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. 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.

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. Loss of Msh-2 function increases measures of allelic recombination in both his-3 and the fluorescent marker gene, indicating that mismatches in asymmetric heteroduplex, as in Saccharomyces cerevisiae, tend to be repaired in the direction of restoration. Furthermore, the presence of substantial numbers of conversion octads in crosses lacking Msh-2 function suggests that the disjunction pathway described in S. cerevisiae is also active in Neurospora, adding to evidence for a universal model for meiotic recombination.

Long-read, whole-genome shotgun sequence data for five model organisms.

Single molecule, real-time (SMRT) sequencing from Pacific Biosciences is increasingly used in many areas of biological research including de novo genome assembly, structural-variant identification, haplotype phasing, mRNA isoform discovery, and base-modification analyses. High-quality, public datasets of SMRT sequences can spur development of analytic tools that can accommodate unique characteristics of SMRT data (long read lengths, lack of GC or amplification bias, and a random error profile leading to high consensus accuracy). In this paper, we describe eight high-coverage SMRT sequence datasets from five organisms (Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, Arabidopsis thaliana, and Drosophila melanogaster) that have been publicly released to the general scientific community (NCBI Sequence Read Archive ID SRP040522). Data were generated using two sequencing chemistries (P4C2 and P5C3) on the PacBio RS II instrument. The datasets reported here can be used without restriction by the research community to generate whole-genome assemblies, test new algorithms, investigate genome structure and evolution, and identify base modifications in some of the most widely-studied model systems in biological research.

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. 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.

Arbitrary single primer amplification of trace DNA substrates yields sequence content profiles that are discriminatory and reproducible.

Single primer amplification is shown to yield a DNA profile that is reproducible when based on the sequence content of the amplicons rather than on the pattern of length polymorphism. The sequence-based profile increases in reliability with increasing numbers of cycles of amplification. This process uses an arbitrarily chosen primer and a low initial annealing temperature in order to amplify sequences from the whole metagenome present in a sample that may contain only trace DNA, and a large number of cycles to select subsets of sequences based on variable amplification efficiency. Using arrays, we demonstrate the utility and limitations of this approach for profiling the large metagenomes typical of soils and the trace DNA present in drug seizures. We suggest that this type of profiling will be most effective once next-generation sequencing and advanced sequence analysis becomes routine.

A rapid wire-based sampling method for DNA profiling.

This paper reports the results of a commission to develop a field deployable rapid short tandem repeat (STR)-based DNA profiling system to enable discrimination between tissues derived from a small number of individuals. Speed was achieved by truncation of sample preparation and field deployability by use of an Agilent 2100 Bioanalyser(TM). Human blood and tissues were stabbed with heated stainless steel wire and the resulting sample dehydrated with isopropanol prior to direct addition to a PCR. Choice of a polymerase tolerant of tissue residues and cycles of amplification appropriate for the amount of template expected yielded useful profiles with a custom-designed quintuplex primer set suitable for use with the Bioanalyser(TM). Samples stored on wires remained amplifiable for months, allowing their transportation unrefrigerated from remote locations to a laboratory for analysis using AmpFlSTR(®) Profiler Plus(®) without further processing. The field system meets the requirements for discrimination of samples from small sets and retains access to full STR profiling when required.

Mutation of msh-2 in Neurospora crassa does not reduce the incidence of recombinants with multiple patches of donor chromosome sequence.

The Neurospora homologue msh-2 of the Escherichia coli mismatch repair gene mutS was mutated by repeat-induced point mutation (RIP) of a 1.9-kb duplication covering 1661bp of the coding sequence and 302 bp 5' of the gene. msh-2(RIP-LK1) exhibited a mutator phenotype conferring a 17-fold increase in the frequency of spontaneous mitotic reversion of his-3 allele K458. In msh-2(RIP-LK1) homozygotes, recombination frequency at the his-3 locus increased up to 2.9-fold over that in msh-2(+) diploids. Progeny of crosses homozygous msh-2(RIP-LK1), like those from crosses homozygous msh-2(+) frequently had multiple patches of donor chromosome sequence, suggesting that patchiness in msh-2(+) crosses is not explained by incomplete repair of heteroduplex DNA by MSH-2. These findings are consistent with data from the analysis of events in a Neurospora translocation heterozygote that suggested multiple patches of donor chromosome sequence arising during recombination reflect multiple template switches during DNA repair synthesis.

Proteinaceous Metabolites from Pyrenophora teres Contribute to Symptom Development of Barley Net Blotch.

ABSTRACT Pyrenophora teres, the causal agent of net blotch of barley (Hordeum vulgare L.), induces a combination of necrosis and extensive chlorosis in susceptible barley cultivars. Cell-free filtrates from both net and spot forms of P. teres; P. teres f. sp. teres, and P. teres f. sp. maculata were found to contain phytotoxic low molecular weight compounds (LMWCs) and proteinaceous metabolites which appear to be responsible for different components of the symptoms induced by the two forms of the pathogen in a susceptible cultivar of barley (cv. Sloop). Proteins induced only brown necrotic spots or lesions similar to those induced by the pathogens 72 h after inoculation. In contrast, LMWCs induced general chlorosis seen 240 h after inoculation but not the localized necrosis. Neither hydrolyzed or heat- or protease-treated proteinaceous metabolites induced the symptoms. This is the first report of the involvement of proteins produced by P. teres in symptom development during net blotch disease of barley.

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. 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.

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%. 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.

Ds-like restless deletion derivatives occur in Tolypocladium inflatum and two foreign hosts, Neurospora crassa and Penicillium chrysogenum.

Single copies of the transposon Restless from Tolypocladium inflatum were introduced into Neurospora crassa and Penicillium chrysogenum. Excision of Restless from its donor site was investigated in N. crassa and in P. chrysogenum using direct selective conditions. In N. crassa, forward selection was also analyzed. Deleted Restless elements were frequently obtained in addition to the expected complete removal of Restless from its donor site. Similar deleted elements were also identified in T. inflatum employing a PCR amplification strategy. These deleted Restless copies strongly resemble maize Ds elements of various types, and direct repeated sequences of 3 to 16 bp were found to flank the truncated regions. In addition Ds1-like Restless elements were identified that carried foreign sequences between the inverted repeats. We discuss how Ds-like Restless elements might be generated by inaccurate excision from an active transposon copy.

Targeting vectors for gene diversification by meiotic recombination in Neurospora crassa.

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.