Role of the oncogene in the mutagenic activity of bovine adenovirus type 3

The mutagenic and carcinogenic effect of two EcoRI-fragments of bovine adenovirus type 3 (BAV-3) DNA inserted into pBR325 has been studied. The C fragment (located between 3,6 and 19,7 map units) contains the viral oncogene, the C fragment (between 44,3 and 63,7 map units) displays no transforming activity. It has been established that oncogene BAV-3 statistically true increases the yield of mutants resistant to 6-mercaptopurine (6MP) in Chinese hamster cells. The C fragment, pBR325 without viral sequences and DNA fragments of different molecular weights from normal Syrian hamster cells have no mutagenic effect. The control over tumor formation in syngenic mice after injection of C3H10T 1/2 and D. C fragments and pBR325 treatment exposed a parallelism between the mutagenic and transforming effect. The study of the combined effect of viral DNA fragments and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) which increases the transforming activity of different carcinogens, shows that the promoter increases the frequency of mutants after viral oncogene treatment and does not induce mutagenic activity of those types of DNA which are unable to transform the cells.     

Restriction mapping and molecular cloning of adenovirus type 4 (subgroup E) DNA

The locations of thirty restriction endonuclease cleavage sites were determined on the genome of adenovirus type 4 (Ad4), the sole member of the subgroup E adenovirions. The restriction endonucleases BglII, EcoRI, HindIII, HpaI, KpnI, SalI, and XbaI cut Ad4 DNA 10, 3, 2, 3, 5, 5 and 3 times, respectively. Orientation of the linear Ad4 map with respect to left and right molecular ends was accomplished by taking advantage of the limited sequence homology between Ad2 and Ad4. Ten non-overlapping fragments of Ad4 DNA representing 98% of the genome, map units 1.6 to 99.6, have been cloned into the plasmid vector pKC7.     

A gene transfer vector-cell line system for complete functional complementation of adenovirus early regions E1 and E4.

The improvements to adenovirus necessary for an optimal gene transfer vector include the removal of virus gene expression in transduced cells, increased transgene capacity, complete replication incompetence, and elimination of replication-competent virus that can be produced during the growth of first-generation adenovirus vectors. To achieve these aims, we have developed a vector-cell line system for complete functional complementation of both adenovirus early region 1 (E1) and E4. A library of cell lines that efficiently complement both E1 and E4 was constructed by transforming 293 cells with an inducible E4-ORF6 expression cassette. These 293-ORF6 cell lines were used to construct and propagate viruses with E1 and E4 deleted. While the construction and propagation of AdRSV beta gal.11 (an E1-/E4- vector engineered to contain a deletion of the entire E4 coding region) were possible in 293-ORF6 cells, the yield of purified virus was depressed approximately 30-fold compared with that of E1- vectors. The debilitation in AdRSV beta gal.11 vector growth was found to correlate with reduced fiber protein and mRNA accumulation. AdCFTR.11A, a modified E1-/E4- vector with a spacer sequence placed between late region 5 and the right inverted terminal repeat, efficiently expressed fiber and grew with the same kinetic profile and virus yield as did E1- vectors. Moreover, purified AdCFTR.11A yields were equivalent to E1- vector levels. Since no overlapping sequences exist in the E4 regions of E1-/E4- vectors and 293-ORF6 cell lines, replication-competent virus cannot be generated by homologous recombination. In addition, these second-generation E1-/E4- vectors have increased transgene capacity and have been rendered virus replication incompetent outside of the new complementing cell lines.     

Subcloning of DNA fragments of the simian adenovirus SA7 oncogene in bacteriophage M13

The XmaI/PstI and XmaI DNA fragments of adenovirus SA7 oncogene and the adjacent region (16.7% of the physical map of SA7 left end DNA) were recloned in M13 bacteriophages mp8 and mp9 in order to obtain the singlestranded fragments EIa and EIb from the DNA region of monkey adenovirus SA7 located on the recombinant plasmid pASP carrying the DNA APstI fragment including the adenovirus SA7 oncogene.     

The mouse adenovirus type 1 contains an unusual E3 region.

