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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 ERIC A. BUTTON B.A., U n i v e r s i t y o f North C a r o l i n a , 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES GENETICS PROGRAMME UNIVERSITY OF BRITISH COLUMBIA We ac c e p t t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA August 1987 ° E r i c A. B u t t o n , 1987 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f G e n e t i c s The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main M a l l Vancouver, Canada V6T 1Y3 Date: August 21, 1987 ABSTRACT The purpose of this study was two-fold. The f i r s t objective was to determine i f Saccharomyces cerevisiae i s a useful system for i n v e s t i g a t i n g the expression of T-DNA ( i t takes several months to obtain s u f f i c i e n t b a c t e r i a - f r e e transformed plant tissue to investigate T-DNA t r a n s c r i p t i o n ) . A short fragment of T-DNA carrying T-DNA gene 7 was cloned into a yeast plasmid i n an attempt to investigate the expression of gene 7 i n yeast. The second objective was to determine the s i g n i f i c a n c e of a heat shock related sequence i d e n t i f i e d i n the 5' region of T-DNA gene 7. Primer extension a n a l y s i s , SI nuclease mapping, and Northern hy b r i d i z a t i o n s i n d i c a t e that 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 from that of t r a n s c r i p t i o n of gene 7 i n crown g a l l tumors. Tr a n s c r i p t i o n i s d i f f e r e n t because the distance 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 s i t e s must be at least 40 nucleotides i n yeast. Therefore, Saccharomyces  cer e v i s i a e does not appear to be a useful system for i n v e s t i g a t i n g the expression of T-DNA. Crown g a l l tumors were subjected to a number of stress agents, inc l u d i n g heat shock, to determine the s i g n i f i c a n c e of the heat shock r e l a t e d sequence i d e n t i f i e d i n gene 7. Primer extension analyses indicate that only cadmium and mercury have a s i g n i f i c a n t e f f e c t on the expression of T-DNA gene 7. Although gene 7 responds to cadmium and mercury, the increase i n t r a n s c r i p t i o n does not appear to be heat shock or metallothionein related, i n d i c a t i n g that another mechanism i s involved i n the enhanced t r a n s c r i p t i o n of T-DNA gene 7 i n crown g a l l tumors. - i i i -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i ABBREVIATIONS v i i i INTRODUCTION 1 MATERIALS AND METHODS 6 Reagents 6 M i c r o b i a l S t r a i n s 8 Tobacco T i s s u e C u l t u r e L i n e s 8 C u l t u r e Media and C o n d i t i o n s 8 Plasmi d s and B a c t e r i o p h a g e s 10 T r a n s f o r m a t i o n o f E. c o l i 10 Large S c a l e I s o l a t i o n o f Plasmid DNA 11 P u r i f i c a t i o n o f Pla s m i d DNA 11 Small S c a l e I s o l a t i o n o f Plasmid DNA 12 R e s t r i c t i o n Endonuclease D i g e s t i o n 13 L i g a t i o n s 14 Agarose E l e c t r o p h o r e s i s o f DNA 14 T r a n s f o r m a t i o n o f Yeast 14 Yeast P l a s m i d P u r i f i c a t i o n 16 I s o l a t i o n fo Yeast RNA 16 S e l e c t i o n o f P o l y A + RNA 17 E n d - L a b e l l i n g o f O l i g o n u c l e o t i d e s 18 Primer E x t e n s i o n A n a l y s i s 19 D e n a t u r i n g U r e a - A c r y l a m i d e Gel 19 S l N u c l e a s e Mapping 19 N o r t h e r n H y b r i d i z a t i o n 19 N i c k T r a n s l a t i o n 21 I s o l a t i o n o f P l a n t Tumor RNA 21 S t r e s s I n d u c t i o n o f P l a n t Tumors 22 RESULTS 23 A. T-DNA Gene 7 i n Yeast 23 Pl a s m i d C o n s t r u c t i o n s 23 Prim e r E x t e n s i o n A n a l y s i s 23 S l N u c l e a s e Mapping 30 N o r t h e r n H y b r i d i z a t i o n s 32 - i v -TABLE OF CONTENTS Page B. Heat Shock-T-DNA Gene 7 34 Heat Shock 34 Heavy Metal Treatment 34 Arsenite Treatment 36 DISCUSSION 40 A. T-DNA Gene 7 i n Yeast 40 B. Heat Shock-T-DNA Gene 7 43 BIBLIOGRAPHY 48 -v-LIST OF TABLES Table P a S e 1 Plasmids and Bacteriophages 10 2 Summary of the Stress Inducted Accumulation of T-DNA Gene 7 RNAs i n Crown G a l l Tumors and Transformed Tobacco Cultures 45 - v i -LIST OF FIGURES F i g u r e Page 1 C o n s t r u c t i o n o f Y-BG.E9 24 2 C o n s t r u c t i o n o f Y-BGE.E9 25 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 Plasmids Y-BG.E9, Y-BGE.E9, Y E p l 3 , and LE.E9 26 4 DNA Sequence of T-DNA Gene 7 27 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' Region o f T r a n s c r i p t 7 28 6 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 RNA Primed w i t h an O l i g o n u c l e o t i d e Complementary t o the 5' Coding Region o f the T-DNA T r a n s c r i p t 7 29 7 SI N u c l e a s e Mapping o f Yeast T-DNA Gene 7 RNA 5' T e r m i n i 31 8 A u t o r a d i o g r a p h o f 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 33 9 The E f f e c t o f a Temperature Shock and the E f f e c t o f Treatment o f Cadmium C h l o r i d e on the E x p r e s s i o n o f T-DNA Gene 7 i n Crown G a l l Tumors 35 10 The E f f e c t o f Heavy M e t a l s on the E x p r e s s i o n o f T-DNA Gene 7 i n Crown G a l l Tumors and Transformed Tobacco C u l t u r e s 37 11 The E f f e c t o f Treatment o f Z i n c C h l o r i d e on the E x p r e s s i o n o f T-DNA Gene 7 i n Crown G a l l Tumors 38 12 The E f f e c t o f Treatment o f Sodium A r s e n i t e on the E x p r e s s i o n o f T-DNA Gene 7 i n Crown G a l l Tumors 39 - v i i -ACKNOWLEDGEMENTS I would l i k e to thank Dr. J . McPherson, without whose support and guidance th i s project would not have been poss i b l e . My thanks also go to Dr. H. Brock for allowing the use of his f a c i l i t i e s and for many he l p f u l d i s c u s s i o n s . I also wish to thank Dr. R. McMaster for his suggestions and guidance. F i n a l l y , I would l i k e to thank J . Button for preparing the figures i n t h i s t h e s i s . - v i i i -ABBREVIATIONS USED DNA - d e o x y r i b o n u c l e i c a c i d cDNA -complementary DNA T-DNA - t r a n s f e r r e d DNA RNA - r i b o n u c l e i c a c i d mRNA -messenger r i b o n u c l e i c a c i d rRNA - r i b o s o m a l r i b o n u c l e i c a c i d A or ATP -a d e n o s i n e t r i p h o s p h a t e C or CTP - c y t i d i n e t r i p h o s p h a t e G or GTP -guanosine t r i p h o s p h a t e T or TTP - t h y m i d i n e t r i p h o s p h a t e HS -heat shock HSE -heat shock element HSTF -heat shock t r a n s c r i p t i o n f a c t o r T i -tumor i n d u c i n g TRP2 - t r y p t o p h a n gene 2 TRP3 - t r y p t o p h a n gene 3 TRIS - t r i s ( hydroxymethyl) aminomethane EDTA - e t h y l e n e diamine t e t r a a c e t i c a c i d SDS -sodium l a u r y l s u l p h a t e DNP - d i n i t r o p h e n o l TBE - t r i s - b o r a t e - E D T A DTT - d i t h i o t h r e i t o l BSA -bo v i n e serum albumin lambda -lambda v i r u s -1-INTRODDCTION The m o l e c u l a r b a s i s f o r crown g a l l , a n e o p l a s t i c t r a n s f o r m a t i o n o f p l a n t c e l l s , i n v o l v e s the t r a n s f e r and i n t e g r a t i o n o f a segment o f the A g r o b a c t e r i u m  t u m e f a c i e n s T i p l a s m i d (T-DNA) i n t o p l a n t n u c l e a r DNA ( C h i l t o n ejt aJL. , 1977; Thomashow et^ a l _ . , 1980; Zambryski e t a l . , 1980). T i plasmids a r e c l a s s i f i e d a c c o r d i n g to the n o v e l amino a c i d s ( o p i n e s ) produced i n the p l a n t tumors ( S c i a k y e t a l . , 1978). The T-DNA o f n o p a l i n e tumors i s i n t e g r a t e d i n t o p l a n t DNA as a c o n t i g u o u s 22 kbp segment (Leemans ej^ a_l . , 1982). T-DNA from o c t o p i n e T i p l a s m i d s c o n t a i n a c o n t i g u o u s 13 kbp segment from the l e f t s i d e o f the T - r e g i o n , TL-DNA (Leemans et a l . , 1982). Some t i s s u e c u l t u r e l i n e s c o n t a i n an 8 kbp fragment from the r i g h t s i d e (TR-DNA) o f the o c t o p i n e T - r e g i o n . TL-DNA i n o c t o p i n e tumors encodes s e v e r a l p o l y a d e n y l a t e d t r a n s c r i p t s whose s y n t h e s i s i s s e n s i t i v e to a m a n i t i n ( W i l l m i t z e r e t a l . , 1981). These f i n d i n g s s u g g e s t t h a t T-DNA genes are t r a n s c r i b e d by the hos t RNA polymerase I I . The 5' f l a n k i n g r e g i o n s o f these genes have some c h a r a c t e r i s t i c s i n common w i t h o t h e r e u k a r y o t i c genes. Most T-DNA genes c o n t a i n a Goldberg-Hogness (TATA) box a t a p o s i t i o n s i m i l a r to t h a t o f o t h e r e u k a r y o t i c genes ( B a r k e r e_t a _ l . , 1983; de Greve et a l . , 1983; Joos et a l . , 1983; K l e e e t a l . , 1984; L i c h t e n s t e i n et a l . , 1984). 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 mRNA s t a r t s i t e (McPherson, 1984). S t u d i e s have i n d i c a t e d t h a t the TATA box i s i n v o l v e d i n s p e c i f y i n g the s i t e o f mRNA i n i t i a t i o n , but sequences upstream o f the TATA box determine b o t h q u a l i t a t i v e and q u a n t i t a t i v e a s p e c t s of t r a n s c r i p t i o n ( B r e a t h n a c h and Chambon 1981; McKnight e_t al., 1981; McKnight et a l . , 1984; Wright et a l . , 1984). The d e l e t i o n or mu t a t i o n o f the TATA box r e s u l t s i n a d e c r e a s e i n t r a n s c r i p t i o n i n i t i a t i o n , and the i n i t i a t i o n o f t r a n s c r i p t s at new s i t e s (Osborne et a l . , 1982; S c h u l z e_t a_ l . , 1982; O r k i n -2-e t a l . , 1983). CAAT sequences have been a l s o i d e n t i f i e d i n some T-DNA genes, 30 t o 50 bases upstream o f the TATA box ( B a r k e r ejt al., 1983). However, no CAAT sequence i s observed i n gene 7. The 0.7 kb t r a n s c r i p t i s r a t h e r abundant compared w i t h the o t h e r mRNAs s p e c i f i e d by o c t o p i n e TL-DNA ( G e l v i n e t a l . , 1982; W i l l m i t z e r e t a l . , 1982). The f u n c t i o n o f T-DNA gene 7 w i t h r e g a r d s t o crown g a l l t u m o r i g e n e s i s i s unknown, whereas s p e c i f i c r o l e s have been a s s i g n e d to most o f the o t h e r TL-DNA encoded genes ( G a r f i n k e l ejt a l . , 1981; Leemans e t a l . , 1982). The d i f f i c u l t y w i t h s t u d i e s o f T-DNA i s t h a t i t takes s e v e r a l months t o o b t a i n s u f f i c i e n t b a c t e r i a - f r e e t r a n s f o r m e d p l a n t t i s s u e t o i n v e s t i g a t e i t s t r a n s c r i p t i o n and i t would be c o n v e n i e n t t o have a r a p i d system i n which t o i n v e s t i g a t e e x p r e s s i o n o f T-DNA sequences. Saccharomyces c e r e v i s i a e i s an e x c e l l e n t 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 c l o n e d e u k a r y o t i c genes and the f u n c t i o n a l e x p r e s s i o n o f s e v e r a l f o r e i g