ABSTRACT
The pre-mRNA splicing factor Prp31p was identified in a screen of temperature-sensitive yeast strains for those exhibiting a splicing defect upon
shift to the non-permissive temperature. The wild-type
PRP31
gene was cloned and shown to be essential for cell viability. The
PRP31
gene is predicted to encode a 60 kDa polypeptide. No similarities with other
known splicing factors or motifs indicative of protein-protein or RNA-protein interaction domains are discernible in the predicted amino
acid sequence. A
PRP31
allele bearing a triple repeat of the hemagglutinin epitope has been generated.
The tagged protein is functional
in vivo
and a single polypeptide species of the predicted size was detected by Western
analysis with proteins from yeast cell extracts. Functional Prp31p is required
for the processing of pre-mRNA species both
in vivo
and
in vitro
, indicating that the protein is directly involved in the splicing pathway.
The removal of intervening sequences from precursor messenger RNAs (pre-mRNAs) is a critical processing step in the maturation of transcripts. The catalytic reactions of splicing require association of the substrate
molecule with numerous
trans
-acting factors to form the spliceosome (for reviews see
1
-
4
and references therein). Five small nuclear RNAs (snRNAs), associated into ribonucleoprotein particles
(snRNPs), assemble into a splicing complex with the pre-mRNA. The U1 snRNA base pairs with the 5' splice site (
5
-
7
), resulting in formation of the commitment complex (
8
-
10
). The U2 snRNP then interacts with the branch point sequence in an ATP-dependent fashion to form the pre-spliceosome (
11
-
15
). The U4/U6 and U5 snRNPs associate with this complex as a single, tri-snRNP particle to form the spliceosome (
11
,
16
-
18
). Conformational rearrangements then occur, allowing cleavage at the 5' splice site and, subsequently, cleavage at the 3' splice site and ligation of the two exons to form the mature mRNA
species.
Current models predict that splicing is catalyzed by the snRNAs and experimental
evidence from both yeast and mammals supports this hypothesis (
19
-
23
). However, the splicing machinery requires a large number of protein factors to
accurately function
in vivo
. More than 30 gene products have been identified as essential for the splicing
reaction in
Saccharomyces cerevisiae
(for reviews see
2
,
24
and references therein). A number of these factors have been identified in
screens of temperature-sensitive strains for those exhibiting a splicing defect at the non-permissive temperature. These are referred to as
PRP
genes, for precursor RNA processing (
25
-
29
). Isolation of suppressors of mutant alleles of the
PRP
genes or of mutated splice sites in pre-mRNAs has revealed a number of novel gene products required for splicing
to occur (
30
-
32
). Still other factors have been identified by assaying for mutations in genes
that in combination with a mutation in one of the snRNAs confer synthetic
lethality (
33
,
34
).
In order to identify novel
trans
-acting proteins required for pre-mRNA splicing in
S.cerevisiae,
we isolated temperature-sensitive mutant strains that accumulate pre-mRNA
in vivo
after shift to the non-permissive temperature of 37oC. Six new genes were identified and were designated
PRP29-34
(
35
). In this report we describe the isolation and characterization of the
PRP31
gene. The 60 kDa gene product is essential for vegetative growth in
S.cerevisiae
. Furthermore, we demonstrate specific inactivation of the
in vitro
splicing activity of extracts derived from a
prp31-1
strain, consistent with a direct requirement for Prp31p in processing of pre-mRNA species. Interactions with a variety of known splicing factors were
examined by genetic and biochemical analyses.
Strains used in this study are listed in Table
1
. Standard media and techniques were utilized for growth and manipulation of
yeast and bacteria (
36
). Transformations were performed by the LiAc method of Ito
et al
. (
37
).
Yeast strain JWY771 was transformed with a library of
Sau
IIIA-digested fragments of yeast genomic DNA cloned in YCp50 (
38
). Cells were plated on selective medium and placed at 23oC for 18-20 h. Seventy five percent of the plates were then shifted to 37oC to select for Ts
+
transformants. The remaining plates were kept at 23oC to estimate the total numbers of transformants examined; these
transformants were then picked, patched and replica-plated to check for growth at 37oC. Fragments to be assayed for complementing activity were subcloned
into pRS316 (
39
). For high copy suppression analysis the 3.0 kb
Hin
dIII fragment was subcloned into pRS426 (
41
).
