Hiroto Shimojo

Structure and gene organization in the transforming Hind III-G fragment of Ad12

The nucleotide sequence of the transforming Hind III-G fragment of Ad12 DNA which encompasses the left 6.8% of the genome has been determined. The fragment was 2320 nucleotides long, and contained a GC cluster at positions 126-155 and a region extremely rich in AT at positions 1098-1142 (number from the leftmost end). Possible coding regions for the two transforming gene products were assigned. The predicted coding region for T antigen g is positions 502-1069 and positions 1144-1373, which are joined by splicing (266 amino acid residues, 30 kd), and that for T antigen f is positions 1845-2126 (94 amino acid residues, 10 kd). The sequence of the Hind III-G fragment was compared with that of the transforming DNA fragment of Ad5 which encompasses the left 8.0% of the genome (2809 nucleotides). There are several discrete regions with significant sequence homology. The comparison suggests that the regions in the left two thirds of the Ad5 and Ad12 transforming DNA fragments (map units 0-4.7% in Ad5 and 0-4.4% in Ad12) bear some resemblance in their gene organizations, and code for proteins containing structurally homologous regions.

Establishment and characterization of rat cell lines transformed by restriction endonuclease fragments of adenovirus 12 DNA

Abstract Rat cells (3Y1) were transformed by the Hin dIII-G fragment of adenovirus 12 DNA and cloned in solft agar cultures, and transformed cell lines (GY1, GY2, and GY3 cells) were established. Part of the Hin dIII-G fragment of adenovirus 12 DNA was the only viral DNA sequence present in GY1 cells. The characteristics of CY1 (a rat cell line transformed by the Eco RI-C fragment of adenovirus 12 DNA), GY1, and WY3 cells (a rat cell line transformed by the whole viral DNA of adenovirus 12) were examined. The growth of each transformed cell line was unlimited as compared to that of untransformed 3Y1 cells. Complement fixation and immunofluorescence suggested that CY1 and GY1 cells contained T antigen, while WY3 cells contained both T antigen and DNA-binding protein. CY1, GY1, and WY3 cells induced tumors in rats after transplantation. In the serum of rats bearing CY1 or GY1 cell tumors only antibody to T antigen was detectable, while antibodies to both T antigen and virus-induced DNA-binding protein were present in the serum of rats bearing WY3 cell tumors. These results show that the transforming gene is located on the left end (7.2%) of adenovirus 12 DNA and suggest that T antigen is among the gene products coded for by this region of the genome.

Induction of DNA synthesis by adenoviruses in contact-inhibited hamster cells

Abstract Highly oncogenic (types 12 and 31), weakly oncogenic (types 3 and 2), and non-oncogenic (types 2 and 5) adenoviruses induced DNA synthesis in contact-inhibited hamster cells. DNAs induced by adenoviruses 2, 7, and 12 in contact-inhibited hamster cells were mainly cellular. High multiplicity of infection was necessary for the induction of DNA synthesis. Adenoviruses 12 and 31 were efficient in the induction of DNA synthesis and the patterns of DNA synthesis induced by these viruses were early and of short duration, whereas adenoviruses 3, 7, 2, and 5 were less efficient in the induction of DNA synthesis and the patterns of DNA synthesis induced by adenoviruses 3 and 7 were late and long-lasting. The patterns of DNA synthesis induced by adenoviruses 2 and 5 were intermediate between these two patterns. Adenoviruses 2 and 5 replicated in hamster cells, and viral DNA detected in DNA induced in hamster cells by adenovirus 2. Adenoviruses 12, 31, 3, and 7 did not replicate in hamster cells and viral DNAs were hardly detected in DNAs induced in hamster cells by adenoviruses 12 and 7.

Incomplete transformation of rat cells by a small fragment of adenovirus 12 DNA.

Rat cells (3Y1) were transformed by the Bpa-H fragment (4.5%, the left end) of adenovirus 12 (Ad 12) DNA, cloned, and transformed cell lines (HY1 to HY7 cells) were established. Most of the sequence of the BpaI-H fragment of Ad 12 DNA was present in HY1 and HY2 cells. The properties of HY (HY1, HY2, HY7), GY1 (a rat cell line transformed by the HindIII-G fragment of Ad 12 DNA), CY1 (a rat cell line transformed by the EcoRI-C fragment), and WY3 (a rat cell line transformed by the whole Ad 12 DNA) cells were examined. The morphology and the growth in Eagles minimal essential medium with 10% fetal calf serum of HY1, HY5, and HY7 cells were similar of CY1 cells, showing the transformed phenotype. However, the following properties of HY1, HY5, and HY7 cells were intermediate between transformed and untransformed cells: (1) the growth in Eagles medium with 2% fetal calf serum; (2) the colony formation in soft agar culture; (3) cellular DNA synthesis in Methocel medium; (4) the tumor growth after transplantation of HY1, HY2, and HY7 cells into rats. Ad 12 T antigens were not detected in HY cells by immunofluorescence. Viral antigen could not be detected in the HY cell extract by complement fixation test. These results indicate that HY cells are incomplete in the transformed phenotype. The significance of incomplete transformation is discussed.

Establishment and characterization of KB cell lines latently infected with adeno-associated virus type 1.

Abstract KB cells latently infected with adeno-associated virus type 1 (AAV1) were cloned. Clones, in which synthesis of AAV1 antigens was induced after superinfection with human adenovirus type 31 (H31), were selected. Without superinfection, these clones were negative for AAV1 antigens and did not contain infectious AAV1. After many passages, these clones still retained the capacity to produce AAV1 antigens and infectious AAV1 after superinfection with H31. Several copies of the AAV1-specific nucleotide sequence were found in the DNA of the cells by analysis of cell DNA-DNA reassociation kinetics. Induction of AAV1 antigens in the cells by superinfection with H31 was enhanced by pretreatment of the cells with either bromodeoxyuridine or iododeoxyuridine.

