Biomedical subjects

H M Temin

Publications and source records attributed to H M Temin.

At least 145 records · Page 8Linked to original sources

PubMed1979

Pheasant virus DNA polymerase is related to avian leukosis virus DNA polymerase at the active site.

The DNA polymerase from Amherst pheasant virus (APV), a member of the pheasant virus species of retroviruses, was compared to the DNA polymerases of avian leukosis viruses (ALV) and a reticuloendotheliosis virus (spleen necrosis virus (SNV)). Immunoglobulin inhibition tests and competition immunoassays showed that APV and ALV DNA polymerases are closely related at their active sites. The determinants common to their active sites are not shared by SNV DNA polymerase. Bu using a species-specific radioimmunoassay, it was shown that both APV and SNV DNA polymerases are grossly different from ALV DNA polymerase. The specificity of the relationship of the active sites of APV and ALV DNA polymerases was confirmed by a heterologous radioimmunoassay. Our data indicate that pheasant viruses are evolutionarily linked to ALV.

Avian Leukosis Virus↗

PubMed1978

Sites of integration of infectious DNA of avian reticuloendotheliosis viruses in different avian cellular DNAs.

The pattern of integration for the infectious DNA of two avian reticuloendotheliosis viruses whose DNA is not inactivated by digestion with the restriction endonuclease, Eco RI was determined. High molecular weight DNA from infected chicken, turkey and pheasant cells was digested with Eco RI, electrophoresed through agarose gels and assayed for infectivity. The same patterns of integration of infectious viral DNA were found for these species of avian cells infected at high or low multiplicities with two reticuloendotheliosis viruses. There were multiple sites of integration in acutely infected cells with concomitant cell death. There was a single site of integration in chronically infected cells with no cell death. There were more integrated infectious viral DNA molecules per cell in acutely infected cells than in chronically infected cells. These results are consistent with the hypotheses that the cell death in the acute phase of infection is a result of the integration of the infectious viral DNA at multiple sites, and that only those cells survive that have the infectious viral DNA integrated exclusively at the single site.

Cell Line↗

PubMed1978

Sites of integration of reticuloendotheliosis virus DNA in chicken DNA.

The pattern of integration of spleen necrosis virus (SNV) DNA in DNA from a large population of SNV-infected chicken cells was studied by nucleic acid hybridization with iodinated viral RNA by the blotting technique of Southern. SNV DNA was found to be integrated at multiple sites in acutely infected chicken cells. Concomitant with the transition from acute to chronic infection, a shift in the pattern of integration was observed. The majority of integrated SNV DNA found in acutely infected cells was absent from chronically infected cells. This result is consistent with the hypothesis that the cell death that occurs after infection of avian cells with reticuloendotheliosis viruses is a consequence of the multiple integrations of the provirus. Viral DNA was also integrated at multiple sites in chronically infected cells. However, infectious viral DNA molecules in chronically infected cells migrated in a uniform manner in agarose gel electrophoresis after EcoRI digestion (which does not cut viral DNA), indicating that not all integrated SNV copies are equally infectious.

Acute Disease↗

PubMed1977

RNA viruses and cancer: Lucy Wortham James Lecture (Basic Science).

Some animal viruses that contain RNA replicate through a DNA intermediate. The molecular details of the replication of these viruses, which are called ribodenoxyviruses, are starting to be known. The ribodenoxyviruses belonging to a single species may either cause sarcomas, leukemia or no disease. The viruses belonging to a single species differ only in whether or not they contain genes for disease formation. In the case of Rous sarcoma virus, the virus causes sarcomas by adding a gene for sarcoma formation to the genome of infected cells. Ribodeoxyviruses appear to undergo different kinds of genetic changes at extraordinarily high rates. In addition, nucleotide sequences related to ribodeoxyvirus RNA are present in the DNA of many uninfected cells. These nucleotide sequences may represent a virus precursor, and ribodeoxyviruses are hypothesized to have evolved from these nucleotide sequences in uninfected cells. These data have led us to hypothesis that non-viral carcinogens act to mutate a cellular gene(s) that is involved in the same types of information transfer and genetic variation as ribodeoxyviruses and thus give rise to the formation of cancer gene(s).

Animals↗

PubMed1977

Infectious DNA of spleen necrosis virus is integrated at a single site in the DNA of chronically infected chicken fibroblasts.

