RESUMEN
Genes regulating recombination in specific chromosomal intervals of Neurospora crassa were described in the 1960s, but the mechanism is still unknown. For each of the rec-1, rec-2, and rec-3 genes, a single copy of the putative dominant allele, for example, rec-2SL found in St Lawrence OR74 A wild type, reduces recombination in chromosomal regions specific to that gene. However, when we sequenced the recessive allele, rec-2LG (derived from the Lindegren 1A wild type), we found that a 10â kb region in rec-2SL strains was replaced by a 2.7â kb unrelated sequence, making the "alleles" idiomorphs. When we introduced sad-1, a mutant lacking the RNA-dependent RNA polymerase that silences unpaired coding regions during meiosis into crosses heterozygous rec-2SL/rec-2LG, it increased recombination, indicating that meiotic silencing of a gene promoting recombination is responsible for dominant suppression of recombination. Consistent with this, mutation of rec-2LG by Repeat-Induced Point mutation generated an allele with multiple stop codons in the predicted rec-2 gene, which does not promote recombination and is recessive to rec-2LG. Sad-1 also relieves suppression of recombination in relevant target regions, in crosses heterozygous for rec-1 alleles and in crosses heterozygous for rec-3 alleles. We conclude that for all 3 known rec genes, 1 allele appears dominant only because meiotic silencing prevents the product of the active, "recessive," allele from stimulating recombination during meiosis. In addition, the proposed amino acid sequence of REC-2 suggests that regulation of recombination in Neurospora differs from any currently known mechanism.
Asunto(s)
Neurospora crassa , Neurospora , Neurospora crassa/genética , Neurospora/genética , Mutación , Mutación Puntual , Heterocigoto , Recombinación Genética , MeiosisRESUMEN
We have built a series of Neurospora crassa strains containing alleles of green fluorescent protein (GFP) to provide a visual phenotype for investigating meiotic recombination. These strains provide a convenient means of screening the Neurospora knockout library for genes involved in genetic recombination. They permit rapid analysis of recombination outcomes by allowing visualization of segregation patterns in a large number of octads from crosses heterozygous for GFP. Using this system the effect of a knockout on gene conversion and/or on crossing over between the fluorescent marker and the centromere can be measured.
Asunto(s)
Proteínas Fluorescentes Verdes/genética , Meiosis , Neurospora crassa/genética , Recombinación Genética , Intercambio Genético , Proteínas Fúngicas/genética , Conversión Génica , Técnicas de Inactivación de Genes/métodos , Heterocigoto , Organismos Modificados GenéticamenteRESUMEN
We have inserted a histone H1-GFP fusion gene adjacent to three loci on different chromosomes of Neurospora crassa and made mating pairs in which a wild type version of GFP is crossed to one with a mutation in the 5' end of GFP. The loci are his-3, am and his-5, chosen because recombination mechanisms appear to differ between his-3 and am, and because crossing over adjacent to his-5, like his-3, is regulated by rec-2. At his-3, the frequencies of crossing over between GFP and the centromere and of conversion of 5'GFP to GFP(+) are comparable to those obtained by classical recombination assays, as is the effect of rec-2 on these frequencies, suggesting that our system does not alter the process of recombination. At each locus we have obtained sufficient data, on both gene conversion and crossing over, to be able to assess the effect of deletion of any gene involved in recombination. In addition, crosses between a GFP(+) strain and one with normal sequence at all three loci have been used to measure the interval to the centromere and to show that GFP experiences gene conversion with this system. Since any gene expressed in meiosis is silenced in Neurospora if hemizygous, any of our GFP(+) strains can be used as a quick screen to determine if a gene deleted by the Neurospora Genome Project is involved in crossing over or gene conversion.
Asunto(s)
Intercambio Genético , Sitios Genéticos , Proteínas Fluorescentes Verdes/genética , Neurospora crassa/genética , Alelos , Cromosomas Fúngicos , Proteínas Fúngicas/metabolismo , Conversión Génica , Proteínas Fluorescentes Verdes/metabolismo , Hemicigoto , Histonas/genética , Mutación , Neurospora crassa/metabolismoRESUMEN
Some organisms, such as mammals, green plants and fungi, require double-strand breaks in DNA (DSBs) for synapsis of homologous chromosomes at pachynema. Drosophila melanogaster and Caenorhabditis elegans are exceptions, achieving synapsis independently of DSB. SPO11 is responsible for generating DSBs and perhaps for the initiation of recombination in all organisms. Although it was previously suggested that Neurospora may not require DSBs for synapsis, we report here that mutation of Neurospora spo11 disrupts meiosis, abolishing synapsis of homologous chromosomes during pachynema and resulting in ascospores that are frequently aneuploid and rarely viable. Alignment of homologues is partially restored after exposure of spo11 perithecia to ionising radiation. Crossing over in a spo11 mutant is reduced in two regions of the Neurospora genome as expected, but is unaffected in a third.
Asunto(s)
Emparejamiento Cromosómico/fisiología , Cromosomas Fúngicos/química , Esterasas/genética , Meiosis/fisiología , Neurospora crassa/genética , Recombinación Genética/fisiología , Secuencia de Aminoácidos , Emparejamiento Cromosómico/efectos de la radiación , Cartilla de ADN , Endodesoxirribonucleasas , Duplicación de Gen , Meiosis/genética , Datos de Secuencia Molecular , Mutación Puntual/genética , Recombinación Genética/genética , Especificidad de la Especie , Esporas Fúngicas/genéticaRESUMEN
Although sequence heterology clearly reduces crossing over in yeast, conflicting studies suggest that mismatches may increase or decrease gene conversion. To investigate this issue in an additional species, we measured the effect of local sequence heterology on conversion in his-3 of Neurospora crassa. Mismatches close to the cog recombination initiator or within his-3 reduce conversion to 70% and 30% of the homologous level, respectively, while heterologous insertions between his-3 and cog increase conversion by 20%. We suggest that, in both Neurospora and yeast, mismatches reduce the efficiency of the establishment and resolution stages of recombination, but substantial heterology may increase the progress of already established events by preventing repair synthesis from switching between templates. These data provide additional support that recombination at his-3 (and perhaps at yeast hotspots) proceeds by a synthesis-dependent strand-annealing mechanism, during which synthesis can switch templates, with the process being more tolerant of sequence mismatch in Neurospora.
Asunto(s)
Conversión Génica , Histidina/química , Neurospora crassa/genética , Alelos , Animales , Disparidad de Par Base , Diploidia , Exones , Técnicas Genéticas , Genotipo , Heterocigoto , Homocigoto , Ratones , Modelos Genéticos , Mutación , Recombinación GenéticaRESUMEN
We have constructed a pair of vectors, pDV2 and pDV3, that enable targeted insertion of exogenous DNA into Linkage Group I of Neurospora crassa at the his-3 locus. Transplaced sequences are inserted between his-3 and the cog(L) recombination hot spot and include his-3 mutations that allow meiotic recombination initiated by cog(L) to be monitored. Selection of correctly placed transforming DNA is based on complementation between different his-3 alleles borne by the plasmids and transformation hosts. The system allows investigation of the effect of any given sequence on recombination as well as diversification of sets of related sequences in vivo for directed evolution of genes.