Table 1
For RNA isolation, yeast strains were grown at 23oC to ~0.5 * 10
8
cells/ml; for temperature shift experiments the cultures were then diluted with
an equal volume of fresh medium and maintained at 23oC or shifted to 37oC for 2 h. Cells from a 10 ml culture were harvested by centrifugation
at 3000
g
for 5 min, washed once with 0.5 vol RE buffer (100 mM LiCl, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA) and suspended in 0.4 ml RE buffer. This solution
was transferred to a fresh tube containing an ~2/3 volume of glass beads and vortexed for 4 min at 4oC. Proteins were removed by sequential extractions with 0.3 ml
equilibrated phenol (United States Biochemicals, Cleveland, OH), 0.3 ml phenol-chloroform-isoamyl alcohol (50:49:1) and 0.3 ml chloroform. RNA
was precipitated at -20oC overnight and suspended in 0.1 ml DEPC-treated dH
2
O.
Analysis of pre-mRNA accumulation was by electrophoresis of RNA on 1% agarose-6% formaldehyde gels as described by Moritz
et al.
(
42
). RNA samples were prepared as in Feinberg and Vogelstein (
43
) except that the sample buffer did not contain glycerol. Thirty to forty
micrograms of RNA were loaded for Northern analysis. Gels were dry blotted to
Nytran Plus membrane (Schleicher & Schuell, Keene, NH). Hybridization with radiolabeled probes has been described
(
44
,
45
). A 2.2 kb
Hin
dIII fragment bearing the
CRY1
and
SNR189
genes was utilized as a probe.
Radiolabeled transcripts were generated using the T7 and T3 promoters
(Stratagene, La Jolla, CA) flanking the
Bgl
II-
Sac
I fragment of
PRP31
in pRS316. These probes were hybridized to a blot of poly(A)
+
RNA. Labeling, hybridization and wash conditions were performed as instructed
by the manufacturer.
A blot of chromosomal DNA separated by CHEF was obtained from Clontech
Laboratories Inc. (Palo Alto, CA) and hybridized with the 3.0 kb
Hin
dIII fragment bearing the
PRP31
gene. Hybridization and wash conditions were performed as per Clontech's
instructions.
The
PRP31
gene was sequenced by the dideoxy chain termination method of Sanger (
46
), using Sequenase enzyme (United States Biochemicals, Cleveland, OH) as per the
manufacturer's instructions. Restriction fragments for sequencing were
subcloned into Bluescript pKS
+
and pSK
+
vectors (Stratagene). Synthetic oligonucleotides used as primers were
synthesized by Operon Technologies (Alameda, CA). Sequence analysis and
homology searches were carried out using the Genetics Computer Group's Sequence
Analysis Software Package (access to this program was courtesy of the
Pittsburgh Supercomputing Center).
A 0.9 kb
Bgl
II-
Sac
I fragment containing the
S.cerevisiae TRP1
gene (
47
) was subcloned into the 3.0 kb
Hin
dIII fragment containing
PRP31
in pRS316 modified such that the
Sac
I and
Eco
RI sites within the polylinker were destroyed. This construct was digested with
Sal
I to release a 1.4 kb fragment (Fig.
2
) and transformed into yeast diploid strain LP112. Sporulation and dissection of
transformants yielded only two viable Trp
-
spore clones. The disruption event was confirmed by Southern analysis of
genomic DNA. To confirm that spore inviability was due to disruption of the
PRP31
gene, a
PRP31
/
prp31
::
TRP1
diploid was transformed with either the 3.0 kb
Hin
dIII fragment or the 1.4 kb
Eco
RI-
Sac
I fragment bearing the
PRP31
gene in pRS316. Transformants were then sporulated and tetrads dissected. All
Trp
+
spore clones were also Ura
+
and 5FOA
s
, indicating that the plasmid-borne gene is essential for vegetative growth of these strains.
