RESUMO
Candida albicans is a diploid dimorphic yeast with no known sexual cycle. The development of a DNA transformation system would greatly improve the prospects for genetic analyses of this yeast. Plasmids were isolated from a Candida Sau3A partial library which complements the ade2-1 and ade2-5 mutations in Saccharomyces cerevisiae. These plasmids contain a common region, part of which, when subcloned, produces ade2 complementation. Among the small number of auxotrophs previously isolated in C. albicans, red adenine-requiring mutants had been identified by several groups. In two of these strains, the cloned Candida DNA transformed the mutants to ADE+ at frequencies of 0.5 to 5 transformants per micrograms of DNA. In about 50% of the transformants, plasmid DNA sequences became stably integrated into the host genome and, in the several cases analyzed by Southern hybridization, the DNA was integrated at the site of the ADE2 gene in one of the chromosomal homologs.
Assuntos
Candida albicans/genética , Candida/genética , Clonagem Molecular , Proteínas Fúngicas/genética , Genes Fúngicos , Genes , Transformação Genética , Candida albicans/enzimologia , Enzimas de Restrição do DNA , Mutação , Hibridização de Ácido Nucleico , Plasmídeos , Saccharomyces cerevisiae/genética , Esferoplastos/metabolismoRESUMO
A method for introducing specific mutations into the diploid Candida albicans by one-step gene disruption and subsequent UV-induced recombination was developed. The cloned C. albicans URA3 gene was disrupted with the C. albicans ADE2 gene, and the linearized DNA was used for transformation of two ade2 mutants, SGY-129 and A81-Pu. Both an insertional inactivation of the URA3 gene and a disruption which results in a 4.0-kilobase deletion were made. Southern hybridization analyses demonstrated that the URA3 gene was disrupted on one of the chromosomal homologs in 15 of the 18 transformants analyzed. These analyses also revealed restriction site dimorphism of EcoRI at the URA3 locus which provides a unique marker to distinguish between chromosomal homologs. This enabled us to show that either homolog could be disrupted and that disrupted transformants of SGY-129 contained more than two copies of the URA3 locus. The A81-Pu transformants heterozygous for the ura3 mutations were rendered homozygous and Ura- by UV-induced recombination. The homozygosity of a deletion mutant and an insertion mutant was confirmed by Southern hybridization. Both mutants were transformed to Ura+ with plasmids containing the URA3 gene and in addition, were resistant to 5-fluoro-orotic acid, a characteristic of Saccharomyces cerevisiae ura3 mutants as well as of orotidine-5'-phosphate decarboxylase mutants of other organisms.
Assuntos
Candida albicans/genética , Genes Fúngicos/efeitos da radiação , Mutação , Raios Ultravioleta , Candida albicans/efeitos da radiação , Enzimas de Restrição do DNA , DNA Fúngico/genética , DNA Fúngico/efeitos da radiação , Genótipo , Métodos , Mapeamento de NucleotídeosRESUMO
A pool of Candida albicans RsaI fragments cloned onto a vector containing pBR322 sequences and the Candida ADE2 gene was used to transform a Candida ade2 mutant to adenine protrophy. A potential autonomously replicating sequence (ARS) in Candida DNA was identified by two criteria: instability of the selectable marker in the absence of selection and the presence of free plasmid in total DNA preparations. Plasmids carrying the ARS transformed C. albicans at a high frequency (200 to 1,000 ADE+ transformants per microgram of DNA), and Southern hybridization analysis of these transformants indicated that multiple copies of the plasmid sequences were present and that, although they were present in high-molecular-weight molecules, these sequences had not undergone rearrangement. Orthogonal field alternation gel electrophoresis indicated that the high-molecular-weight transforming sequences were not associated with any chromosome. The simplest interpretation to account for these data is that the transforming sequences are present as oligomers consisting of head-to-tail tandem repeats. The transformed strains occasionally yield stable segregants in which the transforming sequences are integrated into the chromosome as repeats. The Candida sequence responsible for the ARS phenotype was limited to a single 0.35-kilobase RsaI fragment which is present in one copy per haploid genome.
