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H M Temin

Publications and source records attributed to H M Temin.

At least 73 records · Page 4Linked to original sources
PubMed1988

Evolution of cancer genes as a mutation-driven process.

Cancer is primarily a somatic genetic disease resulting from the accumulation of several precancerous mutations in a cell lineage. The evolution of highly oncogenic retroviruses has been used as a model for the evolution of a cancer cell. The properties of intermediates between one set of replication-competent retrovirus and protooncogene progenitors and the homologous highly oncogenic retrovirus were analyzed to differentiate between selection-driven and mutation-driven models of this evolution. In this case and in some other cases where sufficient data are available, it appears that the intermediates in the evolution of highly oncogenic retroviruses are not transforming, indicating that they were not formed in a purely selection-driven process. Furthermore, analysis of retrovirus mutation rates indicates that there is a high rate of mutation in retrovirus replication such that the evolution of highly oncogenic retroviruses could be mutation-driven. Other evidence is mentioned suggesting that oncogenesis in general is at least partially mutation-driven, although mutational mechanisms are involved that are different from those involved in the evolution of highly oncogenic retroviruses.

Biological Evolution↗
PubMed1988

Retroviruses and the genetics of cancer.

Retroviruses cause cancer by several different mechanisms including addition of an oncogene, addition of a modified viral glycoprotein, activation of a proto-oncogene, transactivation of a proto-oncogene, immune depression, and stimulation of lymphoid cell proliferation. Both the evolution of oncogenes and tumor induction by most retroviruses is multi-step. Study of the evolution of a particular oncogene, v-rel, indicates that this evolution could not have been selection-driven, but that it resulted from the high rate of mutation in retroviruses replication, that is, it was mutation-driven. Argument is made that much other cancer is also mutation-driven.

Humans↗
PubMed1988

v-rel oncoproteins in the nucleus and in the cytoplasm transform chicken spleen cells.

The transforming protein encoded by the v-rel oncogene of the highly oncogenic avian retrovirus reticuloendotheliosis virus strain T (Rev-T) is a 59,000-dalton protein, p59v-rel. The mechanism by which p59v-rel induces transformation of early lymphoid cells is unknown. As a step towards understanding the mechanism of v-rel-induced transformation, we sought to establish the subcellular site of action of p59v-rel. In this report, we show that p59v-rel contains sequences that are necessary for its efficient localization in the nucleus of infected chicken embryo fibroblasts. These v-rel sequences when added to the normally cytoplasmic protein, beta-galactosidase, directed that protein to the nucleus. A mutation in the v-rel nuclear-localizing sequence did not affect the transforming function, although it did alter the nuclear-localizing function. The addition of a supplemental nuclear-localizing sequence from simian virus 40 large T-antigen to v-rel resulted in the expression of a transforming rel protein which was located exclusively in the nucleus of transformed spleen cells, in contrast to wild-type p59v-rel, which was largely cytoplasmic in transformed spleen cells. Our results support the hypothesis that v-rel encodes a protein which can act either in the nucleus or in the cytoplasm to transform spleen cells.

Animals↗
PubMed1988

Transforming viruses spontaneously arise from nontransforming reticuloendotheliosis virus strain T-derived viruses as a result of increased accumulation of spliced viral RNA.

The highly oncogenic avian retrovirus reticuloendotheliosis virus strain T (Rev-T) contains a substitution of the oncogene v-rel for much of env and a deletion of gag and pol relative to the helper virus Rev-A. Replacement of gag and pol sequences in Rev-T suppresses transformation by reducing the accumulation of spliced viral mRNA and v-rel protein in infected cells (C. K. Miller and H. M. Temin, J. Virol 58:75-80, 1986). After infection of spleen cells with viruses containing gag and pol sequences, revertant viruses that are strongly transforming were found. Approximately three-fourths of the revertant viruses appeared structurally the same as the parental virus, and approximately one-fourth of the revertant viruses had large deletions (similar in size and location to the deletion in Rev-T). Two revertant viruses that appeared structurally the same as the parental virus were molecularly cloned. The regions sufficient to change the parental virus to a strongly transforming virus were determined by construction of recombinant viruses. In one revertant virus, the region sufficient for transformation contained a 327-base-pair insertion 5' of the 3' splice site used by Rev-T. In the other revertant virus, the region sufficient for transformation contained a 1-base-pair transition and a deletion of one copy of a 9-base-pair direct repeat, both 3' of the 3' splice site used by Rev-T. These differences resulted in the accumulation of increased levels of subgenomic v-rel mRNA and protein, ultimately leading to transformation.

Animals↗
PubMed1988

Determination of the rate of base-pair substitution and insertion mutations in retrovirus replication.

