Current Genetics (1984) 8:467-470

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© Springer-Vedag 1984

Short communication

Cloning and identification of a DNA fragment coding for the supl gene of Saccharomyces cerevisiae Peter Breining 1 , Andrei P. Surguchov2 , and Wolfgang Piepersberg I 1 Lehrstuhl ftir Mikrobiologieder Universit~itMtinchen, Maria-Ward-Strafela, D-8000 Miinchen 19, Federal Republic of Germany 2 Institute of Experimental Cardiology,USSR Research Centre of Cardiology,Academy of Medical Sciences, Cherepkovskaja 15A, Moscow 121552, USSR

Summary. A plasmid, pYsupl.1, containing a DNA fragment able to suppress the recessive mutant phenotype of the suppressor locus sup1 (allele sup1-ts36) of Saccharomyces cerevisiae was isolated from a bank of yeast chromosomal DNA cloned in cosmid p3030. The complementing gene was localized on a 2.6 kb DNA fragment by further subcloning. Evidence is presented that the cloned DNA segment codes for the sup1 structural gene (chromosome IIR). Key words: Saccharomyces cerevisiae - Cloning - Suppressor

Translational fidehty is controlled by the ribosome and can be influenced by certain antibiotics, mainly aminoglycosides, and by mutations in ribosomal components, as was shown initially for procaryotes (reviewed by Gorini 1974; Piepersberg et al. 1980). Detection of recessive omnipotent suppressor mutations in the yeast Saccharomyces cerevisiae (Inge.Vechtomov and Adrianova 1970; Hawthorne and Leupold 1974) gave the first indication for an analogous control of the accuracy of protein synthesis by the eucaryotic cytoplasmic 80S ribosomes. In yeast such suppressors were mapped at two loci, sup1 (presumably identical to sup45) and sup2 (presumably identical to sup35), on chromosomes II and IV, respectively (Mortimer and Schild 1980). Mutations at both loci cause alterations of various ribosomal functions; sensitivity of growth at either high or low temperatures, and confer aminoglycoside hypersensitivity to the mutant strains (Surguchov et al. 1980, 1981a, 1981b).

Offprint requests to: W. Piepersberg

Current attempts to identify the altered ribosomal components in both supl and sup2 have been unsuccessful. Another, but dominant omnipotent suppressor, SUP46, was shown to correlate with an alteration in one of the proteins of the small (40S) ribosomal subunit in S. cerevisiae (Ishiguro et al. 1981). Several recessive anti. suppressor (asu) mutations of yeast have been classified, in one of which, asu9-1, again an altered 40S subunit protein was demonstrated (Liebmann and Cavenagh 1981). The latter class of mutations could be similar to the misreading restricting, aminoglycoside resistant mutations in Escherichia coli (Gorini 1974; Piepersberg et al. 1980). For further characterization of the genetic organization and the nature of the gene product of the supl locus we isolated a plasmid, pYsupl-1, containing a DNA sequence able to complement supl mutations from a library of cloned genomic DNA orS. cerevisiae (Fig. 1). This could be achieved by genetic complementation of the recessive temperature sensitivity of the supl-ts36 allele and further testing for loss of suppressor phenotype, that is adenine auxotrophy, provoked by the suppressible marker ariel-14, and insensitivity to paromomycin (Surguchov et al. 1981b) in S. eerevisiae strain 7BD244 (see legend to Fig. 1 for details). Several different plasmids, some even smaller than the vector (cosmid p3030; 10.3 kb), were obtained from the same yeast transformant by transformation into E. coll. This in. dicated that extensive recombination of the transformed plasmid with either chromosomal or the resident 2/~m plasmid DNA must have occurred in yeast. The plasmid pYsupl.1 was the largest plasmid obtained and was composed of the unaltered vector plasmid with a 6.3 kb insert in the respective cloning site (Fig. 1A). Testing several subfragments of the inserted yeast DNA for complementation of supl allowed the localization of the

