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Quantitation of tRNAVal/3b in Drosophila melanogaster by RNA-DNA hybridization Larsen, Trina Margaret 1982

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Val QUANTITATION OF tRNA^ IN DROSOPHILA MELANOGASTER JD BY RNA-DNA HYBRIDIZATION by TRINA MARGARET LARSEN B.A. (with d i s t i n c t i o n i n general scholarship) University of C a l i f o r n i a , Berkeley; 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES PROGRAM IN GENETICS UNIVERSITY OF BRITISH COLUMBIA We accept t h i s thesis as conforming the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1982 © Trina M. Larsen, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives. I t i s understood that copying or p u b l i c a t i o n , of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Program i n Genetics The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, B.C., Canada V6T 1W5 DATE ABSTRACT The purpose of t h i s study was two-fold. The f i r s t objective was to determine the optimum h y b r i d i z a t i o n conditions for recombinant plasmids carrying Drosophila tRNA genes. The plasmid DNA was bound to n i t r o -c e l l u l o s e f i l t e r s and hybridized with a homologous tRNA probe. The sec-Val ond objective was to use these plasmids to quantitate l e v e l s of tRNA^ Val i n f l i e s bearing d e f i c i e n c i e s i n the major tRNA^ l o c i . S p e c i f i c p l a s -mids studied were those bearing genes f o r tRNA„, , tRNA. .,, and tRNA,.^  . 3b 4,7 5 Val pDt78R, pDt48, and pDt41R, a l l bearing tRNA,^ genes, were compared under i d e n t i c a l h y b r i d i z a t i o n conditions to determine which DNA annealed tRNA Val 3b most e f f i c i e n t l y . pDt78R was found to anneal 95-100% of the input Val t R N A 3 b u n d e r the widest range of h y b r i d i z a t i o n conditions. Ser A comparison of pDtl6, pDtl7R, pDt27R, and pDt73, a l l bearing tRNA Ser genes, showed that pDtl7R annealed tRNA^ most e f f i c i e n t l y . I n i t i a l stud ies on pDtl7R demonstrated a low o v e r a l l h y b r i d i z a t i o n e f f i c i e n c y of 18%; however, i t was found that adding 21% formamide to the h y b r i d i z a t i o n buffe increased the e f f i c i e n c y of annealing to 85-90%. pDtl2 and pDt39, carrying tRNA^ s genes, exhibited low h y b r i d i z a t i o n e f f i c i e n c i e s of 20 and 15%, re s p e c t i v e l y . Adding 15-20% formamide only increased the h y b r i d i z a t i o n e f f i c i e n c y to 55 and 38%, and these plasmids" were not studied further. Transfer RNA was extracted from 50-300 mg of adult f l i e s and was Val Ser s p e c i f i c a l l y labeled in v i t r o . The l e v e l s of tRNA^ and tRNA^ ^ were quantitated by annealing to pDt78R and pDtl7R immobilized on n i t r o -Val c e l l u l o s e f i l t e r s . The l e v e l of tRNA„, i n the tRNA i s o l a t e d from f l i e s 3 b • * • • V cl-L d e f i c i e n t i n the major tRNA^^ l o c i was examined. The r e s u l t s showed Val that d e l e t i o n of h a l f the major tRNA^ l o c i resulted i n a reduction of Val approximately 50% i n the l e v e l of tRNA^ but did not produce the Minute phenotype; furthermore, the e f f e c t s of d e f i c i e n c i e s at two l o c i were approximately addi t i v e . IV TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS v i i i ABBREVIATIONS ix INTRODUCTION 1 MATERIALS 4 I. Reagents 4 I I . Enzymes 5 I I I . Solutions 5 IV. Plasmids and B a c t e r i a l Strains 7 V. Growth Media 7 VI. Drosophila Strains and Special Chromosomes 8 VII. P u r i f i e d tRNA Isoacceptors 8 METHODS 11 I. Preparation pf Plasmid DNA 11 II . Binding of DNA to N i t r o c e l l u l o s e F i l t e r s 11 I I I . EcoRI Digestion of pDtl7R 11 IV. Extraction of 4S RNA from Drosophila 12 125 V. In v i t r o Labeling of tRNA with I 12 VI. Hybridization of tRNA to Recombinant DNA Plasmids 12 Page RESULTS 14 Val I. Hybridization of Plasmids Containing tRNA^ genes .. 14 Ser I I . Hybridization of Plasmids Containing tRNA^ * genes ... 18 I I I . Hybridization of Plasmids Containing tRNA^ s genes .. 26 IV. Hybridization of Drosophila 4S RNA Extracts 29 DISCUSSION 35 I. P a r t i a l Equilibrium Constants f o r pDNA-tRNA Hybridizations 35 I I . E f f e c t of Concentration on Hybridization 38 I I I . E f f e c t of Time on Hybridization E f f i c i e n c y 39 IV. E f f e c t of Secondary Structure on Hybridization E f f i c i e n c y 39 V. Hybridization of 4S RNA from Drosophila mutants 42 CONCLUSION. 47 BIBLIOGRAPHY 48 APPENDICES 52 v i LIST OF TABLES Table Page 1 Mutant Strains and Special Chromosomes 9 2 Crosses Generating Double Deficiency Heterozygotes 10 Val 3 Amount of tRNA^ i n Wild Type and Deletion Strains 33 4 Equilibrium Constants for pDNA-tRNA Hybridizations 36 LIST OF FIGURES Hybridization Be^Yeen pDt78R and Increasing Amounts of tRNA„, Val Hybridization Between tRNA^ and Increasing amounts of pDt78R Time Cour.s(ji Hybridization Between pDt78R and tRNAn, Jb Hybridizations of pDt41R and;LpDt48 to Increasing Amounts of tRNA^ Comparison of the E f f e c t of Increasing Amounts of Plasmid DNA on the Hybridization E f f i c i e n c i e s of pDt78R, pDt41R, and pDt48 with tRNA^ Ser H y b r i d i z a t i o n Between pDtl7R and tRNAIJ at Increasing Concentrations of Formamide Comparison of Hybridizations Between pDtl7R and Increasing Amounts of tRNA^ i n the Presence and Absence of Formamide Hybridization of pDtl6, pDtl7R, pDt2.7R, and pDt73 to Increasing Amounts of tRNA^ Agarose Gel Electrophoresis of pDtl7R Cut with EcoRI Hybridizations £f pDtl2 and pDt39 to Increasing Amounts of tRNA^ S E f f e c t of Increasing Concentration's of Formamide on Hybridizations of pDtl2 and pDt34 to tRNA^ y S Val Comparison of Levels of tRNA i n Wild Type and Df(3R)84D/Df(3R)D1 5 Drosophila  Comparison of lime Course Hybridizations of pDt78R to tRNA^ 3 and PDtl7R to tRNA^ e r  ACKNOWLEDGEMENTS I would l i k e to thank Dr. R.C. M i l l e r and Dr. G.B. Spiegelman, without whose support and guidance t h i s project would not have been possible. My thanks also go to Dr. G. Tener f or allowing the use of his f a c i l i t i e s , and Dr. I.C. Gillam f o r providing p u r i f i e d Drosophila tRNA and teaching me the procedures for extraction of 4S RNA. I also with to thank Dr. T.A. G r i g l i a t t i and Dr. D. S i n c l a i r for supplying a l l the Drosophila used i n these studies, and for many h e l p f u l d i s -cussions regarding the analysis of my data. ABBREVIATIONS USED DNA - deoxyribonucleic acid pDNA - plasmid DNA ecc DNA - covalently closed c i r c u l a r DNA RNA - r i b o n u c l e i c acid tRNA - transf e r RNA Val tRNA^ - nonacylated v a l i n e 3b tRNA Ser tRNA, 7 - nonacylated serine 4 and/or 7 tRNA 4 » ' Lv s tRNA,.^  - nonacylated l y s i n e 5 tRNA rRNA - ribosomal•RNA 4S RNA - unfractionated 4S RNA ATP - adenosine triphosphate CTP - cyti d i n e triphosphate I-CTP - 5-iodinated CTP_: pDt - recombinant p_lasmid carrying Drosophila tRNA gene pDtR - recloned pDt EcoRI - r e s t r i c t i o n endonuclease i s o l a t e d from Escherichia c o l i SVPD - snake venom phosphodiesterase M9 - M9 s a l t s o l u t i o n RPC-5 - reversed phase chromatography 5 TRIS - T r i s (hydroxymethyl) aminomethane EDTA - ethylene diamine t e t r a a c e t i c a c i d SDS - sodium l a u r y l sulphate 2L, 2R, 3L, 3R - l e f t or r i g h t arm of second or t h i r d chromosome of Drosophila Df - de f i c i e n c y mutation T - t r a n s l o c a t i o n DI - D e l t a mutation M - Minute mutation cpm - counts per minute mCi - m i l l i c u r i e s mM, M - m i l l i m o l a r , molar ng, ug, mg, g - nanogram, microgram, milligram, gram y l , ml, 1 - m i c r o l i t r e , m i l l i l i t r e , l i t r e °C - degrees c e l s i u s hr - hour min - minute GTP, dGTP - guanosine triphosphate or deoxyguanosine triphosphate CTP, dCTP - cy t i d i n e triphosphate or deoxycytidine triphosphate CCA end - the 3' end of a tRNA molecule 1 INTRODUCTION A great deal of contemporary molecular genetic research has focused on the organization and expression of eukaryotic genes. In t h i s regard, genes which encode tRNA isoacceptors are an excellent model system and have received much attention. Drosophila melanogaster produces approxi-mately 90 d i f f e r e n t tRNA species (White et a l . , 1973). There i s some evidence that these genes are d i f f e r e n t i a l l y regulated, r e s u l t i n