From the Frontier Project 3, Proteome Research
Laboratory, Daiichi Pharmaceutical Company, Ltd., 519 Shimo-Ishibashi, Ishibashi-machi, Shimotsuga-gun, Tochigi 329-0512, Japan and § Graduate School of Pharmaceutical Sciences,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
Received for publication, November 7, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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Transcription elongation factor S-II stimulates
cleavage of nascent transcripts generated by RNA polymerase II stalled
at transcription arrest sites. In vitro experiments have
shown that this action promotes RNA polymerase II to read through these
transcription arrest sites. This S-II-mediated cleavage is thought to
be necessary, but not sufficient, to promote read-through in the
in vitro systems. Therefore,
Saccharomyces cerevisiae strains expressing S-II mutant proteins with different in vitro activities were used to
study both the cleavage and the read-through stimulation activities of
S-II to determine which S-II functions are responsible for its biologic
functions. Strains expressing mutant S-II proteins active in both
cleavage and read-through stimulation were as resistant as wild type
strains to 6-azauracil and mycophenolic acid. 6-Azauracil also induced
IMD2 gene expression in both these mutant strains and the
wild type. Furthermore, strains having a genotype consisting of one of
these S-II mutations and the spt4 null mutation grew as
well as the spt4 null mutant at 37 °C, a restrictive
temperature for a strain bearing double null mutations of
spt4 and S-II. In contrast, strains bearing S-II mutations
defective in both cleavage and read-through stimulation had phenotypes
similar to those of an S-II null mutant. However, one strain expressing
a mutant S-II protein active only in cleavage stimulation had a
phenotype similar to that of the wild type strain. These results
suggest that cleavage, but not read-through, stimulation activity is
responsible for all three biologic functions of S-II (i.e.
suppression of 6-azauracil sensitivity, induction of the
IMD2 gene, and suppression of temperature sensitivity of
spt4 null mutant).
Transcription elongation factor S-II, originally purified from
mouse Ehrlich ascites tumor cells, is an RNA polymerase II-stimulating factor in promoter-independent RNA synthesis (1). It has been found in
all eukaryotes thus far investigated, and its primary structure is
highly conserved (2-7). S-II is a unique transcription elongation
factor that promotes transcript elongation through transcription arrest
sites found in genes (8-11). The molecular mechanism of this
phenomenon has been investigated extensively in in vitro
systems, and it has been shown that S-II stimulates the nuclease
activity of RNA polymerase II, which then cleaves the nascent
transcript. Then the 3'-end of the nascent RNA is realigned with the
catalytic site of RNA polymerase II, and the transcription elongation
complex tries reading through the arrest site again (11-14). The
cleavage stimulation activity of S-II can be separated from its
read-through stimulating activity; although the cleavage stimulation
activity is essential, it is not sufficient to promote read-through of
RNA polymerase II in vitro (15).
There have been several reports describing the function of S-II in
Saccharomyces cerevisiae. In several yeast strains bear null
mutations of the genes encoding the transcription elongation machinery,
such as spt4 In this study, the two S-II activities were investigated in an effort
to determine which are responsible for the biologic functions, such as
drug resistance and transcription induction, in S. cerevisiae. Site-directed mutations were introduced into the S-II
gene to generate mutant yeast strains that express S-II mutant proteins
with different levels of cleavage and read-through stimulation
activities in vitro. These strains were then tested to
determine their sensitivity to 6-AU and MPA, their ability to induce
the IMD2 gene in response to 6-AU treatment, and their temperature-sensitive growth retardation in combination with the spt4 null mutation. The results show that
cleavage-stimulating, but not read-through-stimulating, activity
correlates with the biologic functions of S-II, suggesting that the
former activity of S-II is responsible for its functions in
vivo.
