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Regulation of T-DNA gene 7 Button, Eric A. 1987

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REGULATION OF T-DNA GENE 7  By E R I C A.  B.A., U n i v e r s i t y  BUTTON  o f North  Carolina,  A T H E S I S SUBMITTED I N PARTIAL OF THE REQUIREMENTS MASTER  1982  FULFILLMENT  FOR THE DEGREE OF  OF  SCIENCE  in  THE  FACULTY  OF GRADUATE  GENETICS UNIVERSITY  We  accept to  THE  PROGRAMME  OF B R I T I S H COLUMBIA  this  thesis  the r e q u i r e d  UNIVERSITY  Eric  as c o n f o r m i n g standard  OF B R I T I S H  August  °  STUDIES  COLUMBIA  1987  A. B u t t o n , 1987  In  presenting  advanced shall  this  degree  make  thesis  at  the  i t freely  University  available  permission  for  extensive  granted  the  head  by  understood not  be  allowed  Department The  that  of  University  1956  Main  Vancouver, V6T Date:  of  copying without  for  copying  my or my  of  of  British  Canada  1Y3 21,  1987  fulfillment British  reference  of  department  this or  publication written  Genetics  Mall  August  in partial  Columbia  of  and  his this  permission.  the  Columbia,  thesis by  of  requirements I agree  study. for  or  I  further  scholarly  her  thesis  that  for the  Library  agree  purposes  financial  that  may  representatives. for  an  gain  be  It  is  shall  ABSTRACT  The  purpose o f t h i s study was t w o - f o l d .  The f i r s t  determine i f Saccharomyces c e r e v i s i a e  i s a useful  e x p r e s s i o n o f T-DNA ( i t takes s e v e r a l  months to o b t a i n  objective  system f o r i n v e s t i g a t i n g the sufficient  transformed plant  tissue to investigate  of T-DNA c a r r y i n g  T-DNA gene 7 was c l o n e d i n t o a yeast plasmid  to  investigate  T-DNA t r a n s c r i p t i o n ) .  the e x p r e s s i o n o f gene 7 i n y e a s t .  determine the s i g n i f i c a n c e o f a heat shock r e l a t e d 5'  region  bacteria-free  A short  fragment  i n an attempt  The second o b j e c t i v e  was t o  sequence i d e n t i f i e d  i n the  o f T-DNA gene 7.  Primer e x t e n s i o n a n a l y s i s , indicate  was t o  that  SI n u c l e a s e mapping, and Northern  t r a n s c r i p t i o n o f T-DNA gene 7 i n y e a s t i s d i f f e r e n t from that o f  t r a n s c r i p t i o n o f gene 7 i n crown g a l l because the d i s t a n c e  tumors.  Transcription  i s different  between the TATA box and the t r a n s c r i p t i o n  s i t e s must be a t l e a s t 40 n u c l e o t i d e s cerevisiae  hybridizations  i n yeast.  does not appear t o be a u s e f u l  initiation  T h e r e f o r e , Saccharomyces  system f o r i n v e s t i g a t i n g the  e x p r e s s i o n o f T-DNA. Crown g a l l  tumors were s u b j e c t e d t o a number o f s t r e s s  agents,  including  heat shock, t o determine the s i g n i f i c a n c e o f the heat shock r e l a t e d  sequence  identified  cadmium  and  i n gene 7.  Primer e x t e n s i o n a n a l y s e s  indicate  that  only  mercury have a s i g n i f i c a n t e f f e c t on the e x p r e s s i o n o f T-DNA gene 7.  A l t h o u g h gene 7 responds to cadmium and mercury, the i n c r e a s e does not appear to be heat shock or m e t a l l o t h i o n e i n another mechanism i s i n v o l v e d crown g a l l  tumors.  related,  in transcription indicating  that  i n the enhanced t r a n s c r i p t i o n o f T-DNA gene 7 i n  - i i i -  TABLE OF CONTENTS  Page  ABSTRACT  i i  TABLE OF CONTENTS  i i i  L I S T OF T A B L E S  v  L I S T OF FIGURES  v i  ACKNOWLEDGEMENTS  v i i  ABBREVIATIONS  viii  INTRODUCTION  1  MATERIALS  6  AND METHODS  Reagents Microbial Strains Tobacco T i s s u e C u l t u r e Lines C u l t u r e Media and C o n d i t i o n s P l a s m i d s and B a c t e r i o p h a g e s Transformation of E. c o l i L a r g e S c a l e I s o l a t i o n o f P l a s m i d DNA P u r i f i c a t i o n o f P l a s m i d DNA S m a l l S c a l e I s o l a t i o n o f P l a s m i d DNA R e s t r i c t i o n Endonuclease D i g e s t i o n Ligations A g a r o s e E l e c t r o p h o r e s i s o f DNA T r a n s f o r m a t i o n o f Yeast Yeast Plasmid P u r i f i c a t i o n I s o l a t i o n f o Y e a s t RNA S e l e c t i o n o f Poly A RNA End-Labelling of Oligonucleotides Primer E x t e n s i o n A n a l y s i s Denaturing Urea-Acrylamide Gel S l N u c l e a s e Mapping Northern H y b r i d i z a t i o n Nick T r a n s l a t i o n I s o l a t i o n o f P l a n t Tumor RNA S t r e s s I n d u c t i o n o f P l a n t Tumors +  RESULTS A.  T-DNA Gene 7 i n Y e a s t Plasmid Constructions Primer Extension A n a l y s i s S l N u c l e a s e Mapping Northern Hybridizations  6 8 8 8 10 10 11 11 12 13 14 14 14 16 16 17 18 19 19 19 19 21 21 22 23 23 23 23 30 32  -iv-  TABLE OF CONTENTS Page B.  Heat Shock-T-DNA Gene 7 Heat Shock Heavy Metal Treatment A r s e n i t e Treatment  34 34 34 36  DISCUSSION A. T-DNA Gene 7 i n Yeast B. Heat Shock-T-DNA Gene 7  40 40 43  BIBLIOGRAPHY  48  -v-  LIST OF TABLES Table  P a  Se  1  Plasmids and B a c t e r i o p h a g e s  10  2  Summary o f the S t r e s s Inducted Accumulation o f T-DNA Gene 7 RNAs i n Crown G a l l Tumors and Transformed Tobacco C u l t u r e s  45  -vi-  L I S T OF  FIGURES  Figure  Page  1  Construction  o f Y-BG.E9  24  2  Construction  o f Y-BGE.E9  25  3  Restriction YEpl3,  Site  DNA  5  Oligonucleotide Transcript 7  Transcriptase  SI N u c l e a s e  9  10  11  12  t o t h e 5' R e g i o n o f  Extension  Products  from  RNA P r i m e d  C o m p l e m e n t a r y t o t h e 5' C o d i n g  Region  with  an  of the  7  Mapping  Autoradiograph Gene  27  28  T-DNA T r a n s c r i p t  8  Y-BGE.E9,  7  Corresponding  Oligonucleotide  7  Y-BG.E9,  26  S e q u e n c e o f T-DNA Gene  Reverse  o f Plasmids  and LE.E9  4  6  Analysis  29 o f Yeast  T-DNA Gene  o f a n RNA G e l B l o t  7 RNA 5' T e r m i n i  Hybridized  31  t o a T-DNA  7 Probe  33  The E f f e c t o f a T e m p e r a t u r e Shock and t h e E f f e c t Cadmium C h l o r i d e o n t h e E x p r e s s i o n o f T-DNA Gene Tumors  o f Treatment o f 7 i n Crown G a l l  The E f f e c t Crown G a l l  o f H e a v y M e t a l s o n t h e E x p r e s s i o n o f T-DNA Gene Tumors a n d T r a n s f o r m e d T o b a c c o C u l t u r e s  The E f f e c t T-DNA Gene  o f Treatment o f Zinc C h l o r i d e 7 i n Crown G a l l Tumors  The E f f e c t  o f Treatment  T-DNA Gene  7 i n Crown G a l l  on t h e E x p r e s s i o n  o f Sodium A r s e n i t e Tumors  35 7 in 37 of 38  on t h e E x p r e s s i o n  of 39  -vii-  ACKNOWLEDGEMENTS I would l i k e  t o thank Dr. J . McPherson, without whose support and guidance  this project  would not have been p o s s i b l e .  for allowing  the use o f h i s f a c i l i t i e s  a l s o wish to thank Dr. R. McMaster I would l i k e  My thanks a l s o go t o Dr. H. Brock  and f o r many h e l p f u l d i s c u s s i o n s .  f o r h i s s u g g e s t i o n s and g u i d a n c e .  t o thank J . Button f o r p r e p a r i n g  the f i g u r e s i n t h i s  I  Finally,  thesis.  -viii-  ABBREVIATIONS  USED  DNA  -deoxyribonucleic  cDNA  -complementary  T-DNA  -transferred  DNA  RNA  -ribonucleic  acid  mRNA  -messenger  ribonucleic  acid  rRNA  -ribosomal  ribonucleic  acid  A  -adenosine  triphosphate  o r ATP  acid  DNA  C o r CTP  -cytidine  triphosphate  G o r GTP  -guanosine  triphosphate  T o r TTP  -thymidine  triphosphate  HS  -heat  shock  HSE  -heat  shock  HSTF  -heat shock factor  Ti  -tumor  TRP2  -tryptophan  gene  TRP3  -tryptophan  gene 3  TRIS  -tris  element transcription  inducing  2  (hydroxymethyl)  aminomethane EDTA  -ethylene acid  diamine  SDS  -sodium  DNP  -dinitrophenol  lauryl  tetraacetic  sulphate  TBE  -tris-borate-EDTA  DTT  -dithiothreitol  BSA  -bovine  serum  lambda  -lambda  virus  albumin  -1-  INTRODDCTION  The cells,  molecular  involves  tumefaciens  (Sciaky  f o r crown  the transfer  T i plasmid  Thomashow et^ a l _ . , according  basis  (T-DNA) i n t o  amino a c i d s  1978).  plasmids contain  T-region,  TL-DNA  8 kbp fragment TL-DNA synthesis suggest flanking  from the r i g h t  i s sensitive  Most to that  de  et a l . ,  1983; Joos  1984).  from  t h e mRNA  start  o f other  site  transcription  et  al.,  some  et a l . ,  i n specifying  a t new s i t e s  o f the  lines  c o n t a i n an  transcripts  1981).  These  RNA p o l y m e r a s e  (Barker  octopine  whose  findings II.  i n common w i t h (TATA)  T h e 5' other  box a t a  e_t a _ l . , 1 9 8 3 ;  1984; L i c h t e n s t e i n e t  o f T-DNA gene  7 maps 30 b p u p s t r e a m  1984).  Studies  have  indicated  o f mRNA i n i t i a t i o n ,  initiation,  that the  b u t sequences  and q u a n t i t a t i v e  1981; McKnight  The d e l e t i o n  et a l . ,  side  a Goldberg-Hogness genes  from  plant  T-region.  polyadenylated et al.,  into  et al.,  the s i t e  1984).  (Osborne  culture  characteristics  1983; K l e e  i n a decrease i n t r a n s c r i p t i o n  transcripts  T-DNA  from the l e f t  by the host  eukaryotic  (McPherson,  et a l . ,  1982).  1977;  tumors  i s integrated  Some t i s s u e  contain  ( B r e a t h n a c h a n d Chambon  1984; W r i g h t  i n the plant  tumors  o f t h e TATA b o x d e t e r m i n e b o t h q u a l i t a t i v e  of  results  have  A TATA b o x i n t h e 5' r e g i o n  TATA b o x i s i n v o l v e d upstream  produced  (Willmitzer  T-DNA g e n e s  similar  al.,  T i plasmids a r e c l a s s i f i e d  several  are transcribed  position Greve  encodes  ejt aJL. ,  1980).  (TR-DNA) o f t h e o c t o p i n e  to amanitin  o f t h e s e genes  genes.  1982).  of the Agrobacterium  DNA ( C h i l t o n  ( L e e m a n s ej^ a _ l . ,  side  tumors  T-DNA g e n e s  regions  eukaryotic  et a l . ,  of plant  nuclear  a c o n t i g u o u s 13 k b p s e g m e n t  (Leemans  transformation  o f a segment  T h e T-DNA o f n o p a l i n e  i n octopine  that  plant  (opines)  DNA a s a c o n t i g u o u s 22 k b p segment Ti  a neoplastic  and i n t e g r a t i o n  1980; Z a m b r y s k i e t a l . ,  to the novel  et al.,  gall,  aspects  e_t al., 1 9 8 1 ; M c K n i g h t  o r m u t a t i o n o f t h e TATA b o x and the i n i t i a t i o n o f  1982; S c h u l z e_t a _ l . ,  1982; O r k i n  -2-  et  al.,  30  t o 50 b a s e s  CAAT  1983).  sequence The  upstream  i s o b s e r v e d i n gene  by o c t o p i n e  function  genes  The obtain  (Garfinkel  sufficient  transcription investigate excellent and  Mellor al.,  cloned  In  T-DNA g e n e shock  defined  sequence,  foreign  shown  tissue  Most  fragment  yeast  the expression  system  promoter  i s active  months t o  cerevisiae  i s an  eukaryotic  has been  genes  reported  o f these s t u d i e s  et a l . ,  have  1982; V a l e n z u e l a e t  p r o d u c t s encoded  YEpl3  (Edens  by p l a n t  et_ a l ^ . , 1984; T-DNA g e n e 7  (a non-expression v e c t o r ) ,  o f gene  7 i n yeast.  related  homologous  TL-DNA  i n which t o  o f t h e T-DNA c a r r y i n g  shock  1982).  to investigate i t s  sequence  u p s t r e a m o f t h e TATA b o x .  (1982), i n t h e i r  et a l . ,  ( H i t z e m a n ejt a _ l . , 1 9 8 1 ;  i n yeast  plasmid,  mRNAs  o f the other  of cloned genes  that  and p r o c e s s e d  the other  i t takes s e v e r a l  a rapid  1983; T u i t e  DNA s e q u e n c e s  5' C T g G A A t n T T C t A G a ,  H o w e v e r , no  1982).  Saccharomyces  1982).  