Since the E3 region of human adenoviruses codes for a series of proteins that are probably involved in viral pathogenesis, the nucleotide sequence for a 3.6-kilobase DNA fragment in the corresponding region (map units 77 through 89) of the mouse adenovirus type 1 genome has been determined. Analysis of the sequence revealed that the genes for the fiber and for the precursor to the hexon-associated protein, pVIII, that usually flank the E3 region, are well conserved. However, many of the open reading frames contained in the E3 region of human adenoviruses between the pVIII and the fiber genes were absent from the mouse adenovirus type 1 genome.     

Different activities of the adenovirus types 5 and 12 E1A regions in transformation with the EJ Ha-ras oncogene.

We have compared the capacities of the E1A regions of nononcogenic adenovirus type 5 (Ad5) and highly oncogenic Ad12 to cooperate with the EJ bladder carcinoma Ha-ras-1 oncogene in the transformation of primary baby rat kidney cells. Both E1A regions, when cotransfected with the Ha-ras oncogene, transformed the primary cells with a low frequency. Ad5 E1A plus Ha-ras-transformed cells differed in phenotype from cells transformed by Ad12 E1A plus Ha-ras. The cells expressing Ad5 E1A appeared highly transformed and practically failed to adhere to plastic. This phenotype may be due to the virtually complete absence of fibronectin gene expression in these cells. In contrast, the cells expressing Ad12 E1A were flatter and adhered to plastic, whereas fibronectin gene expression was reduced but not absent. The oncogenic potential of the two types of E1A plus ras-transformed cells was tested by their injection into both athymic nude mice and weanling syngeneic rats. The Ad5 E1A plus ras-transformed cells were found to be highly oncogenic in both animal species, whereas the Ad12 E1A plus ras-transformed cells were only weakly oncogenic in both syngeneic rats and nude mice. The difference in oncogenic potential of the Ad5 E1A plus ras- and the Ad12 E1A plus ras-transformed cells is discussed in terms of the different capacities of the Ad5 and Ad12 E1A-encoded proteins to modulate cellular gene expression.     

Inhibition of the cellular response to interferons by products of the adenovirus type 5 E1A oncogene.

Expression of the E1A oncogene of adenovirus type 5 inhibits the response of interferon (IFN)-inducible constructs to Type I (alpha,beta) and II (gamma) IFNs in transient transfection assays. In human cell lines stably expressing E1A mRNA and protein acquisition of an antiviral state and the induction of a number of genes in response to alpha- and gamma-IFNs is inhibited. A short IFN-stimulable response element (ISRE) present in the 5' flanking region of a number of genes mediates induction by alpha- and gamma-IFNs. In cells expressing E1A there is a substantial reduction in the levels of the ISRE-binding factors E and M, inducible by alpha-IFN, and of factor G, inducible by gamma-IFN. In E1A-expressing cells the E alpha subunit of factor E is activated normally in response to alpha-IFN; the defect is in the production or activation of the E gamma subunit. The inhibitory activity of E1A is lost upon deletion of the CR1 domain. The induction of HLA class II genes by gamma-IFN, which involves a different DNA response element(s), and of beta-IFN mRNA in response to double-stranded RNA are also inhibited by E1A. An essential component(s) of a number of signalling pathways must, therefore, be subject, directly or indirectly, to inhibition by E1A.     

The generation and analysis of clones containing bacteriophage T4 DNA fragments

Cytosine-containing DNA of bacteriophage T4 was digested with three restriction endonucleases: endo R · EcoRI, endo R · HindIII and endo R · PstI, and each digestion ligated with a cloning vector to generate three independent collections of T4 DNA-containing clones. The T4 clones were screened for their T4 genetic content by recombinational analysis using amber mutants of T4. Complementation of T4 amber mutant growth and labeling of proteins in vivo provided evidence of expression of specific (g30, g39, g44 and g46) cloned T4 genes.     

Mutant adenovirus type 9 E4 ORF1 genes define three protein regions required for transformation of CREF cells.