g in stage s p e c i f i c v a r i a t i o n s i n the l e v e l of c e r t a i n isoacceptors during the development of wild type f l i e s (White et a l . , 1973). Also, t i s s u e spe-c i f i c r e gulation of tRNA synthesis has been demonstrated i n the p o s t e r i o r s i l k gland of the silkworm, Bombyx mori (Garel e_t a l . , 1970, 1977; Garber and Gage, 1979). In addition, several reports have postulated that tRNA molecules may act d i r e c t l y as regulators of biochemical processes i n eukaryotes (Anderson and G i l b e r t , 1969; Anderson, 1969; Twardzick et a l . , 1971; Jacobson, 1978). A number of Drosophila tRNA genes have been c y t o l o g i c a l l y l o c a l i z e d on the polytene s a l i v a r y gland chromosomes by i n s i t u h y b r i d i z a t i o n ( G r i g l i a t t i et a l . , 1974; Delaney et a l . , 1976; Elder, 1978; K u b l i and Schmidt, 1978; Schmidt et _al. , 1978; Hayashi et a l . , 1980; Schmidt and K u b l i , 1980). Most isoacceptor species show h y b r i d i z a t i o n to more than one chromosomal s i t e , i n d i c a t i n g that the multiple copies of a single tRNA gene are organized into several c l u s t e r s of repeated genes which are dispersed throughout the genome. Many of these genes have been cloned and sequenced (Dunn et_ al_. , 1979b; Hershey and Davidson, 1980; Hosbach et a l . , 1980; Hovemann et al.., 1980; Yen and Davidson, 1980; 2 Robinson and Davidson, 1981; Addison e_t a l . , 1982a), and a few of these cloned sequences have been transcribed i n a homologous system i n v i t r o (Dingermann et a l . , 1981; Rajput e_t al. (in preparation)). However, i t i s not known whether any of these cloned tRNA l o c i are active in vivo. Val tRNA^ was of p a r t i c u l a r i n t e r e s t because of the existence of Val Val Drosophila mutants d e f i c i e n t i n the major tRNA„, l o c i . The tRNA„, jb 3b genes map c y t o l o g i c a l l y to three s i t e s on the r i g h t arm of chromosome 3 at bands 84D3-4, 90BC, and 92B1-9 (Hayashi et a l . , 1980). Grain counts from i n s i t u h y b r i d i z a t i o n with either a p u r i f i e d tRNA or cDNA probe i n d i -cated that the r a t i o of templates at each s i t e i s 5:1:4, res p e c t i v e l y . Val Four s p e c i f i c aspects of the function of these tRNA^ genes were i n -Val vestigated by quantitating the amount of tRNA^ present i n adult f l i e s . F i r s t , do a l l three s i t e s contain t r a n s c r i p t i o n a l l y a c t i v e genes? Val Second, does each contribute tRNA^ to the t o t a l c e l l u l a r pool i n d i r e c t proportion to the number of templates at that s i t e ? T h i r d , does a mech-anism e x i s t to compensate for loss of coding sequences at one or more of the three s i t e s ? F i n a l l y , does loss of 50% of the active templates f o r Val t'RNA^ produce a c h a r a c t e r i s t i c phenotype, such as Minute? In Drosophila, Minutes represent a class of about 50 dominant mutations which share a remarkably uniform syndrome of phenotypic e f f e c t s including prolonged development, small and t h i n thoracic b r i s t l e s , rough eyes, reduced v i a -b i l i t y and f e r t i l i t y of heterozygotes, and recessive l e t h a l i t y . Ritossa et a l . (1966a) hypothesized that Minutes were deletions of the s t r u c t u r a l genes encoding a single tRNA species, and that loss of 50% of a tRNA species resulted i n reduced p r o t e i n synthesis and hence the phenotypes common to Minutes. Val To answer these questions, i n vivo l e v e l s of tRNA., were measured 3D 3 i n normal d i p l o i d s t r a i n s and s t r a i n s which c a r r i e d deletions of the genes at 84D, 92B, and both 84D and 92B. Previous attempts to quantify tRNA l e v e l s i n vivo r e l i e d on chromatography on an RPC-5 matrix (Dunn et a l . , 1979a). While successful, large amounts of input material were required, making the analysis of mutant str a i n s d i f f i c u l t . Therefore, a procedure fo r measuring nanogram quantities of s p e c i f i c tRNAs was developed. The 125 procedure depends on annealing I-labeled tRNA to Drosophila tRNA genes c a r r i e d by recombinant DNA plasmids immobilized on n i t r o c e l l u l o s e f i l t e r s . The behaviour of the recombinant plasmids h y b r i d i z i n g p u r i f i e d tRNA probes was characterized, and optimal h y b r i d i z a t i o n conditions were Val determined. The procedure has been used to measure the amounts of tRNA., j b produced by several Drosophila mutants. The c h a r a c t e r i z a t i o n of the plasmids and the tRNA analysis of these mutants are described here. The Val r e s u l t s indicate that both major s i t e s h y b r i d i z i n g tRNA^ are trans-c r i p t i o n a l l y a c t i v e . A d e l e t i o n of e i t h e r locus reduces the i n vivo Val . . . l e v e l of tRNA„, , and the e f f e c t s of both deletions are addi t i v e . None of the d e f i c i e n c y bearing f l i e s exhibited the Minute phenotype. F i n a l l y , the technique described here should be s e n s i t i v e enough to measure the amounts of s p e c i f i c tRNAs i n i s o l a t e d tissues at d i f f e r e n t developmental stages. MATERIALS Reagents Agarose beads Agarose powder .Albumin, bovine Bromophenol blue 3-mercaptoethanol Casamino acids Cesium chloride (technical grade) Chloramphenicol DEAE c e l l u l o s e - DE22 Dextrose EDTA Ethidium bromide Ge l a t i n Phenol POPOP (p-Bis[2-(5 phenyloxyazolyl)]-benzene) PPO (2,5-Diphenyloxazole) 125 c a r r i e r - f r e e Sodium I (=500 mCi/ml) Sodium pyrophosphate SDS Sucrose TCA Thiamine Thymidine Toluene T r i s T r i t o n Uridine Xylene cyanol Bio-Rad Laboratories Bio-Rad Laboratories Sigma Chemical Co. Bio-Rad Laboratories Bio-Rad Laboratories Difco Laboratories Kawecki Berylco Ind. Inc. Sigma Chemical Co. Whatman Difco Laboratories J.T. Baker Chemical Co. Sigma Chemical Co. J.T. Baker Chemical Co. Matheson, Coleman & B e l l New England Nuclear New England Nuclear Amersham Fisher S c i e n t i f i c Co. BDH Chemicals J.T. Baker Chemical Co. Matheson, Coleman & B e l l Sigma Chemical Co. Calbiochem Fisher S c i e n t i f i c Co. Sigma Chemical Co. J.T. Baker Chemical Co. Sigma Chemical Co. Eastman Kodak Co. 5 I I . Enzymes Lysozyme Nucleotidyl transferase R e s t r i c t i o n endonuclease EcoRI Snake venom phosphodiesterase I I I . Solut ions 1. For preparation of plasmid DNA i ) Tris-HCI Sucrose i i ) T r i t o n X-100 i i i ) D i a l y s i s buffer (TNE) Tris-HCI Sodium chloride EDTA iv) D i a l y s i s buffer (KP^ KHJPO. - KoHP0, 2 4 2 4 EDTA Sigma Chemical Co. provided by Dr. W.R. Addison New England Biolabs Worthington Biochemical 0.05 M pH 8.0 25% 2% 0.02 M pH 7.4 0.02 M 0.001 M 0.5 mM pH 7.4 1 mM 2. Agarose gel electrophoresis i ) Tris-Phosphate buffer (TPE) T r i s 0.04 M pH 8.0 . Sodium phosphate (NaH^O^O.02 M EDTA 0.001 M i i ) Loading buffer Sucrose 40% EDTA 0.25 M • Bromphenol blue 0.02 % Solutions (continued) 3. For preparation of f i l t e r s i ) 6XSSC Sodium chloride 0.9 M Sodium c i t r a t e 0.09 M i i ) EcoRI buffer T r i s 0.09 M pH 7.4 Sodium chloride 0.04 M Magnesium chl o r i d e 0.008 M Gelatin 1 mg/ml 4. For i s o l a t i o n of 4S RNA i ) Sodium acetate 0.14 M pH 4.5 i i ) Sodium acetate 0.14 M pH 4.5 Sodium chloride 0.3 M i i i ) Sodium acetate 0.14 M pH 4.5 Sodium chloride 1.0 M 5. For l a b e l i n g of tRNA i ) SVPD buffer T r i s 0.05 M pH 8.0 Magnesium chloride 0.01 M i i ) Nucleotidyl transferase buffer Glycine 0.05 M pH 9.5 Magnesium sulphate 0.01 M ATP 0.1 mM g-mercaptoethanol 0.005 M i i i ) Stop mix saturated urea xylene cyanol 0.5% bromphenol blue 0.5% 7 I I I . Solutions (continued) 6. For hybridizations i ) 6XSSC SDS 0.1% i i ) Formamide 70% Potassium phosphate 0.06 M (KH £P0 4) Potassium phosphate 0.06 M (K 2HP0 4) Na2EDTA 0.01 M Potassium hydroxide 0.027 M Potassium chloride 0.5 M IV. Plasmids and B a c t e r i a l Strains The construction and i s o l a t i o n of recombinant plasmids containing Drosophila melanogaster tRNA genes has been described (Dunn e_t a l . , 1979b). S p e c i f i c recombinant plasmids used were: pDt78R, pDt48, Val and pDt41R, containing tRNA^ genes; pDtl6, pDtl7R, pDt27R, and Ser pDt73, containing tRNA^ genes; and pDtl2 and pDt39, containing tRNA^ y s genes. V. Growth Media i ) L u r i a broth Tryptone 10 g Yeast extract 5 g Sodium chloride 5 g Sodium hydroxide 1 ml @ 1 M d i s t i l l