t o t h e TATA b o x , a h e a t  contain  genes,  tumorigenesis i s  et a l . ,  the expression  e t al_.,  7, 40 n u c l e o t i d e s  by Pelham  t o most  transformed plant  A short  to investigate  genes  assigned  animals or animal viruses  the multicopy  addition  gall  be c o n v e n i e n t t o h a v e  be s y n t h e s i z e d  into  t o crown  o f T-DNA i s t h a t  of several  with  1982; W i l l m i t z e r  studies  1981; J a c q u e t e t a l . ,  e t a_l. , 1 9 8 4 ) .  an attempt  heat  been  compared  1981; Leemans  expression  i n some T-DNA  ejt al., 1 9 8 3 ) .  ejt a l . ,  1983; S t e p i e n  can a l s o  Rothstein  in  have  1982); however, e x p e r i m e n t s have  genes  was  et al.,  o f T-DNA s e q u e n c e s .  from h i g h e r  ^ t al.,  identified  abundant  regards  for investigating  et a l . , DNA  7 with  bacteria-free  the f u n c t i o n a l  involved  with  expression  also  7.  (Gelvin  roles  and i t would  system  (Henikoff  in  specific  difficulty  been  i s rather  TL-DNA  o f T-DNA gene  unknown, w h e r e a s encoded  have  o f t h e TATA b o x ( B a r k e r  0.7 kb t r a n s c r i p t  specified The  CAAT s e q u e n c e s  I n most  to the heat  regions.  i n controlling  was  identified organisms,  shock  The h e a t the heat  e l e m e n t , as  shock shock  consensus regulated  -3-  e x p r e s s i o n of f o r e i g n genes even when s y n t h e t i c promoter elements a r e used which only have an 8 of 10 match w i t h the HSE B i e n z , 1982).  The heat shock r e l a t e d  has a 7 o f 10 match with the consensus  consensus sequence  sequence of T-DNA gene 7  (Pelham and  (CTTGAAAATTAAGC)  sequence.  The heat shock elements of the D r o s o p h i l a melanogaster hsp 70 and hsp 83 are p r o t e c t e d Wu,  1984b).  from n u c l e a s e s i n the n u c l e i o f heat shocked c e l l s The  HSEs of hsp 70 are the b i n d i n g s i t e s  shock t r a n s c r i p t i o n  factor  p r o t e i n gene t r a n s c r i p t i o n 1985).  (HSTF) t h a t in vitro  (Wu,  1984a;  f o r a D r o s o p h i l a heat  is s p e c i f i c a l l y required  (Parker and T o p o l , 1984;  f o r heat shock  Top e t a l . ,  Recent e v i d e n c e a l s o suggests that the number of heat shock elements  can be a major determinant of the promoter s t r e n g t h of h e a t - i n d u c i b l e genes i n mammalian c e l l s  (Kay ej: al_.,  1986).  The heat shock response of p l a n t s i s s i m i l a r bacteria,  to the response observed i n  f u n g i , i n s e c t s , and mammals ( C r a i g , 1985).  F e a t u r e s o f the heat  shock response t h a t are conserved a c r o s s a broad spectrum o f organisms  include:  (1) the sequences of the 5' f l a n k i n g r e g i o n s of the HS genes which appear to r e g u l a t e e x p r e s s i o n of these genes d u r i n g heat shock, (2) the r a p i d the c e l l ' s  t r a n s c r i p t i o n a l and t r a n s l a t i o n a l machinery to the p r o d u c t i o n o f HS  mRNAs and HS p r o t e i n s , and (3) the s t r u c t u r a l which may 1985).  s w i t c h of  be important f o r t h e i r r o l e  f e a t u r e s of the HS  i n thermal t o l e r a n c e  proteins  (Kimpel and  However, the HS response of p l a n t s i s e a s i l y d i s t i n g u i s h e d  organisms by the c o m p l e x i t y and r e l a t i v e abundance HS p r o t e i n s  (15 to 18 Kd)  v a t i o n i s not y e t known.  ( L i n et a l . ,  It is interesting  a low m o l e c u l a r weight p r o t e i n From a p h y s i o l o g i c a l  1984).  Key,  from o t h e r  of the low m o l e c u l a r weight  The s i g n i f i c a n c e  of t h i s obser-  to note that T-DNA gene 7 encodes  (14Kd).  p o i n t of view, the o n l y b i o l o g i c a l  to HS p r o t e i n s to date i s t h e i r p o s s i b l e r o l e  function  assigned  i n m e d i a t i n g the e x p r e s s i o n of  -4-  thermal  t o l e r a n c e (Kimpel  contribute presumed  t o homeostasis  and Bonner,  I n soybean  synthesis  exact  heat  While there  (A)  and Key,  coordinate By  stresses, induce  altered  range  puffing  protein  proteins  by heat  found  between the  to survive  and Key,  short  1985).  Although  unidentified,  of plants  investigated,  have been  of regulation indicate  seedlings,  and s i n c e  o f the response.  that  very  d u r i n g HS,  the l e v e l  1982).  of actin  heat  and Key,  shock  gene  the t o t a l  r e p r e s e n t over  shock,  as e t h a n o l , a n o x i a , a r s e n i t e ,  of stress  shock  agents  proteins.  (Kimpel  20%  normal  and Key,  1985).  r a p i d l y and  b u t i n many o r g a n i s m s  o r heavy metal  ions w i l l  The D r o s o p h i l a s y s t e m  ( e . g . , DNP, a r s e n i t e ,  patterns (Ellgaard,  poly  on t h e r a p i d and  a r e a new s e t o f p r o t e i n s  t o a heat  mRNAs  E x p r e s s i o n o f t h e heat  i s dependent  transcription  proteins  i n response  1982).  i f at  r e g u l a t e d gene  Since  HS mRNAs  normal  inefficiently  o f an a u x i n  and Key,  and o t h e r o r g a n i s m s  o f HS p r o t e i n  i t is  the chloroplast  but are translated  constant  (Schoffl  heat  i s transported into  soybeans  d u r i n g HS ( S c h o f f l  decline  shock  and L i n d q u i s t ,  has been  remain  role  1986).  I n soybean  fairly  a protective  induced  1984; V e l a s q u e z  (Kimpel  shock  including  1982).  the s y n t h e s i s o f heat  a wide  shock  i n the c e l l s  i n plants  produced such  heat  having  i s thought to  ( A ) RNA p o o l d u r i n g HS, t h e c o n c e n t r a t i o n s o f many  definition,  abundantly  effects  response  and t h e a b i l i t y  o f t h e HS r e s p o n s e  remains  onset  proteins  d u r i n g HS, b u t t h e mRNA l e v e l s  poly  proteins  ejt aL.,  temperatures  et a l . ,  organisms  ( A ) RNAs must  shock  to  other  dramatically  the t o t a l  deleterious  i n f o r m a t i o n on t h e mechanisms  RNA c o n t e n t  poly  (Vierling  unchanged  decrease  shock  shock  an a b s o l u t e c o r r e l a t i o n  lethal  aspects  The h e a t  o f HS p r o t e i n s  one h e a t  mRNAs p e r s i s t  (Schoffl  remains  of  many  from  cellular all  shock  is little  Results  the heat  f u n c t i o n s o f the plant at least  1985).  Schoffl  seedlings,  at otherwise  known t h a t during  1979;  and a c c u m u l a t i o n  treatments the  with  t o counteract or prevent  (Ashburner  1984).  and Key,  release  1972) a n d s y n t h e s i s o f HS  from  other also  responds a n o x i a ) by  proteins  -5-  (Ashburner and Bonner, 1979). of proteins  Arsenite  s i m i l a r to HS p r o t e i n s  and heavy metals seem t o induce a s e t  i n a number o f systems  (Ashburner and  Bonner, 1979; Johnston et^ a l . , 1980; L e v i n s o n ejt a l . , 1980). seedlings  respond  Soybean  to s t r e s s e s such as a r s e n i t e and cadmium as w e l l as heat  shock, a l t h o u g h i n d u c t i o n o f heat shock p r o t e i n s under was not d e t e c t e d (Czarnecka e t a l . , 1984).  other s t r e s s  I t i s not understood why a r s e n i t e  and cadmium i n p a r t i c u l a r induce the heat shock p r o t e i n s o t h e r s t r e s s e s , which  conditions  i n soybean  w h i l e many  are e f f e c t i v e i n many o t h e r organisms, do not appear to  do s o . The  possibility  o f the conserved heat shock sequence  o f T-DNA gene 7  h a v i n g s i g n i f i c a n c e i n i t s response t o s t r e s s r e l a t e d s t i m u l i was i n v e s t i g a t e d .  -6-  MATERIALS AND  METHODS  REAGENTS  Enzymes Restriction or  Bethesda  kinase, BRL.  e n d o n u c l e a s e s were p u r c h a s e d  Research Laboratories  RNase  T l , a n d DNA  Proteinase  was  supplied  was  f r o m Endo  Amersham  by  polymerase  Sigma, r e v e r s e  T4 DNA  I (Klenow  K a n d SI n u c l e a s e were  laboratories.  (Nick  (BRL).  England  ligase,  T4  Biolabs  f r a g m e n t ) were  was  I a n d DNA  also  from Pharmacia,  polymerase  (NEBL)  polynucleotide  f r o m B o e h r i n g e r Mannheim.  transcriptase  DNase  f r o m New  I were  supplied  by  Lysozyme and  Glusulase  supplied  by  Translation K i t ) .  Conditions  f o r each  enzyme  are described  later  i n this  section.  Nucleotides 2'-Deoxyribonucleotide were pH  dissolved  7.0  tration were  by a d d i n g o f each  frozen a[  i n water  32  at  triphosphates  were  to a concentration  a dilute  solution  s t o c k was  ( 0 . 0 5 M)  determined  supplied  o f 10 mM. of Tris  base.  The  dNTPs  adjusted  exact Small  concenaliquots  supplied  complexes  as a q u e o u s s o l u t i o n s  ( 0 . 2 M) were  purchased  and Y[  32  P]-adenosine  containing from  10 u C i / u l .  Vanadyl  BRL.  Phenol Ultra either  0.1  Pure  to  -70°C.  were  ribonucleoside  The pH was  spectrophotometrically.  P]-2'-deoxyribonucleotidetriphosphates  triphosphate  by P h a r m a c i a .  Phenol  M Tris  was  (pH 8.0)  supplied  b y BRL.  or s t e r i l e  HO.  Phenol  was  equilibrated  with  -7-  Chloroform  C h l o r o f o r m was  supplied  by  BDH  Chemicals.  Formamide  Ultra  F o r m a m i d e was  purchased  50 ml w i t h 5 g o f m i x e d b e d ,  mixing for  Pure  2 hours  at  Whatman No.  room  temperature.  1 filter  paper  and  from  BRL.  ion-exchange  The  mixture  dispensed  into  F o r m a m i d e was resin  was  (Bio-Rad  filtered  1 ml  twice  aliquots  and  deionized AG  by  501-X8-10)  through stored  at  -20°C. Formaldehyde Formaldehyde 3.8. then  The  was  supplied  s o l u t i o n was  raised  to  by  deionized  BDH  Chemicals.  w i t h AG  The  pH  501-X8-D m i x e d  of bed  this  solution  resin.  The  was  pH  was  6.8.  Agarose  Ultra  Pure  Agarose  was  purchased  from  BRL.  Acrylamide  Acrylamide  Culture  and  b i s - a c r y l a m i d e were  from  Flow  base  Sigma.  Sigma.  by  Bio  Rad.  Media  Bacto-tryptone, nitrogen  supplied  without  Bacto-yeast amino a c i d s  Ampicillin,  Murashige  Laboratories.  and  extract, were  supplied  t e t r a c y c l i n e , and  Skoog  plant  salt  Bacto-peptone, by  Difco.  and  Bacto-yeast  Amino  c h l o r a m p h e n i c o l were  medium  (MS  m e d i a ) was  acids  were  purchased  obtained  from  from  -8-  Others All  o t h e r chemicals used were o f reagent  grade.  MICROBIAL STRAINS  Bacteria The  following  s t r a i n s of E_. c o l i were used as hosts f o r recombinant  DNA  molecules. E. c o l i  RRl, F», hsdS20, ara-14,  m l l - 1 , sup E44, E. c o l i  JM  101,  F'traD36,  lambda-, was  po A2,  l a c Y , g a l Ks, r s p s L 2 0 , X y l - 5 ,  c o n s t r u c t e d by B o l i v a r  ejt al_. (1977).  ( l a c , p r o ) , sup E, t h i , s t r A , sbcB15, end A,  proAB, l a d , l a c Z M15,  was  hspR4,  c o n s t r u c t e d by Messing  (1981).  Yeast S t r a i n GM-3C-2 was a l p h a , l e u 2 - 3 , 112,  d e s c r i b e d by Fay et a l . (1981).  t r p 1-1,  I t s genotype  h i s 4-519, c y c l - 1 , c y p 3 - l .  TOBACCO TISSUE CULTURE LINES Tumor 1ine  Inciting plasmid  A6S/2 E9 16-12-C  pTiA6 P 6 pTLl T i B  8  CULTURE MEDIA AND E.  0  6  N. tobacum cultivar  x  White B u r l e y a n t h i c nc. X a n t h i nc.  Octopine biosynthes is + +  Reference ( G e l v i n et a l . , 1982) ( G e l v i n et a l . , 1982)  CONDITIONS  coli The  f o l l o w i n g media were used  f o r the growth of E.  LB-  1.0%  Bacto-yeast  0.5%  B a c t o - t r y p t o n e , 0.5%  NaCl, 0.1%  glucose.  extract,  coli:  was  -9-  M9-50 mM — 0.1  mM  Na HP0., 2 4  CaCl ,  10 mM  2  0.001%  25 mM  o  glucose,  thiamine.  For  plates,  Bacto-agar  20  g/1.  E. c o l i  was c u l t u r e d  by  measuring  appropriate  KH.PO.. 2 4  the o p t i c a l  was a d d e d  to the appropriate l i q u i d  at a temperature  d e n s i t y a t 600 nm.  o f 37°C.  