Purification and characterization of Saccharomyces cerevisiae uridine monophosphate kinase

The SOC8 gene was isolated as an extragenic suppressor of cdc8 mutant cells. It has been suggested that SOC8 is allelic with the URA6 gene which was originally identified as a uridine monophosphate kinase. In this article, we describe the purification of the uridine monophosphate kinase from a yeast Saccharomyces cerevisae strain that overproduces the activity 8-fold. The protein was purified through Fast-Flow Q-Separose, phosphocellulose, blue-agarose, and fast protein liquid chromatography Superose 12 columns, and appears homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis. The uridine monophosphate kinase contains a single polypeptide with a molecular weight of 25,000, as evidence by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analysis. The amino acid composition has also been determined. Substrate specificity studies show that the relative activity of nucleoside monophosphates is in order of UMP greater than dUMP, and to a lesser extent, dTMP, GMP, and dGMP. The Km and Vm of UMP, dUMP, and dTMP have been determined.     

Genetic and Molecular Analysis of the Soe1 Gene: A Trna(3)(glu) Missense Suppressor of Yeast Cdc8 Mutations

The CDC8 gene of Saccharomyces cerevisiae encodes deoxythymidylate (dTMP) kinase and is required for nuclear and mitochondrial DNA replication in both the mitotic and meiotic cell cycles. All cdc8 temperature-sensitive mutants are partially defective in meiotic and mitochondrial functions at the permissive temperature. In a study of revertants of temperature-sensitive cdc8 mutants, the SOE201 and SOE1 mutants were isolated. The SOE201 mutant is a disome of chromosome X to which the cdc8 gene maps. Using the chromosome X aneuploids to vary cdc8 gene dosage, we demonstrate that different levels of dTMP kinase activity are required for mitotic, meiotic or mitochondrial DNA replication. The SOE1 mutant contains a dominant suppressor that suppresses five different cdc8 alleles but does not suppress a complete cdc8 deletion. The SOE1 gene is located less than 1.5 cM from the CYH2 gene on chromosome VII and is adjacent to the TSM437-CYH2 region, with the gene order being SOE1-TSM437-CYH2. SOE1 is an inefficient suppressor that can neither suppress the cdc8 hypomorphic phenotype nor restore dTMP kinase activity in vitro. SOE1 is a single C to T mutation in the anticodon of a tRNA(3Glu) gene and thereby, produces a missense suppressor tRNA capable of recognizing AAA lysine codons. We propose that the resultant lysine to glutamate change stabilizes thermo-labile dTMP kinase molecules in the cell.     

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.     

Functional and structural conservation of Schizosaccharomyces pombe dTMP kinase gene.

We describe the isolation and identification of the Schizosaccharomyces pombe dTMP kinase gene by the complementation of a Saccharomyces cerevisiae cell cycle mutant cell, cdc8. The isolated cDNA contains an open reading frame which can encode a protein with the molecular weight of 24,151. The deduced protein sequence is highly conserved among known dTMP kinase sequences from different organisms. The isolated gene should facilitate our study of its enzymatic activity, as well as nucleotide metabolism and cell cycle regulation in this organism.     

Cloning and characterization of complementary DNA encoding human N-acetylglucosamine-phosphate mutase protein

Endomyces fibuliger is a yeast used in the production of Chinese rice wine. It secretes enzymes such as glucoamylase, alpha-amylase and acid protease. Very little is known of the genetics of E. fibuliger. In order to develop a transformation system for this yeast, orotidine-5'-phosphate decarboxylase mutant strains were obtained and characterized. Transformation of the E. fibuliger ura3 mutant F1 with an integrative plasmid that carried the wild-type URA3 gene of E. fibuliger gave complementation of this mutation. The E. fibuliger gene encodes the orotidine-5'-phosphate decarboxylase enzyme consisting of 266 amino acid residues with a 69.4% sequence identity with orotidine-5'-phosphate decarboxylase of Saccharomyces cerevisiae. Our finding that E. fibuliger URA3 complements the ura3 mutation in S. cerevisiae confirms that the URA3 gene of E. fibuliger encodes a protein that exerts a similar function.     