e d H 20 1 I 8 V. Growth Media (continued) i i ) M9S + medium M9S medium (Champe and Benzer, 1962) ' 1 I 15 mg 200 mg 20 mg 10 mg Uridine Thiamine Thymidine VI. Drosophila mutant s t r a i n s and s p e c i a l chromosomes A l l Drosophila used were the generous g i f t of Drs. D. S i n c l a i r and T. G r i g l i a t t i . A synopsis of the mutant s t r a i n s and s p e c i a l chromo-somes i s given i n Table 1. A complete d e s c r i p t i o n i s found i n L i n d s l e y and G r e l l (1968). Two s i t e s on the r i g h t arm of chromo-Val some 3 (3R) contain major c l u s t e r s of tRNA^ genes located at 84D and 92B. In addition, there i s a minor s i t e at 90BC. Cy t o l o g i c a l and experimental evidence indicates that Df ( 3 R ) a n t p ^ S + ^ ^ (hereafter referred to as D.f (3R)84D) lacks the e n t i r e 84D c l u s t e r (Dunn et^ ail_. , 1979a; Duncan and Kaufman, 1975). The Delta (DI) gene has been l o c a l i z e d to the 91E - 92B region of 3R (Lindsley et a l . , 1972). This locus i s haplo-abnormal so that a f l y bearing only one copy of the DI gene exhibits a c h a r a c t e r i s t i c DI phenotype (thickened wing veins and s l i g h t l y roughened eyes). These observations suggested that some DI a l l e l e s might involve deletions which also remove the Val t R N A 3 b § e n e s a t 9 2 B - Table 2 outlines the crosses used to obtain double d e f i c i e n c y heterozygotes. VII. P u r i f i e d tRNA Isoacceptors A l l p u r i f i e d tRNA isoacceptors used were the g i f t of Dr. I.C. Gillam. Table 1. Mutant strains and special chromosomes used Designation of Abbre-chromosome of s t r a i n v i a t i o n Comments Cytology Reference In(3LR)TM3,rip PsepSb b x 3 4 e S e r TM3,Sb Ser Multiply-inverted t h i r d chromosome balancer In(3R)C* Paracentric inversion in 3R used to balance region from Dl_ to t i p of 3R A n t e n n a p e d i a N s + R 1 7 ( A n t p N s + R 1 7 ) Df(3R)84D y-ray-induced revertant of Antp Ns Df(3R)84B1; 84D11-12*** Duncan and Kaufman (1975) D e l t a 5 (DI 5) Df(3R)Dl X-ray induced Df(3R)91F; 92D Lindsley and G r e l l 1968 9 9 Delta (DI ) roughened eye pink-peach T(2,3)D1 o r i g i n unknown roe p Third chromosome con-taining the roughened  eye and pink peach eye colour mutations complex rearrangement including trans-l o c a t i o n of 92B segment to t i p of 2L normal Lindsley and G r e l l (1968) Symbol given i n parenthesis ** See Lindsley and G r e l l (1968) Both In(3R)C,e and In(3R)C, 1(3)a were used i n the present study *** Confirmed by S. Hayashi 10 Table 2. Crosses to construct double deficiency heterozygotes Genotype of^ male parent Progeny genotypes Number Progeny Phenotypes T(2;3)Dl 9/TM3,Sb' Ser 101 DI, Sb, Ser T(2,3)Dl 9/In(3R)C,e T(2;3)D1 9/Df(3R)84D In(3R)C,e/Df (3R)84D 107 119 DI In(3R)C,e/TM3,Sb Ser 112 Sb, Ser, ebony Df(3R)Dl 5/TM3,Sb Ser 79 DI, Sb, Ser, delayed develop-ment 1 Df(3R)D1 5/Df(3R)84D 67 DI, delayed de-velopment Df(3R)Dl 5/In(3R)l(3) a In(3R)C,l(3)ayTM3,Sb Ser In(3R)C,l(3)a/Df (3R)84D 297 277 Sb, Ser * Genotype of female parents = Df(3R)84D/TM3,Sb Ser ^Adults eclosed 1-2 days l a t e r than other progeny classes 11 METHODS I. Preparation of Plasmid DNA Plasmid DNA was prepared as described previously ( M i l l e r et a l . , 1981). Plasmids were analyzed f or p u r i t y by electrophoresis on 0.5% agarose. I I . Binding of DNA to N i t r o c e l l u l o s e F i l t e r s A l l f i l t e r s for h y b r i d i z a t i o n were cut from sheets of Schleicher and Schuell n i t r o c e l l u l o s e (BA84 0.45 micron). Plasmid DNA was d i -luted with 0.5 mM potassium phosphate buffer pH 7.4 containing 1 mM EDTA. The DNA was denatured by heating at 95-99°C u n t i l the increased by 40%. The s o l u t i o n then was c h i l l e d by addition of 4 volumes of 0°-4° 6XSSC and dripped through a pre-wet n i t r o c e l l u l o s e 2 f i l t e r (92.5 cm ) at a rate of 10-20 ml/min. F i l t e r s were a i r dried overnight, baked for 2 hrs at 80°C and cut into small discs for h y b r i d i z a t i o n . I I I . EcoRI Digestion of pDt!7R Plasmid DNA was cut with EcoRI in EcoRI buffer at an enzyme con-centration of 0.17 unit/ug DNA. The dig e s t i o n was done at 37°C over-night (12-16 h r s ) . Average amounts of DNA per dig e s t i o n were 1-2 mg. Reaction volumes va r i e d . 12 E x t r a c t i o n of 4S RNA Drosophila Following growth and c o l l e c t i o n , w ild type and mutant adult Drosophila melanogaster were stored at -70°C. Equal weights of male and female Drosophila were used i n each extraction. T o t a l 4S RNA was extracted from 50-300 mg f l i e s by s c a l i n g down the method of Roe (1975) with modifications as follows. RNA was p u r i f i e d with an excess of DEAE c e l l u l o s e to y i e l d a f i n a l column volume of 0.5 - 1.0 ml. The 4S RNA eluted from the DEAE c e l l u l o s e column was ethanol p r e c i p i t a t e d and c o l l e c t e d on small n i t r o c e l l u l o s e f i l t e r s (0.45 micron) by slow drip f i l t r a t i o n . The 4S RNA was rinsed from the f i l t e r s by suspension i n H^O, dessicated, and stored at -20°C. The y i e l d was 10 yg 4S RNA from 50 mg Drosophila. 125 In v i t r o Labeling of tRNA with I-tRNAs were labeled by the terminal addition method described by Hayashi et a l . (1981). Labeling of unfractionated 4S RNA from Drosophila was two to f i v e - f o l d less e f f i c i e n t than l a b e l i n g of p u r i f i e d tRNAs, but the s p e c i f i c a c t i v i t y was s t i l l high enough to measure accurately nanogram qua n t i t i e s of tRNA. Hybridization of tRNA to Recombinant DNA Plasmids DNA and tRNA were annealed i n 6XSSC 0.1% SDS. When mixtures contained tRNA^ e r a buffered formamide sol u t i o n was added to a f i n a l concentration of 20% formamide. Hybridizations were ca r r i e d out i n one-two ml volumes for 16-24 hrs at 65°C. Each v i a l con-tained three f i l t e r s bearing p u r i f i e d plasmid DNA and one blank f i l t e r . Th e f i l t e r s were shaken vigorously i n s c i n t i l l a t i o n v i a l s during annealing. 13 Unfractionated i~ i" JI-labeled 4S RNA was annealed with three f i l t e r s each of pDtl7R and pDt78R i n the same reaction v i a l . Each tRNA sample was hybridized at f i v e d i f f e r e n t concentrations of RNA to test l i n e a r i t y of annealing with respect to tRNA input. Hybrid-i z a t i o n r e s u l t s were compared only when hybridizations were con-ducted with DNA f i l t e r s cut from the same large f i l t e r . A f t e r h y b r i d i z a t i o n , f i l t e r s were washed three times i n 3XSSC at room temperature, dried 1/2 hr at 80°C, and t h e i r r a d i o -act i v i t y , was counted i n a l i q u i d s c i n t i l l a t i o n spectrometer. Count-ing e f f i c i e n c y was estimated at 65% i n s o l u t i o n and 40% when f i l t e r bound. 14 RESULTS Val Hybridization of Plasmids Containing tRNA„ Genes Jb Optimal h y b r i d i z a t i o n conditions were established f o r each plasmid annealed with i t s homologous tRNA. Plasmids were annealed with p u r i -f i e d , labeled tRNA under conditions of DNA excess to determine the molar equivalents of DNA required to obtain maximum h y b r i d i z a t i o n of input tRNA. Figure 1 describes the r e s u l t s of an experiment i n which plasmid pDt78R was hybridized with ^ " ' i - l a b e l e d tRNA^*^• The average h y b r i d i z a t i o n e f f i c i e n c y of these samples was 95-100%; i . e . , v i r t u a l l y a l l the tRNA i n s o l u t i o n was annealed with the plasmid DNA. A s i m i l a r Val experiment i n which a constant amount of input tRNA^ and increasing amounts of pDt78R were used i s shown i n Figure 2. Maximum hybrid-i z a t i o n was obtained with two or more f i l t e r s per v i a l . Figure 3 Val shows that h y b r i d i z a t i o n between pDt78R and tRNA^ was complete in 8 hours and that the e f f i c i e n c y remained constant i n incubations up to 48 hours. In a l l experiments with pDt78R, a f i v e - f o l d molar excess of DNA was adequate f o r e f f i c i e n t h y b r i d i z a t i o n . Since the s p e c i f i c a c t i v i t y of the tRNA was 1-6 x 10 cpm/ug (counted on f i l -ters) , t h i s h y b r i d i z a t i o n could be used to measure as l i t t l e as one Val ng of tRNA. A control experiment h y b r i d i z i n g tRNA^ to pBR322 displayed a h y b r i d i z a t i o n e f f i c i e n c y of less than 0.2%. When pDt78R . . Val was hybridized with tRNA^ , which d i f f e r s m only 9 nucleotides from Val tRNA^ (Addison et a l . , i n preparation, 1982b) less than 10% of the input tRNA annealed. These r e s u l t s indicate that a l l h y b r i d i z a t i o n was l i m i t e d to the inserted Drosophila DNA and that t h i s h y b r i d i z a t i o n Figure 1. Hybridization between pDt78R and increasing amounts of .