Antibiotics  medium a t  G r o w t h was  monitored  were  t o the  added  concentrations.  Yeast Media (1981).  f o r the c u l t u r e  The f o l l o w i n g  YPD  of yeast  were u s e d  - 2% B a c t o - p e p t o n e ,  YNB - 0.7% B a c t o - y e a s t pH  5.8.  (20 For  h a v e b e e n d e s c r i b e d b y S h e r m a n e_t a l .  i n this  study:  1% B a c t o - y e a s t  extract,  n i t r o g e n base without  YNB was s u p p l e m e n t e d  with  2% g l u c o s e ,  amino a c i d s ,  tryptophan  pH 5.8.  2% g l u c o s e ,  ( 2 0 mg/1) a n d h i s t i d i n e  mg/1).  plates,  agar  Regeneration  Agar  was a d d e d  t o a c o n c e n t r a t i o n o f 2% t o t h e a b o v e  - YNB s u p p l e m e n t e d  with  1 M sorbitol,  media.  2% YPD, a n d 3%  agar. Yeast  Tissue  cultures  tobacco  Murashige-Skook growth  MS m e d i a  crown g a l l (MS) medium  hormones.  tumors  lines  (Murashige  Transformed  containing plant  benzylaminopurine at  mild  shaking.  Cultures  The  plant  were grown a t 30°C w i t h  A6 a n d E9 were g r o w n i n  e t a l . , 1962) a t 25°C  tobacco  cultures  hormones, naphthalene  (0.2 mg/L), a t 25°C.  the appropriate concentrations.  Stress  i n the absence o f  (16-12-C) were  acetic  acid  grown i n  ( 2 mg/L) a n d  i n d u c i n g compounds w e r e  added  -10-  PLASMIDS AND  BACTERIOPHAGES and b a c t e r i o p h a g e s  Plasmids  p r o v i d e d by other workers are l i s t e d i n  Table I.  Table I Name  Reference  Source  pBR322 LE.E9  Bolivar  R. J. J. J.  YEpl3  Broach  Hpall  et a l . (1977) et a l . (1979)  0.8  Kay (U.B.C.) McPherson (U.B.C.) Ngsee (U.B.C.) McPherson (U.B.C.)  TRANSFORMATION OF E . COLI E . c o l i was Higa  (1970).  LB-glucose shaking.  One b a c t e r i a l  media.  by a m o d i f i c a t i o n of the procedure colony was  p i c k e d and i n o c u l a t e d i n 5 ml o f  (0.5 ml) was  i n o c u l a t e d i n t o 50 ml o f  media and incubated at 37°C w i t h shaking  5 x 10  7  cells/ml  (A,  n n  = 0.25).  to a d e n s i t y o f a p p r o x i -  The c u l t u r e was  chilled  minutes and then c e n t r i f u g e d at 4000 g f o r 5 minutes at 4°C. was  d i s c a r d e d and the remaining  CaCl  2  and 10 mM  s u s p e n s i o n was  Tris-Cl  divided  added to the m i x t u r e . from  c e l l s were resuspended  on i c e f o r 10  The  supernatant  i n 0.25 ml o f 100  (pH 8.0) and i n c u b a t e d at 0°C f o r 1 hour.  i n t o 0.1 ml a l i q u o t s and 16 u l of s t e r i l e The c e l l  suspensions  The  mM cell  glycerol  were s t o r e d at -70°C and  was  removed  the f r e e z e r as needed. Plasmid  thawed c e l l and  o f Mandel and  The b a c t e r i a l c e l l s were grown o v e r n i g h t at 37°C w i t h m i l d  The o v e r n i g h t c u l t u r e  LB-glucose mately  transformed  then  DNA  (40-100 ng i n o f 10-100 u l of H 0)  suspension  2  on i c e . The mixture  incubated at 37°C f o r 5 minutes.  was  was  added to 0.1 ml o f the  incubated on i c e f o r 15 minutes  LB-glucose  media (1 ml) was  added and  -li-  the mixture was  i n c u b a t e d f o r 2 hours  at 37°C without  (50 u l to 200 u l ) were spread on s e l e c t i v e media.  shaking.  The  Aliquots  p l a t e s were i n c u b a t e d  at 37°C o v e r n i g h t .  LARGE SCALE ISOLATION OF PLASMID T h i s procedure  was  DNA  d e s c r i b e d by M a n i a t i s et a l . (1982) and  m o d i f i c a t i o n o f the method o f Birnboim and Doly from a 500 ml  c u l t u r e was  g l u c o s e , 25 mM incubated 1% SDS  Tris-Cl  resuspended  (pH 8.0),  f o r 5 minutes at 4°C.  (20 ml) was  minutes. was  An  ice-cold  added and  The  removed and  was  pellet  5 mg/ml lysozome and  s u s p e n s i o n and  s u s p e n s i o n was  transferred tube and  mM  was  N NaOH and  the contents o f the  tube  then incubated on i c e f o r 10  s o l u t i o n of 3 M potassium a c e t a t e and  95% e t h a n o l were added to each DNA  EDTA, and  The mixture was  then c e n t r i f u g e d at 20,000 rpm  supernatant was  bacterial  A s o l u t i o n c o n s i s t i n g of 0.2  the c o n t e n t s were mixed.  10 minutes and  The  i n 10 ml o f a s o l u t i o n c o n t a i n i n g 50  10 mM  added to the c e l l  mixed by g e n t l e i n v e r s i o n .  (1979).  is a  2 M HOAc (15  ml)  i n c u b a t e d on i c e f o r  f o r 20 minutes at 4°C.  to 30 ml Corex tubes.  Two  i n c u b a t e d at -20°C f o r one  The  volumes o f hour.  The  r e c o v e r e d by c e n t r i f u g a t i o n at 10,000 g f o r 20 minutes at room  temperature. p e l l e t was  The  DNA  dissolved  p e l l e t was i n TE  washed with 70% e t h a n o l and  (pH 7.6)  and  air dried.  The  p u r i f i e d by c e n t r i f u g a t i o n i n cesium  c h l o r i d e - e t h i d i u m bromide d e n s i t y g r a d i e n t s .  PURIFICATION OF Plasmid DNA milliliter  PLASMID DNA was  BY CESIUM CHLORIDE GRADIENT CENTRIFUGATION  purified  of p l a s m i d DNA  a c c o r d i n g to M a n i a t i s e t a l . (1982).  isolated, exactly  E t h i d i u m bromide (0.8 ml of 10 mg/ml i n H2O) cesium  chloride solution.  The  1 g o f cesium was  cesium c h l o r i d e  For  c h l o r i d e was  added f o r every 10 ml  s o l u t i o n was  transferred  every added. of to a  -12-  tube s u i t a b l e  for centrifugation  the tube was  filled  Beckman tube  sealer.  i n a Beckman type 50 r o t o r .  w i t h p a r a f f i n o i l and The  tubes were mounted on a stand and  Two  bands were n o r m a l l y v i s i b l e , and  f o r 40 hours  s o l u t i o n was  d i l u t e d with two  E t h i d i u m bromide was  was  The  washed w i t h 70% e t h a n o l . 1 mM  EDTA (pH  supernatant was  The  p e l l e t was  This procedure,  either ampicillin  pellet  was  EDTA, 25 mM  the DNA  i n 10 mM  pellet  Tris-Cl  was  (pH  7.6)  (1979).  LB-glucose  i s a m o d i f i c a t i o n of  media (5 ml) c o n t a i n i n g  (15 ug/ml) was  i n o c u l a t e d with a  c o l o n y and grown o v e r n i g h t at 37°C with m i l d s h a k i n g .  resuspended Tris-Cl  at room temperature  without  resuspended  (50 ug/ml) or t e t r a c y c l i n e  f o r one minute and  A solution  d i s c a r d e d and  DNA  ml p o r t i o n o f the c u l t u r e was  centrifuged  2  spun down a t 10,000 rpm  d e s c r i b e d by M a n i a t i s eji a_l. (1982),  the method of B i r n b o i m and Doly  1.5  and  8.0).  SMALL SCALE ISOLATION OF PLASMID  single bacterial  with  DNA.  incubated at -20°C o v e r n i g h t and  f o r 20 minutes at 4°C.  and  the  was  removed  equilibrated  or t h r e e volumes of H^O  volumes of e t h a n o l were added to p r e c i p i t a t e The mixture  lamp.  the lower band c o n t a i n i n g p l a s m i d DNA  from the s o l u t i o n by repeated e x t r a c t i o n s with n-butanol The  of  at 20°C.  i l l u m i n a t e d with an u l t r a v i o l e t  c o l l e c t e d w i t h an 18 gauge hypodermic n e e d l e .  20XSSC.  remainder  the tubes were s e a l e d u s i n g the  tubes were spun at 50,000 rpm  The  The  the supernatant was  (pH 8.0), and for 5  The  s o l u t i o n of p o t a s s i u m  i n t o a microfuge poured  tube. off.  i n 100 u l a s o l u t i o n c o n t a i n i n g 50 mM lysozyme 4 mg/ml.  The The  tube  was  remaining  g l u c o s e , 10  The mixture was  mM  incubated  minutes.  c o n t a i n i n g 0.2  vortexing.  poured  A  N NaOH and  s o l u t i o n was  a c e t a t e was  1% SDS  (200 u l ) was  added and  s t o r e d on i c e f o r 5 minutes.  added and  the mixture was  mixed  A 150 u l  s t o r e d on i c e f o r  -13-  5 minutes. and  The mixture was  the s u p e r n a t a n t was  p h e n o l / c h l o r o f o r m was at room temperature new  tube.  Two  tube.  ethanol.  pellet  tube was  EDTA (pH  was  d i s c a r d e d and  a i r d r i e d and  The  tube was  the supernatant was  at  4°C  centrifuged  transferred  the s o l u t i o n was  then c e n t r i f u g e d  supernatant was  f o r 5 minutes  An equal volume o f  added and mixed by v o r t e x i n g .  The  The  1 mM  t o a new  volumes of e t h a n o l were added and  temperature.  and  transferred  i n a microfuge  f o r one minute and  -20°C f o r one hour.  The  centrifuged  f o r 10 minutes  the p e l l e t  redissolved  was  i n 10 mM  to a  i n c u b a t e d at at room  washed w i t h  Tris-Cl  (pH  70%  7.6)  8.0).  RESTRICTION ENDONDCLEASE DIGESTION R e s t r i c t i o n endonucleases Eco R l , Bam 500 mM  total  (pH 8.0)  100 mM  Reactions t y p i c a l l y  volume of 50 u l .  Tris-Cl  in this  HI, P v u I I , and Eco RV.  Tris-Cl  reaction.  used  (pH 7.6)  and  A 10X b u f f e r  M g C l , 500 mM  c o n t a i n e d between 0.1 to be d i g e s t e d was  EDTA (pH 8.0).  The  up to 50 u l w i t h s t e r i l e H^O.  restriction  endonuclease  About  by agarose  I f the DNA  was  another r e s t r i c t i o n of p h e n o l . tube.  The  gel  The  of  f o r each enzyme  1 ug o f DNA  dissolved  i n 10  in a mM  c o n c e n t r a t e was  the f i n a l volume  were added per ug o f DNA.  added  was  of the a p p r o p r i a t e  The mixture was  then  extent of the d i g e s t i o n  was  electrophoresis.  to be used  i n a subsequent  d i g e s t i o n ) , the DNA  aqueous l a y e r was  to another microfuge  was  reaction  purified  (a l i g a s e r e a c t i o n  tube.  added and  or  by adding an e q u a l volume  removed and p l a c e d i n another  An equal volume of c h l o r o f o r m was  transferred  and  1 to 5 u n i t s  i n c u b a t e d at 37°C f o r a p p r o x i m a t e l y 2 h o u r s . checked  used  10X b u f f e r  ( o n e - t e n t h of the f i n a l volume of the m i x t u r e ) and brought  I I I , Bgl I I ,  solution consisting  NaCl was  2  The DNA  1 mM  work were Hind  microfuge  the aqueous l a y e r  Sodium a c e t a t e was  was  added to 0.3  M and  -14-  the DNA  was  p r e c i p i t a t e d by adding 2 volumes o f e t h a n o l .  i n c u b a t e d at -20°C f o r 1-12  hours.  c e n t r i f u g a t i o n f o r 10 minutes mately  at 4°C.  1 ml of 70% e t h a n o l was  v o r t e x e d and c e n t r i f u g e d p e l l e t was  a i r dried  The  p r e c i p i t a t e was The  e t h a n o l was  added to the p e l l e t .  f o r 5 minutes.  f o r one hour.  The  collected  was  by  removed and a p p r o x i -  The m i x t u r e was  e t h a n o l was  The p e l l e t was  The m i x t u r e  then  removed and  then d i s s o l v e d  the  i n water  or  Tris-Cl.  LIGATIONS L i g a t i o n r e a c t i o n s were done i n a t o t a l volume of 20 u l u s i n g 10 to 50 ng of v e c t o r DNA  and a three to t e n - f o l d molar excess of i n s e r t  Fragments were l i g a t e d 10 mM  DTT,  and  overhanding-end  1.0 mM  i n a b u f f e r c o n t a i n i n g 66 mM ATP.  T4 DNA  l i g a t i o n s and  i n c u b a t e d at 4°C o v e r n i g h t . treatment  to t r a n s f o r m E.  gels  f o r b l u n t end  10 mM  MgC^,  added (0.1 u n i t f o r  l i g a t i o n s ) and  the m i x t u r e  l i g a t i o n mixtures were used w i t h o u t  further  coli.  AGAROSE GEL ELECTROPHORESIS OF Agarose  l i g a s e enzyme was  1 unit The  Tris-Cl,  fragment.  DNA  (0.7% i n 1 TBE) were prepared f o r e l e c t r o p h o r e t i c  f r a c t i o n a t i o n o f DNA  fragments.  at a v o l t a g e g r a d i e n t of 2-5  E l e c t r o p h o r e s i s was  c a r r i e d out  horizontally  V/cm.  