Search PubMed⌕ Search

Biomedical subjects

H A Lewin

Publications and source records attributed to H A Lewin.

At least 73 records · Page 4Linked to original sources
PubMed1994

Both DQB genes are expressed in BoLA haplotypes carrying a duplicated DQ region.

The objective of this study was to determine whether more than one DQB gene is expressed in three BoLA haplotypes that have a duplicated DQ region. Leukocyte mRNA from three animals genotyped at the BoLA-A, DQB, and DRB3 loci was used as template for reverse transcription-polymerase chain reaction (RT-PCR), cloning, and DNA sequencing. Five DQB alleles were identified. All cDNA clones were 564 base pairs (bp) in length, including 507 bp of nonprimer-derived sequence that contained the coding sequence for the full length of exon 2 and 74 amino acids of exon 3. Three alleles were assigned to the DQB1 locus and two were assigned to DQB2 on the basis of sequence comparisons with previously reported alleles. The expression of DQB1 and DQB2 in individual animals was examined by RT-PCR followed by double digestion of the 564 bp PCR products with Eco O109 I and Dra III in order to discriminate the DQB alleles. Evidence that DQB1 and DQB2 are both transcribed was obtained for three different BoLA haplotypes with DQB duplications, DQB10, DQB11C, and DQB12. These results suggest that the duplication or deletion event that gave rise to the DQB1 and DQB2 genes is a relatively recent event in the evolution of the cattle MHC.

Alleles↗
PubMed1994

A genetic map of nine loci on bovine chromosome 2.

A male-specific genetic linkage map of nine loci on bovine Chromosome (Chr) 2 (BTA2) was constructed from 306 offspring belonging to six paternal half-sib families. Loci studied were the structural genes for liver/bone/kidney alkaline phosphatase (ALPL). Gardner-Rasheed feline sarcoma (v-fgr) oncogene homolog (FGR), alpha-L-fucosidase 1 (FUCA1), and fibronectin 1 (FN1), and the microsatellite loci ARO28, DU17S2, DU17S3, DU17S4, and DU17S5. Genotyping was performed by restriction fragment length polymorphism (RFLP) for structural genes and polymerase chain reaction (PCR) for the microsatellites. Two genetically independent linkage groups were identified. The order of genes in the first linkage group, L31, is (ARO28-FN1)-FGR-FUCA1-ALPL, covering a map distance of 34.1 cM between terminal markers. The second linkage group, L32, consists of DU17S2-DU17S5-DU17S4-DU17S3 and is 41.3 cM in length. Genetic linkage between FN1 and FGR confirms previous physical assignment of these genes to the same synteny group. Currently, the genetic linkage of FN1 and FGR is unique to cattle and thus localizes a site of chromosomal evolution to a 22-cM interval between the two loci.

Animals↗
PubMed1994

The bovine B and C blood group systems are not likely to be the orthologues of human RH: an interesting twist in the comparative map.

We tested the hypothesis that either the bovine B or C blood group system is the orthologue of human RH. A comparative linkage mapping strategy was applied, using blood typing and restriction fragment length polymorphism (RFLP) analysis of four loci linked to RH on HSA1; PGD, FGR, ALPL and FUCA1. Four sires with a total of 255 half-sib offspring were used for the linkage analysis. Strong support for linkage between ALPL, FUCA1 and FGR was obtained for all sire families (lod scores > 11 for all pairwise comparisons). This new linkage group was assigned to bovine synteny group U17 based on previous somatic cell mapping of the FGR locus. The most favoured order is ALPL-FUCA1-FGR (2.18:1), with ALPL and FGR 5.4 CM and 6.3 CM, respectively, from FUCA1. The B and C blood group systems and PGD were genetically independent of each other and all other markers, indicating that neither B nor C is likely to be the bovine orthologue of human RH. However, given available comparative mapping data, there is some chance that the bovine orthologue of RH is on bovine synteny group U6. Although gene order appears to be conserved with humans, the differences in recombination rates between these three loci in cattle, humans and mice strongly suggest that it is not possible to use human map distances to predict map distances in cattle, making it imperative that bovine gene mappers continue to emphasize adding type I markers to the bovine linkage map.

Animals↗
PubMed1994

Polymorphism of bovine MHC class II genes. Joint report of the Fifth International Bovine Lymphocyte Antigen (BoLA) Workshop, Interlaken, Switzerland, 1 August 1992.