S. cerevisiae Mutant Strains--
All yeast strains used in this
study are summarized in Table I. HKY01
and HKY02 are S-II null mutant strains (courtesy of Dr. T. Ito) derived
from YPH499 (24); S-II gene was disrupted by URA3 or
HIS3 gene introduction in these strains, respectively. Plasmid pH1180 was constructed by inserting the 3.4-kb PvuII
fragment of the S. cerevisiae S-II gene into pBluescript
KS(+) (Stratagene, La Jolla, CA). To screen for mutant clones, silent
base-exchange mutations were introduced to form the following new
restriction enzyme recognition sites in each mutant: KasI
sites for Mt1 and Mt2; SacII sites for Mt3, Mt4, Mt8, and
Mt9; an SphI site for Mt5; a BspT104I site for
Mt6; and a PvuI site for Mt7. pMt1b was constructed by
inserting the PstI-NcoI fragment of the
S-II gene amplified by PCR with the primers
5'-AAACTGCAGGATCTGGCGCCAGCGCCCTTAGCTGCAAAGATAGAAG-3' and
5'-GGGGCCAGTTGTTCAGGTCCTAACTGTATGCC-3' (TU-23) into the
PstI-NcoI site of pH1180. pMt1 was constructed by
inserting the PstI-KasI fragment of the S-II gene
amplified by PCR with the primers
5'-CACAGTGTAGTCAGTCCGCATAAGAGCATTCATCATGG-3' (TU-22) and
5'-TAAGGGCGCTGGCGCCAGATCCTTGGCATCG-3' into the
PstI-KasI site of pMt1b. pMt2b was constructed by
inserting the PstI-NcoI fragment of the S-II gene
amplified by PCR with the primers TU-22 and
5'-CATGCCATGGTGGCGCCAGCTGCGGCGTATAAGGCTTGCTTGGC-3' into the PstI-NcoI site of pH1180. pMt2 was constructed by
inserting the KasI-NcoI fragment of the S-II gene
amplified by PCR with the primers 5'-CAACGCACAGGGCGCCACCATAGAAAGG-3'
and TU-23 into the KasI-NcoI site of pMt2b. pMt3b
was constructed by inserting the MfeI-NcoI
fragment of the S-II gene amplified by PCR with the primers
5'-CTATCAATTGCAAACACAATCCGCGGATTTGCCATTGACCAC-3' and TU-23 into the
MfeI-NcoI site of pYSII-2 (2). pMt3 was
constructed by inserting the PstI-NcoI fragment
of pYSII-2 to the PstI-NcoI site of pH1180. pMt4b
was constructed by inserting the PstI-NcoI fragment of the S-II gene amplified by PCR with the primers
5'-AAACTGCAGATCCGCGGATGAACCATTGACCACTGCTTGTACATGTG-3' and TU-23 into
the PstI-NcoI site of pH1180. pMt4 was
constructed by inserting the PstI-SacII fragment
of the S-II gene amplified by PCR with the primers TU-22 and
5'-GGTTCATCCGCGGATCTTGTTTGCAATTG-3' into the
PstI-SacII site of pMt4b. pMt5b was constructed
by inserting the PstI-NcoI fragment of the S-II
gene amplified by PCR with the primers
5'-AAACTGCAGAAGCATGCGGTAACAGATGGGCTTTCTCTTAGAATAG-3' and TU-23
into the PstI-NcoI site of pH1180. pMt5 was
constructed by inserting the PstI-SphI fragment
of the S-II gene amplified by PCR with the primers TU-22 and
5'-CATCTGTTACCGCATGCTTCACATGTACAG-3' into the
PstI-SphI site of pMt5. pMt6 to -9 were obtained
by using a QuikChange site-directed mutagenesis kit (Stratagene) and
pH1180 as a template. Primers used for mutants were
5'-GCCAGGTATGCTATAATTTATTCGAACGTCATATC-3' and
5'-GATATGACGTTCGAATAAATTATAGCATACCTGGC-3' for pMt6,
5'-CAGTCACCGATCGAGCTACATGTGGTAAATG-3' and
5'-CATTTACCACATGTAGCTCGATCGGTGACTG-3' for pMt7,
5'-CAATTGCAAACACAATCCGCGGATAATCCATTGACC-3' and
5'-GGTCAATGGATTATCCGCGGATTGTGTTTGCAATTG-3' for pMt8, and
5'-CAATTGCAAACACAATCCGCGGATGAACC-3' and
5'-GGTTCATCCGCGGATTGTGTTTGCAATTG-3' for pMt9. After the presence of the mutations on the plasmid clones was confirmed by DNA sequencing, the BamHI-PvuI (pMt2) or PvuII (all
other constructs) fragments were excised, and an EZ Yeast
Transformation Kit (Qbiogene, Inc., Carlsbad, CA) was used to introduce
