RESUMO
The Neurospora crassa fmf-1 mutation exerts an unusual 'perithecium-dominant' developmental arrest; fmf-1 x fmf-1+ cross becomes arrested in perithecial development regardless of whether the mutant participates in the cross as the male or female parent. We localized fmf-1 to the LG IL genome segment between the centromere-proximal breakpoint of the chromosome segment duplication Dp(IL)39311 and the centromere. By mapping crossovers with respect to RFLP markers in this region we further localized fmf-1 to an approximately 34-kb-genome segment. Partial sequencing of this segment revealed a point mutation in the gene NCU 09387.1, a homologue of the Schizosaccharomyces pombe ste11+ regulator of sexual development. The fmf-1 mutation did not complement a NCU 09387.1 deletion mutation, and transformation with wild-type NCU 09387.1 complemented fmf-1. S. pombe Ste11 protein (Ste11p) is a transcription factor required for sexual differentiation and for the expression of genes required for mating pheromone signalling in matP and matM cells. If FMF-1 also plays a corresponding role in mating pheromone signalling in Neurospora, then protoperithecia in an fmf-1 x fmf-1+ cross would be unable to either send or receive sexual differentiation signals and thus become arrested in development.
Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Modelos Genéticos , Mutação , Neurospora crassa/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The present study was designed to identify alkaline phosphatases in non-permeabilized hyphal cells of the fungus Neurospora crassa by staining these enzymatic activities with a modified azo dye coupling method. Our strategy allowed the identification of three non-specific alkaline phosphatase activities, one of them possibly being a novel putative enzyme, which is not responsive to either Mg2+ or EDTA. Another alkaline phosphatase activity, whose location in the hyphal cell is regulated by phosphate, is stimulated by Mg2+, inhibited by EDTA, and somehow dependent on the expression of the pho-2+-encoded Pi-repressible alkaline phosphatase.
Assuntos
Fosfatase Alcalina/análise , Hifas/enzimologia , Neurospora crassa/enzimologia , Fosfatase Alcalina/genética , Membrana Celular , Ácido Edético , Histocitoquímica , Neurospora crassa/citologia , Neurospora crassa/genética , Coloração e Rotulagem , Fatores de TempoRESUMO
Repeat-induced point mutation (RIP) is an unusual genome defense mechanism that was discovered in Neurospora crassa. RIP occurs during a sexual cross and induces numerous G : C to A : T mutations in duplicated DNA sequences and also methylates many of the remaining cytosine residues. We measured the susceptibility of the erg-3 gene, present in single copy, to the spread of RIP from duplications of adjoining sequences. Genomic segments of defined length (1, 1.5 or 2 kb) and located at defined distances (0, 0.5, 1 or 2 kb) upstream or downstream of the erg-3 open reading frame (ORF) were amplified by polymerase chain reaction (PCR), and the duplications were created by transformation of the amplified DNA. Crosses were made with the duplication strains and the frequency of erg-3 mutant progeny provided a measure of the spread of RIP from the duplicated segments into the erg-3 gene. Our results suggest that ordinarily RIP-spread does not occur. However, occasionally the mechanism that confines RIP to the duplicated segment seems to fail (frequency 0.1-0.8%) and then RIP can spread across as much as 1 kb of unduplicated DNA. Additionally, the bacterial hph gene appeared to be very susceptible to the spread of RIP-associated cytosine methylation.
