The initial focus of this PhD project was on comparative gene mapping. Comparative gene mapping is facilitated by consensus PCR primers which amplify homologous gene fragments in many species. As a part of an international co-ordinated programme of comparative mapping in pigs, 47 CATS (Comparative Anchor Tagged Sequence) consensus primer pairs for loci located on human chromosomes 9, 10, 20, and 22, were used for amplifying homologous loci in pigs. After optimization of PCR conditions, 23 CATS products have confirmed by comparison with homologous sequences in GenBank. A French somatic cell hybrid panel was used to physically map the 6 porcine CATS products distinguishable from rodent background product, namely ADRA1A, ADRA2A, ARSA, GNAS1, OXT and TOP1. Of these, the map location of ADRA1A and OXT showed inconsistency with the previously recognised conserved relationship between human and pig. The other four loci mapped to positions consistent with known syntenic relationships. Despite low levels of polymorphism, frequently indistinguishable rodent and porcine products in somatic hybrids and some confusion of identity of gene family members, these CATS primers have made a useful contribution to the porcine-human comparative map. The focus of the project then changed to genetic and molecular characterisation of endogenous retroviruses in pigs and their relatives. Pigs are regarded as a potentially good source of organs and tissues for transplantation into humans. However, porcine endogenous retroviruses have emerged as a possible problem as they can infect cultured human cells. Two main types of pig retrovirus, determined by envelope protein, PERV-A and PERV-B, are widely distributed in different pig breeds and a third less common type, PERV-C, has also been recognised. Endogenous retroviruses were analyzed from the Westran (Westmead transplantation) inbred line of pig, specially bred for biomedical research. Thirty-one 1.8 kb env PCR product clones were sequenced after preliminary screening with the restriction enzymes KpnI and MboI. Five recombinant clones between A and B were identified. 55% of clones (17/31) sequenced had stop codons within the envelope protein-encoding region, which would prevent the retrovirus from making full-length envelope protein recognizable by cell-surface receptors of the virus. The endogenous viruses were physically mapped in Westran pigs by FISH (Fluorescence In Situ Hybridisation) using PERV-A and PERV-B envelope clones as probes. Preliminary FISH data suggest that there are at least 22 PERVs (13 PERV-A and 9 PERV-B) and the chromosomal locations of these in the Westran strain are quite different from European Large White pigs. The sequences and mapping results of inbred Westran pig suggest that there are relatively few PERV integration sites compared with commercial pigs and further that a large proportion of clones are defective due to premature stop codons in the envelope gene. To investigate the relationship of endogenous retroviruses in peccaries and pigs, a set of degenerate primers was used to amplify peccary retroviral sequences. The sequences of two putative retroviral clones showed close homology, albeit with a 534 bp deletion, to mouse and pig retroviral sequences. Also, four non-target sequences were amplified from peccary with the degenerate retroviral primers. They are a part of the peccary cofilin gene, a SINE, and a sequence containing a microsatellite. The peccary endogenous retroviral sequences are significant in that they are the first such sequences reported in peccary species and repudiate old claims in the literature that peccaries do not have C-type retroviral sequences.
| Identifer | oai:union.ndltd.org:ADTP/215926 |
| Date | January 2001 |
| Creators | Lee, Jun Heon |
| Publisher | University of Sydney. |
| Source Sets | Australiasian Digital Theses Program |
| Language | English, en_AU |
| Detected Language | English |
| Rights | Copyright Lee, Jun Heon;http://www.library.usyd.edu.au/copyright.html |
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