Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis

All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1Delta strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4beta, 14alpha-dimethyl-4alpha-carboxy-cholesta-8,24-dien-3be ta-ol and 4beta-methyl-4alpha-carboxy-cholesta-8,24-dien-3beta-o l, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.     

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.     

Cloning, characterization and identification of the gene encoding phosphatidylinositol 4-kinase

The vast majority of AIDS-related deaths are associated with opportunistic infections. For fungal infections, there are few effective antifungals, particularly for systemic use. The discovery that very low doses of the bleomycin family of anticancer chemical congeners compromise the integrity of fungal cell walls led to our approach to identify genes that complement-cell wall defects, and develop methods to facilitate the identification of new antifungals targeted to fungal cell walls. This report describes one of the genes cloned by complementation of the blm1-1 mutation of S. cerevisiae using a YCp50-based yeast genomic library. Characterization and identification of the gene were carried out using drug screening tests, Southern hybridization analyses, DNA sequencing and DNA sequence similarity searches in databases. The gene STT4, is essential for viability and encodes a phosphatidylinositol 4-kinase that plays an important role in the phosphatidylinositol-mediated signal transduction pathway required for cell wall integrity. Like blm1-1 mutant strains, stt4 cells arrest mostly in the G2/M phase of the cell cycle. Further studies using this approach should help us understand the role of PI4-K in maintaining fungal cell-wall integrity, identify additional genes affecting potential target structures in cell walls of opportunistic fungal pathogens in AIDS patients, and assist in drug discovery and antifungal drug design.     

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.     

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 the human and Caenorhabditis elegans homologs of the Saccharomyces cerevisiae MSH5 gene

Genetic variation among malaria parasites has important consequences with regard to drug resistance, pathogenicity, immunity, transmission, and speciation. In this regard, malaria parasites have been shown to display a high degree of inter- and intra-species genetic divergence. The nuclear genomes of Plasmodium falciparum, Plasmodium yoelii, and Plasmodium gallinaceum are vastly divergent yet share a similar codon usage and total A/T content of approximately 82%. This is in contrast to other primate-specific species including P. vivax which have an A/T content of approximately 67%. To assess the effects of this evolutionary divergence on the conservation of gene content, organization, and codon usage in the mitochondrial DNA (mtDNA) of malaria parasites, we have cloned and sequenced the mitochondrial genome of Plasmodium vivax, and compared it with the mtDNAs of P. falciparum, P. yoelii, and P. gallinaceum. The P. vivax mitochondrial genome was found to be 5990 base pairs in length, and displayed a gene organization identical to that of P. falciparum, P. yoelii, and P. gallinaceum. Furthermore, there was a remarkable 90% conservation of sequence identity between the mitochondrial genomes of all four species. As an example of intra-species conservation, comparison of mtDNAs from two independently cloned P. falciparum isolates, Malay Camp and C10, revealed only a single nucleotide substitution. A/T content of the P. vivax mitochondrial genome was found to be identical to other species of Plasmodium, hence, we have postulated that the mitochondrial genomes of malaria parasites were refractory to the evolutionary shifts in nucleotide content seen among the nuclear genomes of malaria parasites. Among different Plasmodium species, the second position of mitochondrial codons were found to be the least prone to substitutions and displayed a significant bias in pyrimidines. These aspects of mitochondrial codon usage were distinct from the nuclear genome and may reflect functional aspects of decoding by the mitochondrial translational system.     

Identification of the gene encoding scHelI, a DNA helicase from Saccharomyces cerevisiae

The gene encoding scHelI, a previously characterized DNA helicase from Saccharomyces cerevisiae, has been identified as YER176w, an open reading frame on chromosome V. The gene has been named HEL1 to indicate the DNA helicase activity of the gene product. HEL1 was identified by screening a lambda gt11 yeast protein expression library with antiserum to purified scHelI. Several independent immunopositive clones were isolated and shown to contain portions of HEL1 either by sequencing or by hybridization to a probe containing HEL1 sequences. The HEL1 open reading frame includes the seven conserved helicase motifs, consistent with the DNA helicase activity of scHelI, and the predicted size of the protein is in agreement with the size of purified scHelI. Partially purified cellular extracts from a hel1 deletion mutant strain did not contain scHelI activity. Homology searches revealed protein sequence homology between HEL1 and two previously identified and biochemically characterized yeast helicases, encoded by the DNA2 and UPF1 genes. Haploid hel1 deletion strains were constructed and shown to be viable with growth rates equivalent to those of parental strains. These strains did not differ from the parental strains in ultraviolet light sensitivity or the generation of petite colonies. Furthermore, these haploid deletion strains were capable for mating, the resultant diploid homozygous mutants were viable, capable of sporulation, and the spores displayed no reduction in viability.     

