Copyright 0 1991 by the Genetics Society of America
Interaction of the Yeast Omnipotent SuppressorsSUPI(SUP45)and
SUP2(SUP35)With Non-Mendelian Factors
Adilya R. Dagkesamanskaya and Michael D. Ter-Avanesyan
Institute of Experimental Cardiology, USSR Cardiology Research Center, 121552 Moscow, USSR
Manuscript received October 10, 1990
Accepted for publication March 23, 1991
ABSTRACT
The SUPl and SUP2 genes code for protein factors intimately involved inthe control of translational
accuracy. The disrupted alleles of these genes confer a recessive lethal phenotype in both [psi'] and
[psi-] genetic backgrounds, indicating an essential function for the corresponding proteins. In [psi']
diploids, heterozygous for the SUPl null allele, several dominant phenotypes were evident with slow
growth and inability to sporulate. These dominant phenotypes disappear after transformation with
the multicopy plasmid carrying the wild-type allele of the SUPl gene. Such dominant phenotypes
were not observed for the SUP2 null allele. The incompatibility of multicopy plasmids carrying the
SUP2 gene with guanidine hydrochloride-curable cytoplasmic factor@)was also demonstrated. The
possiblemechanismsof interaction of the SUPl and SUP2 genes with the [psi] determinant are
discussed.
HE study of informational suppression is one of
the most fruitful approaches to the elucidation
of genetic control of translational ambiguity in both
eukaryotic and prokaryotic cells. The best studied
class of informational suppressors in the yeast Saccharomyces cerevisiae is that of nonsense suppressors with
strong codon specificity acting on either ochre(UAA),
amber (UAG) or opal (UGA)mutations. Most of them
are dominant or semidominant. The suppressors belonging to thisclass arise as arule by anticodon
mutation of known or presumed tRNA genes (PIPER
et al. 1976; GOODMAN,
OISON andHALL1977;
BROACH,FRIEDMAN
and SHERMAN
1981). Another,
but less studied, class ofnonsense suppressors are
presumed not to be mutations in the tRNA genes,
because of the lack of codon specificity. The suppressors of this class are usually called omnipotent (HAWTHORNE and LEUPOLD1974; ONO, STEWART
and
SHERMAN 198
1
; LIEBMAN
and ALL-ROBYN
1984; ONO
et al. 1984). It was shown that mutations in some of
these genes may cause and increased level of translational ambiguity (SURGUCHOV
et al. 1980; MASUREKAR
et al. 1981; EUSTICEet al. 1986). The functions of
their gene products in protein synthesis is not known.
Another approach for the search of protein components involved inthe control of translational accuracy
consists in the identification of mutations interacting
with suppressorsof different specificity.Among them,
mutations, both increasing (allosuppressors) and decreasing (antisuppressors)suppressor efficiency were
described (SHERMAN
1982). Besides nuclear mutations, cytoplasmically inherited factors [psi] and [eta]
interacting with different suppressors were identified
T
Generics 128: 5 13-520 (July, 1991)
in several laboratories (for review,seeCOX, TUITE
and MCLAUGHLIN
1988).
The [psi] factor is itself a weak ochre suppressor
(LIEBMAN
and SHERMAN1979). In addition, ochrespecific tRNA-mediated suppressors aswell as certain
frameshift suppressors have a much higher efficiency
of suppression in @si+] strains than in [psi-] strains
(LIEBMAN,
STEWARTand SHERMAN 1975; ONO, STEWART and SHERMAN 1979;
CUMMINS
et al. 1980). The
increased suppression efficiency causes suppressors
to
be lethal in the presence of &si+] factor (COX1971;
GILMORE,
STEWART
and SHERMAN
1971). Another
non-Mendelian factor, [eta'], causes poor growth and
inviability whencombined with several,but not every,
allele of omnipotent suppressors SUP35 and SUP45
(LIEBMAN
and ALL-ROBYN
1984; ALL-ROBYNet al.
1990). The [eta] element does not interact with any
tRNA suppressors tested. Cells can be cured of both
non-Mendelian elements by growth on guanidine hydrochloride (GuHC1)-containing media (COX,TUITE
and MCLAUGHLIN
1988).
