BIOCHEMICAL
Vol. 64, No. 1, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A NEWMITOCHONDRIAL MUTATIONIN SACCHAROMYCES CEREVISIAE* J. J. ELLIOT and A. J. S. BALL Department of Biological Received
February
Sciences, Brock University,
St. Catharines,
Canada
14,1975 SUMMARY
The genetic analysis of a yeast mutant resistant to D(+) glucosamine on glycerol mediumis reported. The mutant phenotype segregates in vegetative diploids, can be enriched for in test mediumand shows non-mendelian inheritance patterns. No cross resistance to several inhibitors of mitochondriogenesis was observed. The new locus is desfgnated as [cry l-r]; catabolite repression resistant yeast. INTRODUCTION Wild type -S. cerevisiae
are poisoned by glucose analogues,such as
D (+) glucosamine and 2 deoxyglucose (1). analogues on non-fermentable communication reports which grows on glycerol
Such yeast, when exposed to these
carbon sources are unable to grow (2).
This
the genetic analysis of a UV induced mutant (GRlO) medium in the presence of glucosamine.
METHODS Yeast strains: D587-4B (a, his1 [rho+]) and D585-1lC (a, lysl [rho+]> were a gift from Dr. Fred Sherman, Department of Genetics, Rochester University. Back crosses were made to strain 4BL (a, lysl [rho']) a derivative of a cross between these parental strains. The mutant, GRlOwas derived from 4B2 (a, his1 [rho+]) also derived from the original parental strain cross. Media: YPD, 1% yeast extract, 2%peptone, 3% dextrose. YPG; substitute glycerol for dextrose in YPD. GGM; the glucosamine resistance test medium which consists of YPG + 0.05% glucosamine HCl. et al. (3) Mating: was according to the procedure of Montenecourt -Ascus dissection: was according to the technique of Johnston and Mortimer (4). The mutant isolation will be described in detail elsewhere. RESULTSANDDISCUSSION Strain
GRlO is one of several isolates
resistance when back crossed to the parental *
which showed variable strain
degrees of
4BL. This variability
This work was supported by Canadian N.R.C. Operating Grant #A6795 awarded to A.J.S.B.
Copyright o 19 75 by Academic Press, Inc. AN n’gh Is of reproduction in any form reserved.
277
BIOCHEMICAL
Vol. 64, No. 1,1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Vegetative
Segregation of Glucoeamine Resistance (GR)
Strain number
4, glucosamine resistant
4BL and 4B2 (parentale)
0
(400)a
GRlO
(mutant)
100
(375)
GR10/4BL
(diploid
loom
(410)
54m
(264)
(GR10/4BL)s (diploid
derivative)
(GR10/4BL)r
derivative)
(diploid
100
(358)
(GR10/4BL)R (enriched diploid)
1ooc
(306)
lOP3r
1ooc
(296)
(enriched haploid)
clones
a numbers in brackets are the total
YPG+clones scored:
and rapid growth in all
m colonies show micro-colony
replicates:
' strong resistance resistance
only.
took the form of micro-colonies diploid,
(1-20/replicate)
YPD grown colonies replicated
derivatives
Petite
(GR).
and then testing
plating
to YPD agar to give 150-200
these colonies for glucosamine resistance
GR10/4BL is less resistant
haploid GRlOwhich is in turn far more resistant 4BL and 4B2. Also, that subsequent vegetative
testing
Any clone
resistance was scored as glucosamine resistant.
It can be seen that the diploid,
sensitive
replication.
colonies (YPG-) were excluded from these totals.
which showedmicro-colony
of
of growing various haploid and diploid
of GRlO in YPD broth,
colonies/plate
the imprint
(5) to GM teat medium. Such micro-
colonies were usually developed 72 hours after Table I shows the results
within
(GR10/4BL)s or resistant
than the
than the parental
strains
growth (in YPD broth)
of
(GR10/4BL)r clones selected from the
of GR10/4BL show an enrichment for sensitivity
(GR> respectively.
