Volume 3 no.9 September 1976

Nucleic Acids Research

Arrangement of transfer-RNA genes in yeast

Horst Feldnan Institut fur Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universitat Miinchen, Goethestrasse 33, 8000 Miinchen 2, GFR

Received 8 July 1976 ABSTRACT

The redundancy and the arrangement of the genes for specific transfer ribonucleic acids in yeast were studied by the hybridization techniques developed by Birnstiel et al., e.g.jl). The redundancy was found to be in the order of 10 genes for tRNpMet tRNpmet, tRNASer. and tRNAPro. High molecular weight yeast DN was 3fractionated by density gradient centrifugation in cesium chloride and the L32P3tRNAs were hybridized to the single fractions. The results together with earlier findings [21 suggest that the cistrons for these tRNAs are arranged in tandem interspersed by 6 to 10 times longer segments of spacer DNA which varies in (G+C) content for the different tRNA species. INTRODUCTION

The arrangement of transfer RNA genes has been studied so far in some eukaryotic organisms, for instance in yeast, e.g. [3j, in Drosophila, e.g.[ 4,5] , and in most detail in Xenopus, e.g.ElJ. It has been shown for Xenopus [Ijthat the tRNA genes are highly redundant and a model has been proposed for the arrangement of the tRNA genes in which one sequence coding for a tRNA is linked to a non-transcribed spacer DNA sequence of relatively low (G+C) content. Such an arrangement is serially repeated to form an isocoding gene cluster. However, little is known on the actual localization of the tRNA genes in eukaryotes and of the control of-their expression. For a number of reasons, yeast seems to be a suitable system for this type of study: in many respects yeast is very similar to higher organisms, extensive genetic studies have been performed, a variety of specific tRNAs have been sequenced, and pure tRNA species can easily be prepared. Also, some of the aspects of tRNA maturation have been studied in yeast [6-8a. We have applied the C Information Retrieval Limited I Falconberg Court London W1 V 5FG England

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Nucleic Acids Research techniques of hybridization developed by Birnstiel et al. [9,lojas an approach to determine the molecular organization of some tRNA genes in yeast, using DNA which was fractionated by density gradient centrifugation in CsCl and 32P-labeled specific tRNA species.

M!TERIALS AND METHODS

32P-labeled tRNAs from yeast were prepared similarly to the procedures outlined in ref. 6 and 8: yeast cells (aS 288 C) were grown in a minimal medium and labeled with 0.05 mCi/ml [32Plphosphate (The Radiochemical Centre, Amersham) for 16 hrs. Bulk tRNA was isolated by extraction of the cells with phenol and purified by chromatography on DEAE-cellulose. From this [11] and [32P]tRNA,Met [12] were prematerial pared as described in the references. The other [32P]tRNAs were prepared similarly to the procedure described in ref. 8: the tRNAs (up to 10 A260-units) were separated in slab gels which were 40 cm long and had slots of 0.4 x 5 cm. Appropriate bands were cut from the gels after radioautography; the tRNA was extracted either electrophoretically for further analyses or extracted from the minced gel slices with the hybridization buffer (6xSSC, 50% formamide). T1-fingerprinting analyses and nucleotide analyses of the tRNAs were performed according to standard procedures [13,14]. Unfractionated tRNA was purified by gel electrophoresis in order to eliminate the ribosomal 5 S and 5.8 S RNAs.

L32PJtRNkMet

Yeast (aS 288 C) DNA was prepared as described in ref. 15. The DNA isolated in this way had an average molecular weight of 12.106 daltons. DNA was sheared to an average molecular weight of 106 daltons by passage through a French pressure cell. Density gradient centrifugations in CsCl were performed as described in ref. 1. Four hundred ag of DNA in 20 ml 0.1 x SSC buffer, pH 8.0, were mixed with 25.2 g CsCl (Merck, Darmstadt, Suprapure) to give an initial density of 1.700 g/ml and centrifuged in a rotor Ti 60 (Spinco, Beckman Instr.) for 61 h at 33 000 and for 42 h at 26 000 rpm. DNA from M.lysodeikticus was added as an internal density marker. Hybridization on filters was carried out similarly as

