(Received for publication, July 7, 1995)
From the
Calmodulin and its target enzymes are important regulators of
numerous cellular processes, including reversible protein
phosphorylation. The calmodulin-dependent protein phosphatase
(calcineurin) has been suggested to play roles in activation of T cells
and in the mating response of yeast. Recently, studies have shown it to
be the target of immunosuppressant drugs such as cyclosporin and
FK-506. In this study, we have cloned the gene for the catalytic
subunit of calcineurin, CnA, from the yeast Schizosaccharomyces
pombe. The gene (named ppb1) has been
mapped to chromosome II by analysis of the hybridization of a genomic
DNA probe to an ordered library. The gene produces a single mRNA
species of 2.5 kilobases, which varies during the cell cycle in
exponentially growing cells. In addition, expression of ppb1
mRA is induced by nitrogen starvation, a
condition that favors mating in S. pombe. The ppb1
gene promoter contains a cis-acting
element for the ste11 transcription factor, and we have shown
that induction of the ppb1
mRNA during
nitrogen starvation is dependent on the ste11 gene product.
Together with earlier studies showing that disruption of the ppb1
gene in S. pombe results in
sterility (Yoshida, T., Toda, T., and Yanagida, M.(1994) J. Cell
Sci., 107, 1725-1735), our studies suggest that the ppb1
gene plays a role in the gene expression
cascade that is essential for mating and sporulation in S.
pombe.
Through the calcium-binding protein calmodulin (CaM), ()a large number of cellular processes are
regulated(1) . Presently, more than 30 different enzymes have
been shown to be regulated in a Ca
/CaM-dependent
manner, making CaM an important mediator of intracellular signal
transduction. Among the targets for CaM are enzymes involved in
glycogen metabolism, cyclic nucleotide metabolism, several protein
kinases, and at least one protein phosphatase.
Reversible protein
phosphorylation is recognized as a fundamental regulatory mechanism in
cells. The CaM-dependent protein phosphatase, calcineurin, has recently
been suggested to play important roles in the control of cell growth
and division(2, 3) , regulation of gene
expression(4, 5) , and in response to mating pheromone
in the yeast Saccharomyces cerevisiae(6, 7) .
The holoenzyme is composed of two subunits, the catalytic subunit
(CnA), and a Ca-binding regulatory subunit (CnB)
structurally related to CaM(8, 9, 10) . While
a great deal is known about calcineurin enzymology, it is only recently
that inroads have been made into understanding specific roles of the
enzyme in vivo.
Recently, it has been shown that calcineurin is a target for immunosuppressant drugs such as FK-506 and cyclosporin(11) . In T cells, it appears to function in the activation of the transcriptional regulator NFAT(4, 5) , a step requisite for T cell activation and inhibitable by immunosuppressants. In Aspergillus nidulans, a filamentous fungus related to yeast, we have shown that the CnA gene is essential for proliferation and that the mRNA is expressed in a cell cycle-dependent manner(2) . In the yeast S. cerevisiae, multiple CnA genes exist, and while it appears they are not essential for vegetative growth, this enzyme appears to play a role in the mating pheromone response pathway(6, 7) . Recently, a CnA homologue was isolated from Schizosaccharomyces pombe and gene disruption suggested that it is not strictly required for vegetative growth, although growth was temperature sensitive in a strain lacking the CnA gene(12) . However, it was found that the lack of a CnA gene caused a sterile phenotype, suggesting that calcineurin is involved in the mating response in S. pombe.
In this study, we have
also cloned the S. pombe CnA gene (designated ppb1). The gene we have isolated is identical
to that observed in the previous study(12) . Hybridization of a ppb1
probe to an ordered phage library
allowed us to determine the location of the ppb1
gene in the S. pombe genome. In addition, we have
examined the expression of ppb1
mRNA. We
observed a moderate change during the cell cycle in exponentially
growing cells. However, when cells are grown in nitrogen-free medium or
allowed to reach saturation, CnA mRNA levels are markedly induced. The S. pombe CnA gene promoter contains a cis-acting element (TR
element) previously shown to be responsive to the ste11
transcription factor(13) . Our
studies show that CnA expression during nitrogen starvation is directly
dependent on ste11
and suggests that CnA may
be an integral component of the signal transduction mechanism that
functions during the mating response in yeast.
