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Characterization of recombinant plasmids carrying Drosophila transfer RNA genes Rajput, Bhanu 1980

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CHARACTERIZATION OF RECOMBINANT PLASMIDS CARRYING DROSOPHILA TRANSFER RNA GENES by BHANUMATI RAJPUT B.Sc. (SUMMA CUM LAUDE) Washington State University, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY UNIVERSITY OF BRITISH COLUMBIA We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JULY, 1980 ©Bhanumati Rajput, 1980 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 m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 a g r e e 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 p u r p o s e s may be g r a n t e d by t h e Head o f my Department o r by h i s 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 o r 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 n o t 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 . D e p a r t m e n t o f |V\\ C R Q & \ O L O G t W The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date i^Yisj C f u l i j ^ \°[10 6 i i A b s t r a c t The purpose of t h i s study was to c h a r a c t e r i z e recombinant p l a s -raids c a r r y i n g D r o s o p h i l a melanogaster tRNA genes. The two groups V a l of recombinant plasmids s t u d i e d were those which c a r r i e d tRNA^ Ser genes and those w i t h tRNA^ y genes. V a l pDt92 and pDt120, both tRNA^ gene-carrying plasmids, were c h a r a c t e r i z e d i n i t i a l l y to determine the number of i n s e r t s they contained and the s i z e of the i n s e r t s . For plasmids c o n t a i n i n g V a l m u l t i p l e i n s e r t s , the i n s e r t which c a r r i e d the tRNA^ gene was al s o determined. These c h a r a c t e r i s t i c s were s t u d i e d by H i n d l l l d i g e s t i o n of the plasmid DNA, agarose g e l e l e c t r o p h o r e s i s , Southern t r a n s f e r onto n i t r o c e l l u l o s e f i l t e r s and h y b r i d i z a t i o n to tRNA^ . I t was found t h a t both, pDt92 and pDt120 contained two i n s e r t s each of s i z e s 0.5kb and 1.7kb,and 2.0kb and 5.*fkb r e s p e c t -V a l i v e l y , w i t h the 0.5kb and 2.0kb fragments c a r r y i n g the tRNA^ genes. pDt92 and pDt120 then were recloned so as to c o n t a i n only the Va l fragments which c a r r i e d the tRNA^ genes, namely the 0.5kb and 2.0kb fragment r e s p e c t i v e l y . Ser pDt92RC and pDt120RC plus three other tRNA^ y gene c o n t a i n i n g plasmids, pDtl6, pDt1?RC and pDt2?RC were f u r t h e r c h a r a c t e r i z e d by the technique of i n s i t u h y b r i d i z a t i o n to study the o r g a n i z a t i o n of these tRNA genes on the Dr o s o p h i l a genome. Four of these p l a s -mids w i t h the exception of pDt17RC h y b r i d i z e d to only one s i t e on the D r o s o p h i l a chromosome. Both, pDt92RC and pDt120RC h y b r i d i z e d i i i to the 90BC s i t e on the r i g h t arm of the t h i r d chromosome; pDt16 andpDt27RC hybridized to the 12DE s i t e on the f i r s t or the X chro-mosome. pDt17RC on the other hand hybridized predominantly to the 12DE s i t e and to a l e s s e r extent to 2}E (2L), 5 6 D (2R), 62D (3L) and 64D (3L) s i t e s . These i n s i t u h y b r i d i z a t i o n r e s u l t s when studied together with those reported by Dunn et a l . (1979h) show that genes f o r a singl e species of tRNA are located on more than one s i t e on the Drosophila genome. i v TABLE OF CONTENTS Page Abstract i i Table of Contents i v List of Tables v i Li s t of Figures v i i Acknowledgements • • ix Abbreviations x Introduction 1 Materials I. Reagents 6 II. Enzymes 7 III. Solutions 7 IV. Bacterial and Drosophila Strains 10 V. Growth Media 10 Methods I. Isolation of recombinant plasmids carrying Drosophila tRNA genes 12 II. Preparation of plasmid DNA 12 III. H i n d l l l digestion of plasmid DNA 13 IV. Agarose gel electrophoresis • 13 V. Size determination of DNA fragments 1if VI. Southern transfer 1*t VII. Isolation and iodination of purified Drosophila tRNAs H VIII. Hybridization,of DNA on nitrocellulose f i l t e r s to L 1 25l] tRNA 15 Page Methods (Continued) IX. Recloning 15 i ) Ligation of DNA 15 i i ) Transformation 15 i i i ) Hogness procedure 16 iv) Single colony isolation 16 v) Checking clones for single inserts • 17 x* In situ hybridization • 17 i) Preparation of squashes of polytene chromosomes • 17 i i ) Preparation of chromosomes on slides for hybridization 17 i i i ) Iodination of plasmid DNA 17 iv) Synthesis and iodination of cRNA • 18 v) In situ hybridization of plasmid DNA and cRNA 19 vi) Autoradiography 19 v i i ) Analysis of autoradiograms « 19 XI. Containment 19 Results I. Characterization of pDt92 and pDt120 20 II. Recloning of pDt92 and pDt120 20 III. In situ hybridization of v a l i n e - 4 and serine-plasmids • 2k Discussion * 35 Conclusion M) Bibliography ^8 v i LIST OF TABLES Table Page I Hi n d l l l fragments and i n situ hybridization results of recombinant plasmids carrying genes for tRNAVal, tRNAVal and tRNA^ 3 6 v i i LIST OF FIGURES Fi g u r e Page 1. Agarose g e l a n a l y s i s and h y b r i d i z a t i o n of H i n d l l l d i g e s t e d PDt92 w i t h £ 2 5 l ] tRNA^ a l 21 2, Agarose g e l a n a l y s i s and h y b r i d i z a t i o n of H i n d l l l d i g e s t e d pDt120 w i t h [ * 2 5 l ] tRNA^ a l 21 3» I s o l a t i o n of re c l o n e s c o n t a i n i n g recombinant Va l plasmids c a r r y i n g tRNA^ genes 23 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 con-t a i n i n g recombinant plasmids c a r r y i n g tRNA^ a l genes 25 5. Checking r e c l o n e s of pDt92 and pDt120 f o r s i n g l e i n s e r t s 26 6. H y b r i d i z a t i o n of [ ] 2 5 l ] tRNA^ a l w i t h H i n d l l l d i g e s t e d DNAs from r e c l o n e s of pDt92 and pDt120 ... 26 7. Agarose g e l a n a l y s i s of H i n d l l l d i g e s t e d p D t l 6 , pDt17RC and pDt27RC 28 8. : H y b r i d i z a t i o n of H i n d l l l d i g e s t e d p D t l 6 , pDt17RC and pDt2?RC w i t h f 2 5 l j tRNA^ e r 28 9. In s i t u h y b r i d i z a t i o n of pDt92RC 29 10. In s i t u h y b r i d i z a t i o n of pDt120RC 29 11. I n s i t u h y b r i d i z a t i o n of p D t l 6 31 12. I n s i t u h y b r i d i z a t i o n of pDt27RC 31 13. I n s i t u h y b r i d i z a t i o n of pDt17RC 3k 1if. L o c a l i z a t i o n of tRNA genes on the poiytene chromosomes of Dr o s o p h i l a 37 15» Agarose g e l a n a l y s i s of H i n d l l l d i g e s t e d V a l V a l plasmids c a r r y i n g genes f o r t R N A ^ or tRNA,, .... kO v i i i Figure Page 16. Hybridization of [ 1 2 5 l ] tRNA^ with Val 3b Hin d l l l digested plasmids carrying genes for tRNA^ 1 or tRNA^ 1 kO r i 17. Hybridization of L125lJ tRNA^al with Hi n d l l l digested plasmids carrying genes for tRNA^ 1 or tRNA7 a l 42 3b k i x ACKNOWLEDGEMENTS I wish to thank Dr. R.C. Miller, J r . and Dr. G.M. Tener for their support and guidance throughout this work. My very special thanks go to Dr. Shizu Hayashi for teaching me the i n situ procedure and for many helpful suggestions regarding that work. I would also li k e to thank Debbie Taylor for technical assistance and Drs. Allen Delaney and Ian Gillam for helpful discussions and ideas. Finally I would like to thank