G’ene, 119 (1992) 65-74 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/92/$05.00

65

GENE 06625

A new family of polymorphic metallothionein-encoding (CUPl) and MTH2 in Saccharumyces cerevisiae (Int~rstr~n

variation;

yeast;

lopper-thion~in;

polymeric

genes;

chromosomal

genes MTHl

DNAs; Southern blot; restriction analysis;

gene conversion)

Gennadi

I. Naumov”,

Elena S. Naumovaa,

Hilkka Turakainen

b and Matti Korhola”

’ Scientific Research Institutefor Genetics and Selection ofIndustrial Microorganisms, Moscow, Russia:” Department of Generics. UniversitJ?of Helsinki, Helsinki, Finland; and ’ Research Laboratories, Alko Ltd, Helsinki, Finland

Received by J.K.C. Knowles: 7 January 1992; Revised/Accepted: 6 May/l1 May 1992; Received at publishers: 1 June 1992

SUMMARY

By pulsed-field gel electrophoresis of chromosomal DNA and hybridization with a cloned MTHI (CUPI) gene, we determined the locations of metallothionein-encoding gene sequences on chromosomes in monosporic cultures of 76 natural strains of Saccharomyces cerevisine. Most of the strains (68) exhibited a previously known location for the MTH sequence on chromosome (chr.) VIII. Seven strains (resistant or sensitive to Cu2’) showed a MTH sequence in a new locus, MTHZ, on chr. XVI. One strain carried an MTH locus on both chromosomes VIII and XVI. Restriction fragment and Southern blot analyses showed that the two MTH loci were very closely related. The strains displayed heterogeneity in the size and structure of their ~TH2 locus. The length of the repeat unit of MTH2 varied: a 1.9-kb or 1.7-kb unit was found, instead of the 2.0-kb unit of the ~TH~ locus. The most resistant strain (resistant to 1.2 mM CuSO,) contained a 0.9-kb repeat unit in addition to those of 1.9 kb and 1.7 kb. All three sensitive (to over 0.3 mM CuSO,) strains with an mth2 locus had a repeat unit of 1.9 kb or 1.7 kb, suggesting the presence of at least two copies of the MTH2 gene, with one always being in the junction area outside of the repeat unit. A monogenic tetrad segregation of 2:2 was usually found in crosses of resistant MTH2 and sensitive mth2 strains. Hybrids between strains with different MTH loci in all combinations showed low ascospore viability, suggesting that the complete lack of an MTH locus may lead to the death of segregants on YPD medium. The MTHl and MTH2 loci were exchangeable. Strains with a high level of Cu2+ resistance were also resistant to Cd2 + . However, these two properties did not cosegregate in heterozygotic hybrids.

INTRODUCTiON

The yeast-amplified metallothionein-encoding gene (n/rTH), responsible for copper resistance and found by Correspondence to: Dr. M. Korhola, Alko Ltd., P.O.Box 350, SF-00101 Helsinki, Finland. Tel. (358-O) 1332263; Fax (358-O) 1332781.

Abbreviations: bp, base pair(s); CAD, gene encoding resistance to cadmium salts (CdR); chr., chromosome; CUP1 (~THlj, gene encoding resistance to copper salts (CL?); kb, kilobase or 1000 bp; MTH, metallothionein; MTH, gene encoding MTH; nt, nucleotide(s); R, resistant/ resistance; ‘, sensitivity/sensitive; S., Saccharomyces.

