Construction of a swine BAC library: application to the characterization and mapping of porcine type C endoviral elements

A porcine bacterial artificial chromosome (BAC) library was constructed usi ng the pBeloBAC11 vector. It comprised 107,520 clones with an average inser t size of 135 kb, representing an almost fivefold coverage of the swine hap loid genome. Screening of the library allowed recovery of one to eight clon es for 142 unique markers located all over the genome, while it failed for only one marker. About 4% chimeric clones were found. The library was also screened for the protease gene of type C porcine endoviral sequences (PERVs ), and 62 clones were recovered, all but two of which contained one proteas e gene. We found 20 protease sequences (PERV-1 to PERV-20) which, despite d iffering by point mutations, were all coding sequences. The most frequent s equence, PERV-2, was 100% similar to a protease sequence expressed in the p orcine PK-15 cell line. Most of the clones harbored envelope genes. Thirty- three BAC clones were mapped by fluorescence in situ hybridization to 22 di stinct locations on 14 chromosomes, including the X and Y chromosomes. Thes e overall results indicate that there is generally one PERV copy per integr ation site. Although PERV sequences were not tandemly arranged, clusters of integration sites were observed at positions 3p1.5 and 7p1.1. Southern blo t experiments revealed 20-30 PERV copies in the Large White pig genome stud ied here, and variations in PERV content among pigs of different breeds wer e observed. In conclusion, this BAC collection represents a significant con tribution to the swine large genomic DNA cloned insert resources and provid es the first detailed map of PERV sequences in the swine genome. This work is the first step toward identification of potential active sites of PERV e lements.