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Fig. IA and B. Cloning and identification of a DNA fragment containing the sup1 (sup45) gene of S. cerevisiae. A A library of yeast DNA from strain $288C (~, SUC2, rnal, gal2, CUP1) partially cleaved with Sau3A and cloned into the hybrid cosmid p3030 (B. Hohn, unpublished) was kindly provided by B. Hohn, Basel. Vector p3030 (10.3 kb) contains the 2 #m plasmid replication origin (Beggs 1978) and the yeast HIS3 gene (wavy lines) inserted into the cosmid pHC79 (Hohn and Collins 1980). The upper right shows part of the right arm of yeast chromosome II with some of the gene loci mapped near sup1 (sup45) (Mortimer and Schild 1980). Eighteen pools of ccoDNA each extracted from about 150 ampicillin resistant transductants of E. coli strain C600 (thi, thr, leu, hsd K, lacY, supE, tonA, X-) (Davis et al. 1980) were transformed separately into S. cerevisiae 7B-D244 (~, leu2-3, Ieu2-112, his3-11, his3-15, ariel-14 (suppressible), sup1-ts36) according to the procedure of Hinnen et al. (1978). Selection of transformants complemented for the recessive sup1 (ts) marker was achieved by plating the spheroplast suspension on regeneration agar (Hinnen et al. 1978) containing adenine (40/~g/ml) and leucine (40 t~g/ml), incubating for 15 h at room temperature before shifting to 37 °C for 3 to 4 days. Temperature resistant and histidine prototrophie transformants grew from only one DNA-pool. Plasmid DNA was reextracted from yeast by the method of Cryer et al. (1975) (methods for transformation of and plasmid preparation from E. coli and for

gene(s) on a 2.6 kb BamHI/XbaI fragment (Fig. 1B). If this fragment was further cleaved by BglII neither part could suppress the sup1 phenotype suggesting that the complementing gene was inactivated. The fragment cloned in pPBM8 (Fig. 1) was unable to complement sup2 phenotypic traits. We conclude that the 2.6 kb seg-

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subcloning plasmid fragments were taken from Davis et al. 1980). Plasmids of various sizes were found in small scale DNA preparations from ampicillin resistant transformants (Kado and Liu 1981). Only the largest plasmid variant (pYsupl-1; 16.6 kb) was able to complement, 100 percent, all the pleiotropic defects caused by the sup1-ts36 allele (temperature sensitivity, suppressor activity, paromomycin hypersensitivity; Surguchov et al. 1979, 1981a). The insert in pYsupl-I was 6.3 kb long (double line). Restriction endonuclease cleavage sites were mapped for: AccI (A) (only the sites not cleaved with SalI are marked), AvaI (A1) (only the sites not cleaved by XhoI are marked), AvalI (A2), BamHI (B), BgllI (Bg), C/aI (C), EcoRI (E), HinclI (Hi) (only the sites not cleaved by either HpaI or SalI; are marked, HindlII (H) HpaI (Hp), XbaI (Xb), XhoI (X) were mapped; no sites for KpnI, PstI, PvuI, and PvulI were found. Parts of the vector p3030 are shown as a single line on both sides. B Several subfragments of the yeast DNA inserted in pYsupl-1 were tested for complementation of supl-ts36. For this purpose fragments were either deleted from pYsupl-1 or cloned into p3030 or into E. coli vectors pACYC177 or pACYC184 (Chang and Cohen 1978). Only plasmids containing the 2.6 kb BarnHI/XbaI fragment subcloned in pPBM8 were able to support growth of yeast strain 7B at high temperature (37 °C) or on high concentrations (1 mg/ml) of paromomycin

m e n t cloned in pPBM8 (Fig. 1B) most likely includes the

supl structural gene. Since pYsupl-1 was too small to be a full length packageable cosmid (Hohn and Collins 1980) and because of the observed instability of plasmid in yeast strain 7B.D244, we chose to isolate further clones with

