Purification and characterization of a DNA helicase from Saccharomyces cerevisiae

A novel DNA helicase, scHelI, has been purified from whole cell extracts of Saccharomyces cerevisiae using biochemical assays to monitor the fractionation. The enzyme unwinds partial duplex DNA substrates, as long as 343 base pairs in length, in a reaction that is dependent on either ATP or dATP hydrolysis. scHelI also catalyzes a single-stranded DNA-dependent ATP hydrolysis reaction; the apparent Km for ATP is 325 microM. The unwinding reaction on circular partial duplex substrates is biphasic, with a fast component occurring within 5 min of the initiation of the reaction and a slow component continuing to 60 min. This is in contrast to the ATP hydrolysis reaction, which exhibits linear kinetics for 60 min. The direction of the unwinding reaction is 5' to 3' with respect to the strand of DNA on which the enzyme is bound. The unwinding reaction is strongly stimulated by the addition of Escherichia coli single-stranded DNA-binding protein when long partial duplex substrates are used. The enzymatic activity of scHelI copurifies with a polypeptide of 135 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide sediments as a monomer in a glycerol gradient in the presence of 0.2 M NaCl.     

Purification and characterization of DNA helicase III from the yeast Saccharomyces cerevisiae

Two forms of DNA helicase activity, Rad3 and ATPase III, were previously purified from the yeast Saccharomyces cerevisiae and characterized. Here, we have identified and purified an additional DNA helicase activity from S. cerevisiae to near homogeneity. This helicase differs from those described previously in its chromatographic behavior, molecular weight, enzymatic properties, and genetic properties. Thus, we named it DNA helicase III. Its apparent molecular mass is about 120 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. DNA helicase III requires a divalent cation Mg2+ or Mn2+, either ATP or dATP, and a single-stranded portion on the duplex substrate. Helicase III moves in the 5'-->3' direction on single-stranded portions of the substrate and unwinds the strand of DNA in the 3'-->5' direction. It also has an intrinsic DNA-dependent ATPase (dATPase) activity that hydrolyzes either ATP or dATP to ADP or dADP and orthophosphate in the presence of DNA. DNA helicase III activity was not affected by either rad3 or radH mutations, suggesting that it is encoded by a gene different from RAD3 and RADH.     

Identification of a poxvirus gene encoding a uracil DNA glycosylase

An open reading frame, BamHI D6R, from the central highly conserved region of the Shope fibroma virus (SFV) genome was sequenced and found to have significant homology to that of uracil DNA glycosylases from a number of organisms. Uracil DNA glycosylase catalyzes the initial step in the repair pathway that removes potentially mutagenic uracil from duplex DNA. The D6R polypeptide was expressed in reticulocyte lysates programmed with RNA transcribed from an expression vector containing the T7 RNA polymerase promoter. A highly specific ethidium bromide fluorescence assay of the in vitro translation product determined that the encoded protein does indeed possess uracil DNA glycosylase activity. Open reading frames from other poxviruses, including vaccinia virus (HindIII D4R) and fowlpox (D4), are highly homologous to D6R of SFV and are predicted to encode uracil DNA glycosylases. Identification of the SFV uracil DNA glycosylase provides evidence that this poxviral protein is involved in the repair of the viral DNA genome. Since this enzyme performs only the initial step required for the removal of uracil from DNA, creating an apyrimidinic site, we suggest that other, possibly virus-encoded, repair activities must be present in the cytoplasm of infected cells to complete the uracil excision repair pathway.     

Cloning and genetic analysis of the gene encoding a new protein kinase in Saccharomyces cerevisiae

The growth of the body was studied in 30 human fetuses ranged from 10 to 22 weeks of gestation. The fetuses were fixed by immersion in 4 percent formaldehyde and the following dimensions were studied: a) lengths: arm, forearm, hand, thigh, leg, foot and crown-rump (sitting height), b) perimeters: head, thorax and abdomen. A covariance matrix was calculated from natural logarithms of all measurements. The relative growth of these measurement was computed by multivariate allometry using a principal components analysis (PCA). All characters were positively correlated with the first principal component which accounted for 94.65 per cent of the total variance. Considering the different measurements in the sequence of the increasing growth rates no one was considered to increase in isometric relationship. PCA showed that the following measurements grew with negative allometry: head perimeter, C-R length, thoracic perimeter, length of the forearm and abdominal perimeter. On the other hand, the following lengths grew with positive allometry: hand, foot, thigh, arm and leg. In conclusion, during the first two trimesters of prenatal life the growth of the body is allometrical. Limbs increase with greater growth rates than the perimeters of the body cavities.     

Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis

The Saccharomyces cerevisiae gene, YFL017C, for a putative acetyltransferase was characterized. Disruption of YFL017C was lethal, leading to a morphology similar to those caused by the depletion of AGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis. The recombinant protein for YFL017C displayed phosphoglucosamine acetyltransferase activities in vitro and utilized glucosamine 6-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine 6-phosphate. These results indicate that YFL017C specifies glucosamine-6-phosphate acetyltransferase; therefore, the gene was designated GNA1 (glucosamine-6-phosphate acetyltransferase). In addition, whereas bacterial phosphoglucosamine acetyltransferase and UDP-N-acetylglucosamine pyrophosphorylase activities are intrinsic in a single polypeptide, they are encoded by distinct essential genes in yeast. When the sequence of ScGna1p was compared with those of other acetyltransferases, Ile97, Glu98, Val102, Gly112, Leu115, Ile116, Phe142, Tyr143, and Gly147 were found to be highly conserved. When alanine was substituted for these amino acids, the enzyme activity for the substituted Phe142 or Tyr143 enzymes was severely diminished. Although the activity of Y143A was too low to perform kinetics, F142A displayed a significantly increased Km value for acetyl-CoA, suggesting that the Phe142 and Tyr143 residues are essential for the catalysis.     

Isolation and characterization of the Saccharomyces cerevisiae DPP1 gene encoding diacylglycerol pyrophosphate phosphatase

Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel lipid signaling pathway. DGPP phosphatase (DGPP phosphohydrolase) is a membrane-associated 34-kDa enzyme from Saccharomyces cerevisiae which catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol. Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1 (diacylglycerol pyrophosphate phosphatase) gene encoding the enzyme. Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in S. cerevisiae. The heterologous expression of the S. cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type S. cerevisiae. DGPP phosphatase possesses a Mg2+-independent PA phosphatase activity, and its expression correlated with the overexpression of DGPP phosphatase activity in S. cerevisiae and in insect cells. DGPP phosphatase was predicted to be an integral membrane protein with six transmembrane-spanning domains. The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases. A dpp1Delta mutant was constructed by deletion of the chromosomal copy of the DPP1 gene. The dpp1Delta mutant was viable and did not exhibit any obvious growth defects. The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold) DGPP. Analysis of the mutant showed that the DPP1 gene was not responsible for all of the Mg2+-independent PA phosphatase activity in S. cerevisiae.     

Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase

We have cloned the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to remove the two C-4 methyl groups leading to cholesterol (animal), ergosterol (fungal), and stigmasterol (plant) biosynthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzy-mosterol, was isolated after mutagenesis. A complementing clone consisting of a 1.35-kb Dra I fragment encoded a 309-amino acid polypeptide (calculated molecular mass, 36.48 kDa). The amino acid sequence shows a C-terminal endoplasmic reticulum retrieval signal KKXX and three histidine-rich clusters found in eukaryotic membrane desaturases and in a bacterial alkane hydroxylase and xylene monooxygenase. The sterol profile of an ERG25 disruptant was consistent with the erg25 allele obtained by mutagenesis.     

Molecular cloning of the actin gene from yeast Saccharomyces cerevisiae

Two overlapping DNA fragments from yeast Saccharomyces cerevisiae containing the actin gene have been inserted into pBR322 and cloned in E.coli. Clones were identified by hybridization to complementary RNA from a plasmid containing a copy of Dictyostelium actin mRNA. One recombinant plasmid obtained (pYA102) contains a 3.93-kb Hindlll fragment, the other (pYA208) a 5.1-kb Pstl fragment, both share a common 2.2-kb fragment harboring part of the actin gene. Cloned yeast actin DNA was identified by R-loop formation and translation of the hybridized actin mRNA and by DNA sequence analysis. Cytoplasmic actin mRNA has been estimated to be about 1250 nucleotides long. There is only one type of the actin gene in S.cerevisiae.     

Isolation and sequence of the gene encoding ornithine decarboxylase, SPE1, from Candida albicans by complementation of a spe1 delta strain of Saccharomyces cerevisiae

The gene encoding ornithine decarboxylase, SPE1, from the pathogenic yeast Candida albicans has been isolated by complementation of an ornithine decarboxylase-negative (spe1 delta) strain of Saccharomyces cerevisiae. Four transformants, three of which contain plasmids with the SPE1 gene, were isolated by selection on polyamine-free medium. The C. albicans ornithine decarboxylase (ODC) showed high homology with other eukaryotic ODCs at both the amino acid and nucleic acid levels.     

Riboflavin biosynthesis in Saccharomyces cerevisiae. Cloning, characterization, and expression of the RIB5 gene encoding riboflavin synthase

Saccharomyces cerevisiae has a monofunctional riboflavin synthase that catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine. We have isolated the gene encoding this enzyme from a yeast genomic library by functional complementation of a mutant, rib5-10, lacking riboflavin synthase activity. Deletion of the chromosomal copy of RIB5 led to riboflavin auxotrophy and loss of enzyme activity. Intragenic complementation between point and deletion mutant alleles suggested that the encoded protein (Rib5p) assembles into a multimeric complex and predicted the existence of a discrete functional domain located at the N terminus. Nucleotide sequencing revealed a 714-base pair open reading frame encoding a 25-kDa protein. Rib5p was purified to apparent homogeneity by a simple procedure. The specific activity of the enzyme was enriched 8500-fold. The N-terminal sequence of the purified enzyme was identical to the sequence predicted from the nucleotide sequence of the RIB5 gene. Initial structural characterization of riboflavin synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a trimer of identical 25-kDa subunits. The derived amino acid sequence of RIB5 shows extensive homology to the sequences of the alpha subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes. In addition, the sequence also shows internal homology between the N-terminal and the C-terminal halves of the protein. Taken together, these results suggest that the Rib5p subunit contains two structurally related (substrate-binding) but catalytically different (acceptor and donator) domains.     

