Helper activity for gene expression, a novel function of the SV40 enhancer.
In this investigation we demonstrate that the enhancer of SV40 possesses an additional function which is a 'helper activity' for a more efficient transfer of viral DNA from the cytoplasm into the nucleus. After DNA transfection into rat-2 cells, the rate of CAT gene expression linked to SV40 promoter/enhancer (pSV2CAT) was approximately 50 fold higher than linked to the tk promoter (pBLCAT2). After direct nuclear microinjection this difference was reduced to a factor of 10. However cytoplasmic injection of the same number of DNA molecules/cell showed again a 50 fold increase for the SV40 promoter/enhancer-CAT construct. This difference was not due to selective degradation of the pBLCAT2 DNA. The 'helper function' did not require the intact 72 bp sequence. In vitro synthesized enhancers lacking certain enhancer motifs (GT-I, TC-II and TC-I sequence) were still effective after cytoplasmic injection whereas an 8 bp deletion (representing a part of the AP-I motif) on the downstream side strongly reduced the helper function after cytoplasmic injection but not the classical transcriptional enhancement after direct nuclear transfer.
Graessmann
M
M
Institut für Molekularbiologie, Berlin, FRG.
Menne
J
J
Liebler
M
M
Graeber
I
I
Graessmann
A
A
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005796:Genes; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection
D004279:DNA, Viral; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase