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

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C H A R A C T E R I Z A T I O N OF RECOMBINANT P L A S M I D S C A R R Y I N G i D R O S P H I L A MELANOGASTER.tRNA. S E R I N E 7 GENES AND T H E I R P R E P A R A T I O N FOR DNA S E Q U E N C I N G by Mark Gregory Spurr B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1977 A T h e s i s Submitted i n P a r t i a l F u l f i l l m e n t of The Requirements For The Degree of Master of Science i n The F a c u l t y of Graduate S t u d i e s ( M i c r o b i o l o g y , U n i v e r s i t y of B r i t i s h Columbia) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard The U n i v e r s i t y of B r i t i s h Columbia Nov. 1979 © MARK GREGORY SPURR 1979 In presenting th i s thesis in par t i a l fu l f i lment of the requirements for an advanced degree at the Univers ity of B r i t i s h Columbia, I agree that the Library shal l make i t f ree ly avai lable for reference and study. I further agree that permission for extensive copying of th i s thesis for scholar ly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publ icat ion of th i s thesis fo r f i nanc ia l gain sha l l not be allowed without my written permission. Department The Univers ity of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 B P 75-51 1 E ABSTRACT Ser S p e c i f i c D r o s p h i l a tRNA 4,7 plasmids were i d e n t i f i e d and 1 2 5 S e r i s o l a t e d by h y b r i d i z a t i o n w i t h p u r i f i e d [ I] tRNA 4,7 mole-c u l e s . Seven c l o n e s were i s o l a t e d c a r r y i n g the D r o s p h i l a Ser tRNA 4,7 gene and were f u r t h e r c h a r a c t e r i z e d by r e s t r i c t i o n endonuclease d i g e s t i o n ; agarose g e l e l e c t r o p h o r e s i s and h y b r i -1 2 5 S e r d i z a t i o n with i n d i v i d u a l p u r i f i e d [ I] tRNA 4,7 molecules. The r e s u l t s show t h a t f i v e d i f f e r e n t DNA fragments have been i s o l a t e d , f o u r which code f o r a s i n g l e , s p e c i f i c i s o a c c e p t o r , and one which appears to code f o r two d i f f e r e n t i s o a c c e p t o r s . Two plasmids which i n i t i a l l y c o n t a i n e d m u l t i p l e Hind I I I i n -s e r t s upon primary i s o l a t i o n were r e c l o n e d to c o n t a i n s i n g l e Ser Hind I I I i n s e r t s c o n t a i n i n g the tRNA 4,7 gene. One of these Ser r e c l o n e d plasmids c o n t a i n e d a s m a l l e r tRNA 4,7 gene c a r r y i n g i n s e r t than d i d i t s o r i g i n a l m u l t i p l e i n s e r t i s o l a t e . Small Ser tRNA 4,7 gene c a r r y i n g r e s t r i c t i o n fragments were l a b e l l e d 3 2 with T4 p o l y n u c l e o t i d e k i n a s e and [ p] ATP, s t r a n d separated, and e l e c t r o e l u t e d , i n p r e p a r a t i o n f o r n u c l e o t i d e sequencing. i i i T A B L E O F C O N T E N T S PAGE ABSTRACT i i LIST OF TABLES. V LIST OF FIGURES AND ILLUSTRATIONS v i ACKNOWLEDGEMENTS v i i i INTRODUCTION 1 MATERIALS AND METHODS 3 B a c t e r i a l S t r a i n s 3 M e d i a S e r 3 I s o l a t i o n o f S i n g l e S p e c i f i c pDt 4,7 Clones 3 H y b r i d i z a t i o n of DNA from B a c t e r i a C o l o n i e s w i t h [ 1 2 5 I ] tRNA 4^7 4 J P r e p a r a t i o n of pDt - S e r i n e plasmid DNA 7 R e s t r i c t i o n Endonuclease Cleavage C o n d i t i o n s 8 Agarose G e l E l e c t r o p h o r e s i s 9 Mo l e c u l a r Weight A n a l y s i s of DNA fragments 9 Recloning of M u l t i p l e I n s e r t Plasmids 10 E l e c t r o e l u t i o n of DNA from Agarose G e l s . . . . ..13 Dephosphorylation of pDtl6 fragments S er C o n t a i n i n g tRNA 7 Genes 14 L a b e l i n g DNA 5' t e r m i n i With P o l y n u c l e o t i d e Kinase and tf[32P] ATP 14 Strand S e p a r a t i o n Gels 15 Containment 16 RESULTS AND DISCUSSION 17 P r i n c i p l e of Procedure 17 C h a r a c t e r i z a t i o n and Mo l e c u l a r Weight c a l c u l a t i o n of the D r o s p p h i l a I n s e r t 17 Ser C h a r a c t e r i z a t i o n o f tRNA 7 plasmid C a r r y i n g S i m i l a r s i z e d Hind I I I i n s e r t s 19 i v PAGE Recloning of pDtl7 and pDt27 31 P r e p a r a t i o n of pDtl6 f o r Sequencing 45 K i n e t i c s of the P o l y n u c l e o t i d e Kinase R e a c t i o n 48 3 2 Strand S e p a r a t i o n of X [ P] ATP L a b e l l e d pDtl6 Fragments 51 REFERENCES 54 V LIST OF TABLES PAGE Table I R e s t r i c t i o n Endonuclease Hind I I I fragments of ! Recombinant plasmids 18 Table I I R e s t r i c t i o n Endonuclease fragments of Recombinant Plasmids C o n t a i n i n g S i m i l a r s i z e Hind I I I fragments..28 v i LIST OF FIGURES AND ILLUSTRATIONS PAGE F i g u r e 1: S i n g l e colony i s o l a t i o n of b a c t e r i a Ser c o n t a i n i n g tRNA 4,7 genes 5 F i g u r e 2: S i z e c o n f i r m a t i o n o f pBR322 and lambda DNA....11 F i g u r e 3: Agarose g e l a n a l y s i s of Hind I I I d i g e s t e d plasmids 20 F i g u r e 4: H y b r i d i z a t i o n o f Hind I I I d i g e s t e d plasmids 125 S e r w i t h [ . I ] tRNA 4,7 22 F i g u r e 5: Agarose g e l a n a l y s i s of Hind I I I d i g e s t e d pDt27 24 F i g u r e 6: H y b r i d i z a t i o n of Hind I I I d i g e s t e d pDt27 i?5 S e r 125 l y s w i t h [ I ] tRNA 7 and [ I] RNA 5 26 F i g u r e 7: Agarose g e l a n a l y s i s of EcoRI and Mbol d i g e s t e d pDt 1,5,81 ...29 F i g u r e 8: Agarose g e l a n a l y s i s of P s t I and P s t I + Hind I I I d i g e s t e d pDt, 1,5,81 32 125 S e r F i g u r e 9: H y b r i d i z a t i o n of [- i ] tRNA 7 wit h P s t I and P s t I + Hind I I I d i g e s t e d pDt 1,5,81 34 F i g u r e 10: Skematic r e p r e s e n t a t i o n o f the r e v e r s e o r i e n t a t i o n of p D t l 36 F i g u r e 11: Agarose g e l a n a l y s i s of Hae I I I and Hae I I I + Hind I I I d i g e s t e d pDt 1,5,81 37 v i i PAGE 125 S e r F i g u r e 12: H y b r i d i z a t i o n of [ i ] tRNA 7 wit h Hae I I I and Hae I I I + Hind I I I d i g e s t e d pDt 1, 5,81 39 F i g u r e 13: Agarose g e l a n a l y s i s of Hae I I I , Hind I I I and Hae I I I + Hind I I I d i g e s t e d pDtl6 and pDt27 41 Ser 12 5 F i g u r e 14: H y b r i d i z a t i o n of [ I ] tRNA 7 wit h Hae I I I , Hind I I I and Hae I I I + Hind I I I d i g e s t e d pDtl6 and pDt27 43 F i g u r e 15: Fragment p r e p a r a t i o n g e l f o r pDtl6 46 F i g u r e 16: K i n e t i c s of the p o l y n u c l e o t i d e k i n a s e r e a c t i o n 4 9 3 2 F i g u r e 17: Po l y a c r y l a m i d e s t r a n d s e p a r a t i o n of # [ P] ATP l a b e l l e d pDtl6 fragments 52 v i i i ACKNOWLEDGEMENTS I would l i k e t o thank the l a b of Dr. G. Tener, and Dr. David Leung o f Dr. M. Smith's l a b f o r t h e i r c o n t r i b u t i o n of ideas and a s s i s t a n c e to t h i s p r o j e c t . A s p e c i a l thanks goes to Dr. R.C. M i l l e r f o r h i s e x c e l -l e n t guidance, m o t i v a t i o n , and a s s i s t a n c e , and t o D.M. T a y l o r f o r her t e c h n i c a l a s s i s t a n c e . X INTRODUCTION S e v e r a l f e a t u r e s of D r o s o p h i l a tRNA genes e s t a b l i s h e d a *-r a t i o n a l e f o r i s o l a t i n g recombinant DNA molecules c o n t a i n i n g these genes. F i r s t , they are e s s e n t i a l f o r normal growth and development of the f l y . (Atwood 1968), Second, evidence i n d i c a t e s t h a t e x p r e s s i o n of these genes i s d i f f e r e n t i a l l y r e g u l a t e d d u r i n g development of w i l d type f l i e s . (White e t a l , 1973), and t h i r d , s e v e r a l r e p o r t s have p o s t u l a t e d t h a t a t l e a s t i n p r o k a r y o t e s some tRNA molecules may a c t d i r e c t l y as regu-l a t o r s of b i o c h e m i c a l processes (Ames and Hartman, 1963; Stent 1964; Sueoka and Kano Sueoko, 1964). Recent s t u d i e s have c l a r i f i e d the l o c a t i o n , number and o r g a n i z a t i o n of the tRNA genes i n D r o s o p h i l a . I t has been r e p o r t e d t h a t a t l e a s t 90 d i f f e r e n t tRNA s p e c i e s are produced i n D r o s o p h i l a (White e t a l , 1973). The l o c i of these tRNA molecules appear to be o r g a n i z e d i n multigene c l u s t e r s . Any one p a r t i c u l a r tRNA molecule may be encoded by s e v e r a l gene c l u s t e r s , s c a t t e r e d on the D r o s o p h i l a map. (Tener e t a l , 1979; Dunn e t a l , 1979; Schmidt e t a l , 1978; K u b l i and Schmidt, 1978; Ser E l d e r , 1978) tRNA 7 genes have been i s o l a t e d on recombinant plasmid DNA molecules i n order t o study t h e i r o r g a n i z a t i o n , and homology by R e s t r i c t i o n endonuclease a n a l y s i s , and nu c l e o -t i d e sequence a n a l y s i s . Ser S i n g l e s p e c i f i c tRNA 7 plasmids were i s o l a t e d d u r i n g 2 c l o n i n g e x p e r i m e n t s r e p o r t e d by Dunn e t a l , (1979). The S e r i s o l a t e d tRNA 7 p l a s m i d s were i d e n t i f i e d by h y b r i d i z a t i o n 125 S e r w i t h s p e c i f i c , p u r i f i e d [ I] tRNA 7 m o l e c u l e s a c c o r d i n g t o t h e p r o c e d u r e o f G r u n s t e i n and Hogness (1975). I n d i v i d u a l p l a s m i d s were c h a r a c t e r i z e d by R. e n d o n u c l e a s e c l e a v a g e , a g a -12 5 r o s e g e l e l e c t r o p h o r e s i s and h y b r i d i z a t i o n w i t h p u r i f i e d [ I] S e r tRNA 7 a c c o r d i n g t o S o u t h e r n (1975). S u b s e q u e n t l y t h e p l a s -3 2 mids were l a b e l l e d w i t h T.4 p o l y n u c l e o t i d e k i n a s e and # [ P i ATP i n p r e p a r a t i o n f o r n u c l e o t i d e s e q u e n c i n g . 3 MATERIALS AND METHODS B a c t e r i a l S t r a i n s E. c o l i SF-8 was obt a i n e d from M. Olson (Olson e t a l . , 1977). The plasmid pBR322 ( B o l i v a r e t a l . , 1977) was pr o v i d e d by H. Boyer. B a c t e r i a c o n t a i n i n g pBR32 2 were grown i n L u r i a broth,and plasmid DNA was obt a i n e d from these b a c t e r i a . E. c o l i SF-8 was transformed w i t h t h i s pBR322 DNA and r e s u l t i n g c o l o n i e s were screened f o r r e s i s t a n c e t o a m p i c i l l i n and t e t r a -c y c l i n e . One colony r e s i s t a n t t o both amp. and t e t . was i s o l -ated and used h e n c e f o r t h as the source of pBR322 DNA. Media B a c t e r i a c o n t a i n i n g plasmids were grown i n L u r i a b r o t h / (lOgm t r y p t o n e , 5gm ye a s t e x t r a c t , 5gm NaCl, per l i t r e pH7.2). P l a t e s were made of L u r i a b r o t h p l u s l O g m / l i t r e , agar. S er I s o l a t i o n of S i n g l e S p e c i f i c pDt 4,7 Clones The S e r i n e plasmids were numbered s e q u e n t i a l l y as they were i s o l a t e d from i n i t i a l c l o n i n g experiments, and were i d e n -t i f i e d as plasmid D r o s o p h i l a tRNA N, pDt N (Dunn e t a l . ) . B a c t e r i a c o n t a i n i n g plasmids c a r r y i n g D r o s o p h i l a tRNA genes were l o c a t e d on r e p l i c a p l a t e s and str e a k e d f o r s i n g l e c o l o n i e s on n u t r i e n t agar i n the absence of any a n t i b i o t i c . 4 Ten c o l o n i e s were p i c k e d from each s t r e a k , and the c o l o n i e s grown up on n i t r o c e l l u l o s e f i l t e r s on n u t r i e n t agar. The c e l l s on the n i t r o c e l l u l o s e were l y s e d by the method d e s c r i b e d by Gr u n s t e i n and Hogness (197 6), and the l i b e r a t e d DNA was anneal-125 S e r ed w i t h [ I] tRNA 4,7 as d e s c r i b e d below. In t h i s way pure c l o n e s of c e l l s were i s o l a t e d which c o n t a i n e d plasmids c a r r y -Ser i n g D r o s o p h i l a tRNA4,7 genes. Any contaminating non-transform-ed c e l l s or c e l l s c o n t a i n i n g d i f f e r e n t D r o s o p h i l a tRNA were e l i m i n a t e d by s t r e a k i n g f o r s i n g l e c o l o n i e s on non s e l e c t i v e Ser media f o l l o w e d by h y b r i d i z a t i o n w i t h l a b e l l e d tRNA4,7. F i g u r e 1 i s a photograph of an autoradiogram of a t y p i c a l h y b r i d i z a -t i o n procedure a t t h i s stage. One f e a t u r e of the f i g u r e should be noted. Not a l l the c o l o n i e s p i c k e d c o n t a i n a plasmid a t t h i s stage of the procedure, c o n f i r m i n g the need f o r s t r e a k i n g on non s e l e c t i v e media. Ser 125 H y b r i d i z a t i o n of DNA from B a c t e r i a C o l o n i e s w i t h [ I] tRNA4,7 N i t r o c e l l u l o s e f i l t e r s prepared as above, were t r e a t e d as d e s c r i b e d by G r u n s t e i n and Hogness (1976). The c e l l s were l y s e d , and the DNA was denatured w i t h 0.5 M NaOH f o r 7-10 min. The f i l t e r s were removed, washed wi t h two changes of 1M T r i s pH7.4., and incubated i n 1.5M NaCl, 0.5M T r i s pH7.4 f o r 5 min. A p a r t i a l vacum was a p p l i e d below the f i l t e r s . They were i n -cubated i n pronase (lmg/ml, 15 min, 22°C, 2xSSC . (0.3M NaCl, 0.03M sodium c i t r a t e ) ) , washed w i t h e t h a n o l , c h l o r o f o r m and 0.3 M NaCl, and baked f o r 3-4 hours a t 80°C, between 3mm paper. 5 FIGURE 1: Single colony i s o l a t i o n of bacteria containing SER tRNA 4, 7 genes. 6 FIGURE 1: C o l o n i e s were stre a k e d on n u t r i e n t agar i n the absence of a n t i b i o t i c . I n d i v i d u a l c o l o n i e s were t r a n s f e r r e d to secon-dary master p l a t e s , r e p l i c a t e d t o n i t r o c e l l u l o s e f i l t e r s and t r e a t e d as d e s c r i b e d (methods). In t h i s photograph c o l o n i e s 125 S e r which d i d not anneal with [ I] tRNA 4,7 show no shadow, i n d i c a t i n g the presence of b a c t e r i a which d i d not c o n t a i n Ser tRNA 4,7 genes. P o s i t i v e c o l o n i e s were p i c k e d f o r subsequent a n a l y s i s . 7 The baked f i l t e r s were s a t u r a t e d w i t h 2xSSC c o n t a i n i n g approx-5 6 125 S e r i m a t e l y 5 x 10 - 1 x 10 cpm [ l]tRNA4,7. The f i l t e r s were p l a c e d between mylar sheets and g l a s s p l a t e s and double wrap-ped i n p o l y v i n y l i d e n e (Saran Wrap). The o p t i m a l i n c u b a t i o n time was s i x and one h a l f hours a t 6 5°C. A f t e r i n c u b a t i o n the f i l t e r s were washed i n 2XSSC th r e e times, incubated f o r 30.min. a t 37°C i n 10 mg/ml RNase A i n 2XSSC, and washed two more times i n 2XSSC, 0.1% SDS. The f i l t e r s were a i r d r i e d and autoradiographed f o r 48 hours u s i n g p r e f l a s h e d X-ray screen f i l m (X-omat) and Dupont screens i n c a s s e t t e s a t -70°C. P r e p a r a t i o n of pDt - S e r i n e plasmid DNA Plasmid c o n t a i n i n g b a c t e r i a were grown to approximately 8 o 6 x 1 0 b a c t e r i a / m l a t 30 C i n L u r i a b r o t h . Chloramphenicol then was added (80mg/ml i n 95% ethanol) to a f i n a l c o n c e n t r a -t i o n of 200 ug/ml, and the c u l t u r e was incubated a minimum of 4 hours. One l i t r e of c e l l s 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 , washed and resuspended i n 10ml 25% sucrose, 0.05 M T r i s pH 8.0. Two ml egg white lysozyme (5mg/ml) was added, and the mixture was incubated a t 4°C f o r 5 min. E i g h t ml 0.25 M EDTA pH 8.0, 0.0625 M EDTA was added. The l y s a t e was c e n t r i -fuged a t 4°C f o r 100 min. a t 25,000 rpm i n a Beckman 42.1 r o t o r . The supernantant was c e n t r i f u g e d to e q u i l i b r i u m i n C s C l and ethidium bromide a c c o r d i n g to Katz e t a l . ( 1 9 7 3 ) . The 8 plasmid DNA was c o l l e c t e d , the ethidium bromide was removed with b u t a n o l , and the s o l u t i o n was d i a l y z e d a g a i n s t 0.02 M NaCl, 0.02 M T r i s pH 8.0, 1- mM EDTA. The s o l u t i o n of p l a s -mid DNA was phenol e x t r a c t e d and ether washed. R e s t r i c t i o n Endonuclease Cleavage C o n d i t i o n s The r e s t r i c t i o n endonucleases EcoRI, Hae I I I , Hind I I I , Mbo I , P s t I, Taq I were purchased from New England B i o l a b s . The DNA was c l e a v e d u s i n g the assay c o n d i t i o n s , m o d i f i e d from those reviewed by Roberts (1979). EcoRI: 100 mM T r i s pH 7.5, 50 mM NaCl., 5mM MgCl 2. 1 u n i t / u g DNA was used i n a d i g e s t i o n a t 37°C f o r 2-4 hours Hae I I I : lOmM T r i s pH7.4, 60 mM NaCl, 7mM MgCl~. 1 unit/ugDNA was used i n a d i g e s t i o n a t 37°C f o r 2-4 hours Hind I I I : lOmM T r i s pH7.4, 60 mM NaCl, 7mM MgCl 2. 1 u n i t / u g DNA was used i n a d i g e s t i o n a t 37°C f o r 2-4 hours. Mbo I: 6mM T r i s pH 7.4, 150 mM NaCl, 6mM MgCl ImM d i t h i o t h r e i t o l . 1 u n i t / u g DNA was used i n a d i g e s t i o n a t 37 C f o r 4 hours Ps t I: 6mM T r i s pH 7.4, 5mM NaCl, 6mM MgCl„ ImM d i t h i o t h r e i t o l . 1 unit/ugDNA was used i n a d i g e s t i o n a t 37 C f o r 4 hours 9 Taq. I: lOmM T r i s pH 7.4, 60mM NaCl, 7mM Mg C l 2 1 u n i t / u g DNA was used i n a d i g e s t i o n a t 5 0°C f o r 3 hours Agarose Gel E l e c t r o p h o r e s i s Gel e l e c t r o p h o r e s i s a n a l y s i s was c a r r i e d out u s i n g 0.5%, 1.0%, 1.3%, 2.0% agarose (Bio-Rad) i n 0.09 M T r i s b o r a t e or 0.04 M T r i s phosphate pH 8.0, 0.02 M Na2PC>4 , ImM EDTA, lug/ml ethidium bromide, i n a S t u d i e r g e l apparatus (McDonell e t a l . , 1977). DNA samples were mixed w i t h 0.2 volume 40% sucrose, 0.025 M EDTA, 0.02% Bromophenol b l u e b e f o r e l o a d i n g the g e l . The e l e c t r o p h o r e s i s was c a r r i e d out a t 4°C f o r 2.5-3.5 hours at 85 v o l t s , 12 m amp. The g e l was exposed t o u l t r a v i o l e t l i g h t and photographed through an orange f i l t e r ( s e r i e s 6) us i n g type 57 p o l a r o i d f i l m . The DNA was t r a n s f e r r e d to n i t r o c e l l u l o s e f i l t e r as des-c r i b e d by Southern (Southern 1975). The f i l t e r s were h y b r i d i -125 S e r zed to [ i]tRNA 7 as p r e v i o u s l y d e s c r i b e d . M o l e c u l a r Weight A n a l y s i s of DNA Fragments The s i z e of DNA fragments r e s u l t i n g from R. endonuclease Hind I I I d i g e s t i o n of recombinant plasmids was determined by l i n e a r r e g r e s s i o n a n a l y s i s , u s i n g Hind I I I cut lambda DNA as a r e f e r e n c e (Murray and Murray, 1975). The a u t h e n t i c i t y of our pBR322 and the s i z e of the lambda Hind I I I fragments as 10 r e p o r t e d by M u r r a y and M u r r a y (1975) was c o n f i r m e d by a g a r o s e g e l e l e c t r o p h o r e s i s w i t h P s t I c l e a v e d 0 X 1 7 4 R.F. amb 3 DNA. S i n c e t h e n u c l e o t i d e s e q u e n c e s o f b o t h pBR322 a n d 0 X 1 7 4 a r e known, one c a n e s t a b l i s h t h e r e l a t i v e s i z e s o f t h e v a r i o u s f r a g m e n t s ( F i g u r e 2 ) . The s p e c i f i c f r a g m e n t s c a r r y i n g t h e D r o s o p h i l a tRNA gene was i d e n t i f i e d by a u t o r a d i o g r a p h y o f t h e RNA-DNA h y b r i d s a t t a c h e d t o t h e n i t r o c e l l u l o s e . R e c l o n i n g o f M u l t i p l e I n s e r t P l a s m i d s P l a s m i d s f o u n d t o c o n t a i n more t h a n one f r a g m e n t r e l e a s e d by d i g e s t i o n w i t h H i n d I I I w e r e r e c l o n e d . L i g a t i o n : 2.5 m i c r o g r a m s o f pBR322 DNA c l e a v e d w i t h H i n d I I I was i n c u b a t e d w i t h 1.4 u n i t s o f b a c t e r i a l a l k a l i n e p h o s p h o t a s e (BAPF W o r t h i n g t o n ) i n 50 u l f o r 60 m i n a t 60°C i n 0.06 M N a C l , 7mM M g C l 2 0 . 