Brenes-Pomales et al. (1955), is one of the most intensively studied markers in molecular biology (Hamer, 1986; Butt and Ecker, 1987). Research on the MTH gene is indeed comprehensive and the following have all been well studied: its cloning, structure, gene expression and significance in copper detoxification (Butt et al., 1984a,b; Ecker et al., 1986; Fogei and Welch, 1982; Hamer et al., 1985), gene conversion, amplification and role of MTH gene in aneuploidy (Fogel and Welch, 1982; Fogel et al., 1983; 1984; Welch et al., 1987; 1990; Whittaker et al., 1988); its amplification caused by c~cinogens (Aladyem et al., 1988); the use of MTH gene and its promoter for the construction

66 TABLE

I

List of key strains

of Saccharomyces

cerevisicre used in the present

Initial

Monosporic

strain

culture

study

Resistance”

Genotype

b

Reference

or

source to cuso,

to CdCl,

(mM)

(mM)

-

X2180-1A

1.0

0.3

MA Ta gal2 mal SE2

-

31-i-7Ba

0.2

0.1

MA Ta trpi a@

L2-43

L2-43-6D

1.2

0.8

HO GAL MAL SUC

Naumov

L3-44

L3-44-7c

0.75

et al. (1983)

M&l-2A

0.5

HO gal4 mai SUC HO gal2 MAL SUC

Naumov

M427

0.2 0.3

Naumov

et al. (1983)

CBS 4054

4054”3B

0.2

0.2

MA Ta gal4 ma1 sue

Naumov

et al. (1983)

M3-33

M3-33-6B

0.3

0.3

HO gal mai WC

Naumov

et al. (1983)

Ml 1-22-10B

0.2

0.2

HO gal2 MAL SUC

Naumov

et al. (1983)

1340-1D

0.7

0.2

HO GAL MAL SW

G.I. Naumov

1753-8-2’

0.75

0.4

MAT GAL MAL

G.I. Naumov

Ml l-22 DBVPG

1340

VKM Y-1753 a Resistance out aa/ required

of yeasts

to copper

and cadmium

were estimated

mg agar; all per ml) containing different concentrations amino acids and bases were added.

b The genes for sucrose, screening

for a change

galactose

and maltose

fermentation

in the color of yeasts growing

on plates with minimal of copper

were used as control

on solid pH indicator

medium

or cadmium

(20 mg glucose/&7

in crosses.

S. Fogel

SUC

after 2 days of incubation

markers

medium

R.K. Mortimer

ade8 ura

et al. (1983)

mg Difco yeast nitrogen at 30°C.

base with-

For auxotrophic

yeasts, the

The ability to ferment these sugars was tested by

with eosin-methylene

blue @MB).

HO, homothallism

gene.

‘ The strain lost its mating type

of industrial vectors (Butt and Ecker, 1987; Henderson et al., 1985; Meaden and Tubb, 1985); and the gene amplification and copper resistance of industrial strains (Fogel and Welch, 1984; Welch et al., 1983). Besides the structural MTHI (CUPl) gene, two regulatory genes needed for copper resistance in yeasts, ACE1 (CUP2) and ACE2, have been identified (Buchman et al., 1989; Butler and Thiele, 1991; Thiele, 1988). The MTHl sequence is located on chr. VIII (Hawthorne and Mo~imer, 1960). While studying genetic poiymo~hism in Saccharomyces cerevisiae strains from different sources, we found MTH sequences at unusual locations in several strains. Here, we describe the molecular and genetic peculiarities of the new MTH2 locus. We found the MTH2 locus to be comprised of repeat units of variable lengths and the MTH2 gene to be a portion of the locus hybridizable to the MTHl copper-metallothionein coding region DNA.

Fig. I. Polymorphism

in MTH genes of monosporic

et al. (1991) and separated by conto~~l~ped time of 60 s and then for 8 h with a switching with ethidium

bromide

to visualiie

MTHl

of natural

(a) Selection of strains Industrial strains or widely used American genetic stock strains of S. cerevisiue are not very suitable for studies of genetic polymorphism because of their limited gene pool. The low fertility of industrial strains makes classical genetic analysis dif%ult. Previously, while carrying out taxonomic studies we have created a large collection of genetic lines of S. cerevisiae strains with different natural origins (Naumov et al., 1983). Monosporic cloning of natural strains leads to the elimination of lethal factors and aneuploidy, and also to the homozygosity of chromosomal sets. In monosporic cultures, it is easy to identify chromosomal DNAs by pulsed-field gel electrophoresis because the band position is usually in accordance with those of common genetic lines (Naumov et al., 1991). Also, the use of highly