Human adenovirus type 9 (Ad9) elicits exclusively estrogen-dependent mammary tumors in rats, and an essential oncogenic determinant for this virus is Ad9 E4 open reading frame 1 (9ORF1), which encodes a 125-residue cytoplasmic protein with cellular growth-transforming activity in vitro. In this study, we engineered 48 different mutant 9ORF1 genes in an attempt to identify regions of this viral protein essential for transformation of the established rat embryo fibroblast cell line CREF. In initial assays with CREF cells, 17 of the 48 mutant 9ORF1 genes proved to be severely defective for generating transformed foci but only 7 of these defective genes expressed detectable amounts of protein. To further examine the defects of the seven mutant proteins, we selected individual cell pools of stable CREF transformants for the wild-type and mutant 9ORF1 genes. Compared to cell pools expressing the wild-type 9ORF1 protein, most cell pools expressing mutant proteins displayed decreased growth in soft agar, and all generated significantly smaller tumors in syngeneic animals. The altered amino acid residues of the seven mutant 9ORF1 polypeptides clustered within three separate regions referred to as region I (residues 34 to 41), region II (residues 89 to 91), and C-terminal region III (residues 122 to 125). By using indirect immunofluorescence, we also assessed whether the mutant proteins localized properly to the cytoplasm of cells. The region I and region II mutants displayed approximately wild-type subcellular localizations, whereas most region III mutants aberrantly accumulated within the nucleus of cells. In summary, we have identified three 9ORF1 protein regions necessary for cellular transformation and have demonstrated that C-terminal region III sequences significantly influence the proper localization of the 9ORF1 polypeptide in cells.     

Solution structure of duplex DNA containing the mutagenic lesion 1,N(2)-etheno-2'-deoxyguanine

We have used high-resolution NMR spectroscopy and molecular dynamics simulations to determine the solution structure of DNA containing the genotoxic lesion 1, N (2)-etheno-2'-deoxyguanosine (epsilonG), paired to dC. The NMR data suggest the presence of a major, minimally perturbed structure at neutral pH. NOESY spectra indicate the presence of a right-handed helix with all nucleotides in anti, 2'-deoxyribose conformations within the C2'-endo/C1'-exo range and proper Watson-Crick base pair alignments outside the lesion site. The epsilonG residue remains deeply embedded inside the helix and stacks between the flanking base pairs. The lesion partner dC is extrahelical and is located in the minor groove of the duplex, where it is highly exposed to solvent. Upon acidification of the sample, a second conformation at the lesion site of the duplex emerges, with protonation of the lesion partner dC and possible formation of a Hoogsteen base pair. Restrained molecular dynamics simulations of the neutral-pH structure generated a set of three-dimensional models that show epsilonG inside the helix, where the lesion is stabilized by stacking interactions with flanking bases but without participating in hydrogen bonding. The lesion counterbase dC is displaced in the minor groove of the duplex where it can form a hydrogen bond with the sugar O4' atom of a residue 2 bp away.     

Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein.

Nuclear extracts from adenovirus type 5 (Ad5) infected HeLa cells were used to study the template requirements for adenovirus DNA replication in vitro. When XbaI digested Ad5 DNA, containing the parental terminal protein (TP), was used as a template preferential synthesis of the terminal fragments was observed. The newly synthesized DNA was covalently bound to the 82 kD preterminal protein (pTP). Plasmid DNAs containing the Ad2 origin sequence or the Ad12 origin sequence with small deletions were analyzed for their capacity to support pTP-primed DNA replication. Circular plasmid DNAs were inactive. When plasmids were linearized to expose the adenovirus origin, both Ad2 and Ad12 TP-free fragments could support initiation and elongation similarly as Ad5 DNA-TP, although with lower efficiency. These observations indicate that the parental terminal protein is dispensable for initiation in vitro. The presence of 29 nucleotides ahead of the molecular end or a deletion of 14 base pairs extending into the conserved sequence (9-22) destroyed the template activity. DNA with a large deletion within the first 8 base pairs could still support replication while a small deletion could not. The results suggest that only G residues at a distance of 4-8 nucleotides from the start of the conserved sequence can be used as template during initiation of DNA replication.