This paper is dedicated to the study of omnipotent
suppressors SUPl and SUP2. Mutations in these genes
have been identified in several laboratories as omnipotent suppressors sup1 and sup2 (INGE-VECHTOMOV
and ANDRIANOVA1970), sup45 and sup35 (HAWTHORNE and LEUPOLD
1974), supQ and supP (GERLACH 1975), frameshift suppressor sufl2 (CULBERTSON,GABER
and CUMMINS
1982), allosuppressors sal4
and sal3 (COX1977), mutation of resistance to novobiocin novl (Pocklington et al. 1990) and cellcycle
and
arresting mutation gstl (KIKUCHI, SHIMATAKE
KIKUCHI 1988). A comparison of their restriction
514
Dagkesamanskaya
A. R.
and M. D. Ter-Avanesyan
maps, nucleotide sequences and complementation
properties has shown that SUPl, SUP45, SAL4 and
NOVl are identical genes asan SUP2, SUP35, SUF12,
SAL3 and GSTl (KUSHNIROVet al. 1988; WILSONand
CULBERTSON
1988; TUITE
et al. 1988; POCKLINGTON
et al. 1990; KIKUCHI,
SHIMATAKE
and KIKUCHI1988).
It was proposed that thesegenes code for protein
factors participating in the elongation step of translaMAICASand FRIESEN
1985; BREINtion (HIMMELFARB,
ING and PIEPERSBERG 1986; KUSHNIROV
et al. 1988;
WILSONand CULBERTSON
1988). Disruption of the
SUP45 and SUFI2 genes revealsthat they are essential
yeast genes(HIMMELFARB,
MAICASand FRIESEN
1985;
POCKLINGTON
et al. 1990; WILSONand CULBERTSON
1988). It is not evident, however, if these disruptions
or [eta+]-carryingstrains. It is
were done in[psi']reasonable to suspect that adisruption that is lethal in
[psi'] or [eta'] might be viable in c[psi-]or [eta-] since
certain salleles ofSUP45 and SUP35 are lethal in [psi']
or [eta'] but viablein [psi-] and [eta-] (Cox 1977;
et al. 1988).
LIEBMAN
and ALL-ROBYN1984; TUITE
In the present study we show that the SUPl gene
disruption has dominant effects in[psi'] diploid stains,
but no effect in diploids cured of this factor. We also
show that multicopy plasmidscarrying the SUP2 gene
are unstable in strains carrying GuHC1-curable cytoplasmic factors. Finally, we report thatSUPl and SUP2
gene disruptions are lethaleven in haploid strains
cured of cytoplasmic determinants by GuHCI.
MATERIALS AND METHODS
Strains, plasmids and media: The strains and plasmids
used in this study are presented in Table 1. SC, YPD, YPG
and sporulation media were prepared and used for cultivation of S. cerevisiae (SHERMAN,
FINKand HICKS1982). For
curing yeast strains of the cytoplasmic determinant [psi] and
[eta], selective or YPD media supplemented with 3-5 mM
GuHCI, were used(TUITE,MUNDY
and Cox 1981). L-broth
medium for Escherichia coli was prepared as described elsewhere (MILLER 1972). Appropriate amounts of amino acids,
nucleic acid bases, or antibiotics were added if necessary.
Agar (2%) was added to prepare solid media. Yeast cells
were grown at 30" and bacteria at 37".
Enzymes: Restriction endonucleases, T4 DNAligases,
DNA polymerase I, and DNAase I were purchased from
Boehringer Mannheim (Mannheim, Federal Republic of
Germany) or NPO "Ferment" (Vilnius, Lithuania). Zymolyase was from Seikagaku Kogyo Co.(Tokyo, Japan). Reaction conditions were those recommended by the manufacturers.
Transformation: Preparation of competent E.coli cells
and the procedurefor E. coli transformation were performed as described elsewhere (MANDELand HICA 1970).
Yeast was transformed by the LiCl method (ITOet al. 1983).