S. cerevisiae strains.

(A, Ien). Then, the chromosomal

with 0.25 M HCl. The filters were baked

(CUP1) probe (a 5-kb, BamHI-Hind111

AND DISCUSSION

Chromosomal

DNA was prepared

as described

by Naumov

homogeneous electric field (CHEF) 1% agarose gel electrophoresis at 200 V for 15 h with a switching time of 90 s. The running buffer was 0.5 x TBE cooled to 14°C. After electrophoresis, the gels were stained

the chromosomes

lose filters after the gels had been treated

cultures

RESULTS

fragment

in PET 13.1; Henderson

DNA was denatured, for 2 h at 80°C.

et al., 1985; Meaden

neutralized

The chromosomal and Tubb,

and transferred

to nitrocellu-

DNA was hybridized

with the

1985) (A right, B, C, D and E). The

probe was prepared mainly according to Maniatis et al. (1982) and labeled with digoxigenin-11-dUTP using the Nonradioactive DNA Labeling Kit (Boehringer Mannheim, FRG). For strain designations see Table I and consult the culture collections: ATCC, American Type Culture Collection (Rockville, MD, USA);CBS, Centraalbureau voor Schimmelcultures (Delft, The Netherlands); CCY, Institute of Chemistry of the Slovak Academy of Sciences (Bratislava, Czechoslovakia); DPVPG, Dipartimento di Biologia Vegetale of the University of Perugia (Perugia, Italy); IFO, Institute of Fermentation (Osaka,

Japan);

NCYC,

National

Collection

of Yeast Cultures

(Norwich,

UK); NRRL,

Northern

Regional

SBY, Section de Bioquimica Instituto National de Investigationes Agrarias (Madrid, Spain); VKM, All-Union Russia); or the authors. TBE is 89 mM Tris base/89 mM boric acidi mM EDTA pH 8.0.

Research

Laboratories

Collection

(Peoria,

of Microorganisms

IL, USA); (Moscow,

I

m

IF0 877

:.

1794

NRRL 12056

CBS 400

DV 30

DBVPG

DBVPG

i

1790 1793

DBVPG

1789

DBVPG

1340 1788

DBVPG DBVPG

CBS 2910

CBS 2909

CBS 2880

XII,

31-l-78

X2180-1A

YNN295

CBS 6325

i

& i

Ksc40

’ Ksc 73

INMIV 493 Ksc2

N19

’ *

NCYC 2576

1 )

GM51

CBS 6328

VKM 503

CBS 5287

CBS 6326

CBS 405

CBS 6329

VKM 1701

! *

1’

::

GIV 51 CBS 403

.

1

d

X2180-1A

YNN 295

m

0

YNN295 31-l-78 X2180-1A ATCC 40490 L2-43-6D L3-44-7C M427 CBS 4054 M435 CCY 28-73 K7 WH 92 NCYC 74 VKM 582 M3-33-60 L2-8 1-7A CBS 6006 Ml04 Ml l-22-108 X2180-1A

YNN 295 X2180-1A 31-l-7B VKM 1703 CBS4411 VKM 1830 CBS 4903 N38 SBY 2592 CBS 5835 N60 M425 Ml80 M479 N39 Ml79 CBS 5370 N37 CBS 308 1 1231-5A

0

a

f

c

I

f

(10)

(8-2)

1831

1755

1753

1752

1742

BC-2 (‘3-2) N149 (10) CBS 3093 CBS 6333 Char.39 N 4-2

YNN 295 X2180-1A VKM 586 VKM 1742 VKM 1752 VKM 1753 VKM 1755 VKM 1831

N 4-2

Char .39

CBS 6333

CBS 3093

N148

BC-2

VKM

VKM

VKM

VKM

VKM

VKM 586

X2180-1A

YNN 295

68 fertile monosporic

cultures

enables

us to obtain intrastrain

hybrids having high ascospore viability. In Table I, the main strains used in our research are listed. All of the strains used are presented in Fig. 1 with some of their electrophoretic karyotypes.

and XV comigrated in this strain (Figs. 2C one strain (1753-8-2) displayed two bands the MTHl probe (Fig. 1A). Hybridization probe confirmed location of one of the MTH XVI (not shown).

