C. Renard

Construction of a swine YAC library allowing an efficient recovery of unique and centromeric repeated sequences.

A swine DNA genomic library was constructed in yeast artificial chromosome (YAC) using the pYAC4 vector and the AB1380 strain. The DNA prepared from two Large White males was partially digested with EcoRI and size selected after both digestion and ligation. The YAC library contained 33792 arrayed clones with an average size of 280 kb as estimated by analysis of 2% of the clones, thus representing a threefold coverage of the swine haploid genome. The library was organized in pools to facilitate the PCR screening. The complexity of the library was tested both for unique and centromeric repeated sequences. In all, 20 out of 22 primer sets allowed the characterization of one to six clones containing specific unique sequences. These sequences are known to be on Chromosomes (Chrs) 1, 2, 5, 6, 7, 8, 13, 14, 15, 17, and X. Eight additional clones carrying centromeric repeat units were also isolated with a single primer set. The sequencing of 37 distinct repeat units of about 340 bp subcloned from these eight YACs revealed high sequence diversity indicating the existence of numerous centromeric repeat unit subfamilies in swine. Furthermore, the analysis of the restriction patterns with selected enzymes suggested a higher order organization of the repeat units. According to preliminary FISH experiments on a small number of randomly chosen YACs and YACs carrying specific sequences, the chimerism appeared to be low. In addition, primed in situ labeling experiments favored the idea that the YACs with centromeric repeat sequences were derived from a subset of metacentric and submetacentric chromosomes.

MOLECULAR GENETIC ANALYSES OF THE MAJOR HISTOCOMPATIBILITY COMPLEX IN PIG FAMILIES AND RECOMBINANTS

Five HLA probes, one corresponding to class I genes, two corresponding to distinct class II light chain genes, DRβ and DQβ, and two to class II heavy chain genes, DRα and DQα, were used to analyse the genomic DNA of the pig. Three informative SLA typed families and four SLA recombinants were studied by Southern blot analysis. About 16 restriction fragments, generated by EcoR 1 or Hind III endonucleases, were revealed for each individual, either with the class I probe or the DRβ probe. The number of restriction fragments which hybridized with the other probes was generally lower. Several restriction fragment‐length polymorphisms were found and these segregated with SLA DRβ‐ and DQα‐like genes are probably tightly clustered within the SLA‐D‐MLR region.