YEAST VOL. 12: 939-941 (1996) A Useful Colony Colour Phenotype Associated with the Yeast Selectable/Counter-Selectable Marker MET15 GREGORY J. COST AND JEF D. BOEKE* Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 N. Wove Street, Baltimore, MD 21205, U.S.A. Received 9 February 1996; accepted 26 February 1996 Strains of Saccharomyces cerevisiae bearing null alleles of the met15 gene are methionine auxotrophs and become darkly pigmented in the presence of Pb2' ions (On0 et al. (1991). Appl. Env. Microbiol. 57, 3183-3186). We describe the cloning of a useful fragment of the MET15 locus which complements both the methionine requirement and the colony colour phenotype. This colony colour phenotype is very useful for genetic screens and may be applicable for use in other yeast species. The combination of the size of METIS, along with its counter-selectabilityand the colour of met15 mutations make this perhaps the most versatile yeast genetic marker. KEY WORDS -methionine; sectored colonies; colour marker; METIS; Saccharomyces cerevisiae Yeast genetics has been greatly facilitated by the use of simple colony-colour phenotypes to reveal the genotype of the organism. The classic example of such an assay in Saccharomyces cerevisiae is the red colour of adel and ade2 mutants, resulting from the accumulation of a naturally occurring coloured byproduct of the biosynthetic pathway of adenine. We here report the characterization of an analogous assay based on the met15 deficiency in the sulfur assimilation pathway of S. cerevisiae, which leads to a nutritional requirement satisfied by methionine or cysteine. Ono et al. (1991) showed that strains lacking functional alleles of the met2 or met15 genes become dark brown to black in the presence of divalent lead ions (Pb2'), ascribed to the formation of a PbS precipitate from the excess HSresulting from this enzymatic deficiency (Figure IA). In addition, the MET15 gene is advantageous for use as a marker because, like URA3, it can be selected both for and against (Singh and Sherman, 1974). We have further examined the potentially useful colony colour phenotype associated with met15 mutations. Yeast strain GRF167 (MATwhis3A200 ura3167) was passaged through ten cycles of galactoseinduced Ty 1 transposition as described by Boeke et al. (1991). Two strains (31-9c and 31-1Oc) isolated from such cycling were found to have *Corresponding author. CCC 0749-503X/96/100939-03 0 1996 by John Wiley & Sons Ltd acquired a requirement for methionine or cysteine; the mutant failed to complement a met15 tester strain. met17 and met25 have been shown to be allelic to met15 (Ono et al., 1991; D'Andrea et al., 1987); here we will refer to this gene only as metl5. The METIS+ locus (GenBank accession no. U17243) was amplified from genomic DNA via long-range polymerase chain reaction (PCR) (GeneAmp XL PCR Kit, Perkin-Elmer), TA cloned (TA Cloning Kit, Invitrogen), and a 2491 base pair SpeI fragment containing MET15 was subcloned into the CEN/ARS shuttle vector pRS413 (Sikorski and Hieter, 1989) to yield pGC3 (available from ATCC, 87440). Transformation of strain 3 1- 1Oc (determined by Southern analysis to have a truncation of the MET15 gene due to insertion of a Tyl element) as well as BY411 (MATa trplA63 his3A200 ade2A ura3A lys2AO metZSAO), (ATCC 200405) with pGC3 resulted in complementation of both the methioninekysteine requirement and the mutant colour phenotype. Transformants grown on nonselective modified LA medium (MLA) exhibit the coloured sectoring of colonies expected of random CEN plasmid loss (Figure 1B). As is true for adel and ade2 mutants, colouration of met15 mutants was first differentiable from wild-type strains after 2-3 days of growth on (rich) MLA medium, and was enhanced after incubation at 4°C for 24 h or more. Yeast G. J. COST A N D J. D. BOEKE 940 Cycles 2 3 4 1 5 6 7 8 9 10 #25 #26 YPD #25 #26 #31 SD (met-) MLA (rich+Pb*+) #25 #26 #31 MLA (ric h+Pb2+) Genomic Locus 315 Spe I 109 Ava I I&(<>-, delta tRNA (Leu) 1036 