Dr. T. G r i g l i a t t i for letting me use some of his f a c i l i t i e s . X A b b r e v i a t i o n s Used ATP - 5' - riboadenosine t r i p h o s p h a t e CTP - 5' - r i b o c y t i d i n e t r i p h o s p h a t e GTP - 5' - riboguanosine triphosphate UTP - 5' - r i b o u r i d i n e t r i p h o s p h a t e RNA - r i b o n u c l e i c a c i d tRNA - t r a n s f e r r i b o n u c l e i c a c i d t R N A V a l - nonacylated v a l i n e tRNA Ser tRNA - nonacylated s e r i n e tRNA rRNA - ribosomal r i b o n u c l e i c a c i d cRNA - complementary RNA DNA - de o x y r i b o n u c l e i c a c i d RNase - rib o n u c l e a s e DNase - deoxyribonuclease Arg, Asn, Asp, - amino a c i d s : a r g i n i n e , asparagine, G l u , l i e , Lys, a s p a r t i c a c i d , glutamic a c i d , i s o l e u c i n e , Met, Ser, V a l l y s i n e , methionine, s e r i n e , v a l i n e H i n d l l l - r e s t r i c t i o n endonuclease i s o l a t e d from Haemophilus i n f l u e n z a e Rd pDt - recombinant plasmid c a r r y i n g D r o s o p h i l a tRNA gene "~ pDtRC - recloned pDt (Recloned pDt was designated as pD'tR by Dunn et a l . (1979b)) RPC - reverse phase chromatography LB - L u r i a broth EDTA - ethylene diamine t e t r a a c e t i c a c i d T r i s - T r i s (hydroxymethyl) aminomethane 2-ME - 2-mercaptoethanol x i A b b r e v i a t i o n s Used DTT cpm F i g . mCi min. u l , ml ug, g mM, M 2L, 2R, 3 L , 3R U.V. d i t h i o t h r e i t o l counts per minute f i g u r e •7. m i l l i c u r i e (10 ^ c u r i e ) minute m i c r o l i t e r (10" l i t e r ) , m i l l i l i t e r (10~ l i t e r ) microgram (10"* gram) , gram -3 m i l l i m o l a r (10 ^ molar), molar l e f t or r i g h t arm of second or t h i r d chromosome of D r o s o p h i l a u l t r a v i o l e t 1 INTRODUCTION Transfer RNAs are very important biological molecules. Besides their central role i n protein synthesis, tRNAs have been shown to have many other functions (Rich et a l . , 1976) . In Drosophila there exists some evidence that tRNA genes are di f f e r e n t i a l l y regulated during development (White et a l . , 1973a) and Atwood (1968) has sug-gested that a class of mutants called Minutes represent lesions i n the tRNA genes. The elucidation of the arrangement, structure and the function of the tRNA genes would help to study some of these aspects. The a v a i l a b i l i t y of extensive genetic information and of many mutants together with the existence of polytene salivary gland chromosomes make Drosophila an ideal system for studying tRNA gene organization and expression. Recent studies indicate that there are approximately 590 (Weber and Berger, 1976) to 750 (Ritossa et a l . , 1966) genes for a l l species of tRNA i n the haploid genome of Drosophila. An estimate of i *00-500 genes for ZfS RNA from i n situ hybridization studies (Elder et a l . , 1980) may be too low. On RPC-5 columns, Drosophila tRNA can be resolved into 63 major and 36 minor peaks (White et a l . , 1973a) giving a total of 99 different species of tRNA. It has been sug-gested (White et a l . , 1973b) that several chromatographically dis-tinct forms of isoaccepting tRNAs have the same nucleotide sequence and are probably products of the same gene i.e., these are homo-geneic species resulting from different degrees of post-transcrip-tional modification. From an analysis of the kinetics of RNA:DNA 2 hybridization on membrane f i l t e r s , Weber and Berger (1976) e s t i -mated that Drosophila tRNA i s made up of about 59 families of kine-t i c a l l y different sequences. A l l these data suggest that there i s an average of 10-13 genes (Weber and Berger, 1976; Ritossa et a l . , 1966) for each tRNA sequence. The technique of i n situ hybridization developed by Gall and Pardue (1969) and John et a l . (1969) allows the identification of a genetic locus without possessing mutants of this particular gene. The only prerequisite i s a pure primary gene product or the gene i t s e l f . Using this technique, DNA sequences known to be highly re-dundant i n Drosophila have been mapped on the chromosomes; 5 S RNA genes occur at 56EF (Wimber and Steffenson, 1970) and those for histones at 39DE (Pardue et a l . , 1977). The salivary gland chromo-somes from the mutant, giant, (Kaufman, 1971) with an increase i n the degree of polyteny, f a c i l i t a t e s the localization of genes of a lower tandem redundancy (i.e., tRNAs) than i s available i n the case of 5 S , 18S and 28S rRNAs and histone genes. Steffenson and Wimber (1971) hybridized total Z fS ^H-RNA to intact chromosomes of D. melanogaster and found that i t hybridized to 68 sites widely distributed on the two chromosomes examined. Most recently Elder et a l . (1980) hybridized 1 2 5 I - l a b e l l e d Z f S RNA to salivary gland polytene chromosomes and identified a total of 63 sites. They believe that these 63 sites represent most of the tRNA sites present i n Drosophila. Many of the sites determined by 3 these two groups agree but there are numerous differences, presum-ably due to the d i f f i c u l t y caused by the low specific activity of the ^H-labelled w>S RNA. A comparison of the kS RNA sites with the locations of Minute mutations suggests that the Minute l o c i do not correspond to the structural genes for tRNA (Elder et a l . , 1980; Tener et a l . , 1980). In situ hybridization studies with single, purified tRNA spe-cies show that a single sequence may be present i n several gene copies at more than one site on the chromosomes. Evidence for this conclusion i s provided by Kubli and Schmidt (1978) who localized genes for tRNA^lu at 52F, % E F and 62A, by T. Schmidt et a l . (1978) who localized tRNA^^ genes at 29D and E, by Elder (1978) who showed that tRNA A r S hybridized to ^ 2A and 84F and tRNA M e t to Zf8AB and 72F-Val 73A and by Dunn et a l . (1979a) who localized the genes for tRNA^ at 8^ D, 90BC and 92B. Most recent evidence i s provided by Hayashi et a l . (1980) who showed that essentially a l l the twelve purified tRNAs tested, hybridized to more than one site on the polytene chromosomes. Whether a l l these tRNA sites truly represent pre-sence of the respective gene depends on the purity of the tRNA used for hybridization. It i s not possible to rule out that at least some sites are the result of traces of other RNAs hybridizing to highly redundant sequences on the DNA ( G r i g l i a t t i et a l . , 197^5 Hayashi et a l . , 1980; Elder et a l . , 1980 ) . The development of techniques for molecular cloning of DNA (Sinsheimer, 1977) has now made i t possible to isolate the tRNA genes i n pure form. This methodology has allowed the study of the organization of tRNA genes i n the genomes of yeast (Beckman et a l . , 1977) , Xenopus l a e v i s (Clarkson et a l . , 1978) , Drosophi la (Yen et a l . , 1977; Dunn et a l . , 1979b) and Bombyx mori (Hagenbuchle et a l . , 1979) . A recombinant p lasmid , pC1T12 (Yen et a l . , 1977) c o n t a i n -i n g a 9.34 kb Drosophi la DNA fragment has been found to conta in a t o t a l of e ight tRNA genes i r r e g u l a r l y spaced wi th in the DNA and coding for a s i n g l e tRNA 4 1 " 2 , three t R N A A s n , one t R N A I l e and three t R N A | y s (0. Schmidt et a l . , 1978; Hovemann et a l . , I 98O) . T r a n -s c r i p t s