PSI+ prion generation in yeast: characterization of the 'strain' difference
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Yeast
Yeast 2001; 18: 489±497.
Research Article
[PSI+] prion generation in yeast: characterization of
the `strain' difference
Natalia V. Kochneva-Pervukhova, Maria B. Chechenova, Igor A. Valouev, Vitaly V. Kushnirov,
Vladimir N. Smirnov and Michael D. Ter-Avanesyan*
Institute of Experimental Cardiology, Cardiology Research Center, 121552 Moscow, Russia
* Correspondence to: Abstract
M. D. Ter-Avanesyan, Institute The yeast cytoplasmically-inherited nonsense suppressor [PSI+] determinant is presumed
of Experimental Cardiology, to be a manifestation of the aggregated prion-like state of the Sup35 protein.
Cardiology Research Center,
Overexpression of the Sup35 protein induces generation of [PSI+] determinants with
3rd Cherepkovskaya Street 15A,
121552 Moscow, Russia.
various suppressor ef®ciency and mitotic stabilities. Here, we demonstrate that the relative
E-mail: ter@cardio.ru frequency of appearance of [PSI+] with different properties depends on the SUP35 allele
used to induce their generation. The difference in properties of [PSI+] determinants was
preserved after their transmission from one yeast strain to another. This difference
correlated with variation in properties of the Sup35 protein. A novel type of prion
instability was observed: some [PSI+] with weak suppressor ef®ciency could convert
spontaneously into strong suppressor determinants. Copyright # 2001 John Wiley &
Received: 22 June 2000 Sons, Ltd.
Accepted: 6 November 2000
Keywords: eRF3; prion strains; [PSI+]; Sup35p; Saccharomyces cerevisiae; yeast
Introduction Weissman, 1997; Prusiner et al., 1998). The con-
temporary concept suggests that they represent a
The [PSI+] genetic determinant of Saccharomyces conformationally altered form (PrPSc) of normal
cerevisiae decreases the ef®ciency of translation host-encoded protein (PrPC), which has acquired an
termination and may be revealed by suppression of ability to convert PrPC into this altered prion form.
nonsense mutations. It exhibits unusual genetic Several models of prion conversion have been
properties, which include a non-Mendelian mode proposed. The heterodimer (template assistance)
of inheritance, an ability to disappear in the model assumes that the prion, existing as a
presence of low concentrations of guanidine hydro- monomer, can form heterodimer complexes with
chloride (GuHCl) and to reappear upon over- normal molecules catalysing their prion rearrange-
expression of the Sup35 protein (Sup35p) (for ment. The two molecules then dissociate and the
review, see Cox et al., 1988; Wickner et al., 1995). cycle repeats. According to this model, the aggrega-
Study of the biochemical properties of Sup35p tion is a secondary process, non-essential for the
showed that it aggregates in the [PSI+] strains, prion conversion (Cohen et al., 1994). The
while being soluble in the strains lacking this nucleated polymerization model considers prions
determinant (Patino et al., 1996; Paushkin et al., as regular polymers, or one-dimensional crystals
1996). These properties are best explained by a that serve as nuclei for further polymerization of
hypothesis that [PSI+] is a phenotypic manifesta- the protein. The conformational rearrangement of
tion of the prion-like state of the Sup35 protein monomers may either occur spontaneously and
(Wickner, 1994). then be ®xed in polymer structure (Jarrett and
Prions are infectious agents responsible for a Lansbury, 1993) or, alternatively, be directly cata-
group of diseases typi®ed by sheep scrapie, bovine lysed by polymer at the moment of accretion
spongiform encephalopathy and human Creutz- (Horwich and Weissman, 1997). As a modi®cation
feld±Jacob disease (for review, see Horwich and of the latter model, it was proposed that the prion
Copyright # 2001 John Wiley & Sons, Ltd.490 N. V. Kochneva-Pervukhova et al.
conformation is adopted concomitantly with assem- although some difference in the rate of Sup35p
bly, via molten oligomeric intermediates (Serio et al., aggregation in cells with various [PSI+] was
2000). revealed (Zhou et al., 1999), the phenomenon of
Sup35p is a yeast member of the eRF3 family of [PSI+] variability is poorly characterized.