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Nucleic Acids Research described in ref. 1 and 9. Total DNA (100 to 250 g) or DNA from single fractions of the gradients were immobilized on Millipore HAWP filters (10 or 25 mm in diameter, respectively) and hybridized with excess tRNA. 40 pg/ml [32P]tRNA (specific activities 2.5 to 8 x 105 cpm/Ag) was used, incubation was for 45 min at 560 C in 6 x SSC, 50% formamide. Non-labeled yeast ribosomal RNA (a gift of G. Pirro of this laboratory) was added at a concentration of 100 pg4ul to the incubation mixtures. RESULTS AND DISCUSSION

High molecular weight DNA from yeast was obtained as reported previously [15]. In CsCl density gradient centrifugation the main band of this DNA was found at a density of 1.698 g/ml corresponding to an average (G+C) content of 38%. In kinetic hybridization experiments, 0.091% of the yeast DNA were found to be complementary to unfractionated tRNA. This represents a haploid genome redundancy of about 360 tRNA 1 daltons cistrons taking the haploid genome in yeast to be 10 in molecular weight f16].The value is in agreement with values reported earlier, e.g. [3J. Our tRNA preparations were essentially free of contaminants, since the same values were obtained in the hybridization experiments without the addition of non-labeled ribosomal RNA. Experiments, in which unfractionated [32P] tRNA was hybridized to homologous native DNA of high molecular weight that had been fractionated by CsCl density gradient centrifugation, gave the result shown in Fig. 1 A. It was found that - contrary to the ribosomal RNAs [3, 17] - a large portion of the tUNA hybridized to main band DNA. In addition, some of the tRNA hybridized to the heavier side and another portion to the light side of the gradient. This pattern remained relatively unchanged, if the native DNA had been sheared to a molecular weight of ca. 106 daltons prior to density gradient centrifugation. A similar finding was reported earlier by Rubin et al. [17]. In order to perform more detailed analyses, several specific [32pJ tRNAs were hybridized to the DNA fractions. Five purified 2381

Nucleic Acids Research

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UZ31 lb

A

ARACTNS

FIGURE 1

Hybridization of yeast [32P]tRNAs to CsCl gradient fractions of homologous native DNA. Absorbance at 260 nm for the DNA, ---o--- radioactivity for [32PJtRNA. The figures above the arrows refer to the average buoyant densities of sequences homologous to the tRNAs based on refractometry measurements (see Table 1). M. lysodeikticus DNA (' = 1.731 g/ml) served as an internal density marker. Pro A: Total tRN., B: tRNA et, C: tRNpMet D: tRNASer, E: tRNA r F: tRNAx. A background of 25 cpm his been deducted. species of [32p] tRNA were used. These were characterized by two-dimensional electrophoreses [83 and by T1-fingerprint analyses (not shown). The primary structures of tRNAMet l11,8J have been reported. The tRN Pro and tRNASer Urpre.Tet tRM tN3Met ndtN2 was one of the isoacceptors for proline; its primary structure is unknown. The aminoacid acceptance of tRN x is not known,

[123,

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r19]

Nucleic Acids Research but according to our fingerprint analysis it was a pure homoof the hybridization exgeneous tRNA species. The results periments are summarized in Fig. 1 and Table 1. As can be seen, different tRNAs hybridized to DNA of differing buoyant density. The two isoaccepting methionine specific tRNAs hybridized near to main band DNK but the two peaks were clearly separated from each other. tRNA Ser hybridized to the heavier side of the DNA gradient, tRNAPro hybridized to the lightest, and tRNAx to the heaviest DNA. The redundancy for a single tRNA species was found to be in the order of 10 cistrons (Table 1). This is in agreement with the occurrence of about 360 tRNA genes and of about 40 to 60 different tRNA species in yeast. In any case, the (G+C) content of a specific tRNA washigher than that of the corresponding tDNA. Furthermore, there wasno strict correlation between the (G+C) contents of these tRNAs and those of the tDNAs. Several conclusions can be reached, if the results obtained here and our earlier findings on the characteristics of yeast tDNA [2] are viewed together. By density gradient centrifuTABLE 1