n genes has been r e p o r t e d ( H e n i k o f f e t a l . , 1981; Jacquet e t a l . , 1982). Most o f these s t u d i e s have i n v o l v e d DNA from h i g h e r animals or anim a l v i r u s e s (Hitzeman ejt a_l. , 1981; M e l l o r ^ t a l . , 1983; S t e p i e n e t a l _ . , 1983; T u i t e e t a l . , 1982; V a l e n z u e l a et a l . , 1982); however, experiments have shown t h a t p r o d u c t s encoded by p l a n t genes can a l s o be s y n t h e s i z e d and p r o c e s s e d i n y e a s t (Edens et_ a l ^ . , 1984; R o t h s t e i n et a_l. , 1984). A s h o r t fragment o f the 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 the m u l t i c o p y y e a s t p l a s m i d , YEpl3 (a n o n - e x p r e s s i o n v e c t o r ) , i n an attempt to i n v e s t i g a t e the e x p r e s s i o n o f gene 7 i n y e a s t . In a d d i t i o n to the TATA box, a heat shock r e l a t e d sequence was i d e n t i f i e d i n T-DNA gene 7, 40 n u c l e o t i d e s upstream o f the TATA box. In most org a n i s m s , heat shock genes c o n t a i n DNA sequences homologous to the heat shock element, as d e f i n e d by Pelham (1982), i n t h e i r promoter r e g i o n s . The heat shock consensus sequence, 5' CTgGAAtnTTCtAGa, i s a c t i v e i n c o n t r o l l i n g the heat shock r e g u l a t e d -3-expression of foreign genes even when synthetic promoter elements are used which only have an 8 of 10 match with the HSE consensus sequence (Pelham and Bienz, 1982). The heat shock related sequence of T-DNA gene 7 (CTTGAAAATTAAGC) has a 7 of 10 match with the consensus sequence. The heat shock elements of the Drosophila melanogaster hsp 70 and hsp 83 are protected from nucleases in the n u c l e i of heat shocked c e l l s (Wu, 1984a; Wu, 1984b). The HSEs of hsp 70 are the binding s i t e s for a Drosophila heat shock t r a n s c r i p t i o n factor (HSTF) that is s p e c i f i c a l l y required for heat shock protein gene t r a n s c r i p t i o n i n v i t r o (Parker and Topol, 1984; Top et a l . , 1985). Recent evidence also suggests that the number of heat shock elements can be a major determinant of the promoter strength of heat-inducible genes i n mammalian c e l l s (Kay ej: al_., 1986). The heat shock response of plants i s s i m i l a r to the response observed in b a c t e r i a , fungi, i n s e c t s , and mammals (Craig, 1985). Features of the heat shock response that are conserved across a broad spectrum of organisms include: (1) the sequences of the 5' flanking regions of the HS genes which appear to regulate expression of these genes during heat shock, (2) the rapid switch of 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 production of HS mRNAs and HS proteins, and (3) the s t r u c t u r a l features of the HS proteins which may be important for th e i r role i n thermal tolerance (Kimpel and Key, 1985). However, the HS response of plants i s e a s i l y distinguished from other organisms by the complexity and r e l a t i v e abundance of the low molecular weight HS proteins (15 to 18 Kd) (Lin et a l . , 1984). The s i g n i f i c a n c e of t h i s obser-vation i s not yet known. It i s i n t e r e s t i n g to note that T-DNA gene 7 encodes a low molecular weight protein (14Kd). From a p h y s i o l o g i c a l point of view, the only b i o l o g i c a l function assigned to HS proteins to date is th e i r possible role in mediating the expression of -4-t h e r m a l t o l e r a n c e (Kimpel and Key, 1985). The heat shock r e s p o n s e i s thought to c o n t r i b u t e t o homeostasis w i t h the heat shock p r o t e i n s h a v i n g a p r o t e c t i v e r o l e presumed t o c o u n t e r a c t or prevent d e l e t e r i o u s e f f e c t s induced by heat shock (Ashburner and Bonner, 1979; S c h o f f l ejt aL., 1984; Ve l a s q u e z and L i n d q u i s t , 1984). In soybean s e e d l i n g s , an a b s o l u t e c o r r e l a t i o n has been found between the s y n t h e s i s and a c c u m u l a t i o n o f HS p r o t e i n s and the a b i l i t y t o s u r v i v e s h o r t heat t r e a t m e n t s a t o t h e r w i s e l e t h a l temperatures (Kimpel and Key, 1985). A l t h o u g h the e x a c t f u n c t i o n s o f the p l a n t heat shock p r o t e i n s remain u n i d e n t i f i e d , i t i s known t h a t a t l e a s t one heat shock p r o t e i n i s t r a n s p o r t e d i n t o the c h l o r o p l a s t d u r i n g heat shock ( V i e r l i n g e t a l . , 1986). While many a s p e c t s o f the HS response o f p l a n t s have been i n v e s t i g a t e d , t h e r e i s l i t t l e i n f o r m a t i o n on the mechanisms o f r e g u l a t i o n o f the r e s p o n s e . R e s u l t s from o t h e r organisms i n c l u d i n g soybeans i n d i c a t e t h a t d u r i n g HS, normal c e l l u l a r mRNAs p e r s i s t i n the c e l l s but a r e t r a n s l a t e d v e r y i n e f f i c i e n t l y i f at a l l ( S c h o f f l and Key, 1982). In soybean s e e d l i n g s , the l e v e l o f a c t i n mRNAs remains unchanged d u r i n g HS, but the mRNA l e v e l s o f an a u x i n r e g u l a t e d gene d e c r e a s e d r a m a t i c a l l y d u r i n g HS ( S c h o f f l and Key, 1982). S i n c e the t o t a l p o l y (A) RNA c o n t e n t remains f a i r l y c o n s t a n t and s i n c e HS mRNAs r e p r e s e n t over 20% of the t o t a l p o l y (A) RNA p o o l d u r i n g HS, the c o n c e n t r a t i o n s o f many normal p o l y (A) RNAs must d e c l i n e ( S c h o f f l and Key, 1982). E x p r e s s i o n o f the heat shock p r o t e i n s i n p l a n t s and o t h e r organisms i s dependent on the r a p i d and c o o r d i n a t e o n s e t o f HS p r o t e i n gene t r a n s c r i p t i o n (Kimpel and Key, 1985). By d e f i n i t i o n , heat shock p r o t e i n s are a new s e t o f p r o t e i n s r a p i d l y and ab u n d a n t l y produced i n response to a heat shock, but i n many organisms o t h e r s t r e s s e s , such as e t h a n o l , a n o x i a , a r s e n i t e , or heavy metal i o n s w i l l a l s o induce the s y n t h e s i s of heat shock p r o t e i n s . The D r o s o p h i l a system responds to a wide range o f s t r e s s agents ( e . g . , DNP, a r s e n i t e , r e l e a s e from a n o x i a ) by a l t e r e d p u f f i n g p a t t e r n s ( E l l g a a r d , 1972) and s y n t h e s i s o f HS p r o t e i n s -5-(Ashburner and Bonner, 1979). Arsenite and heavy metals seem to induce a set of proteins s i m i l a r to HS proteins i n a number of systems (Ashburner and Bonner, 1979; Johnston et^ a l . , 1980; Levinson ejt a l . , 1980). Soybean seedlings respond to stresses such as arsenite and cadmium as well as heat shock, although induction of heat shock proteins under other stress conditions was not detected (Czarnecka et a l . , 1984). It i s not understood why arsenite and cadmium i n p a r t i c u l a r induce the heat shock proteins i n soybean while many other stresses, which are e f f e c t i v e i n many other organisms, do not appear to do so. The p o s s i b i l i t y of the conserved heat shock sequence of T-DNA gene 7 having s i g n i f i c a n c e i n i t s response to stress related s t i m u l i was inve s t i g a t e d . -6-MATERIALS AND METHODS REAGENTS  Enzymes R e s t r i c t i o n e ndonucleases were purchased from New England B i o l a b s (NEBL) or Bethesda R e s e a r c h L a b o r a t o r i e s (BRL). T4 DNA l i g a s e , T4 p o l y n u c l e o t i d e k i n a s e , RNase T l , and DNA polymerase I (Klenow fragment) were a l s o s u p p l i e d by BRL. P r o t e i n a s e K and SI n u c l e a s e were from B o e h r i n g e r Mannheim. Lysozyme was s u p p l i e d by Sigma, r e v e r s e t r a n s c r i p t a s e was from Pharmacia, and G l u s u l a s e was from Endo l a b o r a t o r i e s . DNase I and DNA polymerase I were s u p p l i e d by Amersham ( N i c k T r a n s l a t i o n K i t ) . C o n d i t i o n s f o r each enzyme are d e s c r i b e d l a t e r i n t h i s s e c t i o n . N u c l e o t i d e s 2 ' - D e o x y r i b o n u c l e o t i d e t r i p h o s p h a t e s were s u p p l i e d by Pharmacia. dNTPs were d i s s o l v e d i n water to a c o n c e n t r a t i o n o f 10 mM. The pH was a d j u s t e d to pH 7.0 by ad d i n g a d i l u t e s o l u t i o n (0.05 M) o f T r i s base. The e x a c t concen-t r a t i o n o f each s t o c k was determined s p e c t r o p h o t o m e t r i c a l l y . S m a l l a l i q u o t s were f r o z e n a t -70°C. 32 32 a[ P ] - 2 ' - d e o x y r i b o n u c l e o t i d e t r i p h o s p h a t e s and Y[ P ] - a d e n o s i n e t r i p h o s p h a t e were s u p p l i e d as aqueous s o l u t i o n s c o n t a i n i n g 10 u C i / u l . Vanadyl r i b o n u c l e o s i d e complexes (0.2 M) were purchased from BRL. Phenol U l t r a Pure Phenol was s u p p l i e d by BRL. Phenol was e q u i l i b r a t e d w i t h e i t h e r 0.1 M T r i s (pH 8.0) or s t e r i l e H O . -7-C h l o r o f o r m C h l o r o f o r m was s u p p l i e d by BDH C h e m i c a l s . Formamide U l t r a Pure Formamide was purchased from BRL. Formamide was d e i o n i z e d by m i x i n g 50 ml w i t h 5 g o f mixed bed, ion-exchange r e s i n (Bio-Rad AG 501-X8-10) f o r 2 hours at room temperature. The m i x t u r e was f i l t e r e d t w i c e t h r o u g h Whatman No. 1 f i l t e r paper and d i s p e n s e d i n t o 1 ml a l i q u o t s and s t o r e d a t -20°C. Formaldehyde Formaldehyde was s u p p l i e d by BDH C h e m i c a l s . The pH o f t h i s s o l u t i o n was 3.8. The s o l u t i o n was d e i o n i z e d w i t h AG 501-X8-D mixed bed r e s i n . The pH was then r a i s e d to 6.8. Agarose U l t r a Pure Agarose was purchased from BRL. A c r y l a m i d e A c r y l a m i d e and b i s - a c r y l a m i d e were s u p p l i e d by B i o Rad. C u l t u r e Media B a c t o - t r y p t o n e , B a c t o - y e a s t e x t r a c t , Bacto-peptone, and B a c t o - y e a s t n i t r o g e n base w i t h o u t amino a c i d s were s u p p l i e d by D i f c o . Amino a c i d s were from Sigma. A m p i c i l l i n , t e t r a c y c l i n e , and c h l o r a m p h e n i c o l were purchased from Sigma. Murashige and Skoog p l a n t s a l t medium (MS media) was o b t a i n e d from Flow L a b o r a t o r i e s . -8-Others A l l other chemicals used were of reagent grade. MICROBIAL STRAINS Bacteria The following strains of E_. c o l i were used as hosts for recombinant DNA molecules. E. c o l i RRl, F», hsdS20, ara-14, po A2, lacY, gal Ks, rspsL20, Xyl-5, mll-1, sup E44, lambda-, was constructed by Bolivar ejt al_. (1977). E. c o l i JM 101, ( l a c , pro), sup E, t h i , strA, sbcB15, end A, hspR4, F'traD36, proAB, l a d , lacZ M15, was constructed by Messing (1981). Yeast S t r a i n GM-3C-2 was described by Fay et a l . (1981). Its genotype was alpha, leu2-3, 112, trp 1-1, his 4-519, cy