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Characterization of recombinant plasmids carrying Drosphila melanogaster tRNA Serine7 genes and their… Spurr, Mark Gregory 1979

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CHARACTERIZATION  OF RECOMBINANT PLASMIDS  i D R O S P H I L A MELANOGASTER.tRNA. S E R I N E THEIR PREPARATION  F O R DNA  7  CARRYING  GENES AND  SEQUENCING  by Mark G r e g o r y B.Sc,  A Thesis  Spurr  University of B r i t i s h  Submitted  i n Partial  C o l u m b i a , 1977  F u l f i l l m e n t o f The  R e q u i r e m e n t s F o r The D e g r e e o f M a s t e r o f  Science  in  The  Faculty  (Microbiology,  We a c c e p t  this  o f Graduate  University of B r i t i s h  t h e s i s as conforming  The  Nov.  Columbia  1979  MARK GREGORY  Columbia)  to the required  University of B r i t i s h  ©  Studies  SPURR 1979  standard  In presenting t h i s thesis in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission f o r extensive copying of t h i s thesis f o r s c h o l a r l y purposes may be granted by the Head of my Department or by his representatives.  It i s understood that copying or p u b l i c a t i o n  of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission.  Department The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  BP  75-51  1 E  ABSTRACT  Specific  Drosphila  tRNA  Ser 4,7 p l a s m i d s were i d e n t i f i e d 125  isolated cules.  by h y b r i d i z a t i o n w i t h Seven c l o n e s  purified  were i s o l a t e d  [  S  e  and  r  I] tRNA 4,7 m o l e -  c a r r y i n g the D r o s p h i l a  Ser tRNA 4,7 gene and were f u r t h e r c h a r a c t e r i z e d by r e s t r i c t i o n e n d o n u c l e a s e d i g e s t i o n ; a g a r o s e g e l e l e c t r o p h o r e s i s and h y b r i 125  d i z a t i o n with The  r e s u l t s show t h a t  isolated, and  individual purified five  [  different  S  e  I] tRNA 4,7 DNA  one w h i c h a p p e a r s t o c o d e f o r two d i f f e r e n t contained  molecules.  f r a g m e n t s have b e e n  f o u r w h i c h code f o r a s i n g l e , s p e c i f i c  Two p l a s m i d s w h i c h i n i t i a l l y  r  isoacceptor, isoacceptors.  m u l t i p l e Hind I I I i n -  s e r t s upon p r i m a r y  i s o l a t i o n were r e c l o n e d t o c o n t a i n s i n g l e Ser H i n d I I I i n s e r t s c o n t a i n i n g t h e tRNA 4,7 g e n e . One o f t h e s e Ser r e c l o n e d p l a s m i d s c o n t a i n e d a s m a l l e r tRNA 4,7 gene c a r r y i n g i n s e r t than d i d i t s o r i g i n a l m u l t i p l e i n s e r t i s o l a t e . Small Ser tRNA 4,7 gene c a r r y i n g r e s t r i c t i o n f r a g m e n t s were l a b e l l e d 3 2  with and  T4 p o l y n u c l e o t i d e  kinase  and  electroeluted, i n preparation  [  p] ATP, s t r a n d  f o r nucleotide  separated,  sequencing.  iii  T A B L E  OF  C O N T E N T S  PAGE ABSTRACT  i i  L I S T OF TABLES.  V  L I S T OF FIGURES AND ILLUSTRATIONS  v i  ACKNOWLEDGEMENTS  viii  INTRODUCTION  1  MATERIALS  3  AND METHODS  Bacterial M  e  d  i  Strains  3 S  a  Isolation of Single Hybridization with  [  1 2 5  r  S p e c i f i c p D t 4,7 C l o n e s  o f DNA f r o m B a c t e r i a I]  e  tRNA  3  3  Colonies  4^7  4  Preparation  o f pDt - S e r i n e  p l a s m i d DNA  Restriction  Endonuclease Cleavage Conditions  7 8  Agarose G e l E l e c t r o p h o r e s i s  9  M o l e c u l a r Weight A n a l y s i s  9  Recloning of Multiple Electroelution  o f DNA f r a g m e n t s  Insert  Containing  Strand  tRNA  7  DNA 5' t e r m i n i  and  tf[ P] 32  Separation  Genes  14  With Polynucleotide  14  Gels  15 16 17  of Procedure  calculation  Kinase  ATP  RESULTS AND DISCUSSION Characterization  ..13  fragments  Containment Principle  10  o f DNA f r o m A g a r o s e G e l s . . . .  Dephosphorylation of pDtl6 S er Labeling  Plasmids  and M o l e c u l a r  17 Weight  of the Drospphila Insert Ser C h a r a c t e r i z a t i o n o f tRNA 7 plasmid C a r r y i n g S i m i l a r s i z e d Hind I I I i n s e r t s  17 19  J  iv  PAGE Recloning  of pDtl7  and  Preparation of pDtl6  pDt27  f o r Sequencing  K i n e t i c s of the P o l y n u c l e o t i d e Kinase 32 Strand Separation of X [ P ] ATP L a b e l l e d pDtl6 Fragments REFERENCES  31 45 Reaction  48  51 54  V  L I S T OF  TABLES PAGE  Table  I  Restriction  Endonuclease Hind I I I fragments of  Recombinant p l a s m i d s Table II  Restriction  18  Endonuclease fragments of  Plasmids Containing  !  Similar  Recombinant  s i z e Hind I I I  fragments..28  vi  L I S T OF FIGURES AND ILLUSTRATIONS  PAGE  Figure  1:  Single colony containing  isolation Ser  of b a c t e r i a  tRNA 4,7 genes  5  Figure  2:  S i z e c o n f i r m a t i o n o f pBR322 and lambda DNA....11  Figure  3:  Agarose g e l a n a l y s i s o f Hind  I I I digested  plasmids Figure  4:  20  H y b r i d i z a t i o n o f Hind 125 [ . I ] tRNA 4,7 S  e  r  I I I digested  plasmids  with Figure  5:  22  Agarose g e l a n a l y s i s of Hind  I I I digested  pDt27 Figure  6:  H y b r i d i z a t i o n o f Hind with  Figure  7:  24  i?5 [ I  8:  9:  l  y  s  5  26  p D t 1,5,81  ...29  I I I d i g e s t e d p D t , 1,5,81  P s t I + Hind  10: S k e m a t i c  32 S  e  I I I digested  r  7  with  Pst I  pDt 1,5,81  34  representation of the reverse  orientation Figure  125 and [ I] RNA  125 H y b r i d i z a t i o n o f [i ] tRNA and  Figure  r  7  A g a r o s e g e l a n a l y s i s o f P s t I and P s t I + Hind  Figure  e  pDt27  A g a r o s e g e l a n a l y s i s o f E c o R I and Mbol digested  Figure  S  ] tRNA  I I I digested  of pDtl  36  11: A g a r o s e g e l a n a l y s i s o f Hae I I I and Hae I I I + Hind  I I I digested  p D t 1,5,81  37  vii  PAGE Figure  12:  Hybridization of III  and Hae  125 [ i ] tRNA  I I I + Hind  S  e  r  7  with  Hae  I I I d i g e s t e d pDt 1,  5,81 Figure  13:  39  A g a r o s e g e l a n a l y s i s o f Hae I I I , and  Hae  I I I + Hind  Hind I I I  I I I digested  pDtl6  and  pDt27  41 Ser 12 5  Figure  14:  Hybridization of III,  Hind  [  I I I and Hae  digested pDtl6  I ] tRNA  7  with  Hae  I I I + Hind I I I  and pDt27  Figure  15:  Fragment p r e p a r a t i o n  Figure  16:  K i n e t i c s of the p o l y n u c l e o t i d e  43  g e l f o r pDtl6  46  kinase  reaction  49 32  Figure  17:  Polyacrylamide strand separation ATP l a b e l l e d p D t l 6 f r a g m e n t s  of  # [  P] 52  viii  ACKNOWLEDGEMENTS  I would  like  t o t h a n k t h e l a b o f D r . G. T e n e r , and  Dr. D a v i d L e u n g o f D r . M. S m i t h ' s l a b f o r t h e i r of  ideas  and a s s i s t a n c e  A special  to this  thanks goes  l e n t guidance, motivation, for  contribution  project.  