Biomedical subjects

P J Yeadon

Publications and source records attributed to P J Yeadon.

6 recordsLinked to original sources

PubMed2003

Diversification of exogenous genes in vivo in Neurospora.

We have adapted the meiotic recombination hotspot cog of Neurospora crassa for shuffling exogenous DNA, providing a means of generating novel genes in situ from sequences introduced into chromosomes. Genes to be diversified are inserted between the his-3 locus and cog. Diversification crosses are heterozygous both for alleles of the exogenous DNA and for auxotrophic alleles of his-3. Progeny selected for ability to grow without histidine supplementation are enriched for exchange events within the exogenous DNA. Exchange events initiated by cog can propagate past DNA sequences mismatched for more than 370 bp and complete exchanges in patches of matched sequence as short as 24 bp, parameters that make the system suited for use in the directed evolution of genes for protein engineering. Here we demonstrate the system by shuffling human immunoglobulin kappa chain genes and also endoglucanase genes derived from different species of fungi.

Cellulase↗

PubMed2001

Recombination events in Neurospora crassa may cross a translocation breakpoint by a template-switching mechanism.

To assist investigation of the effect of sequence heterology on recombination in Neurospora crassa, we inserted the Herpes simplex thymidine kinase gene (TK) as an unselected marker on linkage group I, giving a gene order of Cen-his-3-TK-cog-lpl. We show here that in crosses heterozygous for TK, conversion of a his-3 allele on one homolog is accompanied by transfer of the heterologous sequence between cog and his-3 from the other homolog, indicating that recombination is initiated centromere-distal of TK. We have identified a 10-nucleotide motif in the cog region that, although unlikely to be sufficient for hotspot activity, is required for high-frequency recombination and, because conversion of silent sequence markers declines on either side, may be the recombination initiation site. Additionally, we have mapped conversion tracts in His(+) progeny of a translocation heterozygote, in which the translocation breakpoint separates cog from the 5' end of his-3. We present molecular evidence of recombination on both sides of the breakpoint. Because recombination is initiated close to cog and the event must therefore cross the translocation breakpoint, we suggest that template switching occurs in some recombination events, with repair synthesis alternating between use of the homolog and the initiating chromatid as template.

Base Sequence↗

PubMed1999

Polymorphism around cog extends into adjacent structural genes.

The recombination hotspot cog overlaps a highly polymorphic 950-bp region of linkage group I in Neurospora crassa. The sequence of this region in the four strains, Lindegren 25a, Lindegren A, Emerson A and St. Lawrence 74A, each differs from the others by 1.4% or more. Comparison of the sequence of St. Lawrence 74A and Lindegren 25a each side of cog shows a high level of sequence heterology extending in both directions, including the coding sequences for his-3 and a putative gene lpl with homology to yeast lysophospholipase. The St. Lawrence 74A and Lindegren 25a sequences of his-3, centromere-proximal to cog, differ at 14 nucleotides, resulting in six amino-acid variations between the predicted protein sequences. In lpl, distal from cog, the sequences differ at 19 nucleotides leading to five amino-acid differences between the predicted proteins. Sequence heterology between St. Lawrence 74A and Lindegren 25a peaks either side of cog and then declines with distance. At the am locus on linkage group V, heterology is much less but peaks close to a weak recombination hotspot 5' of the coding sequence. Uneven distribution of polymorphism along chromosomes has been explained by a hitch-hiking hypothesis in which selection for advantageous mutations causes local fixation of unselected variation. We suggest that new mutations arising from errors in recombination also contribute to the uneven distribution of polymorphism.

Chromosomes, Fungal↗

PubMed1998

Long, interrupted conversion tracts initiated by cog in Neurospora crassa.

Multiple polymorphisms distinguish Emerson and Lindegren strains of Neurospora crassa within the histidine-3 gene and in its distal flank. Restriction site and sequence length polymorphism in a set of 14 PCR products covering this 6.9-kb region were used to identify the parental origin of DNA sequence information in prototrophic progeny of crosses heterozygous for auxotrophic mutations in his-3 and the silent sequence differences. Forty-one percent of conversion tracts are interrupted. Where the absence of rec-2+ permits activity of the recombination hotspot cog, conversion appears to originate at cog and conversion tracts are up to 5.9 kb long. The chromosome bearing cog(L), the dominant allele that confers a high frequency of recombination, is almost invariably the recipient of information. In progeny from crosses heterozygous rec-2/rec-2+, conversion tracts are much shorter, most are not initiated at cog and either chromosome seems equally likely to be converted. Although 32% of his-3 prototrophs have a crossover that may be associated with conversion, it is suggested that the apparent association between conversion and crossing over at this locus may be due to confounding of coincidental events rather than to a mechanistic relationship.

Alleles↗

PubMed1995

Guest: a 98 bp inverted repeat transposable element in Neurospora crassa.

The region immediately 3' of histidine-3 has been cloned and sequenced from two laboratory strains of the ascomycete fungus Neurospora crassa; St Lawrence 74A and Lindegren, which have different derivations from wild collections. Amongst the differences distinguishing these sequences are insertions ranging in size from 20 to 101 bp present only in St Lawrence. The largest of these is flanked by a 3 bp direct repeat, has terminal inverted repeats (TIR) and shares features with several known transposable elements. At 98 bp, it may be the smallest transposable element yet found in eukaryotes. There are multiple copies of the TIR in the Neurospora genome, similar but not identical to the one sequenced. PCR amplification of Neurospora genomic DNA, using 26 bp of the TIR as a single primer, gave products of discrete sizes ranging from 100 bp to about 1.3 kb, suggesting that the element isolated (Guest) may be a deletion derivative of a family of larger transposable elements. Guest appears to be the first transposable element reported in fungi that is not a retrotransposon.

Base Sequence↗

PubMed1995

The chromosomal region which includes the recombinator cog in Neurospora crassa is highly polymorphic.

The St Lawrence ST74-OR23-IVA and Lindegren Y8743 strains of Neurospora crassa have a different provenance from wild collections and dissimilar cog alleles; that in Lindegren, cogLa (previously designated cog+), is a more efficient recombinator than cogS74A and cogEa (previously cog), the alleles in St Lawrence and Emerson a respectively. Restriction fragment length polymorphisms (RFLPs) and sequence polymorphisms (SPs) were used to map the difference between cogLa and cogS74A to a region that extends from 2.3 to 3.2 kb 3' of the his-3 coding sequence. The DNA sequences from 400 bp 3' of his-3 to 120 bp 3' of the cog region in these strains were found to be homologous but to diverge by 3.5%. The differences include single-base pair changes, short insertion/deletions, differences in the length of poly-T tracts, and three longer sequences present only in St Lawrence: a 98-bp inverted repeat transposable element we have previously called Guest, which has generated a 3-bp direct repeat of the target site present in Lindegren, and 15-bp and 20-bp sequences that have no obvious structural features nor similarity to Guest. Southern analysis of other laboratory strains revealed four major and several minor variants of this region. All strains assayed are descendants of Lindegren A, Lindegren a, Abbott 4A and Abbott 12a, and it is clear that each of these progenitors collected from the wild population had a different variant of the cog region. Sequence divergence of this degree seems remarkable, even in an intergenic region, for fully interfertile strains of a single species.

Chromosome Mapping↗