Sequence analysis of DNA randomly amplified from the Saccharomyces cerevisiae genome

OBJECTIVES: To report the features of malignancies responsible for a chest wall mass and involving the sternum, the sternocostal and/or sternoclavicular joints, the chondrocostal junction and/or the adjacent soft tissues. METHODS: The medical records of patients with a chest wall mass due to malignant disease were reviewed retrospectively. The following data were abstracted from each record: characteristics of the pain and mass, constitutional symptoms, physical findings, laboratory test results, findings from imaging studies (plain radiographs, computed tomography and magnetic resonance imaging of the chest, radionuclide bone scan), histologic features of the biopsy specimen from the chest wall mass and origin of the mass. RESULTS: Seven men and three women with a mean age of 53.1 years were included in the study. A single patient had a history of malignant disease (lymphoma); in the remaining nine patients the chest wall mass was the first manifestation of the malignancy. All ten patients had pain with a mixed time pattern. The mass was located on the sternum in half the patients and in a parasternal location in the other half. Erythrocyte sedimentation rate elevation was found in seven patients, an increased serum level of lactate dehydrogenase in one and a monoclonal immunoglobulin in three. Sternal lesions were visible on plain radiographs in four patients. Computed tomography of the chest consistently disclosed sternal or sternocostal lytic lesions with spread to the adjacent soft tissues; in five cases, enlarged lymph nodes were visible in the anterior part of the mediastinum. Magnetic resonance imaging of the chest did not add to the information provided by computed tomography. Radionuclide uptake on the bone scan was increased, decreased, or normal at the site of the lesion. The cause was Hodgkin's disease in two cases, non-Hodgkin's lymphoma in three, metastatic bone disease in two (from an adenocarcinoma of the lung and a hepatocarcinoma, respectively), multiple myeloma in one, and solitary plasmacytoma in two. CONCLUSION: A chest wall mass can be caused by a known or as yet undiagnosed malignancy. Chest wall involvement due to malignant disease in rare, however. The specific features of sternal metastases, lymphomas involving the sternum, and sternal plasmacytomas are discussed. Nonmalignant chest wall lesions that can manifest as a bulging or swelling of the chest wall are reviewed.     

Isolation of the ALG5 locus encoding the UDP-glucose:dolichyl-phosphate glucosyltransferase from Saccharomyces cerevisiae

Cataract extraction and posterior chamber intraocular lens (PC-IOL) implantation was carried out in 45 glaucoma eyes that had undergone glaucoma filtering surgery (Group A), and in 47 glaucoma eyes in which intraocular pressure (IOP) was well controlled with medication (Group B). To analyze the effects of PC-IOL implantation on the control of IOP and the functioning of the filtering bleb, a life-table analysis using the Kaplan-Meier method was carried out. In Group A the probability that IOP control will not worsen at 2 years was 56 +/- 12 (SE)% in eyes where a functioning filtering bleb had existed preoperatively. The probability that the filtering bleb was not cicatrized at 2 years postoperatively was 44 +/- 11 (SE)%. In Group B the postoperative IOP on the first postoperative day was significantly higher than the preoperative level in primary open angle glaucoma (POAG) eyes, while no such difference was seen in primary angle closure glaucoma (PACG) eyes. The postoperative IOP was significantly lower than the preoperative level from 3 to 6 months postoperatively in POAG eyes and from 1 to 12 months postoperatively in PACG eyes. Medication did not differ significantly pre- and postoperatively. In 64 +/- 11 (SE)% of POAG and 63 +/- 15% of PACG eyes the IOP control did not worsen at 2 years. In 70% of the eyes the IOP control improved postoperatively, and was maintained for 2 years with the probability of 44 +/- 12 (SE)%.     

Identification of a new nuclear gene (CEM1) encoding a protein homologous to a beta-keto-acyl synthase which is essential for mitochondrial respiration in Saccharomyces cerevisiae

We have analysed a new gene, CEM1, from Saccharomyces cerevisiae. Inactivation of this gene leads to a respiratory-deficient phenotype. The deduced protein sequence shows strong similarities with beta-keto-acyl synthases or condensing enzymes. Typically, enzymes of this class are involved in the synthesis of fatty acids or similar molecules. An analysis of the mitochondrial lipids and fatty acids shows no major difference between the wild type and deleted strains, implying that the CEM1 gene product is not involved in the synthesis of the bulk fatty acids. Thus it is possible that the CEM1 protein is involved in the synthesis of a specialized molecule, probably related to a fatty acid, which is essential for mitochondrial respiration.