Assuntos
Candida albicans/genética , Replicação do DNA , Plasmídeos , Clonagem Molecular , Enzimas de Restrição do DNA , Escherichia coli/genética , Teste de Complementação Genética , Vetores Genéticos , Mutação , Hibridização de Ácido Nucleico , Tetra-Hidrofolato Desidrogenase/genéticaRESUMO
Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24-65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet, SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate that SFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with "bypass-Sec14p" mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana , Fosfatidilinositóis/metabolismo , Fosfolipase D/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Compartimento Celular , Divisão Celular , DNA Fúngico , Endossomos/metabolismo , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Dados de Sequência Molecular , Proteínas de Transferência de Fosfolipídeos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Traditionally, the discovery of pharmaceutical and agrochemical products has largely depended on mass screening. Over the years, screen design and screening programs have evolved in terms of the sensitivity with which active material can be identified, the number of samples that can be tested, and the types of molecular targets and cellular functions that can be conveniently assayed. More recently, screens with desirable properties have been developed for a great variety of molecular targets through the exploitation of Saccharomyces molecular biology and genetics. Recent advances have enabled researchers to develop yeast-based screens for agents acting on a number of new therapeutic targets: G-protein linked receptors, cytoplasmic receptors, ion (potassium) channels, novel fungal cell wall enzymes, fungal sterol biosynthesis enzymes, antiviral targets, immunosuppressive targets, cyclic nucleotide phosphodiesterase, oncogenes and the multiple drug resistance (MDR) protein.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Saccharomyces cerevisiae/genética , Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Antivirais/farmacologia , Biotecnologia , Calmodulina/antagonistas & inibidores , Imunossupressores/farmacologia , Transporte de Íons/efeitos dos fármacos , Ligantes , Biologia Molecular , Receptores de Superfície Celular/metabolismoRESUMO
The Saccharomyces cerevisiae cytochrome P450 L1A1 (lanosterol 14 alpha-demethylase)-coding gene was used as a hybridization probe to isolate two HindIII fragments of 2.5 kb and 6.85 kb from a phage lambda library of Candida albicans nucleotide sequences. Restriction endonuclease mapping and Southern blot hybridization experiments indicated that these fragments represent two allelic forms of the same gene. This cloned sequence, when introduced into S. cerevisiae or C. albicans on a multiple copy vector, produced an increase in cytochrome P450 content and resistance to imidazole antifungal agents which are inhibitors of cytochrome P450 L1A1. In addition, the cloned sequence was able to complement a cytochrome P450 L1A1 gene disruption when introduced into S. cerevisiae. These data indicate that the cloned sequence codes for the lanosterol 14 alpha-demethylase cytochrome P450 L1A1 from C. albicans.
Assuntos
Candida albicans/genética , Genes Fúngicos , Genes , Oxirredutases/genética , Candida albicans/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Resistência Microbiana a Medicamentos/genética , Vetores Genéticos , Oxirredutases/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Esterol 14-DesmetilaseRESUMO
2,3-Oxidosqualene (OS) cyclase (OSC) catalyzes the conversion of OS to lanosterol, an essential step in the biosynthesis of sterols. The Candida albicans gene (ERG7) encoding OSC was cloned by complementation of a Saccharomyces cerevisiae OSC mutant (erg7). Two different Erg+ clones were isolated that contain a common overlapping region. The minimum region required for complementation was determined to be approx. 3.2 kb and a single 2.7-kb ERG7 transcript was detected. The cloned Candida ERG7 DNA complemented an additional nonconditional erg7 allele and a temperature-sensitive erg7 mutation. OSC activity was restored in the mutants as determined by [14C]acetate incorporation in vivo as well as incorporation in vitro in cell-free extracts using either [14C]isopentenyl pyrophosphate or [3H]OS as substrate. The level of OSC produced from expression of a single copy of the Candida ERG7 sequence was sufficient to allow growth of the S. cerevisiae erg7 mutants in the absence of exogenous ergosterol. These data support the contention that the Candida ERG7 sequence is the structural gene for OSC.