translation termination factors (Zhouravleva et al., In this work we demonstrate that the relative
1995; Stans®eld et al., 1995) and is composed of the frequencies of appearance of [PSI+] with weak and
amino-terminal region and carboxy-terminal (C) strong suppressor phenotype depend on the SUP35
domain of 253 and 432 amino acids, respectively allele used to induce their generation. We further
(Kushnirov et al., 1988; Ter-Avanesyan et al., 1993, characterize the [PSI+] strains difference and
1994). The evolutionarily conserved C domain of describe [PSI+] with weak suppressor phenotype
Sup35p is responsible for its function in translation that can spontaneously convert into strong suppres-
termination and is essential for cell viability, while sor determinants.
the N-terminal region is neither conserved nor
essential. This region may be further subdivided
into the middle (M) domain, of unknown function, Materials and methods
and the N-terminal (N) domain of 123 amino acids,
required for [PSI+] maintenance (Figure 1). The N Strains and media
domain plays a key role in the [PSI+] phenomenon,
being required for [PSI+] propagation in vivo and The S. cerevisiae strains used were 5V-H19 (MATa
solely responsible for Sup35p prion conversion and ade2-1 SUQ5 can1-100 leu2-3,112 ura3-52 [psix]),
oligomerization into amyloid-like ®brils in vitro c10B-H49 (MATa ade2-1 SUQ5 lys1-1 his3-11,15
(Paushkin et al., 1997a, b; Glover et al., 1997; leu1 kar1-1 cyhr [psix]) (Ter-Avanesyan et al., 1994;
King et al., 1997). Kochneva-Pervukhova et al., 1998), 1B-H67
One of the most intriguing properties of prions is (MATa ade2-1 SUQ5 ura3-52 lys1-1 his3-11,15
leu1 leu2-3,112 kar1-1 cyhr [psi±]) (this work). The
the existence of their different strains. In mammals,
sup35-C allele of the strain 1±5V-H19 encodes a
different prion strains are de®ned by speci®c
truncated Sup35 protein lacking amino acids 1±253
incubation times, distribution of vacuolar lesions
and causes dominant antisuppression and inability
and patterns of PrPSc accumulation (for review, see
to propagate [PSI+] (Kochneva-Pervukhova et al.,
Prusiner et al., 1998). In yeast they can be revealed 1998). This strain contained [PIN+] determinant,
by differences in the suppressor ef®ciency and which allows induction of [PSI+] by overexpression
mitotic stability of independently isolated [PSI+] of Sup35p (Derkatch et al., 1997). We used
determinants (Derkatch et al., 1996). It was shown standard rich (YPD) and synthetic (SC) media for
that at the molecular level mammalian prion strain yeast (Sherman et al., 1986). Non-fermentable
differences correlated with stable variations in the media contained glycerol (24 ml/l) as the sole
prion protein structure (Bessen et al., 1995; Safar carbon source. All solid media contained 2.5%
et al., 1998; Caughey et al., 1998). However, (w/v) agar. Yeast cells were grown at 30uC.
Figure 1. Schematic representation of the Sup35 protein and its fragments. Designations of the SUP35 deletion alleles and
corresponding protein fragments are presented at left. Amino acid numbers are indicated
Copyright # 2001 John Wiley & Sons, Ltd. Yeast 2001; 18: 489±497.Analysis of [PSI+] generation de novo 491
Plasmids recipient strain were scored as cytoductants and
tested for the ability to grow on adenine omission
DNA manipulations were performed using stan-
medium (Ter-Avanesyan et al., 1994). To quantify
dard protocols (Sambrook et al., 1989). A series
the induction of [PSI+], transformants of the 1-5V-
of pEMBL-yex4-based plasmids (Cesareny and
H19 [psix] [PIN+] strain with multicopy plasmids
Murray, 1987) containing either the complete
carrying different SUP35 alleles were crossed with
SUP35 gene or its 3k-deletion alleles (Figure 1) has
the c10B-H19 kar1-1 [psix] [rhox] tester strain
been described previously (Ter-Avanesyan et al.,
carrying the cyhr mutation. Cytoductants were
1993). The pUKC815 plasmid carrying the
selected on glycerol medium containing 3 mg/ml
PGK±lacZ gene fusion, and pUKC817, which is a
cycloheximide and the frequency of [PSI+] induc-
pUKC815 derivative carrying an in-frame TAA
tion was estimated as described by Kochneva-
termination codon at the junction of the PGK and
Pervukhova et al. (1998).
lacZ genes, were described by Stans®eld et al.
