Requirement of Saccharomyces cerevisiae Ras for completion of mitosis.

In the yeast Saccharomyces cerevisiae, Ras regulates adenylate cyclase, which is essential for progression through the G1 phase of the cell cycle. However, even when the adenosine 3',5'-monophosphate (cAMP) pathway was bypassed, the double disruption of RAS1 and RAS2 resulted in defects in growth at both low and high temperatures. Furthermore, the simultaneous disruption of RAS1, RAS2, and the RAS-related gene RSR1 was lethal at any temperature. The triple-disrupted cells were arrested late in the mitotic (M) phase, which was accompanied by an accumulation of cells with divided chromosomes and sustained histone H1 kinase activity. The lethality of the triple disruption was suppressed by the multicopies of CDC5, CDC15, DBF2, SPO12, and TEM1, all of which function in the completion of the M phase. Mammalian ras also suppressed the lethality, which suggests that a similar signaling pathway exists in higher eukaryotes. These results demonstrate that S. cerevisiae Ras functions in the completion of the M phase in a manner independent of the Ras-cAMP pathway.

The Rpb4 subunit of fission yeast Schizosaccharomyces pombe RNA polymerase II is essential for cell viability and similar in structure to the corresponding subunits of higher eukaryotes.

Both the gene and the cDNA encoding the Rpb4 subunit of RNA polymerase II were cloned from the fission yeast Schizosaccharomyces pombe. The cDNA sequence indicates that Rpb4 consists of 135 amino acid residues with a molecular weight of 15,362. As in the case of the corresponding subunits from higher eukaryotes such as humans and the plant Arabidopsis thaliana, Rpb4 is smaller than RPB4 from the budding yeast Saccharomyces cerevisiae and lacks several segments, which are present in the S. cerevisiae RPB4 subunit, including the highly charged sequence in the central portion. The RPB4 subunit of S. cerevisiae is not essential for normal cell growth but is required for cell viability under stress conditions. In contrast, S. pombe Rpb4 was found to be essential even under normal growth conditions. The fraction of RNA polymerase II containing RPB4 in exponentially growing cells of S. cerevisiae is about 20%, but S. pombe RNA polymerase II contains the stoichiometric amount of Rpb4 even at the exponential growth phase. In contrast to the RPB4 homologues from higher eukaryotes, however, S. pombe Rpb4 formed stable hybrid heterodimers with S. cerevisiae RPB7, suggesting that S. pombe Rpb4 is similar, in its structure and essential role in cell viability, to the corresponding subunits from higher eukaryotes. However, S. pombe Rpb4 is closer in certain molecular functions to S. cerevisiae RPB4 than the eukaryotic RPB4 homologues.

Subunits of yeast RNA polymerases: structure and function.

Following isolation of the genes encoding the putative subunits of RNA polymerase in both budding and fission yeasts, combined biochemical and genetic studies, together with a structural approach applicable to large assemblies, have begun to reveal the protein-protein interactions not only between RNA polymerase subunits but also between the RNA polymerases and transcription factors. These protein-protein interactions ultimately lead to control of the activity and specificity of the RNA polymerases.

Responsiveness to exogenous cAMP of a Saccharomyces cerevisiae strain conferred by naturally occurring alleles of PDE1 and PDE2.

The Saccharomyces cerevisiae strain P-28-24C, from which cAMP requiring mutants derived, responded to exogenously added cAMP. Upon the addition of cAMP, this strain showed phenotypes shared by mutants with elevated activity of the cAMP pathway. Genetic analysis involving serial crosses of this strain to a strain with another genetic background revealed that the responsiveness to cAMP results from naturally occurring loss-of-function alleles of PDE1 and PDE2, which encode low and high affinity cAMP phosphodiesterases, respectively. In addition, P-28-24C was found to carry a mutation conferring slow growth that lies in CYR1, which encodes adenylate cyclase, and the slow growth phenotype caused by the cyr1 mutation was suppressed by the pde2 mutation. Therefore P-28-24C is fortuitously a pde1 pde2 cyr1 triple mutant. Responsiveness to cAMP conferred by pde mutations suggests that S. cerevisiae cells are permeable to cAMP to some extent and that the apparent absence of effect of exogenously added cAMP on wild-type cells is due to immediate degradation by cAMP phosphodiesterases.