„„.Val 3b pDt78R DNA was is o l a t e d and bound to n i t r o c e l l u l o s e f i l t e r s as described i n Methods. Hybridization with ''"^"'i-labeled tRNA^^ was ca r r i e d out i n 6XSSC, 0.1% SDS for 24 hrs at 65°C. The molar r a t i o of DNA/RNA ranged between 5-60. The actual amount of DNA i n each h y b r i d i z a t i o n remained constant while input tRNA varied. The average h y b r i d i z a t i o n e f f i c i e n c y was 95%. Figure 2. Hybridization between tRNA^'L and increasing amounts of pDt78R. pDt78R DNA was i s o l a t e d and bound to n i t r o c e l l u l o s e f i l t e r s as described i n Methods. Hybridization to '"'"""'r-labeled tRNA^^" was ca r r i e d Val out i n 6XSSC, 0.1% SDS f o r 16 hrs at 65°C. The amount of input tRNA (- - -) remained constant while the amount of pDt78R DNA varied. The molar r a t i o of DNA/RNA ranged from 10-30. 17 hours hybr id i zed Figure 3. Time course h y b r i d i z a t i o n between pDt78R and tRNA^f 1. jb Val tRNA.^ was hybridized to pDt78R bound to n i t r o c e l l u l o s e f i l t e r s for varying lengths of time. A l l hybridizations were c a r r i e d out in 6XSSC, 0.1% SDS. The molar r a t i o of DNA/RNA was 20 and actual amounts of Val pDt78R and tRNA^ remained constant. The maximum h y b r i d i z a t i o n e f f i c i e n c y was 90%. 18 Val was s p e c i f i c f o r tRNA^ . Therefore, t h i s procedure could be used to quantitate l e v e l s of a s p e c i f i c tRNA.in a complex mixture. Val The h y b r i d i z a t i o n e f f i c i e n c i e s of pDt48 and pDt41R with tRNA^ were investigated under the optimal conditions determined for pDt78R. Figure 4 shows that with constant DNA and increasing amounts of Val tRNA 3 b both pDt48 and pDt41R hybridized less e f f i c i e n t l y than pDt78R. The e f f i c i e n c y of h y b r i d i z a t i o n f o r pDt48 and pDt41R was 53% and 75%, re s p e c t i v e l y . However, by increasing the number of f i l t e r s per v i a l Val while input tRNA remained constant, i t was possible to increase 3b the e f f i c i e n c y of pDt48R to 72% and the e f f i c i e n c y of pDt41R to 100% Val (Figure 5). pDt48 and pDt41R map c y t o l o g i c a l l y to the minor tRNA^ gene locus, 90BC (Dunn et: al_. , 1979b). They d i f f e r i n DNA sequence Val at four nucleotides from the known RNA sequence of tRNA^ (Addison et a l . , i n preparation). Ser Hybridizations of Plasmids Containing tRNA^ Genes Ser Only 18% of labeled tRNA^ annealed to pDtl7R i n i n i t i a l experi-ments. However, adding formamide to the annealing mixture dramatically improved the e f f i c i e n c y of h y b r i d i z a t i o n (Figures 6 and 7). Decreas-ing the r e a c t i o n volume and increasing the h y b r i d i z a t i o n time to 24 hrs also improved the e f f i c i e n c y . Under conditions of 15-20% forma-mide the average h y b r i d i z a t i o n e f f i c i e n c y of pDtl7R with tRNA^ e r was 85-90%. The annealing was l i n e a r with respect to input tRNA through a 10 to 60 f o l d molar excess of DNA. An increase i n the amount of formamide beyond 20% resulted i n a decrease i n h y b r i d i z a t i o n . Ser Labeled tRNA? was also annealed with e i t h e r pDtl6, pDtl7R, pDt27R, 19 Figure 4. Hybridization of pDt41R and pDt48 to increasing amounts of Val tRNA„ . Jb Val tRNA^ was hybridized to either pDt41R or pDt48 bound to n i t r o -c e l l u l o s e f i l t e r s . Hybridizations were c a r r i e d out i n 6XSSC, 0.1% SDS for 16 hrs at 65°C. In both experiments, the r a t i o of DNA/RNA ranged between 5-60, and the amount of input DNA remained constant while input Val tRNA var i e d . Average h y b r i d i z a t i o n e f f i c i e n c i e s were 75% for pDt41R 3b ( 0 - 0 ) and 53% f o r PDt48 (® - .«,). Figure 5. Comparison of the e f f e c t of increasing amounts of plasmid DNA on the h y b r i d i z a t i o n e f f i c i e n c i e s of pDt78R, pDt41R, and pDt48 .Val with tRNA tRNA^f 1 was hybridized to PDt78R ( A ), pDt41R ( 0 ) or pDt48 ( • ) 3b bound to n i t r o c e l l u l o s e f i l t e r s . A l l hybridizations were c a r r i e d out i n Val 6XSSC, 0.1% SDS for 16 hrs at 65°C. Input tRNA b remained constant while the amount of DNA per v i a l v aried. Figure 4 ug pDNA input Figure 6. Hybridization between pDtl7R and tRNA^ e r at increasing con-centrations of formamide. tRNA^ e r was hybridized to pDtl7R bound to n i t r o c e l l u l o s e f i l t e r s at formamide concentrations of 3-21%. Formamide was d i l u t e d into 6XSSC, 0. SDS. Hybridizations were c a r r i e d out at 65°C for 24 hrs. Input amounts of DNA and tRNA remained constant. Figure 7. Comparison of hybridizations between pDtl7R and increasing Ser amounts of tRNA^ i n the presence and absence of formamide. Ser tRNA^ was hybridized to pDtl7R bound to n i t r o c e l l u l o s e f i l t e r s i n the presence ( 0 ) or absence ( • ) of 20% formamide. Formamide was d i l u t e d into 6XSSC, 0.1% SDS. Hybridizations were c a r r i e d out at 65°C for 24 hrs. The molar r a t i o of DNA/RNA was between 5-60. The amount Ser of input DNA remained constant, while input tRNA^ varied. 22 23 or pDt73 i n 20% formamide. Comparison of h y b r i d i z a t i o n e f f i c i e n c i e s Ser showed that pDtl7R hybridized tRNA^ most e f f i c i e n t l y (Figure 8). The other factor found to a f f e c t the e f f i c i e n c y of h y b r i d i z a t i o n Ser between pDtl7R and tRNA^ was the amount of. s u p e r c o i l i n g i n the pDtl7R DNA bound to n i t r o c e l l u l o s e . A plasmid prep containing a high proportion of covalently closed c i r c u l a r (ecc) DNA hybridized Ser labeled tRNA^ l e s s e f f i c i e n t l y than one i n which most of the c i r c l e s were nicked. To circumvent t h i s problem DNA which showed a high pro-portion of ecc when electrophoresed on agarose was cut with EcoRI before binding to n i t r o c e l l u l o s e . In one experiment, uncut pDtl7R (Figure 9, lane 1) was bound to n i t r o c e l l u l o s e and hybridized to Ser labeled, p u r i f i e d tRNA^ . The average h y b r i d i z a t i o n e f f i c i e n c y was 30%. When the same plasmid preparation was p a r t i a l l y digested with EcoRI to y i e l d l i n e a r molecules (Figure 9, lane 2) and then bound to n i t r o c e l l u l o s e , the h y b r i d i z a t i o n e f f i c i e n c y increased to 70%. In both cases, a s i m i l a r percentage of DNA bound to the n i t r o c e l l u l o s e during preparation of f i l t e r s . In a second experiment, pDtl7R was p u r i f i e d and digested to completion with EcoRI to y i e l d a 2.3 and a 4.4 kb fragment (Figure 9, lanes 4 and 5). However, f i l t e r s made with t h i s DNA displayed a low e f f i c i e n c y of 30%. This e f f e c t may be due to the fact that smaller fragments e x h i b i t less stable binding to n i t r o -c e l l u l o s e , and the 2.3 kb fragment containing the gene may be p r e f e r -e n t i a l l y l o s t during the course of the h y b r i d i z a t i o n reaction. To account for v a r i a t i o n s in e f f i c i e n c y , a l l large pDtl7R f i l t e r s were Ser tested with labeled, p u r i f i e d tRNA^ probe before use for experiments invo l v i n g unfractionated 4S RNA. 24 "D CD N ng t R N A s 7 e r input Figure 8. Hybridization of pDtl6, pDtl7R, pDt27, and pDt73 to increasing amounts of tRNA^ e r. pDtl6 ( • ), pDtl7R ( 0 ), pDt27R ( A ) and pDt73 ( A ) were hybrid-Ser ized to p u r i f i e d tRNA ? i n 6XSSC, 0.1% SDS with formamide added to 20%. Reactions were c a r r i e d out for 24 hrs at 65°C. The average h y b r i d i z a t i o n e f f i c i e n c i e s were 62% for pDtl6, 73% for pDtl7R, 66% for pDt27R, and 35% for pDt73. 25 1 2 3 4 5 Figure 9. Agarose gel electrophoresis of pDtl7R cut with EcoRI. Two preparations of the plasmid pDtl7R were cut with EcoRI and e l e c t r o -poresed on 0.5% agarose gels i n Tris-Phosphate buffer (pH 8.0) for 2 hrs at 100 v o l t s . The gel was photographed under u l t r a v i o l e t l i g h t a f t e r s t a i n i n g with ethidium bromide. Lane 1: uncut pDtl7R, Lane 2: pDtl7R p a r t i a l l y digested with EcoRI, Lane 3: A DNA marker, Lane 4: uncut pDtl7R, Lane 5: pDtl7R completely digested with EcoRI. 