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Fig. 2. Physical maps of cloned DNA fragments from yeast chromosome II in comparison to pYsupl-1. The 5.9 kb HindlII fragment from pPBM10 (see Fig. 1) was used to screen a colony bank ofE. coli containing the vecor YEpl3 (Broach et al 1979) with partially Sau3A cleaved yeast DNA cloned into the singleBa mHI site (Nasmyth and Tatchell 1980; kindly provided by B. Hall, Seattle) by colony hybridization (Hanahan and Meselson 1980). Three out of 1196 clones hybridized strongly to the probe. All clones containing a different plasmid, two of which were able to complement for sup1-ts36 (pYsupl-2 and pYsupl-4); the third was called pYCII-3 (for yeast chromosome II). The extent of homology with pYsupl-1 (hatched areas) was confirmed by hybridizing various fragments of pYsupl-1 according to the method of Southern (1975; data not shown). The insert in pYsupl-4 was obviously of the same size as the one cloned in pYsupl-1. The boxed areas are the cloned DNA inserts. The restriction endonuclease cleavage sites are labeled only in the parts not present in pYsupl-1 ; see legend to Fig. 1, P = PstI; the HindllI and Sail sites from the tet region of the vector are indicated in each case

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Fig. 3. Cloning of the supl-ts36 allele. Plasmid pPBM10 was transformed in strain 7B-D244 (cf. Fig. 1). Transformants were obtained by selection for growth at 37 °C or for paromomycin resistance (1,000/~g/ml). Plasmid pPBM10 is not able to replicate autonomously in yeast. It is forced, therefore, to integrate by recombination into the chromosomal site homologous to its insert. Total DNA was extracted from two independently obtained transformants according to the procedure of Cryer et al. (1975). SalI or XhoI fragments were religated and transformed to E. coli C600 selecting for the chloramphenicol resistance of vector pACYC184. The plasmid derived from Sail digests (pPBM24) contained the wild type alleles (SUP1), because they still could complement for the sup1-ts36 mutation in strain 7B-D244. However, the plasmids regained by XhoI cleavage (pPBM25) from both yeast recombinants were no longer able to suppress the supl-ts36 phenotype. Open bar = portion of chromosome II cloned in pACYC184 (see Fig. 1); wavy line = E. coli plasmid; SUP1, sup1-ts36 = wild type and mutant alleles, respectively, of the recessive suppressor; cat = chloramphenicol acetyltransferase gene of pACYC184; (S3A) = one of the Sau3A sites flanking the insert in pYsupl-1 (el. Fig. 1); restriction sites are labelled as in Fig. 1

homology to the insert in pYsupl.1 from another yeast DNA bank in vector Y E p l 3 (Fig. 2). Three independent clones, pYsupl-2, pYsupl-4 and pYCII-3, were obtained. The inserts covered in part or totally the cloned fragment in pYsupl.1. The restriction patterns of the homologous regions were identical (Fig. 2), thereby proving that no large rearrangements due to recombination could have occurred in the fragment cloned in pYsupl.1 during in vitro and in vivo processing. This result was further confirmed by hybridizing the 4.4, 1.15, and 0.65 kb ClaI/SalI fragments of pYsupl-1 (including the full length cloned fragment; see Fig. 1) against various restriction endonuclease digests of total genomic DNA extracted from strain $288C. WithBamHI, EcoRI, and HindlII the main hybridizing bands had lengths of 5.7, 7.3, and 6.1 kb, respectively (data not shown). In order to prove, that the cloned segment of yeast chromosomal DNA in fact coded for the structural gene o f the sup1 locus on chromosome IIR the recessive suppressor allele sup1-ts36 was cloned using a similar approach as given b y Roeder and Fink (1980). As illustrated in Fig. 3 a plasmid, pPBM25, could be recovered which no longer complemented for the supl-ts36 phenotype, though its physical structure was identical to pPBM10 (compare Fig. 1). Since both pPBM 10 and pPBM25 are non-selfreplicating and not selectable via an independent marker in yeast the respective 4.1 kb BamHI/SalI fragments of pPBM24 and pPBM25 (Fig. 3) were subcloned in p3030 (compare legend to Fig. 1). These plasmids replicated autonomously in yeast and expressed histidine prototrophy in his3 m u t a n t strains; again, only the fragment derived from pPBM24 was able to complement the sup1-ts36 mutant