The infectious DNAs of a number of avian leukosis-sarcoma and reticuloendotheliosis viruses were digested with six nucleotide-specific restriction endonucleases, and the digests were tested for infectivity. All of the enzymes inactivated the viral infectivities except for EcoRI, which did not inactivate the infectivity of the DNA of two of the reticuloendotheliosis viruses, spleen necrosis and chick syncytial viruses. The infectious DNA of spleen necrosis virus after digestion with EcoRI had a buoyant density in CsCl solution greater than the density of the high-molecular-weight infectious viral DNA. The infectious EcoRI-digested spleen necrosis virus DNA from chronically infected chicken cells was uniform in size, 10 megadaltons, which indicated a single site of integration. The infectious EcoRI-digested spleen necrosis virus DNA from acutely infected cells was heterogeneous in size, ranging from 8-14 megadaltons, which indicated multiple sites of integration. These results are consistent with the hypothesis that cells that integrate infectious spleen necrosis virus DNA at a single site survive and multiply, whereas cells that integrate infectious viral DNA at additional sites either die or selectively lose or inactivate the DNA in the additional sites.

Acute Disease↗

PubMed1977

Spontaneous mutations affecting the host range of the B77 strain of avian sarcoma virus involve type-specific changes in the virion envelope antigen.

Previously it was shown that the host-range gene of the Bratislava strain of avain sarcoma virus (B77 virus) spontaneously mutates with a very high rate. The wild-type B77 virus called B77 virus-II, mutates either to virus that efficiently infects duck cells (B77 virus-III) or to virus that does not mutate to the ability to infect duck cells (B77 virus-I) (Zarling and Temin, 1976). No significant differences in either the virion envelope glycoproteins or other major virion proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, pseudotypes of B77 virus-I with proteins of a transformation-defective mutant of B77 virus-III formed foci efficiently in duck cells. An alteration in the envelope protein of B77 virus-I was demonstrated by experiments in which B77 firus-I was fused into duck cells with UV-irradiated Sendai virus and formed foci. Neutralization experiments further demonstrated that B77 virus host-range mutants have altered type-specific envelope antigens. Thus, the spontaneous mutations in the host-range gene of B77 virus involve changes in the type-specific virion envelope antigen.

Animals↗

PubMed1977

Formation and structure of infectious DNA of spleen necrosis virus.

The kinetics of formation and the structure of infectious DNA of spleen necrosis virus were determined. Nonintegrated infectious viral DNA first appeared 18 to 24 h after infection of dividing cells and persisted for more than 14 days. The nonintegrated infectious viral DNA was in the form of either a double-stranded linear DNA with a molecular weight of 6 X 10(6), detected in both the cytoplasm and nucleus, or a closed circular DNA of the same molecular weight, detected primarily in the nucleus. Integrated infectious viral DNA appeared soon after the nonintegrated infectious viral DNA and was the predominant form of infectious viral DNA late after infection. Integration of the spleen necrosis virus DNA into the chicken cell genome was demonstrated by three independent criteria. Nucleic acid hybridization indicated that the linear infectious viral DNA had a 5- to 10-fold higher specific infectivity than either the closed circular or integrated infectious viral DNA. Infectious viral DNA did not appear in infected stationary cells, indicating some cellular influence on the formation of infectious viral DNA.

Animals↗

PubMed1977

Inhibition of viral DNA synthesis in stationary chicken embryo fibroblasts infected with avian retroviruses.

Previously, we reported (Fritsch and Temin, J. Virol. 21:119-130, 1977) that infectious viral DNA was not present in spleen necrosis virus-infected stationary chicken cells. However, a stable intermediate was present in such infected stationary cells as evidenced by the appearance of infectious viral DNA shortly after serum stimulation of these cells. After serum stimulation of infected stationary cells, the infectious viral DNA appeared first in the nucleus. In contrast, in infected dividing cells the infectious viral DNA appeared first in the cytoplasm. Significantly reduced amounts of complete plus- or minus-strand viral DNAs were detected by nucleic acid hybridization in stationary chicken cells infected with spleen necrosis virus or Schmidt-Ruppin Rous sarcoma virus compared with the amounts detected in infected dividing cells. These experiments indicated that infected stationary cells did not contain complete noninfectious copies of viral DNA. Furthermore, 5-bromodeoxyuridine labeling and cesium chloride density gradient centrifugation analysis of the infectious viral DNA that appeared after serum stimulation of infected stationary cells indicated that most viral DNA synthesis occurred after addition of fresh serum.

Blood↗

PubMed1977

Nucleotide sequences derived from pheasant DNA in the genome of recombinant avian leukosis viruses with subgroup F specificity.