Isolation and characterization of temperature-sensitive mutations in the RAS2 and CYR1 genes of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains two ras homologues, RAS1 and RAS2, whose products have been shown to modulate the activity of adenylate cyclase encoded by the CYR1 gene. To isolate temperature-sensitive mutations in the RAS2 gene, we constructed a plasmid carrying a RAS2 gene whose expression is under the control of the galactose-inducible GAL1 promoter. A ras1 strain transformed with this plasmid was subjected to ethyl methanesulfonate mutagenesis and nystatin enrichment. Screening of approximately 13,000 mutagenized colonies for galactose-dependent growth at a high temperature (37 degrees) yielded six temperature-sensitive ras2 (ras2ts) mutations and one temperature-sensitive cyr1 (cyr1ts) mutation that can be suppressed by overexpression or increased dosage of RAS2. Some ras2ts mutations were shown to be suppressed by an extra copy of CYR1. Therefore increased dosage of either RAS2 or CYR1 can suppress the temperature sensitivity caused by a mutation in the other. ras1 ras2ts and ras1 cyr1ts mutants arrested in the G1 phase of the cell cycle at the restrictive temperature, and showed pleiotropic phenotypes to varying degrees even at a temperature permissive for growth (25 degrees), including slow growth, sporulation on rich media, increased accumulation of glycogen, impaired growth on nonfermentable carbon sources, heat-shock resistance, impaired growth on low concentrations of glucose, and lithium sensitivity. Of these, impaired growth on low concentrations of glucose and sensitivity to lithium are new phenotypes, which have not been reported for mutants defective in the cAMP pathway.

YGE1 is a yeast homologue of Escherichia coli grpE and is required for maintenance of mitochondrial functions.

The grpE gene is a heat shock gene of Escherichia coli whose product functions as a chaperone to (re)fold proteins. We found a yeast homologue of grpE and designated it YGE1. YGE1 can replace grpE in E. coli, indicating that YGE1 is a functional homologue of grpE. Deletion of YGE1 is lethal. During depletion of the Yge1 product, mitochondria are sequestered in mother cells thereby accumulating cells without mitochondria, suggesting that Yge1 protein plays a pivotal role in maintaining mitochondrial functions.

Effect of Alloiococcus otitidis and three pathogens of otitis media in production of interleukin-12 by human monocyte cell line.

Alloiococcus otitidis is detected in middle ear effusion of otitis media with effusion (OME). Only a limited number of studies are available concerning the immunological profile of A. otitidis. We have studied the ability of A. otitidis and three other representative pathogens of otitis media to stimulate the production of interleukin-12 (IL-12) from a monocytic cell line THP-1. Viable A. otitidis induced the production of IL-12 in THP-1 cells but IL-12 production was reduced if glutaraldehyde-fixed bacteria were used as stimulants. When viable bacteria were physically separated from THP-1 cells during the stimulation period, remarkable reductions of IL-12 secretion were shown after challenge with gram-positive bacteria A. otitidis and S. pneumoniae. When stimulated with soluble extracts of A. otitidis, THP-1 secreted IL-12 in a dose-dependent manner. The subfraction with a molecular mass over 100 kDa showed a strong ability to induce IL-12 production. Our results show that A. otitidis has immunostimulatory capacity with regard to IL-12 production. We also show that soluble antigen(s) of A. otitidis can modulate the immune response in OME.

[The pollen survey and dynamic statistics of patients with allergic rhinitis in Hakodate].

We have investigated the pollen survey (1994-1998) and dynamic statistics of patients with allergic rhinitis (1999-2000) in Hakodate, which is located southern part of Hokkaido. We have noted the pollen dispersion of Cryptomeria japonica, Cupressaceae, white birch, Gramineae and Artemisia. Especially, a lot of dispersion of Cryptomeria japonica has been noted in April. Concerning the dynamic statistics of patients with allergic rhinitis, we have investigated the 192 patients with allergic rhinitis in Hakodate municipal hospital. There has been a lot of pollinosis in March, April, May and September. Frequency of positive reaction to the specific IgE have been 38.0% of house dust, 16.9% of Artemisia, 13.2% of Gramineae, 10.3% of white birch, 9.0% of Cryptomeria japonica and 6.9% of cat in 379 subjects. In conclusion, we have noted that Cryptomeria japonica and white birch in addition to Gramineae and Artemisia are becoming more important antigen in patients with pollinosis in Hakodate, south part of Hokkaido.