t o D r . R.C. M i l l e r  for h i s excel-  a n d a s s i s t a n c e , a n d t o D.M.  her technical assistance.  Taylor  X  INTRODUCTION  Several rationale  features of Drosophila  for isolating  these genes.  First,  and d e v e l o p m e n t  tRNA g e n e s  r e c o m b i n a n t DNA  (Atwood  f o r normal  1968),  indicates  t h a t e x p r e s s i o n o f t h e s e genes  regulated  d u r i n g development  1973), and t h i r d , in prokaryotes lators 1964;  several  of wild  Sueoka Recent  and Kano Sueoko, s t u d i e s have  that at least  in Drosophila  (White e t a l , that at  act directly  (Ames and Hartman,  as r e g u -  1963;  the l o c a t i o n ,  i n Drosophila.  90 d i f f e r e n t  (White e t a l , 1 9 7 3 ) .  number  I t has  The  loci  o f t h e s e tRNA  one p a r t i c u l a r  be e n c o d e d by s e v e r a l  tRNA m o l e c u l e may  Elder,  s c a t t e r e d on t h e D r o s o p h i l a map.  1978)  p l a s m i d DNA  tRNA  by R e s t r i c t i o n  sequence Single  g e n e s have been  Any gene  ( T e n e r e t a l , 1979;  Kubli  and  isolated  molecules i n order to study t h e i r  and homology tide  S c h m i d t e t a l , 1978; Ser 7  and  tRNA s p e c i e s a r e p r o d u c e d  i n multigene c l u s t e r s .  Dunn e t a l , 1979;  Stent  been  m o l e c u l e s a p p e a r t o be o r g a n i z e d  clusters,  least  1964).  clarified  o r g a n i z a t i o n o f t h e tRNA g e n e s reported  is differentially  r e p o r t s have p o s t u l a t e d  of biochemical processes  growth  Second, e v i d e n c e  type f l i e s .  some tRNA m o l e c u l e s may  Schmidt,  1978;  on r e c o m b i n a n t organization,  endonuclease a n a l y s i s ,  and  nucleo-  analysis.  specific  tRNA  *-  molecules containing  they are e s s e n t i a l  of the f l y .  established a  Ser 7 p l a s m i d s were i s o l a t e d  during  2  cloning  e x p e r i m e n t s r e p o r t e d b y Dunn e t a l , (1979). The Ser i s o l a t e d tRNA 7 p l a s m i d s were i d e n t i f i e d by h y b r i d i z a t i o n  125 with to  specific,  purified  the procedure  plasmids  were  [  S  I] tRNA  e  7  r  molecules  o f G r u n s t e i n and Hogness  characterized  by  R.  (1975).  endonuclease  according Individual  cleavage,  aga-  12 5 rose tRNA  g e l e l e c t r o p h o r e s i s and h y b r i d i z a t i o n w i t h p u r i f i e d Ser 7 a c c o r d i n g t o S o u t h e r n (1975). Subsequently the  mids  were  ATP  labelled  with  T.4 p o l y n u c l e o t i d e  i n preparation f o r nucleotide  k i n a s e and  sequencing.  [  plas-  32  # [  I]  Pi  3  MATERIALS AND METHODS  Bacterial E.  coli  1977). by  Strains SF-8 was o b t a i n e d  The p l a s m i d pBR322  H. B o y e r .  Bacteria  f r o m M. O l s o n  containing  provided  pBR32 2 were grown i n L u r i a from t h e s e b a c t e r i a .  E.  SF-8 was t r a n s f o r m e d w i t h t h i s pBR322 DNA and r e s u l t i n g  colonies cycline. ated  eta l . ,  ( B o l i v a r e t a l . , 1977) was  b r o t h , a n d p l a s m i d DNA was o b t a i n e d coli  (Olson  were s c r e e n e d  for resistance  t o a m p i c i l l i n and t e t r a -  One c o l o n y r e s i s t a n t t o b o t h amp. a n d t e t . was  isol-  and u s e d h e n c e f o r t h a s t h e s o u r c e o f pBR322 DNA.  Media Bacteria  containing  (lOgm t r y p t o n e , Plates  5gm y e a s t  p l a s m i d s were grown i n L u r i a extract,  were made o f L u r i a b r o t h  Serine  plus  p l a s m i d s were numbered  were i s o l a t e d f r o m i n i t i a l c l o n i n g tified  as plasmid D r o s o p h i l a  Bacteria  containing  g e n e s were l o c a t e d colonies  lOgm/litre,  pH7.2).  agar.  S er S p e c i f i c p D t 4,7 C l o n e s  Isolation of Single The  5gm N a C l , p e r l i t r e  broth/  as they  e x p e r i m e n t s , and were  tRNA N, p D t N  plasmids carrying  on r e p l i c a p l a t e s  on n u t r i e n t  sequentially  iden-  (Dunn e t a l . ) . Drosophila  and s t r e a k e d  tRNA  for single  a g a r i n t h e a b s e n c e o f any a n t i b i o t i c .  4  Ten  c o l o n i e s were p i c k e d f r o m e a c h  grown up on n i t r o c e l l u l o s e  filters  streak,  and t h e c o l o n i e s  on n u t r i e n t  agar.  The c e l l s  on t h e n i t r o c e l l u l o s e were l y s e d by t h e method d e s c r i b e d by G r u n s t e i n and Hogness 125 [ I ] tRNA  ed w i t h  clones of c e l l s ing  S  (197 6 ) ,  e  and t h e l i b e r a t e d  r  4,7 a s d e s c r i b e d  were i s o l a t e d w h i c h Ser  D r o s o p h i l a tRNA4,7 g e n e s .  ed c e l l s  or c e l l s  below.  e l i m i n a t e d by s t r e a k i n g  I n t h i s way  contained plasmids  Any c o n t a m i n a t i n g  containing different  t i o n procedure be n o t e d . this  stage.  should  a plasmid a t f o r streaking  o f DNA  from B a c t e r i a C o l o n i e s w i t h filters  Ser 125 [ I] tRNA4,7  p r e p a r e d a s a b o v e , were (1976).  treated  The c e l l s  were  a n d t h e DNA was d e n a t u r e d w i t h 0.5 M NaOH f o r 7-10 m i n .  filters  were removed, washed w i t h two c h a n g e s o f 1M  pH7.4., and i n c u b a t e d i n 1.5M N a C l , A partial cubated  hybridiza-  One f e a t u r e o f t h e f i g u r e  as d e s c r i b e d by G r u n s t e i n and Hogness lysed,  Figure  media.  Nitrocellulose  The  of a typical  s t a g e o f t h e p r o c e d u r e , c o n f i r m i n g t h e need  Hybridization  were  tRNA4,7.  