from t h i s plasmid hybr id i zed to the i\2.k reg ion on the Dro-s o p h i l a chromosome (Yen et a l . , 1977) . Dunn et a l . (1979b) undertook to i s o l a t e tRNA genes from Drosophi la on recombinant plasmid DNA molecules by the "shot gun" technique. Such cloned tRNA genes would be u s e f u l as h y b r i d i z a t i o n probes to l o c a l i z e tRNA genes on the Drosophi la genome, they could serve as mater ia l fo r nucleot ide sequence a n a l y s i s to study the s t ruc ture of the tRNA genes and f i n a l l y they could serve as tem-p la tes for t r a n s c r i p t i o n s t u d i e s . The procedure used to get the recombinant plasmids was given by Dunn et a l . (1979b). A t o t a l of 90 c lones of recombinant p l a s -mids conta in ing genes for eleven d i f f e r e n t tRNAs were i s o l a t e d . Before these plasmids could be used for any of the purposes l i s t e d above, they had to be charac ter i zed i n i t i a l l y to determine the number of i n s e r t s they c a r r i e d , the s i z e of the i n s e r t s and the i n s e r t which contained the tRNA gene. Where necessary those; ,p las-mids which c a r r i e d mul t ip le i n s e r t s were r e c l o n e d . Such charac te r -i z a t i o n f o r some of the recombinant plasmids i s o l a t e d was reported by Dunn et a l . (1979b). Some of these plasmids then were used to 5 study the organization of tRNA genes on the Drosophila chromosomes by the technique of i n situ hybridization (Dunn et a l . , 1979b). The purpose of this study was to finish characterization of two groups of recombinant plasmids isolated by Dunn et a l . , (1979b); Ser Val those which contained tRNA. n genes and those with tRNA. genes. In situ hybridization studies on five of these plasmids confirmed the location of their genes on the chromosomes and added to the general picture that i n Drosophila, genes for a single species of tRNA occur at more than one site on the genome. 6 MATERIALS I. Reagents Description ATP Agar Agarose powder Ampicillin Bromophenol blue Casamino acids Cesium chloride (technical grade) Chloramphenicol Dextrose Dithiothreitol E-coli tRNA EDTA Ethidium bromide Gelatin Hydroxyapatite 125 Carrier-free sodium y~L (~500mCi/ml) 2-mercaptoethanol Sodium lauryl sulphate (SDS) Sucrose Tetracycline Source Calbiochem Behring Corp* Difco Laboratories Bio-Rad Laboratories Ayerst Laboratories BDH Chemicals Ltd. Difco Laboratories Kawecki Berylco Industries, Inc. Sigma Chemical Co. Difco Laboratories Calbiochem Sigma Chemical Co. J.T. Baker Chemical Co. Sigma Chemical Co. Fischer Scientific Co. Bio-Rad Laboratories Amersham Searle Co. Bio-Rad Laboratories BDH Chemicals J.T. Baker Sigma Chemical Co. Description Thallium chloride Thiamine Thymidine Toluidine blue 0 Triethanolamine Triton X-100 Tryptophan' Uridine Source ICN. K & K Labs., INC. Calbiochem Worthington Biochemical Corp. Baker Chemical Co. Fischer Scientific Co. Sigma Chemical Co. Fischer Scientific Co. Sigma Chemical Co. II. Enzymes Bacterial alkaline phos-phatase-F DNase I Lysozyme (egg white) Pronase Proteinase K (fungal) RNase-A (Bovine pancreas) RNA Polymerase I (E-coli) Restriction endonuclease Hindl l l Worthington Biochemical Corp. Worthington Biochemical Corp. Sigma Chemical Co. Calbiochem BDH Chemicals Sigma Chemical Co. P-L Biochemicals Inc. New England Biolabs T^ polynucleotide ligase Provided by Dr. R.C. Miller III. Solutions 1• For preparation of plasmid DNA i ) Tris-HCl 0.05M Sucrose 25% pH8.0 i i ) T r i t o n X-100 10% Tris-HCl 0.05M pH8.0 EDTA 0.0625M i i i ) D i a l y s i s buffer (TEN) Sodium chloride 0.02M Tris-HCl 0.02M pH8.0 EDTA 0.001M 2, R e s t r i c t i o n Endonuclease buffers i ) H i n d l l l buffer Sodium chloride 60mM Magnesium chloride 7mM Tris-HCl 10mM pH7.lf G e l a t i n 100 ug/ml 3. Agarose gel electrophoresis i ) Tris-Phosphate buffer T r i s O.O^M pH8.0 Sodium phosphate 0.02M (NaH2P0^) EDTA 0.001M i i ) Loading mix Sucrose 1+0% EDTA 0.025M Bromophenol blue 0.02% k» Southern transfer and Hybridization i ) Denaturing s o l u t i o n Sodium hydroxide 0.2M Sodium chloride 0.6M Thymol blue 20 mg/l i i ) N e u t r a l i z i n g buf fer Sodium ch lo r ide 1.5M T r i s - H C l •> 0.5M pH7.4 i i i ) IPX SSC Sodium ch lo r ide 1.5M Sodium c i t r a t e 0.15M i v ) 2 X S S C +0.5% SDS Sodium c h l o r i d e 0.3M Sodium c i t r a t e 0.03M sodium dodecyl 0.5% sulphate Recloning procedure and Hogness h y b r i d i z a t i o n i ) L i g a t i o n buf fe r T r i s - H C l 50mM pH7.5 Magnesium c h l o r i d e 10mM DDT 1mM ATP 1mM i i ) Sodium ch lo r ide 1.5mM T r i s - H C l 0.5M PH7.4 In s i t u h y b r i d i z a t i o n i ) Beadle and E p h r u s s i ' s Ringers So lu t ion Sodium ch lo r ide 7.50 g Potassium c h l o r i d e 0.35 g Calcium c h l o r i d e 0.31 g Make up the volume to a l i t e r 10 i i ) Formamide buf fer Potassium phosphate 0.06M (KH 2P0^) Potassium phosphate 0.06M (KgHPO^) EDTA 0.01M Potassium hydroxide 0.027M Potassium c h l o r i d e 0.5M Formamide 70% pH 7 . 0 IV. B a c t e r i a l and Drosophi la s t r a i n s i ) E . ctol i - SF8 was obtained from M. Olson (Olson et a l . , 1977) i i ) pBR322 - B a c t e r i a l s t r a i n conta in ing the plasmid pBR322 ( B o l i v a r et a l . , 1977) was provided by H. Boyer. i i i ) Mutant, g i a n t , of Drosophi la melanogaster (Kaufman, 1971) V. Growth Media i ) L u r i a Broth Tryptone 10g Yeast ex t ract 5g Sodium c h l o r i d e 5g D i s t i l l e d water 11 ( vo lumet r ica l ly ) pH7.2 P l a t e s of LB were made with 10 g/1 agar LB agar - a m p i c i l l i n p la tes contained 50 ug/ml a m p i c i l l i n LB agar - t e t r a c y c l i n e p la tes contained 20 ug/ml t e t r a c y c l i n e i i ) M9S medium M9 medium (Champe and Benzer, 1962) p lus Casamino a c i d s 0.2% 11 i i i ) M9S medium plus Uridine 200 ug/ml Thiamine 0.5 ug/ml Tryptophan 50 ug/ml iv) Drosophila growth medium (Lewis, i960) 12 METHODS I. Isolation of recombinant plasmids carrying Drosophila melano-gaster tRNA genes Recombinant plasmids carrying Drosophila melanogaster tRNA genes were constructed by the "shot gun" technique where H i n d l l l cleaved Drosophila DNA was ligated to H i n d l l l cut pBR322 DNA. This work was done by Dunn et a l . (1979b). The plasmids were num-bered sequentially as they were isolated and identified as pJLasmid Drosophila tRNA N, pDtN. II. Preparation of Plasmid DNA i ) pDtl6, pDt2?RC, pDt92 and pDt120 plasmid DNAs were pre-pared as described by Dunn et a l . (1979b). i i ) pDt17RC DNA was prepared by a slight modification of the procedure by Dunn et a l . 0979b). Norgard et a l . (1979) reported about three times greater yield of plasmid DNA when plasmid carry-ing bacteria was grown i n presence of uridine and later treated with chloramphenicol, than would normally be obtained by routine plasmid amplification procedures. Therefore, E-coli containing pDt17RC was grown to about 6.10 bacteria/ml at 3 0 C i n M9S medium plus uridine, thiamine and tryptophan,, Chloramphenicol ( 8 0 mg/ml in 95% ethanol) was added to a f i n a l concentration of 200 ug/ml and the culture was incubated overnight. One l i t e r of the cel l s was collected by centrifugation ( 8 0 0 0 RPM - 15 min.) and the cel l s were resuspended in 5ml 25% sucrose, 0.05M Tris (pH8.0). 2 . 5 ml 0.5M EDTA was added and mixed. 