Isolation and nucleotide sequence of a Saccharomyces cerevisiae protein kinase gene suppressing the cell cycle start mutation cdc25

We have isolated two unlinked yeast genes complementing the cell division cycle mutant cdc25-1, one containing the wild type allele CDC25 and the other acting as an extragenic suppressor of the cdc25-1 lesion if present on a multicopy plasmid. Nucleotide sequence analysis of the suppressor gene has revealed an open reading frame that encodes a 45,000-dalton protein belonging to the protein kinase family. The cdc25-suppressing protein kinase (PK-25) shows 48% sequence similarity to the catalytic subunit (CA) of mammalian cAMP-dependent protein kinase and 27-31% similarity to cyclic nucleotide-independent enzymes, including the yeast CDC28 gene product. The PK-25 gene was targeted by integrative transformation into a chromosomal region unlinked to the CYR2 site, the structural gene of CA. The cdc25-suppressing protein kinase is also functionally different from CA, since cyr2 strains deficient in the free catalytic subunit remain temperature sensitive if transformed with a multicopy plasmid containing the PK-25 gene. Furthermore, a deficiency of the cAMP-binding regulatory subunit (RA) caused by the bcy1 mutation fails to suppress the cdc25 mutation, indicating that PK-25 does not interact with the cAMP receptor protein. Our data suggest that the cdc25 suppressor gene encodes a cAMP-independent protein kinase involved in the control of the cell cycle start.     

Cloning and characterization of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase.

Saccharomyces cerevisiae strains that contain the ery8-1 mutation are temperature sensitive for growth due to a defect in phosphomevalonate kinase, an enzyme of isoprene and ergosterol biosynthesis. A plasmid bearing the yeast ERG8 gene was isolated from a YCp50 genomic library by functional complementation of the erg8-1 mutant strain. Genetic analysis demonstrated that integrated copies of an ERG8 plasmid mapped to the erg8 locus, confirming the identity of this clone. Southern analysis showed that ERG8 was a single-copy gene. Subcloning and DNA sequencing defined the functional ERG8 regulon as an 850-bp upstream region and an adjacent 1,272-bp open reading frame. The deduced 424-amino-acid ERG8 protein showed no homology to known proteins except within a putative ATP-binding domain present in many kinases. Disruption of the chromosomal ERG8 coding region by integration of URA3 or HIS3 marker fragments was lethal in haploid cells, indicating that this gene is essential. Expression of the ERG8 gene in S. cerevisiae from the galactose-inducible galactokinase (GAL1) promoter resulted in 1,000-fold-elevated levels of phosphomevalonate kinase enzyme activity. Overproduction of a soluble protein with the predicted 48-kDa size for phosphomevalonate kinase was also observed in the yeast cells.     

An extra copy of nimEcyclinB elevates pre-MPF levels and partially suppresses mutation of nimTcdc25 in Aspergillus nidulans.

Previous work has shown that nimA encodes a cell cycle regulated protein kinase that is required along with the p34cdc2 histone H1 kinase (MPF) for mitosis in Aspergillus nidulans. We have now identified two other gene products required for mitosis in A.nidulans. nimT encodes a protein similar to the fission yeast cdc25 tyrosine phosphatase and is required for the conversion of pre-MPF to MPF and nimE encodes a B-type cyclin which is a subunit of MPF. A new genetic interaction between nimEcyclinB and nimTcdc25 type genes is reported. Increased copy number of nimEcyclinB can suppress mutation of nimTcdc25 and also lead to increased accumulation of tyrosine phosphorylated p34cdc2 (pre-MPF). This biochemical observation suggests an explanation for the genetic complementation. If nimEcyclinB recruits p34cdc2 for tyrosine phosphorylation to form pre-MPF it follows that increased expression of nimEcyclinB would increase the level of pre-MPF. The increased level of pre-MPF generated may then allow the mutant nimTcdc25 protein to convert enough pre-MPF to MPF and thus permit some mitotic progression. We also demonstrate that correct cell cycle regulation by the p34cdc2 protein kinase pathway is essential for correct developmental progression in A.nidulans.     

