Biomedical subjects

W A Fonzi

Publications and source records attributed to W A Fonzi.

36 records · Page 2Linked to original sources

PubMed1995

Use of URA3 as a reporter of gene expression in C. albicans.

The C. albicans URA3 gene was tested as a reporter of gene expression. An integrating vector was constructed which contained ADE2 as a selectable marker together with a truncated form of URA3 lacking the first three codons. A DNA fragment containing the promoter and the first 90 codons of the C. albicans CEF3 gene was inserted into the unique XhoI site 5' to URA3 in order to provide an in-frame translational fusion. The functionality of the fusion gene was tested following integration of a single copy of the plasmid into the ADE2 locus. The fusion gene was shown to complement a ura3 deletion mutation and to produce orotidine 5'-monophosphate decarboxylase activity (OMP), which is encoded by URA3. Expression of the fusion gene was appropriately regulated by the growth rate and utilized the same transcriptional start sites as the native CEF3 gene. The results demonstrated that URA3 provides a sensitive and versatile reporter gene for use in C. albicans.

Base Sequence↗

PubMed1995

Reduced virulence of Candida albicans PHR1 mutants.

Candida albicans mutants lacking PHR1 exhibit a pH-dependent morphogenic defect which is expressed at pH 7.5, a pH comparable to that of mammalian blood (S. M. Saporito-Irwin, C. E. Birse, P. S. Sypherd, and W. A. Fonzi, Mol. Cell. Biol. 15:601-613, 1995). The in vivo relevance of this expression pattern was tested in a mouse model of systemic candidiasis. A phr1/phr1 mutant was found to be less virulent than an isogenic Phr1+ strain and exhibited altered morphological development in vivo. These results indicate that PHR1 contributes to the virulence of C. albicans.

Animals↗

PubMed1995

PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis.

Candida albicans, like many fungi, exhibits morphological plasticity, a property which may be related to its biological capacity as an opportunistic pathogen of humans. Morphogenesis and alterations in cell shape require integration of many cellular functions and occur in response to environmental signals, most notably pH and temperature in the case of C. albicans. In the course of our studies of differential gene expression associated with dimorphism of C. albicans, we have isolated a gene, designated PHR1, which is regulated in response to the pH of the culture medium. PHR1 expression was repressed at pH values below 5.5 and induced at more alkaline pH. The predicted amino acid sequence of the PHR1 protein was 56% identical to that of the Saccharomyces cerevisiae Ggp1/Gas1 protein, a highly glycosylated cell surface protein attached to the membrane via glycosylphosphatidylinositol. A homozygous null mutant of PHR1 was constructed and found to exhibit a pH-conditional morphological defect. At alkaline pH, the mutant, unlike the parental type, was unable to conduct apical growth of either yeast or hyphal growth forms. This morphological aberration was not associated with defective cytoskeletal polarization or secretion. The results suggest that PHR1 defines a novel function required for apical cell growth and morphogenesis.

Amino Acid Sequence↗

PubMed1994

A hyphal-specific chitin synthase gene (CHS2) is not essential for growth, dimorphism, or virulence of Candida albicans.

In the dimorphic fungus Candida albicans, the CHS2 gene encodes a chitin synthase that is expressed preferentially in the hyphal form. Gene disruption of CHS2 in this diploid asexual fungus was achieved by the "ura-blaster" protocol described for Saccharomyces [Alani, E., Cao, L. & Kleckner, N. (1987) Genetics 116, 541-545]. This involves the sequential disruption of multiple alleles by integrative transformation with URA3 as a single selectable marker. After disrupting the first CHS2 allele, the Ura- phenotype was recovered through cis recombination between repeated hisG sequences that flanked the URA3 marker in the disruption cassette, which was then used again to disrupt further CHS2 alleles. This method of gene disruption is well suited to the mutational analysis of this genetically recalcitrant human pathogen. Three rounds of disruption were required, suggesting that the strain SGY243 is triploid for the CHS2 locus. The resulting homozygous delta chs2::hisG null mutants were viable and made germ tubes with a normal morphology. The germ tubes were formed more slowly than parental strains in serum-containing medium and the germinating cells had a 40% reduction in their chitin content compared to germ tubes of the parent strain. The chitin content of the yeast form was not affected. A prototrophic strain of the chs2 null mutant was not attenuated significantly in its virulence when tested in normal and immunosuppressed mice.