For isolation of genomic
DNA, S. pombe cultures containing 3-5 10
cells per ml were used for each isolation. Genomic DNA was
isolated as described previously(14) .
Small-scale preparation of plasmid and cosmid DNA was performed by the alkaline lysis method as described previously using Wizard DNA Clean-Up resin (Promega)(15) . For larger scale preparations, alkaline lysis followed by precipitation with polyethylene glycol/NaCl was used(15) .
For radiolabeling of DNA hybridization probes,
200 ng of DNA was labeled with [P]dCTP
(Amersham) by the random primer method(15) . Labeled DNA was
separated from unincorporated nucleotides by G-50 Sephadex
chromatography.
For analysis of isolated DNA by Southern blot,
standard procedures were used(15) . DNA was transferred to
Magna Nylon transfer membrane by capillary blotting overnight using 20
SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0)
as the transfer buffer. After transfer, filters were air dried for 30
min, and the DNA cross-linked to the membrane by exposure to UV light
(1200 J/cm
) using a Stratalinker 2400 (Stratagene).
Pre-hybridization and hybridization were as used
previously(2) .
For Northern blot analysis, previously
described methods were used(2) . After transfer to Magna Nylon
membrane, the RNA was cross-linked to the membrane using UV light (1200
J/cm) using a Stratagene Stratalinker 2400.
For
slot-blot analysis, 10 µg of RNA in sterile water was mixed with an
equal volume of SSCF (3 volumes of 20 SSC, 2 volumes of 37%
formaldehyde) and denatured at 68 °C for 15 min. The sample was
chilled in ice, and an equal volume of 15
SSC was added.
Blotting onto Magna Nylon transfer membrane was performed according to
manufacturer's specifications using a Mini-fold slot blot
apparatus (Tyler Research). For quantitation of ppb1
, mRNA 2-fold serial dilutions of total
RNA samples were prepared, applied to transfer membrane, and hybridized
as described above. The autoradiogram was scanned using an Apple Color
One scanner set for 256 levels of gray and 300 dpi resolution. The
hybridization signals were quantified using the program NIH Image
(version 1.55). Determinations of relative mRNA levels were based on
regions of the serial dilution series that showed a corresponding
2-fold difference in the calculated intensity, indicating that the
exposure was in the linear response range of the x-ray film.
Following cross-linking, filters were prehybridized for 2 h at 42
°C in 50% deionized formamide, 5 Denhardt`s, 6
SSC, 0.5% SDS, 100 µg/ml denatured herring sperm DNA
solution(2) . Hybridization was carried out at the same
temperature for 18 h in a fresh solution of the same composition as
that used for prehybridization. Probe concentration was 5
10
cpm/ml. After hybridization, filters were washed five
times in 2
SSC, 0.1% SDS at room temperature, and twice for 15
min in 0.1
SSC, 0.1% SDS at 55 °C. After washing, filters
were covered with Saran Wrap and exposed to Kodak X-OMAT AR at
-70 °C using intensifying screens.
DNA sequencing the
dideoxy-mediated chain termination method was applied as described (18) using a Sequenase version 2.0 kit (U. S. Biochemical
Corp.) and S-dATP (Amersham). Following electrophoresis,
the gel was fixed for 10 min in 10% methanol, 10% glacial acetic acid
and dried under the vacuum at 80 °C for 45 min. The dried gel was
exposed to Kodak X-OMAT AR film at room temperature overnight.
Previous studies have suggested that CnA plays important roles in eukaryotic cells, being required for T cell activation and essential for cell division in A. nidulans(2, 4) . We were therefore interested in cloning the CnA gene of S. pombe as a prelude to studies examining the role of this enzyme in yeast development. To obtain a hybridization probe for screening genomic DNA libraries, we designed oligonucleotides to use in PCR amplification of a portion of an S. pombe CnA homologue (as described under ``Experimental Procedures''). Following PCR amplification, a fragment of the expected size was obtained, which was then subcloned and sequenced. The sequence obtained encoded a predicted peptide 75% identical to human and A. nidulans CnA, indicating that we had obtained a portion of the S. pombe gene by PCR.
The PCR-derived clone was then
used as a probe to screen an ordered P1 phage library (obtained from
Dr. Elmar Meier, (Imperial Cancer Research Fund), London)(16) .