Assuntos
Candida albicans/genética , Transferases Intramoleculares , Isomerases/genética , Alelos , Candida albicans/enzimologia , Cromatografia em Camada Fina , Genes Fúngicos , Teste de Complementação Genética , Isomerases/metabolismo , Mutação , Mapeamento por Restrição , Transformação GenéticaRESUMO
The Saccharomyces cerevisiae ERG24 gene, encoding sterol delta 14 reductase (Erg24p), was cloned by selecting strains carrying sequences on a 2 mu-based vector for resistance to the morpholine fungicide, fenpropimorph (Fp). Four distinct plasmid inserts which conferred Fp resistance (FpR) were recovered (plasmids pML99, pML100, pML101 and pM103). Although Fp is reported to inhibit activity of Erg24p and sterol delta 8-delta 7 isomerase (Erg2p; encoded by ERG2), none of the inserts had restriction maps resembling ERG2. In addition, a 2 mu plasmid overexpression of the ERG2 sequence did not produce FpR. Characterization studies were focused on plasmid pML100, because it was the only plasmid to confer FpR consistently when tested in a number of different genetic backgrounds. Tests with a panel of fungicides indicated that pML100 conferred significant resistance only to compounds (Fp, tridemorph, fenpropidin and azasterol) which have a shared site of action, Erg24p. An insertional disruption of pML100 resulted in an obligate anaerobic phenotype, indicating a lesion in sterol biosynthesis. Sterol analysis of the disrupted mutant demonstrated the accumulation of ignosterol, indicating a loss of Erg24p activity. A SphI-XbaI fragment of pML100 was sequenced, revealing the presence of an ORF encoding a 438-amino-acid protein, which is highly similar to those encoded by two previously reported yeast drug sensitivity genes, sts1+ (Schizosaccharomyces pombe) and YGL022 (S. cerevisiae). Analyses of these genes demonstrated that strains carrying disruptions of sts1+ or YGL022 have ergosterol biosynthesis defects in the enzyme, sterol C-24(28) reductase (Erg4p; encoded by ERG4).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ergosterol/biossíntese , Genes Fúngicos , Família Multigênica , Oxirredutases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico , Resistência a Medicamentos/genética , Dados de Sequência Molecular , Morfolinas/farmacologia , Mutação , Oxirredutases/metabolismo , Plasmídeos , Saccharomyces cerevisiae/enzimologia , Schizosaccharomyces/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Esteroide Isomerases/genética , Esteroide Isomerases/metabolismoRESUMO
The Drosophila melanogaster ecdysone receptor (EcR) is produced in three isoforms, which mediate developmental processes such as metamorphosis. These isoforms were expressed in Saccharomyces cerevisiae to elucidate aspects of receptor transcription activity in a highly defined genetic model system. All three EcR isoforms showed ligand-independent transcriptional activation of an ecdysone reporter gene and the amount of activation correlated with the size of the N-terminal A/B (transactivation) domain present in the isoform: EcR-B1>EcR-A>>EcR-B2. Upon co-expression with ultraspiracle (Usp), transcriptional activation was further increased with EcR-B1 or EcR-A, but was unchanged with EcR-B2 or a truncated EcR lacking the A/B N-terminal domain (EcRDeltaA/B). Thus, the enhanced activity from Usp may depend on the presence of an N-terminal domain of EcR. Co-expression with Usp of several chimeric receptors of the EcR and the mouse androgen receptor (mAR) identified one chimera, composed of the mAR N-terminus and the remainder from EcR (mAR¿EcR-CDEF) that was transcriptionally silent and inducible by Usp. In contrast, the vertebrate homologue, human retinoic acid receptor (RXRalpha), showed ligand-independent transcription when co-expressed with EcRDeltaA/B but not mAR¿ EcR-CDEF. Therefore, RXRalpha does not require its partner to possess an N-terminal domain, yet is intolerant of a heterologous N-terminus. Similarly, the human vitamin D receptor, which has a short N-terminal region, showed greater ligand-independent transcription in the presence of RXRalpha than in the presence of Usp. These results reveal a mechanistic basis for the differential activities among the EcR isoforms, and between Usp and RXRalpha. Furthermore, they provided the foundation for a genetic screen to identify potential insecticides as well as accessory proteins for Usp and EcR.