the fragments separately into HKY01 to create the S-II mutant strains
designated Mt1 to Mt9. When the mutant S-II fragment is introduced by
homologous recombination into the S-II locus, it loses the
URA3 gene instead and is transformed to 5-fluoroorotic
acid-resistant. Therefore, we isolated 5-fluoroorotic acid-resistant HKY01 transformants and performed genomic PCR to screen for the desired mutants with the primers
5'-CACTCGATGATGGGACTACG-3' and 5'-GCGCTAGTAAGACAGATAGG-3'
followed by restriction enzyme digestion. The introduction of the
mutant sequences into the S-II locus was confirmed by Southern blot
analysis. S-II genes were amplified from each S-II mutant strain, and
sites of mutation were confirmed by DNA sequencing. Each mutant strain
bore only the introduced mutation.
A PCR-based gene disruption method (25) was used to prepare all
spt4 Anti-yeast S-II Antibodies--
Recombinant yeast wild type S-II
protein was expressed in Escherichia coli and purified as
described elsewhere (2). Anti-yeast S-II antiserum was prepared at
Asahi Techno Glass Corp. (Chiba, Japan) by injecting a New Zealand
White rabbit with 0.3 mg of the purified recombinant yeast S-II
protein. Anti-yeast S-II antibodies were affinity-purified from the
antiserum as previously described (26).
Western Blot Analysis--
YPH499 was transformed with pRS416
(Stratagene), designated TU100, and used as the wild type strain for
this experiment. Mutant yeast strains Mt1 to Mt9 were also transformed
with pRS416. The resulting nine S-II mutant strains, TU100, and an S-II
mull mutant strain HKY01 were separately grown in SD (ura Indirect Immunofluorescence--
The same yeast strains used in
the Western blot analysis were separately incubated in SD (ura Drug Sensitivity Assay--
The same yeast strains used in the
Western blot analysis were separately incubated in SD (ura Northern Blot Analysis--
The same yeast strains used in the
Western blot analysis were separately incubated in SD (ura Temperature Sensitivity Assay--
TU100 was transformed with
pRS413 (Stratagene) to create TU101. HKY02 was transformed with pRS416
to create TU102. TU101 was used as the wild type strain and TU102 was
used as dst1 Other Methods--
DNA manipulation was performed as described
elsewhere (28). The method of Bradford (29) was used to determine
protein concentration; bovine serum albumin was used as a standard.
Construction of S-II Mutant Strains--
The S. cerevisiae S-II protein consists of three domains: domains I, II,
and III. Domains II and III are joined by a short linker region (30).
Awrey et al. (31) constructed a variety of the S. cerevisiae S-II mutant proteins that span domains II and III. They
then determined the in vitro cleavage and
read-through-stimulating activities of these mutant S-II proteins with
an assay that used a human histone H3.3 gene fragment, which contains
well characterized transcriptional arrest sites, as the template (31).
To see whether both cleavage and read-through stimulating activities of
S-II were responsible for its biologic functions, nine of these mutant S-II proteins, designated Mt1 to Mt9, were selected for further tests.
Results from the in vitro assays conducted by Awrey et al. (31) show that four of the mutant proteins had strong cleavage stimulation but different read-through-stimulating activities (Mt1, -4, -5, and -7); four others (Mt2, -6, -8, and -9) had moderate cleavage
stimulation but different read-through-stimulating activities, and Mt3
had little of either activity (Table II).
