Genes coding for valine transfer ribonucleic acid-3b in Drosophila melanogaster

J. 301. Biol.

(1979) 128, 277-287

Genes Coding

for Valine Transfer Ribonucleic Drosophila melanogaster

Acid-3b

in

ROBERT DUNN, SHIZU HAYASHI, I. C. GILLAM A. D. DELANEY. G. M. TENEK Department of Bioch~emistry University of British Cokmbia Vancouver, B.C., Canada, F6T 1 W.5 '1‘. A. GRICLIATTI,

T. C. KAUFMAN?

AXI)

D. T. SUZUKI

Department of Zoology University of British ColuwLbiu T’6T 7W5 Vancouver, B.C., Canada,

‘I’lle genes for tKNAl;’ were localized to 84D and H2R 011 the polytene chromosomes of .Dro.qophila melanoguster with a possible minor site at WB-(1 by hybridtRNA$“. Vlies carrying ieat,ion G/L &tlr and autoradiography with lz51-labeled it duplicatjion of ttle 841) region had increased amounts (30%) of tRNA.k” irL proportion to the increased number of genes. While a prqxu%ional decrease in tht, alnount of tRNA:i’ in flies bearing a deletion of tile same region was found, tIltA t,otal accept.ancr of \-aline remained at. thtl level follntl in tzlw w11d t,,vpfs.

1. Introduction St,udies on the organization of genes coding for transfer RNAs have become possibk only recently. 111Escherichia co& tRNA genes are located in clusters at several posit)ions in the genome and may contain sequences corresponding to different tRNAs (Smith, 1976). Lt. is also known that the genes for several tRNAs are located in the spactar regions between the 16 S and 23 S ribosomal RNA genes (Lund et cd.> 1976). There art’ about 60 genes coding for tRNA in h’. coli (Smit’h. 1976) while in eukaryotes this number is much larger. In Xenopus Zueoi.s there are estimated to be 43 different tRNA sequences, each represented in the genome by an average of 200 copies (Birnstiel it nl.. 1972; Clarkson et al.. 1973a,b). Clarkson et al. (1973a,b) have proposed a model in nhich the reiterated sequences coding for a single tRNR are arranged in clusters iLnd separated by spacers with an average length of about 760 base-pairs. This model is analogous to the arrangement of the genes coding for ribosomal and 5 8 RNAs in the same organism (Birnstiel et al, 1971 ; R rown et al., 1971). The genes for one species of tRNA, tRNAy, have been shown (Clarknon & Kurer. 1976) to occur in a tract of repeated units 3100 base-pairs in length. t Prosent address:

Department

0022~2830/79~070277-11

$02.00/O

of Zoology,

University of Indiana, Bloomington, Ind. U.S.A. 217 Q 1979 Academic Press Inc. (Londcn) Ltd.

27x

R. DUNN

ET

AI,.

There

is evidence to support the proposal that tRNA genes in Sacchuromyces also occur in clusters with the genes being separated by spacers of about 600 base-pairs (Feldman, 1976). However, genetic studies (Sherman et al., 1973) showed that genes for tRNA TYr fall into eight unlinked groups capable of mutation to a tyrosine-inserting suppressor. Digestion of yeast DNA with endonuclease EcoRI yields eight distinct and separable fragments capable of hybridizing with tRNATY’ (Olson et al., 1977). Seven of these fragments have been separated and successfully introduced into viable bacteriophage lambda and. in all cases but one, contain genes for only tRNATYr. In eukaryotes there are separate tRNAs and tRNA genes in mitochondria and chloroplasts (Borst, 1972). The tRNA genes on yeast mitochondrial DNA are mostly clustered in one region of the genome but some genes are found elsewhere (Martin et al., 1977). Not all are in tight clusters. Electron-microscopic mapping of tRNADNA hybrids with mitochondrial DNA of HeLa cells and Xenopus has shown that the tRNA genes are widely spaced (Angerer et al., 1976; Dawid et al., 1976). Less is known about the organization of genes for tRNAs in Drosophila melanogaster. Chromatography of its tRNA on the RPC-5 system revealed about 100 distinct isoanalysis of the hybridization of tRNA to acceptors (White et al., 1973). Kinetic Drosophila DNA indicated that the tRNAs represent 59 different sequences, each of which is repeated an average of ten times in the genome (Weber & Berger, 1976). In a study of the hybridization of tRNA;YS to larval salivary gland chromosomes (Grigliatti et al., 1974) a single binding site was detected at band 48F-49A, thereby suggesting that all of the genes coding for this tRNA may occur in a cluster as in Xenopus (Clarkson et al., 1973a,b). Recent studies on a plasmid containing DNA from Drosophila which hybridizes to 4 S RNA have shown that four tRNA genes are clustered with spacers of 1.2 to 3.3 x lo3 base-pairs between them (Yen et al., 1977). Three of these genes have similar sequences while the fourth is different. In this paper we report a study in Drosophila on the localization of genes for tRNA;;:,&’ to two sites on the right arm of the third chromosome and on attempts to confirm this localization by studying tRNA levels in flies carrying a genetic duplication or deficiency in one of these regions. cerevisiae