1ml lysozyme (5mg/ml) then was 13 added and the mixture was incubated at l+°C for 10 min. Finally 7ml Triton (2%) was pipetted rapidly into the solution and the lysate was centrifuged at Zf°C for 60 min. at 25,000 RPM. The super-natant (cleared lysate) was collected and centrifuged to equilibrium in CsCl and ethidium bromide according to Bazaral and Helinski (1968)• The plasmid DNA was collected and centrifuged to equilibrium in CsCl and ethidium bromide once again. The plasmid DNA was col-lected, the ethidium bromide was removed with butanol, and the solu-tion was dialyzed against TEN buffer overnight at £f°C. The solu-tion of plasmid DNA was phenol extracted, ether washed and stored at -20°C. III. Digestion of plasmid DNA with restriction endonuclease Plasmid DNA was cut with Hind l l l in Hin d l l l buffer at the enzyme concentration of 1 unit/ug DNA i n the reaction volume of 20-50 u l . The digestion was done at 37°C for 2 hours. IV. Agarose gel electrophoresis 0.5%» 1% or 2% agarose gels were run in Tris-phosphate (pH8.0) buffer with 1 ug/ml ethidium bromide, in a Studier gel apparatus (McDonell et a l , , 1977). DNA samples were mixed with 0.2 volume of loading mix before loading the gel. The electrophoresis was carried out at /f°c for 1.5-3.5 hours at 8 5 V (6.5V/cm). The gel was exposed to U.V. light and photographed through an orange f i l t e r using Type 57 Polaroid film. i ) V. Size determination of DNA fragments The size of DNA fragments resulting from H i n d l l l digestion of recombinant plasmids was determined by linear regression analysis, using Hind l l l cut lambda DNA as a reference (Murray and Murray, 1975) . Linear regression analysis was performed using a HP9810 A calculator made by Hewlett-Packard. VI. Southern transfer The DNA i n the agarose gel was transfered to nitrocellulose paper by the procedure of Southern (1975)• VII. Isolation and iodination of purified Drosophila tRNAs tRNA from adult Drosophila melanogaster was isolated and puri-fied using a number of chromatographic procedures (Dunn et a l . , 1979b) . F i r s t i t was separated on BD-cellulose column (White and Tener, 1 9 7 3 ) » then on Sepharose 6B i n the presence of ammonium sulphate (Holmes et a l . , 1975) . Fractions were further purified on RPC-5 column (Pearson et a l . , 1971) . Purification by similar Val methods of three species of tRNA has been described by Dunn et a l . ( 1978 ) . The system of numbering the isoacceptors of the various tRNAs was taken from White et a l . ( 1 9 7 3 a ) . Amino acids acceptance studies and other c r i t e r i a of purity suggested that the purified tRNAs were not less than 95% pure with no single contami-nant representing more than 2% of the total material. The puri-fied tRNAs were isolated by and obtained from Dr. Ian Gillam. The purified tRNAs were iodinated with by the procedure of Commerford ( 1 9 7 1 ) . 15 VIII. Hybridization of DNA on nitrocellulose f i l t e r to tRNA The f i l t e r with DNA was saturated with 2XSSC containing ap-proximately 5.10^-1.10^ cpm of C 2 5 l ] tRNA. The f i l t e r was placed between mylar sheets and glass plates, wrapped i n Saran wrap and incubated at 65°C for 6 hours. After incubation the f i l t e r was washed three times with 2XSSC, incubated for 30 min. at 37°C i n 10 ug/ml RNase A, washed three times i n 2XSSC, 0.1% SDS and f i n a l l y washed three more times i n 2XSCC. The f i l t e r was dried and auto-radiographed for if8 hours using preflashed X-ray screen film and Dupont screen i n a cassette at -70°C. IX. Recloning procedure i) Ligation 10 ug of pBR322 DNA cut with H i n d l l l was incubated with 1.4 units of bacterial alkaline phosphatase i n 50 ul containing 20 u l of TEN buffer and 5 u l of 1;M Tris pH8.0. This mixture was incubated at 60°C for 60 min. 1+ ug of BAP-treated pBR322 was co-precipitated with 1 ug of Hindl l l cut pDt92 or pDt120:in 0.25M sodium acetate (pH/+.5) and four volumes of 95% ethanol at -20°C overnight. The precipitate was resuspended i n 50 ul of ligation buffer. 20 units of T^ polynucleotide ligase were added and the mixture was incubated at 12°C for k hours. i i ) Transformation 0.2 ul of CaCl 2-treated SF8 cel l s (Dunn et a l . , 1979b) were mixed with 30 ul of 0.1M CaCl 2 and 50 ul of ligated pDt92 or pDt120 i n ligation buffer. The transformation mixture was incubated 16 at if°C for 60 min. after which i t was heat-pulsed at 42°C for 10 min. The culture then was diluted into 10ml of LB and grown for 3 hours at 30°C. The bacteria were plated on LB-ampicillin or LB-tetracycline plates and scored for drug resistance. i i i ) Hogness procedure Bacteria transformed with pDt92 or pDt120 were plated on large (1q.0mm diameter) LB-ampicillin plates to yield approximately 300 colonies per plate. The colonies were replica plated onto nitro-cellulose f i l t e r s on LB-ampicillin plates. The f i l t e r s were treated as described by Grunstein and Hogness (1976). The actual procedure followed was as described by Dunn et a l . (1979b). The f i l t e r s then were hybridized to tRNA^al as described earl i e r . iv) Single colony isolation Colonies containing plasmids carrying tRNA genes were located on master plates and streaked for single colonies on LB agar without any antibiotic. 9-10 isolated colonies were picked from each plate and transferred simultaneously to an LB-ampicillin plate and onto a nitrocellulose f i l t e r on an LB-ampicillin plate. [1 25 I "Veil ^TJ tRNA^ as des-cribed above. Streaking for single colonies on non-selective medium (LB agar) was done to isolate non-transformed colonies (these are ampi-c i l l i n sensitive and appear as tiny s a t e l l i t e colonies around trans-formed, ampicillin resistant colonies on selective LB-ampicillin medium) from the transformed ones. 17 v) Checking clones for single insert carrying tRNA gene Positive colonies containing tRNA genes after second hy-bridization were grown up i n M9S medium; plasmid DNA was isolated, cut with H i n d l l l , run on an agarose gel, transferred to a nitrocel-lulose f i l t e r , (Southern transfer) and hybridized to f 2 5 l ] tRNA^al, X. In situ hybridization of plasmid DNA to polytene chromosomes i) Preparation of salivary gland squashes Polytene chromosomes were prepared from salivary glands of late third instar larvae of the mutant, giant, of Drosophila  melanogaster (Kaufman, 1971) according to the method of Atherton and Gall ( 1 9 7 2 ) . i i ) Preparation of slides Slides with squashes were prepared for i n situ hybridi-zation according to the procedure of Gall and Pardue (1971) with the additional incubation of the preparation i n 2XSSC for 30 min. at 70°C (Bonner and Pardue, 1976) prior to i n i t i a l RNase treatment and the acetylation of squashes (Hayashi et a l . , 1978) to reduce background on autoradiography. i i i ) Iodination of plasmid DNA ( p D t l 6 , p D t 2 7 R C , p D t 9 2 R C , pDt120RC 5 ug of plasmid DNA (dried) was denatured i n 20 u l of 0.3M sodium hydroxide. Enough 1M acetic acid was added to bring the pH up to 4 . 5 - ^ . 7 followed by addition of water to bring the total volume up to 40 u l . The denatured DNA then was iodinated with | ^ 2 ^ l j by a procedure adapted from the methods of Commerford ( 1 9 7 1 ) . 