TRANSFORMATION OF YEAST The  procedure used  d e s c r i b e d by Hinnen  f o r yeast t r a n s f o r m a t i o n was  used  t o those  et a l . (1978), Beggs (1978), Sherman et a l . (1981), and  Orr-Weaver et a l . (1983). formed was  similar  A single  c o l o n y of the yeast s t r a i n  to i n o c u l a t e 5 ml of YEPD, and  the c u l t u r e was  to be  trans-  i n c u b a t e d at  -15-  30°C  overnight  inoculated for by  into  centrifugation  The  resuspended cells  DTT  were  and  incubated  ml  at  (O.D.^Q  added When  the  by  centrifuged sorbitol, for  cells  each  as  cells.  the  tube DNA  at  easily  0.33  were  ml  50  the  cells  cells  were  was  i n c u b a t e d on  added  poured gave  visible  (50-55°C)  on  a YNB  rise  were  appear  are  (1 u g )  i n c u b a t e d on  after  plate  2-3  days  of  was  of  10 mM  to each  embedded  the  under  1.5  More  mg  was  glusulase  hours.  t h e y were h a r v e s t e d  ml  per of  STC  2 >  at  and  8.0)  0.5 was  of  rpm  of  added  YEPD and  solution amino  ml  to  containing 15  was  ml then  acids.  regeneration  30°C.  M  competent  0.3  appropriate  (1  2,000  and  The  and  tube.  u l of  ml  pellet  tube  STC  per  pH  The  solution  at  10  the  entire  the  before.  and  The  within  incubation  dark  minutes,  tube.  as  a phase-contrast  t o 50  CaCl  pellets  removed  centrifuged ml  The  suspension  were  i n 2.5  i c e f o r 15  containing  The  tube  sorbitol  added  tube,  per  temperature.  1M  and  minutes.  added  of  room  i n 0.2  Tris,  centrifuge was  ml  tube  resuspended  10 mM  at  were h a r v e s t e d  refractile.  resuspended  CaCl,,) p e r  to c o l o n i e s  after  i n 2.5  i c e f o r 20  to a s t e r i l e  agar  and  under  shaking  centrifuged  tube.  formed  f o r 5 minutes  u l of plasmid  mixture  and  u l samples  cells  been  cells  sorbitol  examined  was  temperature.  converted to spheroplasts,  10 mM  cells  not  The  tube  to each  culture  grown w i t h m i l d  room  1 M  non-refractile  2,000 rpm  The  per  5-10  and  overnight  then  at  of  added  u l f^O  had  the  - 0.35).  sorbitol  i n 2.5  polyethylene glycol,  Transformants were  been  Tris-Cl, The  regeneration  immediately  had  before.  The  and  was  50  resuspending  10 mM  (20%  with  =  s h a k i n g , and  spheroplasts  Approximately  PEG-TC  glusulase  of  y e a s t were  m i c r o s c o p e , w h i l e normal  5 minutes.  yeast  1 M  Spheroplasts are  i f the  washed  of  with mild  centrifugation  was  ml  ml  f o r 2 minutes  resuspended  diluted  One  The  3,000 rpm  u l of  phase-contrast  of  o f YEPD.  i n 2.5  30°C  microscope.  was  at  then  100  periodically,  by  80  shaking.  approximately 8 hours  were  of  with mild  agar  which  -16-  YEAST PLASMID PURIFICATION Plasmids  were i s o l a t e d  from y e a s t as d e s c r i b e d by L o r i n c z (1984).  medium s i z e d y e a s t c o l o n y ( a p p r o x i m a t e l y placed 8.0),  i n a microfuge  tube  below the l e v e l  3 mm i n diameter) was p i c k e d and  c o n t a i n i n g 200 u l o f 100 mM CaCl2,  1 mM EDTA, 0.1% SDS.  A  10 mM T r i s - C l (pH  Glass beads (0.45 mm diameter) were added to j u s t  o f the l i q u i d  and the contents mixed v i g o r o u s l y on a v o r t e x  mixer f o r 1 minute.  The suspension was e x t r a c t e d w i t h an equal volume o f  Tris-buffered  and then e x t r a c t e d with an equal volume o f c h l o r o f o r m .  phenol  Sodium a c e t a t e was added to 0.3 M, and 2 volumes o f c o l d e t h a n o l were added. After  30 minutes a t -60°C, n u c l e i c a c i d was p r e c i p i t a t e d by c e n t r i f u g a t i o n (10  minutes). used  The a i r d r i e d p e l l e t  to transform E. c o l i  was resuspended  and the e n t i r e DNA s o l u t i o n was  (0.2 ml s u s p e n s i o n ) .  ISOLATION OF YEAST RNA Yeast  RNA was i s o l a t e d as d e s c r i b e d by McNeil  modifications.  A s i n g l e colony o f y e a s t was used  p l u s the a p p r o p r i a t e amino a c i d s .  and Smith (1986) w i t h a few to i n o c u l a t e 5 ml o f YNB  The c u l t u r e was grown w i t h m i l d shaking f o r  1-2 days and 1 ml o f the c u l t u r e was i n o c u l a t e d i n t o 100 ml o f f r e s h YNB media. The  c u l t u r e was then incubated at 30°C w i t h m i l d shaking u n t i l  0.8. was  Cycloheximide  chilled for  c e l l s were t r a n s f e r r e d  and c y c l o h e x i m i d e  4 ° C . The c e l l s  to c e n t r i f u g e tubes  The c e l l s were resuspended (0.1 mg/ml) per tube.  weighed 30 ml Corex tube at  A  S  The c u l t u r e was then  i n t o two 520 ml c e n t r i f u g e b o t t l e s , each h a l f - f u l l o f crushed  1 minutes a t 40°C.  r^O)  W  was added to a c o n c e n t r a t i o n o f 0.1 mg/ml and the c u l t u r e  i n c u b a t e d a t 30°C f o r 5 minutes w i t h m i l d s h a k i n g .  poured  its A^^Q  i c e . The  and c e n t r i f u g e d a t 4000 rpm i n t o about  10 ml o f i c e c o l d  The mixture was t r a n s f e r r e d  to a  and the c e l l s were p e l l e t e d at 4000 rpm f o r 3 minutes  were then immediately  f r o z e n i n a dry i c e / e t h a n o l b a t h .  -17-  Acid-washed g l a s s beads were added to the f r o z e n c e l l wet  weight c e l l s ) f o l l o w e d by 3 ml/g o f i c e - c o l d  pellets  (3g beads/g  RNA e x t r a c t i o n b u f f e r (0.15 M  NaCl, 0.1 M T r i s - H C l ) and 50 u l / g o f Vanadyl r i b o n u c l e o s i d e complexes (VRC: 0.2 M).  The c e l l s  were broken by v o r t e x i n g hard  each f o l l o w e d by 45 seconds o f c o o l i n g on i c e . 9,000 rpm f o r 5 minutes at 40°C.  the  as b e f o r e  supernatant  The  The c e l l s were c e n t r i f u g e d as b e f o r e and  K-digested  acetate The  i n a 37° water bath f o r 60 m i n u t e s .  The aqueous supernatant  to a c l e a n tube and n u c l e i c a c i d was p r e c i p i t a t e d by a d d i n g to 0.3 M, 2.5 volumes of e t h a n o l , and c h i l l i n g  at -20°C  sodium  overnight.  An equal  a t 4°C f o r 16 h o u r s .  RNA p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n (9,000 rpm, 40 minutes, and d i s s o l v e d i n s t e r i l e  sodium a c e t a t e , The  was t r a n s -  i n c o l d e t h a n o l , d r i e d and d i s s o l v e d i n 1 ml o f 20 mM EDTA.  volume o f 4M L i C l was added and the mixture was incubated  4°C)  1 volume o f  p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n (9,000 rpm, 20 m i n u t e s , 40°C)  rinsed  The  supernatant.  o f 0.5% and 0.5 mg/ml,  s o l u t i o n was 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 m y l a l c o h o l (24/24/1). ferred  the p r e v i o u s  K were added t o c o n c e n t r a t i o n s  The mixture was incubated  proteinase  was t r a n s f e r r e d t o a s t e r i l e  E x t r a c t i o n b u f f e r and VRC were added t o  was removed and combined w i t h  SDS and p r o t e i n a s e respectively.  and v o r t e x e d .  intervals,  The mixture was c e n t r i f u g e d a t  The supernatent  30 ml Corex tube and p l a c e d on i c e . the p e l l e t  f o r s i x 15-second  H^O.  A f i n a l ethanol  f o l l o w e d by r i n s i n g w i t h e t h a n o l  precipitation  served  from 0.3M  to d e s a l t the RNA.  p r e c i p i t a t e was a i r d r i e d , d i s s o l v e d i n E^O and s t o r e d at -20°C.  5 mg o f RNA was o b t a i n e d  Up to  from 1 g (wet weight) o f c e l l s .  SELECTION OF POLY A+ RNA Polyadenylated  RNA molecules were i s o l a t e d by chromatography u s i n g o l i g o  dT c e l l u l o s e by a m o d i f i c a t i o n of the procedure o f A v i v and Leder  (1972).  -18-  Oligo  dT  c e l l u l o s e was  Tris,  4 mM  E D T A ) and  built-in-filter. The  run-through  through  the  loading  buffer  applying  200  RNA was  and  ul  were  H 0.  was  RNA  mixture  (100  ug  was  RNA  loading  applied  aliquots)  c o l l e c t e d and  to  was  reapplied  non-polyadenylated.  2 ml  of  a  buffer  small  i n 3 ml  to  column.  The  M  sterile  loaded the  (0.5  of  loading RNA  washed  with  M  KC1.  The  polyadenylated  RNA  sterile  h^O  5 times  and  collecting  fractions.  pooled,  stored  at  precipitated  in ethanol,  and  mM  was  ran  5 ml  eluted  dissolved  a  buffer.  which  0.5  the  10  column w i t h  The  c o l u m n was  NaCl,  of  then  END-LABELING OF The  the  c o l u m n was  fractions  2  equilibrated with  of  by  The  in  sterile  -20°C.  OLIGONUCLEOTIDES  oligonucleotide  (10  p m o l e s ) was  5'-ended  labeled  in a  reaction  (50  32 ul)  containing  [gamma-  (10  u n i t s ) , 50  mM  spermidine, of  2 ul  of  for 0.5  Tris-HCl,  45 M  P]ATP(120 C i  minutes  EDTA and  pH at  7.7,  10  37°C.  heating  of  3000 C i / m m o l e ) , mM  The  to  MgCl ,  5 mM  reaction  was  2  67°C  (10  polynucleotide  dithiothreitol, terminated  by  kinase 0.1  mM  addition  minutes). 32  The ATP  by  grams  labeled  chromatography of  water  EDTA] was mately  for was  15  to  ul  poured  the  tions  were  which  contained  of  and  columns  column  top  of  collected the  buffer  the  was  ml  of  of  H^O  40  ml  of  in microfuge  unincorporated  S e p h a d e x G-25.  was  was  cooled  added  slowly to  the  Column b u f f e r tubes.  The  oligonucleotide  was  for  down t o  and  [10  mM  4  Tris  leading pooled  as  8  temperature, (pH  continuously.  added  8.0),  5  solution  needed  stored  mM  Approxi-  and  and frac-  peak o f r a d i o a c t i v i t y and  P]  c o l u m n s ) were  room  oligonucleotide was  (gamma-  Approximately  enough  column b u f f e r  were p o u r e d was  from  (this  mixture  column.  labeled  separated  column  The  o f f , and  The  a  100  minutes.  added.  200  applied  through  S e p h a d e x G-25  autoclaved the  oligonucleotide  at  -20°C.  -19-  PRIMER EXTENSION ANALYSIS The  o l i g o n u c l e o t i d e was  d e s a l t e d and h y b r i d i z e d  (10^ cpm)  per r e a c t i o n ] with samples of RNA.  i n 250 mM  K C l , 10 mM  specific  RNA,  T r i s - H C l , pH 8.0,  1 mM  A n n e a l i n g was EDTA.  the o l i g o n u c l e o t i d e primer was  [ a p p r o x i m a t e l y 1 ng performed  Following h y b r i d i z a t i o n  extended  The  r e a c t i o n products were denatured  and  to  u s i n g A v i a n myeloma  v i r u s r e v e r s e t r a n s c r i p t a s e u s i n g the c o n d i t i o n s d e s c r i b e d (McKnight 1981).  at 65°C  the l e n g t h s o f the  et a l . , extended  p r o d u c t s e s t i m a t e d by d e n a t u r i n g a c r y l a m i d e g e l e l e c t r o p h o r e s i s .  DENATURING UREA-ACRYLAMIDE Denaturing in  1 X TBE.  GEL  a c r y l a m i d e g e l s (6% and  Samples were denatured  l o a d i n g and  electrophoresis  8%)  c o n t a i n i n g 7 M urea were prepared  i n formamide (100°C, 4 m i n u t e s ) p r i o r  to  (30-40 V/cm).  SI NUCLEASE MAPPING DNA  fragments  were  polynucleotide kinase. and G i l b e r t ,  32  P-labelled  Stand  s e p a r a t i o n was  molecular  S i n g l e s t r a n d s were e l u t e d M NaCl  hybridized  to RNA  and  was  and  7%  from the g e l i n 50 mM The  Tris  pH 8,  20 mM  s i n g l e s t r a n d e d DNA  EDTA,  probes  were  1982).  AND  NORTHERN HYBRIDIZATION  a n a l y z e d by e l e c t r o p h o r e s i s  d e n a t u r a t i o n w i t h formaldehyde  and  T4  as d e s c r i b e d (Maxam  subsequently d i g e s t e d with SI n u c l e a s e as d e s c r i b e d  ELECTROPHORESIS OF RNA RNA  P] ATP  weight.  o v e r n i g h t at room temperature.  (De Greve £t a K ,  GEL  accomplished  32  1980), u s i n g a c r y l a m i d e c o n c e n t r a t i o n s v a r y i n g between 3% and  a c c o r d i n g to the fragment  0.5  at the 5' end u s i n g [gamma-  i n formaldehyde-agarose  gels after  formamide (Lehrach et a l . , 1977;  Maniatis  -20-  et a l . ,  1982).  