Hyperproduction of adenovirus type 12 E1B gene product in monkey cells, using a simian virus 40 vector

Oda, Kinichiro; Fukui, Yoshihiro; Saito, Ikuo; Masuda, Michiaki; Shiroki, Kazuko; Shimojo, Hiroto

doi:10.1128/jvi.45.1.420-427.1983

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“…pBR322 DNA sequence has an inhibitory effect on the replication of pBR322-SV40 recombinant DNA in monkey cells, and the recombinant DNA which efficiently replicated in the cells has this sequence deleted (13). The EtA coding sequences may also be inhibitory to propagation of the recombinant DNA since an SV40 recombinant carrying the Adl2 EtB gene (SV-AdElB-B) propagates very efficiently in monkey cells, as described in the following paper (22). It has been suggested that 70 to 100% of full-length SV40 DNA is suitable for packaging (19); however, the efficiency of packaging may differ significantly within the lengths in this range.…”

Section: Discussionmentioning

confidence: 90%

Expression of adenovirus type 12 E1A gene in monkey cells, using a simian virus 40 vector

Oda¹,

Kato²,

Saito³

et al. 1983

J Virol

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Simian virus 40 (SV40) recombinants carrying the adenovirus type 12 ElA gene were constructed. The SV40 expression vector was constructed by removing most of the VP1 gene and an internal part of the intervening sequence for late 16S RNA and by joining the 5' and 3' splice sites into a small segment. The adenovirus type 12 ElA gene with or without its own promoter was inserted downstream from the SV40 late promoter and the splicing junctions. The recominant DNA was propagated and packaged in monkey cells by cotransfection with an early temperature-sensitive mutant (tsA58) DNA as helper. Immunofluorescent staining of the monkey cells infected with the resulting virus stocks showed that up to 20% of the cells overproduced the ElA gene products in the nuclei. Two-dimensional gel electrophoresis of the products indicated that the products were very similar or identical to the authentic polypeptides synthesized in adenovirus type 12infected human embryo kidney cells. The ElA mRNA was initiated at the SV40 late promoter irrespective of the presence of the EIA promoter and terminated at either the ElA or the SV40 polyadenylation signal. These hybrid mRNAs were correctly spliced in the ElA coding region. MATERIALS AND METHODS Cells and viruses. Established cell lines of African green monkey kidney, CV1 and GC7 (36), and KB cells were cultivated in Eagle medium with 10% calf or 408

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Section: Discussionmentioning

confidence: 90%