Yeast cell-cycle mutantcdc21 is a temperature-sensitive thymidylate auxotroph

Summary Genetic tests with the yeast cell-cycle mutantcdc21 isolated by Hartwell indicate that the CDC21 gene in yeast is the same as the TMP1 gene, whose mutant alleles confer an auxotrophic requirement for thymidine-5-monophosphate (dTMP). Yeast strains carryingcdc21 can grow at 37° in the presence of dTMP provided that they are permeable to this compound. The gene is shown to be linked toade2 on Chr. XV, and a case of intragenic complementation betweencdc21 and anothertmp1 allele is reported.     

Involvement of a type 1 protein phosphatase encoded by bws1+ in fission yeast mitotic control

Fission yeast cdc25+ and wee1+ interact genetically with cdc2+ in the regulation of cell division, respectively as a mitotic activator and inhibitor. cdc25+ is normally essential for mitosis, but this requirement is alleviated in a loss-of-function wee1 mutant background. A plasmid-borne sequence, other than wee1+, that causes a cdc25ts wee1- double mutant to revert to a temperature-sensitive cdc phenotype has been isolated. The gene carried by this plasmid is called bws1+ (for bypass of wee suppression). bws1+ also bypasses the ability of alleles of cdc2 that confer a wee phenotype (cdc2w) to suppress loss-of-function cdc25 mutants. The nucleotide sequence of bws1+ shows that the predicted protein shares 81% amino acid identity with the catalytic subunit of mammalian type 1 protein phosphatase. Thus a genetic screen that might have yielded a protein kinase (wee1+) uncovered a phosphatase that also appears to be involved in the pathway of mitotic control.     

Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation.

The CDC8 gene, whose product is required for DNA replication in Saccharomyces cerevisiae, has been isolated on recombinant plasmids. The yeast vector YCp50 bearing the yeast ARS1, CEN4, and URA3 sequences, to provide for replication, stability, and selection, respectively, was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping. This fragment lies 1 kilobase downstream from the well-characterized sup4 gene, a gene known to be genetically linked to CDC8, thus confirming that the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the lack of CDC8 gene product.     

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.     

Transformation system for an asporogenous methylotrophic yeast, Candida boidinii: cloning of the orotidine-5'-phosphate decarboxylase gene (URA3), isolation of uracil auxotrophic mutants, and use of the mutants for integrative transformation.

An integrative transformation system was established for an asporogenous methylotrophic yeast, Candida boidinii. This system uses a uracil auxotrophic mutant of C. boidinii as the host strain in combination with its URA3 gene as the selectable marker. First, the C. boidinii URA3 gene coding for orotidine-5'-phosphate decarboxylase (ODCase) was cloned by using complementation of the pyrF mutation of Escherichia coli. Next, the host ODCase-negative mutant strains (ura3 strains) were isolated by mutagenesis and selection for 5-fluro-orotic acid (5-FOA) resistance. Five ura3 host strains that exhibited both a low reversion rate and good methylotrophic growth were obtained. All of these strains could be transformed to Ura+ phenotype with a C. boidinii URA3-harboring plasmid linearized within the Candida DNA. The transformants had a stable Ura+ phenotype after nonselective growth for 10 generations. These results and extensive Southern analysis indicated that the linearized plasmid was integrated into the host chromosomal DNA by homologous recombination at the URA3 locus in C. boidinii.     

Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli.

We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid. These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold. The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids. Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s). All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I. The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I. Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid. The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme. Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed.     

Pyp3 PTPase acts as a mitotic inducer in fission yeast.

The p34cdc2 M-phase kinase is regulated by inhibitory phosphorylation of Tyr15, largely through the actions of the p107wee1 tyrosine kinase and p80cdc25 protein tyrosine phosphatase (PTPase). In this study we demonstrate that a second PTPase, encoded by pyp3, also contributes to tyrosyl dephosphorylation of p34cdc2. Pyp3 was identified as a high copy suppressor of a cdc25- mutation. The pyp3 gene encodes a 33 kDa PTPase that is more closely related to human PTP1B and fission yeast pyp1 and pyp2 PTPases than to cdc25. Pyp3 does not share an essential overlapping function with pyp1 or pyp2. We demonstrate that disruption of pyp3 causes a mitotic delay that is greatly exacerbated in cells that are partially defective for cdc25 function and that pyp3 function is essential in cdc25-disruption wee1- strains. Pyp3 PTPase effectively dephosphorylates and activates the p34cdc2 kinase in vitro. We conclude that the pyp3 PTPase acts cooperatively with p80cdc25 to dephosphorylate Tyr15 of p34cdc2.     