As much as 30 ug o f RNA was denatured i n a volume o f 50 u l  c o n t a i n i n g 2.2 M formaldehyde, 50% formamide,  and 1/2 MOPS b u f f e r  (IX MOPS  b u f f e r c o n t a i n e d 40 mM Na MOPS, 10 mM sodium a c e t a t e , 1 mM EDTA, pH 7.9) by h e a t i n g a t 55°C f o r 15 minutes. RNA  samples  Sample l o a d i n g b u f f e r  were loaded on a 1.1% agarose g e l .  (2 u l ) was added  and the  The g e l was made up i n IX MOPS  b u f f e r c o n t a i n i n g 2.2 M formaldehyde and the g e l was run i n IX MOPS a t 0.5 - 1 V/cm for  f o r 6-12 h o u r s . 10 minutes  A f t e r e l e c t r o p h o r e s i s , the g e l was soaked  i n s t e r i l e K^O  and then soaked i n 20X SSC (IX SSC i s 0.15 M NaCl, 0.15 M Na  c i t r a t e ) f o r 30 minutes  p r i o r to t r a n s f e r r i n g the RNA t o n i t r o c e l l u l o s e .  The procedures o f Thomas (1980) were used to t r a n s f e r RNA from agarose gels  to n i t r o c e l l u l o s e  and h y b r i d i z e the RNA t o r a d i o a c t i v e l y  labelled  The N o r t h e r n h y b r i d i z a t i o n apparatus was c l e a n e d out w i t h s t e r i l e chamber was f i l l e d  w i t h 20X SSC.  Two p i e c e s o f f i l t e r  probes.  h^O and the  paper soaked  i n 20X SSC  were l a i d  a c r o s s the box so that the papers j u s t touched the bottom o f each  chamber.  The g e l was then l a i d  on the f i l t e r  p l a c e d around the g e l so that no l i q u i d  paper, and c u t X-ray f i l m was  diffused  n i t r o c e l l u l o s e , which had been soaked i n s t e r i l e soaked  out o f the g e l .  h^O f o r one minute  i n 20X SSC f o r one minute, was p l a c e d on top o f the g e l .  not covered by n i t r o c e l l u l o s e was removed by a r a z o r b l a d e .  and then  The agarose  Two more p i e c e s  of Whatman 3 MM paper were placed on t o p o f the n i t r o c e l l u l o s e , 6 cm s t a c k o f paper t o w e l s .  A piece of  f o l l o w e d by a  A book was p l a c e d on top o f the assemblage and  t r a n s f e r o f RNA to the n i t r o c e l l u l o s e was allowed to proceed f o r about 16 hours.  The n i t r o c e l l u l o s e was then baked a t 80°C f o r 2 h o u r s .  Nitrocellulose  f i l t e r s with bound RNA were p r e h y b r i d i z e d  at 42°C i n s e a l e d p l a s t i c bags c o n t a i n i n g 5X SSC, IX Denhardt's BSA)  solution  f o r 4-16 hours  10 ml o f a mixture o f 50% formamide,  (0.02% F i c o l l ,  0.02% p o l y v i n y l p y r r o l i d o n e , 0.02%  50 mM NaH PO. and Na„HP0. , pH 7.0, and denatured, sheared salmon  sperm  -21-  DNA  (250  ug/ml).  The  p r e h y b r i d i z a t i o n mixture was  h y b r i d i z a t i o n m i x t u r e (10 ml)  which c o n t a i n e d  The  composed of  h y b r i d i z a t i o n mixture was  mixture and  one  part  minutes and  chilled  H y b r i d i z a t i o n was was  of 50% on  The  replaced  with a  r a d i o a c t i v e l y - l a b e l l e d probe.  four p a r t s  dextran s u l f a t e .  The  of  prehybridization  probe was  boiled  for 5  i c e b e f o r e adding i t to the h y b r i d i z a t i o n m i x t u r e .  allowed to proceed  washed i n 2 changes of 0.1%  each time.  the  removed and  filter  was  SDS,  f o r about 2X  SSC  at room temperature  then washed i n two  55°C f o r 20 minutes each.  The  filter  was  18 hours at 42°C.  changes of 0.1X  subjected  The  filter  f o r 5 minutes  SSC,  0.1%  SDS  at  to a u t o r a d i o g r a p h y .  NICK TRANSLATION N i c k t r a n s l a t i o n of DNA Typically 4 x 10^  500  ng  of DNA  - 10 x 10^  was  was  done u s i n g  the Amersham Nick T r a n s l a t i o n K i t .  r a d i o a c t i v e l y labeled  cpm/ug DNA  to a s p e c i f i c  i n a 50 u l r e a c t i o n c o n t a i n i n g  activity  10  u l of  of  nucleo-  32 tide buffer solution at  15°C  (dGTP, dCTP, dTTP, 50  containing for 2 hours.  solution  RNA  extracted  procedure d e s c r i b e d weight) was  P] ATP, The  and  5 u l o f an  r e a c t i o n was  were removed by  enzyme  incubated  running  the  frozen  RNA  from t o t a l  (100  ml)  The  in l i q u i d nitrogen  containing  cell  c a l l u s t i s s u e by a m o d i f i c a t i o n  by McPherson et a l . (1980).  t i s s u e ( f r o z e n powder) was  Heparin.  DNase I.  Unincorporated nucleotides  PLANT TUMOR  was  divided  polymerase I and  [alpha-  through an U l t r o g e l column.  ISOLATION OF  buffer  DNA  uCi of  debris  (10,000 g f o r 5 m i n u t e s ) .  and  10%  t i s s u e (50  ground i n a b l e n d e r .  extracted  1 M Tris,  Callus  SDS,  of g  The  the fresh finely  by homogenization (2 m i n u t e s ) i n 10%  T r i t o n , and  were p r e c i p i t a t e d f o l l o w i n g  0.5  mg/ml  c e n t r i f u g a t i o n at  4°C  -22-  The supernatant phenol:  chloroform  was  filtered  (1:0.5).  through  The aqueous  c h l o r o f o r m and then w i t h c h l o r o f o r m . precipitation  c h e e s e c l o t h and e x t r a c t e d w i t h l a y e r was  N u c l e i c a c i d s were r e c o v e r e d  (2.5 volumes) at -20°C o v e r n i g h t .  RNA  was  by p r e c i p i t a t i n g with L i C l  precipitated  from e t h a n o l  phenol:  by e t h a n o l  The p e l l e t was r e c o n s t i t u t e d  i n 0.1 M NaOAc to which an equal volume o f L i C l recovered  r e - e x t r a c t e d with  (5 M) was  added.  RNA  was  (2.5 M) at -20°C o v e r n i g h t .  The  resulting  (2.5 volumes).  STRESS INDUCTION OF PLANT TDMORS Crown g a l l subjected at  tumors and transformed  to v a r i o u s s t r e s s  c o n c e n t r a t i o n s of 10 ^ M,  tobacco  treatments. 10 ^ M,  c u l t u r e s (16-12-C) were  Cadmium, mercury, z i n c , and a r s e n i t e  10 ^ M, and 10  7  M were added to MS  media  ( p l a n t hormones were added to MS media f o r growth o f 16-12-C) f o r d i f f e r e n t p e r i o d s of time  and RNA  40°C f o r d i f f e r e n t  times  was  isolated.  and RNA was  The p l a n t tumors were heat isolated.  shocked at  -23-  RESULTS  A.  T-DNA Gene  Plasmid  7 i n Yeast  Constructions  Construction as  shown  i n Figures  YEpl3  (Broach  (Fig.  1).  gene  described  Restriction  i n Figure  primer  position  S_. c e r e v i s i a e  extension  strain  7 RNA  transcriptase.  E9  (Fig. 6).  located  that  GM-3C-2  from  added  a n a l y s i s was done o n t h e s e  was u s e d  ( F i g . 5 ) was a d d e d  the center  The s o u r c e  plasmid, 7  containing  plasmids  1  as  electrophoresed  the major  initiation  to t r a n s c r i p t  o f t h e TATA  sequence.  i n a l l the primer  through  site codon  extensions.  octopine  sequencing  tumor  long  o f the open  initiation  that  line  which residue  reading  at a distance  obtained  this  o f 29  when no RNA  4) i n d i c a t e  The 60 n u c l e o t i d e  I t i s thought  reverse  i s the adenosine  The r e s u l t s  stretch.  with  a DNA  i s 45 b a s e s  r e a c t i o n ( F i g . 6, l a n e  60 n u c l e o t i d e  to prime  7 from  cDNA p r o d u c t  o f t h e ATG s t a r t  extension  7 RNA was t h e  ( o l i g o - 7 ) complementary t o  RNA a n d u s e d  corresponds  to the primer  o f T-DNA g e n e  for transcript  transcription  t h e number a n d  ej: a _ l . , 1 9 8 1 ) t r a n s f o r m e d  to yeast  were  obtained  5 ) show t h a t  This  to i d e n t i f y  An o l i g o n u c l e o t i d e p r i m e r  was a n u n e x p e c t e d  appears  done  c o n t a i n i n g gene  a n d a 1,547 bp T-DNA s e g m e n t  (Orr-Weaver  15 n u c l e o t i d e s u p s t r e a m  bases  were  Y-BG.E9 c o n s i s t s o f t h e y e a s t  7 RNA 5' e n d s .  the major  (Fig. 4).  there  method  The r e s u l t s  frame  was  site  The cDNA p r o d u c t s  ( F i g . 6, l a n e  indicates  i n yeast  Analysis  Y-BG.E9 a n d Y - B G E . E 9 .  gel  7 expression  3.  o f T-DNA gene  T-DNA g e n e  Plasmid  Y-BGE.E9 c o n s i s t s o f Y E p l 3  Extension  The  1 a n d 2.  f o r T-DNA gene  et: a J l . , 1 9 7 9 ) , a n d a 2,637 bp T-DNA segment  7 (Fig. 2).  Primer  o f plasmids  that  stretch  60 n u c l e o t i d e  -24-  F i g u r e 1. C o n s t r u c t i o n o f Y-BG.E9 C o n s t r u c t i o n o f yeast plasmid f o r T-DNA gene 7 e x p r e s s i o n i n y e a s t . Y-BG.E9 was c o n s t r u c t e d as f o l l o w s : LE.E9 which i s pBR325 p l u s the EcoRl l e f t j u n c t i o n fragment o f T-DNA/plant DNA sequences o f the E9 tumor l i n e was d i g e s t e d w i t h B g l l l . The 2,637 bp T-DNA fragment c o n t a i n i n g gene 7 was l i g a t e d t o YEpl3 which had been d i g e s t e d with Bam HI (Bam HI and B g l l l generate cohesive ends). The l i g a t i o n r e a c t i o n was then t r e a t e d w i t h Bam Hi to d i g e s t a l l l i g a t i o n products except f o r the recombinant p l a s m i d Y-BG.E9 which c o n s i s t s of YEpl3 p l u s the 2,637 bp T-DNA fragment c o n t a i n ing gene 7. E=EcoRl, H - H i n d l l l , Bg=BglII, B=Bam HI; the dashed l i n e s i n d i c a t e T-DNA sequences.  -25-  F i g u r e 2. C o n s t r u c t i o n o f Y-BGE.E9 C o n s t r u c t i o n o f y e a s t p l a s m i d f o r T-DNA gene 7 e x p r e s s i o n i n y e a s t . Y-BGE.E9 was c o n s t r u c t e d b y d i g e s t i n g L E . E 9 w i t h E c o RV a n d B g l l l . The 1,547 b p T-DNA f r a g m e n t c o n t a i n i n g g e n e 7 was l i g a t e d t o Y E p l 3 w h i c h h a d b e e n d i g e s t e d w i t h Bam HI a n d P v u I I (Bam HI a n d B g l l l h a v e c o h e s i v e e n d s , and P v u I I a n d E c o RV have c o h e s i v e e n d s ) . T h e l i g a t i o n r e a c t i o n was t h e n t r e a t e d w i t h Bam HI t o d i g e s t a l l t h e l i g a t i o n p r o d u c t s e x c e p t f o r t h e r e c o m b i n a n t p l a s m i d Y-BGE.E9 w h i c h i s Y E p l 3 p l u s t h e 1,547 bp T-DNA f r a g m e n t c o n t a i n i n g gene 7. EV=Eco RV, E = E c o R l , H - H i n d l l l , B g = B g l I I , B=Bam H i ; t h e d a s h e d l i n e s i n d i c a t e T-DNA s e q u e n c e s .  -26-  a  b  c  d  e  f  g  h  i  F i g u r e 3. R e s t r i c t i o n s i t e a n a l y s i s o f p l a s m i d s Y - B G . E 9 , Y - B G E . E 9 , Y E p l 3 , a n d LE.E9. DNA was d i g e s t e d w i t h r e s t r i c t i o n enzymes i n d i c a t e d b e l o w a n d t h e p r o d u c t s w e r e a n a l y z e d b y e l e c t r o p h o r e s i s t h r o u g h a 0.7% a g a r o s e g e l i n TBE. The g e l s were s t a i n e d w i t h e t h i d i u m b r o m i d e and p h o t o g r a p h e d i n UV light. ( a ) lambda ( H i n d l l l ) ; ( b ) LE.E9 ( B g l l l ) ; ( c ) Y E p l 3 ( H i n d l l l ) ; ( d ) Y E p l 3 ( B g l l l ) ; ( e ) Y-BGE.E9 ( H i n d l l l ) ; ( f ) Y-BGE.E9 ( B g l l l ) ; ( g ) Y-BG.E9 ( H i n d I I I ) ; ( h ) Y-BG.E9 ( B g l l l ) ; ( i ) l a m b d a ( H i n d l l l ) .  