mouse SacPomb Leishman Tetrahy Giardia Mvannielii M.voltae  KMQQENMKPQEQLTLE PYERDH AVWGVYRP P PKVKN KLREERIKPLEQLTLEPYERDHCIWGRYMRSG-LKK KMQQENMKPQEQLTLEPYERDHAVWGIYRPPPKQKK KMQQENMKPQEQLTLE PYERDH AVWGVYRP P PKVKN KMQEEKIKPQEQLTLEPYERDHCIIVGKYLR HQ KLKKDKLKPLEQVSLEPFERDHAVWGVYRPVKKSKQ KLKEEGLKPEQQLTLEPYERDHAMVLGSYRA TLREHDFKCKEQLDIGEFHRNHAIWGRFRVKA A ILIEGGFDIVDEINIEPFEKDHIMFVGIWKG N IMEQNGFKIVDAINIE PFEKDHMLFVGIWNG Q  ******  *  *  *  *  *  *  **  *  **  *  43  (i).  Direct shotgun cloning from the Sulfolobus genome  acidocaldarius  S o u t h e r n b l o t s of r e s t r i c t i o n e n z y m e d i g e s t e d S .  D N A were probed  w i t h t h e m e t h a n o g e n fibrillarin g e n e o r a n i n t e r n a l 3 0 0 b p g e n e f r a g m e n t a m p l i f i e d b y P C R that r e p r e s e n t s t h e m o s t c o n s e r v e d r e g i o n of t h i s p r o t e i n . T h e s e t w o p r o b e s g a v e v e r y similar hybridization signals. W h e n the  Sulfolobus  D N A w a s p r o b e d with t h e w h o l e  g e n e in b u f f e r w i t h o u t f o r m a m i d e at 6 5 ° C (refer to s e c t i o n 2 . 5 in M a t e r i a l a n d M e t h o d s ) , even  with a  low  stringency w a s h , no signal could be detected  ( d a t a not  shown).  H o w e v e r , w h e n t h e hybridization t e m p e r a t u r e w a s l o w e r e d to 5 2 ° C , after a s i n g l e low s t r i n g e n c y w a s h , a s e t of s i n g l e w e l l d e f i n e d h y b r i d i z a t i o n  Sulfolobus  bands was  detected  g e n o m i c D N A d i g e s t e d b y different restriction e n z y m e s ( F i g u r e 3.2).  in  These  b a n d s r a n g e d in s i z e f r o m 1.55 t o 12 k b a n d g a v e h y b r i d i z a t i o n s i g n a l s c o n s i s t e n t with e a c h c o n t a i n i n g a s i n g l e c o p y of t h e t a r g e t D N A .  T h e D N A s p e c i e s p r e s e n t in t h e s e r e s t r i c t i o n f r a g m e n t s h y b r i d i z i n g t o t h e w e r e p r e s u m e d to b e t h e  Sulfolobus  fibrillarin  gene.  In t h e  probes  Hind Ill-Xba I double  d i g e s t i o n , a 1.7 k b f r a g m e n t r e p r o d u c i b l y h y b r i d i z e d to t h e m e t h a n o g e n fibrillarin p r o b e (Figure 3.2, Xba  l a n e 6 ) . T h e t e r m i n i of t h i s f r a g m e n t w e r e v e r i f i e d b y H i n d III  I s i n g l e d i g e s t i o n s in w h i c h  hybridization.  Sulfolobus  gene  B e c a u s e the  larger  methanogen  size bands were fibrillarin  gene  identified  is 7 0 0  using  and  southern  b p l o n g , t h i s 1.7  kb  D N A f r a g m e n t is l a r g e e n o u g h to c o n t a i n m o s t if n o t all of t h e g e n e .  F o l l o w i n g s i z e f r a c t i o n a t i o n of H i n d I l l - X b a I d i g e s t e d  S. acidocaldarius  genomic  D N A in a 1 % a g a r o s e g e l , t h e p i e c e of a g a r o s e g e l c o n t a i n i n g D N A in t h e 1.7 k b s i z e r a n g e w a s e x c i s e d a n d the D N A e l u t e d . D N A from this p o o l w a s ligated b e t w e e n H i n d III  a n d X b a I s i t e s of t h e p G E M  7(+)  vector a n d transformed  into  E. coli.  the 200  c o l o n i e s f r o m t h i s s i z e s e l e c t e d library w e r e s c r e e n e d u s i n g S o u t h e r n h y b r i d i z a t i o n with  Figure 3.2 Genomic  Genomic Southern hybridization to identify Sufolobus fibrillarin gene  Sulfolobus  D N A w a s d i g e s t e d with different r e s t r i c t i o n e n z y m e s a n d s e p a r a t e d o n a  1 % a g a r o s e g e l . F o l l o w i n g t r a n s f e r to a n y l o n m e m b r a n e , t h e D N A w a s p r o b e d with  M. voltae  fibrillarin g e n e . T h e D N A w a s h y b r i d i z e d in s t a n d a r d 5 x S S P E b u f f e r w i t h o u t f o r m a m i d e at  52  ° C o v e r n i g h t . A f t e r o n e t i m e l o w s t r i n g e n c y w a s h , t h e m e m b r a n e w a s e x p o s e d t o X - r a y fiilm f o r 2 d a y s . ( S o u t h e r n p r o t o c o l r e f e r to M a t e r i a l a n d M e t h o d s s e c t i o n 2 . 5 ).  M. voltae  fibrillarin  g e n e i n s e r t ( a b o u t 7 0 0 b p s ) w a s e x c i s e d f r o m P D # 1 1 6 9 a n d r u n o n t h e S o u t h e r n g e l to s e r v e a s a p o s i t i v e c o n t r o l ( l a n e 1). R e s t r i c t i o n e n z y m e s u s e d f o r d i g e s t i o n s w e r e i n d i c a t e d a b o v e e a c h l a n e s ( l a n e 2 to 8).  45 t h e m e t h a n o g e n fibrillarin g e n e p r o b e a n d f i v e p o s i t i v e c l o n e s w e r e i d e n t i f i e d . G l y c e r o l cell s t o c k s w e r e individually p r e p a r e d for t h e s e five c l o n e s a n d t h e  plasmids were  r e i s o l a t e d to test the  DNA  authenticity.  T o v e r i f y t h a t t h e c l o n e d 1.7  kb  fragment  a c c o u n t e d f o r t h e s i g n a l in t h e S o u t h e r n blot of g e n o m i c D N A , a p p r o x i m a t e l y m o l a r a m o u n t s of g e n o m i c D N A ( 2 0 u g , l a n e 3, f i g u r e 3.3) lane  4-8,  20  experiment.  ng  in  lane  9-13,  figure  3.3)  were  equi-  a n d p l a s m i d D N A s ( 4 0 n g in  u s e d in a  Southern  B o t h g e n o m i c ( l a n e 3) a n d p l a s m i d D N A s ( l a n e 4 - 1 3 )  hybridization  g a v e r i s e to s i g n a l s  of t h e s a m e s i z e a n d s i m i l a r i n t e n s i t y ( F i g u r e 3.3). T h i s p r o v e d t h a t t h e p l a s m i d c l o n e s contain the  1.7  kb fragment  of g e n o m i c  DNA  that h y b r i d i z e s to t h e  methanogen  fibrillarin D N A p r o b e .  To  facilitate  s e q u e n c i n g this  1.7  k b c l o n e , e x o n u c l e a s e III  was  u s e d to  make  u n i d i r e c t i o n a l d e l e t i o n s o n t h i s 1.7 k b D N A (refer to M a t e r i a l a n d M e t h o d s s e c t i o n T h e s i z e s of t h e d e l e t i o n c l o n e s a n d t h e s e q u e n c i n g s t r a t e g y w e r e i l l u s t r a t e d 3.4).  2.6).  (Figure  B o t h s t r a n d s of t h e 1.7 k b D N A f r a g m e n t w e r e s e q u e n c e d a n d o n l y o n e l o n g o p e n  r e a d i n g f r a m e with a l e n g t h of 1.1 k b w a s f o u n d (figure 3.5).  T h i s 1.7 k b s e q u e n c e w a s u s e d f o r a d a t a b a s e s e a r c h u s i n g t h e B L A S T ( A l t s c h u l et al.,  1990)  algorithm  a n d n o h o m o l o g y with k n o w n fibrillarin p r o t e i n g e n e s w a s f o u n d .  T h i s i n d i c a t e d that the s i g n a l g e n e r a t e d  b y t h i s c l o n e in t h e S o u t h e r n  hybridization  r e s u l t e d f r o m f o r t u i t o u s h y b r i d i z a t i o n . S e q u e n c e a l i g n m e n t s b e t w e e n t h e c l o n e d 1.7 fragment homology  and  the  that  methanogen  presumably  fibrillarin  gave  promising hybridizing fragments  rise to  gene the  probe weak  w e r e identified  do  reveal  some  kb  intermittent  cross-hybridization. Since  in t h e a b o v e e x p e r i m e n t s  with  no the  a l r e a d y v e r y l o w s t r i n g e n c y , S o u t h e r n h y b r i d i z a t i o n at e v e n l o w e r s t r i n g e n c y ( l o w e r temperature  and  higher  salt  concentration)  is n o t  expected  to  yield  i n f o r m a t i o n . U s i n g t h e b l a s t x p r o g r a m , s e v e r a l r e g i o n s of t h e t r a n s l a t e d  meaningful  protein  46  kb  5.3 2.8  1.7  Figure 3.3  Southern hybridization to verify the positive clones obtained  A p p r o x i m a t e l y e q u i - m o l a r H i n d III a n d X b a 1 d o u b l e d i g e s t e d  Sulfolobus  genomic D N A and  c l o n e d p l a s m i d D N A w e r e s e p a r a t e d o n a 1% a g a r o s e g e l . F o l l o w i n g transfer to a m e m b r a n e , t h e D N A w a s p r o b e d with 3 0 0 membrane  was  hybridized  in s t a n d a r d  nylon  b p m e t h a n o g e n fibrillarin g e n e f r a g m e n t . 5x  SSPE  buffer  without  formamide  at  The  52  °C  o v e r n i g h t . A f t e r o n e t i m e l o w s t r i n g e n c y w a s h , t h e m e m b r a n e w a s e x p o s e d to X - r a y fiilm f o r 2 d a y s . ( S o u t h e r n p r o t o c o l r e f e r to M a t e r i a l a n d M e t h o d s s e c t i o n 2 . 5 ). S a m p l e s a r e : PD#1169 (methanogen r a n g e o f H i n d III  and Xba  1 digested  Sulfolobus  a g a r o s e g e l , l a n e 3, H i n d III a n d X b a 1 d i g e s t e d 1 3 , H i n d III  lanel,  fibrillarin g e n e c l o n e ) p l a s m i d D N A , I a n e 2 , D N A at t h e 1.7 k b s i z e g e n o m i c D N A (20  Sulfolobus  ug) purified from  an  g e n o m i c D N A ( 2 0 u g ) ; l a n e 4-  a n d X b a 1 d i g e s t e d recombinant p l a s m i d s D N A (lane 4-8, 4 0 n g ; 9-13, 2 0  ng);  l a n e 14 a n d 15, 2 0 0 n g n o n s p e c i f i c 3 kb or 1 kb long D N A fragment a s n e g a t i v e controls .  47  vector s e q .  pb  vector s e q u e n c e  1.7 k b D N A f r a g m e n t  d i r e c t i o n of d e l e t i o n 1.6kb  1.3 k b  1.1  kb  800 bp 500 bp  400 bp 250bp 150 bp  Figure 3.4 Sequencing strategy of one strand of the 1.7 kb DNA fragment T h e 1.7 k b D N A w a s c l o n e d into p G E M 7(+) to g e n e r a t e  the  v e c t o r . P r o m e g a ' s E r a s e - A - B a s e kit w a s u s e d  unidirectional deletion clones. T h e approximate  s i z e s of t h e s e  deletion  c l o n e s a r e f i v e n in t h e f i g u r e a b o v e . U n i v e r s a l f o r w a r d p r i m e r w a s u s e d f o r t h e s e q u e n c i n g of t h e s e c l o n e s . T h e p r i m e r b i n d i n g s i t e (pb) is m a r k e d in t h e d i a g r a m . V e c t o r s i z e is n o t d r a w n to s c a l e .  48 Figure 3.5 contained orf  Sequence of the 1.7 kb DNA clone and the translation of its  R e s t r i c t i o n s i t e s u s e d for c l o n i n g a r e m a r k e d . T h e p r e d i c t e d a m i n o a c i d s e q u e n c e s of t h e 1.1  k b orf a r e d e p i c t e d b e l o w t h e n u c l e o t i d e s e q u e n c e .  56/1 a t g gaa aag Met g l u l y s 116/21 ggt a c t aca gly thr thr 176/41 agt a t a g t c ser i l e v a l 236/61 ata atg cca i l e met p r o 296/81 t a c c t t gga tyr leu gly 356/101 a t e aaa agg i l e l y s arg 416/121 t c t t e a gga ser ser gly 476/141 aat g t t get asn v a l a l a 536/161 gat a t g t t g asp met l e u 596/181 gat t a t t t a asp t y r l e u 656/201 gat ggg a c t asp g l y t h r 716/221 cca ggt tea pro g l y ser 776/241 t t c aaa aca phe l y s t h r 836/261 t t a aag gac l e u l y s asp 896/281 a t t t e g gag i l e ser glu 956/301 gga gaa aga gly g l u arg 1016/321 a t t t t a gaa i l e leu glu 1076/341  g t a aat v a l asn ggt aag gly lys c a t cag his gin t t t ate phe i l e get aaa ala lys gaa aat g l u asn gaa cag glu gin aaa t e c l y s ser gca a t a ala i l e cgc aca arg t h r gag get glu ala tac tac tyr tyr ggt gat g l y asp gtg ata val i l e ate cct i l e pro cct atg p r o met tat ctt t y r leu  tctagataataataacaatcaaataaattataaaaacctcatttctaaaaatact Xba I 86/11 t a t gat g t t aag gga gga gac c c t t a c t e c a t t c t g t t t ace t e a t y r - asp v a l l y s g i y g l y asp p r o t y r s e r i l e l e u phe t h r s e r 146/31 c c g a c t g t g a t g t a t aca cac gag aag g t a a t t c a t ggt gca t t a p r o t h r v a l met t y r t h r h i s g l u l y s v a l i l e h i s g l y a l a l e u 206/51 c t t ggt c t t t a c aac t c t c c t gca aaa e t c t e c t c t aat gac g t c l e u g i y l e u t y r asn s e r pro a l a l y s l e u s e r s e r asn asp v a l 266/71 c c c a t t t a t cac t t g t g g t e g tgg ggt t c t gca t t t a t a gee t c t p r o i l e t y r h i s l e u t r p s e r t r p g l y s e r a l a phe i l e a l a s e r 326/91 t a c g t c t t g ace ggt aag t t t gac cca aaa ace get a t a caa e t a t y r v a l l e u t h r g i y l y s phe asp p r o l y s t h r a l a i l e g i n l e u 386/111 get acg t g g a t a aat gca g t c ccc aca a t g a t a cag a t g a t a t t a a l a t h r t r p i l e asn a l a v a l