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/fisiologia , Receptores de Esteroides/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/metabolismo , Animais , Bioensaio , Clonagem Molecular , Citosol/metabolismo , Proteínas de Drosophila , Humanos , Cinética , Camundongos , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica , Ativação Transcricional , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The desirable features for a screening assay to detect antibacterial antibiotics include 1) high specificity for the desired antibiotic type 2) high sensitivity 3) lack of interference by other compounds likely to be associated with the antibiotic of interest and 4) ease of operation to allow a large number of samples to be tested. These characteristics are largely found in screens employing strains carrying fusions between antibiotic induced promoters and the structural genes for Escherichia coli beta-galactosidase. Screens were designed based upon fusions with three antibiotic induced promoters: the tetracycline induced tetA/tetR promoter from transposon Tn10, the erythromycin induced promoter from the Staphylococcus aureus ermC erythromycin-resistance gene and the chloramphenicol induced promoter from the S. aureus cat86 chloramphenicol-resistance gene. Because there have been no reports of vancomycin induced resistance determinants, a Tn903 random gene fusion pool was screened to isolate a vancomycin induced gene fusion. This gene fusion was induced fairly specifically by glycopeptide antibiotics and the fusion was used as the basis for a glycopeptide screen.
Assuntos
Antibacterianos/análise , Bactérias/efeitos dos fármacos , Clonagem Molecular , beta-Galactosidase/genética , Antibacterianos/farmacologia , Cloranfenicol/análise , Glicopeptídeos/análise , Macrolídeos , Valor Preditivo dos Testes , Regiões Promotoras Genéticas , Reprodutibilidade dos Testes , Tetraciclinas/análise , Vancomicina/análiseRESUMO
The antifungal agents lanomycin and glucolanomycin were isolated from Pycnidiophora dispersa. The compounds were active against species of Candida and dermatophytes but were inactive against Aspergillus fumigatus and Gram-positive and Gram-negative bacteria. The compounds inhibited the cytochrome P-450 enzyme lanosterol 14 alpha-demethylase, and are believed, therefore, to have a mode of action similar to the azole and bis-triazole class of antifungal agents.
Assuntos
Antibacterianos/isolamento & purificação , Antifúngicos/isolamento & purificação , Ascomicetos/metabolismo , Piranos/isolamento & purificação , Aminoglicosídeos , Animais , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Bactérias/efeitos dos fármacos , Candida/efeitos dos fármacos , Ergosterol/biossíntese , Feminino , Fermentação , Camundongos , Piranos/farmacologiaRESUMO
A screen for antibiotics with activity against tetracycline-resistant microorganisms has led to the isolation of Dactylosporangium sp. (ATCC 53693), a producer of several novel tetracycline derivatives. The major fermentation products, dactylocyclines A and B, were purified and MIC values determined against tetracycline-resistant and tetracycline-sensitive Gram-positive bacteria. The dactylocyclines represent the first naturally occurring tetracycline C2 amides which lack cross resistance with tetracycline.
Assuntos
Actinomycetales/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Clortetraciclina/análogos & derivados , Bactérias Gram-Positivas/efeitos dos fármacos , Antibacterianos/química , Clortetraciclina/química , Clortetraciclina/isolamento & purificação , Clortetraciclina/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Espectrofotometria Infravermelho , Resistência a TetraciclinaRESUMO
A new antibacterial agent, lysobactin, has been isolated from a species of Lysobacter (ATCC 53042). The antibiotic was recovered from the Lysobacter cell mass by extraction and reversed phase chromatography. Lysobactin is a dibasic peptide with marked activity against Gram-positive aerobic and anaerobic bacteria.
Assuntos
Antibacterianos/isolamento & purificação , Depsipeptídeos , Bactérias Gram-Negativas/classificação , Antibacterianos/farmacologia , Fermentação , Bactérias Gram-Negativas/metabolismo , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/farmacologiaRESUMO
15G256 gamma is a cyclic lipopeptide antifungal agent discovered in a mechanism of action screen for cell wall acting antifungal agents. The compound shows moderate activity in both greenhouse tests against plant disease caused by pathogenic fungi and in in vitro tests against human fungal pathogens. Microscopic examination of treated fungi suggests that the compound acts by the inhibition of cell wall biosynthesis. However, in vitro inhibition of Neurospora crassa glucan and chitin synthase were only observed at high drug concentrations suggesting that 15G256 gamma may act on a novel cell wall target.