470 p h e n o t y p e o f strain 7B-D244. The mutant locus, therefore, is on the cloned fragment in pPBM25 and offers t h e possibility to elucidate the nature o f the mutational change in the sup1 gene sequence. All attempts to identify the gene product(s) coded for b y the 2.6 kb BarnHI/XbaI fragment cloned in pPBM8 b y translation o f yeast p o l y A + m R N A selected b y hybridization to this DNA segment (Maniatis et al. 1982) in reticulocyte and wheat germ systems failed (data not shown). We conclude (I) that the isolated DNA fragment includes the sup1 structural gene and (II) that it probably codes for a ribosomal protein. Determination o f the nucleotide sequence and the transcriptional product(s) o f the cloned segment will give further insight into the structure o f the gene(s) and its product. A yeast DNA segment complementing mutations in the sup45 locus (presumably identical to sup1) o f S. cerevisiae was also cloned b y J. D. Friesen, Toronto (personal communication), and the fragment identified shows an identical restriction pattern with part o f the insert in pYsupl.1.

Acknowledgements. We thank M. D. Ter-Avanesyan for providing sup1 strain 7B of S. cerevisiae, A. B6ck and V. N. Smirnov for support, J. D. Friesen for communicating unpublished observations, and M. Geier for typing the manuscript. We are grateful to B. Hohn, Basel, and B. Hall, Seattle, for providing libraries of cloned yeast DNA. This work was supported by grants from the Deutsche Forschungsgemeinschaft (to A. P. S. and W. P.).

P. Breining et al.: Cloning of supl Cryer DR, Eccleshall R, Marmur J (1975) In: Prescott DM (ed) Methods in cell biology, vol 12. Academic Press, New York, pp 39-44 Davis RW, Botstein D, Roth JR (1980) Advanced bacterial genetics. A manual for genetic engineering. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Gorini L (1974) In: Nomura M, Tissi&res A, Lengyel P (eds) Ribosomes. Cold Spring Harbor Laboratory, New York, pp 791-804 Hanahan D, Meselson M (1980) Gene 10:63-67 Hawthorne DC, Leupold U (1974) Curr Topic Microbiol Immunol 64:1-47 Hinnen A, Hicks JB, Fink GR (1978) Proc Natl Acad Sei USA 75:1929-1933 Hohn B, Collins J (1980) Gene:291-298 Inge-Vechtomov SG, Andrianova VM (1970) Genetika (USSR) 11:103-115 Ishiguro J, Ono B, Masurekar M, Sherman F, McLaughlin CS (1981) J Mol Bio1147 :391-397 Kado CI, Liu S-T (1981) J Bacteriol 145:1365-1373 Liebmann SW, Cavenagh MM (1981) Curt Genet 3:27-29 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Mortimer RK, Schild D (1980) Microbiol Rev 44:519-571 Nasmyth KA, Tatchell K (1980) Cell 19:753-764 Piepersberg W, Geyl D, Hummel H, B6ek A (1980) In: Osawa S, Ozeki H, Uchida H, Yura T (eds) Genetics and evolution of RNA polymerase, tRNA and ribosomes. University of Tokyo Press, Tokyo, pp 359-377 Roeder GS, Fink GR (1980) Cell 21:239-249 Southern EM (1975) J Mol Biol 98:503-517 Surguchov AP, Berestetskaya YV, Fominykch ES, Pospelova EM, Smirnov VN, Ter-Avanesyan MD, Inge-Vechtomov SG (1980) FEBS Lett 111:175-178 Surguchov AP, Fominykch ES, Smirnov VN, Ter-Avanesyan MD, Mironova LN, Inge-Vechtomov SG (1981a) Biochim Biophys Acta 654:149-198 Surguchov AP, Pospelova EM, Smirnov VN (1981b) Mol Gen Genet 183:197-198

References Beggs JD (1978) Nature 275:104-109 Broach JR, Strathern JN, Hicks JB (1979) Gene 8:121-133 Chang ACY, Cohen S (1978) J Bacteriol 134:1141-1156

Communicated b y C. P. Hollenberg Received January 12 / April 9, 1984