Spo12 Is a Limiting Factor That Interacts with the Cell Cycle Protein Kinases Dbf2 and Dbf20, Which Are Involved in Mitotic Chromatid Disjunction

The DBF2 and DBF20 genes of the budding yeast Saccharomyces cerevisiae encode a pair of structurally similar protein kinases. Although yeast with either gene deleted is viable, deletion of both genes is lethal. Thus, the Dbf2 and Dbf20 proteins are functional alternatives for an essential activity. In contrast to deletions, four different mutant alleles of DBF2 are lethal. Thus, the presence of a nonfunctional Dbf2 protein, rather than the lack of function per se, is inhibitory. Here we present genetic evidence that nonfunctional mutant Dbf2 protein blocks the function of Dbf20 protein by sequestering a common interacting protein encoded by SPO12. Even a single extra copy of SPO12 is sufficient to suppress the dbf2 defect. Since SPO12 appears to encode a limiting factor, it may be a rate limiting cofactor that is involved in the regulation of the Dbf2 and Dbf20 protein kinases. A corollary to the finding that one extra copy of SPO12 can suppress dbf2, is that the acquisition of an extra chromosome VIII, which carries the SPO12 locus, will also suppress dbf2. Indeed, physical analysis of chromosome copy number in dbf2 revertants able to grow at 37° showed that the frequency of chromosome VIII acquisition increased when cells were incubated at the restrictive temperature, and reached a frequency of more than 100-fold the amount in wild-type yeast. This suggested that the dbf2 mutation was not only suppressed by an extra copy of chromosome VIII but also that the dbf2 mutation actually caused aberrant chromosomal segregation. Conventional assays for chromosome loss confirmed this proposal.     

SPO12 and SIT4 suppress mutations in DBF2, which encodes a cell cycle protein kinase that is periodically expressed.

To help clarify the role of DBF2, a previously described cell cycle protein kinase, high copy number suppressors of the dbf2 mutation were isolated. Three open reading frames (ORF) have been identified. One ORF encodes a protein which has homology to a human small nuclear riboprotein, while the remaining two are genes which have been identified previously, SIT4 and SPO12. SIT4 is known to have a role in the cell cycle but the nature of the interaction between SIT4 and dbf2 is unclear. SPO12 has until now been implicated exclusively in meiosis. However, we show that SPO12 is expressed during vegetative growth, moreover it is expressed under cell cycle control coordinately with DBF2. SPO12 is a nonessential gene, but it becomes essential in a DBF2 delete genetic background. Furthermore, detailed analysis of the cell cycle of SPO12 delete cells revealed a small but significant delay in mitosis. Therefore, SPO12 does have a role during vegetative growth and it probably functions in mitosis in association with DBF2.     

cdc2MsB, a cognate cdc2 gene from alfalfa, complements the G1/S but not the G2/M transition of budding yeast cdc28 mutants

The product of the cdc2 gene encodes the p34^cdc2 protein kinase that controls entry of yeast cells into S phase and mitosis. In higher eukaryotes, at least two cdc2 -like genes appear to be involved in these processes. A cdc2 homologous gene has previously been isolated from alfalfa and shown to complement a fission yeast cdc2 ^ts mutant. Here the isolation of cdc2MsB , a cognate cdc2 gene from alfalfa ( Medicago sativa ) is reported. Southern blot analysis shows that cdc2MsA and cdc2MsB are present as single copy genes in different tetraploid Medicago species. cdc2MsB encodes a slightly larger mRNA (1.5 kb) than cdc2MsA (1.4 kb). Both genes were found to be expressed at similar steady state levels in different alfalfa organs. Expression levels of both cdc2Ms genes correlate with the proliferative state of the organs. Complementation studies revealed that in contrast to cdc2MsA, cdc2MsB was not able to rescue a cdc2 ^ts fission yeast mutant. cdc2MsB was also unable to rescue a G2/M-arrested cdc28 ^ts budding yeast mutant which could be rescued by expression of the cdc2MsA gene. Conversely, cdc2MsB but not cdc2MsA was found to complement the G1/S block of another cdc28 ^ts budding yeast mutant. These results suggest that cdc2MsA and cdc2MsB function at different control points in the cell cycle.     