278
(G') and resistance
BIOCHEM’cAL
Vol. 64, No. 1, 1975
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE II Segregation of GR factors
During Sporulation
Cross
4:o r:s
3:l r:s
2:2 r:s
1:3
0:4
Total(')
r:s
r:s
tetrad6
0
1
1
7
1
10
12
0
0
0
0
12
lOP3rp- vs 4BL; (Go x Gs)
0
0
0
0
15
15
lOP3r vs 4BLp-; (GR x Go)
1
6
6
2
1
16
GRlO vs 4BL., (GR x Gs) b (GR10/4BL)R; (GR x Gs)
a all b
tetrad.9 showed 2:2 segregation for the chromosomalmarkers his1 and lysl
superscripts,
R = resistant,
The effect diploid
of selective
S = Sensitive,
enrichment (6) is also shown in Table I.
GRlOf4BL and a haploid derivative
Derivative
were then subcultured
GRclones as described above. addition,
Both strains
all clones showed confluent
resistance
The
lOP3 were grown for 4 days in
GGMbroth and then plated on to YPD agar. and lOP3r respectively,
0 = eliminated.
clones (GR10/4BL)R
to YPD broth and tested for showed 100%resistance
growth on GGMagar, i.e.,
had been converted to confluent
and in
micro-colony
resistance by growth in selective
medium. The results vegetative effects
of Table I, non-Mendelian segregation of mutant factors
clones (haploid and diploid)
of selective
cytoplasmically
(7, 8) together with the enrichment
media (6) are strong indicators
that the GR factor
inherited.
Confirmation of this hypothesis comes from the meiotic segregation data shown in Table II.
All
of the tetrads
reported in Table II showed
Mendelian (2:2) segregation for the chromosomalmarkers his1 and lysl.
279
in
is
BlOCHEMl~l
Vol. 64, No. 1,197s
Tetrads derived from diploid
AND BIOPHYSI~L
GR10/4BL showed a spectrum of segregant
classes together with a spectrum of micro-colony clones.
When the enriched diploid
indicate
showed confluent
numbers (3-30) in resistant
(GR10/4BL)R was sporulated all
tetrads obtained showed 4:0, resistant:sensitive replicates
RESEARCH CO~UNICATIONS
segregation,
growth on CGMplates.
cytoplasmic or mitochondrial
inheritance
of the
and all
These results also of GR. These results
are
not compatible with any simple form of Mendelian inheritance. Our results DNA (mt-DNA). and petite strains
do not prove that the locus of GR is on mitochondrial Treatment of yeast with ethidium bromide eliminates mtDNA (9)
[rho01 derivatives
of 4BL and lOP3r were madeby growing these
in YPD broth + 5 pg/ml ethidium bromide for 48 hours.
produce petite lOP3rp-
cells which are devoid of mtDNA. The result
This should
of crossing
to 4BL and lOP3r to 4BLp- is shown in Table II.
When compared to the two [GR rho+] vs [GS rho+] crosses reported in Table II,
it is clear that elimination
of mtDNA from lOP3r has eliminated the
transmission of GRand the reciprocal
is true for Gs in the 4BLp- cross.
The cumulative evidences of Tables I and 11 make it that glucosamine resistance does not establish
(6
factor)
its uniqueness.
is mitochondriafly
to erythromycin
oligomycin
(5 ug/ml) and found to be sensitive
to be sensitive
resistant
Grlffiths
cry
to these drugs.
strains were used as controls
OLI and OLII strains
(11) and CR, strain
One can therefore
Pnherited but
(5mg/ml), chloramphenicol (5 mg/ml) and
to glucosamine poisoning.
L411, Linnane (lo),
certain
Strain lOP3r was tested for cross
resistance
mitochondrial
fairly
Known
and these proved
Strains used were: ER, strain D22-A16 and D22-A13, Avner and
44-5a, Rank (12).
designate the genotype of GRlO as a, his1 [rho+,
l-r]. REFERENCES
1.