10-iul of 5mM thallium chloride was added to the denatured 18 sample, and the mixture was incubated at 70°C for 10 min. Approxi-mately 1mCi of carrier-free Na [? 2^l] was mixed with 3 ul of 0.4mM Nal before addition to the DNA sample, Iodination was allowed to proceed at 70°C for 30 min. After iodination, 400 ul of 0.03M sodium phosphate and 10 ul of 0.1M sodium sulphite were added and mixed thoroughly. Finally, 135 ug of E-coli tRNA was added as car-r i e r , and the mixture was loaded onto a hydroxylapatite column (0,5X1cm). The column was washed with 25ml of 0.03M sodium phos-phate buffer, pH7.0 and the DNA was eluted with 0.48M sodium phos-phate pH7.0. The 1 2 5 I - l a b e l l e d plasmid DNA was heated at 70°C for 10 min. and then chromatographed on a Sephadex G-25 column (0.9X 40cm) i n 70% formamide buffer, pH7.0. The specific activity ob-n tained was usually around 1-2.10 cpm/ug DNA. iv) Synthesis and iodination of cRNA (pDt17RC) CTP was provided by Dr. Shizu Hayashi. 125n D 2 5 l ] ?I-cRNA was synthesized from pDt17RC DNA by the proce-dure described by Wensink et a l . (1974) with a few modifications. 5 ug of pDt17RC DNA was preincubated with 15 ul of 0.08M Tris-HCl PH8.0, 3 u l of 0.1M MgCl 2, 3 u l of 0.1M 2-ME, 1 unit of E-coli RNA polymerase and 6 ul of water at 37°C for 10 min. 0 . 6 ul each of 10"^ M ATP, GTP and UTP was added and this mixture then was added to about 35 uCi of dessicated {^ 2^lj CTP and incubated at 37°C for 2 hours. Following this reaction, the DNA template was destroyed by digestion with DNase. 200 ul of 0.08M Tris-HCl pH7.8, 47.1 ul E-coli tRNA (100 ug), 152.9 u l water and 1 u l of 10 ug/ml DNase I was added, and the mixture was incubated at 25°C for 20 min. This 19 was followed by phenol (3X) and ether (3X) extractions, cRNA was separated from nucleoside triphosphates by Sephadex G-25 chromato-graphy i n 0.3M sodium acetate pH6.5» The labelled cRNA was ethanol precipitated, and the precipitate was resuspended i n 70% formamide buffer. v) In situ hybridization 70% formamide buffer was used to hybridize to chromosome squashes at 35°C and k5°C respectively for varying periods of time. After hybridization, unbound plasmid DNA or cRNA was removed as described by Gall and Pardue (1971). vi) Autoradiography to G r i g l i a t t i et a l , (197*f), After developing the emulsion, chromo-somes were stained with 0»k% toluidine blue 0 i n 2XSSC. vi i ) Analysis of Autoradiograms graphic representations of the polytene banding pattern of D, mela-nogaster by Lefevre (1976), XI, Containment Bacteria containing recombinant plasmids were handled under B-M containment conditions, as specified by the regulations of the Medical Research and National Science and Engineering Research Councils of Canada, cRNA in The slides were prepared for autoradiography according The labelled bands were identified with the aid of photo-20 RESULTS !• Characterization of pDt92 and pDt120 pDt92 and pDt120 were characterized i n i t i a l l y to determine the number of inserts (i.e, Drosophila DNA fragments) they con-tained, the size of the inserts and the insert which carried the tRNA gene. Both, pDt92 and pDt120 when cut with H i n d l l l and run on agarose gels showed 3 bands each (Figs. 1 and 2 respectively). The sizes of the 3 bands of pDt92 were 0.5kb, 1.7kb and 4.i+kb, whereas the sizes of the 3 bands of pDt!20 were 2.0kb, 4.4kb and 5.4kb. The 4.A>kb band i n each case corresponded to p B R 3 2 2 , therefore p D t 9 2 and p D t 1 2 0 contained two inserts each. Southern transfer to nitrocellulose f i l t e r and hybridization to 0 2 5l] tRNA^al showed that the 0.5kb fragment of pDt92 (Fig. 1) Val and 2.0kb fragment of pDt120 (Fig. 2) contained the tRNA^ genes. II. Recloning of pDt92 and pDt120 pDt92 and pDt120 were recloned to contain only the inserts Val ' which carried the tRNA^ genes i . e . the 0.5kb and the 2.0kb frag-ment respectively. Fig. 3 i s a photograph of an autoradiogram of a hybridization of 025l] V a l tRNA^ to bacterial colonies after the f i r s t Hogness procedure (METHODS). 6 clones out of a total of 300 colonies on that f i l t e r hybridized, a frequency of about 2%. This autoradio-gram represents the result obtained with pDt120; a similar result 21 Fig, 1. Agarose gel analysis and digested PDt92 with [ j 2 5I The plasmid pDt92 and control DNAs were cleaved with Hi n d l l l and were electrophoresed on 1% agarose gel i n T r i s -phosphate buffer (pH8.0) for 2 . 5 hours at 85 volts. The fragments were visualized by staining with ethidium bromide and exposure to U.V. li g h t . Lane 1, A DNA; lane 2, pDt92; lane 3 , p B R 3 2 2 . The agarose gel was treated by the procedure of Southern ( 1 9 7 5 ) . The transfered DNA was incubated with 0 2 5ll tRNAYal J 4 followed by autoradiography. The unmarked lane on the right corresponds to lane 2, i . e . pDt92. hybridization of Hi n d l l l . _..TAVal tRNA. • Fig. 2 . Agarose gel analysis and hybridization of H i n d l l l digested p D t l 2 0 with E 2 5l] tRNA^al. The plasmid pDt120 and control DNAs were cleaved with Hi n d l l l and were electrophoresed on 2% agarose gel i n T r i s -phosphate buffer (pH8.0) for 2 . 75 hours at 85 volts. The gel was stained with ethidium bromide and photographed under U.V. ligh t . Lane 1, A DNA; lane 2 , pBR322; lane 3 , pDt120. The agarose gel was treated by the procedure of Southern (1975) and incubated with [ 1 2 5 l ] tRNA^al. The unmarked lane on the right corresponds to lane 3» i . e . pDt120. F i g . 1 2 3 Fig. 3 * Isolation of reclones containing recombinant plasmids Val carrying tRNA^ genes. Hindll l cut and bacterial alkaline phosphatase treated pBR322 was ligated to Hindl l l cut pDt120. The ligated DNA was used to transform E-col i . Bacteria grown on the nitrocellulose f i l t e r s were treated according to the procedures of Grunstein and Hogness (1976) and incubated with [ [ 2 5 l ] tRNA^al. The f i l t e r s then were washed and developed for autoradiography. This photograph repre-sents a typical autoradiogram after such a procedure. 2 4 was obtained with pDt92, though the frequency was only about 1%. F i g . 4 represents the autoradiogram of the hybridization with b a c t e r i a l colonies a f t e r the second Hogness procedure (METHODS) i . e . a f t e r the pos i t i v e colonies were streaked for i s o l a t i o n on non-selec t i v e medium to eliminate contaminating non-transformed c e l l s . Not a l l the colonies transfered to the f i l t e r contained a plasmid at t h i s point, i n d i c a t i n g that these colonies were not transformed, thus confirming the need for streaking on non-selective medium. 6 clones of pDt120 and 1 clone of pDt92 giving h y b r i d i z a t i o n with tRNA^ a l a f t e r the second Hogness were tested for single i n s e r t s . F i g . 5 shows that one clone of pDt92 and 3 out of 6 clones of pDt120 contained single i n s e r t s , and F i g . 6 shows that these single Val i n s e r t s carried the tRNA^ genes. I I I . In s i t u h y bridization Five recombinant plasmids, pDtl6, pDt17RC, pDt27RC, pDt92RC and pDt120RC were characterized further by i n s i t u h y b r i d i z a t i o n . pDtl6, pDt17RC and pDt27RC have i n s e r t s of sizes 6.7kb, 3.3kb Ser and 6.1kb respectively (Fig. 7) and they a l l carry tRNA, n genes (Fi g . 