2. Materials and Methods (a) Growth

of Drosophila

stocks

The Samarkand strain of D. meZanogaster was grown at 22°C in large plastic cages on with carbon dioxide standard Drosophila medium (Lewis, 1960). Adults were harvested and stored at - 70%. Mutant strains were raised under similar conditions in quarter-pint bottles at 22°C. (b) Preparation

of tRNA

and aminoacylation

with radioactive

oaline

Crude transfer RNA was extracted from adult flies by the phenol method of Kirby (1956) as described by White & Tener (1973). The crude tRNA preparations were further purified on a Bio-Gel A-0.5 m column (1.2 cm x 42 cm). The tRNA preparations were heated to 70°C for 5 min, loaded onto the A-0.5 m column and eluted with distilled water at a flow-rate of 1.5 ml/min. The transfer RNA eluting in the included volume was freezedried and dissolved in 10 mivr-Tris .HCl (pH 7.5), 10 mM-MgCl, for aminoacylation. The preparation of Drosophila aminoacyl-tRNA synthetases and the aminoacylation reactions were carried out as described by White & Tener (1973).

tRX.4;;:’

GENES

TN DHOSOI’Hl

Id.1

(c) Reverse phase chromabgraphy The RPC-5 system described by Pearson et al. (197 1) was preparrd by a modified method. Ohio) (4 ml) was added to 500 ml of 0.45 Adogen 464 (Ashland Chemical Co., Columbus, x-NaCl, 10 mM-MgCl,, 10 mM-sodium acetate (pH 4.5) in a Waring blender. I-\Rer britlf 75 g of Plaskon CTFE-2300 powder (Allied Chemical Inixing to emulsify the Adogen, (lorp, Morristown, N.J.) was added and mixed at high speed for 10 min. The 5uspeusiorr bvas depassed and passed through a 200-mesh stainless steel sieve. To remove fines t.tlt* solids were resuspended twice in 500 ml of the same buffer a11d allowcad to settlr befort, packing irlto a glass-walled high-pressure column (0*9 cam r 21 cm) ,jackcttatl at 37’(’ Un-omatography on RPC-5 was performed with gradients of concentration of Na(.‘l in t 1 Inw-%-lnercapt,oebllttrlol arid IO of 2 buffer systems. System A contained 10 m;cr-M&l,, rn.zl-sodium acetate (pH 4.5). System B contained 1 mu-EDTA, 1 ~n~-2-mercaptoetl~ar~ol end 10 mw-sodium formate (pH 3.8). For determination of radioactivity by scint)illat iota counting samples of the eluate (0.5 ml) were dissolved in a cocktail prepared from ‘l’rit,jll NIOI. xylene and fluors (Lieberman & Moghissi, 1970).

Tile purification of this species by repeated ctraract)erization have been described in detail (e) Labeling

chromat,ograpby in several syst,ems and it,s elsewhere (Dunn eb al., 1978).