Animals↗

PubMed1993

Isogenic strain construction and gene mapping in Candida albicans.

Genetic manipulation of Candida albicans is constrained by its diploid genome and asexual life cycle. Recessive mutations are not expressed when heterozygous and undesired mutations introduced in the course of random mutagenesis cannot be removed by genetic back-crossing. To circumvent these problems, we developed a genotypic screen that permitted identification of a heterozygous recessive mutation at the URA3 locus. The mutation was introduced by targeted mutagenesis, homologous integration of transforming DNA, to avoid introduction of extraneous mutations. The ura3 mutation was rendered homozygous by a second round of transformation resulting in a Ura- strain otherwise isogenic with the parental clinical isolate. Subsequent mutation of the Ura- strain was achieved by targeted mutagenesis using the URA3 gene as a selectable marker. URA3 selection was used repeatedly for the sequential introduction of mutations by flanking the URA3 gene with direct repeats of the Salmonella typhimurium hisG gene. Spontaneous intrachromosomal recombination between the flanking repeats excised the URA3 gene restoring a Ura- phenotype. These Ura- segregants were selected on 5-fluoroorotic acid-containing medium and used in the next round of mutagenesis. To permit the physical mapping of disrupted genes, the 18-bp recognition sequence of the endonuclease I-SceI was incorporated into the hisG repeats. Site-specific cleavage of the chromosome with I-SceI revealed the position of the integrated sequences.

Base Sequence↗

PubMed1993

Cloning and characterization of ECE1, a gene expressed in association with cell elongation of the dimorphic pathogen Candida albicans.

The gene ECE1 (extent of cell elongation 1) was isolated by differential hybridization screening of a Candida albicans cDNA library by using probes derived from populations of yeast cells or hyphae. Expression of this gene was not detected when C. albicans grew as a budding yeast cell but was observed within 30 min after cells had been induced to form hyphae. In all strains tested, regardless of the induction signal, ECE1 expression correlated with the extent of cell elongation. The genomic version of ECE1 was cloned and sequenced. The deduced 271-amino-acid polypeptide consisted of eight tandem repeats of a degenerate 34-amino-acid sequence which contained no discernible homology with other known sequences. An ECE1 null mutant displayed no morphological alterations, which demonstrated that ECE1 is not essential for cell elongation or hypha formation despite the strict morphological association of its expression.

Amino Acid Sequence↗

PubMed1992

Isolation and sequence analysis of the gene for translation elongation factor 3 from Candida albicans.

Elongation factor 3 (EF-3) is a unique and essential component of the translational system in fungi. The gene, CEF-3, encoding elongation factor 3 has been isolated from the dimorphic fungus Candida albicans. A heterologous gene probe containing the coding region of the EF-3 gene from Saccharomyces cerevisiae (YEF-3) was used to screen three Candida albicans genomic DNA libraries. The nucleotide sequences of four partial clones were determined and combined for a full-length of 3,671 base pairs (bp). A continuous open reading frame (ORF) of 3,147 bp encoding a predicted protein of 1,049 amino acids and Mr of 116,739 daltons has been identified. A transcript of 3,400 nucleotides is seen in Northern blot hybridization of Candida albicans total RNA using a CEF-3 gene probe. The single locus CEF-3 gene maps to chromosome 5 in the genome. Comparison of the deduced amino acid sequences of CEF-3 and YEF-3 shows 77.6%. identity. A higher degree of identity, 86.5%, is found when comparing the carboxy-terminal portions of the two proteins. At the nucleotide level, comparison of the coding regions of the two genes exhibit 79% identity while the upstream and downstream regions show 46% and 40% identity, respectively.

Amino Acid Sequence↗

PubMed1992

Fungus-specific translation elongation factor 3 gene present in Pneumocystis carinii.

Historically, Pneumocystis carinii pneumonia has been the most frequent cause of morbidity and mortality in patients with AIDS. Antiprotozoan drugs are effective in the treatment and prophylaxis of P. carinii pneumonia, which lends credence to the widely held view that P. carinii is a protozoan. However, recent genetic evidence suggests that P. carinii should be classified as a fungus. Translation elongation factor 3 (EF-3) is an essential, soluble translation component which is unique to fungal protein synthesis and is not required for protein synthesis in other eukaryotes. We have identified and isolated a gene for EF-3 from P. carinii, adding more evidence for this organism's assignment as a fungus.