8). The plasmid DNAs of pDtl6, pDt27RC, pDt92RC and pDt120RC were l a b e l l e d with [ j 2 ^ l 3 and hybridized to squashes of polytene chro-mosomes. A l l four of these plasmids hybridized to only one s i t e on the Drosophila chromosome. pDt92RC (Fig. 9) and pDt120RC ( F i g . 10) hybridized to the 90BC s i t e on the ri g h t arm of the t h i r d chro-mosome; pDtl6 (Fig. 11) and pDt27RC ( F i g . 12) hybridized to the 12DE s i t e on the X chromosome. 2 5 ••••••• • ••• # Fig. 4. Single colony isolation of bacteria containing recombinant Val plasmids carrying tRNA^ genes. Val Positive colonies containing plasmids carrying tRNA^ genes were picked from the master plates and were streaked for isolation on LB-agar i n the absence of any antibiotic. About 9-10 isolated colonies from the non-selective LB-agar plates were transfered to secondary master plates (LB-ampicillin), replicated to nitrocellu-lose f i l t e r s on LB-ampicillin agar and treated as described i n Fig. 3 and METHODS. This photograph i s an autoradiogram after such a procedure. 26 F i g . 5. Checking r e c l o n e s of pDt92 and pDt120 f o r s i n g l e i n s e r t s . Plasmid DNA was prepared from s e v e r a l p o s i t i v e clones from the secondary master p l a t e s . The recloned plasmid DNAs and c o n t r o l DNAs were cleaved w i t h H i n d l l l and were electrophoresed on 1% agarose g e l i n Tris-phosphate b u f f e r (pH8.0) f o r 2.75 hours at 85 v o l t s . The g e l was s t a i n e d w i t h ethidium bromide and photographed under U.V. l i g h t . Lanes 1-6, s i x d i f f e r e n t r e c l o n e s of pDt120; lane 7, pBR322; lane 8, X DNA; lane 9> a r e d o n e of pDt92;:-lane 10, unrecloned pDt92. Note, that three r e c l o n e s of pDt120 i n lanes 2-4 and one r e d o n e of pDt92 i n lane 9 have s i n g l e i n s e r t s . F i g . 6. H y b r i d i z a t i o n of L. I j tRNA^ w i t h H i n d l l l d i g e s t e d DNAs from r e c l o n e s of pDt92 and pDt120 . The agarose g e l photographed i n F i g . 5 was t r e a t e d by the procedure of Southern ( 1975) and incubated w i t h H 2 5l] tRNA^ a l Lanes 1-10 correspond to lanes 1-10 of F i g . 5 ; i . e . lanes 1-6, re c l o n e s of pDt120; lane 7 , p B R 3 2 2 ; lane 8, ,\ DNA; lane 9> r e -clone of pDt92; lane 10 , unrecloned pDt92. 27 F i g . 6. 1 2 3 4 5 6 7 8 9 ,101 28 F i g . 7. Agarose g e l a n a l y s i s of H i n d l l l d i g e s t e d pDt16, pDt17RC and pDt27RC. The plasmids p D t l 6 , pDt17RC, pDt27RC and c o n t r o l DNAs were cleaved w i t h H i n d l l l and were electrophoresed on 0.5% agarose g e l i n Tris-phosphate b u f f e r (pH8.0) f o r 2.5 hours at 85 v o l t s . The g e l was 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, pDt27RC; lane 2, pDt17RC; lane 3 , pDt16; lane 4» A DNA; lane 5, pBR322. F i g . 8. H y b r i d i z a t i o n of H i n d l l l d i g e s t e d pDt!6, pDt17RC and PDt27RC wi t h £ 2 ^ tRNA| e r. The agarose g e l photographed i n F i g . 7 was t r e a t e d by the pro-cedure of Southern (1975). The t r a n s f e r e d DNA was incubated w i t h P25 1 Ser L IJtRNA^ fo l l o w e d by autoradiography. Lane 1, pDt16; lane 2, pDt17RC; lane 3 , pDt2?RC. 29 Fig. 9. In situ hybridization of pDt92RC. pDt92RC was labelled with N i ] to a specific activity n of approximately 3.5*10 cpra/ug DNA and then hybridized to Drosophila salivary gland chromosomes as described i n METHODS. The photographic emulsion was exposed for 9.5 weeks and on development showed grains over the region 90BC (arrow). Fig. 10. In situ hybridization of pDt120RC. pDt120RC (2.9.10^ cpm/ug) was hybridized as out-lined i n the legend to Fig. 9. Region 90BC (arrow) was labelled after k weeks. 30 F i g . 9. 31 Fig. 11. In situ hybridization of pDtl6. pDtl6 (1.1* IO' ' cpm/ug) was hybridized as described i n the legend to Fig. 9. Site 12DE (arrow) was labelled after 16 days. Fig. 12. In situ hybridization of pDt27RC. pDt27RC (2.5.107 cpm/ug) was hybridized as out-lined i n the legend to Fig. 9. Site 12DE (arrow) was label-led after 27 days. F i g . 11 F i g . 12 3 3 In the case of pDt17RC, I-labelled cRNA was synthesized f i r s t using the plasmid DNA as template. The cRNA then was used to hybridize to squashes of polytene chromosomes. Several advan-tages were observed using this method over the DNA:DNA hybridiza-tion method described earlier: i ) The specific activity achieved by this method (about 2 - 3 . 1 0 7 cpm/ug RNA) was much higher than that obtained by the other method (1-2.10 cpm/ug DNA), allowing grains to be seen i n a matter of few days to a week, as opposed to 3-10 weeks for the DNA-DNA hybridization method. specific because RNA and proteins i f present i n the plasmid preparation also get labelled, contributing to higher back-ground. Unlike the results obtained with the other four plasmids, pDt17RC-cRNA hybridized to more than one site on the Drosophila chromosomes (Fig. 1 3 ) . The highest number of grains was seen at the 12DE site; fewer, but with consistency, grains were also seen at 23E (2L), 56D (2R), 62D ( 3 D and 6^D ( 3 D . i i ) The labelling of cRNA more specific, being restricted to the transcript being made. Commer-ford's method of labelling plasmid DNA with P 2 ^ l l i s not 3k F i g . 13. In s i t u hybridization of pDt17RC. pDt1?RC was used as a template to synthesize cRNA which was la b e l l e d with f 2 ^ l j - C T P to a s p e c i f i c a c t i v i t y of approximately Q 10 cpm/ug RNA and then hybridized to Drosophila s a l i v a r y gland chromosomes as described i n METHODS. The photographic emulsion was exposed for 8 days and on development showed grains over 12DE, 2 3 E , 56D, 62D and 6*fD (arrows). 35 DISCUSSION Val When a l l the tRNA^ gene containing plasmids (Dunn et a l . , 1979b) were studied together as a group, i t was found that they f e l l into four different sized groups (Table I) with pDt23, pDt55, pDt92RC and pDt120RC with insert sizes of 12.0kb, 8.0kb, 0.5kb and 2.0kb respectively being representatives of each of these groups. S e r Similarly, the plasmids containing tRNA. n genes (Dunn et a l . , 1979b) f e l l into five different sized groups (Table I) of insert sizes 3.3kb, If.^kb, 4.7kb, 6.1kb and 6.7kb. These plasmids are classified as tRNAfe£ because both, highly purified tRNA? e r and tRNA^er, hybridize to a l l these plasmids. tRNA^er and tRNA^er are also known to hybridize to identical sites on the Drosophila chro-S e r mosomes (Hayashi et a l . , I98O). It has been shown that tRNA^ and tRNAy have different anticodons (White et a l . , 1975) but se-quencing data indicates that the two tRNAs di f f e r by only a few nucleotides (D. Cribbs, unpublished). Thus these results may be explained most easily by cross-hybridization. Plasmids carrying more than one insert were recloned to con-tain only the inserts with the tRNA genes i n order to assign them unambiguous location on the Drosophila chromosomes by i n situ hy-bridization. in situ hybridization of twelve tRNAs by Hayashi et a l . (I98O) showed that essentially a l l the purified tRNAs hybridized to more than one site on the polytene chromosomes of the salivary glands of D. melanogaster (Fig. 1i+). Sites which were heavily labelled were termed •major' sites, and l i g h t l y labelled sites were called 36 Table I. H i n d l l l Fragments and In Situ Hybridization Results of Recombinant Plasmids Carrying Genes for tRNAyal, tRNA^f1 Ser ^ b and tRNA? £ H » ( Plasmid tRNA Gene Hindlll fragment (Size, kb) Hybridization Site 1 tRNA hybridization at this site2 pDt23 Val -4 12.0 89B minor pDt55 Val -4 8.0 70BC major pDt92RC Val-4 0 .5 90BC minor pDt120RC Val-4 2.0 90BC minor pDtiflRC Val-3b 2.0 90BC minor pDt48 Val-3b 2.4 90BC i minor pDt78RC Val-3b 5.2 84D major P D t 5 Ser-4 ,7 4.4 23E major pDtl6 Ser-4,7 6.7 12DE major pDt17RC Ser-4 ,7 3.3 12DE, 23E, 56D major, minor 62D, 64D pDt27RC Ser-4 ,7 6.1 12DE major pDt73 Ser-4 ,7 4.7 12DE major Plasmid DNA was used for hybridization i n each of these cases except pDt17RC, where cRNA synthesized from pDt17RC was used. tRNA i n situ hybridizations were done by Hayashi et a l . (1980),. 