I$ tR5A,yj’

with. It51

Purified t,RNAi;’ was iodinated with lZ51 bv a procedure aclupt’ed from the methods of Commerford (1971). The tRNA (1 to 2 pg) \t:as incubated in a solut,ion of 0.4 m~l-T1(‘lZ. 0.3 M-sodium acetate (pH 5.0) at 70°C for IO min in a vol. of 20 ~1. Approx. 1 mCi of carrier-free Nalz51 was mixed with 0.8 nmol NaI and addrd to t,lltb RNA solution to bring the volume to approx. 30 ~1. This solution was incubated at 70°C for 30 min. Tllr react,ion mixture was then diluted to 1 ml with 2 x SSCt, 10 ~1 0.1 M-Na,SO, was added and mixed thoroughly. At this point, 2 AzeO units of E. coli tHNA in 50 ~1 of water \VBS added and the mixture loaded onto a column of DEAE-cellulose (0.5 cm x 3 cm). Aft,el washing with 2 x SSC (75 ml) the tRNA was eluted with 10 x SSC. The tRNA (3.0 ml vol.) was mixed with 10 ~1 0.1 M-Na,SO, and incubated at 70°C for 20 min to reduce unstable iodine adducts. The [ 1251]tRNA was then chromatographed on a column (0.9 cm x 40 cm) of Bio-Gel P-10 in 2 x SSC (or in SSAE) and the labeled tRNA collected ill the excluded volume. Its specific activity was normally between 7 x 1O7 and 2 x 1O* disints. min per pg. (f) Hybridization

of iodinated

tRKA2

to Drosophila

salivary

gland chromosomes

Two lots of [1251]tRNA;;’ were used: in 2 x SSC, 157 pg/rnl and 6.8 x IO7 cts/mirr pw /~g; and in 1 x SSAE, 340 pg/ml and 1.9 x lOa cts/min per pg. Chromosomes frorn salivary glands of late third instar larvae of the giant mutant of D. vnelanogaster were prepared fol hybridization in situ according to the method described by Gall & Pardue (1971) with a modification introduced by Bonner & Pardue (1976). Hybridizations were carried ont using 40 ~1 per slide of iodinated tRNA in 2 x SSC for various lengths of time at 65 ‘(.‘. Hybridizations were also carried out using chromosome preparations which had beell acetylated according the the procedure outlined by Hayaslli et al. (1978). In this case the buffer used was SSAE. After hybridizat,ion, the cover slips were removed by dipping t,he slides in the buffer used during hybridization and the slides were transferred to 2 x SS(’ for I5 min. Removal of unbound tRNA, subsequent washings, and dehydration were caarricad out as described by Gall & Pardue (197 I ). Autoradiograph>was carried out according to Grigliatti et al. (1974) and the chromosomes were stained with 0.040:, toluidillr~ t)lue (Baker (‘hemical) in 2 x SSC at pH 7.

Stocks

t RNAy$

(g) Description of duplication. arrd deficiency stocks c-arryirlg a duplication arltl deletion of one of tilt, Irgiolrs itlrnt~ified gem’s (see Kesultx) were const,rnet,ed. Tlir tirletiotl (~tIrolnc)somr

as a sitta of is Df(3R).

280

R. DUNN

ET

AL.

Antp Ns +R17 in which

the material from 84B1,2 through 84D12 inclusive is missing 1975). The origin and cytology of the duplication chromosome ln(3R)dazDfR3LAntpBR is complicated (Kaufman, T. C., Lewis, R. & Wakimoto, B.. unpublished data) and it is sufficient for this paper to indicate that the material from 84B2 through 84D12 is present twice in tile chromosome. Both chromosomes are homozygous lethal and are maintained in balanced stocks with Im(3LR)TM3,vi pp sep h’b br3*’ es Ser (Lindsley & Grell. 1968). All of the ahove cyt,ology is described by Lefevre ( 1975). (Duncan

& Kaufman,

3. Results (a) Hybridization

of iodinated

tRNA:t’

to chromosomes

A typical autoradiogram is shown in Figure 1. Silver grains indicating the binding of radioactive material were consistently found only over two regions, at bands 84D3-4 and 92B on the right arm of the third chromosome; with 557, of the grains over 84D-4 and 45O/, over 92B. Subsequent experiments using shorter periods of hybridization (2 or 4 h), shorter times of exposure (4 or 8 days) or lower concentrations of salt (SSAE) all yielded similar results. A minor binding site was also detected on the third chromosome at bands 90B-C. This region had less than 20:/, of the number of grains found over 84D3-4.