p r o t h r met i l e g i n met i l e l e u 446/131 t t g c c t gga g t t aaa gcg c t t a t a ggt gga cag get a t t c c c t a t l e u pro g l y v a l l y s a l a l e u i l e g i y g i y g i n a l a i l e pro t y r 506/151 a t a t e c gat gca ggg t t a aag t t c t e a acg a t e t a c gga ggc a c t i l e s e r asp a l a g i y l e u l y s phe s e r t h r i l e t y r g l y g l y t h r 566/171 t e c a t t t e a a t t a t a cca ggt aag t t c caa g t c aat gat gat a t a s e r i l e s e r i l e i l e pro g l y l y s phe g i n v a l asn asp asp i l e 626/191 a c t ace c a t cca g t c ccc t t t g t t gag g t a aag g t t a t t aaa c c a t h r t h r h i s p r o v a l pro phe v a l g l u v a l l y s v a l i l e l y s p r o 686/211 agt t a c aac gag a t t ggt gag t t a t a t g t t agg get c c c t g g t t a s e r t y r asn g l u i l e g l y g l u l e u t y r v a l a r g a l a p r o t r p l e u 746/231 aat aat ccc gaa gag act cag aga gcg t a t gac gag aat ggc t g g asn asn pro g l u g l u t h r g i n a r g a l a t y r asp g l u asn g i y t r p 806/251 t t a gca a t a a t a aca gag gaa ggg ggt e t a agg a t a g t c gac aga l e u a l a i l e i l e t h r g l u g l u g i y g l y l e u a r g i l e v a l asp a r g 866/271 aag age ggt ggt gag t g g ate c c c agt age g t a t t g gag t e c a t e l y s ser g i y g i y g l u t r p i l e pro ser ser v a l l e u g l u ser i l e 926/291 gca g t c gaa cag g t t gca g t a t t a gga t a t cca gac cag aaa t g g a l a v a l g l u g i n v a l a l a v a l l e u g i y t y r p r o asp g i n l y s t r p 986/311 gca g t a g t g aaa c t g aag c c a ggt caa agg a c t gat caa aaa gag a l a v a l v a l l y s l e u l y s pro g l y g i n a r g t h r asp g i n l y s g l u 1046/331 aga gat gca g t g aat aag ggc agg a t a aat aag t g g t g g e t a c c t a r g asp a l a v a l asn l y s g l y a r g i l e asn l y s t r p t r p l e u p r o 1106/351  gat aag a t a asp l y s i l e 1136/361 gee t t a agg ala leu arg  g t g t t t g t t gat aat a t g cca t t g a c t age aca gga aag a t a aat aaa e t c v a l phe v a l asp asn met p r o l e u t h r s e r t h r g l y l y s i l e asn l y s l e u aag gat g t g gga a t a ggt t a a agt ggg a t a gac a t t l y s asp v a l g l y i l e g l y OCH  t g t t a a a t t c c t gac  attcacctcaattttttaagtagaactgtatggctagattaatagcctcttgcattttatctaagctcataaacgccata tctcttttggactctattgcacacttctcatggcacggtatatgtataaaccctcccctagcgccaattcctttagcctc cctgagtacgaggaacatagtcatattacagatatacgatccagcactgtggcttatctctgagggaatgttcttagatt tcaggtattcaactaattcttctacaggcaaattagaaaatatcccgtcagctcattcatgtcaattctttgtcccttat atgtgactcccatgttatcaggtatcctggagtacttataatttatagcaattttctcaggagtgatcttagttcttcca aataccaatcctatacctaaaattaaatttaactaataacatcttatactctaaatcaccatttcttcaaactt Hind I I I  50 s e q u e n c e f r o m its c o n t a i n e d 1.1 44%  identity a n d 5 5 %  k b o p e n r e a d i n g f r a m e (orf) w e r e f o u n d t o s h a r e a b o u t  s i m i l a r i t y with  Pseudomonas  oleovorans  medium-chain-fatty-  a c i d - c o A ligase ( G e n b a n k a c c e s s i o n n u m b e r X 6 5 9 3 6 ) . T h e similarities a s r e v e a l e d  by  t h e B L A S T s e a r c h w e r e illustrated in a p p e n d i x I.  T h e E. coli J M 1 0 9  Sulfolobus  H i n d III-  Sulfolobus  DNA  s t r a i n that c o n t a i n s t h e p G E M 7 (+)  v e c t o r h a r b o r i n g t h i s 1.7  kb  X b a I f r a g m e n t w a s g i v e n t h e n u m b e r P D # 1 1 9 3 . S i n c e t h i s 1.7  kb  clone contained  a  1.1  k b orf,  its s e q u e n c e  was  deposited  into  G e n b a n k under accession number U43181.  Degenerate PCR amplification of the Sulfolobus fibrillarin  (ii)  gene fragment  A m u l t i p l e a l i g n m e n t of t h e t e n fibrillarin p r o t e i n s e q u e n c e s ( a s s h o w n in f i g u r e  3.1)  r e v e a l e d h i g h l y c o n s e r v e d s e q u e n c e b l o c k s at a n u m b e r of p o s i t i o n s w i t h i n t h e p r o t e i n . Six  degenerate  sequence  oligonucleotides  blocks. W h e n  were  d e s i g n i n g the  constructed  based  on  these  conserved  o l i g o n u c l e o t i d e s , all p o s s i b l e c o d o n s  were  i n c l u d e d e x c e p t f o r a r g i n i n e . O n l y a r g i n i n e c o d o n s A G A a n d A G G w e r e u s e d in t h e oligonucleotide s e q u e n c e a s data from the  Sulfolobus  g e n o m e project  C G N c o d o n s o n l y a c c o u n t for 4 % of total a r g i n i n e c o d o n u s a g e . T h e s e q u e n c e s are  l i s t e d in T a b l e 2 . 2  in t h e  s u g g e s t that  oligonucleotide  Material a n d M e t h o d s s e c t i o n . O f the  six  o l i g o n u c l e o t i d e s , t h r e e a r e s e n s e , a n d t h r e e a r e a n t i s e n s e . T h e c o m b i n a t i o n of s e n s e a n d a n t i s e n s e p r i m e r s u s e d for a m p l i f i c a t i o n a n d their e x p e c t e d p r o d u c t s i z e s a r e l i s t e d in T a b l e  3.1.  51 Table 3.1  Combinations of PCR primers and their expected product size  PCR reaction number  combination of primers  expected product size (nt)  1  oXW3 a n d oXW5  216  2  oXW3 a n d oXW7  375  3  oXW3 a n d 0XW8  600  4  oXW4 a n d oXW7  150  5  oXW4 a n d 0XW8  350  6  0XW6 a n d 0XW8  200  T h e s e six d e g e n e r a t e  o l i g o n u c l e o t i d e s w e r e u s e d a s p r i m e r s to try t o P C R  fibrillarin g e n e f r a g m e n t s f r o m  Sulfolobus  amplify  g e n o m e . A s a p o s i t i v e c o n t r o l t e m p l a t e in t h e  P C R e x p e r i m e n t s , p l a s m i d P D # 1 1 6 9 (the m e t h a n o g e n fibrillarin g e n e c l o n e ) w a s with  E. coli  Sulfolobus used  as  genomic  a n d the  plasmid D N A was  u s e d in e q u a l  g e n o m i c D N A in t h e P C R a m p l i f i c a t i o n . G e n o m i c D N A o f another  combinations indicating  DNA  of  positive  control.  oligonucleotides  that the  In all  conditions were  both can  controls, give  PCR  products  appropriate  for  of  reactions the  no obviously promising bands were recovered using  D N A a s P C R template.  A c o m p a r i s o n of  products and methanogen  Sulfolobus  with  M. voltae  was  using the  with  p r i m e r s . U n d e r t h e v a r i o u s P C R c o n d i t i o n s u s e d (refer to s e c t i o n 2 . 7 Methods),  molarity  expected  amplification  lengths,  in M a t e r i a l a n d  Sulfolobus  genomic  genomic D N A degenerate  g e n o m i c D N A control P C R products u n d e r o n e  Sulfolobus  PCR  particular DNA  P C R p r o d u c t at t h e a n t i c i p a t e d s i z e w a s o b t a i n e d ( l a n e s 2 , 5, 8,  1 2 , 14). In c o n t r a s t , t h e a m p l i f i c a t i o n s of  six  degenerate  t o u c h d o w n P C R c o n d i t i o n is i l l u s t r a t e d in F i g u r e 3 . 6 . W h e n t h e m e t h a n o g e n u s e d a s template,  mixed  D N A l a c k p r o d u c t s at t h e  was 10,  expected  s i z e s ( l a n e s 3 , 6, 9 , 1 1 , 1 3 , 15). B e c a u s e t h e r e a r e s o m e a m b i g u i t i e s in P C R  reaction  E. coli  genomic  #2 ( u s i n g o X W 3 a n d o X W 7 p r i m e r s ) , P D # 1 1 6 9 p l a s m i d w a s m i x e d with  52  o o c  u TJ  2 o.  CC  o  tr o  Q .  TJ  TJ TJ CD  CD Q . T-Q CM  o o  D_  'E en JS O.  o  ^  TJ  li  JS co CO ~ 2  OT  i  w  m  1 2 3 4 5 6 7 8 9  2  o  o  LO  O IX)  co  CO 2 CO  W 2  1011121314 15  (bp)  600 500 400 300 200  100  Figure 3 . 6 200  ng  A degenerate PCR Gel  M. voltae  or  S. acidocaldarius  units  Taq  p o l y m e r a s e in 1 x T a q b u f f e r in t h e p r e s e n c e of 1 0 0 p m o l e of e a c h p r i m e r , 0 . 1 2 5  DNA  was  PCR  amplified  with 2  mM  d N T P s (final v o l u m e 1 0 0 ul). A f t e r d e n a t u r i n g t h e D N A at 9 5 ° C f o r 5 m i n u t e s , t h e D N A w a s s u b j e c t e d to 2 4 c y c l e s t o u c h - d o w n P C R a m p l i f i c a t i o n . T h e c y c l e p a r a m e t e r s 9 5 ° C 3 0 " , 5 2 ° C - 4 1 ° C (with a half d e g r e e d e c r e a s i n g i n c r e m e n t f o r e a c h c y c l e ) 3 0 " , 1'. A f t e r t h e  t o u c h - d o w n amplification, further  temperature  a t 41 ° C w a s  performed. T h e  p o l y a c r y l a m i d e gel. After ethidium  voltae  30  PCR  c y l c e P C R with a f i x e d products were  b r o m i d e staining, the  annealing  examined  gel w a s  are  72°C  on a  photographed.  5% M.  g e n o m i c D N A w a s u s e d a s a c o n t r o l t e m p l a t e . Its P C R p r o d u c t s a r e l a b e l e d with  M f o l l o w e d b y t h e r e a c t i o n n u m b e r (1 t h r o u g h 6, a s d e s i g n a t e d in t a b l e 3 . 1 ) ,  and  the  e x p e c t e d P C R p r o d u c t s i z e s f o r e a c h c o m b i n a t i o n of p r i m e r s a r e g i v e n in p a r e n t h e s e s ( l a n e s 2 , 5, 8, 1 0 ,  12,  14). W h e n  Sulfolobus  D N A w a s u s e d , the products are  w i t h S f o l l o w e d b y t h e r e a c t i o n n u m b e r ( l a n e s 3 , 6, 9 , ( m e t h a n o g e n fibrillarin g e n e c l o n e ) w a s m i x e d with a n o t h e r c o n t r o l in r e a c t i o n #2  11,  E. coli  13,  15).  labeled  PD#1169 plasmid  genomic D N A and used as  ( P C R with p r i m e r s o X W 3 a n d o X W 7 ) . T h e p r o d u c t s w e r e  s h o w n in l a n e 4. T h e p r o d u c t s f r o m t h e s e c o n d r o u n d P C R a m p l i f i c a t i o n of t h e 3 7 5  bp  r e g i o n D N A p r o d u c e d in P C R r e a c t i o n S 2 w e r e s e p a r a t e d in l a n e 7 . L a n e 1 is 1 0 0  bp  D N A ladder.  53 DNA  and  u s e d a s another  c o n t r o l ( l a n e 4).  product w a s detected. T h e 375 r e a c t i o n #2  In t h i s p a r t i c u l a r c o n d i t i o n , n o 3 7 5  b p r e g i o n of P C R p r o d u c t s in  Sulfolobus  DNA  w a s e x c i s e d f r o m t h e g e l , t h e D N A in it w a s e l e c t r o e l u t e d , p u r i f i e d  bp  PCR and  u s e d f o r a n o t h e r r o u n d P C R a m p l i f i c a t i o n . T h e r e s u l t i n g p r o d u c t s ( l a n e 7) still l a c k a b a n d c o r r e s p o n d i n g t o t h e 3 7 5 b p f r a g m e n t p r e s e n t in M e t h a n o g e n D N A c o n t r o l ( l a n e 5).  T o identify w h e t h e r s o m e  Sulfolobus  fibrillarin g e n e f r a g m e n t s , f i v e  P C R p r o d u c t s with s i z e s c l o s e to c o n t r o l s a r e  Sulfolobus  degenerate  t h e v e c t o r p G E M T . T o test t h e possibility that t h e actually amplified  but the a m o u n t  Sulfolobus  products were  PCR  P C R p r o d u c t s w e r e c l o n e d into  Sulfolobus  fibrillarin  gene  was  of P C R p r o d u c t w a s t o o s m a l l t o v i s u a l i z e , run  on  a  5%  polyacrylamide  gel,  the  p i e c e s of  gel  c o n t a i n i n g D N A at t h e e x p e c t e d p r o d u c t s i z e r a n g e s w e r e c u t o u t , t h e D N A e l u t e d , a n d t h e n s u b j e c t e d t o a s e c o n d r o u n d of P C R a m p l i f i c a t i o n . T h r e e n e w l y a m p l i f i e d p r o d u c t s t h a t r a n at t h e s i z e s of t h e p o s i t i v e c o n t r o l s w e r e a l s o c l o n e d i n t o t h e v e c t o r p G E M T and  sequenced. Comparing  the  amino  acid s e q u e n c e translated  from  the  DNA  s e q u e n c e a f t e r t h e P C R p r i m e r to t h e fibrillarin p r o t e i n s e q u e n c e s f r o m o t h e r s p e c i e s ( s e q u e n c e s a s l i s t e d in f i g u r e 3.1) will i m m e d i a t e l y d e t e r m i n e w h e t h e r t h i s P C R p r o d u c t c o r r e s p o n d s to a fibrillarin  fragment.  S e q u e n c i n g t h e first  100  b p of t h e s e  clones  i n d i c a t e d n o n e c o r r e s p o n d e d to f r a g m e n t s of t h e fibrillarin g e n e ( d a t a n o t s h o w n ) .  (iii)  Raising polyclonal antibodies for methanogen fibrillarin to  detect similar sized proteins in Sulfolobus extracts  B e c a u s e p r o t e i n c o d i n g g e n e s a r e m o r e c o n s e r v e d at t h e a m i n o a c i d l e v e l t h a n the  nucleotide  Sulfolobus  level, further  fibrillarin  protein  experiments  w e r e c o n d u c t e d to a t t e m p t to d e t e c t  using antibodies  prepared  against  the  at the  recombinant  54 methanogen  fibrillarin  protein.  The  o v e r e x p r e s s i o n of  methanogen  fibrillarin,  the  g e n e r a t i o n of p o l y c l o n a l a n t i b o d i e s a n d t h e w e s t e r n blot a n a l y s i s a r e d e s c r i b e d b e l o w .  Plasmids p G E M (Studier  voltae  et  7 + a n d P E T 3 a a n d E. coli s t r a i n s J M 1 0 9  a/., 1990)  and  BL21(DE3)pl_ysS  Methanogen  w e r e u s e d f o r t h e c l o n i n g a n d e x p r e s s i o n of t h e  fibrillarin p r o t e i n . T h e m e t h a n o g e n fibrillarin g e n e w a s a m p l i f i e d b y P C R u s i n g  p r i m e r s o X W 9 a n d o X W 1 0 (refer to t a b l e 2 . 2 ) . T h e 5' p r i m e r , o X W 9 , c o n t a i n s a n N d e 1 s i t e ( C A T A T G ) in w h i c h A T G a l s o s e r v e s a s t h e start c o d o n f o r t h e fibrillarin  protein.  T h i s N d e 1 s i t e is f o l l o w e d b y 2 2 n u c l e o t i d e s e x t e n d i n g into t h e N - t e r m i n a l r e g i o n of t h e fibrillarin  is a n  octamer  s e q u e n c e a d d e d to facilitate the P C R p r o d u c t b e i n g cut b y N d e 1 restriction  enzyme.  The  3'  gene  b e y o n d the  primer,  oXW10,  A T G start  includes  codon;  from  its  5'  to t h e  3'  end  Nde  a  1 site  21-nucleotide  sequence  c o m p l e m e n t a r y t o t h e C - t e r m i n a l of fibrillarin g e n e , a B a m H1 r e s t r i c t i o n s i t e a n d a n additional  4-nucleotide  clamp  to facilitate  digestion. T h e  methanococcus  voltae  fibrillarin g e n e c l o n e w a s kindly p r o v i d e d b y D r . A m i r i ( P h i l i p p s U n i v e r s i t y ,  Marburg,  G e r m a n y ) . A 7 0 0 b p l o n g P C R p r o d u c t w a s o b t a i n e d w h i c h c o r r e s p o n d s t o t h e s i z e of t h e m e t h a n o g e n fibrillarin g e n e . T h i s p r o d u c t w a s t h e n c l o n e d into t h e p E T 3 a v e c t o r using the N d e 1 a n d B a m H 1 transformed fragment  s i t e s in t h e P C R p r i m e r s a n d t h e l i g a t e d p l a s m i d s w e r e  into J M 1 0 9 . T o f a c i l i t a t e  s e q u e n c i n g , t h i s P C R a m p l i f i e d fibrillarin  w a s s u b c l o n e d into t h e v e c t o r p G E M 7 +  between  the B a m H1  gene  and Xba 1  s i t e s . T h e r e s u l t i n g p l a s m i d w a s t r a n s f o r m e d into J M 1 0 9 . T w o o f t h e s e c l o n e s ( f r o m different sequence  PCR  reactions)  errors,  retransformed  into  pET3a strain  were  s e q u e n c e d . F o r the  plasmid containing BL21(DE3)pLysS  this to  PCR  PCR  obtain  P D # 1 1 9 4 . B e c a u s e in t h i s p r o t e i n e x p r e s s i o n s y s t e m , t h e  product  product the  E.coli  final  devoid  was  of  any  isolated  and  expression  strain  host contains an I P T G -  i n d u c i b l e T 7 R N A p o l y m e r a s e a n d t h e f o r e i g n p r o t e i n g e n e is t r a n s c r i b e d f r o m t h e  T7  p r o m o t e r in t h e v e c t o r , I P T G i n d u c t i o n of t h e T 7 R N A p o l y m e r a s e will i n d i r e c t l y i n d u c e  55 the  e x p r e s s i o n of t h e r e c o m b i n a n t  protein  (Studier,  1990).  Figure 3.7 s h o w s the  i n d u c t i o n o f e x p r e s s i o n b y I P T G . O v e r t h e t i m e I P T G w a s a d d e d t o t h e E. coli c u l t u r e , a p r o t e i n b a n d at a s i z e o f 3 0 k D a g r a d u a l l y a c c u m u l a t e d ( l a n e s 3 , 4 , 5 , 6 , f i g u r e 3 . 7 ) . T h e r e c o m b i n a n t m e t h a n o g e n fibrillarin p r o t e i n a c c u m u l a t e s t o a v e r y h i g h l e v e l a f t e r 4 h r s o f I P T G i n d u c t i o n a n d it a c c o u n t s f o r at l e a s t 2 0 % o f t h e t o t a l p r o t e i n in E. coli c e l l l y s a t e a s j u d g e d b y c o o m a s s i e b l u e s t a i n i n g ( l a n e 6 , f i g u r e 3 . 7 ) . In t h e 1 2 . 5 %  SDS-  P A G E , t h e r e c o m b i n a n t protein d i s p l a y s a mobility c o n s i s t e n t with a s i z e of a b o u t 3 0 kDa, somewhat  l a r g e r t h a n t h e p r e d i c t e d 2 6 k D a . In o r d e r t o p u r i f y t h i s  recombinant  p r o t e i n , t h e E. coli c e l l s w e r e l y s e d u s i n g a F r e n c h P r e s s a n d t h e r e s u l t i n g c e l l l y s a t e w a s c e n t r i f u g e d a t 3 0 , 0 0 0 g f o r o n e h o u r . T h e o v e r e x p r e s s e d p r o t e i n w a s f o u n d t o b e in t h e 3 0 , 0 0 0 g pellet fraction (lane 7 a n d 8, figure 3.7), s u g g e s t i n g that t h e r e c o m b i n a n t protein f o r m s inclusion b o d i e s . G u a n i d i n e h y d r o c h l o r i d e o r u r e a w a s u s e d to solubilize the inclusion bodies. W h e n the denaturants were r e m o v e d b y dialysis under various conditions,  t h e protein  always  reprecipitated,  indicating that t h e protein  w a s not  refolding properly.  Because  t h e o v e r e x p r e s s e d protein  c o u l d not b e purified  antibodies w e r e raised against the S D S - d e n a t u r e d form  in t h e n a t i v e  form,  ( r e f e r t o s e c t i o n 2 . 1 0 . 1 in  Material a n d M e t h o d s ) . T w o rabbits w e r e u s e d for t h e i m m u n i z a t i o n a n d after s e v e r a l b o o s t i n j e c t i o n s , a n t i s e r a w e r e o b t a i n e d . T h e s e r a w e r e u s e d in w e s t e r n b l o t s t o p r o b e for a potential  Sulfolobus  r e c o m b i n a n t protein a n d  fibrillarin  M. voltae  protein.  E. coli  total cell e x t r a c t s  containing the  cell lysates w e r e u s e d a s positive controls. T h e  r e s u l t s a r e s h o w n in f i g u r e 3 . 8 .  E v e n w h e n t h e r e c o m b i n a n t protein w a s diluted to 5 to 10 n g (lane 3, figure 3 . 8 B ) , a p r o t e i n b a n d a t t h e s a m e s i z e a s t h e e x p r e s s e d p r o t e i n is still r e c o g n i z e d b y h i g h l y d i l u t e d a n t i b o d i e s , s u g g e s t i n g that t h e a n t i b o d y i s o f h i g h affinity. A n e x t r a c t f r o m E. coli  56  fibrillarin  1  2  3  4  5  6  7  8  Figure 3.7 Induction of Expression by IPTG and the presence of recombinant protein in S-30 pellet fraction C u l t u r e s of s t r a i n P D # 1 1 9 4 w e r e g r o w n in Y T m e d i u m to a n A  6  0  o of 0 . 8 . T h e n I P T G  was  a d d e d to t h e c u l t u r e with f i n a l c o n c e n t r a t i o n of 0.4 m M . A t d i f f e r e n t c h o s e n t i m e p o i n t s , t h e c u l t u r e w a s c o l l e c t e d a n d p r o t e i n s a m p l e s w e r e p r e p a r e d a s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s , s e c t i o n 2 . 8 . P r o t e i n s a m p l e s (listed b e l o w ) w e r e r e s o l v e d b y e l e c t r o p h o r e s i s o n a 1 2 . 5 % S D S - P A G E g e l o n t h e P H A S T S y s t e m . L a n e 1 is t h e L o w M o l e c u l a r W e i g h t L a n e 2 is a p r o t e i n e x t r a c t f r o m 1 0 LI I  E. coli  Marker.  B L 2 1 ( D E 3 ) o v e r n i g h t c u l t u r e . L a n e s 3, 4, 5, 6  a r e e x t r a c t s o f 5 uJ c u l t u r e a f t e r 0 hr, 0 . 5 hr, 2 h r s a n d 4 h r s I P T G i n d u c t i o n , r e s p e c t i v e l y . L a n e 7 a n d l a n e 8 a r e S 3 0 a n d S 3 0 p e l l e t f r a c t i o n s f r o m a b o u t 1 0 uJ f u l l y I P T G i n d u c e d culture.  57  Figure 3.8  Western blot using antibodies raised against methanogen  fibrillarin (A)  12.5% SDS-PAGE gel showing the protein samples for western blot.  L a n e 1 is l o w m o l e c u l a r w e i g h t m a r k e r . L a n e 2 is t h e p r o t e i n e x t r a c t f r o m  E. coli  ul  BL21(DE3)  c u l t u r e of  induction  voltae  overnight culture. L a n e 3 a n d 4 a r e protein extract from  E. coli  e x p r e s s i o n strain  cells (about 30  ug e a c h ) .  f r o m d i f f e r e n t c u l t u r e s ( 2 0 ug  (B)  PD#1194  after 0 . 5  hr a n d  r e s p e c t i v e l y . L a n e 5 a n d 6 a r e t w o total p r o t e i n  lanes 9 and  100  L a n e s 7, 8 a r e  3  hrs  preparations  S. acidocaldarius  e a c h ) . D o u b l e t h e a m o u n t ( 4 0 ug)  protein  ul 100  IPTG of  M.  extract  was loaded  in  10.  Western blot result  T h e s a m e protein  s a m p l e s a s in p a n e l  (A) w e r e s e p a r a t e d  in a  12.5%  SDS-  P A G E g e l f o r w e s t e r n blot (B) with t h e f o l l o w i n g p r o t e i n s a m p l e v a r i a t i o n s : l a n e s 1 a n d 2 (B) c o r r e s p o n d t o l a n e 2 a n d 3 (A) r e s p e c t i v e l y , t h e s a m p l e in l a n e 3 w a s diluted 1 0  4  and 10  7  t i m e s a n d l o a d e d in l a n e s 3 a n d 4 of t h e  SDS-PAGE  f o r w e s t e r n blot (B) r e s p e c t i v e l y . T h e p r o t e i n c o n t e n t s of l a n e s 5 to 1 0 t h e s a m e to t h e  SDS-PAGE  gel  illustrated  in p a n e l  (A).  (top) o r X 2 r a b b i t ( b o t t o m ) a n d s u b s e q u e n t l y i n c u b a t e d with d o n k e y antibody  c o u p l e d with h o r s e r a d i s h  peroxidase  (B)  After transfer  i m m o b i l o n m e m b r a n e , t h e p r o t e i n w a s p r o b e d with a n t i b o d i e s f r o m X 1  secondary  (A)  ( r e f e r to  to  are an  rabbit  anti-rabbit Materials  a n d M e t h o d s , section 2.10.2). T h e primary antibody w a s diluted 1 0 , 0 0 0 fold a n d the s e c o n d a r y antibodies 7,000 fold. Antigen w a s d e t e c t e d by E C L . T h e a b o v e film r e s u l t e d f r o m 3 0 s e c o n d s ' e x p o s u r e .  58 Figure 3.8 (A)  94 67 43 —  30  ( T h e s i z e o f fibrillarin)  Figure 3.8 (B) 1  2  3  4  5  6  7  8  9  10  Probed with X1 Ab 30 kDa ( T h e s i z e of fibrillarin)  Probed with X2 Ab ~+  30 kDa ( T h e s i z e o f fibrillarin)  59 strain B L 2 1 ( D E 3 ) without the e x p r e s s i o n p l a s m i d w a s u s e d a s a n e g a t i v e control (lane 1, f i g u r e 3 . 8 B ) . T h e X 1 a n t i b o d i e s c r o s s - r e a c t with a h i g h m o l e c u l a r w e i g h t p r o t e i n in this extract,  w h i l e t h e X 2 a n t i b o d y c r o s s - r e a c t s with a p r o t e i n o f s i m i l a r s i z e to  the  r e c o m b i n a n t p r o t e i n . B e c a u s e t h e s a m e a n t i g e n p r e p a r a t i o n s w e r e i n j e c t e d into t h e t w o rabbits, the immune  differences  in t h e i r  b a c k g r o u n d of t h e  antibody  rabbits. A  reactivity a r e  likely d u e to the  protein a r o u n d 3 0  kDa was  individual  unambiguously  i d e n t i f i e d in t h e m e t h a n o g e n c e l l e x t r a c t u s i n g a n t i b o d i e s f r o m b o t h r a b b i t s a n d is t h e e n d o g e n o u s m e t h a n o g e n fibrillarin. T h e m e t h a n o g e n fibrillarin g e n e w a s  fortuitously  i d e n t i f i e d d u r i n g s e q u e n c i n g of t h e f l a n k i n g r e g i o n of h m v A g e n e ( A m i r i , 1 9 9 4 ) . T h i s e x p e r i m e n t d e m o n s t r a t e s that t h i s i d e n t i f i e d orf w h i c h w a s f o u n d t o b e h o m o l o g o u s to e u k a r y o t i c fibrillarin  is e x p r e s s e d in m e t h a n o g e n c e l l s .  a p p r o x i m a t e s i z e of fibrillarin w a s i d e n t i f i e d in t h e  However,  Sulfolobus  n o p r o t e i n at  the  protein extract ( l a n e s 7 to  10), i n d i c a t i n g t h i s a n t i b o d y c a n n o t c r o s s r e a c t with t h e p u t a t i v e  Sulfolobus  fibrillarin.  A s a n o t h e r c o n t r o l , t h e m e t h a n o g e n fibrillarin a n t i b o d y w a s u s e d t o p r o b e a y e a s t total c e l l extract (figure 3.9).  