HOCHESTER, R.M. and QUASTEL,J.M.,(Edits) (1963) in "Metabolic Academic Press, New York, N.Y. p 133-142.
280
Inhibitors“,
Vol. 64, No. 1, 1975
BIOCHEMICAL
and BALL, A.J.S.,
AND BIOPHYSICAL
2.
ELLIOT, J.J.
3.
MONTENECOURT, B.S.,
4.
JOHNSTON,J.R. and MORTIMER,R.K.,
(1959) J. Bacterial.
78,272-273.
5.
LFDERBERG, J. and LEDERBERG, E.M.,
(1952) J. Bacterial.
63, 399-406.
6.
RANK, G.H. and BECH-HANSEN, N.T.,
7.
BOLOTIN, M.L., COEN,D., DEUTSCH,J., DUJON, B. and SLONIMSKI, P.P.,(1971) Bull. Inst. Pasteur (Paris) 69, 215-239.
8.
THOMAS,D.Y. and WILKIE, D., 30, 368-372.
(1968) Biochem. Biophys. Res. Comm.
9.
NAGLEY, P. and LINNANE, A.W.,
(1972)
KUO, C.S.,
(1973)
RESEARCH COMMUNICATIONS
Genetics, 2,
LAMPEN, J.O.,
S71.
(1973) J. Bacterial.
(1972) Canadian J. Microbiol,s,
J. Mol. Biol.,
GINGOLD,E.B, SAUNDERS,G. W., LURINS, H.B. and LINNANE, A.W., Genetics 62, 75-79.
11.
AVNER, P.R. and GRIFFITHS, D.E.,
12.
RANK, G.H. and BECH-HANSEN, N.T.,
(1969)
Eur. J. Biochem., 32, 307-311.
(1972) Genetics, 72, 1-15.
281
l-7.
66,181-183.
10.
(1973)
=,233-238.
Vol. 64, No. 1, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A NEWMITOCHONDRIAL MUTATIONIN SACCHAROMYCES CEREVISIAE* J. J. ELLIOT and A. J. S. BALL Department of Biological Received
February
Sciences, Brock University,
St. Catharines,
Canada
14,1975 SUMMARY
The genetic analysis of a yeast mutant resistant to D(+) glucosamine on glycerol mediumis reported. The mutant phenotype segregates in vegetative diploids, can be enriched for in test mediumand shows non-mendelian inheritance patterns. No cross resistance to several inhibitors of mitochondriogenesis was observed. The new locus is desfgnated as [cry l-r]; catabolite repression resistant yeast. INTRODUCTION Wild type -S. cerevisiae
are poisoned by glucose analogues,such as
D (+) glucosamine and 2 deoxyglucose (1). analogues on non-fermentable communication reports which grows on glycerol
Such yeast, when exposed to these
carbon sources are unable to grow (2).
This
the genetic analysis of a UV induced mutant (GRlO) medium in the presence of glucosamine.
METHODS Yeast strains: D587-4B (a, his1 [rho+]) and D585-1lC (a, lysl [rho+]> were a gift from Dr. Fred Sherman, Department of Genetics, Rochester University. Back crosses were made to strain 4BL (a, lysl [rho']) a derivative of a cross between these parental strains. The mutant, GRlOwas derived from 4B2 (a, his1 [rho+]) also derived from the original parental strain cross. Media: YPD, 1% yeast extract, 2%peptone, 3% dextrose. YPG; substitute glycerol for dextrose in YPD. GGM; the glucosamine resistance test medium which consists of YPG + 0.05% glucosamine HCl. et al. (3) Mating: was according to the procedure of Montenecourt -Ascus dissection: was according to the technique of Johnston and Mortimer (4). The mutant isolation will be described in detail elsewhere. RESULTSANDDISCUSSION Strain
GRlO is one of several isolates
resistance when back crossed to the parental *
which showed variable strain
degrees of
4BL. This variability
This work was supported by Canadian N.R.C. Operating Grant #A6795 awarded to A.J.S.B.