Isolation and construction of plasmids: Plasmid DNA
from E. coli was purified by the method of BIRNBOIM
and
DOLY(1979). The p5supl::TRPI plasmid was constructed
by inserting the 1.4-kb EcoRI fragment, carrying the TRPl
gene, derived from plasmid YRp7, into the EcoRI site of
plasmid pPBM16 (Figure 1). The pl7supZ::URA3plasmid
was constructed by replacement of the 0.3-kb HindIII frag-
ment from the internal region of the SUP2 gene of plasmid
pSTR4 by the URA3 HindIII fragment of pILl (Figure 1).
Southernanalysis: Small amounts of high molecular
et al. 1979) and
weight yeast DNA were prepared (STRUHL
cleaved with restriction enzymes. Electrophoresis of DNA
fragments, transfer to nitrocellulose, and hybridization to
nick translated DNA fragments was carried outas described
by MANIATIS, FRITSCH
and SAMBROOK
(1982).
Genetic methock Yeast tetrad analysis procedures have
been described (SHERMAN,
FINKand HICKS1986). SUPl
and SUP2 gene disruptions were made by the approach of
ROTHSTEIN
(1983). The mitotic stability of plasmids was
determined as the percentage of plasmid-carrying cells in
individual colonies of transformants grown on medium selective for maintenance of the plasmid. Cells from the individual transformant colonies werecloned on complete YPD
medium. Individual clones grown on the complete medium
were then scored for the plasmid marker phenotype by the
replica plate method. In the caseof the mitotic stability
typical for the autonomously replicating yeast vectors, the
majority of clones growing on YPD medium were prototrophic for leucine. For extremely unstable transformants,
growth on the selective medium was absent (Figure 2).
RESULTS
The studyof the SUPl gene disruption:The SUPl
and SUP2 gene disruption experiments were performed with the diploid strain H8, heterozygous for
the mating type locus and homozygous for the mutations in the LEU2, URA3, TRPl and HIS3 genes. T o
generate this diploid, the [psi-] strain DBY746 was
crossed with the [psi'] strain AH216 and a segregant
with the genotype MATa leu2-3,112 ura3-52 trpl-289
and his3-11,15 (or his3-Al) was isolated from the
resulting hybrid. The diploid H8 was obtained from
the backcross of thissegregant with the parental strain
DBY746.
In order to disrupt the SUPl gene, the [psi'] diploid
strain H8 was transformed with the mixture of DNA
fragments obtained after digestion
of
plasmid
p5supl::TRpl byBamHI and ClaI restriction endonucleases (Figure 1). Mitoticallystable Trp+ transformants manifested a slow growth rate and inability
to sporulate. For comparison, the sporulation frequency of the original diploid H8 reached 30% after
3 daysof incubation on sporulation medium. The
poor growth of stable transformants was evident on
synthetic glucose medium but was more pronounced
on YPG medium, containing glycerol asa sole carbon
source (Figure 3). It is noteworthy that these transformants with slow growth were unstable and could
segregate large colonies with a frequency of 3-5%.
These large colonies arose mainly due to mitotic homozygotization ofthe SUPl wild-type allele(our unpublished observation).
The plasmid p5supl::TRPl is capable of autonomous replicating due to the presence of the ARSl
sequence into the TRPl locus (TSCHUMPER
and CARBON 1980). Transformation of the diploid H8 with
the intact p5supl::TRPl plasmidyieldedunstable
Suppressors
Omnipotent
in Yeast
515
TABLE 1
Yeast strains and plasmidsused in this study
Source
A. Strains
Genotype
DBY746"
AH2 16"
483/2d
5V-H 19
SL578-SA
SL664-SA
MATa[psi-]
trpl-289
his3-AI
ura3-52
12
leu2-3,I
MATa his3-IlJ5
leu2-3,I
12 pho3 pho5 [psi']
MATa SUQ5 ade2-1 lysl-1 canl-100 [psi-]
MATa SUQ5 ade2-1 canl-100 leu2-3,1 12 ura3-52
[psi']
MATa met8-I leu2-I trp5-48 his5-2 lysl-1 [psi-] [eta']
MATa met8-1 leu2-I trpl-l ade3-26 ilvl-1 his5-2 lysl-I lyr7-I [psi']
[eta-]
MATa met8-1 leu2-I trpl-1 ade3-26
ilvl-1
his5-2
lysl-I tyr7-I [psi-]
leta-1
370bb
B. Plasmids
Yeast
on
pYSUPI
YEpl3-15SUPI'
pPBM 16'
pYST2
PSTR~~
PSTR~~
Y Rp7
PILI'
YED13
genes
D.