T h e a n t i b o d i e s from the two different rabbits r e c o g n i z e d  d i f f e r e n t g r o u p s of y e a s t p r o t e i n s : X 1 a n t i b o d y c r o s s r e a c t s w i t h a 3 0 k D a a n d a 4 0  kDa  proteins, X 2  3.9),  a n t i b o d y c r o s s r e a c t s with a n u m b e r of p r o t e i n s ( l a n e 2 , 3 , f i g u r e  p r e s u m a b l y b e c a u s e of t h e d i f f e r e n t  i m m u n e b a c k g r o u n d s of t h e individual rabbits.  H o w e v e r , a n t i b o d i e s f r o m b o t h r a b b i t s r e c o g n i z e d a p r o t e i n of a s i z e a b o u t 4 0  kDa  ( l a n e 2 a n d 3 , f i g u r e 3.9). It is h i g h l y likely that this p r o t e i n is t h e y e a s t fibrillarin p r o t e i n N O P 1 w h i c h is a r o u n d 3 8 k D a in s i z e . E v e n u s i n g l e s s d i l u t e d p r i m a r y a n t i b o d i e s , n o p r o t e i n of 3 0 k D a in t h e  Sulfolobus  c e l l e x t r a c t c o u l d b e d e t e c t e d ( l a n e 4 t o 7).  60  1 2 3 4 5 6 7 8  1 2 3 4 5 6 7 8  with X2 Ab  Figure 3.9 Western blot to detect yeast fibrillarin P r o t e i n s a m p l e s (listed b e l o w ) w e r e r e s o l v e d b y e l e c t r o p h o r e s i s o n a 1 2 . 5 % S D S - P A G E g e l o n the P H A S T S y s t e m a n d s u b s e q u e n t l y blotted to I m m o b i l o n - P  using a semi-dry method.  T h e b l o t t e d p r o t e i n s w e r e p r o b e d with a n t i s e r u m ( 1 : 1 0 0 0 d i l u t i o n ) f r o m r a b b i t s X 1 X2  (right). A n t i b o d y - a n t i g e n c o m p l e x e s w e r e d e t e c t e d with d o n k e y a n t i - r a b b i t  ( 1 : 5 0 0 0 ) a n d E C L (the w e s t e r n  (left) o r  antibodies  blot p r o c e d u r e w a s d e s c r i b e d in M a t e r i a l s a n d  Methods.  s e c t i o n 2 . 1 0 . 2 ) . T h e film s h o w n r e s u l t e d f r o m 5 s e c o n d s ' e x p o s u r e . T h e s a m p l e s in b o t h b l o t s a r e : l a n e s 1 a n d 8, c e l l e x t r a c t s f r o m  M. voltae ( a b o u t  5 u g e a c h ) ; l a n e 2 is 8  ug  yeast  total c e l l u l a r protein extract ( p r e p a r e d b y boiling y e a s t culture o b t a i n e d f r o m Dr. S a d o w s k i ' s l a b in e q u a l v o l u m e 2 x S D S l o a d i n g b u f f e r a s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s , s e c t i o n 2.8). S.  L a n e 3 is t h e s a m e a s l a n e 2 e x c e p t c o n t a i n i n g 5 ug  acidocaldarius  p r o t e i n (5  ug,  10  ug,  15  ug,  5  ug  p r o t e i n . L a n e s 4 , 5, 6, 7 a r e total  respectively).  61 Anti-fibrillarin nucleolar fibrillarin  antibodies  RNAs. and  The  the  have  been  very  useful  in  studying  eukaryotic  small  a n t i b o d y c o i m m u n o p r e c i p i t a t e s s n o R N A s in a s s o c i a t i o n w i t h  resulting  immunoprecipitated  s u b s e q u e n t R N A s e q u e n c i n g or R T - P C R  small  amplification  R N A s can  be  identified  (Tycowski a n d Steitz,  T y c o w s k i e r a / . , 1993; a n d Morrissey a n d Tollervey, 1993). T h e m e t h a n o g e n  3.7B)  M. voltae  cell  antibody and  as  a n d c o u l d b e u s e d t o s t u d y p o t e n t i a l s n o R N A m o l e c u l e s in m e t h a n o g e n s . with anti-fibrillarin  1989;  fibrillarin  a n t i b o d y r a i s e d in t h i s s t u d y r e a c t s with m e t h a n o g e n fibrillarin s t r o n g l y ( f i g u r e  l y s a t e s w e r e s u b j e c t e d to i m m u n o p r e c i p i t a t i o n  by  e x p e c t e d , fibrillarin w a s s p e c i f i c a l l y p r e c i p i t a t e d b y t h e p o s t - i m m u n e s e r a ( l a n e 7 , f i g u r e 3.9). T o j u d g e t h e s p e c i f i c i t y of c o p r e c i p i t a t e d R N A , i m m u n o p r e c i p i t a t i o n s u s i n g t h e p r e and post immune  s e r a w e r e c a r r i e d out. T h e  immunoprecipitated  R N A s were  end  l a b e l e d a n d d i s p l a y e d o n a n 8 % s e q u e n c i n g g e l . T h e a u t o r a d i o g r a m (not s h o w n in this t h e s i s ) r e v e a l s that the m o c k a n d real i m m u n o p r e c i p i t a t i o n s s h a r e a l a r g e n u m b e r R N A b a n d s of t h e s a m e s i z e s a l t h o u g h t h e R N A b a n d s w e r e m o r e i n t e n s e a n d c l e a r f o r R N A s that w e r e i m m u n o p r e c i p i t a t e d by the post i m m u n e s e r a .  (II)  SECTION TWO: RT-PCR cloning small RNAs from the S.  acidocaldarius extract containing pre-rRNA 5' ETS processing activity In o r d e r t o i d e n t i f y a n d c l o n e o t h e r s n o R N A s p e c i e s in a d d i t i o n t o U 3 , t h e R N A s in the purified S .  acidocaldarius  p r o c e s s i n g fraction w e r e u s e d a s t e m p l a t e s for  amplification.  S i x differently  sized small P C R  fractionated  on  a  5%  polyacrylamide  gel  RT-PCR  products were obtained. T h e y  and  individually  e x c i s e d from  the  were gel,  e l e c t r o e l u t e d , p u r i f i e d , c l o n e d into t h e p G E M T v e c t o r a n d s e q u e n c e d . O n e c l o n e w a s i d e n t i f i e d to b e a P C R artifact b e c a u s e t h e p r i m e r s e q u e n c e s w e r e i n c o r r e c t . A s f o r t h e o t h e r f i v e c l o n e s , t h e y r a n g e in s i z e f r o m 15 to 3 5 n t s with t h e e x p e c t e d p r i m e r  62  1 234 567  30 kDa-  Figure 3 . 1 0 lysate M. voltae  Detection of proteins immunoprecipitated from an M. voltae cell  or S .  acidocaldarius  protein extract  prepared a s Materials a n d  Methods,  s e c t i o n 2 . 8 w a s i m m u n o p r e c i p i t e d with a n t i s e r u m f r o m r a b b i t X 1 ( r e f e r to M a t e r i a l M e t h o d s s e c t i o n 2 . 1 0 . 3 ) . T h e i m m u n o p r e c i p i t a t e d protein s a m p l e s (listed b e l o w )  and were  s e p a r a t e d o n a 1 2 . 5 % S D S - P A G E gel o n the P H A S T S y s t e m . After s e m i - d r y transfer to a n i m m o b i l o n m e m b r a n e , t h e p r o t e i n w a s s e q u e n t i a l l y p r o b e d w i t h 1 : 1 0 , 0 0 0 d i l u t e d X1  s e r a a n d 1:7000 diluted s e c o n d a r y antibody. After E C L d e t e c t i o n , the  w a s e x p o s e d to is  as  membrane  X - r a y film f o r 1 0 s e c o n d s a n d d e v e l o p e d (the w e s t e r n blot p r o c e d u r e  describribed  in  Material  and  Methods  section  2.10.2).  Protein  sample  E. coli p r o t e i n  extract; l a n e 2, 5 u g  p r e c i p i t a t i o n w i t h p r o t e i n A - a g a r o s e b e a d s ; l a n e 6, p r o t e i n s m o c k  immunoprecipitated  a r r a n g e m e n t s a r e : l a n e 1, 5 to 1 0 n g of fully i n d u c e d  M. voltae t o t a l p r o t e i n e x t r a c t ; l a n e 3 , 1 0 ug S. acidocaldarius p r o t e i n e x t r a c t ; l a n e 4, M. voltae F r e n c h P r e s s l y s a t e s o l u b l e f r a c t i o n ( a b o u t 5 ug p r o t e i n ) ; l a n e 5 , m o c k  w i t h p r e - i m m u n e s e r a ; a n d l a n e 7, p r o t e i n s i m m u n o p r e c i p i t e d w i t h s e r a c o n t a i n i n g fibrillarin a n t i b o d i e s ( i m m u n o p r e c i p i t a t e d f r o m t h e s o l u b l e f r a c t i o n of a r o u n d 1 0 0 ug  voltae c e l l  pellet).  M.  63 sequences  p r e s e n t at b o t h e n d s . T w o  of t h e s e c l o n e s w e r e f o u n d t o b e  identical  ( P D # 1 1 9 9 ) . T h e s e q u e n c e s of t h e f o u r c l o n e d P C R p r o d u c t s c o n t a i n i n g t h e  correct  p r i m e r s e q u e n c e s a r e s h o w n in T a b l e 3 . 2 .  D a t a b a s e s e a r c h e s of t h e s e s e q u e n c e s d i d n o t r e v e a l a n y h o m o l o g y w i t h  known  s n o R N A g e n e s . T h e s e s m a l l P C R p r o d u c t s w e r e r a d i o - l a b e l e d a n d u s e d to p r o b e R N A extracted  from  the  p r o c e s s i n g fraction  detected.  Perhaps these  clones are  in n o r t h e r n  breakdown  hybridizations.  p r o d u c t s of  No  mRNA  signal  or  was  precursor  s e q u e n c e s of s t a b l e r R N A or t R N A transcripts that a r e c o p u r i f y i n g with p r o c e s s i n g a c t i v i t y a n d c a n v a r y f r o m o n e p u r i f i c a t i o n to a n o t h e r . T h e s e c l o n e s c a n a l s o b e P C R artifacts. Interestingly, the P D # 1 1 9 9 R N A c l o n e , w h i c h w a s i s o l a t e d twice, c o n t a i n s a n eight-nucleotide s e q u e n c e element  i d e n t i c a l to t h e  Sulfolobus  U 3 box C sequence.  T h i s s e q u e n c e h a s b e e n u n d e r l i n e d in T a b l e 3 . 2 . B o x e s C a n d D a r e p r e s e n t in m o s t of t h e k n o w n s n o R N A s a n d a r e i m p o r t a n t f o r t h e a s s o c i a t i o n of fibrillarin. T h i s s e q u e n c e s i m i l a r i t y m a y b e o n l y f o r t u i t o u s , but it is a l s o p o s s i b l e that t h i s P C R p r o d u c t c o d e s f o r p a r t of a  Sulfolobus  snoRNA.  64  Table 3.2  Cloned RT-PCR product sequences  clone I.D.#  Clone sequence  1  PD#1196  (RACE) C C T A C C G A C T G C G T A  PD#1197  (RACE) C T C T C A A G A T A A C T T A A T G C G I  PD#1198  (RACE) A A C T C T C A T T G A A T C A A G C G T T A T C T C G T  PD#1199  (oJC1) A C G A G A T A A C G C T T G A T T C A A T G A G A G T T A G A G A T G  1  The  primers  Amplification  are marked  at t h e t w o e n d s  (oJC2) I I I  (oJC2)  of t h e c l o n e s . R A C E  of c D N A E n d s ) a n d o J C 1 o l i g o n u c l e o t i d e  (oJC2)  (stands  for R a p i d  s e q u e n c e s a r e l i s t e d in T a b l e  2 . 2 in t h e M a t e r i a l a n d M e t h o d s s e c t i o n . c R A C E i s t h e c o m p l e m e n t a r y primer.  (cRACE)  s t r a n d of R A C E  65  IV  Discussion  A l t h o u g h t h e s t r i n g e n c y of t h e S o u t h e r n e x p e r i m e n t s g e n e t o d e t e c t its an  Sulfolobus  Sulfolobus  authentic  methanogen  fibrillarin  Sulfolobus.  used  fibrillarin  c o u n t e r p a r t is l o w ( a s d e s c r i b e d in R e s u l t s s e c t i o n o n e ) ,  fibrillarin g e n e c o u l d n o t b e i d e n t i f i e d , s u g g e s t i n g t h a t  probe must be very divergent from  Sulfolobus  Alternatively,  oligonucleotides  using methanogen  in t h e  may  lack this  P C R experiments  its p u t a t i v e  gene.  were  h o m o l o g u e in  S i n c e the  based on  degenerate  highly  conserved  fibrillarin s e q u e n c e s , t h e f a i l u r e t o a m p l i f y fibrillarin g e n e f r a g m e n t s f r o m t h e  Sulfolobus  g e n o m e a l s o i m p l i e s that  the  Sulfolobus  m a y not h a v e this g e n e . T h e o b s e r v a t i o n that two  a n t i - m e t h a n o g e n fibrillarin a n t i s e r a c a n r e c o g n i z e y e a s t fibrillarin y e t c a n n o t d e t e c t a n y candidate  protein  acidocaldarius  in  an  acidocaldarius  S.  protein  extract  indicates  p r o b a b l y d o e s not c o n t a i n a h o m o l o g o u s fibrillarin-like  that  protein.  A l l t h r e e s e p a r a t e l i n e s of e v i d e n c e t a k e n t o g e t h e r s u g g e s t t h a t S .  acidocaldarius  l a c k s t h e g e n e f o r f i b r i l l a r i n . If a g e n e l o s t e v e n t h a p p e n e d in t h e e v o l u t i o n p a t h  Sulfolobus, fibrillarin exception  modern  fibrillarin  Sulfolobus  for  Sulfolobus  most  other  snoRNAs,  it m u s t  be  an  It is still  c o n t a i n s a fibrillarin g e n e but it c o u l d n o t b e d e t e c t e d b e c a u s e  little s i m i l a r i t y  with t h e  is a d u p l i c a t e d g e n e a n d  l e s s stringent  and  t h a t it e n c o d e s a U 3 s n o R N A b u t n o t f i b r i l l a r i n .  h a p p e n s to b e p a r a l o g o u s to the Under  to  c e l l will n o t r e t a i n t h i s g e n e . H o w e v e r , s i n c e e u k a r y o t i c  b i n d s to a n d c o e x i s t s with U 3  p o s s i b l e that it s h a r e s  Sulfolobus  S.  methanogen  counterpart.  S. acidocaldarius  M. voltae  conditions, two  One  p o s s i b i l i t y is t h a t  contains only o n e c o p y  which  f l p A g e n e u s e d in t h e c l o n i n g e x p e r i m e n t s .  