Temperature-Sensitive Cdc 7 Mutations of Saccharomyces Cerevisiae Are Suppressed by the Dbf4 Gene, Which Is Required for the G(1)/S Cell Cycle Transition

When present on a multicopy plasmid, a gene from a Saccharomyces cerevisiae genomic library suppresses the temperature-sensitive cdc7-1 mutation. The gene was identified as DBF4, which was previously isolated by complementation in dbf4-1 mutant cells and is required for the G1----S phase progression of the cell cycle. DBF4 has an open reading frame encoding 695 amino acid residues and the predicted molecular mass of the gene product is 80 kD. The suppression is allele-specific because a CDC7 deletion is not suppressed by DBF4. Suppression is mitosis-specific and the sporulation defect of cdc7 mutations is not suppressed by DBF4. Conversely, CDC7 on a multicopy plasmid suppresses the dbf4-1, -2, -3 and -4 mutations but not dbf4-5 and DBF4 deletion mutations. Furthermore, cdc7 mutations are incompatible with the temperature-sensitive dbf4 mutations. These results suggest that the CDC7 and DBF4 polypeptides interact directly or indirectly to permit initiation of yeast chromosome replication.     

Cell-cycle-dependent gene expression studied by two-colour fluorescent detection of a mRNA and histone mRNA

We investigated whether a probe specific for histone H3 mRNA could be used as a marker to study cell-cycle dependency of gene expression by double-fluorescent RNA in situ hybridization (FISH). First, we showed that all S-phase cells in cell cultures having incorporated BrdU revealed histone H3 mRNA expression by RNA FISH, indicating that histone H3 expression is a reliable marker for S-phase cells. Second, we analysed whether the expression of human cytomegalovirus immediate early genes in rat 9G cells, which are known to be induced in an S-phase dependent way by cycloheximide, correlated with the expression of histone H3 mRNA. Double-hybridization experiments with a digoxigenin-labelled probe for IE mRNA and a fluoresceinated probe for histone H3 mRNA revealed that cells expressing IE mRNA also expressed histone H3 mRNA. Third, we examined the cell-cycle dependency of luciferase gene expression in X1 cells. Luciferase mRNA is heterogeneously expressed in X1 cell cultures, but cells expressing luciferase did not necessarily express histone H3 mRNA. This indicates that luciferase gene expression in X1 cells is not induced during S-phase. The results of our study show that histone H3 mRNA expression can be successfully used as a marker to establish cell-cycle dependency of gene expression by double-RNA FISH.     

Cloning and characterization of Chinese hamster homologue of yeast DBF4 (ChDBF4)

The Dbf4 protein is the regulatory subunit of Cdc7 serine/threonine kinase, which is essential for entry into S phase. We report here the cloning and initial characterization of the Chinese hamster homologue of yeast DBF4. The deduced ChDbf4 protein contains 676 amino acids with a predicted molecular mass of 75.8 kDa, and shares extensive identity overall with those of human (68%) and mouse (73%). The ChDBF4 mRNA level was barely detectable in the cells arrested in the quiescent stage (G(0)) by isoleucine starvation. When cells in G(0) were released into the cell cycle, the ChDBF4 mRNA level did not significantly change until the cells reached the G(1)/S boundary, when the level rapidly increased and reached approximately 70% of the maximum level that was observed in mid to late S phase. Interestingly, gamma-irradiation rapidly and transiently downregulated the level of ChDBF4 mRNA in asynchronous cell populations. Since Dbf4-Cdc7 kinase is involved in the regulation of replication initiation, which can be transiently downregulated by irradiation (Larner et al., 1994. Mol. Cell. Biol. 14, 1901, our data raise the possibility that the downregulation of DBF4 (and, thus, the Cdc7 kinase activity) by irradiation may play a role in the cell-cycle checkpoint that functions at the G(1)/S transition and in S phase (Lee et al., 1997. Proc. Natl. Acad. Sci. USA 94, 526).     