(1995). The transformants with all the plasmids
used were selected on uracil omission medium. Determination of the nonsense suppression
ef®ciency
The UAA nonsense suppression levels were deter-
Genetic methods
mined as ratio of b-galactosidase activity in cells
Standard methods of yeast genetics were used transformed with pUKC817 plasmid to that in cells
(Sherman et al., 1986). Nutrition markers were with pUKC815, as described previously (Stans®eld
scored by growth on synthetic complete (SC) media et al., 1995).
lacking speci®c amino acids or nucleic acids bases.
DNA transformation of yeast cells was performed
Preparation, fractionation and analysis of yeast
as described (Gietz et al., 1995). Non-suppressive
cell lysates
petites of the strains c10B-H49, 5V-H19 and
1B-H67 were obtained by ethidium bromide treat- Yeast cultures were grown in liquid YPD medium
ment (Goldring et al., 1970). Yeast strains were or in a medium selective for plasmid marker to an
cured, when necessary, of the [PSI+] determinant OD600 of 1.0 (exponential phase) or, when neces-
by growth on YPD medium supplemented with sary, to OD600 of 3.5 (stationary phase). The cells
3 mM guanidine hydrochloride (GuHCl test) (Tuite were harvested, washed in water and lysed by
et al., 1981). The [psix] colonies of ade2-1 SUQ5 vortexing with glass beads in buffer A (25 mM
carrying strains were identi®ed by their red colour Tris±HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl2,
and adenine requirement because the weak serine- 1 mM dithiothreitol) containing 1 mM phenylmethyl-
inserting tRNA suppressor SUQ5 cannot suppress sulphonyl ¯uoride (PMSF) to limit proteolysis
the ade2-1 ochre mutation in the absence of the degradation. Cell debris was removed by centrifu-
[PSI+] determinant (Cox, 1965). The mitotic gation at 15 000rg for 10 min. To analyse the size
stability of different [PSI+] isolates was determined distribution of Sup35p by differential centrifuga-
as the percentage of [PSI+] cells in their individual tion, the lysates were underlaid with 1 ml 30%
colonies grown on YPD medium. sucrose pads made in buffer A and centrifuged in a
The transfer of [PSI+] in `cytoduction' experi- Beckman SW50 rotor at 45 000 r.p.m. at 4uC for
ments was performed as described (Ter-Avanesyan 30 min. The resulting supernatants, pellets and
et al., 1994; Kochneva-Pervukhova et al., 1998). intermediate fractions were analysed by Western
The recipient strain in such crosses was [psix] blotting. To obtain sedimented material containing
[rhox], while the donor was [rho+]. In addition, Sup35pPSI+, the lysates of 5V-H19 [PSI+] strain
one of the crossed strains carries the kar1-1 were loaded onto a sucrose layer (1 ml, 30%) and
mutation that blocks karyogamy (Conde and Fink, centrifuged at 200 000rg for 30 min at 4uC. The
1976). The strains were mixed together on the sedimented material was resuspended in buffer A
surface of a YPD plate, incubated overnight, and for further use in conversion reactions, performed
then replica-plated to appropriate selective medium as described previously (Paushkin et al., 1997a). A
containing glycerol as the sole carbon source. ratio of Sup35pPSI+ to Sup35NMppsix in mixtures
Respiratory-competent ([rho+]) colonies of the was 1 : 4 in all cases. Protein samples were separated
Copyright # 2001 John Wiley & Sons, Ltd. Yeast 2001; 18: 489±497.492 N. V. Kochneva-Pervukhova et al.