Amino Acid Sequence↗

PubMed1992

A temperature-regulated, retrotransposon-like element from Candida albicans.

A repetitive element was isolated from the genome of Candida albicans. This repetitive element, which we designated alpha, was localized to a 500-bp fragment of genomic DNA. The alpha element was dispersed in the genome and varied in copy number and genomic location in the strains examined. Analyses of various loci containing the alpha element identified a locus containing a composite element. This composite element consisted of two direct repeats of the alpha element separated by approximately 5.5 kb of DNA, a structural arrangement similar to that of retrovirus-like transposable elements. The flanking alpha elements of the composite structure were 388 bp in length and were identical in sequence. They were bounded by the nucleotides 5'-TG. ... CA-3', which were part of a delimiting inverted repeat, a feature conserved in the long terminal repeats of retroviruses and retrovirus-like elements. As in retrovirus-like elements, the entire composite element, including the alpha elements, was transcribed into an approximately unit-length mRNA. The expression of this transcript was greatly increased when cells were grown at 25 versus 37 degrees C. As has been found in many retrotransposons, the composite element was flanked by a 5-bp duplication and varied in both copy number and genomic location in various strains. We conclude that the composite element is a retrotransposon-like element, and we have designated this element Tca1. We suggest that Tca1 may be relevant to the genomic evolution of C. albicans and the pathogenic potential of the organism.

Base Sequence↗

PubMed1989

Structure of a ribosomal protein gene in Mucor racemosus.

As an extension of our analysis of the translational apparatus of Mucor racemosus we have isolated a gene encoding a ribosomal protein of Mucor. Based on a method developed for S. cerevisiae, we identified by hybrid selection and in vitro translation a lambda-Charon 4A clone containing the genomic copy of a Mucor ribosomal protein. The gene consisted of two exons of 57 and 387 nucleotides. The two exons were separated by an 131 nucleotide intron. The processed transcript was 714 nucleotides in length and contained a 25 nucleotide untranscribed leader and an 114 nucleotide untranscribed 3'-end. The protein predicted from the nucleotide sequence contained 148 amino acids and exhibited 61% identity with the S19 ribosomal protein of Xenopus laevis. The promoter region of the gene contained sequences highly homologous to the RPG and Homol1 promoter elements found in S. cerevisiae. Southern blot analysis indicated that the Mucor genome contains three copies of this gene.

Amino Acid Sequence↗

PubMed1989

Regulation of Saccharomyces cerevisiae ornithine decarboxylase expression in response to polyamine.

The mechanism of yeast ornithine decarboxylase (ODC) regulation in response to polyamines was examined. ODC catalyzes the first step of polyamine biosynthesis, the conversion of ornithine to putrescine. ODC activity was modulated approximately 200-fold by varying the availability of polyamines. Variations in ODC activity were associated with parallel changes in the amount of active enzyme molecules. Regulation of ODC activity did not appear to be mediated by changes in the stability of the enzyme. The half-life of ODC was approximately 75 min and was unaffected by the availability of polyamines. Polyamines had no demonstrable effect on the transcriptional or translational stages of ODC expression. The amount of ODC mRNA and the number of ribosomes associated with the mRNA were constant despite the variations in ODC activity. Unlike translationally controlled genes, the regulation of ODC expression did not require either the 5'- or the 3'-untranslated regions of the ODC gene transcript. While gene dosage experiments indicated that negative trans-acting factors are involved in control of ODC expression, these implied factors did not appear to affect translation of ODC mRNA. The results are interpreted to suggest that polyamines regulate ODC expression at a post-translational step prior to assembly of the active form of the enzyme.

Gene Expression Regulation, Enzymologic↗

PubMed1989

Biochemical and genetic characterization of the structure of yeast ornithine decarboxylase.

The ornithine decarboxylase gene of S. cerevisiae encodes a predicted protein of approximately 53 kD highly homologous with the ornithine decarboxylase of other species. However, the native enzyme has been reported as an 86 kD protein. Our molecular sieve analysis indicated a Mr = 110,000 for the native enzyme. SDS-PAGE analysis of [H3]-alpha-difluoromethylornithine labelled enzyme demonstrated a subunit Mr of approximately 50 kD and suggested the native enzyme is a dimer. Genetic analyses support this conclusion. The complementary, ornithine decarboxylase deficient mutations spe 1A and spe 1B were mapped to the enzyme structural gene by linkage analysis and gene conversion mapping. This demonstrated that the mutations exhibit intragenic complementation which suggests protein-protein interactions and an oligomeric structure for the yeast enzyme. We conclude that yeast ornithine decarboxylase is a dimeric enzyme of 53 kD subunits.

Chromatography, Gel↗

PubMed1987

The gene and the primary structure of ornithine decarboxylase from Saccharomyces cerevisiae.

The nucleotide sequence was determined for a 3-kilobase genomic fragment containing the ornithine decarboxylase gene of Saccharomyces cerevisiae. The fragment contained two open reading frames. Gene disruption localized the ornithine decarboxylase gene to a 1398-nucleotide open reading frame. Transcription of the yeast gene initiated at several sites 171 to 211 nucleotides 5' of the translational start site. The 3' end of the transcript extended approximately 300 nucleotides beyond the end of the ornithine decarboxylase coding region and contained two copies of the yeast ARS core sequence. Translation of the ornithine decarboxylase gene appeared to initiate at the first AUG condon of the open reading frame based upon translational fusions with the Escherichia coli beta-galactosidase gene. Since no introns were apparent, the 1398-nucleotide open reading frame was predicted to encode a 466-amino acid protein with a calculated Mr = 52,369. The deduced protein differed significantly in size from previous reports on yeast ornithine decarboxylase, but was very similar in size to mammalian ornithine decarboxylase. When the predicted amino acid sequence of yeast ornithine decarboxylase was compared with that of the mouse enzyme, alignment of the sequences revealed that 40% of the amino acid residues were identical. Chou-Fasman predictions of the secondary structure of the two enzymes indicated that secondary structure was also highly conserved.

Amino Acid Sequence↗

PubMed1986

Trichodermin esterase activity and trichodermin resistance in Mucor racemosus.

Mucor racemosus exhibited inducible phenotypic resistance toward the protein synthesis inhibitor trichodermin. Induction of resistance was elicited by exposure to trichodermin or to cycloheximide. Both adapted and nonadapted cells took up [14C]trichodermin from the medium. Trichodermin was found to be rapidly deacetylated to trichodermol upon entering the cell. Adapted cells deacetylated the drug more rapidly than nonadapted cells both in vivo and in vitro. The trichodermol resulting from deacetylation appeared in the medium, but the growth of adapting cells began well before the total conversion of trichodermin to trichodermol. Based on these data and the observation that trichodermol was a poor inhibitor of Mucor, adaptation appears to result from deacylation of the active antibiotic.

Biological Transport↗

PubMed1985

Expression of the gene for ornithine decarboxylase of Saccharomyces cerevisiae in Escherichia coli.

Diploid cells of Saccharomyces cerevisiae homozygous for the spe1A mutation, which eliminates ornithine decarboxylase activity, were found to sporulate at a greatly reduced frequency in the absence of polyamines. Plasmids which complement the spe1A mutation were isolated by their ability to restore sporulation competence to these cells. Three distinct plasmids were isolated. Each plasmid insert overlapped the same 8.0-kilobase region, and each plasmid restored ornithine decarboxylase activity to spe1A mutants. These plasmids also conferred ornithine decarboxylase activity to Escherichia coli EWH319 from which the ornithine decarboxylase gene is deleted. The plasmid-encoded activity expressed in E. coli resembled S. cerevisiae ornithine decarboxylase in its kinetic characteristics, indicating that the yeast ornithine decarboxylase gene was cloned. Southern blot analysis suggested that ornithine decarboxylase is a single-copy gene in S. cerevisiae. A single 2.1-kilobase transcript was demonstrated by Northern blot analysis.

Escherichia coli↗

PubMed1985

Regulation of protein synthesis factor EF-1 alpha in Mucor racemosus.