We recently described a protocol for determination of retrovirus mutation rates, that is, the mutation frequency in a single cycle of retrovirus replication (J.P. Dougherty and H.M. Temin, Mol. Cell. Biol. 6:4378-4395, 1987; J.P. Dougherty and H.M. Temin, p. 18-23, in J. H. Miller and M. P. Calos, ed., Gene Transfer Vectors for Mammalian Cells, 1987). We used this protocol to determine the mutation rates for defined mutations in a replicating retrovirus by using a spleen necrosis virus-based vector. We determined that the mutation rate for a single base pair substitution during replication of this avian retrovirus is 2 x 10(-5) per base pair per replication cycle and the insertion rate is 10(-7) per base pair per replication cycle. It will be possible to use this protocol to determine mutation rates for other retroviruses.

Base Sequence↗
PubMed1988

Retroviral vector system for the study of cDNA gene formation.

A retroviral vector system was developed to study the retrotransposition of RNAs lacking all cis-acting sequences required for normal retroviral replication. Our experiments indicate that such RNAs can be encapsidated in retroviral proteins, reverse transcribed, and integrated to form functional cDNA genes in infected cells. The frequency of this process, however, was approximately 8 orders of magnitude less than that of normal retroviral replication. The efficiency was limited at each step in this process. Investigation of seven cDNA genes by Southern blot analysis revealed that all of them were truncated at either the 3' or the 5' end or both. These truncations are not seen with natural cDNA genes and raise the question of retroviral involvement in their formation.

Animals↗
PubMed1988

The v-rel oncogene encodes a cell-specific transcriptional activator of certain promoters.

Transformation by the v-rel oncogene of avian reticuloendotheliosis virus strain T (Rev-T) is primarily cell-specific. While v-rel efficiently transforms chicken spleen and bone marrow stem cells in vitro and induces rapid lethal lymphomas in young birds, it does not rapidly transform chicken embryo fibroblasts. The nuclear localization of the v-rel gene product in non-transformed fibroblasts along with its ability to function as a transforming protein in the nucleus of chicken spleen cells suggests that p59v-rel might belong to the family of nuclear oncoproteins and thus may express an immortalizing function in fibroblasts. To gain insight into the specificity of cell transformation by the v-rel oncogene, we determined whether v-rel could immortalize primary rat fibroblasts. Our experiments have shown that, unlike other nuclear oncoproteins, p59v-rel did not immortalize primary rat embryo fibroblasts. However p59v-rel was able to cooperate in a synergistic way with the polyomavirus middle T protein in inducing efficient transformation of established rat fibroblasts by increasing the steady-state level of middle T RNA, indicating that p59v-rel might function as a transactivator. Cotransfection of cells from different species with the v-rel gene along with constructs expressing the chloramphenicol acetyl transferase gene under the control of different promoters revealed that p59v-rel is a cell-specific transcriptional transactivator of certain promoters. Moreover, the extent of cell-specific transactivation by v-rel correlated with its toxic effect in these same cells.

Animals↗
PubMed1987

A promoterless retroviral vector indicates that there are sequences in U3 required for 3' RNA processing.

We constructed a retrovirus vector in which all viral transcriptional signals are deleted during provirus formation. This vector would be safer and more useful in gene-transfer experiments. Construction of this vector involved the deletion of all of the U3 sequence except for 10 base pairs, required for integration, from the right-side long terminal repeat. We found that this deletion resulted in an inability to propagate virus efficiently. When we inserted sequences containing all of the signals for polyadenylylation of simian virus 40 late mRNA at the end of our vector, we were able to propagate virus efficiently. This result indicates that there are sequences in U3 upstream from AAUAAA that are essential for 3' processing of viral RNA.

Animals↗
PubMed1987

Transcription from a spleen necrosis virus 5' long terminal repeat is suppressed in mouse cells.

To determine the block(s) to spleen necrosis virus (SNV) replication in mouse cells, we studied the expression of a dominant selectable marker, neo, or a gene whose product is easily assayed, the chloramphenicol acetyltransferase (cat) gene, in SNV-derived and murine leukemia virus-derived vectors. Using transient (CAT) and stable (Neor phenotype) transfection assays, we showed that the SNV promoter was used in mouse cells only when the 3' SNV long terminal repeat (LTR) was absent. Infection of mouse cells with recombinant SNV viruses was 1% as efficient as infection of permissive dog (D17) cells. The SNV proviruses in mouse cells appeared normal by Southern blot analysis, indicating that their integration probably occurred by normal mechanisms. S1 nuclease analyses of Neor mouse cell clones, each harboring a single recombinant SNV provirus, showed that the selected (internal) promoter was active, but that the 5' SNV LTR promoter was not. However, in the rare (less than 10(-6)) Neor colonies in which expression of the 5' LTR was selected, both promoters were active. Thus, the block to SNV infection of mouse cells is at least at two levels; one is a 100-fold-decreased efficiency at some step(s) up to and including integration, and the other is at transcription.

Animals↗
PubMed1987

Instability of large direct repeats in retrovirus vectors.