Hybridization of the probe to the filter containing P1 phage DNA
resulted in 10 positive signals (Fig. 1). The location of the
signals were mapped by a computer-based analysis system at ICRF and a
cosmid identified based on the P1 phage hybridization pattern. The ppb1 gene was localized to cosmid 32h9, which
maps to chromosome II, between cdc10 and top2 on the S. pombe genetic map(16) . Southern blot analysis of
genomic DNA using the PCR probe showed that a 2.1-kb EcoRI
fragment contained at least part of the CnA gene and that a hybridizing
species of identical size was contained in the 32h9 cosmid. This 2.1-kb EcoRI fragment was subcloned and used to further map the
cosmid. Our mapping results showed that the ppb1
gene was entirely contained within two adjacent HindIII
fragments in the 32h9 cosmid (Fig. 2). The sequence of the ppb1
gene from -867 through the 1st
exon is shown in Fig. 3.
Figure 1: Screening an S. pombe-ordered P1 phage library for a calcineurin A homologue. A P1 phage library was screened with the PCR-derived probe as described under ``Experimental Procedures.'' 12 positive hybridizations were obtained, and this information was sent to Dr. Elmar Maier at ICRF. Based on this hybridization pattern, the cosmid 32h9 of their ordered library was determined to contain a calcineurin A homologue.
Figure 2:
Restriction mapping of the ppb1 gene. Genomic DNA (Gen) and DNA
isolated from the cosmid 32h9 (Cos) were analyzed by Southern
blot as described under ``Experimental Procedures.'' The
samples were digested with either EcoRI (E) or HindIII (H). The DNA was then blotted to nylon
filters and hybridized to the PCR-derived CnA probe. The sizes of the
hybridizing fragments (in kb) are indicated and were identical in both
the cosmid and genomic DNA samples.
Figure 3:
Sequence of 867 bp of 5`-flanking region
of the S. pombe ppb1 gene and the first exon
including the predicted translation start site. The putative ste11 binding site is shown by the underlined italic type,
starting at -137 bp relative to the initiation methionine codon.
The gene that we have cloned is identical to that isolated
previously(12) .
While this work was in progress, we
were in contact with Prof. M Yanagida (Kyoto University) who informed
us that they had also cloned a CnA homologue from S.
pombe(12) . Comparison of our sequence with theirs showed
perfect agreement, indicating that we had cloned the same gene. In that
study, it was found that disruption of the CnA gene in S. pombe was not lethal, as we have also observed (data not shown).
However, other interesting phenotypes were observed. Overexpression
caused a variety of cytological defects in interphase cells, while a ppb1 null strain was sterile, suggesting a
defect in the mating response pathway(12) . Because of these
effects, we were interested in determining whether expression of the ppb1
gene was associated with either cell
cycle progression or the mating response in S. pombe.
To
examine the levels of ppb1 mRNA during the
cell cycle, cells were synchronized using the temperature-sensitive
strain cdc25-22, which arrests cells in G
,
followed by release to permissive conditions and sampling at 20-min
intervals thereafter. Total RNA was prepared, and the levels of ppb1
mRNA were examined by Northern blot
analysis. In addition, the percentage of binucleate cells was monitored
to assess the degree of synchrony and the position of S phase, which
occurs coincident with the maximum of binucleated cells(14) .
The results show that ppb1
mRNA levels vary
slightly during the cell cycle with maximum levels observed conincident
with each S phase (Fig. 4).
Figure 4:
Expression of ppb1 mRNA during the cell cycle in exponentially growing S.
pombe. The levels of S. pombe ppb1
mRNA
were examined during the cell cycle in synchronized exponentially
growing cells. Cells were synchronized by block/release of a cdc25
strain, and total RNA WAS prepared for
Northern blot analysis as described under ``Experimental
Procedures.'' A, to ensure progression was synchronous
and to identify the position of S phase, the percentage of binucleate
cells was determined. Cells were stained with
4`,6`-diamidino-2-phenylindole dihydrochloride, and the percentage of
binucleated cells was determined by microscopic examination of the
sample. For each time point, at least 300 cells were scored. B, Northern blot analysis of total RNA from synchronously
progressing cells. Each lane corresponds to the time points
shown on the graph in A. A single mRNA species of 2.5 kb was
observed.