26 I I I . H ybridization of Plasmids Containing tRNA^ y S Genes The recombinant plasmids pDtl2 and pDt39, both carrying tRNA^ y s . . . Ly s genes, were annealed with increasing amounts of labeled tRNA,.^  i n the absence of formamide. Figure 10 shows the r e s u l t s of one experiment with pDtl2 and two with pDt39. In only one case was the h y b r i d i z a t i o n reaction l i n e a r . The molar r a t i o of DNA/RNA ranged between 5-60 for pDtl2 and 3-36 for pDt39. pDtl2 displayed an i n i t i a l h y b r i d i z a t i o n e f f i c i e n c y of 22% at a DNA excess of 60X but began saturating between 40-20X DNA excess. In one case, pDt39 exhibited s i m i l a r behaviour at 36X DNA excess, with an i n i t i a l e f f i c i e n c y of 17% which dropped o f f between 12~24X DNA excess. The second experiment with pDt39 used twice as much DNA and tRNA per v i a l , although the molar r a t i o s were the same. In t h i s case, h y b r i d i z a t i o n was l i n e a r with an average e f f i c i e n c y of 15% under a l l conditions of DNA excess investigated. Addition of formamide to the h y b r i d i z a t i o n buffer s i g n i f i c a n t l y i n -creased the h y b r i d i z a t i o n e f f i c i e n c i e s f o r these plasmids. Figure 11 shows the e f f e c t of formamide on h y b r i d i z a t i o n with pDtl2 and pDt39. pDt39 displayed a maximum h y b r i d i z a t i o n e f f i c i e n c y of 38% at a forma-mide concentration of 20%. pDtl2 displayed a maximum h y b r i d i z a t i o n e f f i c i e n c y of 55% at a formamide concentration of 15%. Both pDtl2 and pDt39 hybridize c y t o l o g i c a l l y to the major chromosomal locus for tRNA^ y S, 84A-B (S. Hayashi, personal communication). pDt39 has been sequenced and agrees with the known RNA sequence of tRNA^ y S (DeFranco et a l . , i n preparation). The sequence of pDtl2 has not yet been determined. However, i t appears u n l i k e l y that sequence homology or the lack of i t has a major e f f e c t here, since pDtl2 hybridizes more 27 Figure 10. Hybridizations of pDtl2 and pDt39 to increasing amounts of tRNA^ y S. tRNA^ y s was hybridized to eit h e r pDtl2 ( A ) or pDt39 ( t, 0 ) bound to n i t r o c e l l u l o s e f i l t e r s . Hybridizations were c a r r i e d out i n 6XSSC, 0.1% SDS without formamide for 16 hrs at 65°C. The r a t i o of DNA/RNA ranged bet-ween 5-60 for pDtl2 and 3-36 for pDt39. In a l l three experiments the amount of input DNA remained constant while input tRNA^ y S varied. The concentrations of reactants used i n one experiment i n v o l v i n g pDt39 ( 0 ) were twice those i n the other ( • ). Figure 11. E f f e c t of increasing concentrations of formamide on hybrid-izat i o n s of pDtl2 and pDt39 to tRNA^ y S. tRNA^ y S was hybridized to pDtl2 ( 0 ) or pDt39 ( • ) bound to n i t r o -c e l l u l o s e f i l t e r s . Formamide was d i l u t e d into 6XSSC, 0.1% SDS. Hybrid-izati o n s were c a r r i e d out at 65°C for 24 hrs. Input amounts of DNA and tRNA remained constant. F i g u r e 10 5 % formamide 29 e f f i c i e n t l y than pDt39 which i s known to have complete homology with the probe. One factor which may a f f e c t h ybridizations involving Lvs plasmids containing tRNA,- genes i s that i t i s not possible to p u r i f y tRNA,.-7 completely (D. Cribbs, personal communication). This i s o -acceptor co-elutes with a p a r t i a l tRNA^ y s which i s believed to lack the 3' end. The contaminant i s present at a l e v e l of approximately 10%. Although t h i s molecule should not be labeled by CCA end l a b e l i n g and should be eliminated during p u r i f i c a t i o n of the labeled tRNA^ y s } i t may s t i l l be present and i n t e r f e r e with h y b r i d i z a t i o n . IV. Hybridization of Drosophila 4S RNA Extracts In order to determine the amount of a s p e c i f i c tRNA isoacceptor i n a complex mixture, 4S-RNA was extracted and tRNA was labeled spe-. . . 125 c i f i c a l l y with I-CTP as described i n Methods. The labeled tRNA then was annealed with the s p e c i f i c recombinant plasmids pDt78R and pDtl7R under optimum conditions as described above. Annealing was done with at least a ten - f o l d molar excess of DNA. The extract from each type of Drosophila was hybridized at f i v e d i f f e r e n t RNA concen-t r a t i o n s to guarantee l i n e a r i t y of annealing with respect to input. Each v i a l contained both pDt78R and pDtl7R f i l t e r s , so that the amount Val Ser Ser of tRNA„, and tRNA. ., could be measured simultaneously. Since tRNA, 3b 4,7 J 4 Ser and tRNA^ d i f f e r by only three nucleotides (D. Cribbs, personal com-munication) they cannot be distinguished by h y b r i d i z a t i o n , and the extent Ser Ser of annealing to pDtl7R therefore measures the sum of tRNA^ and tRNA^ Val Val This i s i n contrast to tRNA„, and tRNA. which d i f f e r by nine nucleo-3b 4 Ser tides and do not cross h y b r i d i z e . The amount of tRNA _ serves as an 30 in t e r n a l standard since the number of serine templates remained constant i n a l l str a i n s investigated. Since the major serine tRNA locus i s on the X chromosome, equal numbers of male and female Dro- sophila were used to compensate f o r the unequal number of templates in i n d i v i d u a l f l i e s . The amount of tRNA^ y s was not used as an i n t e r n a l standard for evaluation of unfractionated 4S RNA for two reasons. The f i r s t was that pDtl2 and pDt39 both hybridized l e s s e f f i c i e n t l y than pDtl7R. The second was the proximity of the major tRNA,.^  gene locus, Val 85D, to the tRNA„, locus at 84D which i s deleted i n several of the Drosophila mutants of i n t e r e s t . Val Ser The r a t i o . o f tRNA„, /tRNA, _ was calculated f o r w i l d type and f i v e 3b 4,7 d i f f e r e n t mutant genotypes. A comparison of these r a t i o s gives an Val estimate of the decrease i n the amount ..of tRNA„, r e l a t i v e to the 3b Ser amount of tRNA^ ^ i n each mutant (Figure 12). Three to four percent of the input r a d i o a c t i v i t y from a w i l d type Drosophila extract hybrid-ized to pDt78R. This amount agrees with that estimated from r e s u l t s of Val tRNA f r a c t i o n a t i o n by column chromatography f or tRNA^ (White e_t a_l. , 1973). Approximately the same percentage hybridized to pDtl7R on . . . . Val maximum e f f i c i e n c y f i l t e r s ; t h i s indicates that the l e v e l s of tRNA_, JD Ser and tRNA^ ^ i n w i l d type f l i e s are very s i m i l a r . However, for most hybrid i z a t i o n s i n v o l v i n g 4S RNA extracts pDtl7R f i l t e r s used d i d not hybridize with a high e f f i c i e n c y , so the amount of input tRNA hybrid-i z i n g to pDtl7R was as low as 1-2%. Therefore, wild type r a t i o s of Val Ser tRNA^ /tRNA^ ^ varied between 1-4, r e f l e c t i n g the e f f i c i e n c y of pDtl7R f i l t e r s used. 1- N. » 2-CO * < cr cr o. 100 2 0 0 ng input 4 S RNA V a l F i g u r e 12 . C o m p a r i s o n o f l e v e l s o f t R N A ^ i n w i l d t y p e and D f ( 3 R ) 8 4 D / D f ( 3 R ) D 1 3 D r o s o p h i l a . U n f r a c t i o n a t e d 4S RNA from w i l d t y p e ( • ) and D f ( 3 R ) 8 4 D / D f ( 3 R ) D 1 5 ( 0 ) f l i e s was h y b r i d i z e d t o pDt78R and p D t l 7 R as d e s c r i b e d i n M e t h o d s . V a l S e r The r a t i o o f tRNA/,, / t R N A . -, was c a l c u l a t e d f o r each and compared . The 3b 4 , / V a l mean d e c r e a s e i n t he l e v e l o f t R N A ^ d i s p l a y e d h e r e i s 54%. Each r a t i o was c a l c u l a t e d f rom 3 f i l t e r s e a c h o f pDt78R and p D t l 7 R . The minimum amount o f r a d i o a c t i v i t y bound was 400 c p m / f i l t e r and t h e a v e r a g e b a c k g r o u n d p e r v i a l was 20 cpm. 32 One other f a c t o r may a f f e c t the e f f i c i e n c y of h y b r i d i z a t i o n within 4S RNA extracts. The method of l a b e l i n g used depends on e x c i s i n g part of the CCA end of each tRNA molecule with SVPD. The timing of t h i s reaction w i l l be affected by the s t a b i l i t y of the tRNA t a i l , which i n turn i s affected by the percent of GC p a i r i n g between the 5' and 3' ends of the tRNA molecule. Thus, i n a mixture not a l l tRNAs