BOTSTEIN
A. HINNEN
B. Cox
This study
S. LIERMAN
S. LIERMAN
S. LIERMAN
plasmids
Source
SUPI, HIS3,2 pm plasmid origin of replication
SUPI, LEU2, 2 pm plasmid origin of replication
SUP1
SUP2, LEU2, 2 pm plasmid origin of replication
SUP2, LEU2, 2 pm plasmid origin of replication
SUP2
TRPI, ARSI
URA3
LEu2. 2 rrm dasmid oriein of redication
BREININC,
SURCUCHOV
and PIEPERSRERC
(1 984)
P. KASHKIN
W. PIEPERSBERC
TELKOV
et al. ( 1 986)
TELKOV
et al. (1 986)
TELKOV
et al. (1 986)
STINCHCOMR,
STRUHL
and DAVIS(1 979)
V. PESHEHONOV
BROACH.
STRATHERN
and HICKS11 979)
The [psi] status of the strains AH216 and DBY746 was shown in crosses with the tester strain 483/2d [ p s i - ] and with its SUQ5 ade2-1
[psi-] derivative of the opposite mating type by the ability of the SUQ5 (also called SUP16) to suppress the ade2-l ochre mutation, since this
suppressor can suppress the ade2-1 mutation only in the [psi'] genetic background (COX 1965; ONO, STEWARTand SHERMAN1979).
Monogenic segregation for adenine prototrophy suggested the [psi-] status of the strain DBY746. Digenic segregation for Ade' suggested
that AH216 bears the [psi'] determinant.
The [psi-] [eta-] tester strain 370 was subjected before our study to GuHCl treatment to insure that it does not carry these cytoplasmic
determinants.
'YEpl3-15SUPI was obtained by cloning of BamHI-Hindlll SUPl fragment in YEpI3 and pPBMI6 by cloning of EamHI-Clal SUP1
fra ment in pACYC177.
'pSTR4 and pSTR7 were obtained by deletion of various Xhol fragments from the pYST2.
' plLl was obtained by deletion of 2 pm EcoRI fragment from the pFL2 (CHEVALLIER,
BLOCH and LACROUTE
1980).
A
B
A
m 7
PPBM10
M .
P
XCXb
J
I
L
I
1
E B
P
P
C E
E
A*/-
pJsupl::mPl
t
!-
P
XC
E
Xb
i
I
E B
P
-
B
DSm4
I
".
PEP
I
X
P
dl1
,. .
1
S
HPH
I
:
E B S
P
I
r
n
- :
x
H
P
H
P
A0042
p17sup2M?A3
P E P
P
,-
H P
H
J !
s
E B S
4
P
I ~ C U R E 1 .-Construction of the plasmids (represented in linearized form) carrying the supI::TRPI (A) and sup2::URA3 (B) alleles.
I he single line indicates bacterial DNA sequences and the wavy
line indicates the fragmentof LEU2 yeast gene. The dark bar
represents SUPl (A) and SUP2 (B)sequences. The open bar is TRPI
(A) and URA3 (B) yeast genes. T h e striped bar = 2 pm DNA.
Restriction sites: B = EamHI, H = HindllI, P = PstI, X = Xhol, Xb
= Xbal, E = EcoRI, S = Sal1 and C = ClaI.
"
FIGURE2.-A comparison of the stability of the LEU2-pSTR7
s
i
'
]
plasmid in (A) GuHCI-treated and (B) untreated cells of the @
diploid strain H8. Both strains growing on YPD medium were
transferred to selective medium. N o Leu' colonies of the untreated
strain were observed after the second day of incubation on leucine
omission medium.
Trp+ clones that could segregateTrp- colonies during
subculturing in complete nonselective medium. The
growth rate on tryptophanomission medium and the
516
A. R. Dagkesamanskaya and
Ter-Avanesyan
M. D.
FIGURE3.-Dependence of the growth rate of diploids heterozygous for SUPl null allele on the presence of a SUPl-carrying
plasmid or on GuHCI-curable cytoplasmic determinant@).Growth
was measured by comparing the intensities of spots on plates made
by inoculations with suspensions of cells. Plates were incubated for
2 days at 30".Rows B and D representdiploid strains heterozygous
for SUP 1 gene disruption which carry a Yepl3-15SUP I plasmid.
