RESUMO
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.
Assuntos
Genes Fúngicos , Ornitina Descarboxilase/genética , Saccharomyces cerevisiae/genética , Escherichia coli/genética , Regulação da Expressão Gênica , Hibridização de Ácido Nucleico , Plasmídeos , Biossíntese de Proteínas , Transcrição Gênica , Transformação GenéticaRESUMO
Deletion of PHR1, a pH-regulated gene of Candida albicans, results in pH-conditional defects in growth, morphogenesis, and virulence evident at neutral to alkaline pH but absent at acidic pH. Consequently, we searched for a functional homolog of PHR1 active at low pH. This resulted in the isolation of a second pH-regulated gene, designated PHR2. The expression of PHR2 was inversely related to that of PHR1, being repressed at pH values above 6 and progressively induced at more acidic pH values. The predicted amino acid sequence of the PHR2 protein, Phr2p, was 54% identical to that of Phr1p. A PHR2 null mutant exhibited pH-conditional defects in growth and morphogenesis analogous to those of PHR1 mutants but manifest at acid rather than alkaline pH values. Engineered expression of PHR1 at acid pH in a PHR2 mutant strain and PHR2 at alkaline pH in a PHR1 mutant strain complemented the defects in the opposing mutant. Deletion of both PHR1 and PHR2 resulted in a strain with pH-independent, constitutive growth and morphological defects. These results indicate that PHR1 and PHR2 represent a novel pH-balanced system of functional homologs required for C. albicans to adapt to environments of diverse pH.
Assuntos
Apoenzimas/genética , Candida albicans/genética , Desoxirribodipirimidina Fotoliase/genética , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica/fisiologia , Glicoproteínas de Membrana , Sequência de Aminoácidos , Apoenzimas/análise , Candida albicans/citologia , Clonagem Molecular , Desoxirribodipirimidina Fotoliase/análise , Desoxirribodipirimidina Fotoliase/fisiologia , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Fenótipo , RNA Fúngico/análise , RNA Mensageiro/análise , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Mucor/metabolismo , Fatores de Alongamento de Peptídeos/biossíntese , Regulação da Expressão Gênica , Metilação , Mucor/genética , Fator 1 de Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/genética , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Esporos FúngicosRESUMO
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.
Assuntos
Apoenzimas , Candida albicans/metabolismo , Desoxirribodipirimidina Fotoliase , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica , Glicoproteínas de Membrana , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Clonagem Molecular , DNA Complementar/isolamento & purificação , DNA Fúngico/análise , Proteínas Fúngicas/genética , Expressão Gênica , Genótipo , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutagênese , RNA Fúngico/análise , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
Morphological development of the fungal pathogen Candida albicans is profoundly affected by ambient pH. Acidic pH restricts growth to the yeast form, whereas neutral pH permits development of the filamentous form. Superimposed on the pH restriction is a temperature requirement of approximately 37 degrees C for filamentation. The role of pH in development was investigated by selecting revertants of phr2Delta mutants that had gained the ability to grow at acid pH. The extragenic suppressors in two independent revertants were identified as nonsense mutations in the pH response regulator RIM101 (PRR2) that resulted in a carboxy-terminal truncation of the open reading frame. These dominant active alleles conferred the ability to filament at acidic pH, to express PHR1, an alkaline-expressed gene, at acidic pH, and to repress the acid-expressed gene PHR2. It was also observed that both the wild-type and mutant alleles could act as multicopy suppressors of the temperature restriction on filamentation, allowing extensive filamentation at 29 degrees C. The ability of the activated alleles to promote filamentation was dependent upon the developmental regulator EFG1. The results suggest that RIM101 is responsible for the pH dependence of hyphal development.
