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Title: CHARACTERISATION OF A FLOW-SORTED SWINE CHROMOSOME 6 LIBRARY

Author
item GRIMM, DAVID - UNIVERSITY OF MARYLAND
item GOLDMAN, THERESA - USDA, LPSI, PIL
item Holley Shanks, Rhonda
item BUOEN, L - UNIVERSITY OF MINNESOTA
item MENDIOLA, J - UNIVERSITY OF MINNESOTA
item LOUIS, C - UNIVERSITY OF MINNESOTA
item Lunney, Joan

Submitted to: Mammalian Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/27/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Our research is aimed at further defining the area of swine chromosome 6 that contains the gene responsible for Porcine Stress Syndrome (PSS). Animals with PSS exhibit inferior meat quality and will die suddenly in response to stress. The DNA in this region may also contain positive carcass trait genes yet to be identified. In order to investigate the latter possibility, individual copies of swine chromosome 6 were prepared from white blood cells and their DNA was isolated. Fluorescent techniques were used to verify that uncontaminated chromosome 6 DNA was in fact isolated. This DNA was then cloned and searched for the presence of repetitive areas in the sequence called microsatellite markers. Microsatellites are used as reference points along the chromosome to mark an area that has as of yet unidentified DNA sequence. These markers are then arranged by statistical analysis to provide a linear framework of reference points along the chromosome, called a linkage map. Once their location is established, these markers can then be used to follow that region of the chromosome throughout a pedigree and provide clues to the presence of any important genes in that area. The statistical analysis of the results of our search showed that we had isolated two new microsatellites on swine chromosome 6. The addition of more microsatellites to the swine chromosome 6 linkage map will provide the markers necessary to search the region containing the PSS gene for the presence of beneficial carcass trait genes.

Technical Abstract: Swine chromosome 6 enriched libraries, generated with size fractionated DNA isolated from chromosomes sorted by flow cytometry, have been used to develop new chromosome 6 microsatellite markers. Chromosome isolation procedures were established to reproducibly prepare high quality chromosomes from PHA stimulated swine peripheral blood lymphocytes and to sort individual chromosomes after staining with Hoechst 33258 and chromomycin A3. Chromosome purity was verified by fluorescence in situ hybridization (FISH) using painting probes generated by DOP- PCR amplification of as few as 300 sorted chromosomes. Specific staining of swine chromosome 6 was evident from the amplified product. For library construction, DNA was extracted from flow sorted pools representing chromosome 6 and ligated into pBluescript SK 11+. The cloned DNA was PCR amplified using T7 and T3 primers, and 300-700bp DNA fragments were isolated and subcloned into either pBluescript SK 11+ or Lambda ZAP Express to produce the chromosome 6 enriched libraries. The libraries were screened with a radiolabelled poly-(dCA) DNA probe and isolated clones were sequenced to verify the presence of (CA)n repeats. Four microsatellites sequences were isolated, which proved successful with amplification by PCR (S0441, S0442, S0443, and S0444). Mapping for chromosome specificity was performed with these 4 microsatellites using swine reference populations, and linkage analysis showed that S0443 and S0444 mapped to swine chromosome 6, while S0441 and S0442 mapped to chromosomes 2 and 8, respectively.