Next, we tested whether ppb1 mRNA levels vary during changes in the
growth conditions of cells. Since previous studies suggested that ppb1
might be important in responses to
mating pheromone in S. cerevisiae(6, 7) and
that S. pombe lacking calcineurin is sterile (12) , we
were interested in determining if ppb1
mRNA
expression could be induced by conditions that favor transition to the
sexual cycle. Parallel cultures of exponentially growing cells were
shifted to nitrogen-free medium, glucose-free medium, or allowed to
grow to saturation. RNA was isolated, and ppb1
mRNA levels were determined by slot-blot hybridization of
serially diluted total RNA samples. The results indicate that ppb1
mRNA levels are significantly increased
when cells are deprived of nitrogen or allowed to grow to saturation as
compared to histone H2A mRNA (Fig. 5). Scanning densitometry was
used to quantify CnA mRNA levels using these serially diluted RNA
samples. The results indicate that ppb1
mRNA
levels are increased 8-fold in response to nitrogen starvation.
Figure 5:
Expression of ppb1 mRNA under various growth conditions. Wild-type cells were grown
under various conditions, and ppb1
mRNA
levels were examined by slot-blot analysis (panel marked ppb1
). Cells were either grown exponentially (Exp), allowed to reach saturation (Sat), or cultured
for 18 h in minimal medium limited in either nitrogen (-Nit) or glucose (-Glu). As a control,
the same blot was stripped and reprobed with a histone H2A probe (panel marked Histone
H2A).
Examination of the time course of induction showed that levels
increase as soon as 2 h after shifting to nitrogen-free medium, with
maximal levels of expression observed within 8 h after the shift (Fig. 6A). Previous studies have shown that the ste11 transcription factor regulates the
expression of some genes in response to nitrogen starvation. The
expression of ste11
is inhibited by cAMP via
activation of cAMP-dependent protein kinase(13) . Regulation of ste11-dependent genes occurs via a cis-acting element
(TR element) in the 5`-untranslated region of these genes(13) .
Inspection of the ppb1
5`-upstream sequence
revealed a potential TR element starting at position -137
relative to the initiation ATG codon (the TR consensus is TTCTTTGTTY).
The putative element in the ppb1
gene
promoter matches at 9/10 positions and contains a conserved G residue
at position 7 previously shown to be essential for ste11 binding to the element(13) .
Figure 6:
ppb1mRNA expression is dependent on the ste11 transcription factor in S. pombe. A,
exponentially grown S. pombe were shifted to nitrogen-free
medium as described under ``Experimental Procedures'' for the
times indicated. Total RNA was isolated, and cna1 mRNA levels
were determined by slot-blot analysis. ppb1
mRNA rises within 2 h after shifting to nitrogen-free medium and
reaches peak levels by 8 h. The peak level was maintained until 25 h. B, to determine if cAMP affected induction of ppb1
mRNA expression during nitrogen
starvation, exponentially grown cells were treated with (cAMP/Caf.) or
without (Control) cAMP + caffeine as described under
``Experimental Procedures'' and then shifted to nitrogen-free
medium (-N) or allowed to continue exponential growth (Exp). Total RNA was isolated, and ppb1
mRNA levels were determined by slot-blot hybridization. C, to determine if ste11 expression is required for ppb1
induction during nitrogen starvation, ppb1
mRNA levels were examined in a ste11
strain cultured under identical
conditions to the control strain shown in A. The results show
that ppb1
expression during nitrogen
starvation is dependent on ste11.
To determine if the
induction of ppb1 expression during nitrogen
starvation is dependent on ste11
, we examined ppb1
mRNA levels under two different sets of
conditions. First, ppb1
mRNA levels were
examined in cells cultured in the presence or absence of cAMP +
caffeine. This treatment raises intracellular cAMP levels, which would
be expected to inhibit ste11
expression and
in turn the expression of ste11
-dependent
genes. The results show that cells treated in this way fail to increase ppb1
mRNA levels upon nitrogen starvation,
suggesting that ppb1
gene expression requires
expression of the ste11 transcription factor (Fig. 6B). Next, we examined ppb1
mRNA levels in a ste11
null strain.
Using the same experimental protocol as above, we observed that ppb1
mRNA levels did not increase in the ste11
strain upon nitrogen starvation,
suggesting that the TR element present in the ppb1
gene promoter is functional and regulates ppb1
expression in vivo (Fig. 6C).
