Complementary-addressed elimination of E1a sequence of simian adenovirus oncogene SA7 from circular single-stranded DNA of recombinant phage M13

The G fragment of the simian adenovirus SA7 oncogene corresponding to E1a region was cloned into M13mp8 and M13mp9 phages. Single-stranded DNAs of the recombinant phages thus obtained (mp8G and mp9G) partially digested with DNAse II were used to synthesize polyalkylating derivatives capable of specific hybridisation and subsequent alkylation of complementary G sequences of corresponding phage DNAs. After incubation of complementary alkylated DNA in the presence of lysine, the preselected region (G fragment) was specifically eliminated without damaging vector sequences. The method of complementary-addressed cleavage proved to be useful for precise analysis of reactions of polyalkylating derivatives within complementary complexes.     

Oncogene-directed mutagenesis in vivo. Polyalkylating derivatives of short single-stranded polynucleotides, complementary E1-adeno-oncogene, in the normalization of adenovirus-transformed rodent cell lines]

Polyalkylating derivatives of single-stranded polynucleotides (30-200-mers) complementary to the long E1 oncogene sequences of simian adenovirus SA7 cause inherited normalization of SH2 and G11 cells transformed with adenovirus SA7; certain deletions in the integrated proviral E1A oncogene were observed in several cases during this process. The transformed cells are indifferent to reagents noncomplementary to the E1 region. Thus polyalkylating derivatives of single-stranded 30-200-mers act as addressed mutagenes which react in a specific way with the integrated complementary DNA sequences of E1 oncogene in transformed rodent cells and realize oncogene-directed mutagenesis in vivo. During this treatment temporary normalized cells reverting to the initial transformed phenotype are also produced.     

Preparation of single-stranded E1A-oncogene DNA fragments from simian adenovirus SA7 by endonuclease hydrolysis recombinant phage M13 SS-DNA complexed with oligonucleotides, containing restriction sites]

The ss-DNA of the (+) and (-) chains of Ela DNA fragment was obtained by hydrolysis of the recombinant bacteriophages M13 mp8G and mp9G (where G is 1-1750 bp:, E1a region of oncogene SA7) in complexes with the 16 bp oligonucleotides containing AluI and BspRI sites of restriction and sequences complementary to E1a SA7. The obtained fragments overlap the E1a zones associated with the immortalizing potential of SA7.     

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.     

Penetration of oligo/polynucleotides and their polyalkylating derivatives into rat cells transformed by simian adenovirus DNA

The transport regularity of the [32P]-oligo/polynucleotides and their polyalkylating derivatives into SH2 rat cells transformed with SA7 adenovirus DNA was investigated. Derivatives penetrate the SH2 cells and their distribution in the subcellular fractions are proportional to the concentration of reagent in the medium. The transport efficiency of the derivatives is inhibited sharply with cell concentration increase and practically does not depend on the action of cell metabolism inhibitors. The data obtained assumes the mechanism of the derivatives transport to be liquid endocytosis. Being distributed in the cell components the polyalkylating derivatives were accumulated by the cell nuclei up to 10(5)-10(7) molecules per nucleus. Transport efficiency is much greater in the anchored cells than in the suspended ones. Though essential dephosphorylation of the utilized substances is observed in the SH2 cells, part of them maintain native chain length and the 5'-phosphate group after 1 h incubation in nucleic acids obtained from the cell nuclei.     

Comparison of viral RNA sequences in adenovirus 2-transformed and lytically infected cells

The complementary strands of fragments of ³²P-labelled adenovirus 2 DNA generated by cleavage with restriction endonucleases EcoRI or Hpa1 were separated by electrophoresis. Saturation hybridization reactions were performed between these fragment strands and unlabelled RNA extracted from the cytoplasm of adenovirus 2-transformed rat embryo cells or from human cells early after adenovirus 2 infection. The fraction of each fragment strand complementary to RNA from these sources was measured by chromatography on hydroxylapatite. Maps of the viral DNA sequences complementary to messenger RNA in different lines of transformed cells and early during lytic infection of human cells were constructed.Five lines of adenovirus 2-transformed cells were examined. All contained the same RNA sequences, complementary to about 10% of the light strand of EcoRI fragment A. DNA sequences coding for this RNA were more precisely located using Hpa1 fragments E and C and mapped at the left-hand end of the genome. Thus any viral function expressed in all adenovirus 2-transformed cells, tumour antigen, for example, must be coded by this region of the viral genome. Two lines, F17 and F18, express only these sequences; two others, 8617 and REM, also contain mRNA complementary to about 7% of the heavy strand of the right-hand end of adenovirus 2 DNA; a fifth line, T2C4, contains these and many additional viral RNA sequences in its cytoplasm.The viral RNA sequences found in all lines of transformed cells are also present in the cytoplasm of human cells during the early phase of a lytic adenovirus infection. The additional cytoplasmic sequences in the 8617 and REM cell lines also correspond to “early” RNA sequences.     