Mt6 has a mutation in domain II, which binds to RNA polymerase II. Mt1,
-2, and -7 have mutations in the linker region, and the other mutant
proteins have mutations in domain III, which is essential for
transcript cleavage and read-through-stimulating activities as
well as 6-AU sensitivity suppression (31, 32). These mutations were
introduced into the S-II locus of S. cerevisiae by
homologous recombination.
First, Western blot analysis was used to determine the amount of mutant
S-II proteins expressed, and indirect immunofluorescence was used to
determine the cellular localization of the mutant S-II proteins; both
methods used anti-yeast S-II specific antibodies. Results of the
Western blot analysis show that all S-II mutant strains have expression
patterns similar to that of the wild type strain (Fig.
1). In addition to a major band of 42 kDa, which was as large as the recombinant S-II, a faint band (35 kDa)
was observed in all strains. Since this signal was not detected in the
S-II null mutant HKY01 (Fig. 1), it is thought that the smaller band
represents a degradation product of S-II. Results of preliminary studies suggest that the smaller form lacks the carboxyl terminus region2 that is essential for
RNA polymerase II stimulation, read-through stimulation in
vitro, and 6-AU sensitivity suppression in vivo (32).
The band intensity of the Mt4 S-II protein is reproducibly weaker than
the S-II proteins from the other mutant strains (Fig. 1). These data
suggest that, compared with the S-II proteins from the other mutant
strains, the amount of S-II protein expressed by the Mt4 strain was
lower, or the antigenicity of Mt4 S-II protein is weaker. However, it
should be noted that results presented below indicate that the amount
of Mt4 S-II protein expressed was apparently sufficient to perform its
biologic functions (Table II).
The results of indirect immunofluorescence experiments to locate S-II
are shown in Fig. 2. As shown in Fig.
2A, anti-S-II staining of the wild type strain TU100 showed
strong fluorescence that overlapped with nuclear staining, as
previously reported (33). In contrast, no fluorescence was observed in
S-II null mutant strain HKY01 stained with anti-S-II (Fig.
2B) or in wild type (Fig. 2B) and Mt1 to Mt9
(data not shown) cells stained with the same concentration (1.7 µg/ml) of preimmune rabbit IgG. The staining patterns of Mt1 to Mt9
with anti-S-II were indistinguishable from that of the wild type (Fig.
2C) and overlapped with nuclear staining (data not shown).
These results indicate that all of the mutant S-II proteins are
localized in the nucleus as is the wild type S-II protein.
Drug Sensitivity in S-II Mutant Strains--
The S. cerevisiae S-II null mutant is known to be sensitive to 6-AU (2)
and MPA (20). To investigate the biologic effects of S-II mutant
proteins, the sensitivity of each S-II mutant strain to 6-AU and MPA
was tested. Fig. 3 shows that mutant
strains Mt1, -2, -4, -5, -6, -7, and -9 are as resistant to 6-AU as the
wild type is; in contrast, Mt3 and Mt8 are as sensitive to 6-AU as is
the S-II null mutant. Interestingly, despite its inability to promote
read-through in vitro (Table II), Mt5 is as resistant to
6-AU as is the wild type. The sensitivities of mutant strains Mt1 to
Mt9 to 25 µg/ml MPA correlate well with the sensitivities to 6-AU
(Fig. 3). Additionally, introduction of a centromeric plasmid that
contained the S. cerevisiae S-II gene complemented the drug
sensitivities of Mt3 and Mt8 (data not shown). These results suggest
that the read-through-stimulating activity of S-II is not needed for
the suppression of 6-AU and MPA sensitivity.
IMD2 Gene Induction in S-II Mutant Strains--
The
IMD2 gene encodes IMP dehydrogenase, a key enzyme in the
de novo synthesis of guanine nucleotides (34). The activity of IMP dehydrogenase is inhibited by MPA and by 6-azauridine
monophosphate, an active metabolite of 6-AU (20).