Assuntos
Sequência de Bases , Citosina/metabolismo , DNA Fúngico/genética , Metilação , Neurospora crassa/genética , Mutação Puntual , Recombinação Genética , Sequências Repetitivas de Ácido NucleicoAssuntos
Alelos , Animais , Ritmo Circadiano/genética , Códon , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Retroalimentação Fisiológica , Proteínas Fúngicas/genética , Genes Fúngicos , Genes de Insetos , Modelos Biológicos , Mutação , Neurospora crassa/genética , Polimorfismo Genético , TemperaturaRESUMO
A convenient assay to score repeat-induced point mutation (RIP) in Neurospora employs the erg-3 locus as a mutagenesis target. Using this assay we screened 132 wild-isolated Neurospora crassa strains for ability to dominantly suppress RIP. RIP was exceptionally inefficient in crosses with the wild isolates Sugartown (P0854) and Adiopodoume-7 (P4305), thereby suggesting the presence of dominant RIP suppressors in these strains. In other experiments, we found no evidence for dominant RIP suppression by the Spore killer haplotypes Sk-2 and Sk-3.
Assuntos
Cruzamentos Genéticos , Genes Dominantes , Genes Fúngicos , Genes Supressores , Haplótipos , Neurospora crassa/genética , Mutação Puntual , Sequências Repetitivas de Ácido Nucleico , Esporos Fúngicos , TransgenesRESUMO
El presente trabajo describe la construcción de tres vectores de integración y un vector de replicación autonoma de Saccharomyces cerevisiae. Dichos vectores se caracterizan por poseer el gen LEU2 de levadura y los genes de resistencia a ampicilina y tetraciclina que sirven como marcadores geneticos en Escherichia coli. Por otro lado, el gen LEU2 puede ser utilizado en la selección de transformantes de Sacharomyces cerevisiae. Los plásmidos VCp1, VCp2,1, VCp2,2 y VCp2,3 transforman a la levadura en baja frecuencia (aproximadamente 10 transformantes/ug de DNA) y se comportan como vectores de integración verdaderos. Los plásmidos VCp1 y VCp2,1 fueron utilizados para la construcción de dos genotecas de Neurospora crassa a fin de aislar secuencias de replicación autónoma de Neurospora en levadura. A partir de la genoteca en VCp1, se pudo aislar un plásmido portador de un inserto que corresponde a un fragmento de 3,5 kb de DNA del hongo
Assuntos
Técnicas In Vitro , Neurospora crassa/genética , Saccharomyces cerevisiae , Escherichia coli , Biblioteca Gênica , Vetores GenéticosRESUMO
The invertase wild type gene of N. crassa was cloned into the YRp7 yeast vector. This recombinant plasmid was selected by functional complementation of an invertaseless mutant strain of S. cerevisiae. The isolated recombinant plasmid (named pNC2) carries a 7.6 Kb BamHI DNA fragment from N. crassa. The cloned DNA hydbridized with the N. crassa genomic DNA and transformed an invertase mutant of N. crassa Inv- to Inv+. Transformation of N. crassa Inv- to Inv+ seems to take at least two different integration events. One of them involves an integration closely linked to inv locus, and the other one apparently involves as integration of cloned DNA at a genomic site different that the inv locus
Assuntos
Clonagem Molecular/métodos , Genes Fúngicos , Glicosídeo Hidrolases/genética , Neurospora crassa/enzimologia , DNA Fúngico/genética , Neurospora crassa/genética , Plasmídeos , Transformação GenéticaRESUMO
1. Pulse labeling with [35S]-methionine, one-dimensional SDS-polyacrylamide gel electrophoresis and florography were used to study the pattern of protein synthesis in Neurospora crassa mycelia undergoing sexual development. 2. Contact of sexually-competent mycelium with cells of the opposite mating type elicited a rapid and transient increase in the synthesis of two predominant proteins of 58 KDa and yoKDa and 40 KDa localized in the cytosol fraction. 3. Marked changes in the pattern of protein synthesis were also observed in the 12,000 g particulate fraction, predominantly mitochondrial, where the synthesis of a 34 KDa polypeptide was most prominent among others. 4. Poly(A) + RNA extracted from mycelia 2 h after sexual stimulation supported the in vitro synthesis of the 58 KDa and yoKDa major polypeptides synthesized in vivo. 5. No differences were observed in the pattern of protein synthesis of treated cultures and controls 24 h after the sexual stimulus