G CTT  GAA  AAT It)  TTG  TCT  indlll TAT  GCC  CGA  AAT CAT 30  CAC  AGC 40  Met Asn ATG AAT 110 *  Phe TTC  TCG  TCC  CAG 50  CCC  GGC  ATC TAT 60  ATA  TAG  CGC 70  CAA  A l a Asp GCA GAT 120  Thr ACT  Pro Leu A l a Ser Leu Asp CCC TTG GCC TCC CTC GAC 130 140  Leu CTA  Asp GAC 150  Gin Thr CAA ACA 220  Asn AAC  CAC  T r p A l a Cys Glu G l u Phe H e TGG GCA TGC GAA GAG TTT ATC 160 170  Lys Thr AAA ACT  Tyr Gly A l a Ser Pro Gin Leu Glu Thr Gly Glu Val H e TAT GGT GCA TCT CCA CAA TTG GAA ACA GCA GAG GTA ATC 180 190 200 210  Lys AAG  Tyr Leu TAT TTG 240  Tyr G l y Lys Gly Ser Leu TAT GGC AAA GGT TGA CTC 250 260  Glu Glu Leu Ser Phe Thr Thr GAA GAA TTA TCC TTC ACT ACC 310 320  Ala GCC  He ATT 380  Phe TTT  Leu CTG  ATC  GCA 530  TTG  CAT  TAT  TTC 680  ACA  TGT  CAA  AAA 830  Phe TTT  G i n Ser Asn CAA AGC AAC 390  ATC  CGC  ACA CCT 100  Gly Leu Leu GGG CTG CTG 230  TAC ACA 90  CCC CCC 20  AAC  Asn AAT  AGT 80  TAA  Cys TGC  V a l Ser Asn GTT TCA AAT 410  V a l A l a His Gly Ser Met GTA GCC CAT GGA TCG ATG 470 480  CTC  TCA  TCT  CAA TAA 610  ATT  TAT  GTT 620  TTT  CAA  GAT 690  ATC  ATT  AAA ATT 760  ATG  AAA  TGG 770  TTA  TAA 840  CAT  ATT  TAA  Glu GAG 300  Leu Thr Tyr Tyr TTA ACT TAT TAT 370  Val GTC  Pro G l u Lys Gly Phe Leu Arg Cys His Asn Arg CCA GAG AAA GGC TTT CTG AGA TGC CAT AAT CGC 420 430 440  Pro CCA 450  Lys Pro A l a Glu Met AAG CCA GCT GAG ATG I 330 340 PvuII  Tyr Pro H e TAC CCC ATA 460 CTT 540  Lys AAG  He ATA  Tyr Phe Leu TAT TTC CTG 400  AGT  Ser Gin Arg H e TCA CAG CGG ATT 270  Ser STOP AGC TAA GCT 490  ACG GCA 550  ATG  TAC  CAG 560  TTT  GCT  TGG 630  ACT  ATA  CTT TTA 700  CAA  GTA  TAC 710  GAG  TGG  TGG 780  CGA  CCG  ATT TCA 850  TCC  TGG  ATT 860  His Asp Thr His Leu Lys Phe CAT GAC ACA CAC CTC AAA TTT 280 290  Lys Ala Gin Gin Ser Asp AAG GCG CAA CAA AGT GAT 350 360  AGC  TAT  ATC 500  ATA  TAA 570  TCA  GTT  ATA CCT 640  GAC  TTG  TTA 650  GTG  TTT  AAA 720  TTG  AAT  AGC TCA 790  AGC  ACA  CTT 800  AAA  AAG 870  TCA  CCG  CTG  AGA  ATC  AAT  TTA 510  TGT  ATT  ACA  CAT 520  AAT  ATT GAA 580  ATA  TTT  CTG 590  AAT  TTA  AAC 600  TTT  TAT  CAA 660  TAA  ATA  TTT  AAA 670  CTA  ACC ATA 730  AAT  TTT  TAT 740  TTT  TCA  AAT 750  CAA  CAT 810  AAC  GGG  ACC  AAA 820  TCG  AC  TTC  TTGCCGCC | 1,178 Smal  F i g u r e 4. DNA sequence of T-DNA gene 7. The p r e d i c t e d amino a c i d sequence of a p r o t e i n , Mr 14,400 was o b t a i n e d by a n a l y s i s of the open reading frame (Staden, 1980). The p u t a t i v e r e c o g n i t i o n sequence ("TATA box") and the heat shock r e l a t e d sequence (the heat shock consensus sequence i s CT-GAA—TTC-AGare u n d e r l i n e d , the t r a n s c r i p t i o n i n i t i a t i o n s i t e i s i n d i c a t e d ( 1), as are c h a r a c t e r i s t i c p o l y a d e n y l a t i o n s i g n a l s . The n u c l e o t i d e s are numbered from the H i n d l l l s i t e (Base 2,119 i n F i g u r e s 1 and 2 ) . * marks the b e g i n n i n g of p u t a t i v e t r a n s c r i p t i o n i n i t i a t i o n s i t e s of gene 7 i n yeast as determined by SI n u c l e a s e mapping. +  -28-  AAG CTT GAA AAT TAA GCC CCC CCC CGA AAT CAT CGC CAC AGG TCG TCC CAG CCC GGC ATC 10 20 30 40 50 60 Met Asn Phe A l a TAT ATA TAG CGC CAA TAT AGT TTG TCT TAC ACA AAC ACA CCT CAC ATC ATG AAT TTC GCA 70 80 90 100 110 120 3'G TAC TTA oligo  F i g u r e 5. O l i g o n u c l e o t i d e c o r r e s p o n d i n g t o the 5' r e g i o n o f t r a n s c r i p t 7. The DNA sequence r e p r e s e n t s the 5' f l a n k i n g r e g i o n of t r a n s c r i p t 7 showing the l o c a t i o n and p a r t i a l sequence o f the 30-mer 3 -GTACTTAAATCGTCTATGAGGGAACCGGAG-5 . 1  1  5  -29-  1  2 3 45 6  m  <45  F i g u r e 6. R e v e r s e t r a n s c r i p t a s e e x t e n s i o n p r o d u c t s f r o m RNA p r i m e d w i t h a n o l i g o n u c l e o t i d e c o m p l e m e n t a r y t o t h e 5' c o d i n g r e g i o n o f t h e T-DNA t r a n s c r i p t 7 ( F i g u r e 5 ) . RNA s a m p l e s were f r o m y e a s t (GM-3C-2) t r a n s f o r m e d w i t h Y-BG.E9 a n d Y E p l 3 . T h e ^ P - l a b e l l e d e x t e n s i o n p r o d u c t s were s i z e d on a d e n a t u r i n g u r e a - a c r y l a m i d e g e l . The r e s u l t s r e p r e s e n t e x p r e s s i o n f r o m t h e T-DNA c l o n e s : l a n e 1, Y-BG.E9 (2 ug p o l y A s e l e c t e d RNA); l a n e 2, Y-BG.E9 ( 1 0 0 u g t o t a l RNA); C o n t r o l samples: l a n e 3 , Y E p l 3 ( 1 0 0 ug t o t a l RNA); l a n e 4, no RNA; l a n e 5, E9 p l a n t tumor ( 4 0 ug t o t a l RNA); l a n e 6, P - l a b e l l e d H p a l l f r a g m e n t s o f p B R 3 2 2 . The m i g r a t i o n o f t h e 30 b a s e 32 oligonucleotide (oligo-7) i s indicated. +  3 2  P  -30-  s t r e t c h was  the double stranded  cDNA products no RNA  and  RNA  besides  form o f o l i g o - 7 .  The  absence of any  the 60 n u c l e o t i d e s t r e t c h i n the c o n t r o l l a n e s  from yeast  transformed  w i t h YEpl3 ( F i g . 6,  i s not  yeast  r e s u l t s of the p o l y A  transformed  +  s e l e c t e d RNA  with Y-BG.E9 ( F i g . 6,  and  lanes  the t o t a l  1 and  RNA  specific  for  results noted,  from p o l y A  +  s e l e c t e d RNA  though, t h a t the primer  method c o u l d not  and  total  RNA  base upstream of the ATG  from  5' ends.  were i d e n t i c a l .  e x t e n s i o n method was  d e t e c t t r a n s c r i p t i o n products  samples  2, r e s p e c t i v e l y ) show t h a t  t h e r e were a l a r g e number of T-DNA gene 7 RNAs w i t h d i f f e r e n t  that  4,  RNA.  The  one  containing  lanes 3 and  r e s p e c t i v e l y ) demonstrates that the o l i g o n u c l e o t i d e primer yeast  other  I t should  l i m i t e d because  with  initiation  t r a n s l a t i o n i n i t i a t i o n codon and  The be  this  sites  located  downstream from  base.  SI Nuclease Mapping The  SI n u c l e a s e  7 RNAs i n y e a s t .  p r o t e c t i o n method was  SI nuclease  used to map  the 5' ends of T-DNA gene  r e s i s t a n t h y b r i d s were formed between  H i n d l l l / P v u I I T-DNA gene 7 fragment shown i n F i g u r e 4 and preparations.  T h i s method c o u l d d e t e c t  downstream from the ATG in Figure The  each of  transcription initiation  translation initiation  codon.  The  RNA  sites  results  are shown  7.  c o n t r o l s (no RNA,  transformed f o r yeast  2 ug  the  with YEpl3,  RNA.  lane A;  mapping of RNA  fragment of 239  the normal t r a n s c r i p t i o n  from GM-3C-2, lane B; RNA  lane C) demonstrate that the DNA  SI nuclease  F) shows that an RNA  RNA  initiation  isolated  bases was site  and  from GM-3C-2  probe i s not  specific  from E9 p l a n t tumors  protected.  The  (lane  d i s t a n c e between  the PvuII. r e s t r i c t i o n  site  is  239  -31-  A  B C  D  E  F  G  m4M  F i g u r e 7 - SI n u c l e a s e mapping of )'east T-DNA gene 7 RNA 5 ' t e r m i n i . SI n u c l e a s e r e s i s t a n t h y b r i d s were formed between the H i n d l l l / P v u I I T-DNA gene 7 fragment shown i n F i g u r e 4 and 2 ug each of RNA p r e p a r a t i o n s from y e a s t (GM-3C-2) transformed w i t h Y E p l 3 , Y - B G . E 9 , and Y-BGE.E9 ( l a n e s C, D, and E , r e s p e c t i v e l y ) . Lane A , no RNA; lane B, y e a s t RNA w i t h no T-DNA; lane F, RNA from E9 p l a n t tumor RNA (2 u g ) ; lane G , P - l a b e l l e d H p a l l fragments of pBR322. P r o t e c t e d DNA was e l e c t r o p h o r e s e d on 8% a c r y l a m i d e / urea g e l s . 3 2  -32-  bases,  i n d i c a t i n g that  transcription SI and  mapping  Y-BGE.E9 i s shown were  protected  assumed  probe,  that  then  initiation site The  level  shorter  Northern  minor  with RNAs  extension  the shorter  one e n d o f t h e RNA  tumors  tumors.  transformed  some l o n g e r correspond  protected  RNAs  corresponds  minor  to the PvuII  downstream downstream  RNAs c o r r e s p o n d  to i n i t i a t i o n  RNAs  being  from  sites  are expressed at or l e s s ) .  site  correspond from  o r 49 n u c l e o t i d e s  Y-BG.E9  to i n i t i a t i o n  (216 bases  RNA w o u l d  with  RNAs o f 216 b a s e s a n d  a n a l y s i s , and t h e y  end o f t h e 216 b a s e  protected  If i t  o f t h e DNA  to t r a n s c r i p t i o n  the normal  initiation  t h e TATA b o x ( F i g u r e 4 ) .  sites  farther  downstream.  i ffull  length  transcripts  Hybridizations  Northern  hybridizations  were b e i n g  transcribed  Figure  E9 p l a n t  8.  gene  7 probe  Smal  site  (lane  were done  f r o m T-DNA  tumor p o l y 4).  A  The gene  ( 1 , 1 7 8 b a s e s ) o f T-DNA  hybridized obtained  longer  probe  a t a p o s i t i o n 20 n u c l e o t i d e s  i n plant  7 i n E9 p l a n t  D and E , r e s p e c t i v e l y .  by p r i m e r  the other  gene  c o r r e c t l y i d e n t i f i e s the  i s o l a t e d from yeast  b y t h e DNA  than  procedure  o f T-DNA  o f RNAs  These  previously  a much l o w e r  site  i n lanes  protected  as w e l l .  identified  is  initiation  nuclease  shorter  t h e SI n u c l e a s e  t o RNA  approximately  by o t h e r  +  gene  to determine 7 i n yeast.  selected 7 probe gene  RNA was h y b r i d i z e d  extends  7 a s shown  700 b a s e s  The r e s u l t s a r e shown i n  long,  from  the H i n d l l l  i n Figure confirming  ( G e l v i n e t a l . , 1982; W i l l m i t z e r  t o t h e T-DNA  4.  site  t o the  The p r o b e  previous  results  e t a l . , 1982; M c P h e r s o n ,  1984). Hybridizations transformed poly  A  length  +  with  selected  between  t h e gene  7 probe  a n d RNA  i s o l a t e d from  Y-BG.E9 show a t r a n s c r i p t o f 710 b a s e s RNA,  lanes  1 a n d 2, r e s p e c t i v e l y .  transcript i s transcribed  from  T-DNA  gene  This  f o r both  GM-3C-2 total  indicates  7 i n yeast.  that  RNA a n d a  SI n u c l e a s e  full  33-  1 2  3  4  ure 8 - An a u t o r a d i o g r a p h of an RNA g e l b l o t h y b r i d i z e d to a T-DNA gene 7 probe ( H i n d l l l - Smal T-DNA fragment, 1,178 b p , see F i g u r e 4 ) . RNA from y e a s t transformed w i t h Y-BG.E9 was examined. Lane 1, Y-BG.E9 t o t a l RNA (30 u g ) ; lane 2 , Y-BG.E9 p o l y A s e l e c t e d RNA (5 u g ) ; lane 3 , P-labelled H p a l l fragments of pBR322; lane 4 , E9 p l a n t tumor p o l y A s e l e c t e d RNA (5 u g ) . +  3 2  +  -34-  mapping gene  7 i n yeast  yeast (50  or fewer  B. Heat  to determine.  t o compare  since  A residues i n yeast  Gene  S h o c k - T-DNA  effect  crown g a l l  40°C  of a rapid tumors  f o r 1 hour, quantified  were  7, b u t t e r m i n a t i o n s i t e s o f  Transcript  the degree  compared  with  sizes  from  p l a n t s and  of polyadenylation i s different 100-200 i n p l a n t s  - Sagher ^ t  7  temperature  2 hours,  by primer  lane  obtained  6 hours,  1 hour  decrease  These  until  results  they  indicate  that  transcription  o f gene  Metals their  tumors  tumor  obtained  at 40°C,  Transcripts  heat 7.  shock  gene  line  7, a s w o u l d  shocked a t  were  E9 b e f o r e h e a t  identified  heat  be e x p e c t e d  shock  The ( F i g . 9,  previously. 7 RNA l e v e l s  o f crown g a l l  In fact,  heat  g e l ( F i g . 9, l a n e s b - f ) .  a r e no l o n g e r d e t e c t a b l e a f t e r  o f T-DNA gene  Metal  7 from  tumors.were  a s b e f o r e a n d t h e cDNA p r o d u c t s  a DNA s e q u e n c i n g  shock  transcription  Heavy  a n d 24 h o u r s .  to the r e s u l t s  o f heat  o n t h e e x p r e s s i o n o f T-DNA g e n e 7  Crown g a l l  extension analysis  for transcript  b) a r e i d e n t i c a l After  shock  was i n v e s t i g a t e d .  e l e c t r o p h o r e s e d through  results  for  o f gene  Shock The  to  are d i f f i c u l t  sites  1974).  Heat  and  the i n i t i a t i o n  are d i f f i c u l t  al.,  in  identified  shock  decrease  25 h o u r s tumors  f o r a non-heat  o f heat  does  o f tumors  and c o n t i n u e shock.  not increase  decreases shock  gene.  Treatment which  a r e known t o be t o x i c  influence  on t h e a c c u m u l a t i o n  and t r a n s f o r m e d  there  wasn't  hours  o f treatment  to plants  tobacco  a significant with  cultures.  