PubMed Health⌕ Search

Biomedical subjects

D E Catcheside

Publications and source records attributed to D E Catcheside.

At least 19 recordsLinked to original sources
PubMed2001

Structure and function analysis of the calcium-related gene spray in Neurospora crassa.

The spray gene was cloned, and wildtype and mutant alleles were sequenced. Spray(+) has a 3452-bp open reading frame plus seven introns. The spray mutant had a T --> G transversion close to the carboxyl end, creating a stop codon (TGA). The sequence shows no match to genes of known function, but the carboxyl end shows seven transmembrane domains and matches putative membrane proteins of yeast. The most abundant transcript detected was 4.4 kb in size. Repeat-induced point mutagenesis produced the mutant spray phenotype. Electrophysiological analysis showed that ion fluxes in the spray plasma membrane are normal; furthermore, whereas the spray mutant was known to have no organelle-based calcium fluorescence, the cytosol shows a tip-high calcium gradient. The spray mutant is sensitive to calcineurin inhibitors. The results suggest that the SPRAY protein is located in an organellar membrane, regulating the distribution of Ca(2+) via calcineurin.

Amino Acid Sequence↗
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↗
PubMed2000

Methylation of the foreign transposon Restless in vegetative mycelia of Neurospora crassa.

Methylation of foreign and/or repeated sequences in the filamentous fungus Neurospora crassa is believed to be directed against invading transposable elements. To test this hypothesis, the fate of a transposon in N. crassa was investigated. Vectors were constructed which carried the transposon Restless, an active class-II element isolated from the fungus Tolypocladium inflatum. These vectors were introduced into N. crassa strains by protoplast transformation. Two strategies were employed: (1) ectopic multi-copy integration, and (2) site-specific single-copy integration at the his-3 locus. All ectopic transformants exhibited strong methylation as confirmed by Southern hybridization of genomic DNA digested with the methylation-sensitive endonuclease Sau3AI and the methylation-insensitive endonuclease NdeII. Single copies of Restless integrated at the his-3 locus were not methylated. These results are discussed with respect to non-RIP methylation and potential consequences for gene-tagging strategies based on the use of Restless.

Blotting, Southern↗
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↗
PubMed1999

Recombinational landscape across a 650-kb contig on the right arm of linkage group V in Neurospora crassa.

We have established a 650-kb walk spanning the sp to ure-1 interval on Neurospora crassa linkage group V. Loci covered by the walk are, from proximal to distal, sp; rec-2; ure-2; DNA polymerase delta; am; gul-1; ace-5; and ure-1. We have found extensive DNA polymorphism in this region and used this to examine the recombinational landscape. Crossovers are not evenly distributed over the region covered by the contig.

Chromosomes, Fungal↗
PubMed1999

Evidence for negative interference: clustering of crossovers close to the am locus in Neurospora crassa among am recombinants.

In response to a conflict between two mapping studies in the predicted orientation of the allele map with respect to the centromere, Fincham proposed that recombination events at the Neurospora am locus rarely have an associated crossover. Fincham considered that the elevated levels of crossing over between flanking markers in am recombinants resulted from negative interference, an increased probability of a nearby second event, and on this basis predicted a clustering of crossing over near am in these recombinants. In this article we reevaluate the data from three mapping studies of the am locus and report molecular evidence that shows crossovers to be clustered immediately proximal to am in am recombinants.

Crossing Over, Genetic↗
PubMed1998

Analysis of conversion tracts associated with recombination events at the am locus of Neurospora crassa.

In cross B163, heteroallelic am1 am6 and heterozygous for both conventional genetic flanking markers and closer molecular markers, we previously found that the majority on flanker exchanges were remote from events that generated prototrophic recombinants. We report here that natural polymorphisms distinguishing the parents of cross B163 also include sequences within and closely flanking am. Segregation of these markers in B163 prototrophs confirms that the majority of meiotic recombination events at am resulted from gene conversion. Conversion of am6, the distal allele, is more frequent than conversion of am1. Twelve percent of tracts in am6 convertants were discontinuous while 30% of continuous tracts converting am6 extend less than 741 bp.

Alleles↗
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↗
PubMed1996

Gene conversion alone accounts for more than 90% of recombination events at the am locus of Neurospora crassa.