Rows A and C are the same diploid strains without plasmid. The
clones inrows A and B were pregrown on GuHCI-containing
medium.
sporulation frequency of these transformants were
comparablewith those of Trp+transformants carrying
the control plasmid YRp7. The meiotic segregation
of several Trp+ transformants carrying the
p5supl::TRPl plasmid was studied. Tetrads with the
segregation 4 Trp+ : 0 Trp- were observed in every
case. This suggests that the cells of the diploid H8
containing the autonomously replicating plasmid
p5supl::TRPl could sporulate and thereforethis plasmid does not inhibit sporulation.
The genetic analysis of integrative transformants
was hampered by their complete inability to sporulate.
However, we observed that transformation of these
integrants with the plasmid YEpl3-15SUP1 leads to
the restoration of growth and sporulation ability.
Thus, autonomously replicating plasmid withthe wildtype SUP1 gene compensates for thedominant defects
observed in these integrative transformants. This suggests that the growth rate alteration and sporulation
defect in integrative transformants were caused by the
SUPl gene disruption.
The meiotic segregation of sucha transformant that
contains the YEp13-15SUP1 plasmid was studied. A
total of 31 tetrads were isolated with the segregation
4+:0- for the plasmid marker LEU2 and 2+:2- for the
disruption marker TRPI. During repeated subculturing of Trp+ Leu+ spore colonies in complete YPD
medium, we did not observed Trp+ colonies in the
absence of an SUPl gene-carrying plasmid. This suggests that the plasmid loss is lethal fortheSUPldisrupted haploid strains.
The dominant phenotype of the SUPl null allele
disappears not only after transformation with an autonomously replicating plasmid carrying the SUP1
gene, but also during growth of a heterozygous d i p
FIGURE4.-Construction of the diploid strain heterozygous for
the SUP 1gene disruption. (A) The recombination event. For clarity,
only BnmHI (B) and Hind111 (H) sites are indicated. The open bar
corresponds to the SUP 1 gene and the dark bar represents the
TRPl gene. (B) Southern blot analysis of the SUP 1 gene disruption.
DNA from Trp' transformant (lane 1) and the original diploid H8
(lane 2) was digested with BamHl and HindlII, fractionated by
electrophoresis in agarose gels, transferred to nitrocellulose and
then hybridized to the nick translated 2.4-kb EcoRI-XbaI fragment
of the SUP 1 gene isolated from the plasmid pPBM 16 (see Figure
1). The single band corresponding to the 4.8-kb fragment was
observed in the control, while an additional 6.2-kb fragment was
revealed in the diploid heterozygous for the SUP1 null allele, as
expected in the case of an insertion of TRP 1 into the SUP 1.
loid on GuHC1-containingmedium (Figure 3). We
have shown that, when grown on YPD medium s u p
plemented with 3 mM GuHCI, the SUPl gene disrupted diploid H8 segregates large colonieswith a
high frequency. These colonies were Trp+ and were
able to sporulate. The tetrad analysis ofthree of these
clones revealed 2 viable : 2 nonviable spores (a total
of 39 tetrads were studied). All viable segregants were
Trp- and did not carry a disrupted SUPl gene, since
TRPl was used as the disruption marker. These data
confirm our previous conclusion about the recessive
lethality ofSUPl gene disruption. One of the GuHCItreated clones ofthe SUPl gene disrupted diploid H8
was used for the Southern blotanalysisbecauseof
instabilityof the slowly growing psi'] SUPl gene
disrupted strain. Analysis of DNA isolated from the
parental diploid and asupl::TRPI/SUPl heterozygous
diploid indicated that both the wild-type and null
alleles were present in the heterozygote (Figure 4).