0 6 M T r i s pH 8.0. The m i x t u r e was e x t r a c t e d t w i c e w i t h p h e n o l , o n c e w i t h c h l o r o f o r m a n d t h r e e t i m e s w i t h e t h e r . The pDtl7. . o r p D t 2 7 (5ug) was c l e a v e d w i t h R. e n d o n u c l e a s e H i n d I I I a nd a d d e d t o t h e s o l u t i o n c o n t a i n i n g t h e c l e a v e d pBR322 DNA. S o d i u m A c e t a t e was a d d e d t o a f i n a l c o n c e n t r a t i o n o f 0.25 M. F o u r v o l u m e s o f 95% e t h a n o l a t -20°C was a d d e d , a n d t h e m i x t u r e was s t o r e d a t -2 0°C f o r a minimum o f 2 h o u r s . 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 and r e s u s p e n d -e d i n 0.5 m l o f b u f f e r c o n t a i n i n g 0.05 M T r i s pH 7.5, 0.01 M M g C l 2 , 0.01 M d i t h i o t h r e i t o l , 1 mM ATP. Ten u n i t s o f T4 p o l y -n u c l e o t i d e l i g a s e was a d d e d , a n d t h e m i x t u r e was i n c u b a t e d f o r two h o u r s a t 14°C. 11 o FIGURE 2: Size confirmation of pBR322 and lambda DNA 12 FIGURE 2: The a u t h e n t i c i t y of pBR322 and the s i z e of lambda Hind I I I fragments was confirmed by agarose g e l e l e c t r o -p h o r e s i s with P s t I c l e a v e d 0 X 1 7 4 amb 3 DNA. Lane 1 , X DNA d i g e s t e d w i t h Hind I I I ; lane 2, 0 X 1 7 4 R.F. amb 3 d i g e s t e d w i t h P s t I ; lane 3, pBR322 d i g e s t e d w i t h Hind I I I ; lane 4 , X DNA d i g e s t e d w i t h Hind I I I . 13 Transformation: An o v e r n i g h t c u l t u r e of SF-8 was d i l u t e d 9 o 1:50 and grown to approximately 10 b a c t e r i a / m l a t 30 C i n L u r i a b r o t h . Twenty-five ml of c u l t u r e were i c e - c h i l l e d , washed wi t h 10ml 0.01 M NaCl, sedimented, resuspended i n 12 ml0.03 M C a C l 2 and incubated a t 4°C f o r 20 min. The c e l l s then were sedimented and resuspended i n 3ml 0.03 M C a C l 2 . The t r e a t e d c e l l s (0.2ml) were mixed w i t h 0.05 ml of s o l u t i o n con-t a i n i n g 3 ug of the l i g a t e d DNA. The t r a n s f o r m a t i o n mixture was incubated a t 4°C f o r 60 minutes; the temperature was r a i s e d to 42°C f o r 10 minutes. The b a c t e r i a then were d i l u t e d i n t o 10 ml L u r i a b r o t h and grown f o r 2.5 hours a t 30°C. The transformed b a c t e r i a were p l a t e d on L u r i a b r o t h agar and scored f o r drug r e s i s t a n c e . C o l o n i e s which came up on a m p i c i l l i n p l a t e s were r e p l i c a p l a t e d to n i t r o c e l l u l o s e which was t r e a t e d as d e s c r i b e d above. Hy-b r i d i z a t i o n , autoradiography, and s i n g l e colony i s o l a t i o n oper-a t i o n s were performed w i t h about 12 c o l o n i e s f o r each p a r e n t a l plasmid. These were grown i n s u f f i c i e n t q u a n t i t y to g i v e enough plasmid DNA to d i g e s t and analyze on agarose g e l s . A s i n g l e c l o n e w i t h the a p p r o p r i a t e s i n g l e D r o s o p h i l a tRNA gene i n s e r t was chosen. E l e c t r o e l u t i o n of DNA from Agarose Gels P o r t i o n s of agarose g e l s c o n t a i n i n g d e s i r e d DNA fragments were c u t out of s l a b g e l s and p l a c e d i n a v e r t i c a l 5 ml p l a s t i c p i p e t t e , plugged w i t h s i l a t e d g l a s s wool, and covered w i t h 14 e l u t i o n b u f f e r (20mM T r i s - A c e t a t e pH 8.0, ImM EDTA). DNA was e l e c t r o e l u t e d i n t o a d i a l y s i s bag a t 150 v o l t s f o r 8 hours. The e l u t e d DNA was phenol e x t r a c t e d , ether washed s i x times, e t h a n o l p r e c i p i t a t e d w i t h f o u r volumes of 95% ethanol a t -20?C f o r a minimum of 2 hours, d r i e d i n vacuo and resuspended i n d i s t i l l e d water. The p u r i t y of e l u t e d fragments was e s t a b l i s h -ed by e l e c t r o p h o r e s i s on an agarose g e l . Ser . Dephosphorylation of pDt!6 fragments C o n t a i n i n g tRNA 7 Genes The method d e s c r i b e d by G. Korana (1973) was employed. Eleven rpmoles of 5' ends were heat denatured a t 9 0°C f o r 2.5 min., i c e c h i l l e d , 500 mM T r i s pH 8.0 was added, along w i t h 0.1 u n i t s per r e a c t i o n of b a c t e r i a l a l k a l i n e phosphatase F, and incubated a t 60°C f o r 2 hours. The dephosphorylated DNA was phenol e x t r a c t e d , ether washed, et h a n o l p r e c i p i t a t e d and d r i e d i n vacuo. 3 2 L a b e l i n g DNA 5' t e r m i n i w i t h p o l y n u c l e o t i d e Kinase and ;- [ . P ] A T P Both the forward r e a c t i o n f o r f l u s h or r e c e s s e d 5' t e r m i n i and the exchange r e a c t i o n f o r p r o t r u d i n g 5' t e r m i n i d e s c r i b e d by A. Maxim and W. G i l b e r t (personal communication) were u t i -l i z e d . Forward r e a c t i o n : 5 pmoles of 5' dephosphorylated o l i g o n u c -l e o t i d e t e r m i n i were resuspended i n 20 mM T r i s pH 9.5, ImM Spermidine, 0.1 mM EDTA, heat denatured a t 90°C f o r 3.5 min., 15 and i c e c h i l l e d . Kinase b u f f e r (500 mM T r i s pH 9.5, 100 mM MgCl 2/ 50 mM d i t h i o t h r e o t o l , 50% g l y c e r o l ) was added along w i t h 50 pmoles & [ 3 2 P ] ATP (2300 Ci/mmole)i, 20 u n i t s per r e a c t i o n of T4 p o l y n u c l e o t i d e k i n a s e , and the mixture was i n -cubated at 37°C f o r 60 min. Exchange r e a c t i o n : 6 pmoles of 5' dephosphorylated o l i g o n u -c l e o t i d e t e r m i n i were resuspended i n 500 mM i m i d a z o l e - HC1 pH 6.6, 100 mM MgCl 2, 50 mM d i t h i o t h r e o t o l , 1 mM spermidine, 3 2 1 mM EDTA, 5 mM adenosine diphosophate, 7 3 pmoles X[ P ] A T P (700 Ci/mmole), 20 u n i t s T4 p o l y n u c l e o t i d e k i n a s e , and i n -cubated a t 37°C f o r 4 5 min. Both r e a c t i o n s were et h a n o l p r e c i p i t a t e d , and d r i e d i n vacuo. At time 0, 15, 30, 45, 60 min. a 1 u l sample was taken from the r e a c t i o n mixture, s p o t t e d on a p i e c e of 3mm f i l t e r paper, and p r e c i p i t a t e d i n TCA to determine the amount 3 2 of i n c o r p o r a t e d X [ P ] A T P . Strand S e p a r a t i o n Gels A f t e r 5' end l a b e l i n g the double stranded plasmid f r a g -ments, i t was necessary to separate the strands to ensure t h a t 3 2 t h e i r was o n l y one 5' t e r m i n i l a b e l l e d w i t h X [ P ] . ATP p r i o r to running a