Not a l l the c o l o n i e s p i c k e d c o n t a i n  on non s e l e c t i v e  carry-  f o r s i n g l e c o l o n i e s on n o n s e l e c t i v e Ser  o f an a u t o r a d i o g r a m  at this  pure  non-transform-  D r o s o p h i l a tRNA  m e d i a f o l l o w e d by h y b r i d i z a t i o n w i t h l a b e l l e d 1 i s a photograph  DNA was a n n e a l -  vacum was a p p l i e d  i n pronase  0.03M sodium  (lmg/ml,  citrate)),  0.3 M N a C l , and b a k e d  0.5M T r i s  Tris  pH7.4 f o r 5 m i n .  below t h e f i l t e r s .  They were i n -  15 m i n , 22°C,  . (0.3M N a C l ,  2xSSC  washed w i t h e t h a n o l , c h l o r o f o r m and  f o r 3-4 h o u r s  a t 80°C,  between 3mm  paper.  5  FIGURE  1:  Single tRNA  colony isolation SER 4, 7 genes.  of  bacteria  containing  6  FIGURE  1: C o l o n i e s were s t r e a k e d on n u t r i e n t  of  antibiotic.  Individual  dary master p l a t e s , treated  as d e s c r i b e d  colonies  were t r a n s f e r r e d  (methods).  which d i d not anneal w i t h  In t h i s photograph  125 [ I] tRNA 4,7  analysis.  e  Positive colonies  and  colonies  r  show no  t h e presence o f b a c t e r i a which d i d not  tRNA 4,7 g e n e s .  t o secon-  replicated to nitrocellulose f i l t e r s  S  indicating Ser  agar i n t h e absence  were p i c k e d  shadow, contain  f o r subsequent  7  The  baked f i l t e r s  imately  5 x 10  5  were s a t u r a t e d w i t h  - 1 x 10  p l a c e d between m y l a r ped  the  s i x and  filters  one  cpm  sheets  in polyvinylidene  t i m e was  6  [  125  and  2xSSC c o n t a i n i n g a p p r o x S  The  h o u r s a t 6 5°C.  were washed i n  r  The  g l a s s p l a t e s and  (Saran Wrap).  half  e  l]tRNA4,7.  2XSSC  filters double  After  incubation  three times,  incubated  i n 2 X S S C , and  more t i m e s  filters  and  autoradiographed  film  (X-omat) and  8  6x10  t h e n was tion of  f o r 48  added  ug/ml, and  4 hours.  One  litre  resuspended  pH  egg  Two  the mixture EDTA pH fuged  8.0,  a t 4°C  ml  was  white  was  lysozyme  0.0625 M EDTA was min.  -70°C.  approximately  Chloramphenicol  to a f i n a l incubated  collected  i n 10ml  i n c u b a t e d a t 4°C  f o r 100  broth.  ethanol)  of c e l l s  screen  DNA  t h e c u l t u r e was  t i o n , washed and 8.0.  i n cassettes at  C in Luria  (80mg/ml i n 95%  two  were a i r d r i e d  c o n t a i n i n g b a c t e r i a were grown t o o  for  washed  hours u s i n g p r e f l a s h e d X-ray  - Serine plasmid  b a c t e r i a / m l a t 30  o f 200  The  Dupont s c r e e n s  P r e p a r a t i o n o f pDt Plasmid  SDS.  wrap-  optimal incubation  30.min. a t 37°C i n 10 mg/ml RNase A i n 2XSSC, 0.1%  were  25%  by  f o r 5 min.  added.  a t 25,000 rpm  a minimum centrifuga-  sucrose,  (5mg/ml) was  The  concentra-  0.05  M  added,  E i g h t ml l y s a t e was  i n a Beckman  Tris  and  0.25  M  centri42.1  rotor. The and  supernantant  was  centrifuged to equilibrium  e t h i d i u m bromide a c c o r d i n g to Katz  et a l .  (1973).  i n CsCl The  8  p l a s m i d DNA was c o l l e c t e d ,  t h e e t h i d i u m bromide  w i t h b u t a n o l , a n d t h e s o l u t i o n was d i a l y z e d NaCl, mid  0.02 M T r i s  pH 8.0, 1- mM EDTA.  DNA was p h e n o l e x t r a c t e d  R e s t r i c t i o n Endonuclease The  restriction  1 unit/ug  Hae  III:  f r o m New E n g l a n d  pH 7.5, 50 mM N a C l . ,  DNA was u s e d  lOmM T r i s  2  a t 37°C  pH7.4, 60 mM N a C l ,  7mM  MgCl~.  i n a digestion at  f o r 2-4 h o u r s  lOmM T r i s  37°C  I:  MgCl .  i n a digestion  pH7.4, 60 mM N a C l ,  1 u n i t / u g DNA was u s e d  Pst  5mM  2-4 h o u r s  37°C  Mbo I :  Biolabs.  (1979).  1 u n i t / u g D N A was u s e d  Hind I I I :  Hae I I I , H i n d I I I ,  t h e assay c o n d i t i o n s , m o d i f i e d from  those r e v i e w e d by R o b e r t s  for  The s o l u t i o n o f p l a s -  endonucleases EcoRI,  100 mM T r i s  0.02 M  Cleavage C o n d i t i o n s  DNA was c l e a v e d u s i n g  EcoRI:  against  a n d e t h e r washed.  Mbo I , P s t I , T a q I were p u r c h a s e d The  was removed  7mM  MgCl . 2  i n a digestion at  f o r 2-4 h o u r s .  6mM T r i s  pH 7.4, 150 mM N a C l ,  6mM  MgCl  ImM d i t h i o t h r e i t o l .  1 u n i t / u g DNA was  used  a t 37 C f o r 4 h o u r s  i n a digestion  6mM T r i s  pH 7.4, 5mM N a C l ,  dithiothreitol. a digestion  6mM M g C l „  ImM  1 u n i t / u g D N A was u s e d i n  a t 37 C f o r 4 h o u r s  9  Taq.  I:  lOmM T r i s  pH 7.4, 60mM N a C l , 7mM  1 unit/ug  DNA was u s e d  5 0°C  MgCl  2  i n a digestion at  f o r 3 hours  Agarose G e l E l e c t r o p h o r e s i s Gel  electrophoresis  1.0%, 1.3%, 2.0% a g a r o s e 0.04 M T r i s  phosphate  e t h i d i u m bromide, 1977).  DNA  a n a l y s i s was c a r r i e d (Bio-Rad)  out using  i n 0.09 M T r i s b o r a t e o r  pH 8.0, 0.02 M Na PC> , ImM 2  4  i n a Studier g e l apparatus  s a m p l e s were m i x e d  EDTA, l u g / m l  (McDonell e t a l . ,  w i t h 0.2 volume 40% s u c r o s e ,  0.025 M EDTA, 0.02% B r o m o p h e n o l b l u e b e f o r e l o a d i n g The at  e l e c t r o p h o r e s i s was c a r r i e d 85 v o l t s ,  12 m amp.  The g e l was e x p o s e d  and p h o t o g r a p h e d  t h r o u g h an o r a n g e  using  t y p e 57 p o l a r o i d  film.  DNA was t r a n s f e r r e d  c r i b e d by S o u t h e r n zed  M o l e c u l a r Weight The Hind  size  as p r e v i o u s l y  A n a l y s i s o f DNA  o f DNA  fragments  regression analysis,  a reference  filter  (series  filter  The f i l t e r s  were  6)  as deshybridi-  r  resulting  using  (Murray and M u r r a y ,  described.  Fragments  I I I d i g e s t i o n o f recombinant  linear  our  e  to ultraviolet  to nitrocellulose  (Southern 1975).  125 to [ i]tRNA 7 S  the g e l .  o u t a t 4°C f o r 2.5-3.5 h o u r s  light  The  0.5%,  f r o m R.  endonuclease  p l a s m i d s was d e t e r m i n e d by H i n d I I I c u t lambda DNA a s  1975).  The a u t h e n t i c i t y o f  pBR322 and t h e s i z e o f t h e lambda H i n d I I I f r a g m e n t s a s  10  reported gel  by  Murray  the  nucleotide  known, one  can  fragments  2).  Ligation:  found  2.5  incubated  with  2  phenol,  pDtl7. . o r p D t 2 7  III  and  added t o  M.  