bands  can  be  detected  in t h e  methanogen  g e n o m i c D N A u s i n g t h e f l p A g e n e a s a p r o b e , s u g g e s t i n g t h a t a s e c o n d c o p y of t h e fibrillarin  gene  may  exist  in  M. voltae  (Amiri,  1994).  If t h e  putative  duplication  f i b r i l l a r i n g e n e p r e c e d e d t h e initial a r c h a e a s p e c i a t i o n s , t h e s e t w o g e n e s m a y  of  have  66 e v o l v e d t o o d i v e r g e n t l y to b e r e c o g n i z e d a s h o m o l o g o u s . A n o t h e r p o s s i b i l i t y is t h a t b e c a u s e a r c h a e a l i v e in e x t r e m e  environment, constant environmental  m a k e fibrillarin p r o t e i n u n d e r g o p o s i t i v e s e l e c t i o n . in d r a m a t i c a l l y d i f f e r e n t e n v i r o n m e n t : S . °C,  M. voltae  acidocaldarius  and  M. voltae  is a e r o b i c a n d l i v e in p H 4 ,  live 80  is strictly a n a e r o b i c a n d live in n e u t r a l p H , 3 2 ° C - 4 0 ° C ( R o b b et a l . , 1 9 9 5 ) .  T h e c o m p l e t e l y different under  S. acidocaldarius  fluctuations  s e l e c t i o n p r e s s u r e c a n m a k e t h i s p r o t e i n w h i c h is a l r e a d y  positive selection e v o l v e e v e n faster,  s o t h e firillarin  in t h e s e t w o  species  b e c a m e very divergent.  When  the anti-methanogen  fibrillarin a n t i b o d y w a s  u s e d to  coimmunoprecipitate  m e t h a n o g e n s n o R N A s t h a t a s s o c i a t e with f i b r i l l a r i n , a l a r g e n u m b e r of n o n s p e c i f i c RNAs  were  precipitated  antibodies also normally  from  the  methanogen  immunoprecipitate  a  cell  large  extract. amount  Eukaryotic  fibrillarin  of n o n s p e c i f i c  RNAs  i n c l u d i n g 5 S r R N A a n d t R N A s , but s p e c i f i c s m a l l R N A s s u c h a s U 8 , U 1 3 , U 1 5 c a n still b e readily identified from the i m m u n o p r e c i p i t a t e d R N A p o o l ( T y c o w s k i a n d Steitz, T y c o w k i et al.,  1989;  1 9 9 3 ) . F u r t h e r e x p e r i m e n t s , b e y o n d t h e s c o p e of t h i s t h e s i s , t o i m p r o v e  t h e q u a l i t y of t h e i m m u n o p r e c i p i t a t i o n a r e likely t o i d e n t i f y a n d f a c i l i t a t e t h e c l o n i n g of some methanogen s n o R N A genes.  In o r d e r to identify t h e p r o t e i n a n d R N A f a c t o r s r e q u i r e d f o r p r o c e s s i n g of t h e 5' E T S r e g i o n of t h e p r e - r R N A t r a n s c r i p t in S .  acidocaldarius,  w a s purified o n e to two h u n d r e d fold from the S . P o t t e r ( P o t t e r et al.,  a f r a c t i o n with p r o c e s s i n g activity  acidocaldarius  cell free extract b y Dr.  u n p u b l i s h e d d a t a , 1995). T h e purified extract w a s f o u n d to c o n t a i n  f i v e t o s i x p r o t e i n s p e c i e s b y s i l v e r s t a i n i n g , a n d a n u m b e r of s m a l l R N A s v i s u a l i z e d b y 5' [ a  3 2  P ] p C p l a b e l i n g t h e 3 ' e n d of R N A a n d e l e c t r o p h o r e s i n g o n a s e q u e n c i n g g e l  ( P o t t e r e r a / . , 1 9 9 5 b ) . S i l v e r s t a i n i n g of t h e p r o t e i n c o n s t i t u e n t s d i d n o t r e v e a l a n y b a n d c o r r e s p o n d i n g to t h e e x p e c t e d s i z e of fibrillarin. T w o of t h e m a j o r p r o t e i n c o m p o n e n t s  67 of t h e  purified p r o c e s s i n g extract  are the 55  kDa a  a n d |3 s u b u n i t s of h e a t  shock  p r o t e i n s ( H S P 6 0 s ) , a s identified by N-terminal peptide s e q u e n c i n g a n d b y w e s t e r n a n a l y s i s ( D u r o v i c et al.,  1 9 9 6 in p r e p a r a t i o n ) . T h e s e a a n d (3 s u b u n i t s w e r e p r e v i o u s l y  c h a r a c t e r i z e d a s c o m p r i s i n g t h e t h e r m o p h i l i c f a c t o r 5 5 ( T F 5 5 ) in T h e y a r e a b l e to form a n which  blot  18-membered  Sulfolobus  d o u b l e ring s t r u c t u r e c a l l e d a  c a n fold n a s c e n t or m i s f o l d e d p o l y p e p t i d e s (Trent  et  al.,  shibatae.  rosettasome  1991). T h e  TF55  p r o t e i n s a r e h o m o l o g o u s t o t h e H S P 6 0 f a m i l y of e u k a r y a l c y t o s o l c h a p e r o n i n s w h i c h a r e r e s p o n s i b l e f o r p r o t e i n f o l d i n g . P r e v i o u s l y it w a s k n o w n t h a t w h e n e u k a r y o t i c c e l l s a r e s u b j e c t e d to h e a t s h o c k , H S P s e n t e r the n u c l e u s a n d b i n d to t h e p a r t i c l e s , p r e s u m a b l y to h e l p r i b o s o m a l a s s e m b l y ( P e l h a m ,  pre-ribosomal  1986). Therefore,  HSPs  a r e l i k e l y to h a v e s o m e affinity f o r r R N A s . B u t u n d e r n o r m a l c e l l u l a r c o n d i t i o n s , w h e t h e r H S P s h a v e r o l e s in r R N A p r o c e s s i n g a n d w h e t h e r 5' p r e - r R N A l e a d e r p r o c e s s i n g o c c u r o n t h e r o s e t t a s o m e s u r f a c e , r e m a i n s u n c l e a r . A 6 0 k D a p r o t e i n in t h e p u r i f i e d that m i g r a t e s  a b o v e the T F 5 5  doublet on S D S - P A G E  e l e c t r o e l u t e d for s e q u e n c i n g (Potter, 1996,  extract  w a s e x c i s e d from a gel  and  u n p u b l i s h e d data). T h e s e q u e n c i n g result  i n d i c a t e d t h a t t h i s 6 0 k D a b a n d c o n t a i n s m u l t i p l e s m a l l p r o t e i n s p e c i e s . O n e is h i g h l y h o m o l o g o u s to a 7 k D a D N A - b i n d i n g p r o t e i n a n d a n o t h e r is h i g h l y h o m o l o g o u s to a n RNA  h e l i c a s e . S i n c e fibrillarin  cannot  be  identified  in t h e  p r o c e s s i n g activity,  the  o b j e c t i v e o f f u t u r e e x p e r i m e n t s will b e t o c l o n e t h e s e p r o t e i n g e n e s a n d s t u d y t h e i r f u n c t i o n s in t h e p r o c e s s i n g of r R N A .  It h a s b e e n o b s e r v e d that m u l t i p o l y p e p t i d e c e l l u l a r s t r u c t u r e s s u c h a s p r o t e a s o m e s a n d c h a p e r o n e s a l w a y s a s s o c i a t e w i t h a p a n e l of R N A m o l e c u l e s ( D r e y f u s s et 1988). A  U6-like  RNA  was  identified  to b e  among  the  R N A s in a s s o c i a t i o n  al., with  p r o t e a s o m e s ; it w a s s u g g e s t e d that t h e p r o t e a s o m e is a R N A - p r o t e i n p a r t i c l e a n d t h e a s s o c i a t e d R N A s a r e s p e c i f i c ( A r r i g o et a / . , 1 9 8 5 ) . H o w e v e r ,  recent c l o s e examination  h a s r e v e a l e d t h a t t h e R N A s a s s o c i a t e d with p r o t e a s o m e s f r o m v a r i o u s o r g a n i s m s ; with  68 chaperonins--  g r o E L from  E.coli  T. acidophilum,  a n d with t h e r m o s o m e f r o m  have  s i m i l a r s i z e p a t t e r n s . P a r t i a l s e q u e n c i n g of t h e m o s t a b u n d a n t R N A s p e c i e s d e t e r m i n e d t h a t t h e 1 2 0 n u c l e o t i d e s (nts) R N A s p e c i e s a r e 5 S r R N A a n d t h e 8 0 n t s R N A s p e c i e s are t R N A s (Pamnani  era/.,  1994). T h i s s t u d y i n d i c a t e d that t h e r e a r e a l w a y s  many  n o n s p e c i f i c R N A s a s s o c i a t e d with m u l t i p e p t i d e c e l l u l a r s t r u c t u r e s like p r o t e a s o m e s a n d c h a p e r o n e s . A s v i s u a l i z e d b y 5' [ a  Sulfolobus  extract  3 2  P]  p C p l a b e l i n g , t h e d i s t r i b u t i o n of R N A s in t h i s  w i t h 5' r R N A p r o c e s s i n g a c t i v i t y is v e r y s i m i l a r t o t h a t of  R N A s t h a t a s s o c i a t e tightly with t h e p r o t e a s o m e s , t h e r m o s o m e s a n d g r o E L s et al.,  typical  (Pamnani  1 9 9 4 ) . S i n c e T F 5 5 h a s b e e n d i s c o v e r e d t o b e t h e m a j o r p r o t e i n c o m p o n e n t of  t h e p u r i f i e d p r o c e s s i n g a c t i v i t y , a l a r g e n u m b e r of R N A s a r e l i k e l y t o b e n o n s p e c i f i c R N A s that a s s o c i a t e with t h e s e c h a p e r o n e m o l e c u l e s . H o w e v e r , were  identified  in  northern  hybridizations  oligonucleotide probe b a s e d on deduced (Potter e r a / . , 1995; Bachellerie, 1995,  using  Sulfolobus  a  U3  two  cDNA  U20-like  R N A species  probe  snoRNA  and  an  sequence  p e r s o n a l c o m m u n i c a t i o n ) . U 3 , w h i c h is n o r m a l l y  t e n t i m e s m o r e a b u n d a n t t h a n o t h e r s n o R N A s , w a s s h o w n to b e a m i n o r b a n d in t h e total  RNA  of t h e  purified  p r o c e s s i n g activity  (Durovic,  unpublished data,  1995),  i n d i c a t i n g s n o R N A s o n l y a c c o u n t f o r a s m a l l p e r c e n t a g e of t o t a l R N A in t h i s f r a c t i o n . T h i s c o u l d b e a m a j o r o b s t a c l e to c l o n i n g s n o R N A s f r o m t h e R N A p o o l of t h e p r o c e s s i n g activity s i n c e the R T - P C R section 2.7 explain  why  p r o c e d u r e d e s c r i b e d in M a t e r i a l a n d  will i n d i s c r i m i n a t e l y a m p l i f y no  specific s n o R N A was  R N A s from the amplified  by  purified Methods  R N A i n p u t . T h i s is l i k e l y  RT-PCR  in t h e  to  experiments  d e s c r i b e d in R e s u l t s s e c t i o n t w o . T h e r e f o r e , f u t u r e e x p e r i m e n t s s h o u l d a i m t o s e p a r a t e s n o R N A c a n d i d a t e s s e l e c t i v e l y from o t h e r n o n s p e c i f i c R N A s a n d t h e n to identify a n d c l o n e their g e n e s .  them  69 IV  Summary and Perspectives  W i d e l y s p r e a d in e u k a r y o t e s , s n o R N P s a r e i n t i m a t e l y i n v o l v e d in r R N A p r o c e s s i n g , r R N A m o d i f i c a t i o n a n d m a y b e a l s o r i b o s o m e a s s e m b l y . T h e r e c e n t i d e n t i f i c a t i o n of U 3 in  S. acidocaldarius  a n d fibrillarin in  M. voltae  e x p a n d t h e p r e s e n c e of s n o R N P s to  a r c h a e a l s p e c i e s . T h e g o a l of t h e w o r k p r e s e n t e d in t h i s t h e s i s w a s to i d e n t i f y e s s e n t i a l p r o t e i n a n d R N A c o m p o n e n t s , n a m e l y fibrillarin a n d s n o R N A s , of a n a c i t i v e , p u r i f i e d S .  acidocaldarius  5' E T S p r o c e s s i n g f r a c t i o n . S i n c e a r c h a e a l s n o R N P s s h o u l d b e  d i v e r g e n t f r o m their e u k a r y o t i c c o u n t e r p a r t s , c h a r a c t e r i z e d  Sulfolobus  very  s n o R N P s can  b e u s e d a s o u t g r o u p s t o infer e v o l u t i o n a r y i n f o r m a t i o n o n s n o R N P s a n d r e v e a l  their  m o s t c o n s e r v e d a n d t h u s m o s t important s e q u e n c e a n d structural e l e m e n t s .  To  S. acidocaldarius,  c l o n e t h e p u t a t i v e fibrillarin g e n e in  the  M. voltae  fibrillarin  g e n e w a s u s e d a s a h e t e r o l o g o u s S o u t h e r n p r o b e . In a d d i t i o n , p o l y c l o n a l a n t i b o d i e s were prepared against extract.  M. voltae  Finally, degenerate  sequences  in f i b r i l l a r i n  experiments fibrillarin  yielded  fibrillarin  and  Sulfolobus  u s e d to p r o b e a  protein  oligonucleotides were synthesized b a s e d on c o n s e r v e d  a n d w e r e u s e d a s p r i m e r s for P C R a m p l i f i c a t i o n . All t h e s e  negative  gene. Moreover,  results, s u g g e s t i n g that  Sulfolobus  b y s i l v e r s t a i n i n g t h e p r o t e i n s of t h e  d o e s not h a v e  a  purified p r o c e s s i n g  e x t r a c t , a p r o t e i n c o r r e s p o n d i n g to t h e s i z e of fibrillarin c o u l d n o t b e d e t e c t e d .  However,  c h a p e r o n i n T F 5 5 , a 7 k D a D N A binding protein, a putative R N A h e l i c a s e a r e  identified  in t h e  rRNA  unpublished  p r o c e s s i n g active data).  