Polymorphism of the bovine DRB, DQA, DQB, DYA, DOB and DIB genes was investigated using restriction fragment length polymorphism (RFLP) analysis, isoelectric focusing (IEF), class II serology and polymerase chain reaction (PCR) based typing techniques. The simultaneous application of multiple typing techniques and the characterization of multiple genes resulted in a greatly enhanced picture of the bovine class II regions. Thirty-eight class IIa (DR-DQ) and 5 class IIb (DYA-DOB-DIB) haplotypes were defined. It was found that IEF types were associated with DRB3 polymorphism defined by DRB3 PCR-RFLP and DRB3 microsatellite PCR. Serologically defined polymorphism was associated with distinct molecular/IEF motifs and, therefore, DR and DQ specificities could be tentatively distinguished. Although the DR and DQ genes are tightly linked, neither DR nor DQ typing defined all of the class IIa region polymorphism. Furthermore, even the most powerful DRB3 typing technique, DRB3 PCR-RFLP, failed to detect all expressed DRB3 polymorphism. All detected DRB3 polymorphism could, however, be distinguished with a combination of two molecular techniques: DRB3 PCR-RFLP and DRB3 microsatellite PCR. RFLP typing with transmembrane probes detected significantly less polymorphism than typing with cDNA or exon probes. However, the transmembrane probes were useful because they were locus specific. The presence of only 5 of 12 possible class IIb haplotypes was unexpected and indicates that the DYA, DOB and DIB genes are tightly linked.

Animals↗
PubMed1993

Polymorphism in BoLA-DRB3 exon 2 correlates with resistance to persistent lymphocytosis caused by bovine leukemia virus.

The relationship between polymorphism of the bovine lymphocyte Ag (BoLA)-DRB3 gene and resistance and susceptibility to persistent lymphocytosis (PL) caused by bovine leukemia virus (BLV) was investigated. Exon 2 of the BoLA-DRB3 gene was cloned from animals with BoLA haplotypes previously found to be associated with resistance and susceptibility to PL. Sequence analysis revealed the presence of the amino acids Glu-Arg (ER) at putative Ag binding residues 70 and 71 only in BoLA haplotypes associated with resistance to PL. This correlation was confirmed in a case control study (n = 26) using an allele-specific polymerase chain reaction for the detection of ER at residues 70-71. These results provide a molecular basis for Ir gene control of resistance and susceptibility to PL and suggest that the cellular immune response is important in preventing the in vivo spread of BLV infection.

Alleles↗
PubMed1993

Milk and fat yields decline in bovine leukemia virus-infected Holstein cattle with persistent lymphocytosis.

Effects of bovine leukemia virus (BLV) infection on milk and fat yields were studied by using data collected from Holstein cows over a 6-year period. Milk and fat yields in BLV-infected cows with persistent lymphocytosis (PL) declined significantly relative to their BLV-infected non-PL herdmates. Declines were most pronounced in cows older than 6 years. The estimated loss to the dairy industry due to PL is more than $42 million annually. A major histocompatibility complex class I (BoLA-A) allele that has been previously associated with resistance to PL was associated with longevity and realization of milk production potentials, indicating that genetic resistance to PL will have an economic benefit in herds where BLV is endemic.

Age Factors↗
PubMed1993

Order of bovine DRB3, DYA, and PRL determined by sperm typing.

The order and recombination fractions (theta) between the bovine major histocompatibility complex DRB3, DYA, and prolactin (PRL) genes were determined by typing of 254 sperm from a triply heterozygous bull. A recently developed method, primer extension preamplification (PEP), was used to amplify the bovine sperm genome prior to amplification of specific loci by the polymerase chain reaction (PCR). At least 28 copies of the DRB3, PRL, or DYA gene were obtained from 50 cycles of PEP. For sperm typing, alleles of each locus were discriminated by restriction endonuclease cleavage of PCR products and polyacrylamide gel electrophoresis of the restriction fragments. The most likely gene order is PRL-DRB3-DYA, with theta = 0.025 (+/- 0.012) and theta = 0.150 (+/- 0.024), respectively. The odds are 128:1 in favor of this order in comparison with the second most likely order DRB3-PRL-DYA. Our results demonstrate the power of sperm typing in concert with PEP for multilocus gene mapping.

