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Two novel approaches of recombinant PCR technology were employed to graft the complementarity determining regions from a murine monoclonal antibody (mAb) onto human antibody frameworks. One approach relied on the availability of cloned human variable region templates, whereas the other strategy was dependent only on human variable region protein sequence data. The transient expression of recombinant humanized antibody was driven by the adenovirus major late promoter and was detected 48 hrs post-transfection into non-lymphoid mammalian cells. The application of these new approaches enables the expression of a recombinant humanized antibody just 6 weeks after initiating the cDNA cloning of the murine mAb.

A novel eucaryotic vector derived from the transforming region of bovine papilloma virus was established and demonstrated to be highly effective for introducing foreign genes into animal cells. The foreign deoxyribonucleic acid (DNA) is replicated and actively transcribed as an episome, and the transcripts are translated into an authentic gene product. We have constructed a DNA hybrid molecule, BPV69T-rI1, containing the transforming region of bovine papilloma virus DNA and the rat preproinsulin gene I (rI1), and used it to transform susceptible mouse cells. DNA hybridization analysis has demonstrated the presence of multiple unintegrated copies of hybrid DNA molecules, with the bovine papilloma virus 1 DNA segment and the rI1 gene covalently linked in selected transformed cell lines. S1 nuclease analysis revealed the presence of a correctly spliced coding segment of the preproinsulin transcript similar or identical in its electrophoretic mobility to that of messenger ribonucleic acid produced in rat insulinoma cells. Significant levels of a protein immunoreactive with anti-insulin serum were detected by radioimmunoassay in the culture medium of transformed cells. Immunoprecipitation analysis in conjunction with competitive binding to bovine proinsulin established the identity of the protein as that of rat proinsulin.

The viral DNA sequences in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV-1) virions, by full-length linear BPV-1 DNA, or by a defined transforming subgenomic DNA segment of BPV-1 were examined by reassociation kinetics and blot hybridization. In all cases, the transformed cells contained multiple copies of BPV-1 DNA, present exclusively as supercoiled or nicked circular extrachromosomal molecules or as a slowly migrating complex of circular viral DNA molecules. In the transformed cell lines established from cells transfected with full-length linear BPV-1 DNA, there was recircularization of the input DNA which in some cases resulted in the loss of the restriction site used in the linearization of the DNA. In the transformed cell lines established with the defined subgenomic segment there was circularization of the DNA accompanied by the acquisition of new sequences or duplication and rearrangement of the BPV-1 sequences. In contrast to other well-studied virus transformation systems, no integration of the BPV-1 genome into the host chromosome could be detected under conditions sensitive enough to detect 0.1-0.2 viral genome equivalent. It was concluded that maintenance of transformation may be mediated by nonintegrated viral DNA.

We describe a bovine papillomavirus hybrid plasmid containing the neomycin resistance gene from Tn5 inserted into a mammalian cell transcriptional unit. This plasmid is maintained as a stable extrachromosomal element (20 to 100 copies per diploid genome) in mouse cells selected either for the transformed phenotype or for resistance to the aminoglycoside G418. Cells selected for G418 resistance initially display a flat, nontransformed phenotype before exhibiting the gross morphological characteristics of transformation. The delay in the appearance of the transformed phenotype indicated that some intracellular event or series of events subsequent to the establishment of transcriptionally active bovine papillomavirus 1 hybrid plasmid is required for the manifestation of the transformed phenotype.

The DNAs of polyomaviruses JC, BK, and SV40 were analyzed, under a range of nonstringent hybridization conditions, for nucleotide sequence homology. When the hybridizations were performed at T m - 36°, conditions which would detect regions of homology with as much as 26% base mismatch, extensive homology was detected in all gene regions between the JC genome and both BK and SV40 DNAs. The regions of strongest sequence homology between these genomes formed stable duplexes at T m - 21°, indicating at least 85% base match. By two-dimensional cross-hybridization of restriction endonuclease cleavage fragments of JC to both those of BK and SV40 under nonstringent conditions, it was possible to map the homologous DNA fragments of each pair of viruses with respect to each other. The physical maps of these polyomaviruses could be colinearly aligned using the conserved single Eco RI site in each genome as the 0 map position. The region of strongest homology among these three genomes was localized in a narrow segment (0.76 to 0.85 map unit) in the late region, which in the SV40 genome contains the codons for the N-terminal half of the minor viral protein VP2.