sequencing r e a c t i o n . T h i s was accomplished by run-ning a 1:30 c r o s s l i n k e d , 4% p o l y a c r y l a m i d e g e l , (A. Maxam and W. G i l b e r t , p e r s o n a l communication) i n 50 mM T r i s - b o r a t e pH 8.3, 32 2.5 mM EDTA. The P l a b e l l e d plasmid fragment was resuspended 16 i n 0.1 M NaOH, 1 mM EDTA, heat denatured a t 90°C f o r 2 min., immediately loaded onto a p r e e l e c t r o p h o r e s e d g e l , and run o v e r n i g h t a t 150 v o l t s c onstant c u r r e n t . The g e l was wrapped i n p o l y v i n y l i d e n e f i l m (Saran wrap) and autoradiographed f o r 15-60 min. w i t h X-ray no screen f i l m . Separated strands were cut from the g e l and e l e c t r o e l u t e d as d e s c r i b e d above. Containment B a c t e r i a c o n t a i n i n g recombinant plasmids were handled under B-M containment c o n d i t i o n s , as s p e c i f i e d by the r e g u l a -t i o n s of the M e d i c a l Research and N a t i o n a l Sciences and Engin-e e r i n g Research C o u n c i l s . 17 RESULTS AND DISCUSSION P r i n c i p l e of the Procedure E. c o l i c e l l s c o n t a i n i n g recombinant plasmids (Dunn e t . a l . Ser 1979) were i d e n t i f i e d as c a r r y i n g s p e c i f i c D r o s o p h i l a tRNA4,7 12 5 genes by h y b r i d i z a t i o n w i t h p u r i f i e d [ I] l a b e l l e d D r o s o p h i l a Ser tRNA4,7 molecules a c c o r d i n g to the procedure of G r u n s t e i n and Hogness (1975). I n d i v i d u a l plasmids were c h a r a c t e r i z e d by s e v e r a l procedures: R e s t r i c t i o n endonuclease cleavage; agar-ose g e l e l e c t r o p h o r e s i s and h y b r i d i z a t i o n w i t h s p e c i f i c , p u r i -12 5 f i e d [ i ] l a b e l l e d tRNA a c c o r d i n g to Southern (Southern, 1975). C h a r a c t e r i z a t i o n and M o l e c u l a r Weight  C a l c u l a t i o n of the D r o s o p h i l a I n s e r t s Table 1 d e s c r i b e s the seven pBR322 - D r o s o p h i l a DNA Ser recombinant plasmids c h a r a c t e r i z e d , which c a r r y the tRNA4,7 gene. The plasmids were c l e a v e d w i t h R e s t r i c t i o n endonuclease Hind I I I , analyzed by agarose g e l e l e c t r o p h o r e s i s and annealed 125 S e r with [ I] tRNA4,7 molecules a c c o r d i n g to the procedures o f Southern (1975). The s i z e of DNA fragments, r e s u l t i n g from R-endonuclease Hind I I I d i g e s t i o n , was determined by l i n e a r r e g r e s s i o n a n a l y s i s , u s i n g Hind I I I c u t lambda DNA as a r e f e r -ence (Murray and Murray 1975). Table 1 shows the s i z e of the i n s e r t e d fragments generated by Hind I I I cleavage. The symbol 18 TABLE 1 tRNA Plasmid No. Hind I I I Fragments (kb) Ser 4,7 PDt 1 4.4* PDt 5 4.4* PDt 16 6.2* PDt 17 8.6*, 2.0, 1.2, 0.85, 0.56 PDt 17R 3.7* PDt 27 6.2*, 2.5, 2.1, 1.1, 0.9 PDt 27R 6.2* PDt 73 4.7* PDt 81 4.4* Table I R. endonuclease Hind I I I fragments of recombinant plasmids. Each of the recombinant plasmids l i s t e d here was cl e a v e d by R. endonuclease Hind I I I , e l ectrophonesed i n agarose 12 5 Ser g e l s and h y b r i d i z e d w i t h complementary [ I] tRNA 4 , 7 . A l l of the i n s e r t e d Hind I I I fragments from the o r i g i n a l i s o l a t e s are l i s t e d ; the 4.4 kb pBR 322 p a r e n t a l fragment i s omitted. R: s i g n i f i e s t h a t t h e p a r e n t p l a s m i d has b e e n r e c l o n e d t o g i v e a new p l a s m i d c o n t a i n i n g o n l y t h e H i n d I I I f r a g m e n t b e a r i n g t h e tRNA gene. 125 S e r *: r e f e r s t o t h e i n s e r t w h i c h h y b r i d i z e s [ I] tRNA 4 , 7 19 Ser (*) i n d i c a t e s the s i z e of the i n s e r t to which tRNA4,7 anneals. Upon cleavage w i t h Hind I I I , f i v e of the s e r i n e plasmids were shown to c o n t a i n o n l y s i n g l e i n s e r t s of D r o s o p h i l a DNA; two of them c o n t a i n e d 5 i n s e r t s each (pDtl7 and pDt27) (Figure 3,4). The d i f f e r e n c e between the s i z e of the i n s e r t i o n i n pDt 73 and t h a t of p D t l , pDt5 and pDt81 i s s i g n i f i c a n t (Figure 4) pDt27 was of f u r t h e r i n t e r e s t . Upon i n i t i a l i s o l a t i o n from the "shot gun" c l o n i n g experiments, i t was found t h a t Ser l y s pDt27 h y b r i d i z e d both tRNA4,7 and tRNA 5 . A f t e r Hind I I I d i g e s t i o n , agarose g e l e l e c t r o p h o r e s i s , and h y b r i d i z a t i o n with 125 S e r 125 l Y S [ I] tRNA4,7 and I I] tRNA 5 , the same Hind I I I d e r i v e d fragment from pDt27 h y b r i d i z e d both D r o s o p h i l a tRNAs (Figure 5,6) . Ser C h a r a c t e r i z a t i o n of tRNA 7 plasmid C a r r y i n g S i m i l a r s i z e d  Hind I I I i n s e r t s Many of the plasmids were c l e a v e d w i t h d i f f e r e n t R. endo-nucleases, other than Hind I I I , t o ensure the i d e n t i t y of d i f f e r e n t plasmids c a r r y i n g s i m i l a r s i z e d Hind I I I i n s e r t s as i n the case of pDtl6 and pDt27, or p D t l , pDt5 and pDt81 (Table 2) . (i) p D t l , pDt5, pDt81 Homology D i g e s t i o n w i t h R. endonuclease EcoRI i n d i c a t e d t h a t pDt5 and pDt81 were the same i n s e r t and t h a t p D t l was d i f f e r e n t (Figure 7). Mbo I d i g e s t i o n of the same three plasmids i n d i c a t - . ed t h a t they had many s i m i l a r i t i e s (Figure 7). D i g e s t i o n w i t h 2 0 1 23456789 i,4 ti M I 1 I" hi i i fc-4-o FIGURE 3: Agarose gel analysis of Hind I I I digested plasmids. 21 FIGURE 3: The plasmids pDt 1,5,16,17,27,73,81 and c o n t r o l DNAs were cl e a v e d w i t h r e s t r i c t i o n endonuclease Hind I I I and e l e c t r o p h o r -esed on 0.5% agarose i n Tris-phosphate b u f f e r . The fragments were v i s u a l i z e d by s t a i n i n g w i t h ethidium bromide and exposure to u l t r a v i o l e t l i g h t . The number and s i z e s of the D r o s o p h i l a i n s e r t s were determined f o r each plasmid. Lane 1, ADNA; lane 2, p D t l ; l a n e 3, pDt5; lane 4, pDt l 6 ; l a n e 5, p D t l 7 ; l a n e 6, pDt27; lane 7, pDt73; lane 8, pDt81; lane 9, ADNA. 22 23456 78 O FIGURE 4: Hybridization of Hind I I I digested plasmids 125 S E R [ I ] tRNA 4,7 23 FIGURE 4: The agarose g e l photographed i n F i g . 3 was t r e a t e d by the procedure of Southern (1975). The t r a n s f e r r e d DNA was i n -125 Ser cubated w i t h C 13 tRNA 4,7, f o l l o w e d by autoradiography. Lanes 2-8 correspond t o lanes 2-8 of F i g u r e 3; i . e . , lane 2, p D t l ; lane 3, pDt5; lane 4, pDtl6; lane 5, pDt l 7 ; lane 6,pDt27; lane 7, pDt73; lane 8, pDt81. 