The Saccharomyces cerevisiae SOC8-1 gene and its relationship to a nucleotide kinase

The yeast SOC8-1 gene was originally identified by partial complementation of cdc8 mutant strains. We have carried out Bal31 deletion analysis of the SOC8-1 gene to define the minimal size which is required for the complementation of the cdc8 mutation. When the SOC8-1 gene is cloned in a multicopy plasmid, it enables temperature-resistant growth in the cdc8 mutant strain, while the SOC8-1 gene in a single copy plasmid does not. Thus, its suppression of the cdc8 mutant is dosage dependent. The high copy number vector carrying the SOC8-1 gene can complement five different cdc8 alleles, indicating that the suppression is not allele specific. Since CDC8 encodes thymidylate kinase, cells bearing a high copy number plasmid containing SOC8-1 gene were tested for the ability to phosphorylate several nucleoside monophosphates, including UMP, GMP and dTMP. Significantly increased phosphorylation activity was observed, suggesting that SOC8-1 encodes a nucleotide kinase. Both restriction enzyme analysis of the SOC8-1 gene and partial purification of the overproduced kinase in SOC8-1 overproducing strains suggest that SOC8-1 may be allelic with URA6. Consistent with these results, both SOC8-1 and URA6 are located on chromosome XI. Thus, one possible suppression mechanism is that SOC8-1 may provide a trans-acting dTMP kinase activity, bypassing the cdc8 gene defect.     

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.     

Isolation of metabolic genes and demonstration of gene disruption in the phytopathogenic fungus Ustilago maydis

A cDNA library was constructed in the yeast expression vector pYcDE8 using mRNA from the phytopathogenic fungus Ustilago maydis and cDNAs capable of complementing mutations in three yeast genes, URA3, LEU2 and TPI1, were identified. Nucleotide sequence analysis indicated that the cDNA clone, which complemented the yeast ura3 mutation, carries the pyr6 gene encoding orotidine-5'-phosphate decarboxylase. The genomic copy of the pyr6 gene was isolated by hybridization with the cDNA and used to complement a pyr- mutant of U. maydis. One-step gene disruption was demonstrated by transforming U. maydis with a copy of the pyr6 gene interrupted in the coding region by a selectable marker for resistance to hygromycin B.     

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.     

Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation.

A cDNA encoding Cryptococcus neoformans orotidine monophosphate pyrophosphorylase (OMPPase) has been isolated by complementation of the cognate Escherichia coli pyrE mutant. The cDNA was used as a probe to isolate a genomic DNA fragment encoding the OMPPase gene (URA5). By using electroporation for the introduction of plasmid DNA containing the URA5 gene, C. neoformans ura5 mutants could be transformed at low efficiency. Ura+ transformants obtained with supercoiled plasmids containing the URA5 gene showed marked mitotic instability and contained extrachromosomal URA5 sequences, suggesting limited ability to replicate within C. neoformans. Transformants obtained with linear DNA were of two classes: stable transformants with integrated URA5 sequences, and unstable transformants with extrachromosomal URA5 sequences.     

泡盛曲霉抗FOA突变株的筛选与转化

根据泡盛曲霉SG1菌株分生孢子的紫外线致死曲线,选择死亡率为85%～90%的诱变时间诱变分生孢子,然后将其涂布于含FOA(5-flourooroticacid)和尿嘧啶核苷的基本培养基上,选择抗FOA的突变株。经过纯化和回复突变检测后,获得了5株需要尿嘧啶或尿嘧啶核苷才能在基本培养基上生长的稳定突变株。进一步分析鉴定结果表明,这些突变株的URA3基因发生了突变。Northern杂交及RTPCR方法证明这些突变株中URA3基因突变均发生在转录水平上。选择突变株SA5作为受体菌,用含来自黑曲霉的野生型URA3基因的质粒转化该受体菌,结果获得了稳定的转化子。Southern杂交证明野生型URA3基因取代了突变株的ura3基因。     

The orotidine-5′-phosphate decarboxylase gene (URA3) of a methylotrophic yeast,Candida boidinii: Nucleotide sequence and its expression in Escherichia coli

Previously, we cloned a DNA fragment from a genomic library of a methylotrophic yeast, Candida boidinii. This 3.5-kb SalI fragment was capable of complementing the pyrF mutation in Escherichia coli. In this report, we identify this fragment as that harboring an orotidine-5′-phosphate decarboxylase (ODCase) gene (C. boidinii URA3); we have also determined the complete DNA sequence of the C. boidinii URA3 gene. The deduced amino acid sequence of the gene showed homology to ODCase genes from other sources, and it could complement the ura3 mutation of Saccharomyces cerevisiae. The DNA fragment, which harbored the C. boidinii URA3 gene, was able to express ODCase activity in the E. coli pyrF mutant strain without an exogenous E. coli promoter. From nested-deletion analysis, both the 5′-(136 bp) and 3′-(58 bp) flanking regions were shown to be required for pyrF-complementation of the E. coli mutant. The 5′-flanking region had sequences homologous to E. coli promoter consensus sequences (−35 and −10 regions) which may function in the expression of the C. boidinii URA3 gene in E. coli.     