Assuntos
Candida albicans/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Dominantes , Glicoproteínas de Membrana , Fatores de Transcrição , Apoenzimas/genética , Apoenzimas/metabolismo , Divisão Celular/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Proteínas Fúngicas/metabolismo , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Heterozigoto , Concentração de Íons de Hidrogênio , Mutação , Supressão GenéticaRESUMO
Candidiasis is a common infection of the skin, oral cavity and esophagus, gastrointestinal tract, vagina and vascular system of humans. Although most infections occur in patients who are immunocompromised or debilitated in some other way, the organism most often responsible for disease, Candida albicans, expresses several virulence factors that contribute to pathogenesis. These factors include host recognition biomolecules (adhesins), morphogenesis (the reversible transition between unicellular yeast cells and filamentous, growth forms), secreted aspartyl proteases and phospholipases. Additionally, 'phenotypic switching' is accompanied by changes in antigen expression, colony morphology and tissue affinities in C. albicans and several other Candida spp. Switching might provide cells with a flexibility that results in the adaptation of the organism to the hostile conditions imposed not only by the host but also by the physician treating the infection.
Assuntos
Candida albicans/patogenicidade , Candidíase/microbiologia , Candida albicans/genética , Candidíase/fisiopatologia , Humanos , Virulência/genéticaRESUMO
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.
Assuntos
Fosfatase Alcalina/metabolismo , Saccharomyces cerevisiae/enzimologia , Fosfatase Alcalina/isolamento & purificação , Estabilidade de Medicamentos , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Mutação , Fosfatos/farmacologia , Especificidade da Espécie , Esporos Fúngicos/enzimologia , Especificidade por SubstratoRESUMO
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.
Assuntos
Candida albicans/genética , Genes Fúngicos , Sequência de Bases , DNA de Cadeia Simples , Genes Recessivos , Genótipo , Heterozigoto , Homozigoto , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mapeamento por Restrição , Deleção de SequênciaRESUMO
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.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos/efeitos dos fármacos , Ornitina Descarboxilase/genética , Poliaminas/farmacologia , Saccharomyces cerevisiae/genética , Genótipo , Cinética , Ornitina Descarboxilase/biossíntese , Putrescina/farmacologia , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Espermidina/farmacologia , Espermina/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
Genes Fúngicos , Ornitina Descarboxilase , Saccharomyces cerevisiae/enzimologia , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Conversão Gênica , Genes , Substâncias Macromoleculares , Peso Molecular , Mutação , Ornitina Descarboxilase/genética , Conformação ProteicaRESUMO
Over 25 years ago it was noted that the pH of the culture medium influenced germ tube formation of Candida albicans, an opportunistic fungal pathogen. This simple observation has been the stimulus for a number of investigations to discern the mechanisms controlling this response and the significance of this response to the biology of C. albicans. Recent studies have demonstrated that a signaling pathway conserved in several fungal species regulates this morphological response to ambient pH and controls the pH-conditional expression of multiple genes. Significantly, C. albicans responds to the pH of the host niche and this response is critical for virulence.
Assuntos
Candida albicans/patogenicidade , Concentração de Íons de Hidrogênio , Virulência/genética , Candida albicans/genética , Candidíase , Humanos , Infecções Oportunistas/microbiologiaRESUMO
PHR1 and PHR2 encode putative glycosylphosphatidylinositol-anchored cell surface proteins of the opportunistic fungal pathogen Candida albicans. These proteins are functionally related, and their expression is modulated in relation to the pH of the ambient environment in vitro and in vivo. Deletion of either gene results in a pH-conditional defect in cell morphology and virulence. Multiple sequence alignments demonstrated a distant relationship between the Phr proteins and beta-galactosidases. Based on this alignment, site-directed mutagenesis of the putative active-site residues of Phr1p and Phr2p was conducted and two conserved glutamate residues were shown to be essential for activity. By taking advantage of the pH-conditional expression of the genes, a temporal analysis of cell wall changes was performed following a shift of the mutants from permissive to nonpermissive pH. The mutations did not grossly affect the amount of polysaccharides in the wall but did alter their distribution. The most immediate alteration to occur was a fivefold increase in the rate of cross-linking between beta-1,6-glycosylated mannoproteins and chitin. This increase was followed shortly thereafter by a decline in beta-1,3-glucan-associated beta-1, 6-glucans and, within several generations, a fivefold increase in the chitin content of the walls. The increased accumulation of chitin-linked glucans was not due to a block in subsequent processing as determined by pulse-chase analysis. Rather, the results suggest that the glucans are diverted to chitin linkage due to the inability of the mutants to establish cross-links between beta-1,6- and beta-1,3-glucans. Based on these and previously published results, it is suggested that the Phr proteins process beta-1,3-glucans and make available acceptor sites for the attachment of beta-1,6-glucans.