w i l l be digested to the same extent. However, each type of tRNA isoacceptor should be labeled f a i r l y c o n s i s t e n t l y i f uniform l a b e l i n g conditions are maintained. Val A summary of the l e v e l s of tRNA^ measured i n wild type and mutant Drosophila i s presented i n Table 3. As seen i n the t a b l e , mutants carrying chromosome d e f i c i e n c i e s showed reduced l e v e l s of tRNAs. For Val example, the mutant hemizygous for the tRNA^ templates at the 84D s i t e displayed a decrease of 23% r e l a t i v e to controls. The D l ^ mutation which i s missing the 92B s i t e has a mean decrease of 28% i n heterozygotes. The t r a n s l o c a t i o n mutant T(2;3)D1 shows a mean decrease of 12%. From iri s i t u h y b r i d i z a t i o n studies conducted by S. Hayashi, i t appears that t h i s multiply translocated s t r a i n has no Val major loss of tRNA^ templates. It i s i n t e r e s t i n g to speculate that t r a n s p o s i t i o n of t h i s locus may a f f e c t i t s expression. However, a few (10%) templates may be deleted and remain undetected by i n s i t u a h y b r i d i z a t i o n . The double mutant Df(3R)84DVT(2;3)D1 has a mean decrease of 28%, which i s not s i g n i f i c a n t l y d i f f e r e n t from Df(3R)84D alone. The doubly heterozygous mutant Df(3R)84D/Df(3R)D1 5, which Val deletes both major c l u s t e r s of tRNA^^ genes at 84D and 92B, r e s u l t s Val i n a 45% decrease i n the amount of tRNA^K . An analysis of variance Table 3. Amount of tRNA i n various wild-type and d e l e t i o n s t r a i n s Genotype % decrease i n tRNA^f 1 p / p roe p r/roe p 0.0 Df(3R)84D/roe p P 23 ± 3.0 Df(3R)Dl 5/roe p P 28 ± 2.8 T(2;3)Dl 9/roe p P 12 ± 1.25 Df(3R)84D/T(2;3)D1 9 28 ± 2.5 Df(3R)84D/Df(3R)D1 5 45 ± 7.3 Given as mean ± standard error of the mean. 34 (ANOVA) and a two t a i l e d t test show that there i s no s i g n i f i c a n t d i f f e r e n c e between the decreases observed f o r Df(3R)84D and Df (3R)D1"^, or between the sum of these decreases and that observed for the double heterozygote Df(3R)84D/Df(3R)D1 5. These analyses a found i n Appendix B. 35 DISCUSSION The o v e r a l l e f f i c i e n c y of h y b r i d i z a t i o n between pDt recombinant plasmids and t h e i r corresponding tRNAs appears to be determined by three major conditions: the concentration of the n u c l e i c acids used, the length of time of the h y b r i d i z a t i o n reaction, and the secondary structure of the tRNA probe and/or the pDt DNA bound to n i t r o c e l l u l o s e . These variables a f f e c t both the t o t a l amount of tRNA hybridized and the rate at which i t hybridizes. I. P a r t i a l Equilibrium constants for pDNA-tRNA Hybridizations The l i n e a r h y b r i d i z a t i o n data presented i n Figure 1, 4, 8, and 9 can be used to calculate p a r t i a l equilibrium constants f o r the hybrid-i z a t i o n reactions involving each plasmid. These constants are c a l -culated using the formula r l ^ T ^ r n J i A i » and are presented i n Table 4. [DNA] [RNA]' v Several i n t e r e s t i n g trends become obvious when these constants are Val compared. The tRNA^ plasmids demonstrate a c o r r e l a t i o n of the extent of sequence homology between the plasmid and tRNA probe and the . . Val s i z e of the equilibrium constant. pDt78R, which contains a tRNA., J D gene with a sequence i d e n t i c a l to that of the tRNA, has a high equi-11 -1 lib r i u m constant of 9 x 10 ml. mole . pDt41R and pDt48j as was mentioned previously, d i f f e r i n four nucleotides from the sequence Val of the tRNA.^ probe (Addison e_t al_. , i n preparation) . Despite the fac t that the actual h y b r i d i z a t i o n e f f i c i e n c i e s f o r these two pl a s -mids d i f f e r , t h e i r equilibrium constants are approximately the same and are about ha l f that found for pDt78R. 36 T a b l e 4 . E q u i l i b r i u m c o n s t a n t s f o r pDNA-tRNA h y b r i d i z a t i o n r e a c t i o n s P l a s m i d tRNA gene genes i n i n s e r t p r o b e e q u i l i b r i u m [ h y b r i d ] c o n s t a n t [DNA] [RNA] pDt78R pDt41R pDt48 p D t l 6 p D t l 7 R pDt27R pDt73 p D t l 2 pDt39 V a l 3b V a l 3b V a l 3b Ser 4 , 7 Se r 7 Ser 4 , 7 S e r 4 , 7 L y s 5 L y s 5 tRNA. V a l tRNA tRNA. tRNA tRNA' tRNA' tRNA 3b V a l 3b V a l 3b Se r 7 S e r 7 Se r 7 Se r 7 L y s tRNA t R N A ^ S 9 . 0 x 1 0 1 1 m l - m o l e 1 4 .35 x 1 0 1 1 m l - m o l e 1 4 . 2 9 x 1 0 1 1 m l - m o l e 1 4 . 1 9 x 1 0 1 1 m l - m o l e 1 4 . 3 3 x 1 0 U m l - m o l e 1 8 . 1 6 x 1 0 1 1 m l - m o l e " 1 3 . 7 6 x 1 0 1 1 m l - m o l e 1 1.58 x 1 0 1 1 m l - m o l e 1 5 . 2 3 x 1 0 1 0 m l - m o l e 1 The tRNA^ e r plasmids also display equilibrium constants i n 11 -1 the range 4-8 x 10 ml'mole . However, these plasmids d i f f e r i n Val two respects from those carrying tRNA^ genes. The f i r s t i s that pDtl6 and pDt27R contain more than one gene. pDt27R hybridizes with approximately the same e f f i c i e n c y as pDtl7R. However, the equi-librium constant f o r pDt27R i s twice that of pDtl7R - 8.6 x 10""'"' as opposed to 4.33 x 1 0 ^ ml-mole ^. This i s probably a r e f l e c t i o n of Ser the f act that pDt27R contains four tRNA. -, genes where pDtl7R only Ser has one. The other d i f f e r e n c e among the tRNA^ ^ plasmids i s that Ser the sequence of the genes does i n fact vary between that of tRNA^ Ser and tRNA-p . pDt73, which had a s l i g h t l y lower equilibrium constant of 3.76 x 1 0 ^ ml'mole \ has a sequence closer to that of tRNA^ e r. There are three possible nucleotide differences between the sequence Ser Ser of tRNA^ and tRNA^ ; pDt73 d i f f e r s at two of these nucleotides Ser from the sequence of the tRNA^ probe. pDtl7R has an i d e n t i c a l se-quence to that of the probe (D. Cribbs, personal communication). So again, the extent of sequence homology plays a r o l e i n determining th Ser equilibrium constant. However, i n the case of the tRNA^ ^ plasmids t h i s e f f e c t i s complicated by the v a r i a t i o n i n the number of genes per plasmid. Lvs The tRNA,. plasmids display uniformly low equilibrium constants This i s most l i k e l y not a homology e f f e c t since the sequence of pDt39 i s known to be the same as that determined f o r tRNA^ y s (DeFranco e_t a l . i n preparation) and i t i s the least e f f i c i e n t of the two plasmids studied. The low equilibrium constants are i n agreement with the f a c t that the e f f i c i e n c y of h y b r i d i z a t i o n f o r these two plasmids was c o n s i s t e n t l y low. Secondary structure may play a r o l e ; t h i s p o s s i b i l i t y w i l l be discussed l a t e r . E f f e c t of Concentration on H y b r i d i z a t i o n The e f f e c t of concentration on the t o t a l amount of input tRNA hybridized was demonstrated i n two systems. Hybridization between Val pDt78R and increasing amounts of tRNA^ was completely e f f i c i e n t under standard r e a c t i o n conditions of 2 mis 6XSSC, 0.1% SDS per v i a l for 16 hrs at 65°C. Under the same conditions pDt41R and pDt48 hybridized only 75% and 53% of the input tRNA^ 1, re s p e c t i v e l y 3b (Figure 4). However, increasing the number of f i l t e r s per v i a l from two to four (while input tRNA remained constant) increased the hybrid-i z a t i o n e f f i c i e n c y to 100% for pDt41R and 70% for pDt48. This con-centration e f f e c t i s caused by increasing the actual amounts of DNA and tRNA per v i a l . The molar r a t i o of DNA/RNA was i n the same range for the two types of experiment and the volume of buffer per v i a l remained constant. Another type of concentration e f f e c t was observed Ser i n experiments between pDtl7R and tRNA^ . In t h i s case, the volume of h y b r i d i z a t i o n buffer used was decreased while a l l other v a r i a b l e s remained constant. A two-fold increase i n the concentration of reactants with t h i s method also caused an increase i n h y b r i d i z a t i o n e f f i c i e n c y from 18 to 24%. It should be noted that under no conditions did pDt48 hybridize Val 100% of the input tRNA^ . There may be some secondary structure present i n the pDt48 in s e r t which i n t e r f e r e s with h y b r i d i z a t i o n . Val Both pDt41R and pDt48 have been sequenced and the tRNA^^ genes they contain are i d e n t i c a l . These genes d i f f e r at four nucleotides Val from the known