TY - JOUR
T1 - An indicator gene to demonstrate intracellular transposition of defective retroviruses
AU - Heidmann, T.
AU - Heidmann, O.
AU - Nicolas, J. F.
PY - 1988/12/1
Y1 - 1988/12/1
N2 - An indicator gene for detection and quantitation of RNA-mediated transposition was constructed (neo(RT)). It was inserted into a Moloney murine leukemia provirus (Mo-MLV) deleted for the envelope gene to test for intracellular transposition of defective retroviruses [Mo-MLV(neo)(RT)]. Neo(RT) contains the selectable neo gene (which confers resistance to the drug G418), inactivated by a polyadenylylation sequence inserted between the neo promotor and coding sequence. The polyadenylylation sequence is flanked (on the antisense strand of the DNA) by a donor and an acceptor splice site so as to be removed upon passage of the provirus through an RNA intermediate. 3T3 cells transfected with the defective Mo-MLV(neo)(RT) provirus are sensitive to G418. After trans-complementation with Mo-MLV, viral transcripts confer resistance to G418 upon infection of test cells. In the resistant cells, the polyadenylylation sequence has been removed, as a result in most cases of precise splicing of the intronic domain. Retrotransposition of the defective Mo-MLV(neo)(RT) provirus was demonstrated by submitting transfected G418-sensitive clones to selection. Between 1 and 10 G418-resistant clones were obtained per 107 cells. Several possess additional copies, with evidence for precise removal of the intronic domain. By using target test cells in coculture experiments, extracellular intermediates of retrotransposition could not be detected.
AB - An indicator gene for detection and quantitation of RNA-mediated transposition was constructed (neo(RT)). It was inserted into a Moloney murine leukemia provirus (Mo-MLV) deleted for the envelope gene to test for intracellular transposition of defective retroviruses [Mo-MLV(neo)(RT)]. Neo(RT) contains the selectable neo gene (which confers resistance to the drug G418), inactivated by a polyadenylylation sequence inserted between the neo promotor and coding sequence. The polyadenylylation sequence is flanked (on the antisense strand of the DNA) by a donor and an acceptor splice site so as to be removed upon passage of the provirus through an RNA intermediate. 3T3 cells transfected with the defective Mo-MLV(neo)(RT) provirus are sensitive to G418. After trans-complementation with Mo-MLV, viral transcripts confer resistance to G418 upon infection of test cells. In the resistant cells, the polyadenylylation sequence has been removed, as a result in most cases of precise splicing of the intronic domain. Retrotransposition of the defective Mo-MLV(neo)(RT) provirus was demonstrated by submitting transfected G418-sensitive clones to selection. Between 1 and 10 G418-resistant clones were obtained per 107 cells. Several possess additional copies, with evidence for precise removal of the intronic domain. By using target test cells in coculture experiments, extracellular intermediates of retrotransposition could not be detected.
KW - Moloney murine leukemia virus
KW - envelope gene
KW - mobile genetic element
KW - retrotransposition
UR - http://www.scopus.com/inward/record.url?scp=0024121507&partnerID=8YFLogxK
U2 - 10.1073/pnas.85.7.2219
DO - 10.1073/pnas.85.7.2219
M3 - Article
C2 - 2832848
AN - SCOPUS:0024121507
SN - 0027-8424
VL - 85
SP - 2219
EP - 2223
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 7
ER -