e l u c i d a t e their  Future  fraction  experiments  p o s s i b l e interaction  of  Sulfolobus  will b e  to  extract  ( P o t t e r et  clone these  w i t h U 3 a n d to d e t e r m i n e  p r o c e s s i n g of p r e - r R N A at t h e 5" E T S r e g i o n . T h e p r e p a r a t i o n  protein  al.,  1996  genes,  t h e i r f u n c t i o n s in of a n t i b o d i e s  to the  against  m e t h a n o g e n fibrillarin a l s o l a y s t h e f o u n d a t i o n t o s t u d y m e t h a n o g e n s n o R N A s s i n c e fibrillarin  antibodies  can  usually coimmunoprecipitate  s n o R N A s associated  with  70 fibrillarin ( T y c a n d S t e i t z , 1 9 8 9 ; T y c  etal.,  1993; a n d M o r r i s s e y a n d Tollervey, 1993).  R T - P C R w a s c o n d u c t e d to amplify s m a l l R N A s from the purified S .  acidocaldarius  r R N A p r o c e s s i n g f r a c t i o n . B e c a u s e of t h e p r e s e n c e of a l a r g e a m o u n t of n o n s p e c i f i c R N A s a n d R N A degradation products, no obvious s n o R N A s were amplified. experiments  should  aim  to  physically  separate  promising  RNA  species  Future from  n o n s p e c i f i c o n e s a n d s u b j e c t t h e m s e p a r a t e l y for R T - P C R a m p l i f i c a t i o n o r direct R N A sequencing.  71 BIBLIOGRAPHY  Achenbach-Richter and Woese, C R . Archaebacteria."  Altschul,  ( 1 9 8 8 ) " T h e r i b o s o m a l G e n e S p a c e r R e g i o n in  S y s t e m . Appl. Microbiol. 10:211-214  Stephen  F., W a r r e n  Gish,  Webb  Miller,  Eugene  W.  Myers  and  David J .  L i p m a n (1990). " B a s i c local alignment s e a r c h tool." J . M o l . Biol. 2 1 5 : 4 0 3 - 4 1 0  Baserga,  S.  J.  and  Ribonucleoproteins."  Steitz,  J.  A.  (1993)  In T H E R N A W O R L D  "The  Diverse  World  of  Small  E d i t e d b y G e s t e l a n d , R. F . a n d A t k i n s , J .  F. C o l d Spring Harbour Laboratory P r e s s pp 359-381  Bowman,  C.  M.,  J . E.  Dahlberg,  T.  Ikemura, J . Konisky, a n d  " S p e c i f i c I n a c t i v a t i o n of 1 6 S r i b o s o m a l R N A i n d u c e d b y c o l i c i n E 3  M.  Nomura  in vivo."  (1971)  P r o c . Natl.  A c a d . S c i . U . S . A . 68: 9 6 4 - 9 6 8  B r o s i u s , J . , T . J . D u l l , D. D. S l e e t e r a n d H . F. N o l l e r ( 1 9 8 1 ) " G e n e o r g a n i z a t i o n p r i m a r y s t r u c t u r e of a r R N A o p e r o n f r o m  Escherichia coli."  J . Mol. Biol.148:  A m i r i , K. A . ( 1 9 9 4 ) "Fibrillarin-like p r o t e i n s o c c u r in d o m a i n a r c h a e a . . "  and  107-127  J . Bacteriol.  176:  2124-2127  A r i s , J . P . a n d B l o b e l , G . ( 1 9 9 1 ) " c D N A c l o n i n g a n d s e q u e n c i n g of h u m a n fibrillarin, c o n s e r v e d protein r e c o g n i z e d by a u t o i m m u n e  antisera."  a  P r o c . Natl. A c a d . S c i . U . S . A .  88:931-935  Bachellerie,  J - P . , Michot,  B., Nicoloso, M.,  Balakin, A . , Ni, J . a n d  ( 1 9 9 5 a ) " A n t i s e n s e s n o R N A s : a f a m i l y of n u c l e o l a r R N A s with l o n g to r R N A . "  Fournier,  M. J .  complementarities  T I B S 20: 261-264  B a c h e l l e r i e , J . P . , N i c o l o s o , M . , Q u , L. H . , , M i c h o t , B . , C a i z e r g u e s - F e r r e r , M . , C a v a i l l e , J . a n d R e n a l i e r , M . H. ( 1 9 9 5 b ) " N o v e l i n t r o n - e n c o d e d s m a l l n u c l e o l a r R N A s with long sequence  complementarities  to  marure  rRNAs  involved  in  ribosome  biogenesis."  B i o c h e m . Cell Biol. 73:835-843  B e l t r a m e , M . a n d T o l l e r v e y , D . ( 1 9 9 2 ) " I d e n t i f i c a t i o n a n d f u n c t i o n a l a n a l y s i s of t w o binding sites o n yeast pre-ribosomal R N A . " Beltrame, rRNA."  E M B O J . 11: 1 5 3 1 - 1 5 4 2  M . , H e n r y , Y . a n d T o l l e r v e y , D . ( 1 9 9 4 ) " M u t a t i o n a l a n a l y s i s of a n  b i n d i n g site for the  U 3 s n o R N A in t h e 5' e x t e r n a l t r a n s c r i b e d  Nucl. A c i d s R e s . 22: 5 1 3 9 - 5 1 4 7  U3  essential  s p a c e r of y e a s t  pre-  72  B e r g e s , T . , P e r f a l s k i , E . , T o l l e r v e y , D . a n d H u r t , E . C . ( 1 9 9 4 ) " S y n t h e t i c l e t h a l i t y with fibrillain identifies modification."  N O P 7 7 p , a n u c l e o l a r protein r e q u i r e d for p r e - r R N A p r o c e s s i n g a n d  E M B O J . 13:3136-3148  B r o c k , T . D . , B r o c k , K . M . , Belly, R.T. a n d W e i s s R . L . (1972) "Sulfolobus: A N e w of S u l f u r - O x i d i z i n g B a c t e r i a L i v i n g at L o w p H a n d H i g h T e m p e r a t u r e . "  Genus  A r c h . Microbiol.  84: 5 4 - 6 8  B r u c e , A . G . , U h l e n b e c k , O . C . ( 1 9 7 8 ) " R e a c t i o n at t h e t e r m i n i with T 4 R N A l i g a s e . " N u c l . Acids R e s . 5:3665-3677  Caizergues-Ferrer,  M . , Mariottini, P., C u r i e , C , L a p e y r e , B., G a s , N., A m a l r i c , F.  and  A m a l d i , F. (1989) "Nucleolin from X e n o p u s laevis: c D N A cloning a n d e x p r e s s i o n during development."  Cappai.R.,  G e n e s and Devel. 3:324-333  Osborn.A.H.  and  Handman.E.  (1994)  L e i s h m a n i a m a j o r h o m o l o g u e to t h e fibrillarin g e n e . "  "Cloning  and  sequence  of  Mol. B i o c h e m . Parasitol. 64,  a  353-  355 C e c h , T . R . ( 1 9 9 3 ) " S t r u c t u r e a n d M e c h a n i s m of t h e L a r g e C a t a l y t i c a n d G r o u p II i n t r o n s a n d R i b o n u c l e a s e P."  In T H E R N A W O R L D  RNAs: Group I  Edited by Gesteland,  R. F . a n d A t k i n s , J . F . C o l d S p r i n g H a r b o u r L a b o r a t o r y P r e s s p p 2 3 9 - 2 6 9  Chant, J . and  D e n n i s , P . P . (1986) "Archaebacteria: transcription  r i b o s o m a l R N A s e q u e n c e s in H a l o b a c t e r i u m c u t i r u b r u m . "  a n d p r o c e s s i n g of  E M B O J . 5:  1091-1097  C l a r k , M . W . , Y i p , M . L . R., C a m p b e l , J . a n d A b e l s o n , J . ( 1 9 9 0 ) " S S B - 1 of t h e Saccharomyces  cerevisae  Is  a  Nucleolar-specific,  A s s o c i a t e d with the s n R 1 0 a n d s n R 1 1  Silver-binding  Small Nuclear R N A s . "  protein  J . C e l l Biol.  Yeast  That  Is  111:1741-  1751  C h u , S . , A r c h e r , R. H . , Z e n g e l , J . M . a n d L i n d a h l , L. ( 1 9 9 4 ) " T h e R N A of R N a s e  MRP  is r e q u i r e d f o r n o r m a l p r o c e s s i n g of r i b o s o m a l R N A . " P r o c . N a t l . A c a d . S c i . U . S . A . 9 1 : 659-663  D e n n i s , P . P . ( 1 9 8 6 ) " M o l e c u l a r B i o l o g y of A r c h a e b a c t e r i a . "  J . Bacteriol. 168:  471-478  D e n n i s , P . P . ( 1 9 9 1 ) " T h e r i b o s o m a l R N A o p e r o n s of h a l o p h i l i c a r c h a e b a c t e r i a . " General  a n d A p p l i e d A s p e c t s of H a l o p h i l i c  Microorganisms.  V a l e r a , F., P l e n u m P r e s s , N e w York, pp. 2 5 1 - 2 5 7  Edited  by  In  Rodriguez-  73 D o u d n a , J . A . a n d J . W . S z o s t a k ( 1 9 8 9 ) " R N A c a t a l y z e d s y n t h e s i s of strand R N A . "  complementary  Nature 339: 519-524  D r e y f u s s , G . , Matunis, M . J . , P i n o l - R o m a , S . a n d Burd, C . G . (1993) " h n R N P a n d t h e b i o g e n e s i s of m R N A . "  proteins  A n n u . R e v . B i o c h e m . 62: 289-321  D u r o v i c , P . a n d D e n n i s , P . P . ( 1 9 9 4 ) " S e p a r a t e p a t h w a y s f o r e x c i s i o n a n d p r o c e s s i n g of 16S  and  23S  rRNA  hyperthermophilic  from  the  archaebacterium  primary  rRNA  operon  transcript  from  the  S u l f o l o b u s acidicaldarius: similarities to eukaryotic  r R N A p r o c e s s i n g . " M o l . M i c r o b i o l . 13(2): 2 2 9 - 2 4 2  Elela,  S.  A . , Igel,  H.  and Ares,  M.  Jr. (1996)  P r e r i b o s o m a l R N A at a U 3 s n o R N P - D e p e n d e n t S i t e . "  "RNase  III  Cleaves  Eukaryotic  Cell 85:115-124  F o x , G . E . , E . S t a c k e b r a n d t , R. B . H e s p e l l , J . G i b s o n , J . M a n i l o f f , T . A . D y e r , R . S . W o l f e , W . E . B a l c h , R . T a n n e r , L . M a g r u m , L. B . Z a b l e n , R. B l a k e m o r e , R . G u p t a , L . B o n e n , B . J.  Lewis,  D. A . Stahl,  K . R . L u e h r s e n , K. N . C h a n , a n d C . R . W o e s e  p h y l o g e n y of prokaryotes."  (1980) " T h e  Science 209: 457-463  F r a g a p a n e , P., Prislei, S . , Michienzi, A . , Caffarelli., E . a n d B o z z o n i , I (1993) " A novel s m a l l n u c l e o l a r R N A ( U 1 6 ) is e n c o d e d i n s i d e a r i b o s o m a l protein intron a n d o r i g i n a t e s b y p r o c e s s i n g of t h e p r e - m R N A "  Garrett, R . A . , Dalgaard, r R N A operons."  E N B O J . 12: 2 9 2 1 - 2 9 2 8  J . larsen, N. Kjems, J . a n d Mankin,  A . S . (1992)  "Archaeal  T I B S 16:22-26  F o u r n i e r , M . J . a n d M a x w e l l , E . S . (1993) " T h e n u c l e o l a r s n R N A s : c a t c h i n g u p with t h e spliceosomal s n R N A s . " T I B S 18:131-35  Gegenheimer,  P . a n d A p i r i o n , D. (1981) " P r o c e s s i n g of p r o c a r y o t i c r i b o n u c l e i c a c i d . "  Microbiol. R e v . 45: 502-541  G e r b i , S . A . , S a v i n o , R., S t e b b i n s - B o a z , B., J e p p e s e n , C . a n d R i v e r a - L e o n , R. (1990) " A R o l e f o r U 3 S m a l l N u c l e a r R i b o n u c l e o p r o t e i n in t h e N u c l e o l u s ? " In T h e R i b o s o m e : Structure,  Function & Evolution edited  b y Hill, W . E . , M o o r e ,  P. B., Dahlberg, A . ,  S c h l e s s i n g e r , D . , G a r r e t t , R. A . a n d W a r n e r , J . R., A m e r i c a n S o c i e t y for M i c r o b i o l o g y , W a s h i n g t o n , D. C , p p . 4 5 2 - 4 6 9  G e r b i , S . A . (1995) "Small nucleolar R N A . "  B i o c h e m . Cell Biol. 73: 8 4 5 - 8 5 8  74 Gilbert, W . (1986) " T h e R N A World."  Girard,  J - P . , Lehtonen,  Nature 319: 618  H., Caizergues-Ferrer,  M., Amalric,  F., Tollervey,  D. a n d  L a p e y r e , B . ( 1 9 9 2 ) " G A R 1 is a n e s s e n t i a l s m a l l n u c l e o l a r R N P p r o t e i n r e q u i r e d f o r p r e r R N A p r o c e s s i n g in y e a s t "  E M B O J . 11:673-682  Girard.J.P., Feliu.J., Caizergues-Ferrer.M. fibrillarin m R N A s  a n d L a p e y r e , B . (1993) " S t u d y of multiple  r e v e a l s t h a t 3' e n d f o r m a t i o n in S c h i z o s a c c h a r o m y c e s p o m b e  is  s e n s i t i v e to c o l d s h o c k . " N u c l . A c i d s R e s . 2 1 , 1 8 8 1 - 1 8 8 7  Gogarten,  J . P., Kibak,  H . , Dittrich,  P., Taiz,  L., B o w m a n ,  E. J . , Bowman,  B. J . ,  M a n o l s o n , N . F . , P o o l e , R. J . , D a t e , T . , O s h i m a , T . , K o n i s h i , J . , D e n d a , K. & Y o s h i d a , M . (1989) P r o c . Natl. A c a d . S c i . U . S . A . 86: 6 6 6 1 - 6 6 6 5  G r e e n , R a n d J . W . S z o s t a k ( 1 9 9 2 ) " S e l e c t i o n of a r i b o z y m e t h a t is a s u p e r i o r t e m p l a t e in s e l f - c o p y i n g r e a c t i o n . "  S c i e n c e 258: 1910-1915  G u e r r i e r - T a k a d a , C , K. G a r d i n e r , T . M a r s h , N . P a c e , a n d S . A l t m a n . ( 1 9 8 3 ) " T h e R N A moiety  Hampl,  o f r i b o n u c l e a s e P is t h e c a t a l y t i c s u b u n i t of t h e e n z y m e . "  H . , H . S c h u l z e , a n d K. H . N i e r h a u s  Escherichia  coli  5 0 S subuits  involved  Cell 35: 849-857  (1981) " R i b o s o m a l c o m p o n e n t s  in t h e r e c o n s t i t u t i o n  of  from  peptidyltransferase  activity." J . Biol. C h e m . 2 5 6 : 2 2 8 4 - 2 2 8 8  Harlow,  E. and Lane,  D. (1988) "Antibodies, A Laboratory  Manual."  