A
Bgl
II site was introduced downstream of the
Eco
RI site in
PRP31
by oligonucleotide-directed mutagenesis of the 0.6 kb
Hin
dIII-
Eco
RI fragment of
PRP31
in pRS316. A
Bgl
II fragment bearing three tandem repeats of the hemagglutinin epitope (courtesy
of Dr Bruce Futcher) was subcloned into the engineered site. Introduction and
orientation of the epitope was confirmed by restriction enzyme analysis and
sequencing of the construct. The remainder of the
PRP31
gene was then introduced into the tagged fragment by subcloning.
Protein extraction for Western analysis was as described previously (
42
). Samples were loaded onto 3% polyacrylamide (30:0.44) stacking-10% polyacrylamide (30:0.8) running gels. After electrophoresis proteins
were transferred to nitrocellulose. Transfer efficiency and marker protein
migration were determined by staining with Ponceau S solution (Sigma, St Louis,
MO). The blot was then destained with dH
2
O and probed with anti-HA monoclonal antibodies as in Deshmukh
et al.
(
48
). Immunoblots were developed with horseradish peroxidase-conjugated anti-rabbit antibodies using an enzyme chemiluminescence kit (Amersham,
Arlington Heights, IL) as per the manufacturer's instructions.
Cell extracts were prepared according to Lin
et al
. (
49
). Radiolabeled substrate was produced by
in vitro
transcription of a synthetic actin substrate from the SP6 promoter, using [
32
P]UTP at 2 mCi/ml reaction. Splicing assays were conducted at 18oC for 1 h, as described in Lin
et al
. (
49
). Microccocal nuclease digestion of snRNAs was performed as in Tarn
et al.
(
50
). Splicing intermediates and products were separated by denaturing gel
electrophoresis on 6% polyacrylamide (29:1)-8 M urea gels. For biochemical complementation assays equal volumes of
two heat-inactivated
prp
extracts or heat-inactivated
prp
extract and micrococcal nuclease-treated wild-type extract were combined prior to substrate addition. Extracts
were heat inactivated as follows:
prp2-1
, JWY657, 37oC, 30 min;
prp31-1
, JWY2857, 37oC, 30 min; U4
ts
, JWY2419, 42oC, 15 min;
prp24-1,
JWY806, 37oC, 40 min;
prp3-1
, JWY690, 37oC, 40 min.
Strains bearing the
prp31-1
mutant allele are temperature sensitive for growth. In addition, pre-mRNA processing is blocked after a 2 h shift to the non-permissive temperature of 37oC (
35
). RNA was extracted from cells grown to mid-log phase at the permissive temperature of 23oC and then either maintained at 23oC or shifted to 37oC and subjected to Northern analysis using radiolabeled
fragments of the intron-containing
CRY1
and
ACT1
genes as probes. The amount of pre-mRNA was increased and the levels of mRNA were decreased in the strain
initially designated hs29 after a shift to the non-permissive temperature (Fig.
1
, lanes 3 and 4). Subsequent experiments demonstrated that pre-mRNA accumulates in this strain within 30 min after shift (data not
shown).
Hybridization of genomic DNA indicated that the
PRP31
gene is present at single copy in haploid yeast strains, on chromosome XV (data
not shown). An ~1.4 kb message was recognized on a blot of poly(A)
+
RNA upon hybridization with a radiolabeled probe derived from the
Eco
RI-
Sac
I fragment of the
PRP31
gene. The direction of transcription is indicated in Figure
2
.
The
PRP31
gene was determined to be essential by deletion-insertion mutagenesis. The yeast
TRP1
gene was inserted between the
Bgl
II and
Sac
I sites of the
PRP31
gene (Fig.
2
). One wild-type allele of the
PRP31
gene was replaced by this fragment in the wild-type diploid strain LP112. Sporulation of this
PRP31
/
prp31
::
TRP1
diploid strain and tetrad dissection yielded only two viable Trp
-
spore clones in the 35 tetrads examined. This reduced spore viability could be
rescued by introduction of a plasmid-borne copy of the
PRP31
gene prior to sporulation. These data indicate that the
PRP31
gene is required for vegetative growth in
S.cerevisiae
.