sequence of tRNA^ (Addison e_t a l . , i n preparation). The f a c t that pDt41R can hybridize a l l the input tRNA indicates that 39 the lack of homology i s not extensive enough to prevent complete h y b r i d i z a t i o n . ' However, i t most l i k e l y does contribute to the con-centration e f f e c t s observed and may also play a r o l e i n determining the rate of h y b r i d i z a t i o n . I I I . E f f e c t of Time on E f f i c i e n c y of Hybridization Increasing the time of the h y b r i d i z a t i o n r e a c t i o n can also improve the o v e r a l l h y b r i d i z a t i o n e f f i c i e n c y . Although pDt78R . . Val hybridized e s s e n t i a l l y a l l of the input tRNA^ i n 8 hrs, none of the other plasmids investigated hybridized as much tRNA i n as short a time. Under exactly the same h y b r i d i z a t i o n conditions as pDt78R, Ser pDtl7R i n i t i a l l y hybridized an average of 18% of the input tRNA^ Doubling the concentration of DNA and RNA as discussed above i n -creased the e f f i c i e n c y to 24%. When the concentration was doubled and the time of h y b r i d i z a t i o n increased from 16 to 48 hrs the e f f i c i e n c y increased to 50%. However, t h i s s t i l l contrasted sharply with the r e s u l t s obtained f o r pDt78R (Figure 13). IV. E f f e c t of Secondary Structure on Hybridization E f f i c i e n c y A major factor a f f e c t i n g e f f i c i e n t h y b r i d i z a t i o n was found to be the secondary structure of the molecules involved. Two pieces of evidence support t h i s conclusion. The f i r s t i s the dramatic * • SGIT LVS e f f e c t of formamide on hybridizations i n v o l v i n g tRNA^ and tRNA^ . Figures 6 and 7 show that i t was possible to increase the e f f i -Ser ciency of h y b r i d i z a t i o n between pDtl7R and tRNA^ to almost 90% by adding 20% formamide to the buffer. These hybridizations were 40 hours h y b r i d i z e d Figure 13. Comparison of time course hybridizations of pDt78R to tRNA^ 1 and pDtl7R to tRNA^ e r. V a l Hybridizations were c a r r i e d out between pDt78R and tRNA., ( 0 ) and between pDtl7R and tRNA^ e r ( • ) i n 6XSSC, 0.1% SDS. In both cases, the input amounts of pDNA and tRNA remained constant and only the length of time of the h y b r i d i z a t i o n r e a c t i o n v a r i e d . 41 incubated for 24 hours. Although adding formamide and decreasing the volume of buffer used did not have as extensive an e f f e c t on Hi V S hybridizations involving tRNA^ , Figure 10 shows that the e f f i -ciency d i d increase approximately three times above the i n i t i a l r e s u l t s i n Figure 9. Formamide i s a denaturant and has been ex-t e n s i v e l y used i n n u c l e i c acid r e a s s o c i a t i o n experiments. A p r i -mary function i s to place increased constraints on base mis-p a i r i n g so that n u c l e i c acids could be s p e c i f i c a l l y reassociated at low .temperatures (Schmeckpeper and Smith, 1972). In t h i s case, the temperature at which hybr i d i z a t i o n s were ca r r i e d out was not lowered, so the formamide served to denature secondary structure of the tRNA probe and to increase even further the s p e c i f i c i t y of the hybrid-i z a t i o n r e a c t i o n . The second piece of evidence which pinpointed the r o l e of sec-ondary structure i n tRNA-pDNA h y b r i d i z a t i o n lay i n the conformation of pDtl7R i t s e l f . F i l t e r s made from p r i m a r i l y cccDNA hybridized f a r less e f f i c i e n t l y than f i l t e r s made from a mixture of l i n e a r , nicked c i r c u l a r , and cccDNA. Cutting the pDtl7R with EcoRI before binding to n i t r o c e l l u l o s e increased the h y b r i d i z a t i o n e f f i c i e n c y as much as 40%. The e f f e c t of secondary structure demonstrated i n these expe-riments probably also accounts for most of the v a r i a t i o n i n hybrid-i z a t i o n e f f i c i e n c y and rate of h y b r i d i z a t i o n i n the plasmids char-a c t e r i z e d . Secondary structure i n the tRNA molecules v a r i e s accord-ing to GC content, f i d e l i t y of base paring i n the c l o v e r l e a f stem and arms, and the extent of base modification. These factors also a f f e c t secondary structure i n the tRNA gene when denatured. In addition, secondary structure i n the plasmid DNA i s affected by the 42 sequence i n the insert surrounding the tRNA gene and the amount of su p e r c o i l i n g present. A l l these factors must contribute to the differences i n h y b r i d i z a t i o n behaviour observed i n the systems characterized here. V. Hybridization of 4S RNA from Drosophila Mutants The r e s u l t s of h y b r i d i z a t i o n of t o t a l 4S RNA extracted from Drosophila carrying heterozygous d e l e t i o n mutations were presented i n Table 3. Actual data used to c a l c u l a t e the percent decreases i s included i n Appendix A. These indi c a t e that both 84D and 92B Val l o c i contain genes which a c t i v e l y produce tRNA^ . If t r a n s c r i p -t i o n occurred at only one s i t e , f l i e s c a r r y i n g a d e l e t i o n i n one Val homologue of the active s i t e should show a 50% reduction i n tRNA,., Jb l e v e l , while d e l e t i o n of the other s i t e would have no e f f e c t . I t was found, i n f a c t , that the two types of deletions resulted i n . . Val s i m i l a r l e v e l s of tRNA^ synthesis; a 23% decrease of the d i p l o i d l e v e l was associated with the d e l e t i o n of 84D and a 28% decrease with the d e l e t i o n of 92B. This suggests that the number of active genes at 92B i s equal to, i f not greater than, those at 84D. From i n s i t u h y b r i d i z a t i o n data (Dunn e_t a l . , 1979a) i n the d i p l o i d s t r a i n , giant, i t appears that the number of templates complementary Val to tRNA 3 b at 84D i s 25% greater than the number at 92B. These two sets of data can be e a s i l y reconciled. I f , f o r example, the rate Val of t r a n s c r i p t i o n at 92B i s greater than at 84D, the tRNA^ genes at 92B would contribute an equal or greater proportion of the Val tRNA^b to the c e l l u l a r pool. A l t e r n a t i v e l y , the 84D c l u s t e r may 43 contain pseudogenes. Such homologous sequences might anneal with a radioactive tRNA probe but would not be t r a n s c r i p t i o n a l l y active, Measurements of the amount of tRNA^ 1 produced by s t r a i n s 3b haploid for both the 84D and 92B s i t e s showed an average reduction of t h i s tRNA of 45%, suggesting that most, i f not a l l , of the Val active tRNA^ genes can be accounted f o r i n these two l o c i . In support of t h i s hypothesis, i n s i t u h y b r i d i z a t i o n data indicated that 90BC i s a weak s i t e of h y b r i d i z a t i o n , a r e s u l t of e i t h e r the presence of only a few templates i n t h i s region or of incomplete homology between the tRNA and DNA (Dunn et a l . , 1979a). As was mentioned e a r l i e r , pDt41R and pDt48 show four nucleotide d i f f e r -Val ences from the known sequence of tRNA^ , although they have the proper anticodon (Addison ejt a l . , i n preparation). These plasmids map to the 90BC locus. Two other recombinant plasmids containing Val tRNA^ genes have been mapped to 90BC; these genes have been Val sequenced and also d i f f e r from the known sequence of tRNA^ at Val four nucleotides (Addison e_t £il_. , 1982a, b ) . The tRNA^ and Val tRNA^ d i f f e r by 9 nucleotides and show no cross h y b r i d i z a t i o n Val i n s i t u or rn v i t r o . These tRNA^^ pseudogenes may be trans-c r i p t i o n a l l y i n a c t i v e and thus represent n a t u r a l l y occurring mutants. The data on the l e v e l s of tRNA^ 1 observed i n Df (3R)84D/Df (3R)D1 5 ib heterozygotes can be used to test further the Minute hypothesis. I t was proposed that Minute mutations represent small deletions of the tRNA s t r u c t u r a l genes, by analogy with the bobbed mutations (Ritossa et a l , , 1966a, 1966b; Atwood, 1968) which are associated with dele-tions i n rRNA genes. There i s also a s t r i k i n g resemblance between 44 the Minute phenotype and that of the min mutations which are deletions of the 5S RNA genes (Procunier and Tartof, 1975). It has been suggested that t h i s set of phenotypic syndromes could be at-t r i b u t e d to a reduced capacity for protein synthesis. Data i n Table 3 shows that Drosophila with