c l - 1 , cyp3-l. TOBACCO TISSUE CULTURE LINES Tumor I n c i t i n g N. tobacum Octopine 1ine plasmid c u l t i v a r biosynthes i s Reference A6S/2 pTiA6 White Burley + (Gelvin et a l . , 1982) E9 P T i B 6 8 0 6 x a n t h i c nc. + (Gelvin et a l . , 1982) 16-12-C pTLl Xanthi nc. CULTURE MEDIA AND CONDITIONS E. c o l i The following media were used for the growth of E. c o l i : LB- 1.0% Bacto-tryptone, 0.5% Bacto-yeast extract, 0.5% NaCl, 0.1% glucose. - 9 -M9-50 mM Na oHP0., 25 mM KH.PO.. — 2 4 2 4 0.1 mM C a C l 2 , 10 mM g l u c o s e , 0.001% t h i a m i n e . F o r p l a t e s , B a c t o - a g a r was added to the a p p r o p r i a t e l i q u i d medium a t 20 g/1. E . c o l i was c u l t u r e d at a temperature o f 37°C. Growth was mo n i t o r e d by measuring the o p t i c a l d e n s i t y a t 600 nm. A n t i b i o t i c s were added t o the a p p r o p r i a t e c o n c e n t r a t i o n s . Y e a s t Media f o r the c u l t u r e o f y e a s t have been d e s c r i b e d by Sherman e_t a l . ( 1 9 8 1 ) . The f o l l o w i n g were used i n t h i s s t u d y : YPD - 2% Bac t o - p e p t o n e , 1% B a c t o - y e a s t e x t r a c t , 2% g l u c o s e , pH 5.8. YNB - 0.7% B a c t o - y e a s t n i t r o g e n base w i t h o u t amino a c i d s , 2% g l u c o s e , pH 5.8. YNB was supplemented w i t h t r y p t o p h a n (20 mg/1) and h i s t i d i n e (20 mg/1). For p l a t e s , agar was added to a c o n c e n t r a t i o n o f 2% to the above media. R e g e n e r a t i o n Agar - YNB supplemented w i t h 1 M s o r b i t o l , 2% YPD, and 3% a g a r . Ye a s t c u l t u r e s were grown at 30°C w i t h m i l d s h a k i n g . T i s s u e C u l t u r e s The t o b a c c o crown g a l l tumors l i n e s A6 and E9 were grown i n Murashige-Skook (MS) medium (Murashige et a l . , 1962) at 25°C i n the absence o f p l a n t growth hormones. Transformed tobacco c u l t u r e s (16-12-C) were grown i n MS media c o n t a i n i n g p l a n t hormones, naphthalene a c e t i c a c i d (2 mg/L) and b e n z y l a m i n o p u r i n e (0.2 mg/L), at 25°C. S t r e s s i n d u c i n g compounds were added at the a p p r o p r i a t e c o n c e n t r a t i o n s . -10-PLASMIDS AND BACTERIOPHAGES Plasmids and bacteriophages provided by other workers are l i s t e d i n Table I. Table I Name Reference Source Hpall 0.8 pBR322 LE.E9 YEpl3 Broach et a l . (1979) Bolivar et a l . (1977) R. Kay (U.B.C.) J. McPherson (U.B.C.) J. Ngsee (U.B.C.) J. McPherson (U.B.C.) TRANSFORMATION OF E. COLI E. c o l i was transformed by a modification of the procedure of Mandel and Higa (1970). One b a c t e r i a l colony was picked and inoculated i n 5 ml of LB-glucose media. The b a c t e r i a l c e l l s were grown overnight at 37°C with mild shaking. The overnight culture (0.5 ml) was inoculated into 50 ml of LB-glucose media and incubated at 37°C with shaking to a density of approxi-mately 5 x 10 7 c e l l s / m l ( A , n n = 0.25). The culture was c h i l l e d on ice for 10 minutes and then centrifuged at 4000 g for 5 minutes at 4°C. The supernatant was discarded and the remaining c e l l s were resuspended in 0.25 ml of 100 mM C a C l 2 and 10 mM T r i s - C l (pH 8.0) and incubated at 0°C for 1 hour. The c e l l suspension was divided into 0.1 ml aliquots and 16 ul of s t e r i l e g l y c e r o l was added to the mixture. The c e l l suspensions were stored at -70°C and removed from the freezer as needed. Plasmid DNA (40-100 ng in of 10-100 u l of H 20) was added to 0.1 ml of the thawed c e l l suspension on i c e . The mixture was incubated on ice for 15 minutes and then incubated at 37°C for 5 minutes. LB-glucose media (1 ml) was added and - l i -the mixture was incubated for 2 hours at 37°C without shaking. Aliquots (50 u l to 200 u l ) were spread on s e l e c t i v e media. The plates were incubated at 37°C overnight. LARGE SCALE ISOLATION OF PLASMID DNA This procedure was described by Maniatis et a l . (1982) and i s a modification of the method of Birnboim and Doly (1979). The b a c t e r i a l p e l l e t from a 500 ml culture was resuspended in 10 ml of a so l u t i o n containing 50 mM glucose, 25 mM T r i s - C l (pH 8.0), 10 mM EDTA, and 5 mg/ml lysozome and was incubated for 5 minutes at 4°C. A s o l u t i o n consisting of 0.2 N NaOH and 1% SDS (20 ml) was added to the c e l l suspension and the contents of the tube mixed by gentle i n v e r s i o n . The suspension was then incubated on ice for 10 minutes. An i c e - c o l d s o l u t i o n of 3 M potassium acetate and 2 M HOAc (15 ml) was added and the contents were mixed. The mixture was incubated on ice for 10 minutes and then centrifuged at 20,000 rpm for 20 minutes at 4°C. The supernatant was removed and transferred to 30 ml Corex tubes. Two volumes of 95% ethanol were added to each tube and incubated at -20°C for one hour. The DNA was recovered by ce n t r i f u g a t i o n at 10,000 g for 20 minutes at room temperature. The DNA p e l l e t was washed with 70% ethanol and a i r d r i e d . The p e l l e t was dissolved in TE (pH 7.6) and 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 chloride-ethidium bromide density gradients. PURIFICATION OF PLASMID DNA BY CESIUM CHLORIDE GRADIENT CENTRIFUGATION Plasmid DNA was p u r i f i e d according to Maniatis et a l . (1982). For every m i l l i l i t e r of plasmid DNA i s o l a t e d , exactly 1 g of cesium chloride was added. Ethidium bromide (0.8 ml of 10 mg/ml in H2O) was added for every 10 ml of cesium chloride s o l u t i o n . The cesium chloride s o l u t i o n was transferred to a -12-tube s u i t a b l e for ce n t r i f u g a t i o n in a Beckman type 50 rotor. The remainder of the tube was f i l l e d with p a r a f f i n o i l and the tubes were sealed using the Beckman tube s e a l e r . The tubes were spun at 50,000 rpm for 40 hours at 20°C. The tubes were mounted on a stand and illuminated with an u l t r a v i o l e t lamp. Two bands were normally v i s i b l e , and the lower band containing plasmid DNA was c o l l e c t e d with an 18 gauge hypodermic needle. Ethidium bromide was removed from the s o l u t i o n by repeated extractions with n-butanol e q u i l i b r a t e d with 20XSSC. The s o l u t i o n was d i l u t e d with two or three volumes of H^ O and 2 volumes of ethanol were added to p r e c i p i t a t e the DNA. The mixture was incubated at -20°C overnight and spun down at 10,000 rpm for 20 minutes at 4°C. The supernatant was discarded and the DNA p e l l e t was washed with 70% ethanol. The p e l l e t was resuspended in 10 mM T r i s - C l (pH 7.6) and 1 mM EDTA (pH 8.0). SMALL SCALE ISOLATION OF PLASMID DNA This procedure, described by Maniatis eji a_l. (1982), is a modification of the method of Birnboim and Doly (1979). LB-glucose media (5 ml) containing either a m p i c i l l i n (50 ug/ml) or t e t r a c y c l i n e (15 ug/ml) was inoculated with a single b a c t e r i a l colony and grown overnight at 37°C with mild shaking. A 1.5 ml portion of the culture was poured into a microfuge tube. The tube was centrifuged for one minute and the supernatant was poured o f f . The remaining p e l l e t was resuspended in 100 ul a s o l u t i o n containing 50 mM glucose, 10 mM EDTA, 25 mM T r i s - C l (pH 8.0), and lysozyme 4 mg/ml. The mixture was incubated at room temperature for 5 minutes. A s o l u t i o n containing 0.2 N NaOH and 1% SDS (200 ul) was added and mixed without vortexing. The solution was stored on ice for 5 minutes. A 150 u l s o l u t i o n of potassium acetate was added and the mixture was stored on ice for -13-5 minutes. The mixture was centrifuged i n a microfuge for 5 minutes at 4°C and the supernatant was transferred to a new tube. An equal volume of phenol/chloroform was added and mixed by vortexing. The tube was centrifuged at room temperature for one minute and the supernatant was t r a n s f e r r e d to a new tube. Two volumes of ethanol were added and the so l u t i o n was incubated at -20°C for one hour. The tube was then centrifuged for 10 minutes at room temperature. The supernatant was discarded and the p e l l e t was washed with 70% ethanol. The p e l l e t was a i r dried and redissolved in 10 mM T r i s - C l (pH 7.6) and 1 mM EDTA (pH 8.0). RESTRICTION ENDONDCLEASE DIGESTION R e s t r i c t i o n endonucleases used in this work were Hind I I I , Bgl I I , Eco R l , Bam HI, PvuII, and Eco RV. A 10X buffer s o l u t i o n c o n s i s t i n g of 500 mM T r i s - C l (pH 8.0) 100 mM MgCl 2, 500 mM NaCl was used for each enzyme re a c t i o n . Reactions t y p i c a l l y contained between 0.1 and 1 ug of DNA i n a t o t a l volume of 50 u l . The DNA to be digested was dissolved i n 10 mM T r i s - C l (pH 7.6) and 1 mM EDTA (pH 8.0). The 10X buffer concentrate was added (one-tenth of the f i n a l volume of the mixture) and the f i n a l volume was brought up to 50 u l with s t e r i l e H^O. About 1 to 5 units of the appropriate r e s t r i c t i o n endonuclease were added per ug of DNA. The mixture was then incubated at 37°C for approximately 2 hours. The extent of the d i g e s t i o n was checked by agarose gel electrophoresis. I f the DNA was to be used in a subsequent reaction (a l i g a s e reaction or another r e s t r i c t i o n d i g e s t i o n ) , the DNA was p u r i f i e d by adding an equal volume of phenol. The aqueous layer was removed and placed in another microfuge tube. An equal volume of chloroform was added and the aqueous layer was transferred to another microfuge tube. Sodium acetate 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 of ethanol. The mixture was incubated at -20°C for 1-12 hours. The 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 for 10 minutes at 4°C. The ethanol was removed and approxi-mately 1 ml of 70% ethanol was added to the p e l l e t . The mixture was then vortexed and centrifuged for 5 minutes. The ethanol was removed and the p e l l e t was a i r dried for one hour. The p e l l e t was then dissolved in water or T r i s - C l . LIGATIONS Li g a t i o n reactions were done i n a t o t a l volume of 20 u l using 10 to 50 ng of vector DNA and a three to ten-fold molar excess of insert fragment. Fragments were l i g a t e d in a buffer containing 66 mM T r i s - C l , 10 mM MgC^, 10 mM DTT, and 1.0 mM ATP. T4 DNA lig a s e enzyme was added (0.1 unit for overhanding-end l i g a t i o n s and 1 unit for blunt end l i g a t i o n s ) and the mixture incubated at 4°C overnight. The l i g a t i o n mixtures were used without further treatment to transform E. c o l i . AGAROSE GEL ELECTROPHORESIS OF DNA Agarose gels (0.7% in 1 TBE) were prepared for el 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 of DNA fragments. Electrophoresis was c a r r i e d out h o r i z o n t a l l y at a voltage gradient of 2-5 V/cm. TRANSFORMATION OF YEAST The procedure used for yeast transformation was si m i l a r to those