Fm. 1. Hybridization of [ 1251]tRNAy;i to salivary gland chromosomes. The tRNA (speo. act. 6.8 x 10’ cts/min per pg) was hybridized to salivary gland chromosome preparations from the giant mutant. Hybridization was for 8 h at 66°C in 2 x SSC. Two sites are strongly labeled, 84D (a) and 92B (b). A third site, 9OBC (c) is lightly labeled. This preparation was exposed for 16 days. Total magnification, 3200 x .

tRYi,\;;;:’

(b) Mutants

C:EXES

qf Drosoph~a

IN

DROSOI’III

L’s1

I,..1

with changes ill, dosage for I)utatirv

t RNA:,“’

genps

Flies carrying a deletion and others bea,ring a duplication spanning 84D (Duncan & Kaufman. 1975) \vere grown and their t#RNAs extracted. The grnot,yprs of t,hese thrw +*aiIls :lw summarized in Table 1 TABLE

I,ist of the genotypes

1

of sfraiw of Drosophila duplicati0n.s at site MD

with dqficietrcirs

IV

Wait1

All of these stocks carry chrornnsornes RW wiltl-type

identical Oregon-R

Oregon-R backgrounds, ohmmonomos.

(0) ,Tjfect of gere dosage for tRNA:f’

that

on valbe

LH the swr)ntl,

fourth.

and

SC+):

acceptn?lce

Table 2 shows t*he ratios of total valine acceptance to t,hat for I.vsine and alanine as int,ernal controls by preparations of tRNA from the three strains. While the duplication stock showed a. signifiwnt increase in valinc a~cttpt,i~,ll(!c’. t,he deletion did not> she\\- tsht: expectrd dccrrasc. TABLE

2

I~nlit~ acqtatace of tRNA prepared ,from Drosophila stock.9 with n dqficiency or duplication around site 840

Strailr

Ratio of amino acid acceptance

y. , ,f control

(‘alcnlatrdt

100 105 1111

100 90 110

100 103 115

100 90 110

Valinc/lysirle$ Control Deficient) Duplication

0~96 1.01 1.14 Valine/alanine

(‘ontrol Deficiency Ihlplication

0.89 0.92 1.02

$

t These numbers assume tRNA$’ to be 35% of the total valine tRNAs in the control wit,h changes in t,he mutants (see Fig. 4) proportional to the change in thus number of genes. $ These values are the average of 2 experiments each. In this laboratory the difference between replicates in t,he analysis of amino acid uptake is always less than 8% and usually no more than 3qb. $ These valnw arp the average of 4 experiments each.

(d) Effect of gene dosage on the amount oft RNA::’ On RPC-5 chromatography in system A (pH 4-O in the presence of magnesium ion) t#RNAji,“’ and tRNAy::,&’ elute together (Fig. 2(a) and Dunn et al., 1978). System R (pH 3.8. in the absence of magnesium ion) resolves these t(wo species but causes

282

R.

DUNN

ET

AL.

Wild type 3a and 3b 4

IL 5

6

I2

90

120

150

(0)

5

Def ,c,ency

I$ 90

Fractm (b)

no

120

150

(cl

FIG. 2. Chromatography on RPC-5 in system A of valyl-tRNA “&I from flies with duplications of deficiencies at site 84D. tRNAs (4 to 6 A,,, units) from the 3 stocks described in Table 1 were aminoacylated with [‘%]valine (173 mCi/mmol) as described in Materials and Methods. Each preparation of [‘%]valyl-tRNAVal (between 40,000 and 70,000 cts/min) was chromatographed on an RPC-6 column (0.9 cm x 21 cm) at 37% in RPC-5 buffer system A (10 mm-sodium acetate pH 4.0, 10 mM-MgCl,, 1 mM-2-mercaptoethanol). Fractions (0.5 ml) were eluted with a loo-ml linear gradient of NaCl (0.50 M to 0.66 M) at a flow-rate of 15 ml/h. The ratio of peak 3 to peak 4 was calculated from the measured areas under these peaks. (---------) Cts/min per 0.5 ml.