Preparation and characteristics of a new transformed cell line G11 by transfection of NIH/3T3 mouse fibroblasts with DNA from the SA7 oncogene of simian adenovirus

Murine fibroblasts NIH 3T3 were transfected with the plasmid pASP containing simian adenovirus oncogene insertion. Focus forming transformants were cloned with a final dilution technique and a new cell line G11 was created as a result. Transformed status of this cell line is evidenced by changes in morphology, specific cytochemical and adhesion properties, ability to grow in semisolid agar and FCS concentration growth independence. Presence of intact integrated E1a-region of adenovirus SA7 oncogene was shown by blot-hybridization technique. Transformed status of G11 cells can be explained by integration of SA7 oncogene, that is evidenced indirectly by the increased resistance to heat shock.     

Isolation of a cDNA encoding the adenovirus E1A enhancer binding protein: a new human member of the ets oncogene family.

The cDNA encoding adenovirus E1A enhancer-binding protein E1A-F was isolated by screening a HeLa cell lambda gt11 expression library for E1A-F site-specific DNA binding. One cDNA clone produced recombinant E1A-F protein with the same DNA binding specificity as that endogenous to HeLa cells. Sequence analysis of the cDNA showed homology with the ETS-domain, a region required for sequence-specific DNA binding and common to all ets oncogene members. Analysis of the longest cDNA revealed about a 94% identity in amino acids between human E1A-F and mouse PEA3 (polyomavirus enhancer activator 3), a recently characterized ets oncogene member. E1A-F was encoded by a 2.5kb mRNA in HeLa cells, which was found to increase during the early period of adenovirus infection. In contrast, ets-2 mRNA was significantly reduced in infected HeLa cells. The results indicate that E1A enhancer binding protein E1A-F is a member of the ets oncogene family and is probably a human homologue of mouse PEA3.     

Expression of adenovirus type 12 early region 1 in KB cells transformed by recombinants containing the gene.

The adenovirus type 12 (Ad12) early region 1 (E1) gene was introduced into KB cells by using a dominant selection vector, pSV2-gpt, and over 80 Gpt+ KB cell clones were established. Three types of recombinant DNAs (gAE1A, gARC, and gABA) were constructed. They contained the AccI-H, EcoRI-C, and BamHI-A fragments, respectively, of Ad12 DNA in pSV2-gpt. Five of 50 (10%) gABA-transformed cell clones, 12 of 18 (67%) gAE1A-transformed cell clones, and 10 of 18 (56%) gARC-transformed cell clones complemented the growth of Ad5 dl312 (deletion in E1A) and were designated as Gpt+ Ad+ cell clones. In these cell clones at their early passages, recombinant genome sequences were detected in cellular DNA and were expressed. T antigen g (the E1A gene product) was detected by immunofluorescence. The Gpt+ Ad+ cell clones supported the growth of Ad5 deletion mutants in parallel with the expression of Ad12 E1A or E1A plus E1B genes. After infection of Gpt+ Ad+ cell clones with Ad5 dl312, the early genes of dl312 were efficiently transcribed, indicating the expression of the pre-early function of the Ad12 E1A gene. Two clones each from gAE1A-,gARC-, and gABA-transformed cells were subcultured for a long period to determine the stability of the transfecting DNAs. Subculture in a nonselective medium resulted in cells which lost the transfecting DNAs. Subculture in a selective medium resulted in the selection of cells which maintained the gpt gene expression but lost the Ad12 gene expression. These results indicate that the transfecting DNA is present in an unstable state in KB cells.     

Activated H-ras rescues E1A-induced apoptosis and cooperates with E1A to overcome p53-dependent growth arrest.

The adenovirus E1A oncogene products stimulate DNA synthesis and cell proliferation but fail to transform primary baby rat kidney (BRK) cells because of the induction of p53-mediated programmed cell death (apoptosis). Overexpression of dominant mutant p53 (to abrogate wild-type p53 function) or introduction of apoptosis inhibitors, such as adenovirus E1B 19K or Bcl-2 oncoproteins, prevents E1A-induced apoptosis and permits transformation of BRK cells. The ability of activated Harvey-ras (H-ras) to cooperate with E1A to transform BRK cells suggests that H-ras is capable of overcoming the E1A-induced, p53-dependent apoptosis. We demonstrate here that activated H-ras was capable of suppressing apoptosis induced by E1A and wild-type p53. However, unlike Bcl-2 and the E1B 19K proteins, which completely block apoptosis but not p53-dependent growth arrest, H-ras expression permitted DNA synthesis and cell proliferation in the presence of high levels of wild-type p53. The mechanism by which H-ras regulates apoptosis and cell cycle progression is thereby strikingly different from that of the E1B 19K and Bcl-2 proteins. BRK cells transformed with H-ras and the temperature sensitive murine mutant p53(val 135), which lack E1A, underwent growth arrest at the permissive temperature for wild-type p53. p53-dependent growth arrest, however, could be relieved by E1A expression. Thus, H-ras alone was insufficient and cooperation of H-ras and E1A was required to override growth suppression by p53. Our data further suggest that two complementary growth signals from E1A plus H-ras can rescue cell death and thus permit transformation.     