24 1 23 i o FIGURE 5: Agarose gel analysis of Hind I I I digested pDt27 25 FIGURE 5: The p l a s m i d pDt2 7 and c o n t r o l DNA were c l e a v e d w i t h H i n d I I I and were e l e c t r o p h o r e s e d on 0.5% a g a r o s e i n T r i s -p h o s p h a t e b u f f e r . Lane 1, ADNA; l a n d 2, pDt27; l a n d 3, pDt27. 26 27 FIGURE 6: L a n e s 2 and 3 p h o t o g r a p h e d i n F i g u r e 5 were c u t f r o m t h e g e l and t r a n s f e r r e d t o s e p a r a t e n i t r o c e l l u l o s e f i l t e r s by t h e p r o c e d u r e o f S o u t h e r n (1975). L a n e 2, t h e t r a n s -125 S e r f e r r e d DNA was i n c u b a t e d w i t h [ I] tRNA 4,7, f o l l o w e d by a u t o r a d i o g r a p h y ; l a n e 3, t h e t r a n s f e r r e d DNA was i n c u b a t e d 125 L y S w i t h ( I] tRNA 5, f o l l o w e d by a u t o r a d i o g r a p h y . The 6.2 kb f r a g m e n t h y b r i d i z e d b o t h tRNAs. 28 TABLE 2 Plasmid Restriction Fragment No. .-eridonuclease (kb) pDt 1 Hind III 4.4* Pst I 2.34, 1.81*, 1.17 PstI + Hind III 2.14, 1.39*, 0.92, 0. 39, 0. 21 pDt 5 Hind III 4.4* Pst I 3.64* , 0.99, 0.65 PstI;+Hind III 2.14, 1.39*, 0.92, 0. 39, 0. 21 pDt 81 Hind III 4.4* Pst I 3.64* , 0.99, 0.65 PstI J-Hind III 2.14, 1.39*, 0.92, 0. 39, 0. 21 pDt 16 Hind III 6. 2* Hae III 0. 66* , 0.44* Hind III + Hae III 0.66* , 0.44* pDt 27 Hind III / 6.2* Hae III 0.35* Hind III + Hae III 0.35* *: refers to the insert which hybridizes [ I] tRNA 7 Table 2 R. Endonuclease fragments of Recombined Plasmids Containing Similar size Hind III Inserts In order to distinguish different plasmids containing similar sized Hind III inserts, the plasmids were cleaved with other R. endonucleases 125 ser electrophoresed in agarose gels and hybridized with [ I] tRNA 7 . From this table and figures 8, 9, 11, 12, i t was found that pDt 1, 5, ser 81, contained the same tRNA 7 insert, and that pDtl was in reverse orien-tation to pDt 5,81. Also from this table and figures 13, 14, i t was found that pDt 16 and pDt 27 are different from each other, though they contain ser Hind III tRNA 7 inserts of equal size. 29 12345 6 FIGURE 7: Agarose gel analysis of EcoRI and Mbol digested pDt 1, 5, 81. FIGURE 7: The plasmids pDt 1,5,81 were c l e a v e d w i t h R. endonucleases EcoRI and Mbol and e l e c t r o p h o r e s e d on 0.5% agarose i n T r i s -phosphate b u f f e r . Lane 1, pDt5 d i g e s t e d w i t h E c o R I ; lane 2, pDt81 d i g e s t e d w i t h EcoRI; lane 3, p D t l d i g e s t e d w i t h EcoRI; lane 4, pDt5 d i g e s t e d w i t h Mbol; lane 5, pDt81 d i g e s t e d w i t h Mbol; lane 6, p D t l d i g e s t e d w i t h Mbol. The EcoRI d i g e s t i o n s f i r s t suggested t h a t p D t l had a d i f f e r e n t D r o s o p h i l a Hind I I I i n s e r t from pDt 5,81. The Mbol d i g e s t i o n s , although incomplete i n a l l t h r e e w e l l s . s u g g e s t e d t h a t pDt 1,5,81 contained a s i m i -l a r D r o s o p h i l a Hind I I I i n s e r t . 31 R. e n d o n u c l e a s e s P s t I and H i n d I I I r e v e a l e d t h a t t h e i n s e r t i n p D t l was i n t h e o p p o s i t e o r i e n t a t i o n o f t h e i n s e r t i n pDt5 S e r and pDt81; t h e f r a g m e n t h y b r i d i z i n g t o t h e tRNA 7 was a d i f -f e r e n t s i z e i n p D t l compared t o t h e p D t 5 , pDt81 a f t e r P s t I c l e a v a g e , b u t was t h e same s i z e a f t e r P s t I , H i n d I I I d o u b l e d i g e s t i o n ( F i g u r e 8,9). A s k e m a t i c d i a g r a m o f t h e r e v e r s e o r i e n t a t i o n o f p D t l i s shown i n F i g u r e 10. D i g e s t i o n w i t h R. e n d o n u c l e a s e Hae I I I c o n f i r m e d t h e ( C o n c l u s i o n t h a t p D t l was t h e same i n s e r t as pDt5, pDt81, b u t i n t h e o p p o s i t e o r i -e n t a t i o n . I n a l l t h r e e p l a s m i d s t h e f r a g m e n t s h y b r i d i z i n g t o S e r t h e tRNA 7 were t h e same a f t e r Hae I I I , o r Hae I I I , H i n d I I I d o u b l e d i g e s t i o n ( F i g u r e 11,12) : ( i i ) p D t ! 6 , pDt27 Homology: D i g e s t i o n w i t h R. e n d o n u c l e a s e s Hae I I I and H i n d I I I r e v e a l e d t h a t t h e f r a g m e n t s o f p D t l 6 and pDt27 h y b r i d i z i n g S e r tRNA 7 were d i f f e r e n t . ( F i g u r e s 1 3 , 1 4 ) . S i n c e two o f t h e p D t l 6 125 S e r Hae I I I f r a g m e n t s a n n e a l e d w i t h [ I]tRNA 7, i t a p p e a r s t h a t e i t h e r t h e tRNA gene has b e e n c l e a v e d by t h e Hae I I I , o r t h e r e a r e m u l t i p l e c o p i e s o f t h e gene s e p a r a t e d by a Hae I I I s i t e . R e c l o n i n g o f pDt!7 and pDt27 I n many c a s e s c e r t a i n f r a g m e n t s b e a r i n g s p e c i f i c tRNA gen e s were i s o l a t e d o n l y i n c o m b i n a t i o n w i t h o t h e r H i n d I I I f r a g m e n t s . B o t h p D t l 7 and pDt27 c o n t a i n e d f i v e H i n d I I I i n s e r t s . T h e r e -3 2 1 23 456 0 FIGURE 8: Agarose gel analysis of Pst I and Pst I + Hind I I I digested pDt 13S. 33 FIGURE 8: The plasmids p D t l and pDt5 were c l e a v e d w i t h r e s t r i c -t i o n endonucleases P s t I and P s t I + Hind I I I , e l e c t r o p h o r e s e d on 0.5% agarose i n Tris-Phosphate b u f f e r and photographed under u.v. l i g h t a f t e r s t a i n i n g with ethidium bromide. Lane 1, p D t l d i g e s t e d with Hind I I I ; lane 2, p D t l d i g e s t e d w i t h P s t I ; lane 3, p D t l d i g e s t e d w i t h P s t I + Hind I I I ; lane 4, pDt5 d i g e s t e d with Hind I I I ; lane 5, pDt5 d i g e s t e d w i t h Pst I; lane 6, pDt5 d i g e s t e d w i t h P s t I + Hind I I I . 34 1 2 3 4 5 6 725 S e r FIGURE 9: Hybridization of [ I ] tRNA 7 with Pst I, and Pst I + Hind I I I digested pDtl35. 35 FIGURE 9: The a g a r o s e g e l p h o t o g r a p h e d i n F i g . 8 was t r e a t e d by 125 t h e p r o c e d u r e o f S o u t h e r n (1975) and i n c u b a t e d w i t h [ I] S e r tRNA 7. L a n e s 1-6 c o r r e s p o n d t o l a n e 1-6 o f F i g u r e 5; i . e . l a n e 1, p D t l d i g e s t e d w i t h H i n d I I I ; l a n e 2, p D t l d i g e s t e d w i t h P s t I ; l a n e 3, p D t l d i g e s t e d w i t h P s t I + H i n d I I I ; l a n e 4, pDt5 d i g e s t e d w i t h H i n d I I I ; l a n e 5, pDt5 d i ^ g e s t e d w i t h . P s t I ; l a n e 6, pDt5 d i g e s t e d w i t h P s t I + H i n d I I I . 