(b) Chromosomal location of MTH genes After studying the molecular polymorphism of the MTHI (CUPI) gene in industrial strains, Welch et al. (1983) and Fogel and Welch (1984) suggested that, in some strains, a copy of the MTHI locus might have been translocated or transposed to a nonhomologous chromosomal site. Using pulsed-field gel-electrophoresis and Southern blot analysis, we studied the chromosomal location of the MTH gene in 76 strains of S. cerevisiue (Figs. lA-E). As ex-

(c) Segregation studies of MTH genes Hybridization of the MTHZ probe to one chromosomal band does not confirm the presence of only one MTH gene in a strain. Such bands could actually possess more than one gene because chromosomes might comigrate or several unlinked MTH genes could be located on the same chromosome. To clarify the situation, we used recombination analysis. By tetrad analysis, we studied hybrids between sensitive and resistant strains having the new location of

pected, most of the strains carried the MTHl gene in its standard location on chr. VIII. However, eight strains (L243-6D, L3-44-7C, M427-2A, 4054-3B, M3-33-6B, Mll-22-lOB, 1340-1D and 1753-8-2) were found to exhibit a new location for the MTH gene. Hybridization with a GAL4 gene probe (chr. XVI) showed that the new MTH gene was located on chr. XVI in eight strains in spite of the various sizes seen for chromosome XVI (Figs. 2A and B). In strain Mll-22-10B chr. XVI was larger than in other strains (Figs. 2B and C) and hybridization to the ADCl promoter probe (chr. XV) indicated that chromosomes XVI

MTH gene on chr. XVI. Analysis indicated that the copper-resistant character of strains L2-43-6D, L3-447C, M427-2A and 1340-1D had monogenic determination (Table II, hybrid Nos. l-5). Most tetrads gave a regular segregation of 2:2. The presence of six irregular tetrads (3:1, 1:3) out of 86 tetrads studied does not disagree with our conclusion. Meiotic gene conversion is known to occur with a very high frequency in an amplified MTH locus (Fogel and Welch, 1984; Fogel et al., 1984; Welch et al., 1987; 1990). Copper-resistant strains carrying the MTH2 locus on chr. XVI were crossed with each other (Table II,

B

A

C

Fig. 2. Mapping

the MTHZ gene on chr. XVI by Southern gel corresponding

in pET13.1; Henderson et al., 1985; Meaden (D) ADCI promoter (1.5kb HindIII-BamHI preparation

of probes

hybridization

to the hybridization

ADC 1

GAL4

CUP 1

(A) Ethidium bromide-stained

and D). Only hybridizing to to the GAL4 genes on chr.

with the MTHl

ofchromosomal

(CUPJ), GAL4 (chr. XVI) and ADCZ promoter

DNAs with cloned genes: (B) MTHl

(chr. XV) probes.

(5kb, BarnHI-Hind111

fragment

and Tubb, 1985), (C) GAL4 (l.O-kb SalI-PvuII fragment in pALK79; Laughon and Gesteland, 1984) and fragment in pAAH5; Ammerer, 1983). Chromosomal DNA preparation, electrophoresis conditions and

were the same as described

for Fig. 1.