Purification, characterisation and mutagenesis of highly expressed recombinant yeast pyruvate kinase.

Recombinant yeast pyruvate kinase has been purified from a strain of Saccharomyces cerevisiae expressing the enzyme to very high levels. Expression was from a multicopy plasmid under the control of the yeast phosphoglycerate kinase promoter. The gene was expressed in the absence of the genomically encoded pyruvate kinase, using a strain of yeast in which the pyruvate kinase gene has been disrupted by the insertion of the yeast Ura3 gene. The purification procedure minimised proteolytic artefacts and enabled the convenient purification of 15-20 mg enzyme from 11 culture. The purified enzyme was characterised by a high specific activity and by a lack of proteolytic degradation. Two active-site mutants of yeast pyruvate kinase have been produced, expressed and characterised in this system and preliminary results are described.     

On a heuristic point of view concerning the expression of numerous genes during the cell cycle

The current model of the eukaryotic cell cycle proposes that numerous genes are expressed at different times during the cell cycle. The existence of myriad control points for gene expression leads to theoretical and logical problems for cell cycle control. Each expressed gene requires a control element to appear in a cell-cycle specific manner; this control element requires another control element and so on, ad infinitum. There are also experimental problems with the current model based on ineffective synchronization methods and problems with microarray measurements of mRNA. Equally important, the efficacy of mRNA variation in affecting changes in protein content is negligible. An alternative view of the cell cycle proposes cycle-independent, invariant accumulation of mRNA during the cell cycle with decreases of specific proteins occurring only during the mitotic period of the cell cycle.     

A glucose-inducible gene in Schizosaccharomyces pombe,rrg1+, is involved in negative regulation of G2/M progression

A glucose-inducible gene in S. pombe is rrg1+. Its mRNA level is rapidly decreased and increased by glucose-depletion and readdition, respectively. The previous study revealed that the rrg1+ expression was regulated by glucose-dependent mRNA stability control. To understand the significance of the glucose-dependent expression of rrg1+, the cellular function of rrg1+ was explored. Deletion of the rrg1+ gene from the haploid chromosome of S. pombe cells did not lead to cell lethality but brought about cell size reduction, which was accompanied by fast cell proliferation. In accordance with this result, the overexpression of the Rrgl protein under the control of the nmt1 promoter produced elongated cells of G2 delay, and consequently resulted in the slowing-down of cell proliferation. In addition, the rrg1+ mRNA level showed cell-cycle dependent changes, peaking at G2/M. These results demonstrate that Rrg1 might be involved in the negative regulation of cell proliferation and G2/M progression for cell size control.     

Identification of cut8+ and cek1+, a novel protein kinase gene, which complement a fission yeast mutation that blocks anaphase.

The fission yeast Schizosaccharomyces pombe [corrected] temperature sensitivity cut8-563 mutation causes chromosome overcondensation and short spindle formation in the absence of sister chromatid separation. The cut8-563 mutation allows cytokinesis before the completion of anaphase, thus producing cells with a cut phenotype. The cut8+ gene product may be required for normal progression of anaphase. Diploidization occurs at the restrictive temperature, and 60 to 70% of the cells surviving after two generations are diploid. These phenotypes are reminiscent of those of budding yeast (Saccharomyces cerevisiae) ctf13 and ctf14 (ndc10) mutations. The cut8+ gene, isolated by complementation of the mutant, predicts a 262-amino-acid protein; the amino and carboxy domains are hydrophilic, while the central domain contains several hydrophobic stretches. It has a weak overall similarity to the budding yeast DBF8 gene product. DBF8 is an essential gene whose mutations result in delay in mitotic progression and chromosome instability. Anti-cut8 antibodies detect a 33-kDa polypeptide. Two multicopy suppressor genes for cut8-563 are identified. They are the cut1+ gene essential for nuclear division, and a new gene (designated cek1+) which encodes a novel protein kinase. The cek1+ gene product is unusually large (1,309 amino acids) and has a 112-amino-acid additional sequence in the kinase domain. The cek1+ gene is not an essential gene. Protein phosphorylation by cek1 may facilitate the progression of anaphase through direct or indirect interaction with the cut8 protein.     