A connection between pre-mRNA splicing and the cell cycle in fission yeast: cdc28+ is allelic with prp8+ and encodes an RNA-dependent ATPase/helicase.

The fission-yeast gene cdc28+ was originally identified in a screen for temperature-sensitive mutants that exhibit a cell-division cycle arrest and was found to be required for mitosis. We undertook a study of this gene to understand more fully the general requirements for entry into mitosis. Cells carrying the conditional lethal cdc28-P8 mutation divide once and arrest in G2 after being shifted to the restrictive temperature. We cloned the cdc28+ gene by complementation of the temperature-sensitive growth arrest in cdc28-P8. DNA sequence analysis indicated that cdc28+ encodes a member of the DEAH-box family of putative RNA-dependent ATPases or helicases. The Cdc28 protein is most similar to the Prp2, Prp16, and Prp22 proteins from budding yeast, which are required for the splicing of mRNA precursors. Consistent with this similarity, the cdc28-P8 mutant accumulates unspliced precursors at the restrictive temperature. Independently, we isolated a temperature-sensitive pre-mRNA splicing mutant prp8-1 that exhibits a cell-cycle phenotype identical to that of cdc28-P8. We have shown that cdc28 and prp8 are allelic. These results suggest a connection between pre-mRNA splicing and progression through the cell cycle.     

Involvement of cdc13+ in mitotic control in Schizosaccharomyces pombe: possible interaction of the gene product with microtubules.

Previous genetic studies have shown that the fission yeast cdc13+ gene product interacts closely with the cdc2+ protein kinase during mitosis. Here, we have cloned the cdc13+ gene from a S. pombe gene bank by complementation of the temperature-sensitive defect of a cdc13-117 mutant strain. The complementing activity was localized to a 1.9-kb XbaI-NsiI DNA fragment, and nucleotide sequencing revealed a 1446-bp open reading frame. The predicted amino acid sequence contained 482 residues and was not homologous to any protein in a protein database. The cdc13+ gene function was confirmed to be essential for cell division since cells carrying a cdc13 null allele arrested with a cdc phenotype. However, unlike any existing temperature-sensitive cdc13 mutants, cdc13 null mutants arrested in G2 without septa or condensed chromosomes indicating that cdc13+ gene function is required at or prior to the initiation of mitotis. cdc13-117 mutant strains were found to be hypersensitive to the tubulin inhibitor thiabendazole. This observation suggests that the cdc13+ gene product, which is required for mitotic initiation, may interact with microtubules.     

Stf1: Non-Wee Mutations Epistatic to Cdc25 in the Fission Yeast Schizosaccharomyces Pombe

In Schizosaccharomyces pombe, cdc25 is a cell cycle regulated inducer of mitosis. wee1 and phenotypically wee alleles of cdc2 are epistatic to cdc25. Mutant alleles of a new locus, stf1 (suppressor of twenty-five), identified in a reversion analysis of conditionally lethal cdr1-76 cdc25-22 and cdr2-96 cdc25-22 double mutant strains, also suppress both temperature-sensitive and gene disruption alleles of cdc25. These mutants, by themselves, are phenotypically indistinguishable from wild type strains; hence they represent the first known mutations that are epistatic to cdc25 and do not display a wee phenotype. stf1 genetically interacts with other elements of mitotic control in S. pombe. stf1-1 is additive with wee1-50, cdc2-1w and cdc2-3w for suppression of cdc25-22. Also, like wee1- and cdc2-w, stf1- suppression of cdc25 is reversed by overexpression of the putative type 1 protein phosphatase bws1+/dis2+. Interaction with various mutants and plasmid overexpression experiments suggest that stf1 does not operate either upstream or downstream of wee1. Similarly, it does not operate through cdc25 since it rescues the disruption. stf1 appears to encode an important new element of mitotic control.     

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.