Because these data suggest that ste11 regulates ppb1
expression, we decided to directly test whether the TR element
present in the ppb1
gene promoter was
functional in vivo or if ppb1
expression was indirectly regulated by ste11. Two
different reporter constructs were constructed as follows. Fragments
with 5`-end points either at -157 bp or -127 bp relative to
the start of translation and identical 3`-end points(-1) were
synthesized by PCR. Each fragment was first subcloned into
pGEM3Zf(+) and sequenced to ensure no polymerase-induced mutations
had been introduced. The first fragment (-157) contains the TR
element, while the second(-127) starts just after the TR element.
Each fragment was then ligated 5` to the E. coli lacZ gene,
which encodes
-galactosidase. These plasmids also contain an ars sequence, to permit replication in S. pombe, and
the S. cerevisiae URA3 gene, to permit selection of cells
containing the reporter plasmid.
Each of the reporter constructs was
transformed into S. pombe, and ura prototrophs were selected. Cells were then streaked onto filters
overlaid on plates containing normal growth medium (EMM) or medium that
induces mating (malt extract agar). The filters were then removed, and
the presence of
-galactosidase was assayed as described under
``Experimental Procedures.'' As can be seen (Fig. 7),
both constructs direct the expression of low levels of
-galactosidase when cells are grown on EMM. On medium that induces
mating, the -157 construct produced higher levels of
-galactosidase, as indicated by the darker staining. This result
is consistent with the previously observed induction of ppb1
mRNA expression upon nitrogen
starvation. In contrast, the levels of
-galactosidase in cells
containing the -127 construct were unchanged. These data directly
demonstrate that the TR element in the ppb1
promoter is functional in vivo and that the ppb1
gene is directly regulated by the ste11 transcription factor during the mating response.
Figure 7:
The ste11 element present in the ppb1 gene is functional in vivo.
Cells carrying a
-galactosidase reporter construct whose
expression is dependent on fragments, which contain(-157) or
lack(-127) the putative ste11 element, were assayed for
-galactosidase activity as described under ``Experimental
Procedures.'' Activity is shown on a per cell basis and was
determined in growing cells (EMM) and in cells grown on medium
that induces the mating response (MEA (malt extract agar)). As
can be seen, malt extract agar medium induces increased expression in
the construct that contains the TR element(-157) but not in the
one lacking this element(-127), showing that expression of the ppb1
gene during the mating response is
dependent on the ste11 transcription
factor.
In this study, we have cloned the S. pombe homologue
of the calmodulin-dependent protein phosphatase catalytic subunit
(calcineurin A) by screening an ordered P1 phage library with a genomic
fragment produced by PCR. Mapping of P1 phage containing the ppb1 gene allowed mapping of the ppb1
gene to chromosome II between the cdc10 and top1 genes. In the earlier study, the ppb1
gene was said to be localized to
chromosome I, 200 kb away from the sts1
gene(12) . The reason for the discrepancy with our mapping is
unclear. Our sequence exactly matches that of the one from Prof.
Yanagida's group(12) , so it is not another calcineurin
gene, and the cosmid that contains our ppb1
gene was obtained from ICRF after they had interpreted the P1
phage hybridization pattern. Thus, it would seem that one of the
ordered libraries is not correct.
The ppb1 gene product is highly conserved relative to other calcineurin
homologues, being over 70% identical to S.
cerevisiae(6) , human(19) , and Neurospora
crassa(20) calcineurin homologues within the conserved
catalytic domain. Unlike S. cerevisiae, we have no evidence of
other CnA homologues in S. pombe based on low stringency
hybridization or during our original PCR reactions, where we
consistently obtained a single product during amplification when using
degenerate oligonucleotides. However, ppb1
gene disruption was shown not to be lethal (12) . (
)This might suggest that there are redundant CnA genes in S. pombe or that this protein phosphatase is not essential for
vegetative growth. This would be in contrast to our previous studies in A. nidulans, where we observed that CnA is an essential
gene(2) , but consistent with studies in the yeast S.
cerevisiae(6) .
Examination of ppb1 mRNA levels during the cell cycle showed moderate change, similar
to what has been observed in A. nidulans, where CnA mRNA
levels increased prior to S phase(2) . Based on this, it is not
clear why overexpression of ppb1
would have
such drastic cytological effects as previously reported(12) .