Cell density-dependent increase in chromatin-associated ADP-ribosyltransferase activity in simian virus 40-transformed cells

Abstract ADP-ribosyltransferase activity associated with chromatin is two- to tenfold higher in simian virus 40 (SV40)-transformed cells than in untransformed cells. When confluent transformed cells were subcultured, their specific enzyme activity first decreased two- to fourfold and the rapidly increased during the logarithmic phase of growth. This increase ceased or slowed down when the cells entered the stationary phase. In contrast, the activity in the untransformed cells remained low throughout the growth cycle. In SV40tsA-transformed cells (ts = temperature sensitive), this density-dependent increase in the enzyme activity was observed when the cells were cultivated at the permissive temperature, whereas the activity remained low at the restrictive temperature. The enzyme activity did not increase during induction of cellular DNA synthesis in quiescent cells either by addition of fresh medium or by infection with SV40. The chromatin-associated enzyme activity extracted with 1 m NaCl was eluted together with almost all the DNA-binding proteins from a phosphocellulose column with 0.6 m NaCl. The enzyme activity in this fraction from transformed cells, measured with or without added DNA and histones, was higher than that in a similar fraction from untransformed cells, reflecting the difference in the original activities present in the nuclei of these cells. The chain lengths of poly(ADP-ribose) formed by chromatin from SV40-transformed and untransformed cells were not significantly different. These results suggest that the number of initiation sites for ADP-ribosylation is increased in the chromatin of SV40-transformed cells compared to that of untransformed cells.

Transformation of green monkey kidney cells by SV40 genome: The establishment of transformed cell lines and the replication of human adenoviruses and SV40 in transformed cells

Abstract Green monkey kidney cells were transformed by ultraviolet-inactivated adeno 7-SV40 hybrid virus and SV40. Transformed cells were obtained either as piled-up foci in monolayers of normal cells or as surviving colonies among degenerated cells. From these foci or colonies, many transformed cell lines were established. These transformed cells contained SV40 T antigen but not SV40 virion antigen. Approximately 40–50% of SV40 genome was transcribed in 3 transformed cell lines. SV40 could not be detected or rescued from the transformed cells by contact-culture or cell-fusion with green monkey kidney cells, except for one clone which gave variable results. All the transformed cell lines supported the efficient replication of human adenovirus types 2, 7, and 12, while nontransformed cells did not. Transformed cell lines established from foci supported the efficient replication of SV40. However, transformed cell lines established from surviving colonies were resistant to superinfection with SV40 or SV40 DNA.

Isolation and characterization of adenovirus type 12 DNA binding proteins

Abstract Infection of African green monkey kidney cells with type 12 adenovirus results in the production of two single strand specific DNA binding proteins. The molecular weights of these proteins are 60,000 and 48,000. Both proteins are synthesized in the absence of viral DNA replication and neither protein appears to correspond to any polypeptide detected in mature adenovirus virions. Temperature sensitive mutants from three different early, DNA negative, complementation groups (tsA, tsB, and tsC) have been tested for the production of these proteins at permissive and nonpermissive temperatures. Mutants of the tsB and tsC classes produce both DNA binding proteins at 32 and at 40°. TsA mutants produce both proteins at 32° but neither DNA-binding protein can be detected when these mutants are grown at 40°. The properties of adenovirus DNA binding proteins produced by types 2, 5, and 12 adenoviruses are compared.

Isolation and a preliminary characterization of temperature-sensitive mutants of adenovirus 12.

Eighty-eight temperature-sensitive ( ts ) mutants of adenovirus 12, which did not replicate at 38° but did replicate well at 31° were isolated. Thirty-four of these mutants were selected and classified into 13 groups (groups A to M) by complementation tests. Analyses of the defects in viral replication at the nonpermissive temperature (38°) revealed that mutants in 4 groups (groups A, B, C, and D) were defective in the production of hexon, fiber, and penton base, mutants in group E were defective in the production of both fiber and penton base, mutants in group F were defective in the production of both hexon and penton base, a mutant in group G was defective in the production of both hexon and fiber, mutants in group H were defective in the production of only hexon, mutants in group I were defective in the production of only fiber, mutants in group J were defective in the production of only penton base and mutants in 3 groups (groups K, L, and M) were not defective in the synthesis of hexon, fiber, and penton base. These defectiveness were detected by serological reaction and polyacrylamide gel electrophoresis. Immunofluorescent examinations of cells infected with these mutants as well as the examination of heat stability of virions produced at the permissive temperature showed differences among these groups.

Analysis of adenovirus 12 temperature-sensitive mutants defective in viral DNA replication.

Abstract Ten temperature-sensitive mutants of adenovirus 12 defective in viral DNA replication at the nonpermissive temperature (40°) were selected and seven of them were classified into three groups (A, B, and C) by complementation tests in viral DNA synthesis. The mutants in Groups A, B, and C were defective in initiation of viral DNA synthesis at 40°, as revealed by viral DNA chain elongation or ligation and density label in temperature shift-up experiments. Analysis by the M band technique indicated that the mutants in Groups B and C were less defective than the mutant in Group A in formation of viral DNA replication complex at the nonpermissive temperature. Additional differences among mutants in Groups A, B, and C were also shown by temperature shift-down experiments. All these mutants were not defective in formation of T antigen in permissive cells and in induction of cellular DNA synthesis in nonpermissive cells at the nonpermissive temperature. These observations suggest that at least three viral genes are involved at different steps in initiation of viral DNA replication.