36 2.34kb 1.81 1.17 2.14 kb 1.39 • 0.92 0.39 0.21 3.64 kb 0.99 0.65 Skematic representation of the reverse orienta-tion of pDtl. 37 1 23456789 - O FIGURE 11: Agarose gel analysis of Eae I I I and Hae I I I + Bind I I I digested pDt 1>5381 38 FIGURE 1 1 : The p l a s m i d s p D t 1,5,81 w e r e c l e a r e d w i t h Hae I I I a n d Hae I I I + H i n d I I I , e l e c t r o p h o r e s e d o n 2.0% a g a r o s e i n T r i s -p h o s p h a t e b u f f e r a nd p h o t o g r a p h e d u n d e r u.v. l i g h t a f t e r s t a i n i n g w i t h e t h i d i u m b r o m i d e . L a n e 1, p D t l d i g e s t e d w i t h Hae I I I ; l a n e 2, p D t l d i g e s t e d w i t h H i n d I I I ; l a n e 3, p D t l d i g e s t e d w i t h Hae I I I + H i n d I I I ; l a n e 4, p D t 5 d i g e s t e d w i t h Hae I I I ; l a n e 5, p D t 5 d i g e s t e d w i t h H i n d I I I ; l a n e 6, p D t 5 d i g e s t e d w i t h Hae I I I + H i n d I I I ; l a n e 7, p D t 81 d i g e s t e d w i t h Hae I I I ; l a n e 8, p D t 8 1 d i g e s t e d w i t h H i n d I I I ; l a n e 9, p D t 8 1 d i g e s t e d w i t h Hae I I I + H i n d I I I . 39 12 3 4 5 6 789 O 125 S e r FIGURE 12: Hybridization of [ I ] tRNA 7 with Hae I I I , Hind I I I and Hae I I I + Hind I I I digested pDtl}5381 40 FIGURE 12: The agarose g e l photographed i n F i g . 11 was t r e a t e d by the procedure of Southern (1975) and incubated w i t h 125 S e r [ I ] tRNA 7. Lanes 1-9 correspond to lanes 1-9 of F i g u r e 11; i . e . lane 1, p D t l d i g e s t e d w i t h Hae I I I ; lane 2, p D t l d i g e s t e d w i t h Hind I I I ; lane 3, p D t l d i g e s t e d with Hae I I I + Hind I I I ; lane 4, pDt5 d i g e s t e d with Hae I I I ; lane. 5, pDt5 d i g e s t e d w i t h Hind I I I ; lane 6, pDt5 d i g e s t e d with Hae I I I + Hind I I I ; l a n e 7, pDt81 d i g e s t e d with Hae I I I ; lane 8, pDt81 d i g e s t e d w i t h Hind I I I ; lane 9, pDt81 d i g e s t e d w i t h Hae I I I + Hind I I I . 4 1 FIGURE 13: Agarose gel analysis of Hae I I I and Hae I I I + Hind I I I digested pDtl6 and pDt27. 42 FIGURE 13: The plasmids pDtl6 and 27 were c l e a r e d w i t h Hae I I I and Hae I I I + Hind I I I , e l e c t r o p h o r e s e d on 2.0% agarose i n Tris-phosphate b u f f e r and photographed under u.v. l i g h t a f t e r s t a i n i n g w i t h ethidium bromide. Lane 1, pDtl6 d i g e s t e d w i t h Hae I I I ; lane 2, pDtl6 d i g e s t e d w i t h Hae I I I + Hind I I I ; lane 3, pDt27 d i g e s t e d w i t h Hae I I I , lane 4, pDt27 d i g e s t e d with Hae I I I + Hind I I I . 43 o 66 kb .44 .35 .125 Ser FIGURE 14: Hybridization of [ I] tRNA 7 with Hae I I I , and Hae I I I + Hind I I I digested pDt!6 and pDt27, 44 FIGURE 14: The a g a r o s e g e l p h o t o g r a p h e d i n F i g . 13 was t r e a t e d by t h e p r o c e d u r e o f S o u t h e r n (1975) a n d i n c u b a t e d w i t h S e r 12 5 " V - J . [ I ] t R N A 7. L a n e s 1-4 c o r r e s p o n d t o l a n e s 1-4 o f f i g u r e 14; i . e . , l a n e 1, p D t l 6 d i g e s t e d w i t h Hae I I I ; l a n e 2, p D t l 6 d i g e s t e d w i t h Hae I I I + H i n d I I I ; l a n e 3, p D t 2 7 d i g e s t e d w i t h Hae I I I , l a n e 4, p D t27 d i g e s t e d w i t h Hae I I I + H i n d I I I . 45 fore, the plasmid DNA was cleaved with Hind III and was re-cloned with pBR32 2. Recombinant plasmids containing only Hind III fragments carrying the desired tRNA gene were i s o -lated from t h i s preparation and designated pDtNR. I t i s of int e r e s t to note that upon the recloning of pDtl7, 55% of the o r i g i n a l Hind III fragment containing the tRNA gene was recombined out of the pDtl7R Hind III fragment containing the tRNA gene. The o r i g i n a l Hind III fragment containing the tRNA gene i n pDtl7 was 8.3kb, and i n pDtl7R the Hind III fragment was 3.7kb. R. endonucleases Hae III and Hind III confirmed Ser that the pDtl7 Hind III fragment containing the tRNA 7 gene was larger than the pDtl7R Hind III fragment containing the Ser tRNA 7 gene. Preparation of pDt!6 For Sequencing R e s t r i c t i o n endonuclease Hae I I I , Hind III double diges-tion of pDtl6 yielded two fragments 0.66 and 0.44 kb (Figures ser 13,14) which hybridized tRNA 7, y i e l d i n g two fragments of convenient size for sequencing by the method described by A. Maxam and W. G i l b e r t . Four hundred ug of pDtl6 DNA, cleaved with R. endonucleases Hae I I I , Hind III i n tandem were loaded on 2% agarose Tris-borate gels and run at 20 mamp for 48 hours (Figure 15). The desired DNA bands were cut from the gel, electroeluted overnight at 150 v o l t s i n 20 mm Tris-acetate 4 6 1 2 34 567 O FIGURE 15: Fragment preparation gel for pDt!6 47 FIGURE 15; Four hundred micrograms of pDtl6 d i g e s t e d w i t h Hae I I I + Hind I I I , were e l e c t r o p h o r e s e d on 2.0% agarose i n T r i s -borate b u f f e r and photographed under u.v. l i g h t a f t e r s t a i n -i n g w i t h ethidium bromide. Lanes 1-7, pDtl6 d i g e s t e d w i t h Hae I I I + Hind I I I . 48 pH 8.0 phenol e x t r a c t e d , ether washed and e t h a n o l p r e c i p i t a t e d . T r i s - b o r a t e b u f f e r e d g e l s were used to p u r i f y s p e c i f i c r e s t r i c -t i o n fragments, because the phosphate i n T r i s - p h o s p h a t e g e l s was i n s o l u b l e under c o n d i t i o n s a r i s i n g d u r i n g subsequent man-i p u l a t i o n of the fragments. K i n e t i c s of the P o l y n u c l e o t i d e Kinase R e a c t i o n Before attempting to l a b e l 5' t e r m i n i of the two pDtl6 tRNA gene c a r r y i n g fragments, the k i n e t i c s of p o l y n u c l e o t i d e k i n a s e l a b e l l i n g of 5' ends of lambda DNA c l e a v e d w i t h EcoRI which gave 5' extended ends, and Hae I I I which gave 5' b l u n t or f l u s h ends were monitored. Both the forward and exchange kin a s e r e a c t i o n s were assayed w i t h EcoRI c l e a v e d lambda f r a g -3 2 ments, l a b e l l i n g the extended 5' ends wi t h & [ P ] ATP (Figure 16). S i x t y f i v e p ercent of lambda 5' ends were l a b e l -l e d u s i n g the forward k i n a s e r e a c t i o n , and the exchange r e a c -t i o n reached e q u i l i b r i u m a t o n l y 19% of lambda 5' ends i n c o r -p o r a t i n g r a d i o a c t i v i t y . The forward p o l y n u c l e o t i d e k i n a s e r e a c t i o n was used to l a b e l 5' b l u n t ends of lambda Hae I I I fragments. These 5' b l u n t ends were i d e n t i c a l to the 5' b l u n t ends i n the two p D t l 6 , Hae I I I , Hind I I I tRNA gene fragments p r e v i o u s l y i s o l a t e d . 