Biomedical subjects
Publications and source records attributed to K Fujinaga.
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Early virus-specific RNA synthesized in KB cells infected with adenovirus type 7 and virus-specific RNA synthesized in rat embryo cells (71JY1-2) transformed by the adenovirus type 7 HindIII-I.J fragment (left-hand 8.1% of the viral genome) have been mapped on the viral genome. About 25% of the viral genome, four discrete regions, two on each strand of the viral genome, are expressed as "early" mRNA. Almost similar regions in the left-hand 8.1% of the viral genome are transcribed both in KB cells at early times after infection and in 71JY1-2 cells.
Rat cell lines tranformed by viral DNA fragments, EcoRI-C and HindIII-G, of adenovirus type 12 DNA were analyzed for the viral transforming DNA sequences present in cell DNAs. Cell lines transformed by the EcoRI-C fragment of adenovirus type 12 DNA (leftmost 16.5% of the viral genome) contain most of the HindIII-G sequences of the HindIII-G fragment, but at a different frequency depending on the portions of the fragment. The sequence of the AccI-H fragment of adenovirus type 12 DNA (the left part of the HindIII-G; leftmost 4.5% of the viral genome) was detected dominantly in cells transformed by the HindIII-G fragment Southern blot analysis showed that viral DNA sequences are present at multiple integration sites in high-molecular-weight cell DNA from cells transformed by the EcoRI-C or HindIII-G fragment of adenovirus type 12 DNA. These results suggest that most of the HindIII-G sequences in cells transformed by the HindIII-G fragment are present as fragmented forms.
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The viral DNA sequences in cells transformed by adenovirus type-12 (Ad12) DNA fragments were investigated by spot hybridization, a detection by autoradiography of nucleic acid hybrids formed between cell DNAs spotted on a membrane filter and various nick-translated Ad12 DNA fragments. In CY1 cell line, a rat cell line transformed by the EcoRI-C fragment (left hand 16%), all of the HindIII fragments included in the EcoRI-C fragment are shown to be present. In GY1 cell line, a rat cell line transformed by the HindIII-G fragment (left hand 7%), both of the BpaI-H and a part of the BpaI-J, two components consisting of the HindIII-G fragment, were found. A dominant presence of the Ad12 BpaI-H fragment (left hand end 4.5%) of the Ad12 DNA molecule, approximately 60% of the Ad12 transforming DNA sequences (Ad12 HindIII-G), was also shown in GY1. Spot hybridization used in the experiment will be of general use for detecting viral nucleic acid sequences in cells and provides a simple and useful screening method for investigating viral etiology of tumors and transformed cells.
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For attempt to detect an etiological agent, cultures from bovine lymphosarcoma cases (adult form (ALS), calf form (CLS), and thymic form (TLS) were maintained in vitro for over a 18 month period. In two cultures from ALS, bovine leukemia virus (BLV) antigen was constantly detected. On the other hand, BLV antigen remained negative in cultures from two CLS and one TLS cases up to 40 passages. The RNA dependent DNA polymerase activities in these cultures were also negative. Treatment of a culture from CLS (3178) originated from liver tumor with 5'-iodo-2'-deoxyuridine (IdU) and dexamethasone (DXM) resulted in production of an agent serologically and morphologically similar to BLV and in alteration of cell morphology. No virus was detected in culture from TLS after treatment with IdU and DXM.
The fraction of the adenovirus type 5 genome expressed as mRNA in a rat cell line, 5RK, transformed by the HsuI-G restriction fragment was analyzed by saturation hybridization using separated strands of 32P-labeled HindIII-G fragment. It was found that 45 to 50% of G fragment (about 8 X 10(5) daltons) is expressed as mRNA. The size of polyadenylic acid-terminated virus-specific cytoplasmic and nuclear RNA was determined by electrophoresis on polyacrylamide gels containing 98% formamide, followed by hybridization of gel slices with viral DNA. The major virus-specific RNA species present in the cytoplasm is about 14S, whereas the major virus-specific RNA present in the nucleus is 30 to 32S. The large size of nuclear virus-specific RNA suggests that host sequences are covalently linked to the viral transcript.
The oncogenic capacity of the tumour virus lies in a small region of the viral genome, and tumour cells and cells transformed by DNA tumour viruses often contain only a part of the viral genome, the portion including the transforming gene(s). The detection and quantitation of these gene(s) in tumour cell DNA can provide evidence for or against a possible etiological role of the virus in human neoplasia. In this communication, the isolation, purification and identification of the transforming segment of Ad 12 are described.
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32P-Labeled adenovirus-12 (Ad-12) DNA was treated with restriction enzyme (EndoR-Hin dIII) isolated from Haemphilus influenzae (Rd strain) and the resulting 16 specific fragments were separated through gel electrophoresis. This kinetics of renaturation of each of the fragments was measured in the presence of unlabeled Ad-12-transformed hamster embryo cell, clone 9 (Ad-12HE-C19). more than 77% of the viral genome nucleotide sequence was present in Ad-12he-c19 cell DNA with 5 to 10 copies per haploid quantity of cell DNA of each of the sequences of 11 fragments examined; A, C, D, E, F, G, H, I, J (J1+J2), K, and L. However, it is suggested that only a part of the nucleotide sequence in B fragment may b present in the cell DNA.
The presence of multiple copies of a portion of the viral genome was demonstrated in Ad7-transformed cells by a new method based on the kinetic analysis of labeled viral DNA reassociation in the presence of unlabeled transformed cell DNA. DNA-DNA homology measurement utilizing defined DNA fragments (restriction endonuclease digests) as labeled probes supported the above conclusion.
A model and appropriate equations were derived for the quantitative estimation of nucleotide sequence homology between two partially related viral genomes by measurement of the initial rate of reassociation of one labeled DNA in the presence of a second unlabeled DNA. The validity and usefulness of this procedure were demonstrated by the analysis of the reassociation kinetics of labeled adenovirus 7 DNA in the presence of unlabeled adenovirus 2 DNA. Based on DNA reassociation, the extent of homology between adenovirus 2 and 7 genomes was found to be 10 to 12%. The duplex formed between adenovirus 2 and 7 DNA had the appropriate thermal stability for a well-matched DNA-DNA hybrid.