described by Hinnen et a l . (1978), Beggs (1978), Sherman et a l . (1981), and Orr-Weaver et a l . (1983). A single colony of the yeast s t r a i n to be trans-formed was used to inoculate 5 ml of YEPD, and the culture was incubated at -15-30°C o v e r n i g h t w i t h m i l d s h a k i n g . One ml of the o v e r n i g h t c u l t u r e was i n o c u l a t e d i n t o 80 ml of YEPD. The y e a s t were then grown w i t h m i l d s h a k i n g f o r a p p r o x i m a t e l y 8 hours ( O . D . ^ Q = 0.33 - 0.35). The c e l l s were h a r v e s t e d by c e n t r i f u g a t i o n a t 3,000 rpm f o r 2 minutes at room t e m p e r a t u r e . The p e l l e t s were resuspended i n 2.5 ml of 1 M s o r b i t o l per tube and c e n t r i f u g e d as b e f o r e . The c e l l s were then resuspended i n 2.5 ml o f 1 M s o r b i t o l per tube and 10 mg o f DTT and 100 u l o f g l u s u l a s e were added to each tube. The s u s p e n s i o n was i n c u b a t e d a t 30°C w i t h m i l d s h a k i n g , and 5-10 u l samples were removed p e r i o d i c a l l y , d i l u t e d w i t h 50 u l f^O and examined under a p h a s e - c o n t r a s t m i c r o s c o p e . S p h e r o p l a s t s are n o n - r e f r a c t i l e and appear dark under the p h a s e - c o n t r a s t m i c r o s c o p e , w h i l e normal c e l l s a r e r e f r a c t i l e . More g l u s u l a s e was added i f the s p h e r o p l a s t s had not been formed a f t e r 1.5 h o u r s . When the c e l l s had been c o n v e r t e d to s p h e r o p l a s t s , they were h a r v e s t e d by c e n t r i f u g a t i o n a t 2,000 rpm f o r 5 minutes at room t e m p e r a t u r e . The p e l l e t was washed by r e s u s p e n d i n g the c e l l s i n 2.5 ml o f 1M s o r b i t o l per tube and c e n t r i f u g e d as b e f o r e . The c e l l s were resuspended i n 2.5 ml o f STC (1 M s o r b i t o l , 10 mM T r i s - C l , 10 mM CaCl,,) per tube and c e n t r i f u g e d a t 2,000 rpm f o r 5 m i n u t e s . The c e l l s were resuspended i n 0.2 ml o f STC per t u b e . A p p r o x i m a t e l y 50 u l of p l a s m i d (1 ug) was added to 50 u l o f competent y e a s t c e l l s . The m i x t u r e was i n c u b a t e d on i c e f o r 15 m i n u t e s , and 0.5 ml o f PEG-TC (20% p o l y e t h y l e n e g l y c o l , 10 mM T r i s , 10 mM C a C l 2 > pH 8.0) was added to each tube and i n c u b a t e d on i c e f o r 20 m i n u t e s . The e n t i r e s o l u t i o n c o n t a i n i n g the DNA was added to a s t e r i l e c e n t r i f u g e tube, and 0.3 ml of YEPD and 15 ml o f r e g e n e r a t i o n agar (50-55°C) was added to each tube. The s o l u t i o n was then immediately poured on a YNB p l a t e c o n t a i n i n g the a p p r o p r i a t e amino a c i d s . T r a n s f o r m a n t s gave r i s e to c o l o n i e s embedded w i t h i n the r e g e n e r a t i o n agar which were e a s i l y v i s i b l e a f t e r 2-3 days of i n c u b a t i o n at 30°C. - 1 6 -YEAST PLASMID PURIFICATION Plasmids were i s o l a t e d from yeast as described by Lorincz (1984). A medium sized yeast colony (approximately 3 mm in diameter) was picked and placed i n a microfuge tube containing 200 u l of 100 mM CaCl2, 10 mM T r i s - C l (pH 8.0), 1 mM EDTA, 0.1% SDS. Glass beads (0.45 mm diameter) were added to just below the l e v e l of the l i q u i d and the contents mixed vigorously on a vortex mixer for 1 minute. The suspension was extracted with an equal volume of Tri s - b u f f e r e d phenol and then extracted with an equal volume of chloroform. Sodium acetate was added to 0.3 M, and 2 volumes of cold ethanol were added. After 30 minutes at -60°C, nucleic acid 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). The a i r dried p e l l e t was resuspended and the entire DNA s o l u t i o n was used to transform E. c o l i (0.2 ml suspension). ISOLATION OF YEAST RNA Yeast RNA was i s o l a t e d as described by McNeil and Smith (1986) with a few modifications. A single colony of yeast was used to inoculate 5 ml of YNB plus the appropriate amino acids. The culture was grown with mild shaking for 1-2 days and 1 ml of the culture was inoculated into 100 ml of fresh YNB media. The culture was then incubated at 30°C with mild shaking u n t i l i t s A ^ ^ Q W A S 0.8. Cycloheximide was added to a concentration of 0.1 mg/ml and the culture was incubated at 30°C for 5 minutes with mild shaking. The culture was then poured into two 520 ml centrifuge b o t t l e s , each h a l f - f u l l of crushed i c e . The c h i l l e d c e l l s were transferred to centrifuge tubes and centrifuged at 4000 rpm for 1 minutes at 40°C. The c e l l s were resuspended into about 10 ml of ice cold r^O) and cycloheximide (0.1 mg/ml) per tube. The mixture was transferred to a weighed 30 ml Corex tube and the c e l l s were pel l e t e d at 4000 rpm for 3 minutes at 4°C. The c e l l s were then immediately frozen in a dry ice/ethanol bath. -17-Acid-washed glass beads were added to the frozen c e l l p e l l e t s (3g beads/g wet weight c e l l s ) followed by 3 ml/g of ic e - c o l d RNA extraction buffer (0.15 M NaCl, 0.1 M Tris-HCl) and 50 ul/g of Vanadyl ribonucleoside complexes (VRC: 0.2 M). The c e l l s were broken by vortexing hard for s i x 15-second i n t e r v a l s , each followed by 45 seconds of cooling on i c e . The mixture was centrifuged at 9,000 rpm for 5 minutes at 40°C. The supernatent was transferred to a s t e r i l e 30 ml Corex tube and placed on i c e . E x t r a c t i o n buffer and VRC were added to the p e l l e t as before and vortexed. The c e l l s were centrifuged as before and the supernatant was removed and combined with the previous supernatant. SDS and proteinase K were added to concentrations of 0.5% and 0.5 mg/ml, r e s p e c t i v e l y . The mixture was incubated i n a 37° water bath for 60 minutes. The proteinase K-digested s o l u t i o n was extracted with 1 volume of phenol/chloroform/amyl alcohol (24/24/1). The aqueous supernatant was trans-ferred to a clean tube and nucleic a c i d was p r e c i p i t a t e d by adding sodium acetate to 0.3 M, 2.5 volumes of ethanol, and c h i l l i n g at -20°C overnight. The 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 minutes, 40°C) rinsed i n cold ethanol, dried and dissolved i n 1 ml of 20 mM EDTA. An equal volume of 4M L i C l was added and the mixture was incubated at 4°C for 16 hours. The RNA p r e c i p i t a t e was co 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, 4°C) and dissolved i n s t e r i l e H^O. A f i n a l ethanol p r e c i p i t a t i o n from 0.3M sodium acetate, followed by r i n s i n g with ethanol served to desalt the RNA. The p r e c i p i t a t e was a i r dried, dissolved i n E^O and stored at -20°C. Up to 5 mg of RNA was obtained from 1 g (wet weight) of c e l l s . SELECTION OF POLY A+ RNA Polyadenylated RNA molecules were is o l a t e d by chromatography using o l i g o dT c e l l u l o s e by a modification of the procedure of Aviv and Leder (1972). -18-O l i g o dT c e l l u l o s e was e q u i l i b r a t e d w i t h RNA l o a d i n g b u f f e r (0.5 M N a Cl, 10 mM T r i s , 4 mM EDTA) and the m i x t u r e was a p p l i e d to a s m a l l s t e r i l e column w i t h a b u i l t - i n - f i l t e r . RNA (100 ug a l i q u o t s ) was loaded i n 3 ml o f l o a d i n g b u f f e r . The r u n - t h r o u g h was c o l l e c t e d and r e a p p l i e d to the column. The RNA which ran t hrough the column was n o n - p o l y a d e n y l a t e d . The column was washed w i t h 5 ml o f l o a d i n g b u f f e r and 2 ml o f 0.5 M KC1. The p o l y a d e n y l a t e d RNA was e l u t e d by a p p l y i n g 200 u l o f s t e r i l e h^O 5 times and c o l l e c t i n g the f r a c t i o n s . The f r a c t i o n s were then p o o l e d , p r e c i p i t a t e d i n e t h a n o l , and d i s s o l v e d i n s t e r i l e H 20. RNA was s t o r e d a t -20°C. END-LABELING OF OLIGONUCLEOTIDES The o l i g o n u c l e o t i d e (10 p moles) was 5'-ended l a b e l e d i n a r e a c t i o n (50 32 u l ) c o n t a i n i n g [gamma- P]ATP(120 C i o f 3000 Ci/mmole), p o l y n u c l e o t i d e k i n a s e (10 u n i t s ) , 50 mM T r i s - H C l , pH 7.7, 10 mM M g C l 2 , 5 mM d i t h i o t h r e i t o l , 0.1 mM s p e r m i d i n e , f o r 45 minutes at 37°C. The r e a c t i o n was t e r m i n a t e d by a d d i t i o n of 2 u l of 0.5 M EDTA and h e a t i n g to 67°C (10 m i n u t e s ) . 32 The l a b e l e d o l i g o n u c l e o t i d e was s e p a r a t e d from u n i n c o r p o r a t e d (gamma- P] ATP by chromatography through a column of Sephadex G-25. A p p r o x i m a t e l y 8 grams of Sephadex G-25 and 100 ml o f H^O ( t h i s was enough f o r 4 columns) were a u t o c l a v e d f o r 15 m i n u t e s . The m i x t u r e was c o o l e d down to room t e m p e r a t u r e , the water was poured o f f , and 40 ml o f column b u f f e r [10 mM T r i s (pH 8.0), 5 mM EDTA] was added. The columns were poured s l o w l y and c o n t i n u o u s l y . A p p r o x i -m a t e l y 200 u l of column b u f f e r was added to the o l i g o n u c l e o t i d e s o l u t i o n and a p p l i e d to the top of the column. Column b u f f e r was added as needed and f r a c -t i o n s were c o l l e c t e d i n m i c r o f u g e t u b e s . The l e a d i n g peak of r a d i o a c t i v i t y which c o n t a i n e d the l a b e l e d o l i g o n u c l e o t i d e was p o o l e d and s t o r e d a t -20°C. -19-PRIMER EXTENSION ANALYSIS The oligonucleotide was desalted and hybridized [approximately 1 ng (10^ cpm) per reaction] with samples of RNA. Annealing was performed at 65°C i n 250 mM KCl, 10 mM T r i s - H C l , pH 8.0, 1 mM EDTA. Following h y b r i d i z a t i o n to s p e c i f i c RNA, the oligonucleotide primer was extended using Avian myeloma virus reverse transcriptase using the conditions described (McKnight et a l . , 1981). The re a c t i o n products were denatured and the lengths of the extended products estimated by denaturing acrylamide gel elec t r o p h o r e s i s . DENATURING UREA-ACRYLAMIDE GEL Denaturing acrylamide gels (6% and 8%) containing 7 M urea were prepared i n 1 X TBE. Samples were denatured i n formamide (100°C, 4 minutes) p r i o r to loading and electrophoresis (30-40 V/cm). SI NUCLEASE MAPPING 32 32 DNA fragments were P-labelled at the 5' end using [gamma- P] ATP and T4 polynucleotide kinase. Stand separation was accomplished as described (Maxam and G i l b e r t , 1980), using acrylamide concentrations varying between 3% and 7% according to the fragment molecular weight. Single strands were eluted from the gel in 50 mM T r i s pH 8, 20 mM EDTA, 0.5 M NaCl overnight at room temperature. The single stranded DNA probes were hybridized to RNA and subsequently digested with SI nuclease as described (De Greve £t a K , 1982). GEL ELECTROPHORESIS OF RNA AND