69 TABLE

II

Identification

of the MTHZ gene by genetic crosses

Hybrid

Hybrid

No.

phenotype)

Genotype

Number of tetrads segregatingb as CuR:CuS

Ascospore

origin a (parent

viability 0, 1”

3:l

212

of

hybrids 4:o

1:3

96

16

3

0

1

MTH2/mth2

83

20

0

0

0

MTHZjmth2

(Cus) (Cu”)

98

21

2

0

0

MTH2jmth2

98

23

0

0

0

MTHZlmth2

1340-1D (Cu”) x M3-33-6B (Cu’) L2-43-6D (Cu”) x L3-44-7C (Cu”)

90

18

0

0

0

MTHZlmth2

78

0

0

9

0

MTHZIMTH2

L3-44-7C

(CuR) x 1340-1D (CuR)

93

0

0

23

0

MTHZjMTH2

8

L2-43-6D

(Cu”) x M427-2A

96

0

0

21

0

MTHZIMTHZ

” The methods

for cultivation,

1

L2-43-6D

(CuR) x 4054-3B

2

L2-43-6D

(Cu”) x Ml I-22-10B

3 4

L3-44-7C M427-2A

(Cu”) x M3-33-6B (Cu”) x M3-33-6B

5 6 7

(Cu’)

(CuR)

hybridization

and meiotic segregation

yeasts were obtained

by the spore-to-spore

cells and by isolation

of zygotes

spore mating

using a micromanipulator.

h The copper

method

resistance

No. 1 which contained

(Cu’)

mating method

with a micromanipulator.

of segregants

of yeasts were as described

using a micromanipulator. Crosses

between

was tested on plates with minimal

0.9 mM CuSO,.

The composition

of the minimal

hybrid Nos. 6-8). The absence of segregation in tetrads (4:0) showed that strains L2-43-6D, L3-44-7C, M427-2A and 1340-1D had the same MTH2 locus. (d) Restriction analysis Genomic DNA containing the MTH2 locus was digested with restriction enzymes, separated by agarose gel electrophoresis, transferred to nitrocellulose and hybridized to CUP1 probes (Fig. 3). The strains displayed heterogeneity in the size and structure of their MTH locus. The KpnI restriction site present in the MTHl ( = CUPI) repeat unit was missing in the MTH2 locus. Since neither EcoRI nor KpnI cleave within the MTH2 repeat unit, the size of the Kpnl

-/, * Fig. 3. Southern DNA was isolated,

blot analysis

of the MTHl

and MTH2

loci. Genomic

digested with KpnI or EcoRI and analyzed

by South-

ern blotting with the “P-labeled probe MX (2-kb KpnI fragment in PIB176) shown in Fig. 6. The probe was prepared by the in vitro transcription system and labeled with [c(-3ZP]UTP

(Melton

et al., 1984).

Hybrids

heterothallic

before (Naumov of heterothallic

and homothallic

medium

containing

medium

was described

et al., 1986). Hybrids

yeasts were obtained

strains were performed

0.5 mM CuSO,, in the footnotes

of homothallic

by mass mating of

by the haploid

except for plates of segregants

cell-to-

of hybrid

for Table I.

EcoRI or KpnI fragments that hybridized to MTHI probes showed that all three sensitive strains with an MTH2 locus could contain at least two copies of the MTH gene, and the resistant strains could contain 5-20 copies of the MTH gene. Digestion with XbaI showed that all nine strains with an MTHl and/or MTH2 locus had an identical lo-kb fragment representing the left junction (Figs. 4 and 5). This fragment hybridized to the probe containing only the unknown gene (gene X) of the MTHI locus (probe X), but did not hybridize to the probe which contained only the MTH gene (probe M). The size of the X&I fragment representing the right junction was either 20 kb or 4 kb. These fragments contained only the MTH gene, not gene X. Four strains (L3-44-7C, M427-2A, M3-33-6B and Ml l-22-10B) displayed a band of 1.9 kb and two other strains (4054-3B and 1340-1D) a band of 1.7 kb, which hybridized strongly to probe M (Fig. 4). These intense bands represented the basic repeat units ofthe MTH2 locus. The 1.9-kb band also hybridized to probe X, proving that this repeat unit contained both the MTH gene and gene X, as did the 2.0-kb MTHl repeat unit. In strains 4054-3B and 1340-lD, the 1.7-kb unit contained only the MTH gene, possibly as two copies of the gene per unit. In strain L2-43-6D, the 1.7-kb XbaI fragment showed also hybridization to probe X, indicating that in this strain the 1.7-kb unit contained both genes. DNA from the sensitive laboratory strain 3 l-l-7Ba contained a single copy of the MTHI gene and thus displayed only the junction fragments (Fig. 4). All three sensitive wild strains (4054-3B, M3-33-6B and Ml l-22-IOB) with an MTH2 locus displayed three bands that hybridized to the