Sequence analysis of the URA1 gene encoding orotidine-5'-monophosphate decarboxylase of Schizophyllum commune

The URA1 gene (encoding orotidine-5'-monophosphate decarboxylase) of the basidiomycete fungus Schizophyllum commune was mapped to a 1.4-kb BglI-BamHI fragment of two independent phage lambda clones previously isolated from a Schizophyllum genomic library. The fragment was identified by its ability to complement Schizophyllum ura1 mutants via transformation. The complete nucleotide sequence of the fragment containing the URA1 gene was determined. Sequence analysis revealed that the coding region of the URA1 gene encompasses a polypeptide of 279 amino acids (aa) interrupted by two small introns. The deduced aa sequence corresponds to 30.3 kDa and is substantially similar to the sequences of analogous polypeptides from other organisms. No canonical 5'-TATA sequence nor 3'-AATAAA polyadenylation signal are evident in the flanking regions of the URA1 gene.     

Development of an integrative DNA transformation system for the yeast Candida tropicalis.

We developed the alkane and fatty-acid utilizing yeast Candida tropicalis as a host for DNA transformations. The system is based on an auxotrophic mutant host of C. tropicalis which is defective in orotidine monophosphate decarboxylase (ura3). The ura3 host was isolated by mutagenesis and a double-selection procedure that combined nystatin enrichment selection and 5-fluoro-orotic acid resistance selection. As a selectable marker, we isolated and characterized the C. tropicalis URA3 gene. Plasmid vectors that contained the C. tropicalis URA3 gene transformed the C. tropicalis mutant host at a frequency of 10(3) to 10(4) transformants per micrograms of plasmid DNA. Vectors that contained the Saccharomyces cerevisiae URA3 gene could not transform C. tropicalis. DNA transfer was accomplished by modified versions of either spheroplast generation (CaCl2-polyethylene glycol)-fusion or cation (LiCl) procedures developed for S. cerevisiae. Plasmid vectors that had been cut within the C. tropicalis URA3 fragment integrated by homologous recombination at the URA3 locus.     

The adenylate kinase family in yeast: identification of URA6 as a multicopy suppressor of deficiency in major AMP kinase.

The gene URA6 encoding uridylate kinase (UK) from Saccharomyces cerevisiae was isolated as a multicopy suppressor of the respiratory-deficient phenotype of an S. cerevisiae mutant defective in the gene AKY2 encoding AMP kinase (AK). The URA6 gene also restored temperature resistance to two different temperature-sensitive mutations in the gene encoding Escherichia coli AK. By contrast, the gene encoding UK of Dictyostelium discoideum on a multicopy yeast shuttle plasmid, expressed under control of the constitutive yeast AKY2 promoter, failed to complement the deficiency in yeast, although such transformants expressed high UK activity. We show that yeast UK exerts significant AK activity which is responsible for the complementation and is absent in the analogous enzyme from D. discoideum. Since UK also significantly phosphorylates CMP (but not GMP), it must be considered an unspecific short-form nucleoside monophosphate kinase. Wild-type mitochondria lack UK activity, but import AKY2. Since multicopy transformation with URA6 heals the Pet- phenotype of AKY2 disruption mutants, the presence of AKY2 in the mitochondrial intermembrane space is not required to maintain respiratory competence. However, furnishing UK with the bipartite intermembrane space-targeting presequence of cytochrome c1 improves the growth rates of AKY2 mutants with nonfermentable substrates, suggesting that AK activity in mitochondria is helpful, though not essential for oxidative growth.     

Cloning of deoxynucleoside monophosphate kinase genes and biosynthesis of deoxynucleoside diphosphates

The genes encoding four deoxynucleoside monophosphate kinase (dNMP kinase) enzymes, including ADK1 for deoxyadenylate monophosphate kinase (AK), GUK1 for deoxyguanylate monophosphate kinase (GK), URA6 for deoxycytidylate monophosphate kinase (CK), and CDC8 for deoxythymidylate monophosphate kinase (TK), were isolated from the genome of Saccharomyces cerevisiae ATCC 2610 strain and cloned into E. coli strain BL21(DE3). Four recombinant plasmids, pET17b-JB1 containing ADK1, pET17b-JB2 containing GUK1, pET17b-JB3 containing URA6, and pET17b-JB4 containing CDC8, were constructed and transformed into E. coli strain for over-expression of AK, GK, CK, and TK. The amino acid sequences of these enzymes were analyzed and a putative conserved peptide sequence for the ATP active site was proposed. The four deoxynucleoside diphosphates (dNDP) including deoxyadenosine diphosphate (dADP), deoxyguanosine diphosphate (dGDP), deoxycytidine diphosphate (dCDP), and deoxythymidine diphosphate (dTDP), were synthesized from the corresponding deoxynucleoside monophosphates (dNMP) using the purified AK, GK, CK, and TK, respectively. The effects of pH and magnesium ion concentration on the dNDP biosynthesis were found to be important. A kinetic model for the synthetic reactions of dNDP was developed based on the Bi-Bi random rapid equilibrium mechanism. The kinetic parameters including the maximum reaction velocity and Michaelis-Menten constants were experimentally determined. The study on dNDP biosynthesis reported in this article are important to the proposed bioprocess for production of deoxynucleoside triphosphates (dNTP) that are used as precursors for in vitro DNA synthesis. There is a significant advantage of using enzymatic biosyntheses of dNDP as compared to the chemical method that has been in commercial use.     