3 2 14% of lambda 5' b l u n t ends were l a b e l l e d w i t h & [ P ] A T P u t i l i z i n g the forward r e a c t i o n . 49 50 FIGURE 16; 3 2 Graph A r e p r e s e n t s the & [ P] ATP i n c o r p o r a t i o n by 5' t e r m i n i of EcoRI fragments u s i n g the forward p o l y -3 2 n u c l e o t i d e k i n a s e r e a c t i o n . Graph B r e p r e s e n t s the P] ATP i n c o r p e r a t i o n by 5" t e r m i n i of EcoRI fragments u s i n g the p o l y n u c l e o t i d e k i n a s e exchange r e a c t i o n . 51 T h i r t y percent of the 5' b l u n t ends of the 660 base pDt 3 2 16 fragment were end l a b e l l e d w i t h & [ P ] A T P and 4 7% of the 5' b l u n t ends of the 440 base pDtl6 fragment were end l a b e l l e d w i t h & [ 3 2 P ] A T P u s i n g the forward k i n a s e r e a c t i o n . 3 2 Strand S e p a r a t i o n of )([ P ] A T P l a b e l l e d pDt!6 fragments When r e s t r i c t i o n fragments are l a b e l l e d a t both 5' ends, s e p a r a t i o n of t h e i r s t r a n d s e l i m i n a t e s the need f o r secondary r e s t r i c t i o n enzyme cleavage p r i o r to sequence a n a l y s i s . Se-quence i n f o r m a t i o n from one s t r a n d w i l l c o n f i r m the sequence from the other s t r a n d . The pDtl6 440 base fragment was t r e a t -ed with a k a l a i , heat denatured and e l e c t r o p h o r e s e d on a 1:30 c r o s s l i n k e d , 4 percent p o l y a c r y l a m i d e g e l (Maxam and G i l b e r t ) ( F igure 17). The top two dark bands are the separated 440 base s i n g l e s t r a n d s , w i t h s m a l l e r d e g r a d a t i o n products below. The s e p a r a t i o n of these s t r a n d s should prove adequate f o r f u t u r e sequencing experiments. 52 FIGURE 17: Polyaorylamide strand separation of X [ P] ATP l a b e l l e d pDt!6 fragments. 53 FIGURE 17: 3 2 5' t e r m i n i of pDtl6 fragments l a b e l l e d w i t h X[ P] ATP were a l k a l a i , heat denatured and e l e c t r o p h o r e s e d on a 1:30 c r o s s l i n k e d , 4% p o l y a c r y l a m i d e g e l . Lanes 1 and 2, pDtl6 44 0 base fragments. The top two dark bands(a,b) are the 440 base s i n g l e strands w i t h s m a l l e r , l i g h t e r degrada-t i o n products below. 54 BIBLIOGRAPHY Ames, B.N. and Hortman, P.E. The h i s t i d i n e aperon, C o l d S p r i n g Harbor Symp. Quant. B i o l . 28 (1963) 349-356. Atwood, K.C. In Genetic V a r i a t i o n s of D r o s o p h i l i a melanog-a s t e r . ( L i n d s l e y , D.L. and G r e l l , F.H.,eds.), Carnegie I n s t . Publ. No. 627 (1968) 152. B o l i v a r , F., Radriquez, R.L., Greene, P.S. B e t l a c k M.S., Heynecker, H.L. and Bayer, H.W. C o n s t r u c t i o n and charac-t e r i z a t i o n of new c l o n i n g v e h i c l e s I I . A multipurpose c l o n -in g system, Gene 2 (1977) 95-113. Dunn, R., Hayaski, S., G i l l a m , I.C., Delaney, A.D., Tener, G.M., G r i g l i a t t i , T.A., Kaufman, T.C. and Suzuki, D.T. Genes coding f o r v a l i n e t r a n s f e r r i b o n u c l e i c acid-3b i n D r o s o p h i l a melanogas-t e r , J . Mol. B i o l . 128 (1979) 277-287. Dunn, R., Delaney, A.D., G i l l a m , I.C., Hayaski, S., Tener, G.M., G r i g l i a t t i , T., Misna, V., Spurr, M.G., T a y l o r , D.M., M i l l e r , R.C. I s o l a t i o n and c h a r a c t e r i z a t i o n of recombinant plasmids c a r r y i n g D r o s o p h i l a tRNA genes, Gene 7 (3) (1979). E l d e r , R.T., Genes f o r a s i n g l e t r a n s f e r RNA are pr e s e n t a t two chromosomal s i t e s i n D r o s o p h i l a melanogaster, F e d e r a t i o n Proc. 3_7 (1978) 1732. Gr u n s t e i n , M. and Hogness, D.S. Colony h y b r i d i z a t i o n . A method f o r the i s o l a t i o n of c l o n e d DNAs t h a t c o n t a i n a s p e c i f i c gene, Proc. N a t l . Acad. S c i . USA 72 (1975) 3961-3965. Khorana, B i o c h e m i s t r y 12 (1973) 5050 K u b l i , E. and Schmidt, T. The l o c a l i z a t i o n o f tRNA G4" U genes from D r o s o p h i l a malanogaster by " i n s i t u " h y b r i d i z a t i o n , N u c l e i c A c i d s Res. 5 (1978) 1465-1478 Murray, K. and Murray, N.E. Phage lambda r e c e p t o r chromasomes f o r DNA fragments made wit h r e s t r i c t i o n endonuclease I I I of Heamaphilus i n f l u e n z a e and r e s t r i c t i o n endonuclease I of Esch  e n i c h i a c o l i , J . Mol. B i o l . (1975) 551-564. Olson, M.V., Montgomery, D.L., Hopper, A.K., Page, G.S., Horadyski, F. and H a l l , B.D. M o l e c u l a r c h a r a c t e r i z a t i o n o f the t y r a s i n e tRNA genes of ye a s t , Nature 267 (1977) 639-641. Roberts, R.J. R e s t r i c t i o n and m o d i f i c a t i o n enzymes and t h e i r r e c o g n i t i o n sequences, Gene i_ (1978) 183-193. Schmidt, T., Egg, A.H. and K u b l i , E. The l o c a l i z a t i o n of tRNA 2 genes from D r o s o p h i l a melanogaster by " i n s i t u " h y b r i d i z a t i o n , Mol. gen. Genet. 164 (1978) 249-254. 55 Southern, E.M. D e t e c t i o n of s p e c i f i c sequences among DNA fragments separated by g e l e l e c t r o - p h o r e s i s , J . Mol. B i o l . 9j3 (1975) 503-517. Stent, G.S. The operon: on i t s t h i r d a n n i v e r s a r y , Science 144 (1964) 816-820. Sueoka, N. and Kano - Sueoka, T. . A s p e c i f i c . m o d i f i c a t i o n of l e u c y l - tRNA of E s c h e r i c h i a c o l i a f t e r phage T„ i n f e c t i o n , Proc. N a t l . Acad. S c i . USA 52 (1964) 1535-1540. Tener, G. M., Hayaski, S., Dunn, R. Delaney, A.D., G i l l a m , I.C., G r i g l i a t t i , T.A., Kaufman, T.C. and Suzuki, D.T. ^ T r a n s f e r RNA genes o f D r o s o p h i l a melanogaster i n tRNS 2 (J. Abelson, P. Schiemmel and D. S o l i , eds) C o l d S p r i n g Harbor Laboratory, C o l d S p r i n g Harbour. (1979) i n p r e s s . White, B.N., Tener, G.M., Holden, J . , and Suzuki, D.T. A n a l y s i s of tRNAs d u r i n g development of D r o s o p h i l a , Devel. B i o l . 33 (1973) 185-195. White, B.N., Dunn, R., G i l l a m , I.C., Tener, G.M., Armstrong, D.J., Skoog, F., F r i h a r t , C R . and Leonard, N.J. An a n a l y s i s of f i v e s e r i n e t r a n s f e r r i b o n u c l e i c a c i d s from Dros-p h i l a , J . B i o l . Chem. 250 (1975) 515-521. 

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