The study of the SUP2 gene disruption: For obtaining the SUP2 gene disruption the SUPI::URA3
sequence was constructed (see MATERIALS AND METHODS and Figure 1). The integrative plasmid
pl7supP::URA3 was digested by BamHI and XhoI and
used to transform diploid strain H8 to Ura+. The
growth rate and frequency of sporulation of all stable
Ura+ transformants was indistinguishable from that
of the original diploid. Three transformants were
purified by streaking on YPD medium, sporulated,
and tetrads were dissected. All 52 tetrads that were
analyzed contained two viable Ura- spores and two
nonviable spores.Therefore transformation of diploid
Omnipotent Suppressorsin Yeast
H8 by a DNA fragment that carries the SUP2 gene
inactivated by insertion of URA3 leads to recessive
lethality linked to the URA3 marker.
In order to study whether the recessive lethals could
be complemented by the wild-type SUP2 allele, the
integrative Ura' transformants were subjected to repeated transformation by the autonomously replicating pYST2 and pSTR7 plasmids that carry the SUP2
gene and the plasmid marker LEU2. Surprisingly all
selected Leu' transformants were extremely unstable.
Such transformants manifested a slow growth on selective mediumand lost plasmids immediately
on nonselective YPD medium (Figure 2). The diameter of
colonies formed by these transformants on selective
medium at the 4th day of incubation was about 0.25
k 0.02 mm. The diameter of colonies of the same
strain transformed with YEP13 was equal to approximately 0.62 -C 0.03 mm. The genetic analysis ofthese
transformants was hampered by the loss of plasmids
in premeiotic diploid cells prior to sporulation. The
effect of instability was specific to the SUP2 carrying
plasmids, sincethe frequency of the loss of YEP13 and
plasmids that carry the SUPl gene, such as YEp1315SUPl or pYSUP1, in our experiments did not exceed 30%.
After streaking unstable transformants on selective
medium supplemented with 5 mM GuHCI, large colonies arose frequently. The stability of the SUP2 carrying plasmids in these colonies did not differ from
the YEpl3plasmid introduced by the transformation
into the cells of the original diploid (Figure 2). One
of theserapidlygrowingclones
was transferred to
sporulation mediumand 3 1 tetrads were dissected.In
18 tetrads all four spores germinated and segregation
4' : 0- for the plasmid marker LEU2 and 2' : 2- for
the disruption marker URA3 were observed. The plasmid loss was lethal for the Ura' haploid segregants,
while the frequency ofthe loss of the plasmid by Urasegregants reached 30%. Thus, therecessive lethality
that arose as a result of disruption of the SUP2 gene
can be complemented by extra copies of the SUP2
gene.
Southern blot analysisindicated that this integrative
transformant was heterozygous for the sup2::URA3
allele (Figure 5).
Identification of the GuHCl-curable factors interacting with SUPl and SUP2 Aswas shown in the
previous sections,dominant phenotypes ofthe diploid
heterozygous for the SUPl null allele and deleterious
effects of multicopy plasmids carrying the SUP2 gene
canbe cured by growth on GuHCI,whichisalso
known to cure [psi'] and [eta'] elements (TUITE,
MUNDYand Cox 198 1; LIEBMAN
and ALL-ROBYN
1984). It is therefore reasonable to suggest that the
observed effects depend on either one of these cytoplasmically inherited determinants.
517
FIGURE 5.-Construction of the diploid strain heterozygous for
the SUP2 gene disruption. (A) The recombination event. Only XhoI
(X), Sal1 (S) and Hind111 (H) sites are indicated. The open bar
represents the SUP2 gene and the darkbar indicates the URA3
gene. (B)DNA blot hybridimtion analysis of the SUP2 gene disrup
tion.DNAfrom a Ura+ transformant (lane2)and the original
diploid H8 (lane 1) was isolated, digested with XhoI and Sal1 and
hydbridized to the nick-translatedXbaI-XhoI fragment of the SUP2
gene derived from the pSTR4 plasmid (Figure 1). The hybridization with genomic DNA o f s h e control diploid H8 revealed only
one 2.8-kbfragment corresponding to the intact SUP2 gene, while
after hybridization with DNA of the Ura+ transformant an additional 4.0-kb fragment was observed as expected in the case of
insertion of the URA3 gene.
