- 作者: Wen-Jiun Peng, Chau-Ming Chang, Thy-Hou Lin
- 作者服務機構: Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, ROC
- 中文摘要: --
- 英文摘要: nested set of primers. The percentage of positive clonesscreened that contained the DNA-binding sequence ofthe fused Sp1 zinc finger domain was around 13% (5 outof 39 clones). It was found that the Sp1 DNA-bindingsequence was only present in regions that were proxi-mal to one of the long terminal repeats of the integratedviral genome, suggesting that the fusion protein couldselect a target sequence for integration. The host flank-ing sequences determined for all the positive cloneswere also used as queries to perform a BLAST search onthe GenBank mouse EST entries. Although matchingscores for sequences of some of the clones computedwere more significant than others, it was difficult tojudge whether or not the integration in these clones hadbeen targeted to some gene sequences. Most of the inte-gration sites might exist in the introns, since we foundthat the probability of the gene sequences containing anSp1 DNA-binding site was low.A specificity protein 1 (Sp1) zinc finger domain contain-ing two tandem zinc fingers was fused to the C terminusof the integrase (IN) protein of the Moloney murine leu-kemia virus (MuLV). The integrity of the MuLV IN wascompletely preserved, since the fusion was conducted atthe last amino acid residue of the protein. The vectorpMIN-Sp1, which carried the fused MuLV IN-Sp1 zinc fin-ger domain gene, was cotransfected with a wild-typeMuLV vector pMLV-K to NIH/3T3 cells. A nonradioactivereverse transcriptase assay was performed on culturesupernatants collected from the cotransfected cells toconfirm the production of recombinant viruses. The ex-pression of the fusion protein and the integration of theMuLV genome by the fusion protein were confirmed by aNorthern and then a Southern hybridization analysis onthe total RNA or genomic DNA extracted from cellsinfected by viruses collected from the supernatants ofthe cotransfected cells. Regions of the host chromosomethat were selected by the fusion protein as the integra-tion targets were sequenced using the TOPO cloningmethod on a series of PCR products generated with a
- 中文關鍵字: --
- 英文關鍵字: Target integration, Integrase, Sp1 zinc finger domain, Fusion protein, DNA binding, Moloney murine leukemia virus integrase