on a 10±15% SDS±polyacrylamide gel, according to c10B-H49 [psix], using the `cytoduction' procedure
Laemmli (1970), and electrophoretically transferred (see Materials and methods). The crosses of all
to nitrocellulose sheets (Towbin et al., 1979). tested transformants carrying plasmids with differ-
Western blots were probed with polyclonal rabbit ent 3k-deletion SUP35 alleles produced Ade+
anti-Sup35p antibody or antibody against the cytoductants, which were shown to be [PSI+] by
Sup35N2p fragment and developed using the the GuHCl test. These [PSI+] clones differed by
Amersham ECL system. Estimation of relative their suppressor ef®ciency and could be roughly
amount of soluble Sup35p in lysates of different divided into two classes: `strong', with strong
[PSI+] isolates was performed as described by Zhou suppressor phenotype (white on YPD medium,
et al. (1999), with minor modi®cations. and able to grow on adenine omission medium
after 2 days of incubation) and `weak', with weak
suppressor phenotype (pink on YPD medium, and
Results
growing on adenine-free medium on the third or
fourth day of incubation). Remarkably, the relative
Factors in¯uencing the spectrum of [PSI+]
proportion of the weak and strong [PSI+]
strains generated de novo
depended on the Sup35p variant used for their
The overexpression of Sup35p or its N-terminal generation. The increased ef®ciency of [PSI+]
part induces the de novo appearance of the [PSI+] induction by overexpression of short Sup35p was
determinant (Chernoff et al., 1993; Derkatch et al., mainly due to the appearance of weak [PSI+]
1996). This procedure generates various forms (Table 1).
(`strains') of [PSI+] determinant with different
nonsense suppressor ef®ciency and mitotic stability
Strain-speci®c variation of [PSI+] properties
(Derkatch et al. 1996). The C-terminal truncation of
Sup35p greatly increases the frequency of [PSI+] While we roughly divided the [PSI+] strains into
induction (Kochneva-Pervukhova et al., 1998). `strong' and `weak' groups, it was of interest to
Here, we observed that such Sup35p truncation study in more details their distribution by suppres-
also in¯uences properties of the generated [PSI+] sor ef®ciency. For this, [PSI+] induced in the strain
determinants. For the [PSI+] generation, the multi- 1±5V-H19 by overexpression of Sup35NMp were
copy plasmids encoding different C-terminally transferred by cytoduction to the strain 1B-H67,
truncated Sup35p variants were expressed in the bearing the suppressor-assaying plasmid pUKC817
strain 1±5V-H19 [psix] [PIN+] encoding Sup35Cp, (Stans®eld et al., 1995). Cytoductants were selected
which lacks the prionogenic N-terminal region and on the glycerol medium supplemented with cyclo-
thus cannot convert into the prion form. The use of heximide and checked for the ability to grow on
this strain provides two advantages over the wild- adenine omission medium. Ade+ clones were taken
type strain for the quantitative analysis of the for determination of nonsense codon readthrough.
[PSI+] generation. First, the [PSI+] induction in As a reference, the 1B-H67 [psix] recipient tester
transformants of the 1-5V-H19 strain should be strain carrying the pUKC815 plasmid was used. It
solely de®ned by the prion properties of the should be noted that the strain 1B-H67 carries the
plasmid-encoded C-terminally truncated Sup35p SUQ5 ochre suppressor, which increases the level of
variant. Second, this strain allows avoidance of UAA suppression. Among the 51 cytoductants
counterselection of the appearing [PSI+] cells tested, the ef®ciency of UAA suppression ranged
because, in contrast to the strains wild-type for from 5% to 65% (Figure 2). The distribution of
SUP35, the combination of [PSI+] with inducing [PSI+] by suppressor ef®ciency was normal, rather
plasmids is not detrimental in the strains than bimodal. This argues against the existence of
carrying the SUP35-C allele (Dagkesamanskaya only two strains, but is consistent with high number
and Ter-Avanesyan, 1991; Paushkin et al., 1997b). of strains.