The protein synthesis elongation factor EF-1 alpha of Mucor racemosus hyphae contained eight or nine methylated amino acids per molecule, whereas the factor from sporangiospores was nonmethylated. During the course of spore germination, the specific activity of the factor in crude extracts increased sixfold. This increase in activity was accompanied by a constant level of EF-1 alpha-specific mRNA and a constant level of EF-1 alpha protein. Methylation of the protein, however, accelerated during the germination process, in parallel with the increase in specific activity of the factor. We propose that the activity of EF-1 alpha is regulated during germination through methylation of the protein and does not involve transcriptional regulation.

Gene Expression Regulation↗

PubMed1981

A particulate form of alkaline phosphatase in the yeast, Saccharomyces cerevisiae.

A new form of alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) has been identified in the yeast Saccharomyces cerevisiae. Utilizing either synthetic or natural substrates, the enzyme exhibited a broad pH activity curve with maximum activity between 8.5 and 9.0. The enzyme was nonspecific with respect to substrate, attacking a variety of compounds containing phosphomonoester linkages, but has no detectable activity against polyphosphate, pyrophosphate or phosphodiester linkages. The enzyme exhibited an apparent Km of 0.25 mM with respect to p-nitrophenyl phosphate, 0.38 mM with respect to alpha-naphthyl phosphate, and 1.0 mM with respect to 5'AMP. The enzyme is regulated in a constitutive manner and its activity does not increase during phosphate starvation or sporulation, as does the repressible alkaline phosphatase. The enzyme is tightly bound to a particulate fraction of the cell, tentatively identified as the tonoplast membrane. It is not solubilized by treatment with high concentrations of NaCl, KH2PO4 or chaotropic agents. Triton X-100 (0.1%) solubilizes 12% of the particulate activity. This enzyme is differentiated from the other alkaline phosphatases found in yeast by its chromatographic elution DEAE-cellulose, kinetic parameters, heat stability and pH stability, as well as its particulate nature. This particulate alkaline phosphatase was found in every strain examined. It has a significantly lower specific activity in the phoH mutant and a higher activity in the acid phosphatase constitutive mutant A137.

Alkaline Phosphatase↗

PubMed1979

Relationship of glycolytic intermediates, glycolytic enzymes, and ammonia to glycogen metabolism during sporulation in the yeast Saccharomyces cerevisiae.

To identify the factors which control glycogen synthesis in Saccharomyces cerevisiae, we have studied the regulation of glycogen metabolism during sporulation, since in vivo glycogen has been reported to undergo significant changes in concentration during this process. We examined the concentration of a number of key glycolytic intermediates and enzymes in strains that sporulate at different rates and those that are deficient in sporulation. There were no significant changes found in the adenylate energy charge or cyclic AMP levels throughout sporulation. Although significant alterations occurred in the levels of glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, and ATP during sporulation, only the fourfold increase in fructose-1,6-bisphosphate appeared to correlate with glycogen synthesis in all of the strains examined. Only limited changes occurred in the level of a number of glycolytic and gluconeogenic enzymes which were examined during this process. Intracellular glucose content underwent a dramatic 30- to 40-fold increase in sporulating cells. Comparison of strains with different rates of sporulation demonstrated that this increase in glucose content coincides with the time of glycogen degradation in each strain. Both the increase in glucose content and the degradation of accumulated glycogen were not observed in nonsporulating alpha/alpha strains, or in cells incubated in NH(4) (+) supplemented sporulation medium. Although glucose appears to be the direct product of glycogen degradation, a 10-fold increase in a nonspecific alkaline phosphatase occurs at this time, which may be degrading phosphorylated sugars to glucose. All of the strains examined released extracellular glucose while suspended in acetate sporulation medium. It is concluded that most of the changes in the glycolytic pathway that occur during sporulation, with the exception of glycogen degradation and the concomitant increase in intracellular glucose pools, are a response to the transfer to sporulation medium and are independent of sporulation-specific processes. Inhibition of sporulation with ammonium ions resulted in a different pattern of change in all of the glycolytic intermediates examined, including a twofold increase in cyclic AMP levels. Ammonia did not interfere with glycogen synthesis, but prevented sporulation-specific glycogen degradation. The levels of the glycolytic enzymes examined were not affected by ammonia.

Adenine Nucleotides↗