Xba I 2012 Hind 111 2806 Spel I MET 15 pGC3 insert delta 1 C P tRNA (110) 94 1 MET15 MARKER GENE blackened on MLA agar grow at rates indistinguishable from wild type, and are fully viable. After extensive growth, wild-type strains appear slightly yellowed, presumably as a result of the complexation of transient HS - into PbS. The black colour of met15 colonies on MLA medium was more intense when the pH of the medium was adjusted to 7.5 with NaOH before autoclaving and before the addition of agar, glucose, or lead nitrate; this is referred to as MLA-7.5 medium. Growth (colony size) was somewhat lower on MLA-7.5 medium than on MLA, but cells still formed visible colonies in 2-3 days and the more intense colour of these colonies may be easier to see for some applications. The colour phenotype of met15 has several potential uses, including analysis of genetic instability such as plasmid or chromosome loss (Koshland and Hieter, 1987), telomeric silencing, and for haploid and synthetic lethality screens (Blinder et al., 1989; Bender and Pringle, 1991; Geiser et al., 1991). Spontaneous reversion of neither the METZ5::Tyl insertion allele nor the met15-3 allele was observed during our transformation experiments. A complete deletion allele useful for PCR-mediated gene disruption (Baudin et al., 1993; Lorenz et al., 1995) has been constructed. Like ADEl and 2, MET15 provides a colour phenotype, and like URA3 and LYS2, it confers sensitivity to a counterselection agent (methylmercury; Singh and Sherman, 1974). In contrast to the adel and 2 mutations, in which cellular growth is inhibited as a consequence of the toxicity of the coloured byproduct, the pigmentation of cells with PbS does not appear to have a deleterious effect on viability. The MET15 open reading frame is only 1335 base pairs long, making it more practical for molecular biological applications than very large counter-selectable markers such as LYS2 (4178 bp), or the large colour marker ADE2 (2700 bp). Thus, MET15 may be the most versatile marker available for yeast molecular genetics. Finally, analogous genes in Candida Schizosaccharomyces and other microorganisms are likely to be similarly useful. ACKNOWLEDGEMENT Supported in part by a grant from the National Institutes of Health to J.D.B. REFERENCES Baudin, A,, Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F. and Cullin, C. (1993). A simple and efficient method for direct gene deletion in Succhuromyces cerevisiue. Nucl. Acids Res. 21, 3329-3330. Bender, A. and Pringle, J. (1991). Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Succhuromyces cerevisiue. Mol. Cell Biol. 11, 1295-1305. Blinder, D., Bouvier, S. and Jenness, D. 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Ono, B., Ishii, N., Fujino, S. and Aoyama, I. (1991). Role of hydrosulfide ions in methylmercury resistance in Sacchuromyces cerevisiae. Appl. Env. Microbiol. 57, 3183-31 86. Sikorski, R. S. and Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Succhuromyces cerevisiue. Genetics422, 19-27. Singh, A. and Sherman, F. (1974). Association of methionine requirement with methylmercury resistant mutants of yeast. Nature 247, 227-229. Figure 1. (A) Lines 25, 26 and 31: three strains of Saccharomyces cerevisiue which have undergone one through ten cycles of induced retrotransposition grown on YPD (rich) medium, SD (minimal) medium supplemented with the appropriate amino acids excluding methionine, and on MLA (rich, lead nitrate) medium. The methionine auxotrophy was acquired between the eighth and ninth cycles of induction of transposition. MLA agar is as reported by Ono et al. (1989), with the substitution of Pb(NO,), for Pb(oAc), and the addition of lead nitrate after autoclaving, to agar which is at 45-50°C. (B) Colonies of strain 31-1Oc displaying colour sectoring due to random loss of pGC3 on nonselective MLA medium. (C) The MET15 genomic locus. The black box indicates the open reading frame. The flanking positions of degenerate 6 elements and tRNA genes are indicated.