EFFECTS OF PROVIRUS INTEGRATION IN THE TPL-1 ETS-1 LOCUS IN MOLONEY MURINE LEUKEMIA VIRUS-INDUCED RAT T-CELL LYMPHOMAS - LEVELS OF EXPRESSION, POLYADENYLATION, TRANSCRIPTIONAL INITIATION, AND DIFFERENTIAL SPLICING OF THE ETS-1 MESSENGER-RNA/

The Tpl-1 locus was defined as a genomic DNA region which is targeted by provirus insertion during progression of Moloney murine leukemia vi rus-induced rat T-cell lymphomas. Using a panel of 156 (Mus musculus X Mus spretus) X Mus musculus interspecific backcross mice, we mapped T pl-1 to mouse chromosome 9 at a distance of 1.2 +/- 0.9 centimorgans f rom the Ets-1 proto-oncogene (S. E. Bear, A. Bellacosa, P. A. Lazo, N. A. Jenkins, N. G. Copeland, C. Hanson, G. Levan, and P. N. Tsichlis, Proc. Natl. Acad. Sci. USA 86:7495-7499, 1989). In this report, we pre sent evidence that all the known Tpl-1 provirus insertions occurred im mediately 5' of the first exon of Ets-1 (exon A) and that the earlier detected distance between Tpl-1 and Ets-1 was due to the high frequenc y of meiotic recombination in the region between the site of provirus integration and exon III. Northern (RNA) blot analysis of polyadenylat ed RNA from normal adult rat tissues and Moloney murine leukemia virus -induced T-cell lymphomas and hybridization to a Tpl-1/Ets-1 probe der ived from the 5' end of the gene revealed two lymphoid cell-specific R NA transcripts, of 5.5 and 2.2 kb. Sequence analysis of a near-full-le ngth (4,991-bp) cDNA clone of the 5.5-kb RNA revealed a 441-amino-acid open reading frame encoding a protein identical to the human and mous e Ets-1 proteins with the exception of five and nine species-specific conservative amino acid differences, respectively. The steady-state le vel of the Tpl-1/Ets-1 RNA and of the Ets-1 protein was modestly eleva ted in tumors carrying a provirus in the Tpl-1 locus. The relative rat io of the two Ets-1 transcripts, which were shown to arise by differen tial polyadenylation, was not affected by provirus insertion. Moreover , the major site of transcriptional initiation, which was localized by primer extension 250 bp upstream of the 5' end of the Ets-1 cDNA clon e, was shown to be identical in normal cells and tumors carrying a pro virus in the Tpl-1 locus. Finally, the differential splicing of Ets-1 exon VII was shown by RNase protection to occur at a rate of 15 to 26% and to remain unaffected by provirus insertion. The subtlety of these effects, in contrast to the strong growth selection of cells with a p rovirus in the Tpl-1/Ets-1 locus, suggests that provirus insertion may affect the fine regulation of the gene, perhaps during cell cycle pro gression.