Analysis of integration sites of Jaagsiekte sheep retrovirus in ovine pulmonary adenocarcinoma

Abstract

Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumor of sheep caused by Jaagsiekte sheep retrovirus (JSRV). To test the hypothesis that JSRV insertional mutagenesis is involved in the oncogenesis of OPA, we cloned and characterized 70 independent integration sites from 23 cases of OPA. Multiple integration sites were identified in most tumors. BLAST analysis of the sequences did not disclose any potential oncogenic motifs or any identical integration sites in different tumors. Thirty-seven of the integration sites were mapped to individual chromosomes by PCR with a panel of sheep-hamster hybrid cell lines. Integration sites were found on 20 of the 28 sheep chromosomes, suggesting a random distribution. However, four integration sites from four different tumors mapped to chromosome 16. By Southern blot hybridization, probes derived from two of these sites mapped to within 5 kb of each other on normal sheep DNA. These sites were found within a single sheep bacterial artificial chromosome clone and were further mapped to only 2.5 kb apart, within an uncharacterized predicted gene and less than 200 kb from a mitogen-activated protein kinase-encoding gene. These findings suggest that there is at least one common integration site for JSRV in OPA and add weight to the hypothesis that insertional mutagenesis is involved in the development of this tumor.

FIG. 1.

Schematic diagram of the ls/hs PCR technique used to amplify JSRV integration sites.

FIG. 2.

Southern blot analysis of sheep DNA hybridized with chromosome 16 integration sites 175/10 and 177/7 showing colocalization of these two sites. Each lane consists of 10 μg of genomic DNA digested with BamHI (B), EcoRI (E), PstI (P), or SacI (S). The first panel was hybridized with flank probe 175/10, the second was hybridized with 177/7, and the third was hybridized with both probes. The molecular weight (MW) standard is HindIII-digested phage lambda DNA.

FIG. 3.

Ovine BAC (OBAC) clone 383R6C1 contains integration sites 175/10 and 177/7. (a) Schematic diagram of BAC subclone (GenBank accession no. AY523942) showing PstI, SacI, and EcoRI restriction sites in relation to the integration sites. Arrows indicate the orientation of the two integration sites. (b) Alignment of the BAC clone with the orthologous HSA5q11.2 genomic sequence represented according to Build 33 of the human genome sequence according to the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/mapview). Predicted genes are in dark grey, and confirmed genes are in light grey. Boxes indicate the relative positions of BLASTn hits from randomly sequenced BAC subclones.

FIG. 4.

PCR assay specific for integration site 175/10 showing that it is present in a restricted region of the tumor only. A PCR assay was carried out with 250 ng of genomic DNA 175 LTi, from which integration site 175/10 was cloned; genomic DNA 175 LTii, made from a nearby (approximately 1 cm apart) piece of the same lung tumor; and genomic DNA 175 Kid, made from kidney tissue from the same animal. Plasmid p175/10, containing integration site 175/10, was used as the positive control. -ive, negative control.