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K J Beh

Publications and source records attributed to K J Beh.

At least 19 recordsLinked to original sources
PubMed2001

Molecular analysis and nematode resistance association of a polymorphism at the 5' end of the sheep IgE gene.

Previous work using Southern analysis of genomic DNA detected a polymorphism at the 5' end of the sheep and cattle IgE gene. Identical length differences found between fragments following digestion with restriction enzymes indicated that the basis for the polymorphism was an insertion/deletion event. To characterise the polymorphism, the entire cattle and sheep Cvarepsilon genes were sequenced including 668bp of 5' untranslated DNA. Sequence comparison revealed a high degree of similarity between the ovine and bovine genes at both the nucleotide and amino acid level. A feature of the 5' untranslated DNA was the presence of an 87bp repeat starting at -365 upstream of the Cvarepsilon start site. PCR primers were designed to span most of the 5' untranslated sequence, including the repeat unit, and used to amplify genomic DNA from a panel of 40 sheep. Three alleles were found with frequencies of 0.7, 0.29, 0.01 which were identical to the Southern analysis results. Sequencing of the two commonest alleles revealed the basis for the polymorphism was a 36bp deletion from the 87bp repeat. Association studies in a sheep selection flock phenotypically assessed for parasite resistance found a highly significant association between one of the IgE alleles and resistance to the intestinal nematode parasite Trichostrongylus colubriformis (P=0.005). Attempts to confirm this finding in two other flocks using linkage analysis and genotype association failed to find any significant associations between the IgE polymorphism and resistance to either T. colubriformis or Haemonchus contortus.

Alleles↗
PubMed2001

An enhanced linkage map of the sheep genome comprising more than 1000 loci.

A medium-density linkage map of the ovine genome has been developed. Marker data for 550 new loci were generated and merged with the previous sheep linkage map. The new map comprises 1093 markers representing 1062 unique loci (941 anonymous loci, 121 genes) and spans 3500 cM (sex-averaged) for the autosomes and 132 cM (female) on the X chromosome. There is an average spacing of 3.4 cM between autosomal loci and 8.3 cM between highly polymorphic [polymorphic information content (PIC) > or = 0.7] autosomal loci. The largest gap between markers is 32.5 cM, and the number of gaps of > 20 cM between loci, or regions where loci are missing from chromosome ends, has been reduced from 40 in the previous map to 6. Five hundred and seventy-three of the loci can be ordered on a framework map with odds of > 1000 : 1. The sheep linkage map contains strong links to both the cattle and goat maps. Five hundred and seventy-two of the loci positioned on the sheep linkage map have also been mapped by linkage analysis in cattle, and 209 of the loci mapped on the sheep linkage map have also been placed on the goat linkage map. Inspection of ruminant linkage maps indicates that the genomic coverage by the current sheep linkage map is comparable to that of the available cattle maps. The sheep map provides a valuable resource to the international sheep, cattle, and goat gene mapping community.

Animals↗
PubMed1997

Production and verification of an anti-ovine IgE monoclonal antibody.

Recombinant mouse/sheep IgE was used in the production of an anti-IgE monoclonal using conventional hybridoma techniques. The specificity of hybridomas secreting anti sheep IgE monoclonal antibodies was verified using a number of assays including competitive ELISAs, ability to induce mediator release from mast cells, and IgE binding using western blotting. Immunohistochemical staining demonstrated the binding of putative anti-IgE monoclonals to the sheep mast cell surface. The isotype of the antibody was IgG1:K.

Animals↗
PubMed1996

A tandemly repetitive DNA sequence is present at diverse locations in the genome of Ostertagia circumcincta.

A novel repetitive DNA sequence in the sheep parasitic nematode Ostertagia circumcincta was cloned and sequenced. This 1.2-kb sequence (Oc1B) was not found in the closely related cattle parasite Ostertagia ostertagi, nor in the more distantly related sheep parasites Haemonchus contortus or Trichostronylus colubriformis. Sequences similar to Oc1B were found at various genomic locations and contained a pair of 33-bp direct repeats. Oc1B also contained a single copy of a 218-bp sequence (designated OcREP) which was present in 100 to 200 copies in the O. circumcincta genome and mostly organized in distinctive tandem arrays. The dual organizational pattern of OcREP as both a satellite-like sequence as well as interspersed as single copies amongst dissimilar sequences adds to the growing evidence for the fluidity of the parasitic nematode genome, and of eukaryotic genomes in general.