IMD2 gene transcription can be induced by the addition of
6-AU or MPA to the culture medium (22), and this induction depends on
S-II. This induction is thought to be important for the development of
6-AU and MPA resistance. Therefore, the ability of 6-AU to induce
IMD2 gene transcription in the S-II mutant strains was
tested. The wild type TU100 strain, the S-II null mutant HKY01 strain,
and the mutant strains Mt1 to Mt9 transformed with pRS416 were
incubated in a synthetic medium in the presence or absence of 75 µg/ml 6-AU for up to 2 h, and Northern blot analysis was used to
determine the amount of IMD2 mRNA produced by each
strain. As shown in Fig. 4, mutant
strains Mt1, -2, -4, -5, -6, -7, and -9 induced IMD2 gene
expression as well as the wild type strain did, whereas mutant strains
Mt3 and Mt8 were as transcriptionally inactive as the S-II null mutant. These data illustrate that the biologic ability to induce the IMD2 gene transcription correlates positively with the
in vitro cleavage stimulation activity exhibited by the S-II
mutant proteins.
Temperature Sensitivity of the Yeast Strains Bearing S-II Mutations
and spt4 Null Mutation--
SPT4 gene encodes a subunit of
the transcription elongation factor DSIF (DRB
sensitivity-inducing factor) (35).
A null mutant of the DST1 gene, which encodes S. cerevisiae S-II, shows temperature sensitivity when combined with
the spt4 null mutation, whereas the spt4 null
mutant is not temperature-sensitive (17). To see whether mutant S-II
proteins can suppress the temperature-sensitive phenotype of the
dst1 At a transcriptional arrest site in a gene, S-II stimulates the
nuclease activity of RNA polymerase II into cleaving several bases from
the nascent transcript and causes the realignment of the 3' end of the
nascent transcript with the active site of the polymerase, and the
transcription elongation complex tries to read through the arrest site
again (11-14). Results from previous studies have shown that this
cleavage stimulation activity of S-II is necessary, but not sufficient,
to promote read-through in vitro (15). To clarify this
observation, S. cerevisiae S-II mutant strains were used to
investigate the roles of the cleavage and read-through stimulation
activities of S-II in vivo. A 6-AU sensitivity assay (Fig.
3), RNA analysis for IMD2 induction by 6-AU (Fig. 4), and
rescue from temperature-dependent growth inhibition of
separate strains carrying one of the S-II mutations and an spt4 null mutation (Fig. 5) were used to assess S-II
function. The results show that S-II mutant proteins that were active
in both cleavage stimulation and read-through in vitro also
suppressed 6-AU sensitivity, induced IMD2 gene expression,
and suppressed dst1 The results of the present study also imply the presence of some as yet
unidentified transcriptional read-through factors. Since the mutant
S-II protein Mt5, which lacks read-through activity in
vitro, exhibited full biologic activity, it is possible that once
the nascent transcript is cleaved by RNA polymerase II, other read-through factors might promote read-through in place of a read-through-incompetent S-II protein in Mt5 strain. Awrey et al. (31) suggested that a conformational change in the ternary elongation complex (RNA polymerase II-DNA template-nascent transcript) is required for read-through after the cleavage of the nascent transcript. It has been suggested that S-II and Rpb9 influence the
conformational change necessary for read-through (31). Thus, Rpb9 is a
good candidate for a read-through factor other than S-II. Mutant yeast
strains sensitive to 6-AU that bear the Mt5 mutation may be good tools
for identifying this putative read-through factor. Another possible
approach would be to use an in vitro read-through assay
system containing the S-II Mt5 protein to isolate read-through-promoting factors biochemically. These studies, which are
currently under way, are the next logical steps in understanding the
regulation of transcription elongation by RNA polymerase II in
eukaryotic cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, rpb9
, and ctk1
,
S-II is indispensable for cell proliferation under certain culture
conditions (16-18). Although Kulish et al. (19) reported
that S-II promotes read-through of RNA polymerase II in vivo
as well as in vitro, it remains to be elucidated whether
S-II functions in vivo through its cleavage-stimulating activity, its read-through-stimulating activity, or both. S-II also
plays a role in the development of
6-AU1 or MPA resistance in
yeast (2, 20) and in the expression of the pyrimidine-specific
5'-nucleotidase SDT1 gene; it is also involved in the
induction of the IMP dehydrogenase IMD2 gene and the
polyamine transporter TPO1 gene in response to treatment
with 6-AU or MPA (21-23). Stimulation of expression of these genes is thought to be necessary for developing 6-AU and MPA resistance in
S. cerevisiae.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in this study
strains. PCR with the plasmid pRS416 (Stratagene) as
a template was used to prepare the DNA fragment containing the URA3 gene used to disrupt the SPT4
gene. The primers used were
5'-ATTCATTACTATTATACATGTGATATCAGAACGGAAGGTTAGATTGTACTGAGAGTGCAC-3' and
5'-TTACACCTGGCCACATTCAGTTTGGCAAAAGCGAACGAGGCTGTGCGGTATTTCACACCG-3'. The resulting fragment was separately introduced to YPH499,
HKY02, and Mt1 to -9 using an EZ Yeast Transformation Kit.