NORTHERN HYBRIDIZATION RNA was analyzed by electrophoresis in formaldehyde-agarose gels a f t e r denaturation with formaldehyde and formamide (Lehrach et a l . , 1977; Maniatis -20-et a l . , 1982). As much as 30 ug of RNA was denatured i n a volume of 50 u l containing 2.2 M formaldehyde, 50% formamide, and 1/2 MOPS buffer (IX MOPS buffer contained 40 mM Na MOPS, 10 mM sodium acetate, 1 mM EDTA, pH 7.9) by heating at 55°C for 15 minutes. Sample loading buffer (2 ul) was added and the RNA samples were loaded on a 1.1% agarose g e l . The gel was made up i n IX MOPS buffer containing 2.2 M formaldehyde and the gel was run in IX MOPS at 0.5 - 1 V/cm for 6-12 hours. After electrophoresis, the gel was soaked i n s t e r i l e K^O for 10 minutes 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 ) for 30 minutes p r i o r to t r a n s f e r r i n g the RNA to n i t r o c e l l u l o s e . The procedures of Thomas (1980) were used to transfer RNA from agarose gels to n i t r o c e l l u l o s e and hybridize the RNA to r a d i o a c t i v e l y l a b e l l e d probes. The Northern h y b r i d i z a t i o n apparatus was cleaned out with s t e r i l e h^O and the chamber was f i l l e d with 20X SSC. Two pieces of f i l t e r paper soaked i n 20X SSC were l a i d across the box so that the papers just touched the bottom of each chamber. The gel was then l a i d on the f i l t e r paper, and cut X-ray f i l m was placed around the gel so that no l i q u i d d iffused out of the g e l . A piece of 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 h^O for one minute and then soaked i n 20X SSC for one minute, was placed on top of the g e l . The agarose not covered by n i t r o c e l l u l o s e was removed by a razor blade. Two more pieces of Whatman 3 MM paper were placed on top of the n i t r o c e l l u l o s e , followed by a 6 cm stack of paper towels. A book was placed on top of the assemblage and transfer of RNA to the n i t r o c e l l u l o s e was allowed to proceed for about 16 hours. The n i t r o c e l l u l o s e was then baked at 80°C for 2 hours. N i t r o c e l l u l o s e f i l t e r s with bound RNA were prehybridized for 4-16 hours at 42°C i n sealed p l a s t i c bags containing 10 ml of a mixture of 50% formamide, 5X SSC, IX Denhardt's solution (0.02% F i c o l l , 0.02% poly v i n y l p y r r o l i d o n e , 0.02% BSA) 50 mM NaH PO. and Na„HP0. , pH 7.0, and denatured, sheared salmon sperm -21-DNA (250 ug/ml). The prehybridization mixture was removed and replaced with a h y b r i d i z a t i o n mixture (10 ml) which contained the r a d i o a c t i v e l y - l a b e l l e d probe. The h y b r i d i z a t i o n mixture was composed of four parts of p r e h y b r i d i z a t i o n mixture and one part of 50% dextran s u l f a t e . The probe was b o i l e d for 5 minutes and c h i l l e d on ice before adding i t to the h y b r i d i z a t i o n mixture. Hybridization was allowed to proceed for about 18 hours at 42°C. The f i l t e r was washed i n 2 changes of 0.1% SDS, 2X SSC at room temperature for 5 minutes each time. The f i l t e r was then washed i n two changes of 0.1X SSC, 0.1% SDS at 55°C for 20 minutes each. The f i l t e r was subjected to autoradiography. NICK TRANSLATION Nick t r a n s l a t i o n of DNA was done using the Amersham Nick T r a n s l a t i o n K i t . T y p i c a l l y 500 ng of DNA was r a d i o a c t i v e l y labeled to a s p e c i f i c a c t i v i t y of 4 x 10^ - 10 x 10^ cpm/ug DNA i n a 50 u l reaction containing 10 u l of nucleo-32 t i d e buffer (dGTP, dCTP, dTTP, 50 uCi of [alpha- P] ATP, and 5 u l of an enzyme so l u t i o n containing DNA polymerase I and DNase I. The reaction was incubated at 15°C for 2 hours. Unincorporated nucleotides were removed by running the s o l u t i o n through an U l t r o g e l column. ISOLATION OF PLANT TUMOR RNA RNA was extracted from t o t a l c a l l u s tissue by a modification of the procedure described by McPherson et a l . (1980). Callus tissue (50 g fresh weight) was frozen in l i q u i d nitrogen and ground in a blender. The f i n e l y divided tissue (frozen powder) was extracted by homogenization (2 minutes) i n buffer (100 ml) containing 1 M T r i s , 10% SDS, 10% T r i t o n , and 0.5 mg/ml Heparin. The c e l l debris were p r e c i p i t a t e d following c e n t r i f u g a t i o n at 4°C (10,000 g for 5 minutes). -22-The supernatant was f i l t e r e d through cheesecloth and extracted with phenol: chloroform (1:0.5). The aqueous layer was re-extracted with phenol: chloroform and then with chloroform. Nucleic acids were recovered by ethanol p r e c i p i t a t i o n (2.5 volumes) at -20°C overnight. The p e l l e t was reconstituted i n 0.1 M NaOAc to which an equal volume of L i C l (5 M) was added. RNA was recovered by p r e c i p i t a t i n g with L i C l (2.5 M) at -20°C overnight. The r e s u l t i n g RNA was p r e c i p i t a t e d from ethanol (2.5 volumes). STRESS INDUCTION OF PLANT TDMORS Crown g a l l tumors and transformed tobacco cultures (16-12-C) were subjected to various stress treatments. Cadmium, mercury, z i n c , and arsenite at concentrations of 10 ^ M, 10 ^ M, 10 ^ M, and 10 7 M were added to MS media (plant hormones were added to MS media for growth of 16-12-C) for d i f f e r e n t periods of time and RNA was i s o l a t e d . The plant tumors were heat shocked at 40°C for d i f f e r e n t times and RNA was i s o l a t e d . - 2 3 -RESULTS A. T-DNA Gene 7 i n Yeast  P l a s m i d C o n s t r u c t i o n s C o n s t r u c t i o n o f pl a s m i d s f o r T-DNA gene 7 e x p r e s s i o n i n y e a s t were done as shown i n F i g u r e s 1 and 2. Pl a s m i d Y-BG.E9 c o n s i s t s o f the y e a s t p l a s m i d , Y Epl3 (Broach et: aJl., 1979), and a 2,637 bp T-DNA segment c o n t a i n i n g gene 7 ( F i g . 1 ) . Y-BGE.E9 c o n s i s t s o f YEpl3 and a 1,547 bp T-DNA segment c o n t a i n i n g 1 gene 7 ( F i g . 2 ) . R e s t r i c t i o n s i t e a n a l y s i s was done on these p l a s m i d s as d e s c r i b e d i n F i g u r e 3. Primer E x t e n s i o n A n a l y s i s The primer e x t e n s i o n method was used to i d e n t i f y the number and p o s i t i o n o f T-DNA gene 7 RNA 5' ends. The source o f T-DNA gene 7 RNA was the S_. c e r e v i s i a e s t r a i n GM-3C-2 (Orr-Weaver ej: a _ l . , 1981) t r a n s f o r m e d w i t h Y-BG.E9 and Y-BGE.E9. An o l i g o n u c l e o t i d e primer ( o l i g o - 7 ) complementary t o T-DNA gene 7 RNA ( F i g . 5) was added t o y e a s t RNA and used to prime r e v e r s e t r a n s c r i p t a s e . The cDNA p r o d u c t s were e l e c t r o p h o r e s e d through a DNA s e q u e n c i n g g e l ( F i g . 6 ) . The r e s u l t s o b t a i n e d f o r t r a n s c r i p t 7 from o c t o p i n e tumor l i n e E9 ( F i g . 6, l a n e 5) show t h a t the major cDNA pro d u c t i s 45 bases l o n g which i n d i c a t e s t h a t the major 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 the a d e n o s i n e r e s i d u e l o c a t e d 15 n u c l e o t i d e s upstream o f the ATG s t a r t codon o f the open r e a d i n g frame ( F i g . 4 ) . T h i s c o r r e s p o n d s to t r a n s c r i p t i n i t i a t i o n at a d i s t a n c e o f 29 bases from the c e n t e r o f the TATA sequence. The r e s u l t s o b t a i n e d when no RNA was added to the primer e x t e n s i o n r e a c t i o n ( F i g . 6, lane 4) i n d i c a t e t h a t t h e r e was an unexpected 60 n u c l e o t i d e s t r e t c h . The 60 n u c l e o t i d e s t r e t c h appears i n a l l the primer e x t e n s i o n s . I t i s thought t h a t t h i s 60 n u c l e o t i d e -24-Figure 1. Construction of Y-BG.E9 Construction of yeast plasmid for T-DNA gene 7 expression i n yeast. Y-BG.E9 was constructed as follows: LE.E9 which is pBR325 plus the EcoRl l e f t junction fragment of T-DNA/plant DNA sequences of the E9 tumor l i n e was digested with B g l l l . The 2,637 bp T-DNA fragment containing gene 7 was l i g a t e d to YEpl3 which had been digested with Bam HI (Bam HI and B g l l l generate cohesive ends). The l i g a t i o n reaction was then treated with Bam Hi to digest a l l l i g a t i o n products except for the recombinant plasmid Y-BG.E9 which consists of YEpl3 plus the 2,637 bp T-DNA fragment contain-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 by d i g e s t i n g LE.E9 w i t h Eco RV and B g l l l . The 1,547 bp T-DNA fragment c o n t a i n i n g gene 7 was l i g a t e d to Y E p l 3 which had been d i g e s t e d w i t h Bam HI and PvuII (Bam HI and B g l l l have c o h e s i v e ends, and PvuII and Eco RV have c o h e s i v e e n d s ) . 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 the l i g a t i o n p r o d u c t s except f o r the recombinant p l a s m i d Y-BGE.E9 which i s YEpl3 p l u s the 1,547 bp T-DNA fragment c o n t a i n i n g gene 7. EV=Eco RV, E=EcoRl, H - H i n d l l l , Bg=BglII, B=Bam H i ; the dashed l i n e s i n d i c a t e T-DNA sequences. -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-BG . E 9 , Y-BGE . E 9 , Y E p l 3 , and L E . E 9 . 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 and the p r o d u c t s were a n a l y z e d by 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 bromide and p h o t o g r a p h e d i n UV l i g h t . ( 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 ) lambda ( H i n d l l l ) . G CTT GAA AAT TAA GCC CCC CCC CGA AAT CAT CGC CAC AGC TCG TCC CAG CCC GGC ATC TAT ATA TAG CGC CAA It) 20 30 40 50 60 70 i n d l l l Met Asn Phe Ala Asp Thr Pro Leu Ala Ser Leu Asp Leu Asp TAT AGT TTG TCT TAC ACA AAC ACA CCT CAC ATC ATG AAT TTC GCA GAT ACT CCC TTG GCC TCC CTC GAC CTA GAC 80 90 100 110 * 120 130 140 150 Trp Ala Cys Glu Glu Phe H e Lys Thr Tyr Gly Ala Ser Pro Gin Leu Glu Thr Gly Glu Val He Gin Thr Asn TGG GCA TGC GAA GAG TTT ATC AAA ACT TAT GGT GCA TCT CCA CAA TTG GAA ACA GCA GAG GTA ATC CAA ACA AAC 160 170 180 190 200 210 220 Asn Gly Leu Leu Tyr Leu Tyr Gly Lys Gly Ser Leu Ser Gin Arg He His Asp Thr His Leu Lys Phe Lys Glu AAT GGG CTG CTG TAT TTG TAT GGC AAA GGT TGA CTC TCA CAG CGG ATT CAT GAC ACA CAC CTC AAA TTT AAG GAG 230 240 250 260 270 280 290 300 Lys Glu Glu Leu Ser Phe Thr Thr H e Lys Pro Ala Glu Met Lys Ala Gin Gin Ser Asp Leu Thr Tyr Tyr Val AAG GAA GAA TTA TCC TTC ACT ACC ATA AAG CCA GCT GAG ATG AAG GCG CAA CAA AGT GAT TTA ACT TAT TAT GTC 310 320 I 330 340 350 360 370 PvuII Ala H e Phe Gin Ser Asn Tyr Phe Leu Cys Val Ser Asn Pro Glu Lys Gly Phe Leu Arg Cys His Asn Arg Pro GCC ATT TTT CAA AGC AAC TAT TTC CTG TGC GTT TCA AAT CCA GAG AAA GGC TTT CTG AGA TGC CAT AAT CGC CCA 380 390 400 410 420 430 440 450 Phe Leu Tyr Pro H e Val Ala His Gly Ser Met Ser STOP TTT CTG TAC CCC ATA GTA GCC CAT GGA TCG ATG AGC TAA GCT AGC TAT ATC ATC AAT TTA TGT ATT ACA CAT AAT 460 470 480 490 500 510 520 ATC GCA CTC AGT CTT TCA TCT ACG GCA ATG TAC CAG CTG ATA TAA TCA GTT ATT GAA ATA TTT