The [PSI+] phenotype is not manifested in the The [PSI+] isolates obtained also differed by
strain 1±5V-H19 due to the presence of non-prion their mitotic stability. [PSI+] isolates with strong
Sup35Cp (Ter-Avanesyan et al., 1994). To monitor suppressor phenotype were 100% stable during
the [PSI+] generation, we transferred cytoplasm growth on YPD medium, while [PSI+] isolates
from the 1±5V-H19 transformants to a tester strain, with weak suppression segregated [psix] subclones
Copyright # 2001 John Wiley & Sons, Ltd. Yeast 2001; 18: 489±497.Analysis of [PSI+] generation de novo 493
Table 1. The frequency and properties of [PSI+] generated de novo depend on the structure of plasmid SUP35
alleles
Total number Number of `Strong' [PSI+]
Inducing of [rho+] Number of [PSI+] `strong' [PSI+] cytoductants `Strong' [PSI+] among all [rho+]
allele cytoductants cytoductants [PSI+] (%) (%) cytoductants (%)
sup35-N1 2700 1068 39 39.6t6.9 3.7t0.5 1.4t0.2
sup35-N2 1980 260 11 13.1t0.9 4.2t1.9 0.56t0.29
sup35-NM 12000 65 13 0.54t0.13 20.0t0.8 0.11t0.02
sup35-DS 45000 202 63 0.45t0.24 31.2t1.4 0.14t0.07
SUP35 40800 89 53 0.22t0.03 59.6t4.1 0.13t0.03
The frequency and spectrum of [PSI+] induced in the strain 1±5V-H19 by multicopy plasmids carrying indicated SUP35 alleles were estimated as
described in Results. In each case, where it was possible, 100 respiratory-competent Ade+ cytoductants were examined by the GuHCl test. All
tested Ade+ cytoductants appeared due to [PSI+] acquisition. The [PSI+] induction data represent averages from three independent
transformants. The standard deviation is indicated.
with a frequency of494 N. V. Kochneva-Pervukhova et al.
Table 2. Mitotic stability of different [PSI+] strain
Type of [PSI+] [PSI+] isolate Number of clones Number of [psix] clones % of [psix] clones
43 806 0 0
Strong 51 1419 0 0
77 1034 0 0
45 994 0 0
53 917 4 0.4t0.1
Weak 55 798 37 4.6t0.3
67 826 2 0.2t0.1
79 405 2 0.5t0.1
Percentage of [PSI+] loss was calculated as described in Results. Data represent averages from three independent subclones of each [PSI+] isolate.
The standard deviation is indicated.
(Zhou et al., 1999). This is likely to be below the Discussion
physiological range, since Sup35p is essential. By
our data, the decrease of Sup35p below 5% of its The protein-only concept for prions presumes that
wild-type levels causes growth inhibition and cell the differences between their strains can only result
death (I. A. Valouev, unpublished). It is noteworthy from conformational variations of prion protein.
that the experiments described above were per- Indeed, the structural difference of PrPSc was
formed with yeast cultures in stationary phase of demonstrated for different strains of mammalian
growth, which could be an important factor prion (Caughey et al., 1998; Safar et al., 1998).
increasing the Sup35p aggregation. Thus, the phenomenon of prion strains reveals the
The use of cells harvested in the stationary phase ability of prion proteins to exist not in just two, but
was due to observation that this allows distinguish- in multiple alternative self-propagating conforma-
ing more clearly [PSI+] and [psix] cells by Sup35p tions. Such an ability is interesting in itself, but it
aggregation than in the exponential phase. There- also gives an important insight into the debate
fore, the experiment was reproduced with the cells about the models for prion conversion: the exis-
harvested in exponential phase, which gave remark- tence of strains is more compatible with variants of
ably different results: the soluble Sup35p of the the nucleated polymerization model than with the
strain with `strong' [PSI+] constituted 6% and with heterodimer model. Indeed, it appears unlikely that
`weak' [PSI+] 25% of its amount in the [psix] strain many different prion conformations can stably exist
(Figure 3). It should be noted that the [psix] and self-reproduce as monomers. In contrast, in the
reference level of Sup35p in this case was about polymerization model the alternative conformations
three-fold higher than in stationary-phase cells. would be stabilized by intermolecular interactions
The Sup35p from `weak' and `strong' [PSI+] and reproduced along the length of the prion
strains was also distinguished by its ability to seed polymer. The strain variation was observed for
the prion conversion of Sup35ppsix in vitro. Lysate both the conventional [PSI+] of S. cerevisiae and
of [psix] transformant of the strain 5V-H19 with arti®cial [PSI+] based on N-terminal domain of
multicopy plasmid encoding Sup35NMp was mixed Sup35p from yeast P. methanolica (Derkatch et al.,
with sedimented material obtained from the lysates 1996; Kushnirov et al., 2000). This supports the