Cold  Spring  Harbour Laboratory P r e s s  H a r t s h o r n e , T . a n d A g a b i a n , N (1994) " A c o m m o n c o r e structure for U 3 s m a l l n u c l e o l a r RNAs."  Nucl. A c i d s R e s . 22: 3354-3364  H e n r i q u e z , R . , B l o b e l , G . a n d A r i s , J . P . ( 1 9 9 0 ) " I s o l a t i o n a n d S e q u e n c i n g of N O P 1 " J . Biol. C h e m . 265: 2 2 0 9 - 2 2 1 5  Henry,  Y . , W o o d , H., Morrissey, J . P., Petfalski,  (1994)  " T h e 5'  e n d of y e a s t  u p s t r e a m c l e a v a g e site."  5.8S  rRNA  E., Kearsey, S., a n d Tollervey,  is g e n e r a t e d  by exonucleases  from  D. an  E M B O J . 13: 2 4 5 2 - 2 4 6 3  H i g g i n s . D . G . a n d S h a r p . P . M . (1989). "Fast a n d sensitive multiple s e q u e n c e on a microcomputer." C A B I O S vol. 5,151-153.  alignments  75 H u g h e s , J . M . X . a n d A r e s , M . J . ( 1 9 9 1 ) " D e p l e t i o n of U 3 s m a l l n u c l e o l a r R N A inhibits c l e a v a g e in t h e 5' e x t e r n a l t r a n s c r i b e d s p a c e r of y e a s t p r e - r i b o s o m a l R N A a n d i m p a i r s formation of 1 8 S r i b o s o m a l R N A . "  E M B O J . 10: 4 2 3 1 - 4 2 3 9  H u g h e s , J . M . X . (1996) "Functional b a s e - p a i r i n g interaction b e t w e e n highly c o n s e r v e d e l e m e n t s of U 3 small nucleolar R N A a n d small subunit R N A . " J . M o l . Biol. 2 5 9 : 6 4 5 654  Hui,  I. a n d D e n n i s , P . P . ( 1 9 8 5 ) " C h a r a t e r i z a t i o n  of t h e r i b o s o m a l R N A g e n e c l u s t e r in  Halobium cutirubrum." J . B i o l . C h e m . 2 6 0 : 8 9 9  Iwabe, N., K u m a , K - l . , H a s e g a w a , M . O s a w a , S . a n d Miyata, T . (1989)  "Evolutionary  r e l a t i o n s h i p of a r c h a e b a c t e r i a , e u b a c t e r i a , a n d e u k a r y o t e s i n f e r r e d f r o m  phylogenetic  t r e e s of d u p l i c a t e d g e n e s . "  P r o c . Natl. A c a d . S c i . U S A 8 6 : 9 3 5 5 - 9 3 5 9  J a n s e n , R. P . , Hurt, E . C , K e r n , H . , L e h t o n e n , H . , C a r m o - F o n s e c a , M . , L a p e y r e , B. and Tollervey,  D . ( 1 9 9 1 ) " E v o l u t i o n a r y c o n s e r v a t i o n of t h e h u m a n  fibrillarin a n d its f u n c t i o n a l e x p r e s s i o n in y e a s t . "  nucleolar  protein  J . Cell Biol. 113, 7 1 5 - 7 2 9  J a n s e n , R., T o l l e r v e y , D. a n d Hurt, E . C . (1993) " A U 3 s n o R N P protein with h o m o l o g y t o s p l i c i n g f a c t o r P R P 4 a n d G B d o m a i n s is r e q u i r e d f o r r i b o s o m a l R N A p r o c e s s i n g . " E N B O J . 12: 2 5 4 9 - 2 5 5 8  Jarmolowski, A . , Zagorski, J . , Li, H. V .  a n d F o u r n i e r , M . J . ( 1 9 9 0 ) " I d e n t i f i c a t i o n of  e s s e n t i a l e l e m e n t s in U 1 4 R N A o f S a c c h a r o m y c e s c e r e v i s i a e . "  Jong,  A. Y.-S.,  Clark,  "Saccharomyces Binding Proteins."  M. W . , Gilbert,  cerevisiae  E M B O J . 9: 4 5 0 3 - 4 5 0 9  M . , O e h m , A . a n d C a m m p b e l , J . L.  S S B 1 Protein  (1987)  a n d its R e l a t i o n s h i p t o N u c l e o l a r R N A -  M o l . a n d C e l l . B i o l . 7: 2 9 4 7 - 2 9 5 5  J o y c e , G . F . a n d O r g e l , L . E . (1993) " P r o s p e c t s for U n d e r s t a n d i n g t h e O r i g i n of t h e R N A W o r l d . " in T H E R N A W O R L D e d i t e d b y G e s t e l a n d . , R. F . a n d A t k i n s . , J . F . C o l d S p r i n g Harbor Laboratory Press pp. 25  K a s s , S . , T y c , K., Steitz, J . A . a n d S o l l n e r - W e b b , B. ( 1 9 9 0 ) " T h e U 3 S m a l l  Nucleolar  R i b o n u c l e o p r o t e i n F u n c t i o n s in t h e F i r s t S t e p of P r e r i b o s o m a l R N A P r o c e s s i n g . "  Cell  60: 8 9 7 - 9 0 8  Keeling, P . L . , Charlebois, R . L a n d Doolittle.W.F. eubacterial form a n d eukaryotic content"  (1994) " A r c h a e b a c t e r i a l  genomes:  C u r r . O p i n . in G e n e t , a n d D e v e l . 4 : 8 1 6 - 8 2 2  76 King, T. C , Sirdeshmukh,  R. a n d S c h l e s s i n g e r , D. (1984) " R N a s e  III  o b l i g a t e f o r m a t u r a t i o n b u t n o t f o r f u n c t i o n of E s c h e r i c h i a c o l i p r e - 2 3 S  cleavage  rRNA."  is  Proc.  Natl. A c a d . S c i . U S A 81: 185-188  K i s s - L a s z i o , Z . , Henry, Y . „ B a c h e r r e , J - P . , C a i z e r g u e s - F e r r e r , M . , a n d K i s s , T . (1996) "Site-Specific R i b o s e Methylation Nucleolar R N A s . "  of P r e r i b o s o m a l R N A : A N o v e l F u n c t i o n f o r S m a l l  Cell 85: 1077-1088  K i s s , T . a n d Filipowicz, W . (1993) "Small nucleolar R N A s e n c o d e d b y introns of the h u m a n cell c y c l e regulatory g e n e R C C 1 . "  E M B O J . 12: 2 9 1 3 - 2 9 2 0  K j e m s , J . a n d G a r r e t t , R. A . ( 1 9 8 8 ) " N o v e l S p l i c i n g M e c h a n i s m f o r t h e R i b o s o m a l R N A Intron in t h e A r c h a e b a c t e r i u m D e s u l f u r o c o c c u s m o b i l i s "  Cell 54: 693-703  K r i n k e , L . a n d W u l f f , D . L. ( 1 9 9 0 ) " T h e c l e a v a g e s p e c i f i c i t y of R n a s e III "  Nucl. Acids  R e s . 18: 4 8 0 9 - 4 8 1 5  Kruger,  K., P . J . G r a b o w s k i , A . J . Z a u g , J . S a n d s D . E . G o t t s c h l i n g , a n d T . R . C e c h  (1982) "Self-splicing R N A : autoexcision i n t e r v e n i n g s e q u e n c e of T e t r a h y m e n a . "  a n d autocyclization  of t h e r i b o s o m a l R N A  Cell 31: 147-157  L a f o n t a i n e , D., V a n d e n h a u t e , J . a n d Tollervey, D. (1995) " T h e 1 8 S r R N A Dimlp  is r e q u i r e d for p r e - r i b o s o m a l  R N A p r o c e s s i n g in y e a s t . "  dimethylase  G e n e s & D e v e l . 9:  2470-2481  L a n e , B. G . , O f e n g a n d , J . , a n d G r a y , M . W . (1995) " P s e u d o u r i d i n e a n d 0 - m e t h y l a t e d 2  n u c l e o t i d e s : s i g n i f i c a n c e o f t h e i r s e l e c t i v e o c c u r a n c e in r R N A d o m a i n s t h a t f u n c t i o n in r i b o s o m e - c a t a l y z e d s y n t h e s i s of t h e p e p t i d e b o n d s in p r o t e i n s . "  Biochemie 77:7-15  L a p e y r e , B., B o u r b o n , H . a n d Amalric, F. (1987) "Nucleolin, the major nucleolar of g r o w i n g  eukaryotic  sequence."  Lapeyre,  cells: A n u n u s u a l protein structure  revealed  protein  b y the nucleotide  P r o c . Natl. A c a d . S c i . U . S . A . 8 4 : 1 4 7 2 - 1 4 7 6  B . , Mariottini,  P., Mathieu,  C ,  Ferrer,  P., Amaldi,  F., Amalric,  F. a n d  C a i z e r g u e s - F e r r e r , M . ( 1 9 9 0 ) " M o l e c u l a r c l o n i n g o f X e n o p u s fibrillarin, a c o n s e r v e d U 3 small nuclear ribonucleoprotein disease."  r e c o g n i z e d b y a n t i s e r a f r o m h u m a n with  autoimmune  M o l . a n d C e l l . Biol. 10: 4 3 0 - 4 3 4  L a r s e n , N. H., Letters, H., Kjems, J . a n d Garrett, R . A . (1986) "Evolutionary  Divergence  Between  t h e R i b o s o m a l R N A O p e r o n s of H a l o c o c c u s m o r r h u a e a n d D e s u l f u r o c o c c u s  mobilis."  S y s t e m . A p p l . M i c r o b i o l . 7: 4 9 - 5 7  77 L e e , W - C , X u e , Z . a n d M e l e s e , T . (1991) " T h e N S R 1 Specifically Motifs"  Binds Nuclear  localization  G e n e E n c o d e s a P r o t e i n that  Sequences and  h a s two  RNA  Recognition  J . of C e l l B i o l . 1 1 3 : 1 - 1 2  L e s s e r , C . F. a n d Guthrie, C . (1993) "Mutations S p e c i f i c i t y : I m p l i c a t i o n for t h e A c t i v e S i t e . "  Leverette,  in U 6 s n R N A T h a t A l t e r S p l i c e S i t e  Science 262:1982-1988  R. D . , A n d r e w s , M . T . a n d M a x w e l l E . S . ( 1 9 9 2 ) " M o u s e U 1 4  s n R N A Is a  P r o c e s s e d I n t r o n of t h e C o g n a t e h s c 7 0 H e a t S h o c k P r e - M e s s e n g e r R N A . "  Cell 71:  1215-1221  L i , H . V . , Z a g o r s k i , J . a n d F o u r n i e r , M . J . ( 1 9 9 0 ) " D e p l e t i o n of U 1 4 S m a l l N u c l e a r ( s n R 1 2 8 ) D i s r u p t s P r o d u c t i o n of 1 8 S r R N A in S a c c h a r o m y c e s c e r e v i s i a e . "  RNA  Mol. and  C e l l . B i o l . 10: 1 1 4 5 - 1 1 5 2  L y g e r o u , Z . , Mitchell,  P., Petfalski,  E., Seraphin, B., a n d Tollervey,  D.  (1994)  "The  P O P 1 g e n e e n c o d e s a p r o t e i n c o m p o n e n t c o m m o n to t h e R N a s e M R P a n d R N a s e ribonucleoproteins."  P  G e n e s & D e v . 8: 1 4 2 3 - 1 4 3 3  M a s e r , R. L. a n d C a l v e t , J . P . ( 1 9 8 9 ) " U 3 s m a l l n u c l e a r R N A c a n b e p s o r a l e n c r o s s l i n k e d in v i v o t o t h e 5 ' - e x t e r n a l t r a n s c r i b e d s p a c e r of p r e - r i b o s o m a l R N A . "  P r o c . Natl.  A c a d . S c i . U S A 86: 6 5 2 3 - 6 5 2 7  Maxwell,  E . S . a n d Fournier, M . J . (1995) " T h e small nucleolar R N A s "  Annu.  Rev.  B i o c h e m . 35: 897-934  M i t c h e l l , P . , P e t f a l s k i , E . , a n d T o l l e r v e y , D . ( 1 9 9 6 ) " T h e 3 ' e n d of y e a s t 5 . 8 S  r R N A is  g e n e r a t e d b y a n e x o n u c l e a s e p r o c e s s i n g m e c h a n i s m . " G e n e s & D e v . in p r e s s M o r r i s s e y , J . P. a n d Tollervey,  D. (1993) " Y e a s t s n R 3 0  Is a S m a l l N u c l e o l a r  RNA  R e q u i r e d f o r 1 8 S r R N A S y n t h e s i s . " M o l . a n d C e l l . B i o l . 13: 2 4 6 9 - 2 4 7 7  M o u g e y , E . B . , O ' R e i l l y , M . , O s h e i m , Y . , M i l l e r , O . L. J . , B e y e r , A . a n d S o l l n e r - W e b b , B . ( 1 9 9 3 a ) " T h e t e r m i n a l b a l l s c h a r a c t e r i s t i c of e u k a r y o t i c  r R N A transcription  units  in  c h r o m a t i n s p r e a d s a r e r R N A p r o c e s s i n g c o m p l e x e s . " G e n e s & D e v e l . 7: 1 6 0 9 - 1 6 1 9  Mougey,  E.  B.,  Pape,  L.  K.  and  Sollner-Webb,  B.  (1993b)  "A U 3  small  nuclear  ribonucleoprotein-requiring  p r o c e s s i n g e v e n t in t h e 5' e x t e r n a l t r a n s c r i b e d s p a c e r of  Xenopus  M o l . C e l l . B i o l . 13: 5 9 9 0 - 5 9 9 8  precursor rRNA."  78 Noller, H . F . a n d J . B . C h a i r e s (1972) " F u n c t i o n a l m o d i f i c a t i o n of 1 6 S r i b o s o m a l R N A by kethoxal."  P r o c . Natl. A c a d . S c i . U . S . A . 6 9 : 3 1 1 5 - 3 1 1 8  Noller, H. F. (1991) " R i b o s o m a l R N A a n d translation."  A n n u R e v . B i o c h e m . 60: 191-  227  Noller,  H.  F., V,  Hoffarth  a n d L. Z i m n i a k  t r a n s f e r a s e to p r o t e i n e x t r a c t i o n m e t h o d s . "  (1992)  "Unusal  resistance  of  peptidyl  Science 256: 1416-1419  N o l l e r , H . F . ( 1 9 9 3 a ) " O n t h e O r i g i n of t h e R i b o s o m e : C o e v o l u t i o n o f S u b d o m a i n s o f t R N A and rRNA."  In T H E R N A W O R L D  E d i t e d b y G e s t e l a n d , R. F . a n d A t k i n s , J . F.  C o l d Spring Harbour Laboratory Press 137-156  Noller,  H . F . (1993b) "Peptidyl T r a n s f e r a s e : Protein, Ribonucleoprotein, or R N A ? "  J.  Bacteriol. 175: 5 2 9 7 - 5 3 0 0  Nicolso,  M., Caizergues-Ferrer,  M., Michot,  B., A z u m ,  M - C . a n d Bachellerie, J - P .  ( 1 9 9 4 ) " U 2 0 , a N o v e l S m a l l N u c l e o l a r R N A , Is E n c o d e d in a n Intron o f t h e  Nucleolin  G e n e in M a m m a l s " M o l e c u l a r a n d C e l l u l a r B i o l o g y 14: 5 7 6 6 - 5 7 7 6  N o m u r a , M . , S . M i z u s h i m a , M . O z a k i , P. T r a u b a n d C . V . Lowry (1969) "Structure a n d f u c t i o n of r i b o s o m e s a n d their m o l e c u l a r c o m p o n e n t s . "  Cold Spring Harbour  Symp.  Q u a n t . Biol. 34: 4 9 O s t e r g a a r d , A , L a r s e n , N., Letters, H., K j e m s , J . a n d Garrett, R. (1987) " A r i b o s o m a l RNA  operon  a n d its f l a n k i n g  thermoautotrophicum,  region from the A r c h a e b a c t e r i u m  Marburg  evolutionary implications."  