Recombination between viral and cellular genes can give rise to new strains of retroviruses. For example, Rous-associated virus 61 (RAV-61) is a recombinant between the Bryan high-titer strain of Rous sarcoma virus (RSV) and normal pheasant DNA. Nucleic acid hybridization techniques were used to study the genome of RAV-61 and another RAV with subgroup F specificity (RAV-F) obtained by passage of RSV-RAV-0 in cells from a ring-necked pheasant embryo. The nucleotide sequences acquired by these two independent isolates of RAV-F that were not shared with the parental virus comprised 20 to 25% of the RAV-F genomes and were indistinguishable by nucleic acid hybridization. (In addition, RAV-F genomes had another set of nucleotide sequences that were homologous to some pheasant nucleotide sequences and also were present in the parental viruses.) A specific complementary DNA, containing only nucleotide sequences complementary to those acquired by RAV-61 through recombination, was prepared. These nucleotide sequences were pheasant derived and were not present in the genomes of reticuloendotheliosis viruses, pheasant viruses, and avian leukosis-sarcoma viruses of subgroups A, B, C, D, and E. They were partially endogenous, however, to avian DNA other than pheasant. The fraction of these nucleotide sequences present in other avian DNAs generally paralleled the genetic relatedness of these avian species to pheasants. However, there was a high degree of homology between these pheasant nucleotide sequences and related nucleotide sequences in the DNA of normal chickens as indicated by the identical melting profiles of the respective hybrids.

Alpharetrovirus↗

PubMed1976

The DNA provirus hypothesis.

I have discussed the observations and experiments that led to the formulation and establishment of the provirus hypothesis and the DNA provirus hypothesis, which includes RNA-directed DNA synthesis for the formation of the provirus. I have also discussed some aspects of the present status of our knowledge of the mechanism of formation of the DNA provirus both to point out the work remaining to be done and to illustrate hypotheses for the origins of ribodeoxyviruses and the origins of other animal enveloped RNA viruses and of animal small DNA viruses. Finally, I have indicated that I do not believe that infectious viruses cause most human cancers, but I do believe that viruses provide models of the processes involved in the etiology of human cancer.

Avian Leukosis Virus↗

PubMed1976

Incorporation of noncomplementary nucleotides at high frequencies by ribodeoxyvirus DNA polymerases and Escherichia coli DNA polymerase I.

The fidelity of DNA synthesis with synthetic homopolymer templates by two ribodeoxyvirus DNA polymerases and E. coli DNA polymerase I was examined by nearest neighbor frequency analyses. The experiments were designed to favor the incorporation of noncomplementary nucleotides, and the reactions were carried out only for short periods of time. All incorporations were template dependent. The frequency of incorporation of noncomplementary nucleotides next to complementary nucleotides by all three of these DNA polymerases significantly varied, depending on the conditions, from almost none (less than 0.2%) to 100% of the incorporation of the complementary nucleotide. In general, there was more incorporation of noncomplementary nucleotides in the presence of Mn2+ than in the presence of Mg2+ and with ribohomopolymer templates than with deoxyribohomopolymer templates. With a heteropolymer RNA template, the infidelity of DNA synthesis by one of the ribodeoxyvirus DNA polymerases was also increased by the presence of Mn2+ in the DNA synthesizing reaction. These findings indicate that, under appropriate conditions, any DNA polymerase has the potential to make a significatn number of errors in DNA synthesis and that altered conditions for DNA polymerase action might play a role in spontaneous mutation.

Base Sequence↗

PubMed1976

Lack of infectivity of the endogenous avian leukosis virus-related genes in the DNA of uninfected chicken cells.

The infectivity of the avian leukosis virus-related genes in the DNA of four genetically distinct types of chicken cells was determined. Infectious DNA of Rous-associated virus-O(RAV-O) was obtained from V- chicken cells which were experimentally infected with RAV-O and from V+tvbs chicken cells, which spontaneously produced RAV-O and were sensitive to exogenous RAV-O infection. However, infectious DNA of RAV-O was not obtained from uninfected V- chicken cells or from V+tvbr chicken cells, which spontaneously produced a low titer of RAV-O but were resistant to exogenous RAV-O infection. No detectable amplification of the RAV-O related DNA sequences in the V+tvbs cells was found by hybridization of RAV-O 125I-labeled RNA to the DNAs of V+tvbs and uninfected V- cells. These results indicate that the endogenous avian leukosis virus-related genes in uninfected V- and V+tvbr cells differ from the RAV-O proviruses in RAV-O-infected V- and V+tvbs cells. The lack of infectivity of the DNA of V+tvbr cells is consistent with the hypothesis that the endogenous RAV-O genome in V+tvbr cells is linked to a cis-acting control element, which results in its inefficient expression.

Animals↗

PubMed1976

Avian leukosis viruses of different subgroups and types isolated after passage of Rous sarcoma virus-Rous-associated virus-0 in cells from different ring-necked pheasant embryos.