For identification of the cytoplasmically inherited
factor a haploid segregant of a rapidly growing clone
of the GuHCI-treated,SUPldisrupted diploid H8 was
taken. This segregant was Trp- and therefore did not
carry the sup1::TRpl allele. In order toscore for [psi]
this segregant was crossed with the SUQS [psi-] tester
strain 483/2d and the efficiencyof the SUQS was
examined inthe meiotic progeny. Sinceresulting d i p
loid was heterozygous for the ade2-1 mutation and
SUQS suppressor,digenic segregation for adenine prototrophy was expected in the presence of [psi'] while
monogenic segregation would suggest the [psi-] genetic background. A total of 29 tetrads of this diploid
were isolated with the segregation 2 Ade' : 2 Ade-.
The analogous test performed for a segregant of the
SUPl disrupted strain H8 transformed with the plasmid YEpl3-15SUPl (as mentioned above, the SUPldisrupted diploid H8 is unable to sporulate in the
absence of this plasmid) revealed the digenic segregation for adenine prototrophy: 5(2 Ade' : 2 Ade-),
nonparental ditype; 14(3 Ade' : 1 Ade-), tetratype
and 3(4 Ade' : 0 Ade-), parental ditype. The data
obtained indicate that the SUPldisrupteddiploid H8
bears the [psi'] determinant, while its rapidly growing
derivative selectedafter cloning on GuHCI-containing
medium did not carry this cytoplasmically inherited
factor.
Although diploid H8 carried the [psi'] factor, one
cannot conclude that theeffects observed were caused
by this cytoplasmic element. Actually, other GuHCIcurable factors, including [eta'], could also be present
in this strain and might be the realcauseof
the
518
A. R. Dagkesamanskaya and M. D. Ter-Avanesyan
harmful effects. To discriminate between thesepossibilities the following approach was used. Haploid segregants of the [psi-] SUPl-disrupted diploid transformed with the plasmid YEp13-15SUPl were isolated. Several of thesesegregants
of appropriate
mating type carrying theSUPl null allele and the
plasmid YEP 13-15SUPl were thencrossed with tester
strains SL578-3A [psi-] [eta']; SL664-3A [psi'] [eta-]
and 370 [psi-] [eta-].
The growth rate andsporulation
ability of the resulting diploids, heterozygous for the
SUPl gene disruption, were analyzed. Only clones of
the hybrids, derivedfrom the cross with the [psi']
[eta-]tester strain and which have lost the YEp1315SUPl plasmid, express slow growth rate and inability to sporulate. Thus, the dominanteffects observed
for diploids heterozygous for SUPl null allele depend
on the presence of the [psi'] determinant.
T h e [psi'] strain 5V-H19 as well as the psi'] SUP2
disrupted strain H8 transformed with the SUP2 carrying multicopy plasmids pYST2 and pSTR7 grew
slowly on leucine omission medium and was extremely
unstable. The [psi-] colonies of several transformants
of the strain 5V-H19 were selected by the pink color
after thegrowth on leucineless medium supplemented
with 5 mM GuHCl. T h e growth rateand plasmid
stability of the [psi-] derivatives of transformants was
indistinguishable from that of the original [psi'] strain
5V-H 19 transformedwith the plasmid YEpl3.
SL664-3A and SL578-3A tester strains characterized for [psi] and [eta]determinants were also used
for transformation with plasmids pYST2 and pSTR7.
Onlytransformants of the SL664-3A [psi'] [eta-]
strain expressed poor growth on theselective medium
and were extremely unstable in nonselective conditions. T h e transformation of the SL578-3A [psi-]
[eta'] strain did notlead to visible inhibition of growth
on selective medium. It was observed, however, that
thesetransformantswere
also unstable. T h e frequency of pSTR7 plasmid loss varied between 60 and
90% for different transformants. Growth on GuHClcontaining medium decreased the frequency of plasmid loss for some clones presumably cured of the
[eta'] determinant up to 20-30%.
out that theheterozygous SUPlnull mutation showed
two dominant effects in the [psi'] diploid transformants: slow growth and inability to sporulate. T w o
hypotheses that explain the dominant effects of the
SUPl gene disruption could be proposed.
The first
one is: the null allele of the SUPl gene (sup1::TRPl)
codes for an anomalous protein that is toxic for yeast.