chromosome deletions r e s u l t i n g i n loss of the e n t i r e 84D gene c l u s t e r from one chromosome and loss of the 92B c l u s t e r from the other are e f f e c t i v e l y haploid for the tRNA^ 1 genes. Df (3R) 84D/Df (3R)D1 5 heterozygotes exhibited the expected Dl_ phenotype as w e l l as somewhat reduced v i a b i l i t y and pro-longed development. However, the l a t t e r t r a i t s are general pheno-typ i c c h a r a c t e r i s t i c s of d e f i c i e n c y mutants, and i t i s important to emphasize that no diagnostic features of the M phenotype are d i s -played by f l i e s bearing any combination of the 84D and 92B dele-ti o n s . Thus, i t appears that loss of 50% of the templates coding for a s i n g l e tRNA species does not e l i c i t the M syndrome. This ob-servation s e r i o u s l y undermines the very basis of the Minute hypo-thesis . F i n a l l y , i t must be asked whether any mechanism ex i s t s to compensate for loss of a large portion of the templates for a single tRNA species. In Drosophila i t i s w e l l documented that loss of rRNA genes i s compensated for by s e l e c t i v e a m p l i f i c a t i o n of the templates on the non-deleted chromosomes (Endow, 1980). Compensation has also been observed f o r the loss of 5S RNA genes (Procunier and Tartof, 1975) and more recently for the histone genes (Chernyshev et a l . , 1980). Osley and Hereford (1981) have demonstrated that trans-c r i p t i o n i n yeast c e l l s containing a d u p l i c a t i o n of the histone H2A 45 and H2B genes was dose dependent, but the increased t r a n s c r i p -t i o n a l capacity was o f f s e t by an increased rate of histone mRNA degradation. Thus, the histone mRNA pool s i z e was i d e n t i c a l i n du p l i c a t i o n and normal s t r a i n s . This form of compensation did not extend to the mRNA transcribed from a non-histone gene also c a r r i e d on the duplicated segment, which implies that the compensation mechanism s p e c i f i c a l l y regulates the s i z e of the histone gene mRNA pools. Thus, compensation for changes i n template number may be a general property associated with redundant l o c i and might occur by a v a r i e t y of mechanisms. However, r e s u l t s of experiments with dele-Val tions of the tRNA^ l o c i suggest that the e f f e c t s of these deletions Val . . . on tRNA^b synthesis are additive. Therefore, i t appears that no Val mechanism ex i s t s to compensate for loss of tRNA^^ s t r u c t u r a l genes. This i s i n d i r e c t contrast to the systems discussed above. However, i n a previous study (Dunn et a l . , 1979a), i t was found that the l e v e l of t o t a l v a l i n e acceptance was the same i n wild type and the Df(3R)84D s t r a i n . This could occur i n one of two ways. There could be a s l i g h t increase i n the rate or frequency of tr a n s c r i p t i o n s from Val a l l v a l i n e tRNA genes. Since tRNA^^ represents approximately 30% of the t o t a l v a l i n e acceptance (White et a l . , 1973), a s l i g h t increase Val (~5%) i n tRNA„, production would occur i n the mutant s t r a i n s ; t h i s 3b would not be detected by f i l t e r h y b r i d i z a t i o n . A l t e r n a t i v e l y , trans-Val . . . . c r i p t i o n of tRNA^ genes could be s e l e c t i v e l y increased, since t h i s isoacceptor can respond, with reduced e f f i c i e n c y , to the same codon Val as tRNA^ (Dunn et aJL. , 1978). In any event, compensation of t o t a l v a l i n e acceptance occurs by a mechanism which does not s e l e c t i v e l y 46 increase the deleted template. Thus, a more complicated mechanism than e x i s t s f o r other redundant l o c i appears to be involved. 47 CONCLUSION The h y b r i d i z a t i o n system described has been demonstrated to hybrid-. . . 125 lze e f f i c i e n t l y nanogram qua n t i t i e s of I-labeled tRNA. Hybridization conditions can be manipulated to achieve a high maximum h y b r i d i z a t i o n e f f i c i e n c y . The system has been applied to measure the changes i n Val tRNA^D l e v e l s i n mutant Drosophila, and these measurements have provided V a l information about the function of d i f f e r e n t tRNA^ gene cl u s t e r s i n vivo Other possible applications of the technique include measurement of i n v i t r o t r a n s c r i p t s from the recombinant plasmids carrying tRNA genes and measurement of tRNA l e v e l s i n s p e c i f i c tissues of Drosophila. Thus, further i n s i g h t into the functioning of the tRNA genes i n Drosophila can be obtained. 48 BIBLIOGRAPHY Addison, W.R., A s t e l l , C.R., Delaney, A.D., Gillam, I.e., Hayashi, S., M i l l e r , R.C., Rajput, B., Smith, M., Taylor, D.M., Tener G.M. (1982a) The structures of genes h y b r i d i z i n g with tRNAY from Drosophila melanogaster. J. B i o l . Chem. 257:670-673. Addison, W . 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APPENDIX A M u t a n t r a t i o t R N A , * 1 / t R N A , D f ( 3 R ) 8 4 D p r oe p r Df (3R)D1 roe p c o n t r o l 1. 64 1. ,42 mutant 1. ,50 1. ,49 c o n t r o l 2 . 46 2. ,30 mutant 1. ,99 1. ,99 c o n t r o l 1. ,63 1. ,55 mutan t 1. ,46 1, ,30 c o n t r o l 1. ,64 1. ,42 mutant 1. ,35 1. .50 c o n t r o l 2. ,46 2. ,30 mutant 1. ,75 1. ,86 c o n t r o l 1. ,63 1. .55 mutant 1. ,18 1. ,18 c o n t r o l 1. ,59 1. ,57 mutant ,89 ,88 c o n t r o l 1. .64 1, .42 mutant 1. ,40 1, .46 c o n t r o l 2, .46 2, .30 mutant 1. .84 1, .78 c o n t r o l 1, .63 1 .55 mutant 1. .27 1 .20 c o n t r o l 1, .59 1 .57 mutant .92 .92 2 . 3 6 1.64 2 .24 2 . 0 0 1.72 1.23 2 . 3 6 1.60 2 .24 1.72 1.26 1.46 .89 2 . 3 6 1.62 2 .24 1.86 1.72 1.19 1.46 .91 2 .37 2 .00 2 .31 1.97 1.82 1.37 2 .37 1.52 2 .31 2 .00 1.82 1.25 1.45 .91 2 .37 2 .31 1.92 1.82 1.11 1.45 .89 % A v g . % A v e r a g e D e c r e a s e d e c r e a s e 2 .18 1. 99 16 1.82 1. 69 2 .28 2 . 32 15 2 . 3 0 1. 99 1.68 1. 68 21 1.29 1. 33 2 .18 1. 99 24 1.67 1.53 2 .28 2 . 32 17 2 .16 1. 94 1.68 1. 68 25 1.39 1. 25 1.43 1. 50 40 .95 88 2 .18 1. 99 25 1.51 1. 50 2 .28 2 . 32 23 1.60 1. 80 1.68 1. 68 27 1.32 1. 22 1.43 1. 50 39 .90 91 APPENDIX A (continued) r a t i o tRNA,|* /tRNA! Mutant T(2,3)D1 9 control 1. 64 1. 42 2.36 2.37 p roe p mutant 1. 52 1. 53 2.28 1.83 control 2. 46 2. 30 2.24 2.31 mutant 1. 80 2. 25 1.86 2.20 cont r o l 3. 98 4. 29 4.17 4.56 mutant 4. 05 3. 51 3.71 4.50 control 1. 59 1. 57 1.46 1.45 mutant 1. 08 1. ,29 1.36 1.25 T(2;3)D1 9 c o n t r o l 84 .85 .77 .92 Df (3R)84D mutant ,68 ,64 .68 .66 cont r o l ,93 1. ,0 1.08 1.07 mutant ,65 ,68 .69 .74 control 1. ,59 1. ,57 1.46 1.45 mutant 1. .02 1, .15 1.14 1.22 Df (3R)84D control 1, .64 1. .42 2.36 2.37 Df(3R)D1 5 mutant 1, .62 1 .56 1.44 1.72 control 3 .98 4.29 4.17 4.56 mutant 2 .00 1 .76 1.86 2.04 control 1 .59 1 .57 1.46 1.45 mutant .75 .73 .67 .68 control 1 .48 1 .49 1.53 1.60 mutant .71 .70 .74 .76 % Avg. % Average Decrease decrease 2.18 1.99 9 1.93 1.82 2.28 2.32 12 2.13 2.05 4.64. 4.33 11 3.26 3.81 1.43 1.50 15 1.41 1.28 .86 .86 25 .59 .65 1.06 1.03 33 .70 .69 1.43 1.50 25 1.07 1.12 2.18 1.99 20 1.65 1.60 4.64 4.33 54 2.28 1.99 1.43 1.50 53 .69 .704 1.66 1.55 54 .70 .72 APPENDIX B - S t a t i s t i c a l Analysis Df(3R)84D Df(3R)Dl 5 T(2;3)D1 9 ffiffigff ffigffi Y 22.6 28.5 11.8 27.6 45.3 n ZY 158 114 47 83 181 n 7 4 4 3 4 I. ANOVA A. Comparison between Df(3R)84D and Df(3R)Dl 5 Y-Y Among groups 1 , 0 r i 0 F s Y-Y Within groups Y-Y Total df SS MS 1 89 89 20 799 40 21 888 2.23 F.05[l,20] = 4.35 .'. Fs « F.05[l,20] - no s i g n i f i c a n t difference B. "Comparison between Df (3R)84D + Df (3R)D1 5 and jj^Sl? 4" Df(3R)D1-v v A df SS MS Fs Y-Y Among groups —r — — & & F 1 190 190 1.57 Y-Y Within groups 14 1697 121 Y-Y Total 15 1887 F.05[l,14] = 4.60 .-. Fs « F.05[l,14] - no s i g n i f i c a n t difference This ANOVA was calculated using a subgroup of Df(3R)84D to correct f o r differences i n sample s i z e . 55 A two t a i l e d t - t e s t of the hypothesis that the sum Df (3R)84D7roe "PP + Df (3R)Dl 5/roe p p does not d i f f e r - s i g n i f i c a n t l y from Df(3R)84D7Df(3R)D1 5 o r I 5A + V *C I " 0 A B C Df(3R)84D/roe p P Df(3R)Dl 5/roe p P Df(3R)84D/Df(3R)D1 5 22.6 28.5 45.3 7 4 4 74.30 51.67 283.58 S E = / S 2 A ' + S 2 S 2 = 9 72 1 - _A- B + C nA nB t = YA + YB + Y C — = .597 SE df = n A + n B + n c - 3 = 12 '.05112] = 2 - 1 7 9 ••• fc« t . 0 5 [ 1 2 ] - no s i g n i f i c a n t difference 

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