Sequence analysis of the complementing DNA revealed a potential open reading
frame (ORF) of 1485 bp. Surprisingly, this ORF extends nine codons upstream of
the second
Sac
I site and 40 codons downstream of the
Eco
RI site. The
Eco
RI-
Sac
I fragment was able to complement the
prp31
::
TRP1
null allele, as well as the
prp31-1
Ts
-
allele (data not shown), indicating that: (i) the C-terminal portion of Prp31p is not required for stability or function of
the protein; (ii) some portion of the N-terminal segment of the protein may not be required or translation may
initiate from a codon other than the first methionine residue. Primer extension
analysis mapped the 5'-ends of the
PRP31
transcripts to nucleotides -60, -65 and -73 5' of the first ATG of the ORF, indicating that this
first ATG is most likely the initiation codon for the wild-type
PRP31
gene (data not shown). Thus complementation by the
Eco
RI-
Sac
I subclone may result from use of a cryptic promoter in the plasmid vector and a
different ATG initiation codon. The inferred amino acid sequence of the
PRP31
gene product does not contain any obvious motifs or homologies to sequences
currently in the GenBank Database (version 8).
The gene is predicted to encode a product of ~60 kDa. The
PRP31
gene was epitope tagged by insertion of a triple hemagglutinin epitope at the 3'-end of the gene, 32 bp downstream of the
Eco
RI site. To determine whether the tagged allele was functional the construct was
subcloned into the
HIS3
-marked plasmid pRS313 and introduced into JWY2964 (
prp31
::
TRP1
[
PRP31
/pRS316]). Transformants that had lost the
URA3
-containing helper plasmid were then selected by plating His
+
transformants on 5-FOA. The Ura
-
His
+
colonies obtained demonstrated no temperature sensitivity or other growth defects. The tagged protein could be detected by Western blot
analysis (Fig.
3
) of cell extracts derived from yeast strains bearing
PRP31-HA
on a CEN-based plasmid. The tagged protein migrates at ~65 kDa, in agreement with the size predicted if translation
initiation occurs at the first methionine residue in the ORF, however, post-translational modification of a smaller gene product, initiating from a
downstream methionine residue, could yield similar results.
Many strains defective in pre-mRNA processing have been shown to be temperature sensitive for splicing
in vitro
as well as
in vivo
. This result is taken as evidence that the mutations in these strains affect a
gene product with a direct role in splicing
per se
, rather than exhibiting an indirect effect due to a defect in transcription,
translation or transport of splicing factors (
52
). In order to determine whether Prp31p is directly required for splicing
extracts were prepared from the
prp31-1
strain JWY2857. These extracts are capable of splicing an
in vitro
transcribed synthetic actin substrate, resulting in formation of the lariat
intron-3' exon intermediate and the lariat intron and mature mRNA products
(Fig.
4
A, lane 1). Upon pre-incubation at increased temperatures prior to substrate addition, the
prp31
extract is no longer able to splice the actin substrate (Fig.
4
A, lane 2). Accumulation of pre-mRNA was observed, with no intermediate species being detected, consistent
with the
in vivo
splicing defect. Heat inactivation of
prp31
extracts is consistent with a direct involvement of Prp31p in the splicing
pathway. Furthermore, since no intermediate species were observed
in vitro
following heat inactivation of the extract or
in vivo
following a temperature shift, the Prp31p protein must play a role prior to the
first cleavage reaction. An additional role in subsequent stages of splicing
cannot be ruled out at this time.
While heat inactivation of extracts derived from a
prp31-1
strain is most likely due to a loss of Prp31p function, the possibility formally
exists that the extract is generally deficient for splicing activity following
incubation at elevated temperatures. To ascertain that the
in vitro
splicing defect is due to specific inactivation of Prp31p, biochemical
complementation assays were performed. Heat-inactivated
prp31
extract was combined with an equal volume of heat-inactivated extract derived from a
prp2-1
strain prior to addition of radiolabeled substrate, splicing was allowed to
proceed and the reactions were analyzed for formation of splicing intermediates
and products. Prp2p is required for the first cleavage reaction of splicing but
is not required for spliceosome assembly (
53
). Combining the two heat-inactivated extracts restored splicing activity (Fig.