Increases in cell size at START caused by hyperactivation of the cAMP pathway in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae, passage through START, which commits cells to a new round of cell division, requires growth to a critical size. To examine the effect of hyperactivation of the cAMP pathway on cell size at START, a strain was constructed that is able to respond to exogenously added cAMP. In the presence of cAMP, this strain showed increased cell volume at bud emergence, suggesting that the critical cell size necessary for START is increased. In addition, a mutation that results in unregulated cAMP-dependent protein kinase (bcy1) caused increased cell size at START. These results indicate that hyperactivation of the cAMP pathway causes increases in cell size through cAMP-dependent protein kinase. Cells carrying a hyperactive allele of CLN3 (CLN3-2) also showed increased size at START in the presence of cAMP. These cells retained resistance to alpha factor, however, suggesting that increases in cell size by cAMP are not due to a reduction of Cln3 activity. The observed increases in cell size due to hyperactivation of the cAMP pathway suggest that cell size modulation by nutrient conditions may be associated with a change of the activity of the cAMP pathway.

Functional analysis of RNA polymerase II Rpb3 mutants of the fission yeast Schizosaccharomyces pombe.

The RNA polymerase II (Pol II) of Schizosaccharomyces pombe is composed of 12 subunits. Subunit Rpb3 has sequence homology with the N-terminal domain of the prokaryotic alpha subunit, which plays a key role in RNA polymerase assembly. Together with the Rpb2 (the beta homologue) and Rpb11 (the second alpha homologue) subunits, Rpb3 constitutes a core subassembly (Rpb2-Rpb3-Rpb11) which corresponds to the the alpha2beta assembly intermediate of prokaryotic RNA polymerase. For the functional mapping of Rpb3, we made a collection of 12 heat-sensitive (Ts) or cold-sensitive (Cs) S. pombe mutants, each carrying a single mutation in one of the four conserved regions of Rpb3. The altered functions of six representative Pol II mutants containing the mutant Rpb3 were analyzed in vitro using an improved version of the GAL4-VP16 activator-dependent transcription system catalyzed by S. pombe cell extracts. The transcription activity by the extracts from Rpb3 mutants decreased to varying extents after heat treatment; but the extracts from Rpb3 mutants which had mutations in the eukaryote-specific conserved regions B and C regained their activity by the addition of GAL4-VP16, to a larger extent than those from the region A and D mutants. We propose that both terminal regions (A and D) play important roles in RNA polymerase assembly, while the central portion (regions B and C) is involved in activated transcription.

Mutant farnesyltransferase beta subunit of Saccharomyces cerevisiae that can substitute for geranylgeranyltransferase type I beta subunit.

The protein farnesyltransferase (PFT) beta-subunit gene of Saccharomyces cerevisiae, DPR1, was randomly mutagenized by PCR to construct a mutant DPR1 gene library on a high-copy plasmid. The library was screened for suppression of the temperature sensitivity conferred by a mutation in the protein geranylgeranyltransferase type I (PGGT-I) beta-subunit gene, CAL1. A mutant DPR1 gene was identified whose product contained a single amino acid change of Ser-159 to Asn. This mutant gene also suppressed a cal1 disruption even on a low-copy plasmid, suggesting that the product (designated S159N) can substitute for PGGT-I beta subunit in vivo. Its ability to act as a PFT is not drastically reduced, since the mutant gene still complemented a dpr1 disruption. Results of in vitro assays demonstrate that the mutant enzyme has increased activity to farnesylate, a substrate for PGGT-I. On the other hand, the ability to farnesylate its own substrate is reduced. The increased ability to utilize the PGGT-I substrate is due to its increased affinity for the protein substrate. In addition, the mutant enzyme shows a severalfold increase in the sensitivity to a peptidomimetic inhibitor that acts as a competitor of the protein substrate. These results point to the importance of the beta subunit of PFT for the binding of a protein substrate and demonstrate that Ser-159 of DPR1 product is critical for its substrate specificity.

Characterization of a staurosporine- and temperature-sensitive mutant, stt1, of Saccharomyces cerevisiae: STT1 is allelic to PKC1.

Staurosporine is an antibiotic that specifically inhibits protein kinase C. Fourteen staurosporine- and temperature-sensitive (stt) mutants of Saccharomyces cerevisiae were isolated and characterized. These mutants were divided into ten complementation groups, and characterized for their cross-sensitivity to K-252a, neomycin, or CaCl2. The STT1 gene was cloned and sequenced. The nucleotide sequence of the STT1 gene revealed that STT1 is the same gene as PKC1. The STT1 gene conferred resistance to staurosporine on wild-type cells, when present on a high copy number plasmid. STT1/stt1::HIS3 diploid cells were more sensitive to staurosporine than STT1/STT1 diploid cells. Analysis of temperature-sensitive stt1 mutants showed that the STT1 gene product functioned in S or G2/M phase. These results suggest that a protein kinase (the STT1 gene product) is one of the essential targets of staurosporine in yeast cells.