Nucleotide composition of tRNAs, buoyant densities and (G+C) contents of tDNAs, and redundancy of tRNA genes in yeast. tRNA

Nucleotide comp. (moles %) ) Cp up Ap GP

unfract 20.7 28.1 26.2 24.9 Ser 2 Met 1 Met 3 Pro x

18.8 21.3 26.7 20.9 22.1

32.9 33.3 25.3 28.6 26.0

22.4 32.0 25.3 24.4

25.9 13.4 22.7 23.1 29.8 22.1

tRNA hybri- gene dized redun-

%DNA

tDNA

buoyant

(G+C)

density

(g/ml)b

54.3

I 1.706

55.3 65.3 50.6 51.0 55.8

1.698 11.692 1.700 1.699 1.696 1.692 1.706

c

46 to 32 40 39 36 32 46

d

0.091

0.0026 0.0030 0.0032 0.0021 0.0049

dancy e 360

10 11 12 8 19

a)the values for minor nucleotides were included into the values b for the corresponding parental nucleotides. from refractometry measurements (cf.also Fig. 1) c)determined c)calculated from the buoyant densities by the formula of d Schildkraut et al. [20J. with excess tRNA under saturating conditions. e)hybridization )based on a molecular weight of 1010 daltons for the haploid

yeast genome and of 26500 daltons for a tRNA.

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Nucleic Acids Research gations in Cs2SO4 it was found f2] that yeast tRNA-tDNA hybrids had a ratio of DNA to RNA of 6 to 10, irrespective whether the tDNA had been isolated from DNA with an average molecular weight of 106 or 107 daltons. The lower (G+C) content of the tDNAs as compared to the tRNA cistrons shows that these must be linked to DNA lower in (G+C) content than the tRNM cistrons. Our data are compatible with the assumption that the tRNA cistrons are linked to non-transcribed spacer DNA. The comparison of the values obtained for the different tRNA species (Fig. l and Table 1) indicates that the spacers should be different since the base compositions of the corresponding DNA vary considerably. The noncoincident hybridization of different specific tRNAs with high molecular weight DNA and our earlier findings 12 J would strongly suggest that the cistrons for these tRNAs are arranged in separate gene clusters interspersed by spacer DNA. The data also allow a rough estimation of the lengths of the spacer DNA which on the average should be about 6 to 10 times the length of a tRNA cistron. An accurate calculation would afford much more information. For example, the actual lengths and the base compositions of the rRNA cistrons are unknown: the tRNA precursor molecules that could be isolated from pulse-labeled yeast cells contain 100 to 145 nucleotide residues but they do not seem to correspond to the original transcription products E 6). It was noticed, however, that they had a somewhat higher average (G+C) content than mature tRNA. Altogether, we favour a model for the arrangement of the genes for the yeast tRNAs which is similar to the model proposed by Birnstiel et al. [l11 for Xenopus. We suggest that the tRNA cistrons are arranged in tandem, with an about tenfold repetition, interspersed by 6 to 10 times longer spacer DNA which varies in (G+C) content for the different tRNA species. However, some further points have to be considered in this context. The finding that yeast suppressor strains which for example insert tyrosine in response to U-A-A or U-A-G, have been mapped at different loci (for a review see ref. 21), and that in some cases suppression was shown to be mediated by tRNAs C22,231. do not speak against a clustered arrangement of tRNA genes. 2384

Nucleic Acids Research It is still unknown whether all of the suppressor tRNAs are in fact modified tyrosine specific tRNAs [231. On the other hand, it cannot be excluded that within a cluster two or more out of the cistrons could be closely linked as to form a tandem precursor molecule during transcription. Until now, however, we have not observed tRNA precursors of this type in yeast. Furthermore, it remains unknown, whether the clustered tRNA cistrons are really isogenic. The possibility exists that within a cluster one or the other gene for a different tRNA may be included. It is also possible that for some tRNA species or in addition to the tandem repeats, singular tRNA genes do exist. The small extra peaks found in the hybridization patterns (Fig. 1) may be an indication for this. Some of the problems mentioned here are being currently investigated by other methods in our laboratory.