Cloning and characterization of the gene encoding human osteoprotegerin/osteoclastogenesis-inhibitory factor

Cri-du-chat is a human contiguous gene deletion syndrome resulting from hemizygous deletions of chromosome 5p. Here we describe the isolation from within this interval of the human Semaphorin F (SEMAF) gene, a member of a family of proteins that has been implicated in axonal pathfinding. The human SEMAF gene covers at least 10% of the deleted region and defines a new class within this large gene family characterized by the presence of seven type 1 thrombospondin repeats. Prominent expression of murine semaphorin F (Semaf) was observed in the mouse brain, consistent with a role for semaphorin F as a signaling molecule that guides axons or migrating neuronal precursors during development. The known functions of semaphorins and the interesting pattern of expression for Semaf suggest that haploinsufficiency for SEMAF may disrupt normal brain development and might lead to some of the features of Cri-du-chat.     

Expression, purification, and characterization of choline kinase, product of the CKI gene from Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) is the product of the CKI gene. Choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine by the CDP-choline pathway. The yeast enzyme was overexpressed 106-fold in Sf-9 insect cells and purified 71.2-fold to homogeneity from the cytosolic fraction by chromatography with concanavalin A, Affi-Gel Blue, and Mono Q. The N-terminal amino acid sequence of purified choline kinase matched perfectly with the deduced sequence of the CKI gene. The minimum subunit molecular mass (73 kDa) of purified choline kinase was in good agreement with the predicted size (66.3 kDa) of the CKI gene product. Native choline kinase existed in oligomeric structures of dimers, tetramers, and octomers. The amounts of the tetrameric and octomeric forms increased in the presence of the substrate ATP. Antibodies were raised against the purified enzyme and were used to identify choline kinase in insect cells and in S. cerevisiae. Maximum choline kinase activity was dependent on Mg2+ ions (10 mM) at pH 9.5 and at 30 degrees C. The equilibrium constant (0.2) for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 6.26 kcal/mol, and the enzyme was labile above 30 degrees C. Choline kinase exhibited saturation kinetics with respect to choline and positive cooperative kinetics with respect to ATP (n = 1.4-2.3). Results of the kinetic experiments indicated that the enzyme catalyzes a sequential Bi Bi reaction. The Vmax for the reaction was 138.7 micromol/min/mg, and the Km values for choline and ATP were 0.27 mM and 90 microM, respectively. The turnover number per choline kinase subunit was 153 s-1. Ethanolamine was a poor substrate for the purified choline kinase, and it was also poor inhibitor of choline kinase activity. ADP inhibited choline kinase activity (IC50 = 0.32 mM) in a positive cooperative manner (n = 1.5), and the mechanism of inhibition with respect to ATP and choline was complex. The regulation of choline kinase activity by ATP and ADP may be physiologically relevant.     

A mutation in a purported regulatory gene affects control of sterol uptake in Saccharomyces cerevisiae

Aerobically growing wild-type strains of Saccharomyces cerevisiae are unable to take exogenously supplied sterols from media. This aerobic sterol exclusion is vitiated under anaerobic conditions, in heme-deficient strains, and under some conditions of impaired sterol synthesis. Mutants which can take up sterols aerobically in heme-competent cells have been selected. One of these mutations, designated upc2-1, gives a pleiotropic phenotype in characteristics as diverse as aerobic accumulation of sterols, total lipid storage, sensitivity to metabolic inhibitors, response to altered sterol structures, and cation requirements. During experiments designed to ascertain the effects of various cations on yeast with sterol alterations, it was observed that upc2-1 was hypersensitive to Ca2+. Using resistance to Ca2+ as a screening vehicle, we cloned UPC2 and showed that it is YDR213W, an open reading frame on chromosome IV. This belongs to a fungal regulatory family containing the Zn(II)2Cys6 binuclear cluster DNA binding domain. The single guanine-to-adenine transition in upc2-1 gives a predicted amino acid change from glycine to aspartic acid. The regulatory defect explains the semidominance and pleiotropic effects of upc2-1.     

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.     

Cloning and chromosomal localization of a mouse cDNA with homology to the Saccharomyces cerevisiae gene zuotin

The eukaryotic DnaJ homologs form a family of proteins with diverse functions. One member of the family, the Saccharomyces cerevisiae gene zuotin, was isolated for its ability to bind Z-DNA. Here, we have isolated a mouse cDNA called ZRF1 (for zuotin-related factor1) with significant homology to zuotin. The DnaJ domain and candidate phosphorylation sites of zuotin and ZRF1 are highly conserved. ZRF1 gene is localized on chromosome 5. The structural similarity of zuotin and ZRF1 suggests conservation of function of this DnaJ subfamily.     

Cloning and characterization of the gene encoding a new DNA methyltransferase from Neisseria gonorrhoeae

A HindIII fragment of N. gonorrhoeae MS11 DNA coding for DNA methyltransferase (MTase) activity was cloned and expressed in E. coli AP1-200-9 cells. The sequence of 4681 bp was determined, and its analysis revealed two open reading frames (ORFs) sharing some similarity with known DNA MTases. ORF1 encodes an active N4mC MTase (M.NgoMV). The enzyme modifies only one strand of double stranded DNA and preferentially recognises the sequence GCCHR although it is able to methylate other sites. The exact recognition sequence cannot be precisely defined due to a relaxed specificity. The second ORF shows high homology to 5mC Mtases, but we were unable to demonstrate DNA methylating activity of its product either in vivo or in vitro.