strain:  transcription  signals,  Methanobacterium R N A structure  and  S y s t e m . A p p l . M i c r o b i o l . 9: 1 9 9 - 2 0 9  P a c e , N . R . a n d B u r g i n , A . B . ( 1 9 9 0 ) " P r o c e s s i n g a n d E v o l u t i o n o f t h e r R N A s " In T h e R i b o s o m e : Structure. Garret,  Function a n d Evolution.  Edited  b y Hill, W .  E . , Darlberg,  A.,  R. A . , M o o r e , P . B . , S c h l e s s i n g e r , D. a n d W a r n e r , J . R., A m e r i c a n S o c i e t y for  Microbiology, Washington, D . C . , pp. 417-425  Parker,  K.  A.  a n d Steitz,  Ribonucleoprotein with P r e - r R N A "  J . A.  (1987)  "Structural  Analyses  of t h e  Human  Particle R e v e a l a C o n s e r v e d S e q u e n c e A v a i l a b l e for B a s e  U3  Pairing  M o l . a n d C e l l . B i o l . 7: 2 8 9 9 - 2 9 1 3  P e c u l i s , B . A . a n d Steitz, J . A . (1993) "Disruption of U 8 N u c l e o l a r s n R N A Inhibits 5 . 8 S a n d 2 8 S r R N A P r o c e s s i n g in t h e X e n o p u s O o c y t e . "  Cell 73: 1233-45  79 P i p e r , P . W . , B e l l a t i n , J . A . , a n d L o c k h e a r t , A . ( 1 9 8 3 ) " A l t e r e d m a t u r a t i o n of s e q u e n c e s at t h e 3 ' t e r m i n u s of 5 S g e n e t r a n s c r i p t s in a lacks a R N A processing endonuclease."  Saccharomyces cerevisiae  m u t a n t that  E M B O J . 2: 3 5 3 - 3 5 9  Potter, S . , Durovic, P., a n d D e n n i s , P . P . (1995) " R i b o s o m a l R N A P r e c u r s o r P r o c e s s i n g b y a E u k a r y o t i c U 3 S m a l l N u c l e o l a r R N A - l i k e M o l e c u l e in a n A r c h a e o n "  Science  268:  1056-1060  Potter,  S . , D u r o v i c , P., R u s s e l l , A . , W a n g , X . , d e J o n g - W o n g , D. a n d D e n n i s , P.  (1995)  "Preriobosomal  RNA  endonuclease mediated  processing  in  Archaea:  p r o c e s s i n g of p r e c u r s o r 1 6 S  Sulfolobus acidocaldarius."  characterizing  r R N A in t h e  of  the  P.  RNP  thermoacidophile  B i o c h e m . C e l l Biol. 73: 8 1 3 - 8 2 3  Prislei, S . , M i c h i e n z i , A . , Presutti, C , F r a g p a n e , P. a n d B o z z o n i , I (1993) " T w o different s n o R N A s a r e e n c o d e d in i n t r o n s of a m p h i b i a n a n d h u m a n L1 r i b o s o m a l p r o t e i n g e n e s . " Nucl. A c i d s R e s . 21: 5824-5830  Q u , L - H . , N i c o l o s o , M., Michot, B., A z u m , M - C , C a i z e r g u e s - F e r r e r , M., Renalier, and  Bachellerie,  complementarity  J-P  (1994)  Reiter,  W.,  a  novel  small  nucleolar  RNA  Palm,  P.,  and  Zillig,  W.  (1988)  "Analysis  S u l f o l o b u s i n d i c a t e s that a r c h a e b a c t e r i a l  of  transcription  promoters are  13  M-H. nt.  Essential  role  of a  in  the  homologous  Nucl. Acids R e s . 16:1-19  R e i t e r , W . , H u d e p o h l , U . , a n d Z i l l i g , W ( 1 9 9 0 ) " M u t a t i o n a l a n a l y s i s of a n s e l e c t i o n in vitro."  a  Nucl. A c i d s R e s . 22: 4073-4081  t o e u k a r y o t i c p o l II p r o m o t e r s . "  promoter:  with  t o 2 8 S r R N A , in e n c o d e d in a n intron of r i b o s o m a l p r o t e i n L 5 g e n e in  chicken and mammals."  archaebacterium  "U21,  TATA  b o x for t r a n s c r i p t i o n  efficiency  archaebacterial and  start-site  P r o c . Natl. A c a d . S c i . U . S . A . 87: 9 5 0 9  R o b b , F T . , Place, A . R . , Sowers, K.R., Schreier, H.J., D a s S a r m a , S. a n d Fleischmann, E.M.  (1995)  "Archaea,  a  laboratory  manual."  Cold  Spring  Harbor  Laboratory  P r e s s , p p 13  S a m a h a , R. R., G r e e n , R. a n d N o l l e r , H . F . ( 1 9 9 5 ) " A b a s e p a i r b e t w e e n t R N A a n d r R N A in t h e p e p t i d y l t r a n s f e r a s e c e n t e r of t h e r i b o s o m e . "  23S  Nature 377:309-314  S a m b r o o k , J . , Fritsch, E . F., a n d Maniatis, T . (1989) " M o l e c u l a r c l o n i n g : a  laboratory  manual."  N.Y.  2 n d ed. C o l d Spring Harbor Laboratory Press, C o l d Spring Harbor,  80 S a v i n o , R . a n d G e r b i , S . A . ( 1 9 9 0 ) "In v i v o d i s r u p t i o n of X e n o p u s U 3 s n R N A  affects  r i b o s o m a l R N A p r o c e s s i n g . " E M B O J . 9: 2 2 9 9 - 2 3 0 8  S c h e e r , U . , T h i r y , M . a n d G o e s s e n s , G . ( 1 9 9 3 ) " S t r u c t u r e , f u n c t i o n a n d a s s e m b l y of t h e nucleolus."  T r e n d s in C e l l B i o l o g y 3 : 2 3 6 - 2 4 1  S c h i m m a n g , T., Tollervey, nucleolar  protein  D.,  K e r n , H., Frank,  r e l a t e d to m a m m a l i a n  R N A a n d is e s s e n t i a l for viability."  Schmitt,  R. a n d Hurt, E . C . ( 1 9 8 9 ) " A  fibrillarin  is a s s o c i a t e d w i t h s m a l l  yeast  nucleolar  E M B O J . 8:4015-4024  M . E . a n d C l a y t o n , D . A . ( 1 9 9 3 ) " N u c l e a r R n a s e M R P is r e q u i r e d f o r c o r r e c t  p r o c e s s i n g of p r e - 5 . 8 S  r R N A in  Saccharomyces  cerevisize."  M o l . C e l l . Biol. 13: 7 9 3 5 -  7941  S o l l n e r - W e b b , B. (1993) "Novel Intron-Encoded S m a l l Nucleolar R N A s . "  C e l l 75:  403-  405  Sontheimer,  E . J . a n d Steitz, J . A . (1993) " T h e U 5 a n d U 6  A c t i v e S i t e C o m p o n e n t s of t h e S p l i c e s o m e . " S c i e n c e 2 6 2 :  Small Nuclear  RNAs  as  1989-1996  S r i v a s t a v a , A . K. a n d S c h l e s s i n g e r , D . ( 1 9 9 0 ) " r R N A P r o c e s s i n g in E s c h e r i c h i a c o l i " In T h e R i b o s o m e : Structure.  F u n c t i o n a n d E v o l u t i o n . E d i t e d b y Hill, W .  E., Darlberg, A.,  G a r r e t , R . A . , M o o r e , P . B . , S c h l e s s i n g e r , D . a n d W a r n e r , J . R., A m e r i c a n S o c i e t y f o r Microbiology, Washington, D.C., pp. 426-434  S u n , S . a n d W o o l f o r d , J . L. J r . ( 1 9 9 4 ) " T h e y e a s t N O P 4 g e n e p r o d u c t is a n  essential  n u c l e o l a r p r o t e i n r e q u i r e d for p r e - r R N A p r o c e s s i n g a n d a c c u m u l a t i o n of 6 0 S  ribosomal  subunits."  E M B O J . 13: 3 1 2 7 - 3 1 3 5  S t a e h e l i n , T . , D . M a g l o t t , a n d R. E . M o n r o ( 1 9 6 9 ) " O n t h e c a t a l y t i c s i t e of t r a n s f e r : a p a r t of t h e 5 0 S r i b o s m e s t r u c t u r e . "  peptidyl  Cold Spring Harbor S y m p . Quant.  Biol.  34: 3 9 - 4 8  S t r o k e , I. L. a n d W e i n e r , A . M . ( 1 9 8 9 ) " T h e 5' e n d of U 3 s n R N A c a n b e c r o s s l i n k e d in v i v o t o t h e e x t e r n a l t r a n s c r i b e d s p a c e r of rat r i b o s o m a l R N A p r e c u r s o r s . " 210:  J . M o l . Biol.  497-512  S t u d i e r , F . W . , R o s e n b e r g , A . H . , D u n n , J . J . a n d D u b e n d o r f f , J . W . ( 1 9 9 0 ) " U s e of T 7 RNA 185:  P o l y m e r a s e to Direct 60-89  E x p r e s s i o n of C l o n e d G e n e s . "  Methods  in  Enzymology  81  Turley.S.J.,  T a n . E . M . and  Pollard,K.M.  (1993)  "Molecular  a n a l y s i s o f U 3 s n o R N A - a s s o c i a t e d m o u s e fibrillarin."  cloning  and  sequence  Biochim. Biophys. Acta  1216,  119-122  T h o m p s o n , L. D . , B r a n d o n , L. D . , N i e u w l a n d t ,  D. T . a n d D a n i e l s , C . J . ( 1 9 8 9 ) " T r a n s f e r  R N A i n t r o n p r o c e s s i n g in t h e h a l o p h i l i c a r c h a e b a c t e r i a . "  C a n . J . Microbiol. 35: 36-42  T o l l e r v e y , D . ( 1 9 8 7 ) " A y e a s t s m a l l n u c l e a r R N A is r e q u i r e d f o r n o r m a l p r o c e s s i n g of pre-ribosomal R N A . "  E M B O J . 6: 4 1 6 9 - 4 1 7 5  T o l l e r v e y , D., L e t h t o n e n , H., C a r m o - F o n s e c a , M . a n d Hurt, nucleolar R N P protein N O P 1  E . C . (1991) "The  small  (fibrillarin) is r e q u i r e d for p r e - r R N A p r o c e s s i n g in y e a s t . "  E M B O J . 10: 5 7 3 - 5 8 3  T o l l e r v e y , D . , L e t h o n e n , H . , J a n s e n , R., K e r n , H . a n d H u r t , E . C . ( 1 9 9 3 ) Sensitive Mutations  Demonstrate  "Temperature-  R o l e s for Y e a s t F i b r i l l a r i n in P r e - r R N A P r o c e s s i n g ,  P r e - r R N A Methylation, a n d R i b o s o m e A s s e m b l y . " Cell 72: 4 4 3 - 4 5 7  T r e n t , J . D.,  Nimmesgern,  E . , W a l l , J . S . , Hartl,  F - U . a n d H o r w i c h , A . L. ( 1 9 9 1 ) " A  molecular c h a p e r o n e from a thermophilic archaebacterium protein t-complex polypeptide-1."  Tycowski,  K.  T.,  S h u , M.  D.  is r e l a t e d t o t h e  eukaryotic  Nature 354:490-493  and  Steitz,  J . A.  (1993)  "A small  nucleolar  RNA  p r o c e s s e d f r o m a n i n t r o n of t h e h u m a n g e n e e n c o d i n g r i b o s o m a l p r o t e i n S 3 "  is  Genes  and Devel. 7:1176-1190  T y c o w s k i , K. T . , S h u , M . D . a n d S t e i t z , J . A . ( 1 9 9 4 ) " R e q u i r e m e n t f o r U22  S m a l l N u c l e o l a r R N A in 1 8 S  R i b o s o m a l R N A Maturation."  Intron-Encoded  Science 266:1558-  1561  Van  N u e s , R. W . , V e n e m a , J . , Rientjes, J . M . J . , D i r k s - M u l d e r ,  ( 1 9 9 5 ) " P r o c e s s i n g of e u k a r y o t i c  pre-rRNA:  the  role  of t h e  A and Raue,  transcribed  H.  A.  spacers."  B i o c h e m . Cell Biol. 73: 789-801  W i s e , J . A . ( 1 9 9 3 ) " G u i d e s to t h e H e a r t of t h e S p l i c e s o m e . " S c i e n c e 2 6 2 :  1978-1979.  W o e s e , . C R . a n d O l s e n , G . J . (1986) "Archaebacterial P h y l o g e n y : P e r s p e c t i v e s o n the Urkingdoms."  S y s t e m . A p p l . M i c r o b i o l . 7: 1 6 1 - 1 7 7  82 Woese,  C . R . , Kandler,  organisms:  O , Wheelis,  M . L . (1990)  P r o p o s a l for the d o m a i n s A r c h a e a ,  "Towards  Bacteria,  a  natural  and Eucarya."  system  of  P N A S 87:  4576-4579  W o e s e , C R . a n d N . R . P a c e (1993) "Probing R N A structure, comparative  analysis."  function a n d history by  in T h e R N A W o r l d , e d i t e d b y G e s t e l a n d , R . F . a n d A t k i n s , J . F .  C o l d Spring Harbor Laboratory Press, C o l d Spring Harbor pp. 91-117  83  Appendix I BLAST Search Results: >sp:ALKK_PSEOL MEDIUM-CHAIN-FATTY-ACID--COA ACYL-COA SYNTHETASE). Length = 546  LIGASE (EC 6.2.1.-)  (MEDIUM-CHAIN  P l u s S t r a n d HSPs: Score = 205 (94.1 b i t s ) , Expect = 3.5e-41, Sum P(5) = 3.5e -41 I d e n t i t i e s = 40/97 (41%), P o s i t i v e s = 54/97 (55%), Frame = + 1 Query: Sbjct: Query: Sbjct:  712 NGWFKTGDLAIITEEGGLRIVDRLKDVIKSGGEWIPSSVLESIISEIPAVEQVAVLGYPD 891 P + AV+ +GWF TGD+A I +G + I DR KD+IKSGGEWI + LESI 469 410 DGWFSTGDVATIDSDGFMTICDRAKDIIKSGGEWISTVELESIAIAHPHIVDAAVIAARH 892 QKWGERPMAWKLKPGQRTDQKEILEYLRDAVNKGRI 1002 +KW ERP+ + P E+ Y D V + +1 470 EKWDERPLLIAVKSPNSELTSGEVCNYFADKVARWQI 506  Score = 97 (44.5 b i t s ) , Expect = 3.5e-41, Sum P(5) = 3.5e-41 I d e n t i t i e s = 16/36 (44%), P o s i t i v e s = 27/36 (75%), Frame = + 1 Query: Sbj c t :  163 LSSNDVIMPFIPIYHLWSWGSAFIASYLGAKYVLTG 270 LS+ D I+P +P++H+ +WG+ + A+ +GAK VL G 217 LSARDTILPWPMFHVNAWGTPYSAAMVGAKLVLPG 252  Score = 81 (37.2 b i t s ) . Expect = 3.5e-41, Sum P(5) = 3.5e-41 I d e n t i t i e s = 14/35 (40%), P o s i t i v e s = 22/35 (62%), Frame = + 1 Query: Sbjct:  997 RINKWWLPDKIVFVDNMPLTSTGKINKLALRKDVG 1101 ++ +W +PD +FV+ +P TGKI K LR+ G 500 KVARWQIPDAAIFVEELPRNGTGKILKNRLREKYG 534  Score = 52 (23.9 b i t s ) , Expect = 3.5e-41, Sum P(5) = 3.5e-41 I d e n t i t i e s = 10/20 (50%), P o s i t i v e s = 13/20 (65%), Frame = + 1 Query: Sbjct:  19 DVKGGDPYSILFTSGTTGKP 78 DV + S+ +TSGTTG P 173 DVDENEASSLCYTSGTTGNP 192  Score = 36 (16.5 b i t s ) , Expect = 3.5e-41, Sum P(5) = 3.5e-41 I d e n t i t i e s = 5/19 (26%), P o s i t i v e s = 12/19 (63%), Frame = +1 Query: Sbjct:  388 KALIGGQAIPYNVAKSISD 444 + ++GG A P ++ + +D 298 RVWGGSACPASMIREFND 316  •  

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