URA+ transformants were selected on SD (ura
)
agar plates (0.67% (w/v) yeast nitrogen base without amino acids; 2%
(w/v) glucose; 0.1 mg/ml each histidine, adenine sulfate, tryptophan,
and lysine; and 0.25 mg/ml leucine; 2% bactoagar), and Southern blot
analysis was used to confirm gene disruption.
) medium at
30 °C until they reached midlog phase growth, and then cells were
collected by centrifugation. A CellLytic Y solution (Sigma) containing
1 mM phenylmethylsulfonyl fluoride, 5 mM
benzamidine, 10 µg/ml leupeptin, and 2 µg/ml pepstatin A was used
to lyse each strain separately. Fifty micrograms of protein from the
crude lysate was loaded in each lane of a SDS-polyacrylamide gel and
then blotted to a Hybond-P membrane (Amersham Biosciences). The blot
was incubated with affinity-purified anti-yeast S-II antibodies at
4 °C for 16 h and then with anti-rabbit IgG (donkey) conjugated
to horseradish peroxidase (Amersham Biosciences) at 20 °C for 1 h. ECL Plus (Amersham Biosciences) was used for detection. The
Kaleidoscope Prestained Standard protein ladder (Bio-Rad) was used to
estimate molecular weights.
)
medium at 30 °C until they reached midlog phase growth, and then
0.18 volume of formaldehyde and 0.13 volume of 1 M
potassium acetate buffer (pH 6.5) were added to the medium. Each
culture was incubated at 23 °C for 1 h. Cells were then
collected by centrifugation and resuspended in 0.1 M
potassium acetate buffer (pH 6.5) containing 5% formaldehyde and
incubated at 23 °C for 1 h. The cells were then collected by
centrifugation and washed once with SHA buffer (1 M
sorbitol, 0.1 M HEPES-Na, pH 7.2, and 5 mM
NaN3) and resuspended in SHA. Cells were then treated with
Zymolyase 100T (Seikagaku Corp., Tokyo, Japan) and transferred onto
ADCELL slides (Erie Scientific Co.; Portsmouth, NH). Cells were fixed
in methanol at
20 °C for 6 min and then in acetone at
20 °C
for 30 s. Fixed cells were incubated with affinity-purified
anti-yeast S-II antibodies or preimmune rabbit IgG followed by
incubation with anti-rabbit IgG (goat) conjugated to the Alexa-488
fluorescent dye (Molecular Probes, Inc., Eugene, OR) for detection. The
nuclei were stained with 4',6-diamidino-2-phenylindole.
) medium at
30 °C for overnight and then diluted with fresh medium to
A600 = 0.0425. The diluted cultures were
incubated until the A600 reached 0.17 and then
diluted 10-, 100-, 1000-, or 10000-fold, and 7 µl of each diluted
culture was spotted onto SD (ura
) plates containing 75 µg/ml 6-AU,
25 µg/ml MPA, or no drug. The plates were incubated at 30 °C for 4 days.