Two WD repeat-containing TATA-binding protein-associated factors in fission yeast that suppress defects in the anaphase-promoting complex.

The general transcription factor IID consists of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). Here we report the isolation of two related TAF genes from the fission yeast Schizosaccharomyces pombe as multicopy suppressors of a temperature-sensitive mutation in the ubiquitin-conjugating enzyme gene ubcP4(+). The ubcP4(ts) mutation causes cell cycle arrest in mitosis, probably due to defects in ubiquitination mediated by the anaphase-promoting complex/cyclosome. One multicopy suppressor is the previously reported gene taf72(+), whereas the other is a previously unidentified gene named taf73(+). We show that the taf73(+) gene, like taf72(+), is essential for cell viability. The taf72(+) and taf73(+) genes encode proteins homologous to WD repeat-containing TAFs such as human TAF100, Drosophila TAF80/85, and Saccharomyces cerevisiae TAF90. We demonstrate that TAF72 and TAF73 proteins are present in the same complex with TBP and other TAFs and that TAF72, but not TAF73, is associated with the putative histone acetylase Gcn5. We also show that overexpression of TAF72 or TAF73 suppresses the cell cycle arrest in mitosis caused by a mutation in the anaphase-promoting complex/cyclosome subunit gene cut9(+). These results suggest that TAF72 and TAF73 may regulate the expression of genes involved in ubiquitin-dependent proteolysis during mitosis. Our study thus provides evidence for a possible role of WD repeat-containing TAFs in the expression of genes involved in progression through the M phase of the cell cycle.

Isolation and characterization of temperature-sensitive mutations in the gene (rpb3) for subunit 3 of RNA polymerase II in the fission yeast Schizosaccharomyces pombe.

Subunit 3 (Rpb3) of eukaryotic RNA polymerase II is a homologue of the alpha subunit of prokaryotic RNA polymerase, which plays a key role in subunit assembly of this complex enzyme by providing the contact surfaces for both beta and beta' subunits. Previously we demonstrated that the Schizosaccharomyces pombe Rpb3 protein forms a core subassembly together with Rpb2 (the beta homologue) and Rpb11 (the second alpha homologue) subunits, as in the case of the prokaryotic alpha2beta complex. In order to obtain further insight into the physiological role(s) of Rpb3, we subjected the S. pombe rpb3 gene to mutagenesis. A total of nine temperature-sensitive (Ts) and three cold-sensitive (Cs) S. pombe mutants have been isolated, each (with the exception of one double mutant) carrying a single mutation in the rpb3 gene in one of the four regions (A D) that are conserved between the homologues of eukaryotic subunit 3. The three Cs mutations were all located in region A, in agreement with the central role of the corresponding region in the assembly of prokaryotic RNA polymerase; the Ts mutations, in contrast, were found in all four regions. Growth of the Ts mutants was reduced to various extents at non-permissive temperatures. Since the metabolic stability of most Ts mutant Rpb3 proteins was markedly reduced at non-permissive temperature, we predict that these mutant Rpb3 proteins are defective in polymerase assembly or the mutant RNA polymerases containing mutant Rpb3 subunits are unstable. In accordance with this prediction, the Ts phenotype of all the mutants was suppressed to varying extents by overexpression of Rpb11, the pairing partner of Rpb3 in the core subassembly. We conclude that the majority of rpb3 mutations affect the assembly of Rpb3, even though their effects on subunit assembly vary depending on the location of the mutation considered.

Identification of the domain of Saccharomyces cerevisiae adenylate cyclase associated with the regulatory function of RAS products.

Various truncated CYR1 genes of Saccharomyces cerevisiae were fused to efficient promoters and expressed in Escherichia coli and S. cerevisiae cells with or without the RAS genes. The catalytic domain of adenylate cyclase encoded by the 3'-terminal 1.3 kb region of the open reading frame of the CYR1 gene produced cyclic AMP, irrespective of the presence of RAS genes. The product of the 3'-terminal 2.1 kb region of CYR1 showed guanine nucleotide-dependent adenylate cyclase activity and produced a large amount of cAMP in the presence of the RAS gene. Thus, the domain encoded by the 0.8 kb region adjacent to the catalytic domain is associated with the regulatory function of the RAS products. The cyr1 RAS1 RAS2 cells carrying the 3'-terminal 1.3 kb region of CYR1 were unable to respond to environmental signals such as sulfur starvation and temperature shift, but the cyr1 cells carrying the 2.1 kb region and at least one RAS gene were able to respond to these signals. The environmental signals may be transferred to the adenylate cyclase system through the RAS products.