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.     

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.     

Mutations in the Saccharomyces cerevisiae CDC1 gene affect double-strand-break-induced intrachromosomal recombination.

To isolate Saccharomyces cerevisiae mutants defective in recombinational DNA repair, we constructed a strain that contains duplicated ura3 alleles that flank LEU2 and ADE5 genes at the ura3 locus on chromosome V. When a HO endonuclease cleavage site is located within one of the ura3 alleles, Ura+ recombination is increased over 100-fold in wild-type strains following HO induction from the GAL1, 10 promoter. This strain was used to screen for mutants that exhibited reduced levels of HO-induced intrachromosomal recombination without significantly affecting the spontaneous frequency of Ura+ recombination. One of the mutations isolated through this screen was found to affect the essential gene CDC1. This mutation, cdc1-100, completely eliminated HO-induced Ura+ recombination yet maintained both spontaneous preinduced recombination levels and cell viability, cdc1-100 mutants were moderately sensitive to killing by methyl methanesulfonate and gamma irradiation. The effect of the cdc1-100 mutation on recombinational double-strand break repair indicates that a recombinationally silent mechanism other than sister chromatid exchange was responsible for the efficient repair of DNA double-strand breaks.     

Expression of a Trichoderma reesei beta-xylanase gene (XYN2) in Saccharomyces cerevisiae.

The XYN2 gene encoding the main Trichoderma reesei QM 6a endo-beta-1,4-xylanase was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 699-bp open reading frame that encodes a 223-amino-acid propeptide. The XYN2 gene, located on URA3-based multicopy shuttle vectors, was successfully expressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II (ADH2) and phosphoglycerate kinase (PGK1) gene promoters and terminators, respectively. The 33-amino-acid leader peptide of the Xyn2 beta-xylanase was recognized and cleaved at the Kex2-like Lys-Arg residues, enabling the efficient secretion and glycosylation of the heterologous beta-xylanase. The molecular mass of the recombinant beta-xylanase was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 27 kDa. The construction of fur1 ura3 S. cerevisiae strains allowed for the autoselection of the URA3-based XYN2 shuttle vectors in nonselective complex medium. These autoselective S. cerevisiae strains produced 1,200 and 160 nkat of beta-xylanase activity per ml under the control of the ADH2 and PGK1 promoters in rich medium, respectively. The recombinant enzyme showed highest activity at pH 6 and 60 degrees C and retained more than 90% of its activity after 60 min at 50 degrees C.     

Decreased UV mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae.

Summary A DNA replication mutant of yeast, cdc8, was found to decrease UV-induced reversion of lys2-1, arg4-17, tyr1 and ura1. This effect was observed with all three alleles of cdc8 tested. Survival curves obtained following UV irradiation in cdc8 rad double mutants show that cdc8 is epistatic to rad6, as well as to rad1; cdc8 rad51 double mutants seem to be more sensitive than the single mutants. Since UV-induced reversion in cdc8 rad1 and cdc8 rad51 double mutants is like that of the cdc8 single mutants, we conclude that CDC8 plays a direct role in error-prone repair. To test whether CDC8 codes for a DNA polymerase, we have purified both DNA polymerase I and DNA polymerase II from cdc8 and CDC+ cells. The purified DNA polymerases from cdc8 were no more heat labile than those from CDC+, suggesting that CDC8 is not a structural gene for either enzyme.     

One-step gene disruption by cotransformation to isolate double auxotrophs in Candida albicans

Summary The Candida albicans LEU2 gene was disrupted by substituting lambda DNA for a small deletion within the LEU2 gene. Cotransformation with a selectable URA3 ARS vector was used to introduce a linear fragment containing the disruption into the genome of a C. albicans ura3 deletion mutant. Cotransformants containing the lambda DNA were identified by colony hybridization and the URA3 plasmid was subsequently cured. Leu2 disrupted heterozygotes were detected by Southern hybridization and one disruptant was subsequently treated with UV irradiation. Only one leu2 ura3 mutant (SGY-484) was isolated out of 11,000 mutagenized cells. SGY-484 was transformed to Leu⁺ with either the C. albicans or Saccharomyces cerevisiae LEU2 gene. Southern hybridization analysis revealed that the mutant is not homozygous for the disruption; the leu2 mutation reverts and is most likely a point mutation. Unexpectedly, an ade2 ura3 mutant was isolated from the same mutagenesis.