The  p r e c i p i t a t e was i n 0.5 2  two  ml  0.01  nucleotide  was  of M  was  stored  at  min  at  mixture and  cleaved  a  14°C.  carrying  the of  with  the  released  at  0.05  was  ATP.  the  M  extracted  times  R.  with  Ten  mixture  NaCl, twice  ether.  endonuclease cleaved  pH  and  hours. resuspend-  7.5,  u n i t s of was  of  added, 2  Hind  pBR322  concentration  Tris  III  phosphotase  -20°C was  M  Hind  60°C i n 0.06  the  final  with  c e n t r i f u g a t i o n and  1 mM  added, and  fragment  f o r a minimum o f  c o l l e c t e d by  was  various  alkaline  three  ethanol  buffer containing  ligase  hours at  The  -2 0°C  dithiothreitol,  are  0X174  the  cleaved  bacterial  added t o 95%  DNA.  cellulose.  solution containing was  3  autoradiography  p B R 3 2 2 DNA  chloroform  Four volumes of  mixture  MgCl ,  8.0.  amb  recloned.  u l f o r 60  (5ug)  the  for  pH  Sodium A c e t a t e  0.25  nitro  I I I were  i n 50  the  by  agarose  Plasmids  u n i t s of  once w i t h  s i z e s of  fragments  identified the  by  R.F.  0X174  c o n t a i n more t h a n one  1.4  Tris  The  DNA.  specific  to  confirmed  b o t h pBR322 and  relative  micrograms of  MgCl 0.06 M  with  to  Hind  (BAPF W o r t h i n g t o n ) 7mM  The  attached  digestion with  was  I cleaved  of M u l t i p l e I n s e r t  Plasmids  ( 1 9 7 5 ) was  sequences of  t R N A g e n e was  RNA-DNA h y b r i d s  Recloning  Pst  e s t a b l i s h the  (Figure  Drosophila  ed  Murray  electrophoresis with  Since  by  and  0.01 T4  M poly-  incubated  11  o  FIGURE  2:  Size  confirmation  of pBR322  and  lambda  DNA  12  FIGURE 2: The a u t h e n t i c i t y Hind  o f pBR322  I I I f r a g m e n t s was c o n f i r m e d  phoresis with  I I I ; l a n e 2,  t h e s i z e o f lambda  by a g a r o s e  P s t I c l e a v e d 0 X 1 7 4 amb  d i g e s t e d w i t h Hind with PstI;  and  0 X 1 7 4  3 DNA.  Lane 1 , X D N A  R.F. amb  l a n e 3, pBR322 d i g e s t e d w i t h H i n d  X DNA d i g e s t e d w i t h Hind I I I .  gel electro-  3 digested  I I I ; lane 4 ,  13  Transformation: 1:50  An o v e r n i g h t c u l t u r e o f SF-8 was  and grown t o a p p r o x i m a t e l y 10  Luria broth.  ml0.03 M C a C l  and r e s u s p e n d e d  treated  cells  taining  3 ug o f t h e l i g a t e d  The  (0.2ml) were m i x e d  cells  i n 3ml 0.03 M C a C l .  The  w i t h 0.05 ml o f s o l u t i o n  con-  2  i n c u b a t e d a t 4°C f o r 60  t h e t e m p e r a t u r e was  t o 42°C  raised  t h e n were d i l u t e d  on L u r i a  C o l o n i e s which  bridization,  into  came up on a m p i c i l l i n treated  to digest  were  resistance.  as d e s c r i b e d  plated  above.  Hy-  colony i s o l a t i o n  12 c o l o n i e s  were grown i n s u f f i c i e n t  enough p l a s m i d DNA  b r o t h and  p l a t e s were r e p l i c a  a u t o r a d i o g r a p h y , and s i n g l e  These  f o r each  quantity  single  oper-  parental  to give  and a n a l y z e on a g a r o s e g e l s .  clone w i t h the appropriate  i n s e r t was  10 ml L u r i a  minutes.  The t r a n s f o r m e d b a c t e r i a  a t i o n s were p e r f o r m e d w i t h a b o u t plasmid.  f o r 10  b r o t h a g a r and s c o r e d f o r d r u g  t o n i t r o c e l l u l o s e w h i c h was  A  D r o s o p h i l a tRNA gene  chosen.  E l e c t r o e l u t i o n o f DNA  from Agarose  P o r t i o n s of agarose g e l s were c u t o u t o f s l a b g e l s pipette,  The  i n 12  DNA.  grown f o r 2.5 h o u r s a t 30°C.  single  resuspended  t r a n s f o r m a t i o n m i x t u r e was  bacteria  plated  sedimented,  ice-chilled,  and i n c u b a t e d a t 4°C f o r 20 m i n .  2  t h e n were s e d i m e n t e d  minutes;  o a t 30 C i n  bacteria/ml  T w e n t y - f i v e ml o f c u l t u r e were  washed w i t h 10ml 0.01 M N a C l ,  The  9  diluted  Gels  containing  and p l a c e d  plugged with s i l a t e d  desired  DNA  in a vertical  g l a s s wool,  fragments  5 ml  plastic  and c o v e r e d w i t h  14  elution  buffer  (20mM T r i s  was  electroeluted  The  eluted  DNA  was  into  - A c e t a t e pH  a dialysis  phenol  bag  extracted,  8.0,  ImM  a t 150  volts  a minimum o f 2 h o u r s , d r i e d  distilled  water.  The  purity  DNA  f o r 8 hours.  e t h e r washed s i x t i m e s ,  e t h a n o l p r e c i p i t a t e d w i t h f o u r v o l u m e s o f 95% for  EDTA).  i n vacuo  of eluted  and  e t h a n o l a t -20?C resuspended  fragments  was  in  establish-  ed by e l e c t r o p h o r e s i s on an a g a r o s e g e l . Ser . C o n t a i n i n g tRNA 7 Genes  D e p h o s p h o r y l a t i o n o f pDt!6 f r a g m e n t s The method d e s c r i b e d Eleven min.,  by  G.  Korana  (1973) was  rpmoles o f 5' ends were h e a t d e n a t u r e d ice chilled,  500  mM  Tris  pH  8.0  was  u n i t s per r e a c t i o n of b a c t e r i a l  alkaline  and  i n c u b a t e d a t 60°C f o r 2 h o u r s .  The  was  phenol extracted,  for  2.5  phosphatase  F,  dephosphorylated  DNA  e t h e r washed, e t h a n o l p r e c i p i t a t e d  and  i n vacuo.  L a b e l i n g DNA Both and  a t 9 0°C  added, a l o n g w i t h  0.1  dried  employed.  5' t e r m i n i w i t h p o l y n u c l e o t i d e K i n a s e and  the forward r e a c t i o n  the exchange r e a c t i o n  by A. Maxim and W.  Gilbert  3 2  ;- [ . P ] A T P  f o r f l u s h o r r e c e s s e d 5'  for protruding  5'  termini  termini  described  ( p e r s o n a l c o m m u n i c a t i o n ) were  uti-  lized. Forward  reaction:  leotide  t e r m i n i were r e s u s p e n d e d  Spermidine,  0.1  mM  5  p m o l e s o f 5' d e p h o s p h o r y l a t e d o l i g o n u c i n 20 mM  EDTA, h e a t d e n a t u r e d  Tris  pH  9.5,  a t 90°C f o r 3.5  ImM min.,  15  and  ice chilled.  