) medium at
30 °C until midlog phase, and then 6-AU to give 75 µg/ml was
added. After 0, 0.5, or 2 h, cells were collected by
centrifugation, and a hot phenol method (27) was used to extract total
RNA. Fifteen micrograms of total RNA was loaded onto each lane of an
agarose gel containing formaldehyde for Northern blot analysis. The
probes were the PCR products amplified from yeast genomic DNA
with 5'-GTGGTATGTTGGCCGGTACTACCG-3' and 5'-TCAGTTATGTAAACGCTTTTCGTA-3'
as primers for IMD2 (22) and 5'-TGATAACGGTTCTGGTATGTG-3' and
5'-TAGTCAGTCAAATCTCTACCG-3' as primers for actin. The AlkPhos
Direct Labeling System with CDP-Star (Amersham Biosciences) was used to
label the probes with alkaline phosphatase, and the probes were
hybridized with the blots at 55 °C for 16 h. The blots were
exposed to x-ray films, and the developed films were scanned with a
GS-700 calibrated densitometer (Bio-Rad) to quantify the amount of
transcript. The signal of the actin mRNA in each sample was used to
normalize the band intensity, and the IMD2 signal of the
TU100 following a 2-h induction by 6-AU was used as a relative
measurement unit.
in this experiment. Yeast strains
spt4
and Mt1spt4
to Mt9spt4
were transformed with pRS413. These strains and dst1
spt4
were separately incubated in SC (ura
his
) medium (0.67% (w/v)
yeast nitrogen base without amino acids, 2% (w/v) glucose, and 0.06%
(w/v) complete supplement mixture (
his,
leu,
ura,
trp) from
Qbiogene Inc., 0.1 mg/ml each leucine, tryptophan, and lysine, and 0.04 mg/ml adenine sulfate) at 30 °C for 16 h and then diluted to
A600 = 0.085 with YPDA medium (1% (w/v) yeast
extract, 2% (w/v) polypeptone, 2% (w/v) glucose, and 0.04 mg/ml
adenine sulfate). The diluted cultures were incubated until the
A600 reached 0.17 and then diluted 10-, 100-, 1000-, and 10,000-fold; 7 µl of each culture was then spotted onto
YPDA plates. Plates were then incubated at either 30 or 37 °C
(restrictive temperature for dst1
spt4
double null mutant) for 5 days.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Summary of the phenotypes of S-II mutants
, very sensitive or marginal
induction for in vivo activities.
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Fig. 1.
Expression of S-II mutant proteins.
Yeast strains were incubated in SD (ura ) medium at 30 °C until
they reached midlog phase, and then cells were collected, lysed, and
used for Western blot analysis with anti-yeast S-II antibody. Purified
recombinant S-II was used as a positive control. The numbers
represent the S-II mutant strain numbers 1-9. WT, wild type
TU100 strain; KO, S-II null mutant HKY01 strain.
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Fig. 2.
Cellular localization of S-II mutant
proteins. S-II proteins and yeast nuclei were visualized as
described in "Experimental Procedures." DAPI, nuclear
staining with 4',6-diamidino-2-phenylindole. Bars, 10 µm.
A, wild type cells stained with anti-S-II antibody.
B, S-II null mutant cells stained with anti-S-II antibody
and wild type cells stained with preimmune rabbit IgG as negative
controls. C, results for S-II mutant strains Mt1 to Mt9.
Only S-II stainings are shown.
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Fig. 3.
Drug sensitivity of S-II mutant strains.
Yeast strains were spotted onto SD (ura ) plates containing 75 µg/ml
6-AU, 25 µg/ml MPA, or no drug as described under "Experimental
Procedures." The plates were then incubated at 30 °C for 4 days.
WT, wild type TU100 strain; KO, S-II null mutant
HKY01 strain.
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Fig. 4.