Copyright o 19 75 by Academic Press, Inc. AN n’gh Is of reproduction in any form reserved.
277
BIOCHEMICAL
Vol. 64, No. 1,1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Vegetative
Segregation of Glucoeamine Resistance (GR)
Strain number
4, glucosamine resistant
4BL and 4B2 (parentale)
0
(400)a
GRlO
(mutant)
100
(375)
GR10/4BL
(diploid
loom
(410)
54m
(264)
(GR10/4BL)s (diploid
derivative)
(GR10/4BL)r
derivative)
(diploid
100
(358)
(GR10/4BL)R (enriched diploid)
1ooc
(306)
lOP3r
1ooc
(296)
(enriched haploid)
clones
a numbers in brackets are the total
YPG+clones scored:
and rapid growth in all
m colonies show micro-colony
replicates:
' strong resistance resistance
only.
took the form of micro-colonies diploid,
(1-20/replicate)
YPD grown colonies replicated
derivatives
Petite
(GR).
and then testing
plating
to YPD agar to give 150-200
these colonies for glucosamine resistance
GR10/4BL is less resistant
haploid GRlOwhich is in turn far more resistant 4BL and 4B2. Also, that subsequent vegetative
testing
Any clone
resistance was scored as glucosamine resistant.
It can be seen that the diploid,
sensitive
replication.
colonies (YPG-) were excluded from these totals.
which showedmicro-colony
of
of growing various haploid and diploid
of GRlO in YPD broth,
colonies/plate
the imprint
(5) to GM teat medium. Such micro-
colonies were usually developed 72 hours after Table I shows the results
within
(GR10/4BL)s or resistant
than the
than the parental
strains
growth (in YPD broth)
of
(GR10/4BL)r clones selected from the
of GR10/4BL show an enrichment for sensitivity
(GR> respectively.
278
(G') and resistance
BIOCHEM’cAL
Vol. 64, No. 1, 1975
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE II Segregation of GR factors
During Sporulation
Cross
4:o r:s
3:l r:s
2:2 r:s
1:3
0:4
Total(')
r:s
r:s
tetrad6
0
1
1
7
1
10
12
0
0
0
0
12
lOP3rp- vs 4BL; (Go x Gs)
0
0
0
0
15
15
lOP3r vs 4BLp-; (GR x Go)
1
6
6
2
1
16
GRlO vs 4BL., (GR x Gs) b (GR10/4BL)R; (GR x Gs)
a all b
tetrad.9 showed 2:2 segregation for the chromosomalmarkers his1 and lysl
superscripts,
R = resistant,
The effect diploid
of selective
S = Sensitive,
enrichment (6) is also shown in Table I.
GRlOf4BL and a haploid derivative
Derivative
were then subcultured
GRclones as described above. addition,
Both strains
all clones showed confluent
resistance
The
lOP3 were grown for 4 days in
GGMbroth and then plated on to YPD agar. and lOP3r respectively,
0 = eliminated.
clones (GR10/4BL)R
to YPD broth and tested for showed 100%resistance
growth on GGMagar, i.e.,
had been converted to confluent
and in
micro-colony
resistance by growth in selective
medium. The results vegetative effects
of Table I, non-Mendelian segregation of mutant factors
clones (haploid and diploid)
of selective
cytoplasmically
(7, 8) together with the enrichment
media (6) are strong indicators
that the GR factor
inherited.
Confirmation of this hypothesis comes from the meiotic segregation data shown in Table II.
All
of the tetrads
reported in Table II showed
Mendelian (2:2) segregation for the chromosomalmarkers his1 and lysl.
279
in
is
BlOCHEMl~l
Vol. 64, No. 1,197s
Tetrads derived from diploid
AND BIOPHYSI~L
GR10/4BL showed a spectrum of segregant
classes together with a spectrum of micro-colony clones.