ACKNOWLEDGEMENTS The excellent technical assistance of Mrs. M. Redler is gratefully acknowledged. The Deutsche Forschungsgemeinschaft (SFB 51) has supported this work. REFERENCES

1 A2g -unit is the amount of material in 1 ml solution which give an absorption of 1 at 260 nm in a 1 cm light path. SSC buffer is 0.15 M NaCl, 0.015 M Na-citrate, pH 7.3. 1 Clarkson, S.G. and Birnstiel, M.L. (1973), Cold Spring Harb. Symp.Quant.Biol. 2XXVIII, pp. 451-459 2 Pirro, G. and Feldmann, H. (1973), Z.Physiol.Chem. 356, 1703-1708 3 Schweizer, E., McKechnie,C,and Halvorson, H.O. (1969), J.Mol.Biol. 40, 261-277 4 Ritossa, F.M. and Spiegelman, S. (1965) Proc. Natl. Acad. Sci. (U.S.) 53, 737-745 5 Grigliatti, T.A., qhite, B.N., Tener, G.M., Kaufman, T.C., Holden, J.J., and Suzuki, D.T. (1973), Cold Spring Harb. Symp.Quant.Biol. XXXVIII, pp. 461-474 6 Blatt, B. and Feldmann, H. (1973), FEBS lett. 37, 129-133 7 Pirro, G. and Feldmann, H. (1973) Z. Physiol. Chem. 356, 264 8 Fradin, A., Gruhl, G. and Feldmann, H. (1975), FEBS lett. 50, 185-189 9 Birnstiel, M.L., Sells, B.H., and Purdom, I.F. (1972), J. Mol. Biol. 63, 21-39 10 Clarkson, G.S., Birnstiel, M.L., and Purdotn, I.F. (1973), J.Mol.Biol. 79. 411-429 2385

Nucleic Acids Research Meixner, E., Thesis Universitgt m4nchen 1976 Gruhl, H. and Feldmann, H. (1975), FEBS lett. 57, 145-148 Sanger, F., Brownlee, G.G., and Barrell, B.C. (1965), J.Mol.Biol. 13, 373-398 14 Barrell, B.G., in: Procedures in Nucl. Acid Res., Vol. 2 (Cantoni, G.L. and Davies, D.R., eds.) pp. 751-779, Harper and Row, New York, 1971 15 Pirro, G. and Feldmann, H. (1975). Z.Physiol.Chem. 356, 1683-1701 16 Bhargava, M.M. and Halvorson, H.0. (1971), J.Cell.Biol. 40, 49, 423 - 429 17 Rubin, G.M. and Sulston, J.E. (1973), J.Mol.Biol.79,521-530 18 Simsek, M. and RajBhandary, U.L. (1972), Biochem. Biophys. Res. Commun. 49, 508-515 19 Zachau, H.G., DUtting, D., and Feldmann, H. (1966), Angew. Chem. 78, 392-393 20 Schildkraut, C.L., Marmur, J., and Doty, P. (1962), J.Mol.Biol. 4, 430 - 443 21 Hawthorne, D.C. and Leupold, U. (1974), in: Current Topics in Microbiology and Immunology, 64, W. Arber et al. eds. (Berlin, Heidelberg, New York, Springer Verlag), pp. 1-47 22 Capecchi, M.R., Hughes, S.H., and Wahl, A.M. (1975), Cell 6, 269-277 23 Gesteland, R.F., Wolfner, M., Grisafi, P., Fink, G., Botstein, D., and Roth, J.R. (1976), Cell 7, 381-390.

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Arangement of transfer-RNA -genes in yeast.

Volume 3 no.9 September 1976 Nucleic Acids Research Arrangement of transfer-RNA genes in yeast Horst Feldnan Institut fur Physiologische Chemie, Ph...
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