IMD2 induction by 6-AU. Yeast strains
were incubated in SD (ura ) medium at 30 °C until they reached
midlog phase growth, and then 6-AU was added to 75 µg/ml to each
culture. After 0, 0.5, or 2 h, cells were collected, and total RNA
was extracted for Northern blot analysis with IMD2 and actin
genes as probes. WT, wild type TU100 strain; KO,
S-II null mutant HKY01 strain. A, Northern blot analysis
results. The numbers represent the S-II mutant strain
numbers 1-9. B, graphic representation of data shown in
A. The signals of the actin mRNA was used to normalize
the band intensities; one mRNA amount unit represents the amount of
IMD2 mRNA present in the wild type strain TU100 after
2 h of induction.
spt4
double null mutant strain, S-II
mutant strains carrying the spt4 null mutation were
constructed and tested for growth at 37 °C. The results are shown in
Fig. 5. The
dst1
spt4
double null mutant strain showed
clear temperature sensitivity. Mt1spt4
,
Mt4spt4
, Mt5spt4
, Mt7spt4
,
and Mt9spt4
grew as well as spt4
at
37 °C, indicating that the Mt1, -4, -5, -7, and -9 S-II mutant
proteins suppress temperature sensitivity as well as does the wild type
S-II protein. However, the read-through activities of these five S-II
mutant proteins vary and do not correlate with their suppression
activities. Mt2spt4
and Mt6spt4
showed
moderate temperature sensitivity, indicating that the suppression activities of Mt2 and Mt6 proteins are weaker than that of the wild
type S-II protein. Mt3spt4
and Mt8spt4
were
temperature-sensitive, showing that the Mt3 and Mt8 proteins lack
suppression activity. These results indicate that the biologic ability
to suppress temperature-restricted growth correlates positively with
the in vitro cleavage stimulation activities of the S-II
mutant proteins.
View larger version (111K):
[in a new window]
Fig. 5.
Temperature sensitivity of spt4
and S-II double mutant strains. Yeast strains were spotted
onto YPDA plates as described under "Experimental Procedures" and
incubated at either 30 or 37 °C for 5 days.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
spt4
temperature-dependent growth inhibition. In contrast, those
S-II mutant proteins inactive in vitro did not exhibit any of these biologic functions. Surprisingly, S-II mutant protein Mt5,
which has strong cleavage stimulation activity but almost no
read-through activity in vitro, performed all biologic
functions. Overall, the cleavage-stimulating activities, but not
read-through stimulating activities of each mutant S-II protein,
corresponded well with their biologic activities. The Mt2, Mt3, Mt6,
and Mt8 S-II mutant proteins performed biologic functions not as well as wild type S-II protein (Table II). But the amounts of Mt2, -3, -6, and -8 proteins expressed were as great as the amount of wild type S-II
(Fig. 1), and all mutant proteins were localized in the nucleus as the
wild type protein was (Fig. 2). Thus, the low biologic activities of
Mt2, -3, -6, and -8 S-II mutant proteins were not caused by
insufficient protein expression or by cellular mislocalization. These
results, summarized in Table II, strongly suggest that the cleavage
stimulation activity of S-II alone is responsible for its biologic
functions, whereas the read-through stimulation activity of S-II is
dispensable. Previously, S-II has been regarded as a transcriptional
read-through factor, and its read-through activity has been assumed to
be important for its biologic functions; however, the results of the
present study do not support this notion. It is possible, however, that
the S-II mutant proteins that are inactive in an in vitro
read-through assay have sufficient read-through activity in the cell.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. T. Ito for HKY01 and HKY02, critical reading of the manuscript, and helpful discussions; Dr. M. Nakanishi-Matsui for critical reading of the manuscript and helpful discussions; and Steven E. Johnson for editing the manuscript.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 81-285-51-2382; Fax: 81-285-51-2226; E-mail: nakanfnt@daiichipharm.co.jp.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M211384200
2 N. Adachi and T. Nakanishi, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: 6-AU, 6-azauracil; MPA, mycophenolic acid.
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