Avian leukosis viruses of subgroups A and F (RAV-A and RAV-F) arose at a low rate after passage of Rous sarcoma virus-Rous-associated virus-0, which is subgroup E, in cells from ring-necked pheasant embryos. In cells of two embryos, all of the viruses isolated after virus passage were RAV-F. However, in cells of a third embryo, both RAV-A and RAV-F were isolated. In addition, there sometimes were type-specific differences among the different isolates of RAV-A and RAV-F from the cells of single embryos. These results indicate that the RAV-A and RAV-F probably arose by recombination of viral and cellular genes, that different ring-necked pheasant embryo may have different endogenous avian leukosis virus-related nucleotide sequences, and that recombination at different sites in these endogenous sequences might give rise to type-specific differences among the RAV-A and RAV-F.

Animals↗

PubMed1976

RNA polymerase activity in purified virions of avian reticuloendotheliosis viruses.

An RNA polymerase activity that synthesizes a U-rich RNA hydrogen bonded to a large viral RNA molecule was found in the cores of virions of avian reticuloendotheliosis viruses (REV). The RNA polymerase activity was separable from the DNA polymerase activity of REV virions. The 5'-terminus of the newly synthesized RNA was A. In addition, a tRNA nucleotidyl transferase activity, which added -CpCpA ends to tRNA, appears to be present in the REV virions.

Animals↗

PubMed1975

Replication of reticuloendotheliosis viruses in cell culture: chronic infection.

After an initial acute infection with cell killing, chicken or duck embryo fibroblasts infected in culture with reticuloendotheliosis viruses set up a chronic infection with no cell killing or morphological transformation. Essentially all of the chronically infected cells produced virus. The virus production was not sensitive to cytosine arabinoside or mitomycin C as was virus production in an acute infection. The chronically infected cells had a strong group-specific resistancto the c.p.e. of superinfecting reticuloendotheliosis viruses. However, they were sensitive to vesicular stomatitis virus and avian leukosis-sarcoma viruses. After double infection, single cells produced reticuloendotheliosis virus and avian leukosis-sarcoma virus.

Alpharetrovirus↗

PubMed1975

Infectious DNA from cells infected with Rous sarcoma virus, reticuloendotheliosis virus or Rous-associated virus-O.

We have described an efficient and quantitative assay for infectious DNA of the avian ribodeoxyviruses and have applied this method to study the possible existence of infectious viral DNAs in uninfected cells. Infectious DNA from cells infected with RSV or REV consisted of a single unit of DNA with a specific infectivity of 10(-5)-10(-6). The minimum molecular weight of RSV DNA required for infection of chicken cells was about 6 million, while the minimum molecular weight of infectious REV DNA was about 20 million. This difference may reflect complementation of the RSV DNA by endogenous avian leukosis virus-related DNA in uninfected chicken cells. Uninfected chicken cells do not contain infectious DNA for an REV, nor for a strongly transforming or a nontransforming avian leukosis virus.

Animals↗

PubMed1975

Purification and properties of spleen necrosis virus DNA polymerase.

DNA polymerase was purified to apparent electrophoretic homogeneity from virions of spleen necrosis virus (SNV). (SNV is a member of the reticuloendotheliosis group of avian ribodeoxyviruses). The SNV DNA polymerase appears to consist of a single polypeptide with a molecular weight of 68,000. The SNV DNA polymerase has a preference for Mn2+ for DNA synthesis with an RNA template and Mg2+ for DNA synthesis with a deoxyribohomopolymer template. At the optimum concentrations of divalent cation, the relative rates of DNA synthesis by SNV DNA polymerase with different template.primers were similar to the relative rates of DNA synthesis by an avian leukosis virus DNA polymerase, with the exception of a lower relative rate of DNA synthesis by SNV DNA polymerase with SNV RNA. However, in contrast to DNA synthesized by the avian leukosis virus DNA polymerase with a SNV RNA template, DNA synthesized by SNV DNA polymerase with an SNV RNA template did not hybridize to the SNV RNA. SNV DNA polymerase has RNase H activity which is antigenically distinct from the RNase H activity of avian leukosis-sarcoma virus DNA polymerase.

Animals↗

PubMed1975

High spontaneous mutation rate of an avian sarcoma virus.

Three genetically distinct types of chicken sarcoma virus Bratislava 77 (B77 virus) differing in their ability to infect duck cells were identified. B77 virus type I does not infect duck cells; B77 virus type II has a low efficiency of infection of duck cells; and B77 virus type III has a high efficiency of infection of duck cells. B77 viruses type I and III are produced by spontaneous mutation during the growth of B77 virus type II in chicken cells. The spontaneous mutation of B77 virus type II TO B77 virus type III occurs with a high rate (approximately 1 mutation per 50 infected cell generations), requires cell replication, and neither occurs during the synthesis of viral DNA on an RNA template nor during the transcription of progeny viral RNA from the provirus. The rate of spontaneous mutation of B77 virus type II to B77 virus type I is greater than the rate of spontaneous mutation of B77 virus type II to B77 virus type III.

Animals↗