The most resistant strain (L2-43-6D; 1.2 mM CuSO,) studied here contained repeat units of both 1.9 kb and 1.7 kb (Fig. 4). In addition, it had an extra unit of 0.9 kb hybridizing to probe M but not to probe X, suggesting that this strain contained some extra copies of the metallothionein gene. These extra copies might be responsible for the increased resistance of this strain to copper. Strain 1753-8-2, containing the MTH locus on both chr. XVI and chr. VIII (Fig. lA), also had two EcoRI re-

kb 23

-

9467

-

44-

striction fragments (10 kb and 20 kb) hybridizing to the probes (Fig. 5). The IO-kb EcoRI fragment showed a weaker hybridization signal than the 20-kb fragment, as did chr. XVI compared to chr. VIII (Fig. lA), suggesting that the lo-kb EcoRI fragment originates from the MTH2 locus

iT

2.3

-

20

-

9 -ml

Fig. 4. Southern DNA-Hind111

blot analysis

marker

bands

of XbaI-digested

genomic

made with probe X (1.3-kb, XbuI-KpnI fragment probe M (0.7-kb, XbaI-KpnI fragment described

DNA.

Phage 1.

and lengths are given. Hybridizations

were

in pIB176, panel A) or

in pIBI76,

panel B) prepared

as

in the legend to Fig. 3.

probes (Fig. 4). Two were the junction fragments and one was the repeat unit (1.9 kb or 1.7 kb), indicating that the strains contained more than one copy of the MTH gene. Xbal

@ kb 23

-

9.4

-

6.7

-

4.4

-

on chr. XVI and the 20-kb fragment from the MTHl locus on chr. VIII. Wowever, there were no common repeat units between strain 1753-8-2 and strain X2180-1A (Fig. 5) suggesting that the MTHl locus of strain 1753-8-2 was not identical to the ~THl locus found in strain X2 180- 1A, but rather originated from the ~TH2 focus by transposition. The restriction maps of MTHI and NTH2 loci are shown in Fig. 6.

Sau3A

(e) Genetic segregation of MTHZ and MTH2 loci Incomplete tetrad and random spore analyses showed monogenic segregation in crosses between copper-resistant MTH2 strains and a copper-sensitive mthl strain (Table III, hybrid Nos. 9 and 10). Surprisingly, phenotypic segXbal

ECORI

Sau3A

EcoRl

*

*

2.3-

-

Fig. 5. Southern and analyzed

blot analysis

by Southern

of strain 1753-8-2

blotting

cont~ning

with ““P-labeled

both MTHI

and ME%’ loci. Genomic

DNA was isolated.

probe X (panel A) or probe M (panel B) prepared

as described

digested with restriction in the legend for Fig. 3.

enzymes

71 MTHIIMTHZ (L2-43-6D xX2180-1A and 1D x X2180-1A) crosses by colony hybridization

probe MX. Most of the segregants (200 altogether) showed a clear hybridization to the probe. We isolated genomic DNA from colonies (including two sensitive segregants of hybrid Nos. 11 and 12) showing very faint or no hybridization, digested it with XbaI, separated it in an agarose gel, transferred the DNA to a nitrocellulose filter, and hybridized with probe M and probe X. All segregants studied had both junction fragments (see Fig. 6) indicating that all seg-