c

1

4and5

)eflclency

Wild type I-

30 -

3b )-

I-

) -

6 ,?I

20 -

3u J. . 80

c,,.A

80

100 120 140 EC

100 120 140 160

Fractmn na

(0)

(bl

Cc)

Va’ from flies with duplications FIG. 3. Chromatography on RPC-5 in system B of valyl-tRNA or deficiencies at site 84D. tRNAs (4 to 6 A,,, units) from the 3 stocks described in Table 1 were aminoacylated with [3H]valine (824 mCi/mmol) as described in Materials and Methods. Each preparation of [3H]valyl-tRNAVa’ (between 47,000 and 69,000 cts/min) was chromatographed on an RPC-5 column (0.9 cm >: 21 cm) at 37°C in buffer system B. Fractions (0.5 ml) were eluted with a loo-ml linear gradient of NaCl (0.55 M to 0.75 M) at a flow-rate of 16 ml/h. The areas under the 3 major peaks were determined, from which the ratios of tRNAy2’ and tRNA$’ to RNAE were calculated (Table 3).

t,RNA;;’

GENES

IN

DROSOPHILA

L’S:%

t,Rn’A;;,“’ to be incompletely resolved from tRNAz” (Fig. 3(a) and Goodman et (11.. 1978). As can be seen in Figure 3 (and summarized in Table 3) the ratio of tRNA:i’ to the remained constant in the three stocks. The same elution diagrams corn bined tRNAz:k or and Table also show that the quantity of tRNA,, vs’ in relation to either tRNA:z’ t,RKAz$ in the mutant stocks differed from t’he control value. Figure 2 shows elution profiles of valyl-tRNAa yvs’ from the three stocks run on RPC-5 in system A. The rat,ios are also shown in Table-S. Tlrr of levels of combined valyl-tRNAT&’ to valyl-tRNAla’ _ ratios for the aneuploid stocks again differed from that in the wild type.

4. Discussion Conclusions derived from the results of hybridization between an RNA probe and DKA depend upon the purity of the RNA sample used. The isolation and characterizaused here has been described elsewhere (Dunn et al., 1978). In tion of the tRNA:t’ brief, the RNA was obtained by chromatography on a series of four columns. The unit, a value within the range exproduct accepted 1760 pmoles of valine per A,,, pected for a pure acceptor. On a fifth chromatographic system it ran as a single peak. Also its nucleoside composition showed close to one residue of each of several modified bases per molecule and it contained no traces of C, or U, which are found in tRNA:t’ Part of this highly purified sample of tRNA:t’ was labeled t’o high and tRNAp’. specific activity by reaction with radioactive iodine and hybridized to slides carrying prepared Drosophda larval salivary gland chromosomes. Figure 1 shows that two major sites on the right arm of the third chromosome were labeled. Even when conditions of hybridization were varied these two bands, 84D and 92B, were consistently labeled. If the DNA sequences at different chromosome positions have an equal probability of binding homologous RNA, relative grain counts should reflect the proporbion of genes coding for tRNAj6&’ in the two bands. Including the third less consistently labeled band the ratio of grains over 84D. 92B and 90B-C was 5 :4:+9. There are several possible explanations for the weak hybridizat’ion at this last position. One possibiliby is that it was labeled 1)~ t,races of a contaminating RlYA which is itI too low a concentration t’o be detectable otherwise. Alternatively tRNAr:l may havr i\ degree of homology with another DNA sequence at this site and bind weakly to it.. Both possibilities are minimized to some extent since shorter periods of incubation also gave labeling at this posit’ion. A third possibility is that’ this site may represent R single copy of the gene for tRNAy:l, whereas t,he two major sites represent multipIt, copi~. Thus. we conclude that clusters of genes for t,RNAI:’ occur at- two major sites on the right arm of t#he third chromosome wit’h a possiblra minor site at R positiotl t wtwrn the t.wo. If’ region 84D is indeed the site of 50 to 550;; of the genes coding for tRNAT:‘. a deletion or duplication of that region should specifically alter the level of this isoaccepting species by a predictable amount) (assuming that the levels of t,RNAs ark proportional to gene dosage). Indeed, flies bearing the duplication gave tRNA that was found to have a 15 to 1976 increase in valine acceptance over the control. HOWever. the stock carrying the deletion also exhibited a 3 to 59/i, increase in valine acceptance and not the expected decrease. These levels of acceptance are greater t#hnll thoscb exptact,ed from the change in bhe gene dosage. If‘ w ignore the minor site at BOB-C R model for thr dist,ribution of gents fi)r

1.14 1.39 0.88

TABLE

3

0.31 0.31 0.31

peaks in Figs 2 and 3.