MgCl /  50 mM  2  with  Kinase b u f f e r  dithiothreotol,  50 p m o l e s & [ P ]  r e a c t i o n o f T4 cubated  a t 37°C f o r 60  cleotide 6.6,  50%  glycerol)  mM  pH  was  (2300 Ci/mmole)i,  20  9.5,  100  added units  mM  along per  t h e m i x t u r e was  in-  min.  6 p m o l e s o f 5' d e p h o s p h o r y l a t e d o l i g o n u -  t e r m i n i were r e s u s p e n d e d 100  Tris  p o l y n u c l e o t i d e k i n a s e , and  Exchange r e a c t i o n :  pH  ATP  3 2  (500 mM  MgCl , 2  50 mM  i n 500  mM  dithiothreotol,  imidazole 1 mM  HC1  spermidine, 32  1 mM  EDTA, 5 mM  (700 C i / m m o l e ) , cubated  adenosine  20 u n i t s T4  a t 37°C f o r 4 5  Both  diphosophate,  min.  r e a c t i o n s were e t h a n o l p r e c i p i t a t e d , At  taken  the r e a c t i o n mixture,  of  from  paper,  time  and  15,  30,  precipitated  X [  incorporated  Strand  0,  P] ATP  p o l y n u c l e o t i d e k i n a s e , and i n -  i n vacuo.  filter  7 3 p m o l e s X[  45,  60 min.  dried  a 1 u l sample  s p o t t e d on  i n TCA  and  was  a piece of  to determine  the  3mm amount  32  P ]A T P .  Separation Gels  After  5'  end  ments, i t was  labeling  the double  stranded plasmid  frag-  necessary to separate the strands to ensure  that  32  t h e i r was  o n l y one  5'  termini  to running a sequencing n i n g a 1:30 W. 2.5  Gilbert, mM  crosslinked,  labelled with  reaction. 4%  T h i s was  X [  P ] . ATP  accomplished  p o l y a c r y l a m i d e g e l , (A. Maxam  prior by  run-  and  p e r s o n a l c o m m u n i c a t i o n ) i n 50 mM T r i s - b o r a t e pH 8.3, 32 EDTA. The P l a b e l l e d p l a s m i d f r a g m e n t was resuspended  16  in  0.1  M NaOH, 1 mM  immediately overnight  loaded onto  a t 150  volts  in polyvinylidene 15-60 cut  min.  from  EDTA, h e a t d e n a t u r e d  film  w i t h X-ray  t h e g e l and  a t 90°C f o r 2  a p r e e l e c t r o p h o r e s e d g e l , and constant current. ( S a r a n wrap) and  no  screen f i l m .  electroeluted  The  g e l was  min., run wrapped  autoradiographed f o r Separated  s t r a n d s were  as d e s c r i b e d a b o v e .  Containment Bacteria u n d e r B-M  c o n t a i n i n g recombinant  containment  conditions,  t i o n s of the M e d i c a l Research eering  Research  Councils.  and  as  p l a s m i d s were specified  by  handled the  regula-  N a t i o n a l S c i e n c e s and  Engin-  17  RESULTS AND  Principle  of the  E. c o l i 1979)  DISCUSSION  Procedure  cells  containing  were i d e n t i f i e d  recombinant  as c a r r y i n g  specific  g e n e s by h y b r i d i z a t i o n w i t h p u r i f i e d Ser tRNA4,7  plasmids  (Dunn  et.al. Ser D r o s o p h i l a tRNA4,7  12 5 [ I] l a b e l l e d D r o s o p h i l a  m o l e c u l e s a c c o r d i n g t o t h e p r o c e d u r e o f G r u n s t e i n and  Hogness  (1975).  I n d i v i d u a l p l a s m i d s were c h a r a c t e r i z e d  several procedures:  Restriction  endonuclease  by  cleavage; agar-  o s e g e l e l e c t r o p h o r e s i s and h y b r i d i z a t i o n w i t h s p e c i f i c , p u r i 12 5 fied [ i ] l a b e l l e d tRNA a c c o r d i n g t o S o u t h e r n ( S o u t h e r n , 1 9 7 5 ) . Characterization  and M o l e c u l a r W e i g h t  C a l c u l a t i o n of the D r o s o p h i l a I n s e r t s T a b l e 1 d e s c r i b e s t h e s e v e n pBR322 - D r o s o p h i l a recombinant gene. Hind  The  plasmids c h a r a c t e r i z e d , which c a r r y  I I I , a n a l y z e d by 125  with  [  Southern  S  (1975).  regression  a g a r o s e g e l e l e c t r o p h o r e s i s and  annealed  r  The  Hind  size  o f DNA  u s i n g Hind  and M u r r a y 1 9 7 5 ) .  fragments  fragments,  I I I d i g e s t i o n , was  analysis,  (Murray  inserted  endonuclease  I] tRNA4,7 m o l e c u l e s a c c o r d i n g t o t h e p r o c e d u r e s  R-endonuclease  ence  e  Ser tRNA4,7  the  p l a s m i d s were c l e a v e d w i t h R e s t r i c t i o n  DNA  resulting  determined  I I I c u t lambda DNA  by  from  linear  as a  refer-  T a b l e 1 shows t h e s i z e o f  g e n e r a t e d by H i n d  III cleavage.  of  The  the  symbol  18  TABLE 1  Plasmid  tRNA Ser  4,7  Table  I  Hind  No.  III  PDt  1  4.4*  PDt  5  4.4*  PDt  16  6.2*  PDt  17  8.6*,  PDt  17R  3.7*  PDt  27  6.2*,  PDt  27R  6.2*  PDt  73  4.7*  PDt  81  4.4*  Fragments  2.0, 1.2,  (kb)  0.85,  2.5, 2.1, 1.1,  0.56 0.9  R. e n d o n u c l e a s e H i n d I I I f r a g m e n t s o f r e c o m b i n a n t plasmids. Each o f the recombinant plasmids l i s t e d  c l e a v e d by R. e n d o n u c l e a s e H i n d I I I ,  and h y b r i d i z e d w i t h c o m p l e m e n t a r y  of t h e i n s e r t e d Hind I I I fragments are  listed;  R:  signifies give  t h e 4.4  a new  bearing  that  kb pBR  [  Ser I ] tRNA 4 , 7 .  from the o r i g i n a l  322 p a r e n t a l  fragment  t h e p a r e n t p l a s m i d has been  plasmid  t h e tRNA  containing  only  refers  A l l  isolates  i s omitted.  recloned  the Hind  to  I I I fragment  gene.  125 *:  was  electrophonesed i n agarose 12 5  gels  here  to the insert  which  hybridizes  [  S  e  r  I ] tRNA 4 , 7  19  Ser (*)  indicates  the s i z e of the i n s e r t  Upon c l e a v a g e w i t h H i n d  III, five  shown t o c o n t a i n o n l y s i n g l e o f them c o n t a i n e d 5 i n s e r t s The d i f f e r e n c e that of pDtl,  anneals.  of the serine plasmids  inserts each  t o w h i c h tRNA4,7  o f D r o s o p h i l a DNA;  ( p D t l 7 and pDt27)  between t h e s i z e o f t h e i n s e r t i o n pDt5 and pDt81 i s s i g n i f i c a n t  were two  (Figure 3,4).  i n pDt 73 and  (Figure  4)  pDt27 was o f f u r t h e r i n t e r e s t . Upon i n i t i a l i s o l a t i o n t h e " s h o t gun" c l o n i n g e x p e r i m e n t s , i t was f o u n d t h a t Ser lys pDt27 h y b r i d i z e d b o t h tRNA4,7 and tRNA 5 . A f t e r H i n d I I I from  digestion, 125 [  agarose S  e  I] tRNA4,7 and  fragment  gel electrophoresis, 125  r  I  l  I] tRNA  f r o m pDt27 h y b r i d i z e d  Y  and h y b r i d i z a t i o n  with  S  5 , t h e same H i n d  III derived  b o t h D r o s o p h i l a tRNAs  (Figure  5,6) . Ser C h a r a c t e r i z a t i o n o f tRNA Hind  7  plasmid Carrying Similar  sized  III inserts Many o f t h e p l a s m i d s were c l e a v e d w i t h d i f f e r e n t  nucleases, different in  other than Hind plasmids carrying  I I I , t o ensure similar  sized  t h e c a s e o f p D t l 6 and p D t 2 7 , o r p D t l ,  R.  endo-  the i d e n t i t y of Hind  III inserts  pDt5 and pDt81  as  (Table  2) . (i)  pDtl,  p D t 5 , pDt81  D i g e s t i o n w i t h R. and  Homology endonuclease  pDt81 were t h e same i n s e r t Mbo  EcoRI i n d i c a t e d  and t h a t p D t l was  (Figure  7).  