4
A, lane 7). Microccocal nuclease treatment of an extract derived from wild-type strain BJ2168 depletes the extract of snRNAs and rendered this
extract unable to splice
in vitro
(Fig.
4
A, lane 6). Extract depleted in this way was able to complement heat-inactivated
prp31
extract (Fig.
4
A, lane 8). This complementation confirms that the (sn)RNA components of the
prp31
extract are still intact upon heat inactivation. Taken together these results
are consistent with a model in which the temperature sensitivity of the
prp31
extract is a result of the specific inactivation of an exchangeable protein
factor directly required for pre-mRNA splicing
in vitro
as well as
in vivo
.
Identification of interactions between gene products, either genetically or
biochemically, can provide information about the potential functions of
splicing factors. To assess potential interactions between Prp31p and other PRP
proteins biochemical complementation assays were performed. Extracts made from
strains bearing mutations in gene products that are components of the same
snRNP particle or are required to interact during splicing may be unable to
complement each other in
in vitro
biochemical complementation assays. These experiments thus provide a mechanism
for identifying potential interactions between components of the splicing
machinery. Heat-inactivated
prp31
extracts were combined with equal volumes of heat-inactivated extracts derived from different
prp
strains, splicing substrate was then added and the reactions were analyzed for
formation of splicing intermediates and products as described above. Strains
were chosen to represent factors present on different snRNP components or that
act at different steps in the assembly and function of the spliceosome.
Extracts were made from
spp2-1
,
prp3-1
,
prp6-1
,
prp8-1
,
prp11-1
,
prp16
,
prp18
,
prp22-1
,
prp24-1
and U4
ts
strains. Efficient spliceosome assembly requires the function of the
PRP3
(J. Anthony, E. M. Weidenhammer and J. L. Woolford Jr, in preparation),
PRP6
(
54
),
PRP8
(
55
),
PRP11
(
56
-
58
) and
PRP24
(
26
) gene products. Mutations in or deletions of any of these genes result in
accumulation of pre-mRNA
in vitro
. Spp2p is required after spliceosome assembly for the first cleavage reaction (
32
), while Prp16p, Prp18p and Prp22p are required at later stages of splicing (
59
-
61
). The strain bearing the U4
ts
mutant allele was identified in the same screen that identified the
prp31-1
strain (
35
). Integration and mapping analyses indicated that the temperature sensitivity
of the strain was due to a mutation in the
SNR14
gene, which encodes U4 snRNA (J. Roy and J. L. Woolford Jr, unpublished
observations). Each of these heat-inactivated extracts was able to complement the
in vitro
splicing defect of a heat-inactivated
prp31
extract. Examples of this complementation are shown in Figure
4
B. Lanes 1-4 show the
in vitro
splicing activity of extracts derived from a
prp31-1
, a
prp3-1
, a
prp24-1
and a U4
ts
strain; in lanes 5-8 the extracts were heat-inactivated prior to substrate addition. Equal volumes of the heat-inactivated extracts were combined, splicing substrate was
added and the reaction mixes were analyzed for formation of intermediates and
products. Lanes 9-11 demonstrate that all combinations of heat-inactivated extracts were capable of complementing the
prp31-1
defect. Similar results were obtained with extracts derived from each of the
other mutant strains.
To further examine potential interactions between the
PRP31
gene and other
PRP
genes high copy suppression analysis was utilized. Suppression of a mutant
phenotype by overexpression of an unlinked gene is indicative of potential
interactions or functional relationships between two gene products (for a
review see
62
). Such interactions have been defined between
PRP3
and
PRP4
(
63
),
PRP2
and
SPP2
(
63
,
32
) and
PRP21
and
PRP9
(
57
). The
PRP31
gene was cloned into the 2 [mu] high copy plasmid vector pRS426. This construct was introduced into
prp 2-prp6
,
prp8
,
prp9
,
prp11
,
prp16
,
prp17
-
prp27
,
prp32
and
prp34
strains and growth of the transformants was monitored at 23, 30, 32, 35 and 37oC. In all cases increased dosage of the
PRP31
gene had no effect on the temperature sensitivity of these strains (data not
shown). In addition, the
PRP18
and
PRP28
genes were introduced into a
prp31-1
strain on a high copy plasmid vector. Again, no suppression of the temperature-sensitive defect was observed.