of 5V-H19 carrying either `strong' or `weak' [PSI+]. idea that the ability to exist in multiple forms is a
general property of prions, rather than a speci®c
In both cases Sup35NMp was convertible to an
trait of the mammalian prions. However, the
aggregated (Figure 4) and protease-resistant (data
evidence for the existence of yeast prion strains is
not shown) form, but the amount of converted not as strong as for mammalian prions, for which it
material was about two-fold lower when the was shown that their strain-speci®c differences are
reaction was seeded by the lysate of cells with preserved upon transmission from one animal to
`weak' [PSI+]. No Sup35NMp aggregation was another. In this paper, we reinforce the evidence for
observed in a control experiment without addition the [PSI+] strain variation being related solely to
of the Sup35pPSI+ seeds. Sup35p by showing that genetic properties of
Copyright # 2001 John Wiley & Sons, Ltd. Yeast 2001; 18: 489±497.Analysis of [PSI+] generation de novo 495
Figure 4. Conversion of Sup35NMppsix to an aggregated
form caused by mixing with aggregated Sup35p isolated from
cells with `weak' and `strong' [PSI+]. Western blots were
probed with antibody against Sup35N2p. Experiment: lysate
of [psix] cells expressing Sup35NMp were mixed with [PSI+]
sedimented material containing Sup35p, incubated for 20 min
Figure 3. [PSI+]-dependent Sup35p aggregation. `Weak' and and analysed as described. Control: analysis of Sup35NMp in
`strong' [PSI+] were transferred by cytoduction from the [psix] lysate after 2 h of incubation. Cytosol, sucrose and
strain c10B-H49 to the strain 5V-H19 [psix] and the levels pellet: supernatant, intermediate fraction and sedimented
of soluble Sup35p in corresponding cytoductants were material obtained after centrifugation of lysates and mixes
compared with that of Sup35p in the strain 5V-H19 [psix].
A. Distribution of Sup35p between the soluble and
Sup35p properties, rather than to any additional
aggregated forms in lysates of cells harvested at stationary mutations in other genes. The extent of Sup35p
phase of growth. Cytosol, sucrose and pellet: supernatant, aggregation should be proportional to the rate of its
intermediate fraction and sedimented material, obtained prion conversion, and indeed Sup35pPSI+ seeds
after centrifugation of lysates. Blots were immunostained from `strong' strains caused more ef®cient prion
for Sup35p. B. Semi-quantitative dot±blot analysis of soluble conversion of Sup35ppsi± in vitro. It is also interest-
Sup35p in lysates of different [PSI+] isolates. Lysates were ing that the level of soluble Sup35p greatly
prepared from cells harvested at stationary or exponential depended on the growth phase of [PSI+] cells,
phase of growth. The total protein from soluble fraction of being much lower at the stationary phase. In the
each lysate was adjusted to 0.66 mg/ml, serially diluted in `strong' [PSI+] cells, this level constituted, in
four-fold increments and applied to nitrocellulose mem-
comparison to [psix] cells, 6% at the exponential
brane. Blots were probed with antibody af®nity puri®ed
against the Sup35N2p fragment. Equal levels of the total
phase and only 1% at stationary phase. This may be
protein in dilutions were con®rmed by staining the same explained by a decreased rate of synthesis of soluble
membranes by Ponceau S, a non-speci®c protein stain (not Sup35p in stationary cells. On the other hand, the
shown) demand for functional Sup35p in stationary cells is
also likely to be decreased.
[PSI+] determinants independently isolated in one Genetic properties allowed us to roughly divide
yeast strain are reproducible in another yeast strain. [PSI+] into two classes: `strong', mitotically stable
We observed that the suppressor ef®ciency of [PSI+] determinants with strong suppressor pheno-
[PSI+] strains correlated with the extent of Sup35p type, and `weak', mitotically unstable [PSI+] with
aggregation. Similar observations were made earlier weak suppressor phenotype. Quantitative analysis
for conventional and `arti®cial' [PSI+] strains of the suppressor ef®ciency of independently
(Zhou et al., 1999; Kushnirov et al., 2000). Such a obtained [PSI+] has shown that they represent a
correlation may be anticipated, since the stop codon heterogeneous group and may differ from each
readthrough should be inversely related to the levels other more than 10-fold. The [PSI+] distribution by
of soluble Sup35p. This observation indicates that suppressor ef®ciency suggested that the number of
the [PSI+] phenotype variation is related to the [PSI+] strains is greater than two and may be
Copyright # 2001 John Wiley & Sons, Ltd. Yeast 2001; 18: 489±497.496 N. V. Kochneva-Pervukhova et al.
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