The adenylate cyclase/protein kinase cascade regulates entry into meiosis in Saccharomyces cerevisiae through the gene IME1.

Entry into meiosis in Saccharomyces cerevisiae cells is regulated by starvation through the adenylate cyclase/cAMP-dependent protein kinase (AC/PK) pathway. The gene IME1 is also involved in starvation control of meiosis. Multicopy IME1 plasmids overcome the meiotic deficiency of bcy1 and of RASval19 diploids. Double mutants ime1 cdc25 and ime1 ras2 are sporulation deficient. These results suggest that IME1 comes after the AC/PK cascade. Furthermore, the level of IME1 transcripts is affected by mutations in the AC/PK genes CDC25, CYR1 and BCY1. Moreover, the addition of cAMP to a cyr1-2 diploid suppresses IME1 transcription. The presence in a bcy1 diploid of IME1 multicopy plasmids does not cure the failure of bcy1 cells to arrest as unbudded cells following starvation and to enter the G0 state (thermotolerance, synthesis of unique G0 proteins). This indicates that the pathway downstream of the AC/PK cascade branches to control meiosis through IME1, and to control entry into G0 and cell cycle initiation, independently of IME1.

Amino acid substitutions that convert the protein substrate specificity of farnesyltransferase to that of geranylgeranyltransferase type I.

Protein farnesyltransferase (FTase), a heterodimer enzyme consisting of alpha and beta subunits, catalyzes the addition of farnesyl groups to the C termini of proteins such as Ras. In this paper, we report that the protein substrate specificity of yeast FTase can be switched to that of a closely related enzyme, geranylgeranyltransferase type I (GGTase I) by a single amino acid change at one of the three residues: Ser-159, Tyr-362, or Tyr-366 of its beta-subunit, Dpr1. All three Dpr1 mutants can function as either FTase or GGTase I beta subunit in vivo, although some differences in efficiency were observed. These results point to the importance of two distinct regions (one at 159 and the other at 362 and 366) of Dpr1 for the recognition of the protein substrate. Analysis of the protein, after site directed mutagenesis was used to change Ser-159 to all possible amino acids, showed that either asparagine or aspartic acid at this position allowed FTase beta to function as GGTase I beta. A similar site-directed mutagenesis study on Tyr-362 showed that leucine, methionine, or isoleucine at this position also resulted in the ability of mutant FTase beta to function as GGTase I beta. Interestingly, in both position 159 and 362 substitutions, amino acids that could change the protein substrate specificity had similar van der Waals volumes. Biochemical characterization of the S159N and Y362L mutant proteins showed that their kcat/Km values for GGTase I substrate are increased about 20-fold compared with that of the wild type protein. These results demonstrate that the conversion of the protein substrate specificity of FTase to that of GGTase I can be accomplished by introducing a distinct size amino acid at either of the two residues, 159 and 362.

Reconstitution of the GTP-dependent adenylate cyclase from products of the yeast CYR1 and RAS2 genes in Escherichia coli.

Plasmids carrying the CYR1 gene of yeast Saccharomyces cerevisiae, which encodes adenylate cyclase, were introduced into the cya mutant strain of Escherichia coli. The transformants had a GTP-independent adenylate cyclase activity but did not produce cAMP. The E. coli transformant carrying the yeast RAS2 or RAS2val19 gene had no adenylate cyclase activity. Transformant cells carrying both CYR1 and RAS2 produced GTP-dependent adenylate cyclase and cAMP, and those carrying CYR1 and RAS2val19 produced GTP-independent adenylate cyclase and a large amount of cAMP. Production of cAMP in the transformant carrying CYR1 and either RAS2 or RAS2val19 was confirmed by staining colonies on maltose-MacConkey plates and by measuring induction of beta-galactosidase by isopropyl beta-D-thiogalactopyranoside. Mixing a crude extract from the E. coli transformant carrying CYR1 with a crude extract from cells carrying RAS2 reconstituted the GTP-dependent adenylate cyclase. Reconstitution of the GTP-dependent adenylate cyclase was observed by mixing the plasma membrane fraction of yeast CYR1 ras1 ras2 bcy1 mutant and a crude extract from the E. coli transformant carrying RAS2 or by mixing a crude extract from the E. coli transformant carrying CYR1 and the membrane fraction of yeast cyr1 RAS1 RAS2 BCY1 mutant. The data suggest that the yeast GTP-dependent adenylate cyclase consists of catalytic and regulatory subunits encoded by the CYR1 and RAS2 genes, respectively.