We have used closely flanking molecular markers located approximately 4 kb distal and 6 kb proximal of the am locus to investigate the incidence of crossover events associated with the generation of prototrophic recombinants in a cross heteroallelic am1 am6. Ninety-three percent of prototrophs were generated by events that did not recombine the molecular markers, indicating that simple conversion accounts for the formation of most prototrophs and that associated crossovers are much less frequent (approximately 0.07) than estimated previously using more distant flanking markers. This suggests that conversion and crossing over during meiosis may arise from distinct mechanisms or that if, as is widely supposed, conversion and crossing over result from alternate modes of resolution of Holliday junctions then, at least for the am locus of Neurospora, the mode of resolution is strongly biased in favor of retaining the parental association of flanking sequences. Because estimates of the association of conversion and crossing over based on more distant gene markers are similar for yeast and Neurospora (approximately 0.35), our observation may have general significance.

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 orientation of gene maps by recombination of flanking markers for the am locus of Neurospora crassa.

Fincham (1967), Smyth (1973 b) and Rambosek and Kinsey (1983) have each generated fine-structure maps of the am gene of Neurospora crassa. Each map had a consistent linear order of alleles but the assignment of an orientation with respect to other linkage group-V loci differed. Fincham found the end marked by the am6 allele to be at the distal end of the locus, Smyth found am6 to be at the proximal end while the data of Rambosek and Kinsey did not suggest an orientation. Smyth s orientation has been adopted as the standard, but not unreservedly. We have aligned the genetic and physical maps of the am gene, showing that am6 is at the distal end, supporting Fincham's orientation. However, we suggest that an assumption used to orient fine structure genetic maps is flawed and that the conflicting orientation between these three studies follows from the different choice of flanking markers.

Chromosome Mapping↗
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↗
PubMed1991

The initiation site for recombination cog is at the 3' end of the his-3 gene in Neurospora crassa.

Recombination at his-3 in Neurospora crassa is thought to be initiated through a site designated cog which lies in the his-3 to ad-3 interval of linkage group I. Fragments of the his-3 gene were used to transform various his-3 mutant alleles to prototrophy in order to link the genetic map to the nucleotide sequence. It was established that cog is at the 3' end of his-3 and is therefore not the his-3 promoter. This suggests that cog may be dissimilar to a number of yeast recombinators which are associated with promoters of transcription.

Alleles↗
PubMed1988

Cloning and expression of the pneumococcal neuraminidase gene in Escherichia coli.

A gene bank of Sau3AI-generated Streptococcus pneumoniae DNA fragments was constructed in Escherichia coli K-12 by cloning into the BamHI site of the cosmid vector pHC79. One clone capable of cleaving the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid was isolated. This activity was inhibited by treatment with a mouse antiserum raised against purified pneumococcal neuraminidase. The recombinant plasmid purified from this clone (designated pJCP301) contained approximately 3.0 kb of pneumococcal DNA. Western-blot analysis indicated that E. coli K-12[pJCP301] produced a 98-kDa polypeptide which reacted with antineuraminidase serum.

Blotting, Southern↗
PubMed1985

Cloning of the ARO cluster gene of Neurospora crassa and its expression in Escherichia coli.

We have constructed a phage, lambda Ncl, which comprises a 4.0 kb HindIII insert of Neurospora DNA into the immunity region of the vector lambda 598. lambda Ncl complements the aroD6 mutation of E. coli, permitting the formation of galaxy plaques on medium lacking aromatic supplements, and transforms an aro-9 qa-2 Neurospora mutant to prototrophy at a low frequency. Low levels of 5-dehydroquinate hydrolyase (E.C.4.2.1.10.), with properties unlike those of the catabolic isoenzyme that is coded by qa-2, are present in E. coli aroD6 cell lysates following infection with lambda Ncl. lambda Ncl does not hybridize with qa-2 DNA and it is concluded that it contains at least the aro-9 region of the pentafunctional aro cluster gene.

Amino Acids↗
PubMed1981

Genes in Neurospora that suppress recombination when they are heterozygous.

Genes that suppress recombination when heterozygous have been found distributed as a polymorphism in wild and laboratory populations of Neurospora crassa. Three alleles, ssE, ssS and ssC, are associated, respectively, with th the three wild types Emerson, St. Lawrence 74A and Costa Rica A. It is proposed that ss (synaptic sequence) genes modulate recombination by determining the pairing closeness of DNA duplexes in the vicinity of the nit-2 locus. When heterozygous, ss suppresses recombination 2- to 20-fold within the nit-2 locus, which it adjoins, but crossing over in intervals flanking nit-2 is not affected. The magnitude of suppression depends upon the ss alleles involved, and ss acts multiplicatively with rec-1; together, these genes modulate recombination within the nit-2 locus over a range exceeding 100-fold. The ss effect is not attributable to gross chromosomal rearrangement, but could be due to small inversions or insertions, such as transposable elements.

Genes, Bacterial↗