Probe M

x

SK

E

sx

S

I

K

X

SX

S

II

I

-_

-I

K

S

X I

..I

1340with

_

regants had at least one copy of both the MTH2 gene and gene X (not shown). This supports the theory that the MTH locus contains some essential sequences. Fig. 6. Restriction denotes the location location

maps

of the MTHZ and MTH2 loci. The thick line

of the metallothionein

of the gene, X. The probes

(f) Intergene conversion of MTH2 and mthl Segregants from hybrid L2-43-6D (CuR) x 31-l-7Ba (Cu”) (Table III, hybrid No. 9) with one or two spores germinated from each ascus were assayed for copper resistance or sensitivity on plates containing 0.9 mM CuSO,. The intensity of the Southern hybridization of the segregants (Fig. 7) allowed us to clearly differentiate sensitive and resistant alleles of MTH loci. Double recombinants MTH2/mthl were not obtained among the 16 segregants studied. However, we found a conversion of resistant allele MTH2 to sensitive allele mth2 in segregants N9-8A, N98B, N9-8-3 and N9-1lA and a conversion of sensitive allele mthl into resistant allele MTHl in two segregants N9- 1 IB and N9- 12A (Fig. 7). To determine which parent MTH genes were represented by the new alleles, mth2 and MTHl, we studied some of the segregants in more detail. The isolated, EcoRI-digested DNA fragments were hybridized to probe MX (Fig. 8). The resistant parent, L2-43-6D and the resistant segregants, 10A and llB, displayed a 25kb EcoRI fragment hybridizing to the probe. The sensitive segregants displayed only the 5-kb EcoRI fragment hybridizing to the probe, as did DNA from the sensitive parent strain (31-l-7Ba) containing the MTHl locus. Digestion with XbaI also produced parental types of frag-

gene. The thin line denotes the CUP1 DNA

made from cloned

are

shown above the map of the MTHI locus. E, EcoRl; K, Kpnl; S, Sau3A; X, XbaI.

regation was absent in hybrids MTHZ/MTHl (Table III, hybrid Nos. 11, 12); nearly all segregants were copperresistant. There are two alternative explanations for the latter data: (i) determinants MTHl and MTHZ are allelic or (ii) expected sensitive spores with recombinant genotype mthl ‘mth2’ (without MTHl and MTH2 loci at all) cannot survive on complete medium. The first explanation is in contradiction with the location of the MTHI and MTH2 genes on different chromosomes: chr. VIII and XVI (Figs. 1 and 2). In accordance with the literature data (Ecker et al., 1986; Hamer et al., 1985), the death of spores having genotypes mthl’ and mth2” is probably not caused by an absence of MTH sequence, but a lack of some closely located genes. Almost all MTH2(mthZ)/MTHl (mthl) crosses have actually shown an ascospore viability of 44-76x (Naumov et al., 1983; Table III in the current study). We further studied segregants from MTH2/mthl (L243-6D x 31-l-7Ba and L3-44-7C x 31-l-7Ba) and

TABLE

III

Studies of hybrids

heterozygotic

Hybrid

Hybrid

No.

phenotype)

for two genes, MTHl and MTH2

origin (parent

Ascospore viability

Number

of asci

segregating”

Random

Relevant

spore

genotype

segregation CuR:CuS

of hybrids

as CuR:CuS

% 212

4:o

3:o

2:l

1:2

9

L2-43-6D

(CuR) x 3 l- I-7Ba (Cu’)

46

1

0

0

3

2

18: 17

MTHZ/mthl

10 11

L3-44-7C L2-43-6D

(Cu”) x 31-l-7Ba (Cu”) (CuR) x X2180-1A (CuR)

69 70

2 0

0 6

0 29

11 1

6 0

40:35 113:l

MTHZlmthl MTHZIMTHI

12

1340-1D (CuR) x X2180-1A

73

0

8

30

1

0

12O:l

MTHZ/MTHl

a The copper

resistance

No. 9 which contained

of segregants 0.9 mM C&O,.