0.83 1.08 0.57

100 123 71

in the RPC-5

0.73 0.90 0.52

by chromatography

tRNA,, Vd as O/oof control

measuredf

tRNA?‘/tRNAVa!db 18,

isoacceptors

t Amounts of tRNAs obtained by measuring the area under the chromatographic $ These data from Fig. 3. 5 These data from chromatograms such as t.hat. in Fig. 2.

Control Duplication Deficiency

system A$

in buffer

Chromatography

tRNA;:‘/tRNA::;

system Bf

0.29 0.28 0.29

strain

in buffer

of tRNA;,a’

Control Duplication Deficiency

Drosophila

Chromatography

Amounts system

100 130 69

tRN.A;;i GENES

IN

DROSOPHI

I,.-1

2S.i

tRNAz;’ may be formalized as shown in Figure 4. The wild-type chromosomes carr! live (or a multiple of that number) copies of the gene at 84D and four (or the same multiple) copies at 92B. The expected changes in number of these genes in the two chromosomal va,riants are as shown. Since tRNAz::,&’ represents only about 35”,, of the total valine acceptance (Fig. 3(a)), the changes in acceptance of valine between tRNAs from either mutant and the wild-type flies is expected to he about lOq6. As indicated in Table 2, the actual acceptances by tRNA from both variants was greater than anticipated. These results suggest the existence of possible control mechanisms for tRNA production which respond either to the absolute amount8 of a given accept801 activity in a cell or to the ratio of various isoaccept,om.

Total

% of wld type

Wild type

I8

100

Duphcotlon

23

128

84 D(5umts)

92 8 (4 units1

72

Deflcmcy --I

A--.

Definitive studies of the changes caused by deletion or duplication require detrrminations of levels of tRNAzt;,&’ itself. No single chromatographic system t’ried gavel complete resolution of all seven valine isoacceptors. Changes were therefore estimated from the results of chromatography in two systems. System B (pH 3.8 in the absenccb of magnesium ion) separated tRNAzi,&’ and tRNA:t’ (Fig. 3). The latter was incomplet,ely resolved from the minor species tRNAp’. Comparison of the areas under these peaks with those under the combined peak of tRNAl$ showed t’hat the relative content of tRNA:t’ was constant between control and mutant stocks (Table 3), while the relative levels of tRNAz:’ increased and decreased, respectively, by 20 to 300,, in the duplication and deletion stocks. Chromatography in system ,4 (pH 4.0, 10 matmagnesium ion) (see Materials and Methods, section (c)) resolved the species tRNAy:’ and tRNAi” but caused both species of tRNAz*’ to elute together (Fig. 2). Table 3 shows the ratios obtained for each stock for the areas under peaks 3 and 4 and thei! tRNA1”’ and tRNAz*’ remained constant average values. The proportions of tRNA:z’, in all three stocks (Table 3) and in the wild type the relative amounts of tRNA:i’ and tRNA;;’ were, respectively, 0.29 and 0.73 that of the combined tRNAz:k (Table 3). The tRNA:t’ value is slightly in error since it also includes the minor component teRNAT (Fig. 3). I n experiments not presented here this value was amended by rt’chromatographing in system B a sample of combined tRNAp’ from wild-type flies separated on system A. The ratio of tRNA:i’ to combined tRNAi” was 0.27. Thus. tRNA;;’ contributes 27% to the total valine acceptance of the tRNAy’ peak in wild remains constant in the mutants (Fig. 3) type flies. The ratio of tRNA~~l/tRNA~l and allows calculat’ion of the ratios of tRNA:i’ to tRNAI*‘. Table 3 shows that the

286

FL. DUNN

ET

AL.

