Molecular, genetic and physical mapping of the porcine genome

The goals of the pig genome project are to characterize the molecular basis of agricultural traits and to shed light on the evolution and organization of the mammalian genome. The first objective of this thesis was to physically assign cosmid clones that contain genetically mapped microsatellites. Individual cosmids were cytogenetically localized to chromosomes 1, 6, 13, 14, and five cosmids were mapped to chromosome 7. The physical assignment of these cosmids was consistent with their previously assigned genetic location, integrating these markers. The second objective was to characterize a cosmid clone comprised of tandem copies of a 3.3 kb higher-order repeat. The DNA sequences of three independent higher-order repeats are >99% identical to each other and the total expanse of the array is approximately 2.2 Mb. The higher-order repeats are comprised of ten monomers, whose DNA sequence is on average 79% identical to each other. A cloned 3.3 kb higher-order repeat unit hybridized specifically to the centromere of chromosome 9. The organization of monomer sequences into chromosome-specific higher-order repeats is a characteristic of the human and swine centromeres, which suggests that the evolutionary mechanisms that create higher-order repeats is conserved. The third thesis objective was to use differential display-reverse transcription PCR (DDRT-PCR) to identify longissimus muscle cDNAs that are differentially expressed between a line of pigs selected for increased 200-day weight and a non-selected control line. A 590-bp cDNA product was sequenced based on its greater abundance in the select line. The DDRT-PCR product corresponds to the 3′-end of the pig homolog of the 16-kDa cAMP-regulated phosphoprotein gene (ARPP-16), which was RT-PCR amplified and sequenced. The porcine homolog of the 19-kDa cAMP-regulated phosphoprotein gene (ARPP-19) was also cloned and sequenced and only differs from ARPP-16 by the addition of 16 N-terminal amino acids. In all tissues studied ARPP-19 mRNA was detected by RT-PCR amplification. Semi-quantitative RT-PCR verified that onlyARPP-16 is up-regulated in the select line. The relative increase in ARPP-16 mRNA expression in the select line animals indicates that this gene may play a role in the molecular pathways regulating postnatal skeletal muscle growth.