Synthetic lethality is another indication of interactions between gene products
involved in a common cellular pathway (for a review see
62
). Combinations of mutant alleles of several genes encoding splicing factors
exhibit synthetic lethality, including:
prp3-1
with
prp4-1
(
65
);
prp24-1
with
prp28-1
(
27
); various combinations of
prp5
,
prp9
,
prp11
and
prp21
mutant alleles with each other and with mutant U2 snRNA (
57
,
58
);
mud1
and
mud2
with mutant U1 snRNA (
33
); mutant alleles of the
SLU
genes with mutant U5 snRNA and with alleles of the
PRP16
and
PRP18
genes (
34
). The
prp31-1
allele was tested for synthetic lethality with
spp2-1
,
prp3-1
,
prp4-1
,
prp8-1
,
prp18
, U4
ts
and
prp24
mutant alleles. In all cases double mutant spore clones were obtained,
indicating that the
prp31-1
mutation does not exacerbate the mutant phenotypes of the other genes tested.
Although these analyses with Prp31p have not yet revealed associations of the
protein with other splicing factors, such interactions cannot be ruled out
based on the negative results described here. High copy suppression is likely
to occur in an allele-specific manner. Furthermore, only those mutant proteins that result in
reduced levels of activity may be suppressible. Mutant factors that completely
obstruct progression of the splicing pathway are not likely to be suppressible
by overexpression of interacting factors. Similarly, synthetic lethality and
in vitro
non-complementation of mutant alleles of genes required for splicing is likely
to be observed under conditions in which the mutations affect a domain of the
components required for interaction. More open ended genetic selections for
suppressors of
prp31
alleles or screens for mutations synthetically lethal with
prp31
may yield information about factors with which Prp31p is associated. Isolation
of alleles of the
PRP31
gene bearing mutations in different regions of the sequence may yield
substrates that are differentially affected in protein-protein and/or protein-RNA interactions and so would be useful for further defining the
requirement for this protein in splicing.
Experiments to determine the potential snRNP association of Prp31p, as well as
to define the function of this product in splicing, are in progress.
We are grateful to Dr Craig Peebles and members of our laboratory for critical
comments on this manuscript. This work was supported by Public Health Service
grant GM-38782 from the National Institutes of Health to J.L.W. Computer analyses
accessed through the Pittsburgh Supercomputing Center were funded by grant DMB
890100P.
