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

Simian virus 40 (SV40) recombinants carrying the adenovirus type 12 E1A 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 E1A gene with or without its own promoter was inserted downstream from the SV40 late promoter and the splicing junctions. The recombinant 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 E1A 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 12-infected human embryo kidney cells. The E1A mRNA was initiated at the SV40 late promoter irrespective of the presence of the E1A promoter and terminated at either the E1A or the SV40 polyadenylation signal. These hybrid mRNAs were correctly spliced in the E1A coding region.     

Plasmids for the cloning and expression of full-length double-stranded cDNAs under control of the SV40 early or late gene promoter

Okayama and Berg (1) have recently described a technique for the high efficiency cloning of full-length dscDNAs. We have constructed eukaryotic expression vectors compatible both with this technique (and with classical techniques) for dscDNA cloning. The vectors are such that recombinants obtained contain dscDNAs in the correct orientation downstream from a block of sequence comprising either the SV40 early or late gene promoter linked to a pair of splice sites from a rabbit beta-globin gene. A sequence encoding an SV40 polyadenylation site follows the dscDNA. We have used our vectors to make a library from chicken oviduct polyA(+) RNA using the Okayama and Berg technique. Ovalbumin recombinants occur in the library at the expected frequency and a high proportion contain full length copies of the ovalbumin mRNA. However, a similar result was not obtained for conalbumin recombinants. When recombinants are introduced into eukaryotic cells by either calcium phosphate coprecipitation or protoplast fusion, expression of chicken ovalbumin or conalbumin may be detected by indirect immunofluorescence. Under optimal conditions (use of SV40 late promoter and cos 7 cells) ovalbumin protein could be detected when the ovalbumin recombinant was present in only 2% of the protoplasts used for fusion. This suggests that colony banks obtained using our vectors could be screened in batches of 50 by protoplast fusion followed by a search for expression of a given protein using indirect immunofluorescence.     

A direct selection strategy for shotgun cloning and sequencing in the bacteriophage M13.

A new cloning strategy is described which utilizes direct selection of recombinants for shotgun sequencing in the filamentous bacteriophage M13. Direct selection is accomplished by insertional inactivation of the M13 gene X protein, a powerful inhibitor of phage-specific DNA synthesis when overproduced. An extra copy of gene X was inserted into the intergenic region of M13 and placed under the control of the bacteriophage T7 gene 10 promoter and RBS. Random fragments are cloned into the EcoRV cloning site of the new gene X cistron and recombinants are selected in an E. coli male strain producing T7 RNA polymerase. Cloning efficiencies obtained with M13-100 or phosphatase-treated M13mp19 vector are comparable. The direct selection capability of M13-100 was demonstrated to have the following advantages: (a) consistently achieved high ratios of recombinants to religated vector in the libraries, averaging about 500:1 (0.2% background), and (b) the elimination of the need for phosphatase treatment of the vector to reduce background. The direct selection strategy significantly improves the efficiency of shotgun library construction in M13, and should therefore facilitate the cloning aspects of large scale sequencing projects.