ed t h a t  t h e y had many s i m i l a r i t i e s  that  different  I d i g e s t i o n o f t h e same t h r e e p l a s m i d s (Figure 7).  pDt5  indicat-.  Digestion with  20  1 23456789 i,4  o  ti M  I 1 I" hi fc-4-  i i  FIGURE  3:  Agarose  gel  analysis  of  Hind  I I I digested  plasmids.  21  FIGURE 3: The  plasmids  cleaved with esed  p D t 1,5,16,17,27,73,81 a n d c o n t r o l  r e s t r i c t i o n endonuclease Hind  on 0.5% a g a r o s e i n T r i s - p h o s p h a t e  were v i s u a l i z e d b y s t a i n i n g w i t h to u l t r a v i o l e t l i g h t . i n s e r t s were d e t e r m i n e d lane  2, p D t l ;  6, pDt27; l a n e  lane  I I I and e l e c t r o p h o r -  buffer.  ethidium  3, p D t 5 ; l a n e  7, p D t 7 3 ; l a n e  4, p D t l 6 ;  The f r a g m e n t s  bromide and exposure  The number a n d s i z e s f o r each plasmid.  DNAs were  of the Drosophila L a n e 1, lane  8, p D t 8 1 ; l a n e  9,  ADNA;  5, p D t l 7 ; ADNA.  lane  22  2 3 4 5 6 78 O  FIGURE  4:  Hybridization 125 [ I ]  of S  tRNA  E  R  4,7  Hind  I I I digested  plasmids  23  FIGURE 4: The  agarose g e l photographed  the procedure of Southern 125 cubated  with  C  lane  lane  The t r a n s f e r r e d  DNA was i n -  Ser 13 tRNA 4 , 7 , f o l l o w e d by  L a n e s 2-8 c o r r e s p o n d pDtl;  (1975).  i n F i g . 3 was t r e a t e d by  to lanes  2-8 o f F i g u r e  3, pDt5; l a n e 4, p D t l 6 ;  7, pDt73; l a n e  8, p D t 8 1 .  lane  autoradiography. 3; i . e . , l a n e  5, p D t l 7 ;  lane  2,  6,pDt27;  24  1o 23  i  FIGURE 5:  Agarose  gel  analysis  of Hind  III  digested  pDt27  25  FIGURE  5:  The Hind  plasmid  I I I and were  phosphate  buffer.  pDt2 7 and  c o n t r o l DNA  electrophoresed Lane  1, ADNA;  were  on  0.5%  land  2,  cleaved  agarose pDt27;  in  land  with Tris3,  pDt27.  26  27  FIGURE  6:  Lanes the by  2 and  3 photographed  g e l and t r a n s f e r r e d the procedure  i n Figure  to separate  of Southern  (1975).  DNA  was  incubated  autoradiography;  125 with kb  (  lane L  I ] tRNA  fragment  y  3,  with  [  cut  nitrocellulose L a n e 2,  125 ferred  5 were  S  I ] tRNA  the transferred  e  the  filters trans-  r  4,7, DNA  from  followed  was  by  incubated  S  5,  f o l l o w e d by  hybridized  both  autoradiography.  tRNAs.  The  6.2  28 TABLE 2  Plasmid No. pDt 1  pDt 5  pDt 81  pDt 16  pDt 27  *:  Hind III  4.4*  Pst I  2.34, 1.81*, 1.17  PstI + Hind III  2.14, 1.39*, 0.92, 0.39, 0. 21  Hind I I I  4.4*  Pst I  3.64* , 0.99, 0.65  PstI;+Hind I I I  2.14, 1.39*, 0.92, 0.39, 0.21  Hind I I I  4.4*  Pst I  3.64* , 0.99, 0.65  PstI J-Hind I I I  2.14, 1.39*, 0.92, 0.39, 0. 21  Hind III  6. 2*  Hae III  0. 66*, 0.44*  Hind I I I + Hae III  0.66* , 0.44*  Hind III  6.2*  Hae I I I  0.35*  Hind III + Hae III  0.35*  /  refers to the insert which hybridizes [  Table 2  Fragment (kb)  Restriction .-eridonuclease  I] tRNA  7  R. Endonuclease fragments of Recombined Plasmids Containing Similar size Hind I I I Inserts  In order to d i s t i n g u i s h d i f f e r e n t plasmids containing similar sized Hind III i n s e r t s , the plasmids were cleaved with other R. endonucleases 125 ser electrophoresed i n agarose gels and hybridized with [ I] tRNA 7 . From t h i s table and figures 8, 9, 11, 12, i t was found that pDt 1, 5, ser 81, contained the same tRNA  7  i n s e r t , and that pDtl was i n reverse orien-  tation to pDt 5,81. Also from t h i s table and figures 13, 14, i t was found that pDt 16 and pDt 27 are d i f f e r e n t from each other, though they contain ser Hind I I I tRNA 7 inserts of equal size.  29  12345 6  FIGURE  7:  Agarose pDt  1,  gel 5,  analysis  81.  of EcoRI  and  Mbol  digested  FIGURE  7:  The  plasmids  p D t 1,5,81 were c l e a v e d w i t h  E c o R I and Mbol and e l e c t r o p h o r e s e d phosphate b u f f e r . pDt81 d i g e s t e d w i t h lane  lane  4, pDt5 d i g e s t e d w i t h M b o l ;  Mbol;  lane  first  suggested  insert  lane  Drosophila  Hind  lane  with  The E c o R I d i g e s t i o n s Drosophila  Hind I I I  The Mbol d i g e s t i o n s , a l t h o u g h t h a t p D t 1,5,81 c o n t a i n e d  III insert.  2,  EcoRI;  5, pDt81 d i g e s t e d  t h a t p D t l had a d i f f e r e n t  in a l l three wells.suggested lar  EcoRI;  3, p D t l d i g e s t e d w i t h  6, p D t l d i g e s t e d w i t h M b o l .  f r o m p D t 5,81.  endonucleases  on 0.5% a g a r o s e i n T r i s -  L a n e 1, pDt5 d i g e s t e d w i t h EcoRI;  R.  incomplete a  simi-  31  R.  endonucleases  in  pDtl  and  was  i n the  pDt81;  ferent  Pst  the  size  fragment  cleavage,  but  digestion  (Figure  R.  was  of  the  same  entation.  the  III revealed  hybridizing  same  8,9).  A  Hae  to  size  the  pDt5,  of the  pDt5,  tRNA  7  pDt81  Pst  I,  diagram  the  but  the  insert Ser  Hind of  III  the  in  pDt5  a  dif-  Pst  double  that  with pDtl  opposite  fragments  I  reverse  Digestion  i n the  insert  was  after  the (Conclusion  pDt81,  a l l three plasmids  that  the  i n F i g u r e 10.  III confirmed as  to  after  skematic  i s shown  insert  In  Hind  compared  pDtl  endonuclease  was  and  opposite orientation  i n pDtl  orientation  I  ori-  hybridizing  to  Ser the  tRNA  double :(ii)  7  were  the  digestion  pDt!6,  (Figure  pDt27  Digestion  same a f t e r  Hae  I I I , or  R.  endonucleases  Hae  I I I and pDt27  are  the  tRNA  multiple  annealed  gene  has  with  been  copies of  the  of  pDt!7  and  pDt27  I n many  cases  certain  Recloning  were  isolated  Both  pDtl7  and  only  [  I]tRNA  c l e a v e d by  gene  e  the  fragments  bearing  with  contained five  other  Hind  III  two  of  the  pDtl6  r  7,  s e p a r a t e d by  i n combination  pDt27  S  Hind  hybridizing  Since  125  either  III  Homology:  with  I I I fragments  I I I , Hind  11,12)  r e v e a l e d t h a t t h e f r a g m e n t s o f p D t l 6 and Ser tRNA 7 were d i f f e r e n t . ( F i g u r e s 13,14).  