+
Present address: Department of Medicine, Division of Infectious Diseases,
University of California, San Diego, CA 92093, USA
Strain
Genotype
Source
JWY771
MAT
a
prp31-1 his4-519 ura3-52 lys2 cry2
::
LEU2
65
DBY1034
MAT
a
his4-519 ura3-52 lys2-801
D. Botstein
JWY2861
MAT
a
prp31-1 trp1 ura3-52
This study
LP112
MAT
a
/MAT[alpha]
can1-100/can1-100 his3-11,15/his3-11,15 leu2-3,112/leu2-3,
112 trp1-1/trp1-1 ura3-1/ura3-1 ade2-1/ade2-1
J. Friesen
JWY2964
prp31
::
TRP1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 ade2-1
[PRP31/pRS316]
This study
JWY2857
MAT
a
prp31-1 ade1 leu2-3,112 trp1 ura3-52
This study
JWY657
MAT
a
prp2-1 ura3-52 ade2 His
-
65
JWY630
MAT
a
prp3-1 ade1,2 ura1 tyr1 his7 lys2 gal1
J. Abelson
JWY688
MAT
a
prp6-1 leu2-3,112 lys2 tyr1
65
JWY715
MAT
a
prp8-1 leu2-3,112 his7 Lys
-
65
JWY775
MAT
a
prp16 his4 ura3-52 lys2
[Delta]
trp1
65
JWY803
MAT
a
prp22-1 ade2-101 his3
[Delta]
200 ura3-52 lys2-801
J. Abelson
JWY806
MAT
a
prp24-1 ade2-101 his3
[Delta]
200 ura3-52 lys2-801
J. Abelson
BJ2168
MAT
a
leu2 ura3-52 trp1 prb1-1122 pep4-3 prc1-407 gal2
E. Jones
JWY675
MAT
a
prp3-1 ura3-52 ade1
65
JWY701
MAT
a
prp4-1 ura3-52 his7 ade1
65
JWY732
MAT
a
prp5-1 ura3-52 lys2 his7
65
JWY784
MAT
a
prp6 his4-519 ura3-52 lys2 cry2
::
LEU2
65
JWY666
MAT
a
prp8-1 ura3-52 ade2 his7
65
JWY664
MAT
a
prp9-1 ura3-52 ade2 His
-
65
JWY623
MAT
a
prp11-1 ura3-52 leu2-3,112 trp1 lys2 gal1 tyr1
65
JWY792
MAT
a
prp17 ade2-101 his3
[Delta]
200 ura3-52 lys2-801
J. Abelson
JWY794
MAT
a
prp18 ade2-101 his3
[Delta]
200 ura3-52 lys2-801
65
JWY796
MAT
a
prp19 ade2-1 his3
[Delta]
200 ura3-52 lys2-801 leu2
65
JWY798
MAT
a
prp20 ade2-101 his3
[Delta]
200 ura3-52 tyr1
65
JWY800
MAT
a
prp21 ade2-101 his3
[Delta]
200 ura3-52 tyr1
65
JWY802
MAT
a
prp22 ade2-101 his3[Delta]200 ura3-52 tyr1
65
JWY804
MAT
a
prp23 ade2-101 his3
[Delta]
200 ura3-52 tyr1 lys2-801
65
JWY806
MAT
a
prp24 ade2-101 his3
[Delta]
200 ura3-52 lys2-801
65
JWY808
MAT
a
prp25 ade2-101 his3
[Delta]
200 ura3-52 tyr1
65
JWY810
MAT
a
prp26 ade2-101 his3
[Delta]
200 ura3-52 tyr1
65
JWY812
MAT[alpha]
prp27 ade2-101 his3
[Delta]
200 ura3-52 lys2-801
65
JWY2871
MAT
a
prp31-1 his3 leu2-3,112 lys2 ura3-52
This study
JWY2885
MAT
a
prp32 leu2 lys2 ura3-52 His
-
This study
JWY2419
MAT
a
snr14
ts
lys2-801 ura3-52 leu2-3,112 his4 trp1
[Delta]
67
JWY2402
MAT[alpha]
prp18 ura3-52 lys2-801 his3
67
JWY695
MAT
a
prp4-1 leu2-3,112 lys2 his7 tyr1 ade2
65
JWY40
MAT
a
his3
[Delta]
200 leu2
[Delta]
1 lys2-801 trp1
[Delta]
101 ura3-52 spp2
[Delta]
1
::
LEU2 +
pM2 (spp2-1)
32
SC252
MAT
a
ura3-52 ade1 leu2-3,112
J. Hopper
JWY2862
MAT[alpha]
prp31-1his3 his4-519 lys2 ura3-52
This study
JWY2865
MAT
a
prp31-1 ade1 leu2-3,112 lys2 ura3-52
This study
JWY2866
MAT
a
his4-519 lys2 ura3-52
This study
JWY2867
MAT[alpha]
his3 leu2-3,112 ura3-52
This study
JWY2868
MAT[alpha]
prp31-1 ade1 his3 his4-519 ura3-52
This study
hs87 (2b)
MAT
a
prp34-1 trp1 leu2-3,112 lys2-801 his3
67
JWY721
MAT[alpha]
prp11-1 his7 tyr1 ade2 leu2-3,112
65
REFERENCES
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