37 S4 S7 m n i — i — i G3 l 1 2L R2 K2 I 1 r M3 M2 Z4" | K2 T3j K 5 K2 G3 G3 54° G3 i rn V4 2R n n M3Z4 V3a L2 161 V4 M3 M2 KS, V3b R 2 S2b K5 S2b rAZl *3 M 2 " ° Y T 3 V4 V3b v j . j V4 I i i 'Y V3a V4 V4 A '.11,1 .-fl i Ji  (!) *J >«1«I .'I I » " « " ' » *h> <' I •> IVI I HI I V3bT4 S2b I—i—I I m 3 R * sum (:t i « ? n I i f l i u n n i w n u i n w i i : ;• F i g . 14. L o c a l i z a t i o n of tRNA genes on the polytene chromosomes of D r o s o p h i l a (S. Hayashi). In s i t u h y b r i d i z a t i o n of about twelve p u r i f i e d , £|2^l] tRNAs was performed by Hayashi et a l . ( 1 9 8 0 ) . These r e s u l t s are p l o t t e d on the composite photographs of polytene chromosomes prepared by Lefevre ( 1976 ) . Standard amino a c i d a b b r e v i a t i o n s are used to de-signate s i t e s f o r the corresponding tRNA genes. The r e l e v a n t ones are: V3b, tRNA 7* 1; v<+, tRNA 7* 1; Sk, S 7 , tRNA^ e£. The h e a v i e r , l a r -ger type designates 'major' s i t e s and the l i g h t e r , s m a l l e r p r i n t designates 'minor' s i t e s . The l i n e s above the chromosomes cover regions where Minute mutants map. 38 'minor' s i t e s . t R N A ^ x h y b r i d i z e d to two major s i t e s , 8i*D and 92B and one minor s i t e 90BG, a l l these s i t e s being present on the r i g h t V a l arm of the t h i r d chromosome (3R)» On the other hand, tRNA^ hy-b r i d i z e d to 56D (2R), 70BC ( 3 L ) , the two major s i t e s and 84D (3R)» 89B ( 3 R ) , 90BC (3R) and 92B ( 3 R ) , the many minor s i t e s ; a l l these s i t e s being d i s t r i b u t e d on d i f f e r e n t arms and d i f f e r e n t chromosomes. Ser S i m i l a r l y the major s i t e s f o r tRNA^ r,, 12DE (X chromosome), 23E (2L) and the minor s i t e s , 56D (2R), SkT> (3L) l i e on d i f f e r e n t chromosomes. The confidence t h a t can be placed i n the r e s u l t s of the tRNA i n s i t u h y b r i d i z a t i o n depends c r i t i c a l l y on the s p e c i f i c i t y of bi n d i n g of the tRNA to the corresponding gene and on the p u r i t y of the l a b e l l e d tRNA probe. For these reasons, the tRNA i n s i t u data reviewed above was not completely c o n v i n c i n g . F i r s t , i t was p o s s i -b l e t h a t some of the tRNA h y b r i d i z a t i o n s i t e s , p a r t i c u l a r l y the minor s i t e s , d i d not represent the presence of a tRNA gene. There are s e v e r a l p o s s i b i l i t i e s as to what the minor s i t e s could represent i ) they could represent u n r e l a t e d genes which p o s s i b l y occur as m u l t i p l e copies and correspond to contaminants i n the tRNA probes used f o r h y b r i d i z a t i o n , i i ) they could represent genes of r e l a t e d sequences which have some homology to the tRNAs used as probes, or i i i ) they could indeed represent s i n g l e or a few copies of the tRNA gene which i s present i n a g r e a t e r number at the r e -'.v s p e c t i v e major s i t e s and which i s t r u l y homologous to the > tRNA probe used f o r h y b r i d i z a t i o n . 39 A l l these p o s s i b i l i t i e s would cause formation of fewer grains at the minor sites compared to the major sites. Second, the sharing of the major and the minor sites of hy-Val bridization by the tRNA isoacceptors was ambiguous. Again, several explanations are possible: i) there could have been contamination of one valine isoac-ceptor with another, i i ) there possibly exists some sequence homology between tRNA^al and tRNA V a l, or 3D k ' i i i ) genes for both these isoacceptors exist at the shared s i t e . Val There i s some evidence for sequence homology between tRNA^ and tRNA^al (Figs. 1 5 - 1 7 ) ; plasmids containing tRNA^ 1 genes hybridize strongly to [^2^l] tRNA^al on Southern transfer and weakly to D 2 5lJ tRNA^al. Also, plasmids with tRNA^al genes hybridize strongly to ["25l] tRNA7*1 and weakly to [J 25l) tRNA^al. In situ hybridization with the plasmid DNAs was undertaken i n order to discriminate among the various p o s s i b i l i t i e s stated above and to confirm the sites determined by tRNA i n situ hybridi-zation. Table I summarizes the hybridization sites for various Val Val Ser plasmids carrying the tRNA^ genes, tRNA^b genes and the tRNA^ j, genes. The data represented i n this table i s collective data from work reported here and that reported by Dunn et a l . ( 1979b ) . It was observed i n general, that a plasmid carrying a single insert hybridized preferentially to only one of the tRNA-sites, determined by Hayashi et a l . ( 1 9 8 0 ) , for that respective tRNA. Other tRNA-ko F i g . 15. Agarose gel analysis of H i n d l l l digested plasmids V a l V a l carrying genes for tRNA^ or tRNA^ . Five valine-4 plasmids, p D t 2 3 , p D t 5 5 , p D t 9 2 R C , pDt120RG and pDtllO, three valine-3b plasmids, pDt/f1RC, pDti f8 and pDt78RC and control DNAs were cleaved with H i n d l l l and were electrophoresed on 1% agarose gel i n Tris-phosphate buffer (pH8.0) for 2.75 hours at 85 v o l t s . The gel was stained with ethidium bromide and photographed under U.V. l i g h t . Lane 1, pDtllO; lane 2 , pDt120RC; lane 3, pDt92RC; lane if, pDt55; lane 5, pDt23; lane 6, pBR322; lane 7, A DNA; lane 8, pDt78RC; lane 9, pDt/+8; lane 10, pDtiflRC. F i g . 16. Hybridization of H°lJ tRNA^f 1 with Hind I I I d i -Val gested plasmids carrying genes for tRNA,, or . m T A V a l *° tRNA^ . The agarose gel photographed i n F i g . 15 was treated by the procedure of Southern (1975). The transfered DNA was incubated with 0 2^lj tRNA^ 1 followed by autoradiography. Lanes 1-10 correspond to lanes 1-10 of F i g . 15; i . e . lane 1, pDt110; lane 2, pDt120RC; lane 3, pDt92RC; lane 4, pDt55, lane 5, pDt23; lane 6, pBR322; lane 7, X DNA; lane 8, pDt78RC; lane 9, pDtq.8; lane 10, pDtlflRC. F i g . 15 F i g . 16. 