This anomalous protein may, for example, compete
with the normal one encoded by the wild-type allele
of the SUPl gene. A similar situation has been described for a null allele of the RNA1 gene of S .
cerevisiae (ATKINSON
and HOPPER1987). This hypothesis, however, seems to be scarcely probable since
transformation of the &si']
diploid strain H8 with the
autonomously replicating plasmid p5supl::TRPl did
not noticeably alter the growth rate and sporulation
efficiency. The second hypothesis suggests that the
complete inactivation of one of the two SUPl wildtype alleles of a diploid strain leads to a decrease of
the gene productlevel, and hence to alteration of cell
physiology.
One of the main results of this study is that dominant effects of the SUPl null mutation depend on the
presence of a cytoplasmic determinant that can be
cured by GuHCl. It appears thatthis determinant was
not [eta'] and might represent [psi'].
It is therefore
possible to suggest thatthe [psi] determinant can
inactivate a significant proportion of the SUPl encoded protein.
T h e SUP2 null allele, unlike the SUPl one,does not
confer any dominant phenotypes in either [psi']
or
[psi-] heterozygous diploids. Therefore,the SUP2
gene product does not interact with the [psi] determinant in the samemanner as theSUPlprotein.
Nevertheless, we have observed another type of interaction of the SUP2 gene with a GuHCI-curable cytoplasmic determinant. It was demonstrated that transformants of [psi'] [eta-]strains, which carry multicopy
plasmids with the SUP2 gene, grow slowly on selective
medium and are extremely unstable in nonselective
conditions. It was also observed that the presence of
the [eta'] determinant also led to some instability of
transformants, which carry the SUP2-containing plasmid. However, the stability of this plasmid was not SO
DISCUSSION
dramatically lowin strains with [psi-] [eta'] background as in [psi'] [eta-]strains. We suggest that extra
Previous studies have shown that both SUPl and
copies
of the SUP2 wild-type allele are toxic for &si']
SUP2 omnipotent suppressors are single copy essential
and
to
a lesser extent for [eta'] yeast cells. Therefore,
yeast genes (HIMMELFARB,
MAICASand FRIESEN1985;
selection
would occur for growthof low plasmidCOPYet al. 1990; WILSONand CULBERTSON
POCKLINGTON
number
segregants.
That, however, would increase
1988). In these studies, however, diploid strains not
the
plasmid
instability
which in turn would slow the
characterized in relation to [psi] and [eta]background
growth
of
transformants.
Recently it was observed
were used. In this paper we present evidence that null
that
plasmid-mediated
amplification
of the SUP2 wildalleles ofSUP1 and SUP2 genes produce recessive
type
allele
causes
the
suppression
of
ochre, amber or
lethality in both [psi'] and &si-] genetic background.
opal
mutations
in
yeast
(CHERNOFF
et
al. 1988; KUSHIt is well known that null alleles of various genes
NIROV
et
al.
1990).
This
finding
suggests
thatthe
are recessive.We were, however, surprised to find
Omnipotent Suppressors in Yeast
combination of different factors increasing translational ambiguity, namely the [psi] determinant and
extra copies ofthe SUP2 gene, leads to ahigh level of
inaccuracy incompatible with cell viability.
It is worth noting thatthe [eta'] butnot psi']
determinant is known to interact with some sup1 and
sup2 mutant alleles (LIEBMAN
and ALL-ROBYN1984).
A lethal interaction of [psi] determinant with allosupressors which are alleles of SUP1 and SUP2,was also
observed, but it is not possible to exclude the possibility that the effect depended on thesimultaneouspresence of the [eta*] element (COX, TUITEand MCLAUCHLIN1988). We also cannot rule out the possibility that the effects observed in this study depend
on unknownGuHC1-curable factors present in our
strains simultaneously with[psi] determinant.
The authors are grateful to W. PIEPERSBERG
for providing us
with the plasmid pPBM16, to P. K. KASHKINfor the plasmid YEpl315SUPI and to V. PESHEHONOV
for the plasmid PILI. We are also
grateful to J. ALL-ROBYN
and S. W. LIEBMAN
for yeast strains 370,
SL664-3Aand SL578-3A,helpful discussion and critical reading of
the manuscript.
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Communicating editor: M. CARISON