We have observed that expression of a truncated, CaM-independent form
of mouse CnA in S. pombe has no effects on cells. It is
possible, therefore, that overexpression of the full-length protein
might have other effects due to binding to CaM or other target
proteins. In the previous study, the authors apparently used the normal
version of the thiamine-regulated nmt1 promoter present in the
plasmid pREP1, and so it is possible that the exceptionally high levels
of expression obtained using this promoter might have nonspecific
effects. Further, more detailed studies should be able to resolve this
issue.
We have found that the ppb1 mRNA is
induced in response to nitrogen starvation, which is a primary signal
in regulating the mating response in yeast(21) . Several genes
are known to be induced by nitrogen starvation, of which a subset has
been shown to be dependent on the ste11 transcription factor,
itself a nitrogen starvation-induced
gene(13, 22, 23, 24) . The ste11 gene product regulates expression by a 10-bp cis-acting element
(TTCTTTGTTY) known as a TR element. Inspection of the ppb1
gene promoter revealed that it contains
a putative ste11 regulatory element, matching at 9/10
positions. Our observation that nitrogen starvation does not induce ppb1
mRNA expression in a strain lacking a
functional ste11 gene supports the conclusion that the ppb1
gene does contain a functional TR
element and is regulated during nitrogen starvation by the ste11 transcription factor. Lack of this element results in a loss of
the nitrogen starvation-dependent increase in expression, clearly
showing that nitrogen starvation-induced ppb1
expression is directly dependent on the ste11 transcription factor.
Significantly, a protein phosphatase in S. cerevisiae homologous to the vaccinia virus VH-1 gene
product and S. pombe cdc25 protein phosphatase, has also been
shown to be induced by nitrogen starvation(25) . Calcineurin
levels also increase in response to treatment of S. cerevisiae cells with the -factor-mating pheromone(7) . The
precise role either for the VH-1 phosphatase homologue or the
calcineurin gene in the S. pombe mating response is unknown.
It has been shown that mating and sporulation are inhibited by at least
two protein kinases. The S. pombe cAMP-dependent protein
kinase (the pka1 gene of S. pombe) has been
previously shown to inhibit ste11 gene expression, in turn
inhibiting the expression of ste11-dependent genes required
for mating(13) . A second protein kinase, pat1/ran1, also inhibits mating in S.
pombe(26) . In this case the effect is mediated via the mei2 gene, which is indispensable for the mating response.
Thus, the role of protein phosphatases may be to antagonize protein
kinases, which might otherwise inhibit the mating response. It will be
important to determine if substrates either for pka1 or pat1 kinases are dephosphorylated by calcineurin.
In S.
cerevisiae, it has been suggested that CnA antagonizes the mating
pheromone response pathway. This conclusion is based on the
demonstration that cells lacking the cna1 and cna2 genes are unable to recover from -factor-induced
arrest(6) . This apparent disparity in roles for CnA in the two
yeast may be a consequence of the different modes of mating. In S.
pombe, mating is induced by starvation, while in S. cerevisiae it occurs in rich medium(21, 27) . In addition,
the expression of the CnA homologues has not been examined in S.
cerevisiae, so it is unknown if CnA expression increases during
the mating response as we have shown in S. pombe.
It is possible that the role of CnA is as part of a gene expression cascade involved in regulating the expression of genes specific to the sexual cycle. It has been shown in T cells that CnA activity is required for T cell activation(4) . In this system, the role of CnA is to dephosphorylate the factor NFAT, which is an upstream regulator of interleukin gene expression(4, 5) . It is possible, therefore, that CnA is a regulator of gene expression, downstream of ste11, and responsible for controlling other genes essential for the mating response.
Consistent with this is the observation
that ppb1 mRNA levels remain elevated during
nitrogen starvation, maintaining peak levels 24 h after the switch to
nitrogen-poor media. Not all nitrogen starvation-induced genes have
this response. The res2
gene is induced
within 6 h by nitrogen starvation, but levels decline by 12 h (23) . In addition, ppb1
mRNA is
increased in haploid cells, suggesting that CnA may have a role early
in the mating response pathway, prior to formation of the zygote.
Finally, it has been shown that the loss of the calcineurin gene causes
sterility in S. pombe(12) . This suggests that
calcineurin plays an essential role in the sexual cycle of this
organism. The observation that ppb1
mRNA
levels are induced by nitrogen starvation and remain elevated for a
prolonged period suggests that CnA may be required throughout mating,
meiosis, and sporulation. Future studies will examine the specific role
of the ppb1
gene in each of these processes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) YSPPPB1.