1 2 3 4 5 6 7 8 9 10 42 1 2 3 4 5 6 7 8 9 1 0 An agarose gel identical to that photographed in Fig. 15 was treated by the procedure of Southern (1975). The transfered DNA was incubated with O^l] tRNA^al followed by autoradiography. Lane 1, pDt110; lane 2, pDt120RC; lane 3 , pDt92RC; lane 4, pDt55; lane 5, pDt23; lane 6, pBR322; lane 7, X DNA; lane 8, pDt78RC; lane 9, pDt48; lane 10, pDt41RC. 43 s i t e s might have been l a b e l l e d a l s o , but under the c o n d i t i o n s used, g r a i n s at those s i t e s could not be d i s t i n g u i s h e d above background. I f the h y b r i d i z a t i o n at the p r e f e r e n t i a l s i t e was due to a d d i t i o n a l homology o f f e r e d by the f l a n k i n g sequences ( i . e . i n a d d i t i o n to the homology due to the gene), then the h y b r i d i z a t i o n s i t e denotes the s i t e on the D r o s o p h i l a chromosome from which the i n s e r t o r i g i n a t e d . H y b r i d i z a t i o n at a given s i t e would t h e r e f o r e confirm the presence of the tRNA gene at t h a t s i t e . T h i s p a t t e r n of h y b r i d i z a t i o n a l s o made i t p o s s i b l e to see i f plasmids wi t h d i f f e r e n t s i z e d i n s e r t s but c a r r y i n g the same tRNA gene h y b r i d i z e d to the same s i t e or to a d i f f e r e n t s i t e . Such i n f o r m a t i o n would throw some l i g h t on the o r g a n i z a t i o n of tRNA genes i n D r o s o p h i l a . In s i t u h y b r i d i z a t i o n data on the f i v e s e r i n e plasmids (Table I) confirmed the presence of tRNA. % genes at 12DE and 23E, the two Ser major tRNA^ y s i t e s according to Hayashi et a l . (1980). The four plasmids which h y b r i d i z e d to 12DE had d i f f e r e n t s i z e d i n s e r t s , sug-Ser g e s t i n g the presence of m u l t i p l e copies of tRNA, tj gene at t h i s s i t e . In f a c t , E l d e r et a l . (1980) have estimated 5 copies of a p u r i f i e d 4S RNA gene at 12E and 2 copies of the same gene at 23EF. The h y b r i d i z a t i o n p a t t e r n of pDt17RC was d i f f e r e n t from other plasmids i n that i t h y b r i d i z e d to more than one s i t e . I t should be noted t h a t cRNA was used f o r h y b r i d i z a t i o n i n t h i s case i n s t e a d of plasmid DNA (METHODS), but t h i s d i f f e r e n c e may not be the reason f o r h y b r i d i z a t i o n at m u l t i p l e s i t e s because such a trend was not observed f o r s e v e r a l other plasmids where cRNA was used f o r h y b r i -d i z a t i o n (S. Hayashi, personal communication). The other p o i n t to kk note i s that pDt17RC-cRNA not only hybridized to a l l the tRNA^ ? sites (12DE, 2 3 E F , %D, 64D) but also to 62D, a site which has not Ser been previously characterized as a tRNA^ r , s i t e . It i s conceivable that there was fragmentation of the i n i t i a l transcript and frag-ments containing the gene and/or flanking sequences hybridized to the various sites. 12DE and 23E have already been established as tRNA. t gene sites (above). 56D and GkD are minor tRNA. n sites according to Hayashi et a l . (1980) and the fact that grains were seen at these sites by two independent methods lends c r e d i b i l i t y Ser to the presence of tRNA^ r, genes at these sites. Additional sup-port for this observation i s provided by Elder et a l . (1980) who reported weak hybridization at 5 6 D and 6 4 D E with ifS RNA. Hybridi-zation at 6 2 D site i s a l i t t l e more d i f f i c u l t to explain. H i n d l l l digestion and agarose gel analysis (Fig. 7) confirms that pDt17RC has only one insert, so the hybridization at 62D site i s not due to the presence of an extra fragment. It i s possible that frag-ments containing the flanking sequences hybridized to 62D s i t e , which must mean that the flanking region represents some sequence which i s present at multiple sites on the genome of Drosophila. It could be another tRNA gene which was not detected by the probe of 12 tRNAs used by Dunn et a l . (1979b) or i t could be some other small RNA species (Elder et a l . , 1980). In s i t u hybridization results of Valine-^, and Valine-3b plas-Val raids (Table I) confirmed the presence of tRNA, genes at.the 70BC h Val and 89B sites and tRNA^ gene at the 84D si t e . Additional evi-Val dence for the presence of tRNA^ genes at the 84D site i s provided h5 by the s t u d i e s of Dunn et a l . (1979a) on the e f f e c t of d u p l i c a t i o n V a l and d e l e t i o n i n the 84D r e g i o n on the l e v e l of t R N A ^ i n f l i e s . 90BC i s an i n t e r e s t i n g s i t e i n that two plasmids from each group h y b r i d i z e d to t h i s s i t e . There was some evidence given e a r l i e r ( F i g s . 15-17) that t h i s s h a r i n g of s i t e s may be due to sequence V a l V a l homology between t R N A ^ and tRNA^ • But i f the assumption, t h a t the a d d i t i o n a l sequence homology provided by the f l a n k i n g r e g i o n s causes the plasmid DNA to p r e f e r e n t i a l l y h y b r i d i z e to one s i t e on the D r o s o p h i l a chromosome, i s c o r r e c t , only the fragment which o r i -g inated from the s i t e should be able to h y b r i d i z e t h e r e . A f r a g -ment which had o r i g i n a t e d from another s i t e but w i t h sequence homo-logy ( e i t h e r complete or p a r t i a l ) only i n the coding r e g i o n would not be able to h y b r i d i z e to the s i t e i n question because i t would not have the proper f l a n k i n g sequences. Therefore h y b r i d i z a t i o n of fragments c o n t a i n i n g e i t h e r tRNA^ J J or tRNA* gene to the 90BC s i t e must represent presence of both of these genes at the 90BC s i t e . Indeed DNA sequence data on pDt92RC (Carolyn A s t e l l , unpub-l i s h e d ) and pDt120RC (author, unpublished),the two v a l i n e - i f p l a s -mids which h y b r i d i z e to the 90BC s i t e , shows the presence of a tRNA gene whose sequence i s very s i m i l a r to that of D r o s o p h i l a V a l tRNA^ ( B i l l Addison, unpublished). No such sequence data i s yet a v a i l a b l e on the two valine - 3 b plasmids which h y b r i d i z e to the V a l 90BC s i t e nor i s the sequence of D r o s o p h i l a t R N A ^ yet known. E l d e r et a l . (1980) have estimated that about 9 copies of kS RNA gene occur at the 90C s i t e , a l a r g e enough number to accomodate genes f o r two species of tRNA. 46 Another feature of importance to note i s t h a t of the seven v a l i n e plasmids t e s t e d , f i v e h y b r i d i z e d to t h e i r r e s p e c t i v e minor s i t e s (Table I ) as determined by Hayashi et a l . (1980) . T h i s proves t h a t the minor s i t e s do represent the presence of tRNA genes at those l o c i . This o b s e r v a t i o n i s s u b s t a n t i a t e d by E l d e r et a l . (1980) who estimated about 1-3 copies of a tRNA gene at such weakly l a b e l l e d s i t e s . Conclusion The i n s i t u h y b r i d i z a t i o n study presented here confirmed the presence of tRNA£ ^ genes at 12DE, 23E and p o s s i b l y at 56D and 64D and t R N A ^ 1 genes at 70BC, 89B and 90BC s i t e s . I n s i t u h y b r i -d i z a t i o n w i t h plasmid DNAs a l s o confirmed t h a t minor s i t e s of hy-b r i d i z a t i o n seen w i t h tRNA - i n s i t u h y b r i d i z a t i o n represent the presence of a few copies of tRNA genes at those l o c i . These r e s u l t s along w i t h others reported elsewhere ( K u b l i and Schmidt, 1978; T. Schmidt et a l . , 1978; E l d e r , 1978; Hayashi et a l . , 1980) show t h a t genes f o r many i n d i v i d u a l tRNAs occur as m u l t i p l e copies i n more than one c l u s t e r at s i t e s w idely s c a t t e r e d on the chromosomes. A c l u s t e r at a s i t e may c o n t a i n genes f o r a s i n g l e tRNA species or i t may c o n t a i n genes f o r more than one tRNA species (Yen et a l . , 1977; 0 . Schmidt et a l . , 1978; Hovemann et a l . , 1980) . 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