The papillomaviruses, which are widely distributed in nature, typically induce benign skin tumours (warts) in their natural hosts (reviewed in ref. 1). These viruses are members of the papovavirus group, as are the polyomaviruses2. In contrast to polyomaviruses such as SV40, polyoma and BK (reviewed in ref. 3), the papillomaviruses have not been extensively studied, primarily because of the lack of a suitable tissue culture system. In general, the proliferative skin changes induced by the papillomaviruses are limited to the epidermal cells, and the viruses have a very narrow host range. However, some bovine papilloma viruses (BPV) are exceptional in that they induce fibropapillomas in their natural host (cows); these BPV can also induce cellular transformation in tissue culture4–6 and fibroblastic tumours in heterologous animals, including hamsters7, mice5 and horses8,9. BPV DNA from virions can also transform rodent cells and is tumorigenic in hamsters10. Recently, a tissue culture focus assay11 in mouse cells (NIH 3T3 and C127) has been described for those BPV which are the aetiological agents of fibropapillomas in cows. The virally transformed cells, which did not contain infectious virus, grew in soft agar and were tumorigenic in nude (nu/nu) mice. We12 have recently molecularly cloned the entire genomes of BPV-1 and BPV-2 (ref. 13) in Escherichia coli K12 using the certified plasmid vector pBR322 (ref. 14). We now demonstrate that these cloned DNAs can transform NIH 3T3 and C127 cells. Further, a molecularly cloned fragment which contains 69% of the BPV-1 genome can also induce cellular transformation.

Publisher Summary This chapter describes the construction of NP–BPV recombinant DNA and isolation of such recombinant DNA transformants that produce influenza virus NP. To construct NP–BPV recombinant DNA, the Bam HI cleaved full-length NP DNA was inserted into the Bgl II site located between the MT promoter and the SV40 polyA addition site of cloned vector DNA containing the pML2 moiety (a derivative of pBR322). The recombinant DNA containing the NP DNA inserted in the sense transcription orientation was selected and partially digested to open either of the two remaining Bam HI sites: one located within the NP gene and the other located downstream of the SV40 polyA addition sequence. The NP–BPV recombinants that contained BPV DNA inserted at the Bam HI site located downstream of the SV40 polyA site were selected. The chapter illustrates the final construct of NP–BPV DNA in which the transcription orientation of the BPV insert is opposite to the transcription direction of the NP gene.

We constructed several retroviruses which transduced a mutant dihydrofolate reductase gene that was resistant to methotrexate inhibition and functioned as a dominant selectable marker. The titer of dihydrofolate reductase-transducing virus produced by virus-producing cells could be increased to very high levels by selection of the cells in increasing concentrations of methotrexate. Helper virus-free dihydrofolate reductase-transducing virus was also generated by using a broad-host-range amphotropic retroviral packaging system. Cell lines producing helper-free dihydrofolate reductase-transducing virus with a titer of 4 X 10(6) per ml were generated. These retroviral vectors should have general utility for high-efficiency transduction of genes in cultured cells and in animals.

The papillomaviruses are widespread in nature and induce benign squamous epithelial tumors in a wide variety of higher vertebrates. The proliferative lesions induced by most papillomaviruses are limited to the squamous epithelium. There is a subgroup of papillomaviruses, however, which includes the bovine papillomaviruses types 1 and 2 (BPV-1 and BPV-2), the ovine papillomavirus, and the deer papillomavirus which induce fibropapillomas consisting both of a proliferative dermal fibroblastic component as well as a proliferative squamous epithelial component. The full vegetative cycle of the papillomaviruses is expressed only in terminally differentiating squamous epithelial cells comprising the papilloma. Although these self-limiting tumors generally regress, some papillomas may undergo malignant progression.

Summary Foetal mouse glial cultures were inoculated with murine K papovavirus and subjected to serial subcultivation. Two cell lines were developed. The first of these, KVBCG2A, remained positive for viral infectivity and K virus capsid (V) antigen for over 30 subcultivations. Productive infection was not abolished by serial subcultivation in the presence of antiviral antibody. The second cell line, KVBCG1B, became negative for infectious virus and K virus V antigen, could be cloned from single cells and produced tumours in mice. Sera from tumour-bearing animals produced nuclear fluorescence of KVBCG1B cells and K virus-infected mouse embryo cells but did not react with uninfected mouse embryo cells or with cells infected by polyoma virus. DNA hybridization studies confirmed the presence of K virus DNA in KVBCG1B cells and suggested integration of the viral genome into host chromosomal DNA. K virus produces both persistent infection and cell transformation in glial cultures derived from its natural host.

The low transformation efficiency of Bacillus subtilis 168 lysogenic for phages ø105 or SPO2 is shown to result from the induction of lytic phage replication in competent cells. Lysogenic competent cells have a higher rate of spontaneous prophage induction than noncompetent cells. Mutants of ø105 and SPO2 which form lysogens resistant to spontaneous induction were isolated, and these lysogens exhibited higher transformation levels than those formed by wild-type phage. These results suggest that the physiological state of competence in B. subtilis promotes prophage derepression leading to cell death and the loss of potential transformants.