(CuR)

was tested on plates with minimal The composition

of minimal

medium

medium

containing

was as described

0.5 mM CuSO,, in footnotes

except for plates of segregants for Table 1.

of hybrid

72

Fig. 7. Translocations sponding

of mth and MTH alleles in segregants

to the hybridization

and the MTHl

of chromosomal

probe used and its preparation

A

DNAs

of hybrid

L2-43-6D

(MTH2/mthl).

x 3 l-l-7Ba

with the MTHZ cloned gene (right). Chromosomal

were the same as described

Ethidium

bromide-stained

DNA preparation,

gel (left) corre-

electrophorcsis

conditions,

for Fig. 1.

EcoRl

Xbal

B

KD

r23--

--q 2320-

Fig. 8. Southern 0.9 mM CuSO, in Fig. 6.

blot analysis was isolated,

of segregants

from the cross of L2-43-6D

(MTHZ)

with 31-l-7Ba

digested with EcoRI (panel A) or XbaI (panel B) and analyzed

ments (Fig. 8). These results showed that the sequences MTHl and MTH2 loci were exchangeable.

in

(g) Recombination of MTH and CAD genes Although yeast metallothionein binds cadmium and zinc in vitro (Winge et al., 1985) in addition to copper, the metallothionein MTHl gene by itself is not able to provide yeasts with resistance to heavy metals. Only the MTH gene

(mthl).

by Southern

Genomic

DNA from segregants

blotting with the “P-labeled

growing

on

probe, MX, shown

located on plasmids, in combination with certain genetic backgrounds, can provide cadmium and silver resistance in yeast cells (Ecker et al., 1986; Jeyaprakash et al., 1991; Macreadie et al., 1991). Our strain, L2-43-6D, with a high level of copper resistance, was also tolerant to cadmium (Table I). We tried to examine the relations of the MTH2 and CADx genes by analysis of the heterozygotic hybrid, CuRCdR x Cu”Cd”

73 (Table II, hybrid No. 1). Tetrad analysis showed monogenie determination of cadmium resistance which did not cosegregate with copper resistance. Three types of tetrads were observed: 2P, lN, 11T (six irregular tetrads lCdR: 3Cd”(2); 1 CuR: 3 Cu” (1); 3 CuR: 1 Cu” (3) were excluded). A nonmetallothionein gene CAD2, of cadmium resistance has been described (Tohoyama et al., 1990) and it cannot be excluded that CAD2 and CADx could be regulatory genes which interact with both MTH and mth alleles. (h) Conclusions (1) Screening

copper-activated

expression

of independent

origin allowed us to find a new type of MTH gene polymorphism: an unusual location on chr. XVI. Also, theMTH gene polymorphism studied by Welch et al. (1983) and Fogel and Welch (1984) was found in the new MTH2 gene; i.e, a difference in the number of tandemly amplified MTH units and in the size of the basic repeat unit. Obviously, the inbred monosporic nature of the strains did not allow the detection of the type of gene polymorphism associated with the difference between heterozygotic and aneuploid strains; i.e., the detection of the number of the same MTH2 clusters located on different homologous chromosomes. (2) Our results demonstrated another common property ofMTH genes in animals and yeasts; i.e., the ability to form multiple gene families. (3) Intragene conversion occurring in an amplified MTHZ locus with high frequency (Fogel and Welch, 1984; Fogel et al., 1984; Welch et al., 1987; 1990) and, especially, the intergene conversion of MTH2 and mthl genes described by us cannot be explained by any simple recombination mechanism. (4) Discovery of the MTH gene family in S. cerevisiae presents new opportunities plification, gene conversion

to study their regulation, and translocation.

am-

protein.

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ACKNOWLEDGEMENTS

Kluyveromyces

We wish to thank Drs. Pirkko Suominen, Dennis Thiele and James Haber for their interest in the present research.

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