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 using the pBeloBAC11 vector. It comprised 107,520 clones with an average insert size of 135 kb, representing an almost fivefold coverage of the swine haploid genome. Screening of the library allowed recovery of one to eight clones 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 protease gene. We found 20 protease sequences (PERV-1 to PERV-20) which, despite differing by point mutations, were all coding sequences. The most frequent sequence, PERV-2, was 100% similar to a protease sequence expressed in the porcine PK-15 cell line. Most of the clones harbored envelope genes. Thirty-three BAC clones were mapped by fluorescence in situ hybridization to 22 distinct locations on 14 chromosomes, including the X and Y chromosomes. These overall results indicate that there is generally one PERV copy per integration site. Although PERV sequences were not tandemly arranged, clusters of integration sites were observed at positions 3p1.5 and 7p1.1. Southern blot experiments revealed 20-30 PERV copies in the Large White pig genome studied here, and variations in PERV content among pigs of different breeds were observed. In conclusion, this BAC collection represents a significant contribution to the swine large genomic DNA cloned insert resources and provides 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 elements.
Rogel-Gaillard
C
C
Laboratoire de Radiopathologie et d'Etude du Génome, Département de Génétique Animale, Jouy-en-Josas, France. rogel@biotec.jouy.inra.fr
Bourgeaux
N
N
Billault
A
A
Vaiman
M
M
Chardon
P
P
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004247:DNA / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics*; D017988:Gammaretrovirus / Q000235:genetics*; D015698:Genomic Library; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013552:Swine / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics
D014764:Viral Proteins; D004247:DNA; D010450:Endopeptidases