CTG AAT TTA AAC 530 540 550 560 570 580 590 600 TTG CAT CAA TAA ATT TAT GTT TTT GCT TGG ACT ATA ATA CCT GAC TTG TTA TTT TAT CAA TAA ATA TTT AAA CTA 610 620 630 640 650 660 670 TAT TTC TTT CAA GAT ATC ATT CTT TTA CAA GTA TAC GTG TTT AAA TTG AAT ACC ATA AAT TTT TAT TTT TCA AAT 680 690 700 710 720 730 740 750 ACA TGT AAA ATT ATG AAA TGG GAG TGG TGG CGA CCG AGC TCA AGC ACA CTT CAA TTC CAT AAC GGG ACC AAA TCG 760 770 780 790 800 810 820 CAA AAA TTA TAA TAA CAT ATT ATT TCA TCC TGG ATT AAA AGA AAG TCA CCG AC TTGCCGCC 830 840 850 860 870 | 1,178 Smal Figure 4. DNA sequence of T-DNA gene 7. The predicted amino acid sequence of a protein, Mr 14,400 was obtained by analysis of the open reading frame (Staden, 1980). The putative recognition sequence ("TATA box") and the heat shock related sequence (the heat shock +consensus sequence is CT-GAA—TTC-AG-are underlined, 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 is indicated ( 1), as are c h a r a c t e r i s t i c polyadenylation s i g n a l s . The nucleotides are numbered from the H i n d l l l s i t e (Base 2,119 in Figures 1 and 2). * marks the beginning of putative 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 in yeast as determined by SI nuclease 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 Ala 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 5 o l i g o Figure 5. Oligonucleotide corresponding to the 5' region of t r a n s c r i p t 7. The DNA sequence represents the 5' flanking region of t r a n s c r i p t 7 showing the loc a t i o n and p a r t i a l sequence of the 30-mer 31-GTACTTAAATCGTCTATGAGGGAACCGGAG-51 . -29-1 2 3 45 6 m <45 F i g u r e 6. 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 RNA primed w i t h an o l i g o n u c l e o t i d e complementary t o the 5' c o d i n g r e g i o n o f the T-DNA t r a n s c r i p t 7 ( F i g u r e 5 ) . RNA samples were from y e a s t (GM-3C-2) t r a n s -formed w i t h Y-BG.E9 and Y E p l 3 . The ^ 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 from the 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 (100 ug t o t a l RNA); C o n t r o l samples: l a n e 3,YEpl3(100 ug t o t a l RNA); lane 4, no RNA; l a n e 5, E9 p l a n t tumor (40 ug t o t a l RNA); lane 6, 3 2 P - l a b e l l e d H p a l l fragments o f pBR322. The m i g r a t i o n o f the 30 base 3 2 P o l i g o n u c l e o t i d e ( o l i g o - 7 ) i s i n d i c a t e d . -30-s t r e t c h was the double stranded form of oligo-7. The absence of any other cDNA products besides the 60 nucleotide str e t c h i n the control lanes containing no RNA and RNA from yeast transformed with YEpl3 ( F i g . 6, lanes 3 and 4, r e s p e c t i v e l y ) demonstrates that the oligonucleotide primer is not s p e c i f i c for yeast RNA. The r e s u l t s of the poly A + selected RNA and the t o t a l RNA samples from yeast transformed with Y-BG.E9 ( F i g . 6, lanes 1 and 2, r e s p e c t i v e l y ) show that there were a large number of T-DNA gene 7 RNAs with d i f f e r e n t 5' ends. The r e s u l t s from poly A + selected RNA and t o t a l RNA were i d e n t i c a l . It should be noted, though, that the primer extension method was l i m i t e d because t h i s method could not detect t r a n s c r i p t i o n products with i n i t i a t i o n s i t e s located one base upstream of the ATG t r a n s l a t i o n i n i t i a t i o n codon and downstream from that base. SI Nuclease Mapping The SI nuclease protection method was used to map the 5' ends of T-DNA gene 7 RNAs in yeast. SI nuclease r e s i s t a n t hybrids were formed between the H i n d l l l / P v u I I T-DNA gene 7 fragment shown i n Figure 4 and 2 ug each of RNA preparations. This method could detect 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 downstream from the ATG t r a n s l a t i o n i n i t i a t i o n codon. The r e s u l t s are shown in Figure 7. The controls (no RNA, lane A; RNA from GM-3C-2, lane B; RNA from GM-3C-2 transformed with YEpl3, lane C) demonstrate that the DNA probe i s not s p e c i f i c for yeast RNA. SI nuclease mapping of RNA i s o l a t e d from E9 plant tumors (lane F) shows that an RNA fragment of 239 bases was protected. The distance between the normal 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 and the PvuII. r e s t r i c t i o n s i t e is 239 - 3 1 -A B C D E F G m4M F igu re 7 - SI nuc lease mapping of )'east T-DNA gene 7 RNA 5 ' t e r m i n i . SI nuc lease r e s i s t a n t hybr ids 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 igu re 4 and 2 ug each of RNA p repa ra t i ons from yeast (GM-3C-2) t ransformed w i th Y E p l 3 , Y - B G . E 9 , and Y-BGE.E9 ( lanes C, D, and E, r e s p e c t i v e l y ) . Lane A, no RNA; lane B, yeast RNA w i t h no T-DNA; lane F, RNA from E9 p lan t tumor RNA (2 u g ) ; lane G, 3 2 P - l a b e l l e d H p a l l fragments of pBR322. Pro tec ted DNA was e lec t rophoresed on 8% a c r y l a m i d e / urea g e l s . -32-b a s e s , i n d i c a t i n g t h a t the SI n u c l e a s e p r o c e d u r e c o r r e c t l y i d e n t i f i e s 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 of T-DNA gene 7 i n E9 p l a n t tumors. SI n u c l e a s e mapping o f RNAs i s o l a t e d from y e a s t t r a n s f o r m e d w i t h Y-BG.E9 and Y-BGE.E9 i s shown i n l a n e s D and E, r e s p e c t i v e l y . RNAs o f 216 bases and s h o r t e r were p r o t e c t e d by the DNA probe w i t h some l o n g e r minor RNAs b e i n g p r o t e c t e d as w e l l . These l o n g e r minor RNAs c o r r e s p o n d to i n i t i a t i o n s i t e s i d e n t i f i e d p r e v i o u s l y by primer e x t e n s i o n a n a l y s i s , and they a r e e x p r e s s e d at a much lower l e v e l than the s h o r t e r p r o t e c t e d RNAs (216 bases or l e s s ) . I f i t i s assumed t h a t one end o f the RNA c o r r e s p o n d s to the PvuII s i t e o f the DNA probe, then the o t h e r end o f the 216 base RNA would c o r r e s p o n d to t r a n s c r i p t i o n i n i t i a t i o n at a p o s i t i o n 20 n u c l e o t i d e s downstream from the normal i n i t i a t i o n s i t e i n p l a n t tumors or 49 n u c l e o t i d e s downstream from the TATA box ( F i g u r e 4 ) . The s h o r t e r p r o t e c t e d RNAs c o r r e s p o n d to i n i t i a t i o n s i t e s f a r t h e r downstream. N o r t h e r n H y b r i d i z a t i o n s N o r t h e r n h y b r i d i z a t i o n s were done t o determine i f f u l l l e n g t h t r a n s c r i p t s were b e i n g t r a n s c r i b e d from T-DNA gene 7 i n y e a s t . The r e s u l t s a r e shown i n F i g u r e 8. E9 p l a n t tumor p o l y A + s e l e c t e d RNA was h y b r i d i z e d t o the T-DNA gene 7 probe ( l a n e 4 ) . The gene 7 probe extends from the H i n d l l l s i t e t o the Smal s i t e (1,178 b a s e s ) of T-DNA gene 7 as shown i n F i g u r e 4. The probe h y b r i d i z e d to RNA a p p r o x i m a t e l y 700 bases l o n g , c o n f i r m i n g p r e v i o u s r e s u l t s o b t a i n e d by o t h e r ( G e l v i n e t a l . , 1982; W i l l m i t z e r e t a l . , 1982; McPherson, 1984). H y b r i d i z a t i o n s between the gene 7 probe and RNA i s o l a t e d from GM-3C-2 tr a n s f o r m e d w i t h Y-BG.E9 show a t r a n s c r i p t of 710 bases f o r both t o t a l RNA and p o l y A + s e l e c t e d RNA, lane s 1 and 2, r e s p e c t i v e l y . T h i s i n d i c a t e s t h a t a f u l l l e n g t h t r a n s c r i p t i s t r a n s c r i b e d from T-DNA gene 7 i n y e a s t . SI n u c l e a s e 33 -1 2 3 4 ure 8 - An autorad iograph 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 bp, see F igu re 4 ) . RNA from yeast t ransformed wi th Y-BG.E9 was examined. Lane 1, Y-BG.E9 t o t a l RNA (30 ug ) ; lane 2, Y-BG.E9 po ly A + s e l e c t e d RNA (5 ug ) ; lane 3 , 3 2 P - l a b e l l e d H p a l l fragments of pBR322; lane 4 , E9 p lan t tumor po ly A + s e l e c t e d RNA (5 ug ) . -34-mapping i d e n t i f i e d the i n i t i a t i o n s i t e s o f gene 7, but t e r m i n a t i o n s i t e s o f gene 7 i n y e a s t a r e d i f f i c u l t t o d e t e r m i n e . T r a n s c r i p t s i z e s from p l a n t s and y e a s t are d i f f i c u l t t o compare s i n c e the degree o f p o l y a d e n y l a t i o n i s d i f f e r e n t (50 or fewer A r e s i d u e s i n y e a s t compared w i t h 100-200 i n p l a n t s - Sagher ^ t a l . , 1974). B. Heat Shock - T-DNA Gene 7  Heat Shock The e f f e c t o f a r a p i d temperature shock on the e x p r e s s i o n o f T-DNA gene 7 i n crown g a l l tumors was i n v e s t i g a t e d . Crown g a l l tumors.were heat shocked a t 40°C f o r 1 hour, 2 h o u r s , 6 h o u r s , and 24 h o u r s . T r a n s c r i p t s were i d e n t i f i e d and q u a n t i f i e d by primer e x t e n s i o n a n a l y s i s as b e f o r e and the cDNA p r o d u c t s were e l e c t r o p h o r e s e d through a DNA s e q u e n c i n g g e l ( F i g . 9, l a n e s b - f ) . The r e s u l t s o b t a i n e d f o r t r a n s c r i p t 7 from tumor l i n e E9 b e f o r e heat shock ( F i g . 9, l a n e b) a r e i d e n t i c a l to the r e s u l t s o b t a i n e d p r e v i o u s l y . A f t e r 1 hour o f heat shock at 40°C, gene 7 RNA l e v e l s d e c r e a s e and c o n t i n u e to d e c r e a s e u n t i l they are no l o n g e r d e t e c t a b l e a f t e r 25 hours o f heat shock. These r e s u l t s i n d i c a t e t h a t heat shock o f crown g a l l tumors does not i n c r e a s e t r a n s c r i p t i o n o f T-DNA gene 7. In f a c t , heat shock o f tumors d e c r e a s e s t r a n s c r i p t i o n o f gene 7, as would be expe c t e d f o r a non-heat shock gene. Heavy M e t a l Treatment M e t a l s which are known to be t o x i c to p l a n t s at h i g h l e v e l s were t e s t e d f o r t h e i r i n f l u e n c e on the a c c u m u l a t i o n o f T-DNA gene 7 RNAs i n crown g a l l tumors and t r a n s f o r m e d tobacco c u l t u r e s . As seen i n F i g u r e 9 ( l a n e s g - n ) , t h e r e wasn't a s i g n i f i c a n t i n c r e a s e i n t r a n s c r i p t i o n of gene 7 u n t i l a f t e r 24 —4 hours o f treatment w i t h cadmium (10 M) at which time t h e r e was a t w o - f o l d -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 of a temperature shock and the e f f e c t o f trea t m e n t of cadmium c h l o r i d e on the e x p r e s s i o n o f T-DNA gene 7 i n crown g a l l tumors. C u l t u r e s were heat shocked at 40°C f o r 1 hour, 2 ho u r s , 6 h o u r s , and 1 day and RNA was i s o l a t e d as d e s c r i b e d i n methods. C u l t u r e s were a l s o grown on media c o n t a i n i n g cadmium c h l o r i d e ( 1 0 - 4 M ) f o r 1 hour, 2 h o u r s , 6 h o u r s , 1 day, 2 days and 20 days and 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 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 complementary to the 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 are shown i n the f i g u r e . Lane a, 3 2 P - l a b e l l e d Hpa I I fragments o f pBR322; la n e b, crown g a l l tumor l i n e E9 w i t h no tre a t m e n t ; lane c, E9 at 40°C f o r 1 hour; lane d, 40°C f o r 2 hou r s ; lane e, 40°C f o r 6 h o u r s ; l a n e f , 40°C f o r 1 day; lane g, 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 h, A6 t r e a t e d w i t h 10~ 4M CdCl2 f o r 1 hour; l a n e i , 1 0 - % CdCl2 f o r 2 hou r s ; l a n e j , 10~ 4M C d C l 2 f o r 6 hou r s ; lane k, 10~ 4M C d C l 2 f o r 1 day; lane 1, gamma 3 2 P - o l i g o n u c l e o t i d e T7; lane m, 10 - 4M C d C l 2 f o r 2 days; lane n, 10~ 4M C d C l 2 f o r 10 days; lane o, 3 2 P - l a b e l l e d Hpa I I fragments of pBR322. -36-i n c r e a s e i n t r a n s c r i p t i o n i n crown g a l l tumors. A f t e r 10 days o f tr e a t m e n t w i t h cadmium, gene 7 RNAs accumulated to 3 times the normal l e v e l . D i f f e r e n t -4 -7 c o n c e n t r a t i o n s o f cadmium r a n g i n g from 10 M to 10 M were a l s o t e s t e d to determine t h e i r e f f e c t s on gene 7 t r a n s c r i p t i o n ( F i g . 