Screening by tRNA primer extension analysis of porcine kidney mRNA libraries defines a novel endogenous porcine retroviral long terminal repeat.
BACKGROUND
We have taken advantage of the common requirement of all eukaryotic retroelements for a specific tRNA primer to initiate DNA synthesis and applied a previously described in vitro screening methodology to the analysis of in vivo porcine tissues for transcriptionally active retroviral sequences.


METHODS
A series of 18-base pair (bp) 3' tRNA oligomers complementary to established primer binding sites for a variety of vertebrate retroviruses, retrotransposons, and retroposons were applied to primer extension analysis of kidney poly(A) mRNA. Primer extension products are predicted to represent "strong stop" signals characteristic of the initial stages of retroviral transcription.


RESULTS
Several extension products were cloned, sequenced, and analyzed as probes for screening the porcine genome for potentially active retroviral sequences. We used this strategy to identify and clone a 655-bp 5' long terminal repeat of a porcine retrovirus with significant homology to the simian sarcoma virus. This transcriptionally active virus has an 82-bp U5 region, a conserved AATAAA polyadenylation sequence, a 39-bp repeat reminiscent of other retroviral enhancers, and a unique glycine primer binding site.


CONCLUSIONS
Our results suggest that tRNA primer cloning can effectively identify novel retroviral elements.
Hoopes
C W
CW
Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, USA.
Platt
J L
JL
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D015723:Gene Library; D007668:Kidney / Q000737:chemistry* / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D012343:RNA, Transfer / Q000235:genetics*; D012351:RNA, Transfer, Gln / Q000235:genetics; D012352:RNA, Transfer, Glu / Q000235:genetics; D012354:RNA, Transfer, His / Q000235:genetics; D012357:RNA, Transfer, Lys / Q000235:genetics; D012361:RNA, Transfer, Pro / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D013552:Swine; D020079:Terminal Repeat Sequences
D017931:DNA Primers; D012351:RNA, Transfer, Gln; D012352:RNA, Transfer, Glu; D012354:RNA, Transfer, His; D012357:RNA, Transfer, Lys; D012361:RNA, Transfer, Pro; D012343:RNA, Transfer