at high  o f T-DNA gene As s e e n  —4 ( 1 0 M) a t w h i c h  were  7 RNAs i n c r o w n  tested gall  i n Figure 9 (lanesg-n),  increase i n transcription  cadmium  levels  time  o f gene  7 until  after  t h e r e was a t w o - f o l d  24  -35-  a  b  c  d  e  f  g  h  i  j  k  l  m  n  o  67  F i g u r e 9 - The e f f e c t o f a t e m p e r a t u r e s h o c k a n d t h e e f f e c t o f t r e a t m e n t o f cadmium c h l o r i d e on t h e e x p r e s s i o n o f T-DNA g e n e 7 i n c r o w n g a l l t u m o r s . C u l t u r e s were h e a t s h o c k e d a t 4 0 ° C f o r 1 h o u r , 2 h o u r s , 6 h o u r s , a n d 1 d a y and RNA was i s o l a t e d as d e s c r i b e d i n m e t h o d s . C u l t u r e s were a l s o grown on m e d i a c o n t a i n i n g cadmium c h l o r i d e ( 1 0 M ) f o r 1 h o u r , 2 h o u r s , 6 h o u r s , 1 d a y , 2 d a y s a n d 20 d a y s a n d RNA was i s o l a t e d . Reverse t r a n s c r i p t a s e e x t e n s i o n p r o d u c t s f r o m c r o w n g a l l tumor RNA ( 2 0 u g ) p r i m e d w i t h o l i g o n u c l e o t i d e T7 c o m p l e m e n t a r y t o t h e 5' c o d i n g r e g i o n o f T-DNA t r a n s c r i p t 7 a r e shown i n t h e f i g u r e . L a n e a , P - l a b e l l e d Hpa I I f r a g m e n t s o f pBR322; l a n e b , c r o w n g a l l tumor l i n e E9 w i t h no t r e a t m e n t ; l a n e c , E9 a t 4 0 ° C f o r 1 h o u r ; l a n e d , 4 0 ° C f o r 2 h o u r s ; l a n e e, 4 0 ° C f o r 6 h o u r s ; l a n e f , 4 0 ° C f o r 1 d a y ; l a n e g , c r o w n g a l l tumor l i n e A6 w i t h no t r e a t m e n t ; l a n e h , A6 t r e a t e d w i t h 10~ M CdCl2 f o r 1 hour; l a n e i , 1 0 - % CdCl2 f o r 2 h o u r s ; l a n e j , 10~ M C d C l f o r 6 h o u r s ; l a n e k, 1 0 ~ M C d C l f o r 1 day; l a n e 1, g a m m a P - o l i g o n u c l e o t i d e T 7 ; l a n e m, 1 0 M C d C l f o r 2 days; l a n e n, 10~ M C d C l f o r 10 d a y s ; l a n e o, P - l a b e l l e d Hpa I I f r a g m e n t s o f pBR322. - 4  3 2  4  4  4  2  2  32  - 4  2  4  3 2  2  -36-  increase with  in transcription  cadmium, g e n e  i n crown g a l l  7 RNAs a c c u m u l a t e d  tumors.  to 3 times  -4 concentrations determine  o f cadmium r a n g i n g  their  effects  on gene  from  10  gene  indicate that  7 transcription When  crown g a l l  —4  were  of  level.  Different  tested to  ( F i g . 10, l a n e s  c-g).  The  -7 M t o 10 M cadmium a l l i n c r e a s e  10 d a y s  treated with  treatment  of  treatment.  concentrations  of mercury  ranging  —8  from  10  gene  7 transcription  various  o f 10  three-fold after tumors  the normal  7 transcription  concentrations  10 d a y s  -7 M t o 10 M were a l s o  -4 results  After  M t o 10  M  ( F i g . 10, l a n e s  h - l ) , there  f o ra l l concentrations.  concentrations  of zinc  was a t h r e e - f o l d i n c r e a s e i n  When t u m o r s  ( F i g . 11), there  were  was no e f f e c t  treated on gene  with 7  transcription. The  effect  of metals  on g e n e  culture  16-12-C was a l s o  tobacco  c u l t u r e was t r a n s f o r m e d  mosaic of  v i r u s enhancer  T-DNA gene  gall  tumors.  When  dramatic  increase  Arsenite  Treatment  Crown g a l l  appears  element  with  plasmid  upstream  7 increased  i n transcription  tumors were  a n d gene t o have  o f gene  on t r a n s c r i p t i o n  7.  The 1 6 - 1 2 - C  100-fold.  The t r a n s c r i p t  the normal  treated with  to various  7 RNAs were q u a n t i f i e d .  no e f f e c t  times  i s unknown a t t h i s  subjected  m-o).  tobacco  DNA c o n t a i n i n g a C a u l i f l o w e r  o f T-DNA gene  t h e 16-12-C c u l t u r e s w e r e  transcription  i n the transformed  i n v e s t i g a t e d ( F i g . 10, l a n e s  7 i n t h e 16-12-C c u l t u r e i s f i v e  cadmium,  arsenite  7 transcription  level  either  The r e a s o n  level  i n crown  mercury or for this  time.  concentrations  As shown i n F i g u r e o f T-DNA g e n e  7.  o f sodium 12, a r s e n i t e  -37-  a b c d e f  g h i j k l m n o p  67  F i g u r e 10 - T h e e f f e c t o f h e a v y m e t a l s o n t h e e x p r e s s i o n o f T-DNA g e n e 7 i n crown g a l l tumors and t r a n s f o r m e d t o b a c c o c u l t u r e s . C u l t u r e s were grown on m e d i a c o n t a i n i n g cadmium c h l o r i d e ( 0 , 1 0 ^ M t o 1 0 ^ M) a n d m e r c u r i c c h l o r i d e ( 0 , 1 0 " M t o 10~^M) f o r 10 d a y s a f t e r w h i c h t i m e RNA was i s o l a t e d as d e s c r i b e d i n m e t h o d s . Reverse t r a n s c r i p t a s e e x t e n s i o n products f r o m c r o w n g a l l tumor RNA ( 1 0 u g ) p r i m e d w i t h o l i g o n u c l e o t i d e T7 c o m p l e m e n t a r y t o t h e 5' c o d i n g r e g i o n o f T-DNA t r a n s c r i p t 7 a r e shown i n t h e figure. The 3 2 . p - i b l l e d e x t e n s i o n p r o d u c t s were s i z e d on d e n a t u r i n g -  -  8  a  e  urea-acrylamide gels. 32 - l a b e l l e d molecular  Lane  a, gamma-32p- ligonucleotide  T7; l a n e b,  0  P  lane  c, crown  10 M  CdCl ;  _ 5  E9  with  2  gall lane  weight  tumor e, w i t h  no t r e a t m e n t ;  markers  line 10  (Hpa I I fragments  E9 t r e a t e d _ 6  M CdCl ; 2  with  lane  l a n e h , E9 t r e a t e d  o f pBR322);  10~^M C d C l 2 ;  f , with  w i t h 10' -4  10  - 7  lane  d, w i t h  M CdCl ; 2  l a n e g,  M  HgCl25 Lane i , w i t h 10 M H g C l ; l a n e j , w i t h 1 0 ~ M H g C l ; l a n e k, w i t h 1 0 " M H g C l ; l a n e 1, 10 M H g C l ; l a n e m, t r a n s f o r m e d t o b a c c o c u l t u r e l i n e 16-12-C t r e a t e d w i t h 10 M H g C l ; l a n e n , 16-12-C t r e a t e d w i t h 1 0 " M C d C l ; l a n e o, 16-12-C w i t h no t r e a t m e n t ; l a n e p , 3 2 p _ ] _ t j ^ j J-J- f g t s o f pBR322. _ 5  6  7  2  2  2  - 8  2  _ 4  4  2  2  a  e  e c  H  p  a  r  a  m  e  n  -38-  1 2 34 5 6 7 8 67  m*  Figure  11 - T h e e f f e c t  o f treatment  o f z i n c c h l o r i d e on t h e e x p r e s s i o n o f  T-DNA gene 7 i n c r o w n g a l l t u m o r s . C u l t u r e s were grown o n m e d i a c o n t a i n i n g z i n c c h l o r i d e ( 0 , 1 0 " M t o 1 0 ~ M ) f o r 10 d a y s a f t e r w h i c h t i m e RNA was i s o l a t e d as d e s c r i b e d i n m e t h o d s . Revere t r a n s c r i p t a s e e x t e n s i o n products f r o m c r o w n g a l l tumor RNA ( 2 0 u g ) p r i m e d w i t h o l i g o n u c l e o t i d e T7 c o m p l e m e n t a r y t o t h e 5' c o d i n g r e g i o n o f T-DNA t r a n s c r i p t 7 a r e shown i n t h e figure. L a n e 1, P - l a b e l l e d Hpa I I f r a g m e n t s o f pBR322; l a n e 2, c r o w n g a l l tumor l i n e A6 w i t h no t r e a t m e n t ; l a n e 3, A6 t r e a t e d wi t h 1 0 M Z n C l ; l a n e 4, w i t h 1 0 ~ M Z n C l ; l a n e 5, w i t h 1 0 " M Z n C l ; l a n e 6, w i t h 1 0 ~ M Z n C l ; l a n e 7, A6 w i t h no t r e a t m e n t ; l a n e 8, E9 w i t h no t r e a t m e n t . 7  4  3 2  _ 4  2  5  6  2  2  7  2  -39-  1  2  3  4  5  F i g u r e 12 - The e f f e c t of treatment of sodium a r s e n i t e on the e x p r e s s i o n of T-DNA gene 7 i n crown g a l l t u m o r s . C u l t u r e s were grown on media c o n t a i n i n g sodium a r s e n i t e ( 0 , 10~ M to 1 0 M ) f o r 10 days a f t e r which time RNA was i s o l a t e d as d e s c r i b e d i n methods. Reverse t r a n s c r i p t a s e e x t e n s i o n p r o d u c t s from crown g a l l tumor RNA (20 ug) primed w i t h o l i g o n u c l e o t i d e T7 c o m p l e mentary to the 5 ' c o d i n g r e g i o n of T-DNA t r a n s c r i p t 7 are shown i n the figure. Lane 1, crown g a l l tumor l i n e A6 w i t h no t r e a t m e n t ; l a n e 2 , A6 t r e a t e d w i t h 1 0 M a r s e n i t e ; lane 3 , w i t h 10~->M a r s e n i t e ; l a n e 4 , w i t h 10~^M a r s e n i t e ; lane 5 , w i t h 1 0 M a r s e n i t e . 7  _4  _ 4  - 7  -40-  DISCUSSION  A.  T-DNA Gene Primer  indicate  extension  that  tion  gall  analysis  begin  sites.  TATA  distance  I n most  cription  start  from  downstream  have been However,  point  that  cells varies  from  and p r i m e r  a r em u l t i p l e  transcrip-  sites  o f gene  initiation  site  7 in  i n crown  t h e TATA b o x .  i n a l l yeast  i n contrast  to higher  and t h e c o r r e s p o n d i n g  o f higher  genes  studied  eukaryotic  systems,  setof t r a n s c r i p t i o n  f r o m 4 0 t o 150 bp ( S e n t e n a c  t h e TATA b o x h a s a s e p a r a t i o n from  that o f  mapping  i s s i m i l a r to that  observed  i s v a r i a b l e , ranging eukaryotic  t h e normal from  o f t h e promoter  1982).  SI n u c l e a s e there  hybridizations  i sdifferent  transcription initiation  b e t w e e n a TATA s e q u e n c e  sites  7 i n yeast  show t h a t  downstream  sequences  and H a l l ,  initiation  The major  mapping, and N o r t h e r n  tumors.  7 i n yeast  the s t r u c t u r e  eukaryotes.  1982).  o f gene  o r 49 n u c l e o t i d e s  yeast  (Sentenac  7 i n crown g a l l  20 n u c l e o t i d e s  tumors, In  the  o f gene  initiation  yeast  a n a l y s i s , SI n u c l e a s e  t r a n s c r i p t i o n o f T-DNA g e n e  transcription extension  7 i n Yeast  25 t o 30 bp ( B r e a t h n a c h  and H a l l ,  from  the trans-  a n d Chambon,  1981). It loose  has been spatial  (McNeil  sequences  for  very  f o r the yeast  1986).  This  (+ 15 b a s e - p a i r s ) . located  (McNeil  subset  between  likely  within  and S m i t h ,  a specific  spacing  that  r e l a t i o n s h i p between  and Smith,  base-pairs  gene  shown  that  iso-l-cytochrome  t h e TATA s e q u e n c e s  distance There  also  Each  exists  four  region  McNeil  sequences  and p o s s i b l y  i s thought  and Smith  T-A-T-A a n d s t a r t s i s n o t a s i n g l e intervening  determine  from  i sa  start  sites  100 t o 60 five  TATA  o f the iso-l-cytochrome  TATA s e q u e n c e  o f mRNA s t a r t s .  there  a n d t h e mRNA  relationship varies  t h e 5' n o n - c o d i n g 1986).  c gene  fixed  t o be r e q u i r e d  suggest  that the  distance  the spacing,  c  and i t  either  is  directly  -41-  or  through a s e r i e s  suggest that  of s p e c i f i c spacing f a c t o r s .  the TATA box i n yeast does not s e l e c t  measuring a f i x e d d i s t a n c e initiator  element c l o s e  however, that  to the i n i t i a t i o n  GAL al.,  and M a j o r s , 1987).  1  in yeast.  These s t u d i e s  initiation  s i t e s by I n s t e a d , an  c o n t a i n s the s i g n a l indicate,  s e n s i t i v i t y at  When the h e x a n u c l e o t i d e i s d e l e t e d  indicate  for  TATA sequence i n y e a s t from the  under i n d u c i n g c o n d i t i o n s  the f u n c t i o n a l  yeast  (West ejt  importance o f the TATA box  The TATA box appears t o be d i r e c t l y i n v o l v e d  transcription  Chen and S t r u h l  Photoprinting studies  marks a f u n c t i o n a l  1 gene, e x p r e s s i o n decreases 3 0 - f o l d 1984).  site itself  1986).  the t r a n s c r i p t i o n - d e p e n d e n t a l t e r a t i o n i n l i g h t  h e x a n u c l e o t i d e 5' ATATAA3  (Selleck  initiation  from i t (Chen and S t r u h l ,  d e t e r m i n i n g where t r a n s c r i p t i o n b e g i n s .  the  In c o n t r a s t ,  i n the s e l e c t i o n o f  s i t e s i n y e a s t , a l t h o u g h the exact f u n c t i o n  o f the  TATA box i s unknown. A TATA box i n the 5' r e g i o n o f T-DNA gene 7 maps 30 bp upstream from the normal mRNA s t a r t s i t e i n crown g a l l  tumors.  SI n u c l e a s e mapping shows  that  the  mRNA s t a r t s i t e s o f gene 7 i n yeast b e g i n 49 n u c l e o t i d e s downstream from  the  TATA box.  