Recombination hotspots in Neurospora crassa controlled by idiomorphic sequences and meiotic silencing.

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 10 kb region in rec-2SL strains was replaced by a 2.

Fluorescent Protein as a Tool for Investigating Meiotic Recombination in Neurospora.

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.

Meiotic Recombination in Neurospora crassa Proceeds by Two Pathways with Extensive Holliday Junction Migration.

Analysis of thousands of Δmsh-2 octads using our fluorescent recombination system indicates that, as in other filamentous fungi, symmetric heteroduplex is common in the his-3 region of Neurospora crassa. Symmetric heteroduplex arises from Holliday junction migration, and we suggest this mechanism explains the high frequency of His+ spores in heteroallelic crosses in which recombination is initiated cis to the his-3 allele further from the initiator, cog+. In contrast, when recombination is initiated cis to the his-3 allele closer to cog+, His+ spores are mainly a result of synthesis-dependent strand annealing, yielding asymmetric heteroduplex.

Use of fluorescent protein to analyse recombination at three loci in Neurospora crassa.

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.

Chromosome pairing and meiotic recombination in Neurospora crassa spo11 mutants.

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.

Sequence heterology and gene conversion at his-3 of Neurospora crassa.

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%.