Retrovirus vectors were constructed with large (0.85- to 1.3-kilobase-pair) direct repeats in their genomes. Deletions involving the direct repeats occurred at a high frequency. Deletions occurred both when the direct repeats were in tandem and when they were separated by additional sequences. These deletions occurred during virus replication.

Base Sequence↗
PubMed1987

Lack of competition results in efficient packaging of heterologous murine retroviral RNAs and reticuloendotheliosis virus encapsidation-minus RNAs by the reticuloendotheliosis virus helper cell line.

We constructed recombinant reticuloendotheliosis virus (Rev)-derived and murine leukemia virus-derived vectors to characterize the specificity of packaging retroviral RNAs in Rev proteins. Using this approach, we further localized the Rev encapsidation sequence (E) to a 144-nucleotide region and determined that there are sequences in both the 5' and 3' halves of this region which are necessary in cis for viral replication. We found that the Rev E, like the murine leukemia virus E (psi), is position independent (R. Mann and D. Baltimore, J. Virol. 54:401-407, 1986). Also, a 156-nucleotide region of the Rev intron enhanced replication in a cis-acting fashion in the presence, but not in the absence, of helper virus. Finally, we showed that packaging of E- and heterologous retroviral genomes occurred efficiently in the Rev helper cell in the absence of competing E-containing (E+) viral RNAs.

Base Sequence↗
PubMed1986

Comparison of promoter suppression in avian and murine retrovirus vectors.

Previously, we described "promoter suppression" in infectious retrovirus vectors with two genes and an internal promoter. Here, we examined several parameters of promoter suppression and found that the amount of suppression in an integrated retrovirus vector was dependent both on whether the vector was derived from spleen necrosis virus or murine leukemia virus and on which internal promoter was present in the vector. Murine leukemia virus vectors showed less suppression than analogous spleen necrosis virus vectors. Furthermore, the amount of suppression was not dependent on either the relative strengths of the promoters nor the distance between the promoters. Moreover, we found that in vectors in which one promoter was suppressed, there was an inverse correlation between the DNaseI sensitivity of the chromatin surrounding a promoter and the suppression of its expression.

Chromatin↗
PubMed1986

Different localization of the product of the v-rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T.

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus that transforms early lymphoid cells in vivo and in vitro, but REV-T does not transform chicken embryo fibroblasts (CEF). Using antisera to p59v-rel, the v-rel oncogene product of REV-T, we show that p59v-rel is expressed at equal levels and is a phosphoprotein in REV-T infected spleen cells and CEF. Biochemical fractionation and immunofluorescence of REV-T infected nontransformed CEF show that p59v-rel is loosely associated with the nucleus. However, in REV-T transformed spleen cells p59v-rel is primarily a cytoplasmic protein. MSB-1 cells, a Marek's disease virus transformed T cell leukemic line, and E26 virus transformed myeloid cells show nuclear staining of p59v-rel when they are infected by REV-T. Our results indicate that there is a correlation between a cytoplasmic localization of p59v-rel and transformation by REV-T, and they suggest that p59v-rel cannot transform cells in which it assumes solely a nuclear location.

Animals↗
PubMed1986

Retroviruses and evolution.

A wide variety of different genetic elements and DNA sequences have used reverse transcription in their replication or transposition. Some of these elements may be evolutionarily related and show a continued evolution to autonomy from the cell genome. The evolution of a cancer reflects a similar drive for autonomy from the organism.

Biological Evolution↗
PubMed1986

Nondefective spleen necrosis virus-derived vectors define the upper size limit for packaging reticuloendotheliosis viruses.

We constructed a nondefective retrovirus vector based on spleen necrosis virus (SNV), a replication-competent reticuloendotheliosis virus. We introduced different DNA sequences into this vector and studied the ability of the resulting viruses to replicate in chicken embryo fibroblasts. The replication efficiency of SNV-derived viruses decreased with increasing virus size. Viruses larger than 9.4 kilobases (kb) were rapidly overgrown by replication-competent deletion mutants. The size restriction for the efficient replication of nondefective SNV-derived viruses prevented the production of viruses larger than 10.0 kb. Analysis of the kinetics of virus particle release indicated that the size restriction occurred during virus encapsidation.

Capsid↗
PubMed1986

Insertion of several different DNAs in reticuloendotheliosis virus strain T suppresses transformation by reducing the amount of subgenomic mRNA.

The highly oncogenic retrovirus reticuloendotheliosis virus (Rev) strain T (Rev-T) has, relative to its helper virus Rev strain A, a substitution of the oncogene v-rel for most of the env gene and a large deletion of gag and pol sequences. When the helper virus sequences that are deleted in Rev-T are replaced, the recombinant virus is nontransforming (I. S. Y. Chen and H. M. Temin, Cell 31: 111-120, 1982). We show that suppression of transformation occurs when several different DNA sequences are inserted in Rev-T and that suppression is correlated with a reduction in the amount of v-rel mRNA and v-rel protein in infected cells. The reduced amount of v-rel protein is insufficient for transformation.

Animals↗