10, l a n e s c - g ) . The -4 -7 r e s u l t s i n d i c a t e t h a t c o n c e n t r a t i o n s o f 10 M to 10 M cadmium a l l i n c r e a s e gene 7 t r a n s c r i p t i o n t h r e e - f o l d a f t e r 10 days o f t r e a t m e n t . When crown g a l l tumors were t r e a t e d w i t h c o n c e n t r a t i o n s o f mercury r a n g i n g —4 —8 from 10 M to 10 M ( F i g . 10, l a n e s h - l ) , t h e r e was a t h r e e - f o l d i n c r e a s e i n gene 7 t r a n s c r i p t i o n f o r a l l c o n c e n t r a t i o n s . When tumors were t r e a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f z i n c ( F i g . 11), t h e r e was no e f f e c t on gene 7 t r a n s c r i p t i o n . The e f f e c t o f metals on gene 7 t r a n s c r i p t i o n i n the 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 16-12-C was a l s o i n v e s t i g a t e d ( F i g . 10, l a n e s m-o). The 16-12-C tobac c o c u l t u r e was t r a n s f o r m e d w i t h p l a s m i d DNA c o n t a i n i n g a C a u l i f l o w e r mosaic v i r u s enhancer element upstream o f T-DNA gene 7. The t r a n s c r i p t l e v e l o f T-DNA gene 7 i n the 16-12-C c u l t u r e i s f i v e times the normal l e v e l i n crown g a l l tumors. When the 16-12-C c u l t u r e s were t r e a t e d w i t h e i t h e r mercury or cadmium, t r a n s c r i p t i o n o f gene 7 i n c r e a s e d 1 0 0 - f o l d . The re a s o n f o r t h i s d r a m a t i c i n c r e a s e i n t r a n s c r i p t i o n i s unknown at t h i s time. A r s e n i t e Treatment Crown g a l l tumors were s u b j e c t e d to v a r i o u s c o n c e n t r a t i o n s o f sodium a r s e n i t e and gene 7 RNAs were q u a n t i f i e d . As shown i n F i g u r e 12, a r s e n i t e appears to have no e f f e c t on t r a n s c r i p t i o n o f T-DNA gene 7. -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 - The e f f e c t of heavy metals on the e x p r e s s i o n o f T-DNA gene 7 i n crown g a l l tumors and transformed 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 media c o n t a i n i n g cadmium c h l o r i d e ( 0 , 1 0 - ^ M to 1 0 - ^ M) and m e r c u r i c c h l o r i d e ( 0 , 10" 8M t o 10~^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 (10 ug) primed w i t h o l i g o n u c l e o t i d e T7 comple-mentary t o the 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 are shown i n the f i g u r e . The 3 2 . p - i 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 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 s . Lane a, g a m m a - 3 2 p - 0 l i g o n u c l e o t i d e T7; l a n e b, 3 2 P - l a b e l l e d m o l e c u l a r weight markers (Hpa I I fragments o f pBR322); l a n e c, crown g a l l tumor l i n e E9 t r e a t e d w i t h 10~^M CdCl2; l a n e d, w i t h 10 _ 5M C d C l 2 ; lane e, w i t h 10 _ 6M C d C l 2 ; l a n e f , w i t h 1 0 - 7M C d C l 2 ; l a n e g, E9 w i t h no tr e a t m e n t ; lane h, E9 t r e a t e d w i t h 10' - 4 M HgCl25 Lane i , w i t h 1 0 _ 5 M H g C l 2 ; lane j , w i t h 10~ 6M H g C l 2 ; la ne k, w i t h 10" 7M H g C l 2 ; l a n e 1, 10 - 8 M H g C l 2 ; lane m, transformed tobacco 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 1 0 _ 4 M H g C l 2 ; lane n, 16-12-C t r e a t e d w i t h 10" 4M C d C l 2 ; l an e o, 16-12-C w i t h no tre a t m e n t ; lane p, 3 2 p _ ] _ a t j e ^ e c j H p a J - J - f r a g m e n t s o f pBR322. -38-1 2 3 4 5 6 7 8 67 m * F i g u r e 11 - The e f f e c t o f treatment o f z i n c c h l o r i d e on the e x p r e s s i o n o f T-DNA gene 7 i n crown g a l l tumors. C u l t u r e s were grown on media c o n t a i n i n g z i n c c h l o r i d e (0, 10" 7M to 10~ 4M) 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. Revere 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 comple-mentary to the 5' co 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 are shown i n the f i g u r e . Lane 1, 3 2 P - l a b e l l e d Hpa II fragments of pBR322; l a n e 2, crown g a l l tumor l i n e A6 w i t h no tr e a t m e n t ; lane 3, A6 t r e a t e d wi th 1 0 _ 4 M Z n C l 2 ; l a n e 4, w i t h 10~ 5M Z n C l 2 ; lane 5, w i t h 10" 6M Z n C l 2 ; lane 6, w i t h 10~ 7M Z n C l 2 ; lane 7, A6 w i t h no tr e a t m e n t ; lane 8, E9 w i t h no t r e a t m e n t . - 3 9 -1 2 3 4 5 F igu re 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 tumors. Cu l tu res 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~ 7M to 10 _ 4 M) fo r 10 days a f t e r which t ime RNA was i s o l a t e d as desc r ibed in methods. Reverse t r a n s c r i p t a s e e x t e n s i o n products from crown g a l l tumor RNA (20 ug) primed w i th o l i g o n u c l e o t i d e T7 comple-mentary to the 5 ' coding reg ion of T-DNA t r a n s c r i p t 7 are shown i n the f i g u r e . Lane 1, crown g a l l tumor l i n e A6 w i th no t rea tment ; lane 2, A6 t r ea ted w i th 1 0 _ 4 M a r s e n i t e ; lane 3 , w i th 10~->M a r s e n i t e ; lane 4 , w i th 10~^M a r s e n i t e ; lane 5 , w i th 1 0 - 7 M a r s e n i t e . -40-DISCUSSION A. T-DNA Gene 7 i n Yeast Primer e x t e n s i o n a n a l y s i s , SI n u c l e a s e mapping, and N o r t h e r n h y b r i d i z a t i o n s i n d i c a t e t h a t t r a n s c r i p t i o n of T-DNA gene 7 i n y e a s t i s d i f f e r e n t from t h a t 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 tumors. SI n u c l e a s e mapping and pri m e r e x t e n s i o n a n a l y s i s o f gene 7 i n y e a s t show t h a t t h e r e a r e m u l t i p l 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 . The major 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 o f gene 7 i n ye a s t b e g i n 20 n u c l e o t i d e s downstream from the normal i n i t i a t i o n s i t e i n crown g a l l tumors, or 49 n u c l e o t i d e s downstream from the TATA box. In y e a s t the s t r u c t u r e of the promoter i s s i m i l a r to t h a t o f h i g h e r e u k a r y o t e s . TATA sequences have been observed i n a l l y e a s t genes s t u d i e d (Sentenac and H a l l , 1982). However, i n c o n t r a s t to h i g h e r e u k a r y o t i c systems, the d i s t a n c e between a TATA sequence and the c o r r e s p o n d i n g s e t o f 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 i s v a r i a b l e , r a n g i n g from 40 to 150 bp (Sentenac and H a l l , 1982). In most e u k a r y o t i c c e l l s the TATA box has a s e p a r a t i o n from the t r a n s -c r i p t i o n s t a r t p o i n t t h a t v a r i e s from 25 t o 30 bp (Bre a t h n a c h and Chambon, 1981). I t has been shown t h a t f o r the y e a s t i s o - l - c y t o c h r o m e c gene t h e r e i s a l o o s e s p a t i a l r e l a t i o n s h i p between the TATA sequences and the mRNA s t a r t s i t e s ( M c N e i l and Smith, 1986). T h i s d i s t a n c e r e l a t i o n s h i p v a r i e s from 100 to 60 b a s e - p a i r s (+ 15 b a s e - p a i r s ) . There a l s o e x i s t s f o u r and p o s s i b l y f i v e TATA sequences l o c a t e d w i t h i n the 5' non-coding r e g i o n o f the i s o - l - c y t o c h r o m e c gene (M c N e i l and Smith, 1986). Each TATA sequence i s thought to be r e q u i r e d f o r a s p e c i f i c s u b s e t of mRNA s t a r t s . M c N e i l and Smith suggest t h a t the s p a c i n g between T-A-T-A and s t a r t s i s not a s i n g l e f i x e d d i s t a n c e and i t i s ve r y l i k e l y t h a t i n t e r v e n i n g sequences determine the s p a c i n g , e i t h e r d i r e c t l y -41-or through a series of s p e c i f i c spacing f a c t o r s . In contrast, Chen and Struhl suggest that the TATA box in yeast does not select i n i t i a t i o n s i t e s by measuring a fixed distance from i t (Chen and St r u h l , 1986). Instead, an i n i t i a t o r element close to the i n i t i a t i o n s i t e i t s e l f contains the sig n a l for determining where t r a n s c r i p t i o n begins. Photoprinting studies i n d i c a t e , however, that the transcription-dependent a l t e r a t i o n i n l i g h t s e n s i t i v i t y at the hexanucleotide 5' ATATAA31 marks a functional TATA sequence i n yeast (Selleck and Majors, 1987). When the hexanucleotide i s deleted from the yeast GAL 1 gene, expression decreases 30-fold under inducing conditions (West ejt a l . , 1984). These studies indicate the functional importance of the TATA box in yeast. The TATA box appears to be d i r e c t l y involved i n the s e l e c t i o n of 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 in yeast, although the exact function of the TATA box is unknown. A TATA box i n the 5' region of 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 nuclease mapping shows that the mRNA s t a r t s i t e s of gene 7 i n yeast begin 49 nucleotides downstream from the TATA box. This confirms previous r e s u l t s which indicate that a TATA box must be at least 40 nucleotides upstream of 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 for t r a n s c r i p t i o n in yeast (Chen and Stru h l , 1986). These facts suggest that when foreign DNA i s to be expressed in yeast, the distance between the i n i t i a t i o n s i t e and the TATA box in the foreign gene is c r i t i c a l . I f the TATA box is less than 40 bp upstream from the ATG t r a n s l a t i o n i n i t i a t i o n codon, then i t is highly u n l i k e l y that a complete, functional protein w i l l be tra n s l a t e d . The major 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 T-DNA gene 7 in yeast begin after the ATG t r a n s l a t i o n i n i t i a t i o n codon which means that the trans-l a t i o n reaction is prevented. -42-The p o s s i b l e r e a s o n f o r the abnormal e x p r e s s i o n o f f o r e i g n genes i