When the enriched diploid
indicate
showed confluent
numbers (3-30) in resistant
(GR10/4BL)R was sporulated all
tetrads obtained showed 4:0, resistant:sensitive replicates
RESEARCH CO~UNICATIONS
segregation,
growth on CGMplates.
cytoplasmic or mitochondrial
inheritance
of the
and all
These results also of GR. These results
are
not compatible with any simple form of Mendelian inheritance. Our results DNA (mt-DNA). and petite strains
do not prove that the locus of GR is on mitochondrial Treatment of yeast with ethidium bromide eliminates mtDNA (9)
[rho01 derivatives
of 4BL and lOP3r were madeby growing these
in YPD broth + 5 pg/ml ethidium bromide for 48 hours.
produce petite lOP3rp-
cells which are devoid of mtDNA. The result
This should
of crossing
to 4BL and lOP3r to 4BLp- is shown in Table II.
When compared to the two [GR rho+] vs [GS rho+] crosses reported in Table II,
it is clear that elimination
of mtDNA from lOP3r has eliminated the
transmission of GRand the reciprocal
is true for Gs in the 4BLp- cross.
The cumulative evidences of Tables I and 11 make it that glucosamine resistance does not establish
(6
factor)
its uniqueness.
is mitochondriafly
to erythromycin
oligomycin
(5 ug/ml) and found to be sensitive
to be sensitive
resistant
Grlffiths
cry
to these drugs.
strains were used as controls
OLI and OLII strains
(11) and CR, strain
One can therefore
Pnherited but
(5mg/ml), chloramphenicol (5 mg/ml) and
to glucosamine poisoning.
L411, Linnane (lo),
certain
Strain lOP3r was tested for cross
resistance
mitochondrial
fairly
Known
and these proved
Strains used were: ER, strain D22-A16 and D22-A13, Avner and
44-5a, Rank (12).
designate the genotype of GRlO as a, his1 [rho+,
l-r]. REFERENCES
1.
HOCHESTER, R.M. and QUASTEL,J.M.,(Edits) (1963) in "Metabolic Academic Press, New York, N.Y. p 133-142.
280
Inhibitors“,
Vol. 64, No. 1, 1975
BIOCHEMICAL
and BALL, A.J.S.,
AND BIOPHYSICAL
2.
ELLIOT, J.J.
3.
MONTENECOURT, B.S.,
4.
JOHNSTON,J.R. and MORTIMER,R.K.,
(1959) J. Bacterial.
78,272-273.
5.
LFDERBERG, J. and LEDERBERG, E.M.,
(1952) J. Bacterial.
63, 399-406.
6.
RANK, G.H. and BECH-HANSEN, N.T.,
7.
BOLOTIN, M.L., COEN,D., DEUTSCH,J., DUJON, B. and SLONIMSKI, P.P.,(1971) Bull. Inst. Pasteur (Paris) 69, 215-239.
8.
THOMAS,D.Y. and WILKIE, D., 30, 368-372.
(1968) Biochem. Biophys. Res. Comm.
9.
NAGLEY, P. and LINNANE, A.W.,
(1972)
KUO, C.S.,
(1973)
RESEARCH COMMUNICATIONS
Genetics, 2,
LAMPEN, J.O.,
S71.
(1973) J. Bacterial.
(1972) Canadian J. Microbiol,s,
J. Mol. Biol.,
GINGOLD,E.B, SAUNDERS,G. W., LURINS, H.B. and LINNANE, A.W., Genetics 62, 75-79.
11.
AVNER, P.R. and GRIFFITHS, D.E.,
12.
RANK, G.H. and BECH-HANSEN, N.T.,
(1969)
Eur. J. Biochem., 32, 307-311.
(1972) Genetics, 72, 1-15.
281
l-7.
66,181-183.
10.
(1973)
=,233-238.
