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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. (1989). Constitutive mutants in the yeast pheromone response:
ordered function of the gene products. Cell 56,
479436.
Boeke, J. D., Eichinger, D. J. and Natsoulis, G. (1991).
Doubling Ty 1 element copy number in Succhuromyces
cerevisiue: host genome stability and phenotypic
effects. Genetics 129, 1043-1052.
D’Andrea, R., Surdin-Kerjan, Y., Pure, G. and Cherest,
H. (1987). Molecular genetics of met17 and met25
mutants of Succhuromyces cerevisiue: intragenic
complementation between mutations of a single
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Geiser, J., van Tuinen, D., Brockerhoff, S., Neff, M. and
Davis, T. (1991). Can calmodulin function without
binding calcium? Cell 65, 949-959.
Koshland, D. and Hieter, P. (1987). Visual assay for
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Lorenz, M. C., Muir, R., S., Lim, E., McElver, J.,
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Gene 158, 113-117.
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.
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