Clone-specific high-frequency retrotransposition of a recombinant virus containing a VL30 promoter in SV40-transformed NIH3T3 cells.
A recombinant virus, containing the promoter of a VL30 LTR and tagged with the neomycin gene as a selection and indicator marker, was constructed to investigate transposition events in NIH3T3 cells after SV40 transformation. This retroviral construct was transfected into psi/CRE packaging cells, and pseudovirions were used to infect NIH3T3 cells. Clones resistant to G418 bearing single-copy integrations of the recombinant virus were isolated and transformed by SV40 virus. Transpositions were detected through RFLPs with a neomycin probe and 'retrotransposition' was further confirmed by inverse PCR and DNA sequencing of transposed and parental copies. We found that: (1) retrotransposition of this recombinant virus occurred with a high frequency in a parental clone transformed with SV40 virus suggesting that the frequency of retrotransposition depended on the initial site of provirus integration; (2) the transposition frequency was independent of the transcription level of the recombinant construct; and (3) analysis of transposition-positive transformants showed that the high transposition frequency appeared to be associated with the induction of endogenous reverse transcriptases.
Tzavaras
T
T
Laboratory of General Biology, Medical School, University of Ioannina, GR 45 110 Ioannina, Greece.
Kalogera
C
C
Eftaxia
S
S
Saragosti
S
S
Pagoulatos
G N
GN
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002472:Cell Transformation, Viral / Q000235:genetics*; D015342:DNA Probes; D017930:Genes, Reporter; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D018626:Retroelements; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
D015342:DNA Probes; D018626:Retroelements