Nup211, the fission yeast homolog of Mlp1/Tpr,is involved in mRNA export

Synthetic lethal mutants have been previously isolated in fission yeast Schizosaccharomyces pombe, which genetically interact with spmex67, in order to identify the genes involved in mRNA export. The nup211 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex2, under synthetic lethal condition. We showed that Nup211, fission yeast homolog of Mlpl/Mlp2/Tpr, is essential for vegetative growth and Nup211-GFP proteins expressed at endogenous level are localized mainly in nuclear periphery. The accumulation of poly(A)⁺ RNA in the nucleus is exhibited when expression of nup211 is repressed or over-expressed. These results suggest that the Nup211 protein plays a pivotal role of mRNA export in fission yeast.     

Yeast gene SRP1 (serine-rich protein). Intragenic repeat structure and identification of a family of SRP1-related DNA sequences

We have isolated and sequenced a yeast gene encoding a protein (Mr 24,875) very rich in serine (SRP) and alanine residues that accounted for 25% and 20% of the total amino acids, respectively. The SRP1 gene is highly expressed in culture conditions leading to glucose repression (Marguet & Lauquin, 1986), the amount of SRP1 mRNA representing about 1 to 2% of total poly(A)+ RNA. A repetitive structure of eight direct tandem repeats 36-base long, also reflected in the amino acid sequence, was found in the second half of the open reading frame. The consensus amino acid sequence of the repeat was Ser-Ser-Ser-Ala-Ala-Pro-Ser-Ser-Ser-Glu-Ala-Lys. Replacing the genomic copy of the cloned gene with a disrupted SRP1 gene indicated that the SRP1 gene was not essential for viability in yeast, but several SRP1-homologous sequences were found within the yeast genome, raising the possibility that the disrupted SRP1 gene is rescued by one of the other SRP-homologous sequences. Complete separation of yeast chromosomes by contour-clamped homogeneous field electrophoresis indicated that, apart from chromosome V, which carries the SRP1 gene, 12 chromosomes have SRP-related sequences with various degrees of homology. These sequences were located on chromosomes XV, VII and XI under stringent conditions of hybridization (tm -20 degrees C), and observed on chromosomes I, II, III, IV, VI, VIII, X, XI and XII, only under low-stringency conditions (tm -40 degrees C). Northern blot analysis of both the wild type and SRP1-disrupted strains indicated that along with SRP1 at least one more member of the SRP family was transcribed to a 0.7 kb (1 kb = 10(3) bases) polyadenylated RNA species clearly distinct from the SRP1-specific mRNA (1 kb long). Analyses of the SRP1 repeat domain suggested a model for the divergent evolution of the repeats in the SRP1 sequence.     

Sequence of the nucleoprotein gene from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae

Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA which was sequenced. Two non-overlapping open reading frames (ORFs) were identified. The largest, encoding a polypeptide of 382 amino acids (relative molecular mass (Mr) 43,483), was shown to be the viral nucleoprotein gene. The second ORF, found 3' to the larger ORF, encodes a polypeptide of 78 amino acids (Mr 9068) which has yet to be assigned to a viral product. The nucleoprotein gene was expressed in yeast cells under the control of two types of yeast promoters: the constitutive PGK promoter, and the inducible GAL1 promoter. Yeast cells containing recombinant plasmids, with the nucleoprotein gene in the correct orientation, produced a polypeptide of Mr 47,000, identical to the viral product, that reacted with a specific monoclonal antibody.