Animals↗
PubMed1993

Detection of bovine leukemia virus proviral DNA in individual cells.

We have developed a method of analyzing individual cells to detect proviral DNA of the bovine leukemia virus (BLV) using flow cytometry and PCR. Individual cells of the BL3* cell line, which contain multiple integrated copies of the BLV provirus, and the uninfected cell line BL3(0), were sorted into wells of a 96-well plate. Following cell lysis, portions of the BLV envelope (ENV) and cellular prolactin (PRL) genes were amplified simultaneously using PCR. Viral and cellular products of first-round PCR were amplified separately in a second round of PCR using "heminested" primers. Separation of the PCR products by polyacrylamide gel electrophoresis yielded distinct fragments of the predicted sizes. The operational sensitivity of this method for the detection of virus was > 90% when testing single infected cells. In addition, we were able to reliably amplify DNA from a single BL3* cell among as many as 10(5) BL3(0) cells and established that the sensitivity for detecting a single infected cell among 20, 100, or 1000 uninfected cells was at least 90%. Estimates of low percentages of infected cells were obtained by applying probability theory to results of experiments conducted on wells containing more than one cell. Using these methods, B lymphocytes obtained from the peripheral blood of BLV-infected cattle were tested for proviral DNA. BLV ENV was identified in 76.9 +/- 4.9% of single B cells tested from a seropositive animal with persistent lymphocytosis (PL), but in only 0.033 +/- 0.009% of B cells from another seropositive cow without PL.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Close linkage between bovine prolactin and BoLA-DRB3 genes: genetic mapping in cattle by single sperm typing.

The major histocompatibility complex and prolactin (PRL) genes are syntenic in humans and cattle but the genetic distance between these loci has not been determined for either species. In this study, the sperm typing technique was used to measure the recombination frequency between the bovine lymphocyte antigen (BoLA)-DRB3 and PRL loci. A total of 300 sperm were typed from one doubly heterozygous bull for segregation of DRB3 and PRL alleles. Sperm typing was performed using the polymerase chain reaction (PCR) and restriction enzyme cleavage of the PCR products, followed by resolution of the restriction fragments in polyacrylamide gels. Digestion with the restriction endonuclease RsaI allowed the unambiguous discrimination of alleles for both loci. The maximum likelihood estimation of the recombination fraction theta = 0.04, with a 95% confidence interval of 0.01 to 0.07. Close linkage between PRL and DRB3 has important implications for marker-assisted selection in animal breeding since PRL has been shown to be closely linked to a locus that affects milk yield, and BoLA loci influence susceptibility to a number of infectious diseases. Our results demonstrate the general applicability of the sperm typing procedure for gene mapping in species other than humans and provide an example of how parallel efforts to map the genomes of agriculturally important species of animals can have a positive impact on the development of a primary human linkage map.

Alleles↗
PubMed1992

Extensive polymorphism of the BoLA-DRB3 gene distinguished by PCR-RFLP.

A polymerase chain reaction (PCR)-based method is described for typing of alleles of the bovine lymphocyte antigen (BoLA)-DRB3 gene. A total of 30 DRB3 alleles were distinguished by digestion of PCR amplification products of BoLA-DRB3 exon 2 with RsaI, BstYI and HaeIII (PCR-RFLP). All restriction fragment patterns, with the exception of one HaeIII pattern, were consistent with restriction sites that were found among 14 previously sequenced DRB3 alleles. The PCR-RFLP typing method was evaluated on 168 genomic DNA samples collected from animals of 10 cattle breeds, 48 of which were typed in the Fourth International BoLA Workshop for BoLA-DRB and -DQ by conventional restriction fragment length polymorphism (RFLP) analysis using heterologous and homologous DNA probes. Thirty-one DRB/DQ haplotypes containing 23 DRB3 alleles were identified among the 48 workshop animals analysed. Using PCR-RFLP, 11 DRB3 alleles were identified in 18 workshop animals for which DRB RFLPs were not informative. PCR-RFLP typing of additional animals revealed five new DRB3 alleles, of which three contained a putatively located three basepair deletion in the identical position as found for the sequenced allele DRB*2A. PCR-RFLP was shown to be a rapid and sensitive method for the detection of polymorphism in a functionally relevant domain of the BoLA-DRB3 gene and should be useful for studying the evolution of DRB polymorphism in cattle and other Bovidae.

Alleles↗