n y e a s t i n p r e v i o u s s t u d i e s may have been due to the f a c t t h a t the d i s t a n c e r e q u i r e m e n t of the TATA box i n y e a s t i s d i f f e r e n t than i n o t h e r e u k a r y o t e s . When the r a b b i t B - g l o b i n gene was c l o n e d i n y e a s t , the B - g l o b i n t r a n s c r i p t s from y e a s t were about 20-40 n u c l e o t i d e s s h o r t e r at the 5' end than normal g l o b i n mRNA (Beggs ej: a l _ . , 1980). I t was suggested t h a t the abnormal 5' t e r m i n i o f the B-g l o b i n t r a n s c r i p t s r e p r e s e n t e d e i t h e r 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 r e c o g n i z e d by a y e a s t RNA polymerase or d e g r a d a t i o n o f l o n g e r t r a n s c r i p t s o r i g i n a t i n g at the promoter s i t e or even f u r t h e r upstream. The TATA box i n the B - g l o b i n gene maps 28 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 i n i t i a t i o n s i t e . The major i n i t i a t i o n s i t e s o f the B - g l o b i n gene i n y e a s t are 46 and 65 n u c l e o t i d e s downstream from the TATA box. F o r t u n a t e l y , the ATG i n i t i a t i o n codon was s t i l l downstream from 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 s , so normal t r a n s l a t i o n was s t i l l a p o s s i b i l i t y . In t h i s c a s e , however, the gene was not s p l i c e d p r o p e r l y and i t was c o n c l u d e d t h a t the y e a s t system was inadequate f o r the stud y o f t r a n s c r i p t i o n and p r o c e s s i n g o f the B - g l o b i n gene. In most e u k a r y o t e s , t r a n s c r i p t i o n i n i t i a t i o n i s c o n f i n e d t o a s h o r t r e g i o n . In y e a s t , however, t h e r e can be many s i t e s a t which t r a n s c r i p t s a r e i n i t i a t e d (Faye e t a l . , 1981; Hsu and Schimmel, 1984; Joh n s t o n and D a v i s , 1984; Z a l k i n e_t a_l. , 1984). SI n u c l e a s e mapping o f the mRNA 5' t e r m i n i f o r the TRP2 and TRP3 y e a s t genes showed t h a t t h e r e were m u l t i p l e c l u s t e r s o f 5' ends f o r b o t h TRP2 and TRP3 mRNA ( Z a l k i n e t a l . , 1984). I t i s not s u r p r i s i n g t h e n t h a t t h e r e are m u l t i p l 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 when gene 7 i s c l o n e d i n y e a s t . In a r e c e n t paper ( H i r s c h and Beggs, 1984), i t was r e p o r t e d t h a t T-DNA had been c l o n e d i n y e a s t . N o r t h e r n h y b r i d i z a t i o n s were done to i d e n t i f y T-DNA t r a n s c r i p t s w i t h v a r i o u s probes, but no RNA s i m i l a r i n s i z e to the 0.7 kb mRNA -43-was i d e n t i f i e d . As for other T-DNA t r a n s c r i p t s , the pattern of t r a n s c r i p t i o n was complex. RNAs i d e n t i f i e d by Northern hybridizations appeared to be c h a r a c t e r i s t i c a l l y larger than expected RNA lengths. Results i n th i s i n v e s t i -gation indicate that a f u l l length t r a n s c r i p t i s transcribed from gene 7 i n yeast, although t r a n s c r i p t i o n i n i t i a t i o n and termination are d i f f e r e n t than i n crown g a l l tumors. The gene 7 t r a n s c r i p t was most l i k e l y detected i n t h i s i n v e s t i g a t i o n because a s p e c i f i c probe for gene 7 was used. In conclusion, 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 than i n crown g a l l tumors. S p e c i f i c a l l y , t r a n s c r i p t i o n i n i t i a t i o n i s d i f f e r e n t because the distance 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 s i t e s must be at least 40 nucleotides in yeast. Although yeast is an excellent system for i n v e s t i g a t i n g the expression of some cloned eukaryotic genes, i t does not appear to be a useful system for i n v e s t i g a t i n g the expression of T-DNA. B. Heat Shock - T-DNA Gene 7 By d e f i n i t i o n , HS proteins are a new set of proteins r a p i d l y and abundantly produced i n response to heat shock. The appearance of heat shock proteins is dependent on t r a n s c r i p t i o n a l a c t i v a t i o n of the heat shock genes. This induc-t i o n i s immediate and increases the concentration of hsp mRNA from undetectable (less than one molecule per c e l l ) to a thousand molecules per c e l l within an hour of heat shock (Schlesinger e_t al_. , 1982). Eukaryotic HSP genes have one or more copies of a 14-base-pair (bp) sequence, referred to as a heat shock promoter element (HSE), upstream from 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 (Bienz, 1985; Craig, 1985; Pelham, 1985). A heat shock sequence s i m i l a r to the HSE was i d e n t i f i e d in the promoter region of T-DNA gene 7. Although untreated crown g a l l tissue contains s i g n i f i c a n t l e v e l s of gene 7 RNAs, there is at least one case where a plant heat shock gene i s expressed at -44-s i g n i f i c a n t l e v e l s p r i o r to heat shock ( C z a r n e c k a ejt a_l. , 1984). When c l o n e d cDNAs to heat shock ( h s ) - i n d u c e d mRNAs were used to a s s e s s whether v a r i o u s s t r e s s e s i n d u c e d the a c c u m u l a t i o n o f p o l y ( A ) RNAs i n soybean s e e d l i n g s , one s e t of p o l y (A) RNAs which were p r e s e n t at v a r i a b l e l e v e l s i n c o n t r o l ( n o n-s t r e s s e d t i s s u e ) t i s s u e were i n c r e a s e d some 5- to 1 0 - f o l d by heat shock and o t h e r s t r e s s agents such as cadmium and a r s e n i t e ( C z a r n e c k a et_ al_. , 1984). A summary o f the i n f l u e n c e o f a number o f s t r e s s a g e n t s , i n c l u d i n g heat shock, on T-DNA gene 7 RNA l e v e l s based on primer e x t e n s i o n a n a l y s i s i s p r e -s e n t e d i n T a b l e 2. The d a t a p r e s e n t e d here demonstrate t h a t o n l y cadmium and 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 of T-DNA gene 7. Cadmium and mercury i n c r e a s e the e x p r e s s i o n o f gene 7 3 - f o l d a f t e r 10 days o f t r e a t -ment. A r s e n i t e , z i n c , and heat shock do not induce the a c c u m u l a t i o n o f gene 7 RNAs to l e v e l s above t h a t of normal l e v e l s i n crown g a l l tumors. In f a c t , when crown g a l l tumors were s u b j e c t e d to a temperature of 40°C, gene 7 RNA l e v e l s d e c r e a s e d u n t i l a f t e r one day when they were no l o n g e r d e t e c t a b l e . In soybean s e e d l i n g s the c o n c e n t r a t i o n s of many normal p o l y ( A ) RNAs d e c l i n e s a f t e r 2 hours o f heat shock (Kimpel and Key, 1985). F u r t h e r m o r e , when t r a n s -formed c a l l u s t i s s u e i s exposed to h i g h t e m p e r a t u r e s , n o p a l i n e s y n t h a s e (a non-heat shock gene) t r a n s c r i p t s d e c r e a s e to a v e r y low l e v e l i m m e d i a t e l y a f t e r heat shock (Spena et_ a l . , 1985). These f a c t s suggest t h a t T-DNA gene 7 i s not a heat shock gene. Due to the f a c t t h a t T-DNA gene 7 responded o n l y to cadmium and mercury, the p o s s i b i l i t y o f gene 7 b e i n g a m e t a l l o t h i o n e i n gene was i n v e s t i g a t e d . M e t a l l o t h i o n e i n s (MTs) are a f a m i l y o f low m o l e c u l a r weight, c y s t e i n e - r i c h p r o t e i n s o f about 6,000 to 7,000 m o l e c u l a r weight which are w i d e l y d i s t r i b u t e d i n n a t u r e ( K a g i e_t a_l. , 1984). They a r e e x p r e s s e d i n many d i f f e r e n t t i s s u e s and c e l l types when these are exposed to heavy metals such as cadmium, z i n c , -45-Treatment No Treatment 1 hour (25°C) 2h 6h 1 day 2d lOd Heat shock 40°C(E9) IX .75X .5X .25X OX — — C d C l 2 10 _ 4M(A6) IX IX IX IX 2X 2X 3X C d C l 2 10 - 4M(E9) IX 3X HgCl 2 10 _ 4M(E9) IX 3X Z n c l 2 10"4M(A6) IX IX Arsenite 10"4M(A6) IX IX C d C l 2 10 - 4M(16-12-C) 5X 100X HgC12 1 0" 4M(16-12-C) 5X 100X Table 2 Summary of the stress induced accumulation of T--DNA gene 7 RNAs in 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 = estimated r e l a t i v e l e v e l from primer extension analyses as determined by a densitometer; — = not analyzed. -46-copper and mercury ( K a g i and Nordberg, 1979). The i n d u c t i o n o f these p r o t e i n s i s r e g u l a t e d m a i n l y at the l e v e l of t r a n s c r i p t i o n (Durnam and P a l m i t e r , 1981; K a r i n e_t a_l_. , 1984; Mayo ejt a l _ . , 1982). A l t h o u g h the gene 7 p r o t e i n i s a low m o l e c u l a r weight p r o t e i n (14Kd), t h i s p r o t e i n i s not c y s t e i n e r i c h ( l e s s than 2% o f the amino a c i d c o n t e n t are c y s t e i n e s ) . In a d d i t i o n , a computer a n a l y s i s was done to determi n e i f t h e r e were any sequences s i m i l a r to the consensus sequence r e s p o n s i b l e f o r metal r e g u l a t i o n ( 5 1 - TGCGCCCGCTTC-3') ( C a r t e r et a l . , 1984). No sequence s i m i l a r to the m e t a l consensus sequence was i d e n t i f i e d i n the 5' r e g i o n o f gene 7, These f a c t s suggest t h a t T-DNA gene 7 i s not a m e t a l l o t h i o n e i n gene. In an attempt to i d e n t i f y DNA sequences o f f u n c t i o n a l importance i n the 5' r e g i o n of T-DNA gene 7, the t r a n s f o r m e d tobacco c u l t u r e l i n e , 16-12-C, was t r e a t e d w i t h mercury and cadmium. The 5' f l a n k i n g r e g i o n o f T-DNA gene 7 i s d e l e t e d upstream o f the H i n d l l l s i t e ( F i g . 4) i n t h i s tobacco c u l t u r e l i n e . I t s h o u l d be noted t h a t t h e r e i s a l s o a C a u l i f l o w e r mosaic v i r u s enhancer element upstream o f T-DNA gene 7 i n t h i s c u l t u r e l i n e . The normal t r a n s c r i p t l e v e l o f T-DNA gene 7 i n the 16-12-C c u l t u r e i s f i v e times the normal l e v e l o f t h a t i n crown g a l l tumors. When the 16-12-C c u l t u r e s were t r e a t e d w i t h e i t h e r mercury or cadmium, t r a n s c r i p t i o n o f gene 7 i n c r e a s e d 1 0 0 - f o l d . The r e a s o n f o r t h i s d r a m a t i c i n c r e a s e i s unknown at t h i s time, but prima f a c i e i t appears t h a t the me t a l s are d i r e c t l y i n f l u e n c i n g the C a u l i f l o w e r mosaic v i r u s enhancer element to i n c r e a s e t r a n s c r i p t i o n o f gene 7. In c o n c l u s i o n , i t does not appear t h a t T-DNA gene 7 i s a heat shock gene. A l t h o u g h gene 7 responds to cadmium and mercury, the i n c r e a s e i n t r a n s c r i p t i o n does not appear to be heat shock or m e t a l l o t h i o n e i n r e l a t e d . As f o r the importance o f DNA sequences i n the 5' r e g i o n o f gene 7, the d e l e t i o n o f the 5' r e g i o n o f gene 7 i s c o m p l i c a t e d by the presence of a C a u l i f l o w e r mosaic -47-enhancer element. 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