presence of the deletion decreased the level of tRNAj6&’ by 31% and the duplication increased it by 30%, values close to the 28% predicted by the model (Fig. 4). This quantitative agreement supports the proposal that genes for tRNAT::,&’ occur at two major loci, one of which is at 84D. The simple proportionality between the dosage of genes and their product, tRNA:t’, parallels that described by Grell (1962) for the protein xanthine dehydrogenase in rosy mutants of Drosophila. A number of attempts have been made to determine the absolute number of in vitro both in solution and on cellulose nitrate tRNA”*’ genes by hybridization discs. Although initial studies indicated the presence of 12 to 13 genes for tRNA:t’, subsequent studies to be reported elsewhere have shown the kinetics of hybridization of the purified tRNAs to be complex. A recent study (Yen et al., 1977) on the structure of a plasmid incorporating a segment of Drosophila DNA containing genes for tRNAs indicated that tRNA genes in Drosophila may not be as closely clustered as they are in Xenopus (Clarkson et al., 1973a,b; Clarkson & Kurer, 1976). We intend to investigate further the organization of tRNAzt:,&’ genes by a similar approach. The work reported in this paper was supported by grants Council of Canada (MT-1279), the National Research Council National Cancer Institute (contract 6051).

from the Medical Research of Canada (A-1764) and the

REFERENCES Angerer, L., Davidson, N., Murphy, W., Lynch, D. 85 Attardi, G. (1976). Cell, 9, 81-90. Birnstiel, M. L., Chipchase, M. & Speirs, J. (1971). Progr. Nucl. Acid Res. Mol. Biol. 11, 351-389. Birnstiel, M. L., Sells, B. H. & Purdom, I. F. (1972). J. Mol. Biol. 63, 21-39. Bonner, J. J. & Pardue, M. L. (1976). Chromosoma, 58, 87-99. Borst, P. (1972). Annu. Rev. Biochem. 41, 333-376. Brown, D. D., Wensink, P. C. & Jordan, E. ( 197 1). Proc. Nat. Acad. Sci., U.S.A. 68, 3175-3179. Clarkson, S. G. & Kurer, V. (1976). Cell, 8, 183-195. Clarkson, S. G., Birnstiel, M. L. & Serra, V. (1973a). J. Mol. Biol. 79, 391-410. Clarkson, S. G., Birnstiel, M. L. & Purdom, 1. F. (1973b). .7. Mol. Biol. 79, 41 I-429. Commerford, S. L. (1971). Biochemistry, 10, 19!#3&2000. Dawid, I. B., Klukas, C., Ohi, S., Ramirez, J. L. & Upholt, W. B. (1976). In The Genetic Function of Mitochondrial DNA (Saccone, C. & Kroon, A. M., eds), pp. 3-13, NorthHolland Publishing Company, Amsterdam. Duncan, I. W. & Kaufman, T. C. (1975). Genetics, 80, 733-752. Dunn, R. J., Addison, W., Gillam, I. C. & Tener, G. M. (1978). Canad. .I. Biochem. 56, 618-623. Feldman, H. (1976). Nucl. Acids Res. 3, 2379-2386. Gall, J. G. & Pardue, M. L. (1971). In Methods in Enzymology, (Grossman, 1. & Moldave, K., eds), vol. 21, pp. 470-480, Academic Press, New York and London. Goodman, H. M., Olson, M. V. & Hall, B. C. (1977). Proc. Nat. Acad. Sci., U.S.A. 74, 5453-5457. Grell, E. H. (1962). 2. T/ererbungslehre, 93, 371-377. Grigliatti, T. A., White, B. N., Tener, G. M., Kaufman, ‘I’. C. & Suzuki, D. T. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 3527-3531. Hayashi, S., Gillam, I. C., Delaney, A. D. & Tener, G. M. (1978). J. Histochem. Cytochem. 26, 677-679. Kirby, K. 8. (1956). Biochem .J. 64, 405-408. Lefevre, G. (1975). In The Genetics and Biology of Drosophila (Ashburner, M. & Novitski, E., eds), vol. la, pp. 31-66, Academic Press, New York.

tl%NA:;;’

GENES

TS nROsOPHtI..-l

28;

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