Assuntos
Apoenzimas/genética , Candida albicans/genética , Desoxirribodipirimidina Fotoliase/genética , Proteínas Fúngicas , Glucanos/metabolismo , Glicoproteínas de Membrana , beta-Galactosidase/genética , beta-Glucanas , Sequência de Aminoácidos , Apoenzimas/metabolismo , Candida albicans/enzimologia , Parede Celular/química , Parede Celular/metabolismo , Reagentes de Ligações Cruzadas , Desoxirribodipirimidina Fotoliase/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Solubilidade , beta-Galactosidase/metabolismoRESUMO
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.
Assuntos
DNA/análise , Ornitina Descarboxilase/genética , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA/metabolismo , Ornitina Descarboxilase/análiseRESUMO
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.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Esterases/metabolismo , Mucor/efeitos dos fármacos , Sesquiterpenos/farmacologia , Tricodermina/farmacologia , Transporte Biológico , Resistência Microbiana a Medicamentos , Indução Enzimática , Inativação Metabólica , Mucor/metabolismo , Fatores de Tempo , Tricodermina/metabolismoRESUMO
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.
Assuntos
Candida albicans/genética , Elementos de DNA Transponíveis , DNA Fúngico/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Bases , Evolução Biológica , Southern Blotting , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
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.
Assuntos
Clonagem Molecular , Genes Fúngicos , Mucor/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular/métodos , DNA Fúngico/genética , Escherichia coli/genética , Vetores Genéticos , Halobacterium/genética , Dados de Sequência Molecular , Mapeamento por Restrição , Proteínas Ribossômicas/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Xenopus laevisRESUMO
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.
Assuntos
Candida albicans/genética , Fatores de Alongamento de Peptídeos/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Fatores de Alongamento de Peptídeos/química , Mapeamento por Restrição , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência do Ácido NucleicoRESUMO
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.
Assuntos
Candida albicans/genética , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Genes Reporter , Orotidina-5'-Fosfato Descarboxilase/genética , Sequência de Bases , Candida albicans/enzimologia , Carboxiliases/genética , Dados de Sequência Molecular , Fatores de Alongamento de Peptídeos/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas de Saccharomyces cerevisiae , Transformação GenéticaRESUMO
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.
Assuntos
Sulfato de Amônio/farmacologia , Glicogênio/metabolismo , Glicólise , Saccharomyces cerevisiae/metabolismo , Nucleotídeos de Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Glucose/metabolismo , Hexosefosfatos/metabolismo , Fosfoenolpiruvato/metabolismo , Esporos FúngicosRESUMO
The pH of the environment has been implicated in controlling the yeast-hypha transition and pathogenesis of Candida albicans. Several C. albicans genes, including PHR1 and PHR2, are pH dependent in their expression. To investigate the mechanism of pH-dependent expression, we have cloned and characterized PRR1 (for pH response regulator). PRR1 is homologous to palF, a component of the pH response pathway in Aspergillus nidulans. Expression of PRR1 was itself pH dependent, being maximal at acid pH but reduced severalfold at alkaline pH. In a prr1 null mutant the alkaline-induced expression of PHR1 was completely abolished. Conversely, expression of PHR2 was no longer repressed at alkaline pH. A prr1 null mutant exhibited no morphological abnormalities at either pH; however, it lost the ability to form hyphae on medium 199 and on 10% serum plates. The ability to filament on serum was not restored by forced expression of PHR1, indicating that additional PRR1-dependent genes are required for hyphal development. These developmental genes appear to be distinct from those controlled by the developmental regulator EFG1, since the EFG1-dependent gene HWP1 was expressed normally in the prr1 null mutant. We conclude that PRR1 encodes a component of the pH-dependent response pathway in C. albicans and that this pathway regulates the expression of multiple components of hyphal development.