T h i s confirms p r e v i o u s r e s u l t s which i n d i c a t e  must be a t l e a s t 40 n u c l e o t i d e s upstream o f the t r a n s c r i p t i o n for  transcription  when f o r e i g n initiation  i n yeast  DNA i s t o be expressed  unlikely  that  The major t r a n s c r i p t i o n the ATG t r a n s l a t i o n  lation reaction  i s prevented.  a TATA box  initiation  These f a c t s  suggest  site that  i n y e a s t , the d i s t a n c e between the  than 40 bp upstream from the  codon, then i t i s h i g h l y  begin a f t e r  1986).  s i t e and the TATA box i n the f o r e i g n  TATA box i s l e s s  translated.  (Chen and S t r u h l ,  that  gene i s c r i t i c a l .  ATG t r a n s l a t i o n  a complete, f u n c t i o n a l  initiation  initiation  I f the  initiation protein  w i l l be  s i t e s o f T-DNA gene 7 i n y e a s t  codon which means that  the t r a n s -  -42-  The  p o s s i b l e reason  f o r t h e abnormal  in  previous  of  t h e TATA b o x i n y e a s t  rabbit were  s t u d i e s may h a v e  B-globin  about  20-40 n u c l e o t i d e s  ej: a l _ . , 1 9 8 0 ) .  globin  transcripts  a yeast  the maps  site  i n yeast,  shorter  represented  either  sites  downstream  from  t h e TATA b o x .  downstream  from  the transcription  and  i t was c o n c l u d e d  transcription  I n most In  o f the B-globin  a possibility.  yeast,  (Faye  In this that  however,  gene  1984).  TRP3 y e a s t  genes  mapping  showed  there  that  TRP2 a n d TRP3 mRNA  (Zalkin  there  transcription  aremultiple  5' t e r m i n i o f t h e B-  transcripts  initiation  sites,  originating at  site.  gene  The major  so normal  c o d o n was  still  t r a n s l a t i o n was  t h e gene was n o t s p l i c e d  properly  f o r the study o f  gene.  i s confined  a t which  1984; J o h n s t o n  were m u l t i p l e  to a short  transcripts and D a v i s ,  o f t h e mRNA 5' t e r m i n i clusters  for  sites  when gene  region.  are initiated 1984; Z a l k i n t h e TRP2 a n d  o f 5' e n d s  I t i s not surprising  initiation  recognized  a r e 46 a n d 65 n u c l e o t i d e s  initiation  e t a l . , 1984).  sites  t h e ATG i n i t i a t i o n  o f the B-globin  SI n u c l e a s e  yeast  g l o b i n mRNA  initiation  s y s t e m was i n a d e q u a t e  e t a l . , 1981; Hsu and Schimmel,  e_t a_l. ,  i n yeast  c a n be many s i t e s  from  T h e TATA b o x i n t h e B - g l o b i n  however,  transcription  there  o f longer  initiation  case,  normal  requirement  When t h e  transcripts  the abnormal  transcription  Fortunately,  the yeast  and p r o c e s s i n g  eukaryotes,  that  i n yeast  the d i s t a n c e  eukaryotes.  the B-globin  o f the t r a n s c r i p t i o n  initiation  still  that  i n other  f u r t h e r upstream.  upstream  o f f o r e i g n genes  a t t h e 5' e n d t h a n  or degradation  o r even  28 n u c l e o t i d e s  than  I t was s u g g e s t e d  RNA p o l y m e r a s e  promoter  due t o t h e f a c t  i sdifferent  g e n e was c l o n e d  (Beggs  by  been  expression  then  for  both  that  7 i s cloned i n  yeast. In a r e c e n t been  cloned  transcripts  paper  i n yeast. with  ( H i r s c h and Beggs, Northern  various  1984),  i t was r e p o r t e d  h y b r i d i z a t i o n s were done  p r o b e s , b u t no RNA s i m i l a r  that  to i d e n t i f y  i n size  T-DNA h a d T-DNA  t o t h e 0.7 k b mRNA  -43-  was  identified.  was  complex.  As f o r other T-DNA t r a n s c r i p t s ,  RNAs i d e n t i f i e d  characteristically gation indicate  that a f u l l  tumors.  by Northern h y b r i d i z a t i o n s appeared to be  l a r g e r than expected RNA l e n g t h s . length t r a n s c r i p t  yeast, although t r a n s c r i p t i o n crown g a l l  the p a t t e r n o f t r a n s c r i p t i o n  Results  i s transcribed  i n this  investi-  from gene 7 i n  i n i t i a t i o n and t e r m i n a t i o n a r e d i f f e r e n t  The gene 7 t r a n s c r i p t was most l i k e l y d e t e c t e d  than i n  i n this  i n v e s t i g a t i o n because a s p e c i f i c probe f o r gene 7 was used. In c o n c l u s i o n , t r a n s c r i p t i o n of T-DNA gene 7 i n yeast i s d i f f e r e n t crown g a l l  tumors.  Specifically, transcription  initiation  is different  the d i s t a n c e between the TATA box and the t r a n s c r i p t i o n i n i t i a t i o n be a t l e a s t 40 n u c l e o t i d e s for  investigating  i n yeast.  Although yeast  because  s i t e s must  i s an e x c e l l e n t  system  the e x p r e s s i o n o f some cloned e u k a r y o t i c genes, i t does not  appear t o be a u s e f u l system f o r i n v e s t i g a t i n g  B.  than i n  the e x p r e s s i o n o f T-DNA.  Heat Shock - T-DNA Gene 7 By d e f i n i t i o n , HS p r o t e i n s a r e a new s e t o f p r o t e i n s r a p i d l y and abundantly  produced  i n response to heat shock.  dependent tion  on t r a n s c r i p t i o n a l  i s immediate  (less  The appearance of heat shock p r o t e i n s i s  a c t i v a t i o n o f the heat shock genes.  This  induc-  and i n c r e a s e s the c o n c e n t r a t i o n o f hsp mRNA from u n d e t e c t a b l e  than one molecule per c e l l ) t o a thousand molecules per c e l l w i t h i n an  hour o f heat shock ( S c h l e s i n g e r e_t al_. , 1982).  E u k a r y o t i c HSP genes have one  or more c o p i e s o f a 14-base-pair (bp) sequence, r e f e r r e d promoter (Bienz,  element  (HSE), upstream from the t r a n s c r i p t i o n  1985; C r a i g , 1985; Pelham,  the HSE was i d e n t i f i e d  1985).  to as a heat initiation  A heat shock sequence  shock  site  s i m i l a r to  i n the promoter r e g i o n o f T-DNA gene 7.  A l t h o u g h u n t r e a t e d crown g a l l  tissue contains s i g n i f i c a n t  levels  o f gene 7  RNAs, t h e r e i s at l e a s t one case where a p l a n t heat shock gene i s e x p r e s s e d a t  -44-  significant cDNAs  to heat  stresses set  levels  stressed other  tissue)  tissue  agents  o n T-DNA  gene  sented  i n Table  2.  have  mercury  ment. RNAs  after  callus  non-heat  until  after  tissue  shock  possibility  cell  et_ al_. ,  1984).  including  heat  i s pre-  cadmium a n d  o n t h e e x p r e s s i o n o f T-DNA  gene  7.  7 3-fold  do n o t i n d u c e  levels  after  10 d a y s  of treat-  the a c c u m u l a t i o n  i n crown g a l l  tumors.  of 40°C,  Cadmium  In gene  o f gene  (Kimpel  et_ a l . ,  p o l y ( A ) RNAs  and Key, 1985).  to high  7 RNA  decrease 1985).  to a very  These  facts  when  nopaline synthase  low l e v e l suggest  In  declines  Furthermore,  temperatures,  7  fact,  one d a y when t h e y were no l o n g e r d e t e c t a b l e .  that  T-DNA 7 being  gene  trans(a  immediately  that  7 responded  T-DNA  gene 7  o f low m o l e c u l a r  6,000 t o 7,000 m o l e c u l a r  when t h e s e  o n l y t o cadmium a n d m e r c u r y ,  a m e t a l l o t h i o n e i n gene was  (MTs) a r e a f a m i l y  ( K a g i e_t a_l. , 1 9 8 4 ) . types  shock and  extension analysis  demonstrate  one  gene.  o f gene  o f about  by heat  agents,  subjected to a temperature  i s exposed  (Spena  to the fact  nature  shock  seedlings,  only  shock  gene) t r a n s c r i p t s  shock  not a heat  proteins  and  here  various  i n control (non-  (Czarnecka  on p r i m e r  cloned  that  o f normal  were  Metallothioneins  in  based  presented  effect  tumors  levels  o f a number o f s t r e s s  and h e a t  that  o f heat  shock  heat  Due the  at variable  When  whether  i n soybean  s e e d l i n g s t h e c o n c e n t r a t i o n s o f many n o r m a l  2 hours  formed  RNAs  i n c r e a s e d some 5- t o 1 0 - f o l d  levels  The d a t a  zinc,  decreased  soybean  present  ejt a_l. , 1 9 8 4 ) . to assess  as cadmium a n d a r s e n i t e  7 RNA  above  when c r o w n g a l l  is  such  of poly(A)  i n c r e a s e t h e e x p r e s s i o n o f gene  to levels  after  were  a significant  Arsenite,  levels  (Czarnecka  ( h s ) - i n d u c e d mRNAs were u s e d  summary o f t h e i n f l u e n c e  mercury  shock  the accumulation  shock,  and  to heat  ( A ) RNAs w h i c h were  stress  A  shock  induced  of poly  prior  weight  They a r e e x p r e s s e d  a r e exposed  investigated.  weight,  which  cysteine-rich  are widely  distributed  i n many d i f f e r e n t  t o heavy metals  such  tissues  as cadmium,  zinc,  -45-  2d  Treatment  No Treatment (25°C)  1 hour  Heat shock 40°C(E9)  IX  .75X  CdCl  2  10 M(A6)  IX  IX  CdCl  2  10 M(E9)  IX  3X  _4  -4  2h  .5X IX  6h  1  day  .25X  OX  —  IX  2X  2X  lOd  —  3X  HgCl  2  10 M(E9)  IX  3X  Zncl  2  10" M(A6)  IX  IX  IX  IX  _4  4  Arsenite CdCl  10" M(A6) 4  10 M(16-12-C)  5X  100X  " M(16-12-C)  5X  100X  -4  2  HgC12  Table  10  4  2  Summary of the s t r e s s induced a c c u m u l a t i o n of T--DNA gene 7 RNAs i n crown g a l l tumors and transformed tobacco c u l t u r e s . A6 = crown g a l l tumor l i n e A6; E9 = crown tumor l i n e E9; 16-12-C = transformed tobacco c u l t u r e . X = e s t i m a t e d r e l a t i v e l e v e l from primer e x t e n s i o n analyses as determined by a d e n s i t o m e t e r ; — = not a n a l y z e d .  -46-  copper is  and mercury  regulated  molecular 2%  1984;  weight  o f t h e amino  was  done  1984). the  acid  No s e q u e n c e  In  treated  with  deleted  upstream  element level that  for that  In  there  importance  1  (less  than  analysis  - TGCGCCCGCTTC-3') ( C a r t e r  consensus  suggest that  sequence  et a l . ,  was i d e n t i f i e d i n  T-DNA g e n e  of functional  i s also  ( F i g . 4) i n t h i s a Cauliflower  7 i n this  culture  culture  7 i s not a  i s unknown a t t h i s  i t does  not appear  that  sequences  shock  were  treated  100-fold.  with  either  The r e a s o n  facie  mosaic  level of  i t appears  virus  enhancer  7. T-DNA gene  7 i s a heat  the increase  or m e t a l l o t h i o n e i n  i n t h e 5' r e g i o n  7 i s complicated  line.  enhancer  the normal  time, but prima  t o cadmium a n d m e r c u r y ,  t o be h e a t  virus  7is  The n o r m a l t r a n s c r i p t  i n f l u e n c i n g the Cauliflower  t r a n s c r i p t i o n o f gene  o f T-DNA g e n e  times  7 increased  1 6 - 1 2 - C , was  tobacco c u l t u r e  line.  i s five  i m p o r t a n c e i n t h e 5'  line,  mosaic  t h e 16-12-C c u l t u r e s  t r a n s c r i p t i o n o f gene  7 responds  o f DNA  o f gene  rich  s i m i l a r to the consensus  T h e 5' f l a n k i n g r e g i o n  site  When  are directly  conclusion, gene  proteins  i s a low  I n a d d i t i o n , a computer  (5  sequences  7 i n t h e 16-12-C  dramatic increase  not appear  region  a n d cadmium.  tumors.  to increase  Although  DNA  o f T-DNA gene  gall  the metals  7 protein  i s not cysteine  7, t h e t r a n s f o r m e d t o b a c c o c u l t u r e  that  o r cadmium,  element  does  be n o t e d  o f T-DNA g e n e  this  facts  o f the H i n d l l l  upstream  mercury  These  to i d e n t i f y  mercury  i n crown  regulation  s i m i l a r to the metal  o f T-DNA g e n e  should  protein  were a n y s e q u e n c e s  f o r metal  o f these  gene.  an a t t e m p t  region  A l t h o u g h t h e gene  are cysteines).  i f there  7,  The i n d u c t i o n  o f t r a n s c r i p t i o n (Durnam a n d P a l m i t e r , 1981;  (14Kd), t h i s  content  o f gene  metallothionein  It  protein  responsible  5' r e g i o n  1979).  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