Hae  Hae  i t appears Hae a  I I I , or  Hae  III  specific Hind  that  III  III inserts.  there  site.  tRNA  genes  fragments. There-  3 2  1 23 456 0  FIGURE 8:  Agarose digested  gel pDt  analysis 1 S. 3  of  Pst  I and  Pst  I + Hind  III  33  FIGURE  8: The p l a s m i d s p D t l  tion on  endonucleases PstI  0.5%  agarose  after  1, p D t l d i g e s t e d  with  lane  lane  staining Hind  3, p D t l d i g e s t e d  pDt5 d i g e s t e d I;  and P s t I  with  Hind  6, pDt5 d i g e s t e d  with  restric-  I I I , electrophoresed  b u f f e r and p h o t o g r a p h e d with  I I I ; lane with  I I I ; lane with  cleaved  + Hind  i n Tris-Phosphate  u n d e r u.v. l i g h t  PstI;  and pDt5 were  ethidium  bromide.  2, p D t l d i g e s t e d  PstI + Hind  I I I ; lane  5, pDt5 d i g e s t e d  P s t I + Hind I I I .  Lane with 4,  with Pst  34  1 2 3 4 5 6  725 FIGURE  9:  Hybridization and  Pst  of I + Hind  [ I ]  S  tRNA  I I I digested  e  7  r  with pDtl 5. 3  Pst  I,  35  FIGURE  9:  The  agarose  g e l photographed  i n Fig.  8 was  t r e a t e d by  125 the  procedure Ser  tRNA  7.  i.e.  lane  digested III;  lane  gested  of Southern  Lanes  1-6  1, p D t l with 4,  with.PstI;  correspond  digested  PstI; pDt5  (1975) a n d i n c u b a t e d  lane  6,  with  Hind  3, p D t l  digested lane  to lane  with pDt5  1-6  Hind  with  I I I ; lane  digested  [  of Figure  I I I ; lane  digested  with  with  2,  I]  5;  pDtl  PstI  +  Hind  5, p D t 5 d i ^  PstI  + Hind I I I .  36  2.34kb 1.81 1.17 2.14 kb 1.39 • 0.92 0.39 0.21  Skematic  representation  tion  pDtl.  of  3.64 0.99 0.65  kb  of  the  reverse  orienta-  37  1 23456789  FIGURE  11:  Agarose Bind  gel  analysis  I I I digested  of Eae pDt  1>5 81 3  -  I I I and  O  Hae  III +  38  FIGURE  11: The  Hae  p l a s m i d s pDt  I I I + Hind  1,5,81 w e r e  I I I , electrophoresed  phosphate b u f f e r and p h o t o g r a p h e d staining Hae  I I I ; lane  digested Hae  with  with  I I I ; lane  ethidium 2, p D t l Hae  with  with  I I I ; lane  pDt81 d i g e s t e d  bromide. digested  I I I + Hind  Hae  I I I + Hind  with  2.0%  Lane with  with  Hae  agarose  u n d e r u.v.  light  I I I and in Trisafter  1, p D t l d i g e s t e d  Hind  I I I ; lane  7, p D t  with  I I I + Hind I I I .  I I I ; lane  3,  with pDtl  4, p D t 5 d i g e s t e d  Hind  I I I ; lane  8, p D t 8 1 d i g e s t e d Hae  on  I I I ; lane  5, p D t 5 d i g e s t e d  digested Hae  cleared with  Hind  81  6,  with  pDt5  digested  I I I ; lane  9,  39  12 3 4 5 6 789 O  FIGURE  12:  Hybridization Hind  I I I and  of Hae  125 [ I ]  S  tRNA  I I I + Hind  e  7  r  with  I I I digested  Hae I I I , pDtl 5 81 }  3  40  FIGURE  12: The a g a r o s e g e l p h o t o g r a p h e d  by  the procedure of Southern  125  S  [  I ] tRNA  11;  i . e . lane  e  III;  7.  III;  L a n e s 1-9  Hind  correspond  III;  lane  to lanes  Hae  with  Hind  III;  lane  I I I + Hind  III.  III;  lane  Hae  III;  lane  Hae  III;  of Figure 2,  pDtl  Hae I I I  l a n e . 5, pDt5  6, pDt5 d i g e s t e d w i t h  l a n e 7, pDt81 d i g e s t e d w i t h Hind  III;  1-9  3, p D t l d i g e s t e d w i t h  4, pDt5 d i g e s t e d w i t h  pDt81 d i g e s t e d w i t h Hae  (1975) and i n c u b a t e d  1, p D t l d i g e s t e d w i t h  lane  digested with + Hind  treated  r  digested with + Hind  i n F i g . 11 was  Hae I I I  lane  9, pDt81 d i g e s t e d  8, with  41  FIGURE  13:  Agarose +  Hind  gel  analysis  I I I digested  of pDtl6  Hae  I I I and and  pDt27.  Hae  III  42  FIGURE  13: The  and  Hae  plasmids  I I I + Hind  pDtl6 III,  and  e l e c t r o p h o r e s e d on  Tris-phosphate  buffer  after  with ethidium  staining  w i t h Hae l a n e 3, w i t h Hae  III;  l a n e 2,  27 were c l e a r e d w i t h Hae I I I  and  bromide.  III,  l a n e 4,  in  light  L a n e 1, p D t l 6  p D t l 6 d i g e s t e d w i t h Hae  III.  agarose  p h o t o g r a p h e d u n d e r u.v.  pDt27 d i g e s t e d w i t h Hae I I I + Hind  2.0%  digested  I I I + Hind  III;  pDt27 d i g e s t e d  43  o  66 kb .44 .35  FIGURE  14:  Hybridization and  Hae  of  I I I + Hind  .125  [  I]  tRNA  I I I digested  Ser 7  with pDt!6  Hae I I I , and  pDt27,  44  FIGURE  14:  The by  the  12 5  agarose g e l photographed procedure Ser  of  Southern  i n F i g . 13  (1975) and  was  treated  incubated  with  "V-J.  [  I]tRNA  7.  14;  i . e . , lane  Lanes 1,  digested  w i t h Hae  with  I I I , lane  Hae  1-4  pDtl6  correspond  lanes  d i g e s t e d w i t h Hae  I I I + Hind 4,  to  I I I ; lane  3,  1-4  of  I I I ; lane pDt27  p D t 2 7 d i g e s t e d w i t h Hae  figure 2,  pDtl6  digested  I I I + Hind I I I .  45  f o r e , the plasmid DNA was c l e a v e d w i t h Hind I I I and was r e cloned w i t h pBR32 2.  Recombinant plasmids c o n t a i n i n g only  Hind I I I fragments c a r r y i n g the d e s i r e d tRNA gene were i s o l a t e d from t h i s p r e p a r a t i o n and d e s i g n a t e d pDtNR.  I t i s of  i n t e r e s t to note t h a t upon the r e c l o n i n g of p D t l 7 , 55% o f the o r i g i n a l Hind I I I fragment c o n t a i n i n g the tRNA gene was recombined out o f the pDtl7R Hind I I I fragment c o n t a i n i n g the tRNA gene.  The o r i g i n a l Hind I I I fragment c o n t a i n i n g the tRNA  gene i n pDtl7 was 8.3kb, and i n pDtl7R the Hind I I I fragment was 3.7kb.  R. endonucleases Hae I I I and Hind I I I confirmed Ser t h a t the pDtl7 Hind I I I fragment c o n t a i n i n g the tRNA 7 gene  was l a r g e r than the pDtl7R Hind I I I fragment c o n t a i n i n g the Ser tRNA 7 gene.  P r e p a r a t i o n o f pDt!6 F o r Sequencing R e s t r i c t i o n endonuclease Hae I I I , Hind I I I double d i g e s t i o n o f pDtl6 y i e l d e d two fragments 0.66 and 0.44 kb ser  (Figures  13,14) which h y b r i d i z e d tRNA 7, y i e l d i n g two fragments o f convenient s i z e f o r sequencing by the method d e s c r i b e d by A. Maxam and W. G i l b e r t .  Four hundred ug o f pDtl6 DNA,  cleaved  w i t h R. endonucleases Hae I I I , Hind I I I i n tandem were loaded on 2% agarose T r i s - b o r a t e g e l s and run a t 20 hours  (Figure 15).  