Animals↗
PubMed1994

Characterization of a tandemly repetitive DNA sequence from Haemonchus contortus.

Genomic DNA from the sheep parasitic nematode Haemonchus contortus was shotgun cloned in the plasmid vector pUC18. Recombinants which gave the strongest hybridization signals to 32P-radiolabelled genomic DNA were selected as representatives of the repetitive component of the parasite DNA. One repetitive sequence which hybridized only with DNA from H. contortus and not with DNA from two other sheep nematodes (Trichostrongylus colubriformis and Ostertagia circumcincta) was further characterized by sequencing and dot blot analysis. A related repeat was found in the closely related species Haemonchus placei. Experiments to determine the genomic organization of the repeat showed that it existed in a multi-copy number tandem array. This is the first report on the characterization of repetitive DNA in sheep parasite nematodes.

Animals↗
PubMed1994

A middle-repetitive DNA sequence element in the sheep parasitic nematode, Trichostrongylus colubriformis.

A novel repetitive DNA sequence in the sheep parasitic nematode Trichostrongylus colubriformis was cloned and sequenced. A 1.1 kb repetitive sequence (Tc15) which hybridized with DNA from T. colubriformis but not with DNA from two other parasitic nematodes, Haemonchus contortus and Ostertagia circumcincta, or sheep was further characterized. Southern blot analysis showed that the repeat hybridized to a range of fragments in restriction digested T. colubriformis DNA and existed in multiple copy number tandem arrays. However, to define clearly the repetitive monomeric unit further screening of phagemid libraries containing BamH I restriction fragments using a subclone of Tc15 as a probe was carried out. Restriction map and sequence data were compiled for 3 clones containing a 145 bp highly repetitive sequence (designated TcREP) which shared homology with the original pTc15 clone. TcREP hybridized to a tandemly repeating sequence monomer of 145 bp in T. colubriformis DNA which was cloned from various genetic environments in the T. colubriformis genome. TcREP homologous sequences were also found in the genomes of two other species of the same genus (Trichostrongylus axei and Trichostrongylus vitrinus) but not in a fourth species (Trichostrongylus rugatus).

Animals↗
PubMed1994

Cellular inflammatory responses in skin of sheep selected for resistance or susceptibility to fleece rot and fly strike.

Leucocyte populations were examined in normal and inflamed skin of sheep bred for resistance (R) or susceptibility (S) to bacterial fleece rot and the common sequela, body strike caused by the dipteran parasite Lucilia cuprina. No differences between R and S lines were found in numbers of neutrophils accumulating in acute inflammatory lesions induced by activated complement, leukotriene B4, interleukin (IL)-1 beta, tumour necrosis factor-alpha, IL-8 or endotoxin from Pseudomonas aeruginosa. T19+ (alpha gamma delta T cell subset) lymphocytes and eosinophils were more prevalent in skin of sheep from the S line whereas IgE+ cells were more prevalent in skin of sheep from the R line. In an unrelated population of sheep, animals with low fleece rot scores had more intense neutrophil migration into inflammatory lesions induced by all the mediators examined than did animals with high fleece rot scores. IgE+ cells were more prevalent in animals with low fleece rot scores, although in contrast to R and S lines, T19+ cells tended to be elevated in this group of animals. The results suggest that defence mechanisms associated with IgE+ cells in skin may play an important role in resistance to fleece rot and fly strike.

Animals↗
PubMed1994

Expression of a recombinant sheep IgE gene.

An ovine cosmid library was screened with a bovine C epsilon gene probe. IgE and IgA constant region genes (C epsilon and C alpha, respectively) were isolated within a single 26 kb cosmid insert. The C epsilon gene was cloned into an expression vector which contained the rearranged VDJ genomic segment encoding an anti-(4-hydroxy-3-iodo-5-nitrophenyl) acetic acid (anti-NIP) VH chain. This chimeric construct was transfected into murine hybridoma cells producing L chain with anti-NIP antibody specificity and stable transfectomas secreting chimeric ovine-murine IgE anti-NIP antibodies were obtained. Chimeric antibody, affinity purified from culture supernatants of transfected cells on a NIP-sepharose column, was characterised by SDS-PAGE and shown to contain H and L chains of expected size. These experiments demonstrated the power of advanced molecular genetic techniques to generate immunological reagents against low abundance immunoglobulin isotypes thus facilitating monoclonal antibody production for further immunological studies.

Animals↗