Assuntos
Antifúngicos/farmacologia , Parede Celular/efeitos dos fármacos , Lactonas/farmacologia , Fenóis/farmacologia , Parede Celular/metabolismo , Quitina Sintase/antagonistas & inibidores , Glucosiltransferases/antagonistas & inibidores , Humanos , Neurospora crassa/efeitos dos fármacosRESUMO
Research on the ergosterol biosynthetic pathway in fungi has focused on the identification of the specific sterol structure required for normal membrane structure and function and for completion of the cell cycle. The pathway and its end product are also the targets for a number of antifungal drugs. Identification of essential steps in ergo-sterol biosynthesis could provide new targets for the development of novel therapeutic agents. Nine of the eleven genes in the portion of the pathway committed exclusively to ergosterol biosynthesis have been cloned, and their essentiality for aerobic growth has been determined. The first three genes, ERG9 (squalene synthase), ERG1 (squalene epoxidase), and ERG7 (lanosterol synthase), have been cloned and found to be essential for aerobic viability since their absence would result in the cell being unable to synthesize a sterol molecule. The remaining eight genes encode enzymes which metabolize the first sterol, lanosterol, to ultimately form ergosterol. The two earliest genes, ERG11 (lanosterol demethylase) and ERG24 (C-14 reductase), have been cloned and found to be essential for aerobic growth but are suppressed by mutations in the C-5 desaturase (ERG3) gene and fen1 and fen2 mutations, respectively. The remaining cloned genes, ERG6 (C-24 methylase), ERG2 (D8AE7 isomerase), ERG3 (C-5 desaturase), and ERG4 (C-24(28) reductase), have been found to be nonessential. The remaining genes not yet cloned are the C-4 demethylase and the C-22 desaturase (ERG5).
Assuntos
Ergosterol/biossíntese , Genes Fúngicos/genética , Saccharomyces cerevisiae/genética , Clonagem Molecular , Lanosterol/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Esqualeno/metabolismo , Fatores de TempoAssuntos
Antibacterianos , Antibacterianos/isolamento & purificação , Peptídeos/isolamento & purificação , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Resistência Microbiana a Medicamentos , Espectroscopia de Ressonância Magnética , Micrococcus/análise , Micrococcus/classificação , Peptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Inibidores da Síntese de Proteínas , Espectrofotometria Infravermelho , Espectrofotometria UltravioletaRESUMO
Auxotrophic and prototrophic control strain pairs of Candida albicans constructed by molecular biology methodologies were evaluated for pathogenicity in a systemic mouse model. Mutants that were auxotrophic for adenine, uracil, and heme each showed a lowered level of pathogenicity relative to control strains. It can be concluded from these experiments that decreased pathogenicity in each case is due to the auxotrophic mutation, because mutant and control strains were constructed so as to differ at a single locus. These observations suggest that new therapeutic agents for Candida infections might be designed based upon the inhibition of biosynthetic pathways that, in some cases, might be absent from the host.
Assuntos
Candida albicans/patogenicidade , Candidíase/microbiologia , Animais , Candida albicans/genética , Modelos Animais de Doenças , Feminino , Genótipo , Camundongos , Mutação , VirulênciaRESUMO
The induction of vancomycin resistance in enterococci containing the vanA gene cluster is thought to be controlled by a two-component sensor-response regulator system encoded by vanR and vanS. Eight inducing compounds were identified by screening a panel of more than 6,800 antibiotics and synthetic compounds including the three tested glycopeptides (vancomycin, avoparcin, and ristocetin), two other cell wall biosynthesis inhibitors (moenomycin and bacitracin), two cyclic peptide antibiotics (antibiotic AO341 beta and polymyxin B), and a macrocyclic lactone antibiotic (moxidectin). Induction activity by structurally unrelated antibiotics suggests that the induction signal is not a structural feature of vancomycin.