mamp f o r 48  The d e s i r e d DNA bands were c u t from the  g e l , e l e c t r o e l u t e d o v e r n i g h t a t 150 v o l t s i n 20 mm  Tris-acetate  46  1 2 34  567 O  FIGURE  15:  Fragment  preparation  gel  for  pDt!6  47  FIGURE 15; Four  h u n d r e d m i c r o g r a m s o f p D t l 6 d i g e s t e d w i t h Hae I I I  + Hind  I I I , were e l e c t r o p h o r e s e d on 2.0% a g a r o s e  borate  buffer  ing  and p h o t o g r a p h e d u n d e r u.v. l i g h t  w i t h e t h i d i u m bromide.  Hae I I I + H i n d I I I .  L a n e s 1-7, p D t l 6  in Trisafter  stain-  digested with  48  pH  8.0  phenol extracted,  e t h e r washed and  T r i s - b o r a t e b u f f e r e d g e l s were u s e d tion was  fragments, because the  Kinetics  the  of the  Before  kinase  attempting  w h i c h g a v e 5' flush  kinase  restric-  during  gels  s u b s e q u e n t man-  fragments.  Polynucleotide  labelling  specific  phosphate i n Tris-phosphate  Kinase  to l a b e l  5'  tRNA gene c a r r y i n g f r a g m e n t s , t h e  or  to p u r i f y  i n s o l u b l e under c o n d i t i o n s a r i s i n g  i p u l a t i o n of  ethanol p r e c i p i t a t e d .  of  5'  ends o f  Reaction  t e r m i n i of the kinetics  lambda DNA  e x t e n d e d e n d s , and  ends were m o n i t o r e d .  Hae  pDtl6  polynucleotide  cleaved with  EcoRI  I I I w h i c h g a v e 5'  blunt  Both the  r e a c t i o n s were a s s a y e d w i t h  of  two  forward  and  EcoRI c l e a v e d  exchange  lambda  frag-  32 ments, l a b e l l i n g (Figure led  16).  using  tion  the  the  extended  Sixty five forward  5'  ends w i t h  percent  kinase  of  porating The label  5'  lambda 5'  r e a c t i o n , and  reached e q u i l i b r i u m at only  19%  & [  of  the  lambda  P]  ends were exchange 5'  labelreac-  ends i n c o r -  radioactivity. forward blunt  polynucleotide  ends o f  kinase  lambda Hae  b l u n t ends were i d e n t i c a l  to the  r e a c t i o n was  I I I fragments. 5'  I I I , Hind  I I I tRNA gene f r a g m e n t s p r e v i o u s l y  14%  of  5'  lambda the  b l u n t ends were l a b e l l e d w i t h  forward  reaction.  used  These  b l u n t ends i n t h e  Hae  utilizing  ATP  to 5'  two  pDtl6,  isolated. 32 & [  P] ATP  49  50  FIGURE  16;  Graph A r e p r e s e n t s by  5' t e r m i n i  of  the  EcoRI  32 & [ P] ATP  fragments using  incorporation the forward  poly32  nucleotide  kinase  reaction.  ATP  i n c o r p e r a t i o n by  the  polynucleotide  5"  Graph B r e p r e s e n t s  termini  kinase  of  exchange  EcoRI  the  fragments  reaction.  P] using  51  Thirty  percent of the  5' b l u n t ends o f t h e  660  base  pDt  3 2  16  f r a g m e n t were end  the  labelled with  5' b l u n t ends o f t h e  l a b e l l e d with  &  [  3  2  )([  When r e s t r i c t i o n  restriction  base pDtl6  3 2  fragments are  prior  from  The  pDtl6  strand.  17).  base s i n g l e The  heat  denatured  The  t o p two  strands, with  sequencing  fragments  a t both  440  c o n f i r m the  5'  dark  bands a r e t h e  a  experiments.  treat1:30  Gilbert)  separated  smaller degradation products strands should prove  Se-  sequence  e l e c t r o p h o r e s e d on (Maxam and  ends,  secondary  b a s e f r a g m e n t was  4 percent polyacrylamide g e l  separation of these  future  and  end  t o sequence a n a l y s i s .  strand w i l l  (Figure  pDt!6  of  reaction.  s t r a n d s e l i m i n a t e s the need f o r  one  crosslinked,  kinase  labelled  quence i n f o r m a t i o n f r o m  ed w i t h a k a l a i ,  4 7%  f r a g m e n t were  P ] A T P labelled  enzyme c l e a v a g e  the o t h e r  P ] A T P and  u s i n g the forward  P ] A T P  Strand Separation of  separation of t h e i r  440  & [  440 below.  adequate f o r  52  FIGURE  17:  Polyaorylamide labelled  pDt!6  strand  separation  fragments.  of  X [  P]  ATP  53  FIGURE 17: 32 5' t e r m i n i o f p D t l 6 ATP were a l k a l a i , 1:30  cross linked,  pDtl6 the tion  heat  denatured  440 b a s e s i n g l e below.  The t o p two d a r k  strands with  with  X[  P]  and e l e c t r o p h o r e s e d on a  4% p o l y a c r y l a m i d e g e l .  44 0 b a s e f r a g m e n t s .  products  fragments l a b e l l e d  L a n e s 1 and 2, bands(a,b) a r e  smaller, lighter  degrada-  54  BIBLIOGRAPHY  Ames, B.N. and Hortman, P.E. The h i s t i d i n e a p e r o n , C o l d S p r i n g H a r b o r Symp. Quant. B i o l . 28 (1963) 349-356. Atwood, K.C. I n G e n e t i c V a r i a t i o n s o f D r o s o p h i l i a melanogaster. ( L i n d s l e y , D.L. a n d G r e l l , F . H . , e d s . ) , C a r n e g i e I n s t . P u b l . No. 627 (1968) 152. B o l i v a r , F., R a d r i q u e z , R.L., G r e e n e , P.S. B e t l a c k M.S., H e y n e c k e r , H.L. and B a y e r , H.W. C o n s t r u c t i o n and c h a r a c t e r i z a t i o n o f new c l o n i n g v e h i c l e s I I . A m u l t i p u r p o s e c l o n i n g s y s t e m , Gene 2 (1977) 95-113. Dunn, R., H a y a s k i , S., G i l l a m , I . 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Colony h y b r i d i z a t i o n . method f o r t h e i s o l a t i o n o f c l o n e d DNAs t h a t c o n t a i n a gene, P r o c . N a t l . A c a d . S c i . USA 72 (1975) 3961-3965. Khorana,  B i o c h e m i s t r y 12 (1973)  A specific  5050  K u b l i , E . a n d S c h m i d t , T. The l o c a l i z a t i o n o f tRNA 4 " genes f r o m D r o s o p h i l a m a l a n o g a s t e r by " i n s i t u " h y b r i d i z a t i o n , N u c l e i c A c i d s R e s . 5 (1978) 1465-1478 G  U  M u r r a y , K. a n d M u r r a y , N.E. Phage lambda r e c e p t o r chromasomes f o r DNA f r a g m e n t s made w i t h r e s t r i c t i o n e n d o n u c l e a s e I I I o f H e a m a p h i l u s i n f l u e n z a e and r e s t r i c t i o n e n d o n u c l e a s e I o f E s c h e n i c h i a c o l i , J . M o l . B i o l . (1975) 551-564. O l s o n , M.V., Montgomery, D.L., H o p p e r , A.K., Page, G.S., H o r a d y s k i , F. a n d H a l l , B.D. 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