Hybrids of human and monkey adenoviruses (adeno-adeno hybrids) that can reproduce in monkey cells: biological and molecular genetic peculiarities

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.     

Functional complementation of the adenovirus E1B 19-kilodalton protein with Bcl-2 in the inhibition of apoptosis in infected cells.

Expression of the adenovirus E1A oncogene induces apoptosis which impedes both the transformation of primary rodent cells and productive adenovirus infection of human cells. Coexpression of E1A with the E1B 19,000-molecular-weight protein (19K protein) or the Bcl-2 protein, both of which have antiapoptotic activity, is necessary for efficient transformation. Induction of apoptosis by E1A in rodent cells is mediated by the p53 tumor suppressor gene, and both the E1B 19K protein and the Bcl-2 protein can overcome this p53-dependent apoptosis. The functional similarity between Bcl-2 and the E1B 19K protein suggested that they may act by similar mechanisms and that Bcl-2 may complement the requirement for E1B 19K expression during productive infection. Infection of human HeLa cells with E1B 19K loss-of-function mutant adenovirus produces apoptosis characterized by enhanced cytopathic effects (cyt phenotype) and degradation of host cell chromosomal DNA and viral DNA (deg phenotype). Failure to inhibit apoptosis results in premature host cell death, which impairs virus yield. HeLa cells express extremely low levels of p53 because of expression of human papillomavirus E6 protein. Levels of p53 were substantially increased by E1A expression during adenovirus infection. Therefore, E1A may induce apoptosis by overriding the E6-induced degradation of p53 and promoting p53 accumulation. Stable Bcl-2 overexpression in HeLa cells infected with the E1B 19K- mutant adenovirus blocked the induction of the cyt and deg phenotypes. Expression of Bcl-2 in HeLa cells also conferred resistance to apoptosis mediated by tumor necrosis factor alpha and Fas antigen, which is also an established function of the E1B 19K protein. A comparison of the amino acid sequences of Bcl-2 family members and that of the E1B 19K protein indicated that there was limited amino acid sequence homology between the central conserved domains of E1B 19K and Bcl-2. This domain of the E1B 19K protein is important in transformation and regulation of apoptosis, as determined by mutational analysis. The limited sequence homology and functional equivalency provided further evidence that the Bcl-2 and E1B 19K proteins may possess related mechanisms of action and that the E1B 19K protein may be the adenovirus equivalent of the cellular Bcl-2 protein.     

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.     

Hybrids of Human and Monkey Adenoviruses (Adeno-Adeno Hybrids) That Can Reproduce in Monkey Cells: Biological and Molecular Genetic Peculiarities

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.     

Restriction of single-stranded M13 DNA using synthetic oligonucleotides: the structural requirement of restriction enzymes

A targeted ss (single stranded) DNA cleavage technique is reported which involves the use of synthetic oligomers complementary to the ss M13 DNA polylinker. BamHI, SmaI, and KpnI restriction enzymes were tested with a partial duplex DNA formed from ss M13 DNA and a nested series of fragments derived from a synthetic 21-mer which were complementary to the polylinker region. These enzymes require up to two flanking nucleotides in addition to the hexameric recognition site for efficient cleavage. This technique could be useful for effecting unique cleavages of DNA with enzymes which generally give a large number of fragments and for strategies of ss DNA manipulation.     

Expression of adenovirus E1a and E1b gene products and the Escherichia coli XGPRT gene in KB cells

The recombinant plasmid pSV2-gpt, which contains the Escherichia coli XGPRT gene under the control of a simian virus 40 early promoter, was modified to contain the type 2 adenovirus (Ad2) XhoI-C (0 to 15.5 map units) restriction endonuclease fragment. Plasmid (pLB206) DNA was introduced into human KB cells by Ca2+-mediated DNA transfection, and transformants were selected in medium containing xanthine, aminopterin, and mycophenolic acid, as a consequence of expression of the dominant, selectable XGPRT gene. A series of 13 gpt+ cell lines were isolated and tested for their ability to complement Ad5 deletion mutants in E1a (H5dl312) and E1b (H5dl315). Four classes of gpt+ KB cell lines were identified, including clones constitutively expressing both E1a and E1b, only E1a, or only E1b or not expressing either E1a or E1b. DNA and RNA filter transfer hybridization analysis substantiated the conclusions that those cell lines capable of complementing viral host range mutants contained the appropriate viral DNA sequences and cytoplasmic polyadenylated RNA species. DNA filter transfer hybridization studies also revealed that the transfected vector DNA was stably integrated into chromosomal DNA in the KB transformants and the number of integrated sites ranged from 1 to 3. The gpt+ KB cell line that only expressed E1b gene functions only contained viral E1b gene sequences; those cell lines that expressed neither E1a nor E1b gene function contained only small or no regions of Ad2 DNA. When weaned off the selective medium, transformed KB cell lines stably maintained their inserted DNA in the absence of selective pressure and could easily be adapted to growth in suspension culture.