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Biomedical subjects

H M Temin

Publications and source records attributed to H M Temin.

At least 37 records · Page 2Linked to original sources
PubMed1993

Rate and mechanism of nonhomologous recombination during a single cycle of retroviral replication.

Oncogenes discovered in retroviruses such as Rous sarcoma virus were generated by transduction of cellular proto-oncogenes into the viral genome. Several different kinds of junctions between the viral and proto-oncogene sequences have been found in different viruses. A system of retrovirus vectors and a protocol that mimicked this transduction during a single cycle of retrovirus replication was developed. The transduction involved the formation of a chimeric viral-cellular RNA, strand switching of the reverse transcription growing point from an infectious retrovirus to the chimeric RNA, and often a subsequent deletion during the rest of viral DNA synthesis. A short region of sequence identity was frequently used for the strand switching. The rate of this process was about 0.1 to 1 percent of the rate of homologous retroviral recombination.

Animals↗
PubMed1993

Recent advances in retrovirus vector technology.

Retroviral vectors are widely used for the study of retroviral replication and to introduce DNA into somatic cells. An exciting new approach in retroviral vector technology is the use of internal ribosome entry sites from picornaviruses to provide stable expression of multiple genes. In addition, strategies are being developed that target the expression of retroviral vectors to specific cell populations.

Animals↗
PubMed1993

Alteration of location of dimer linkage sequence in retroviral RNA: little effect on replication or homologous recombination.

Retrovirus particles contain a dimer of retroviral genomic RNA. A defined region of the retrovirus genome has previously been shown to be important for both dimerization and encapsidation. To study the importance of the position of this encapsidation and dimerization signal for retroviral replication and homologous recombination, we used a previously described spleen necrosis virus-based helper cell system. This system allows retroviral vectors with multiple genetic markers to be studied after a single cycle of retroviral replication. The sequence responsible for dimerization, the encapsidation/dimer linkage sequence (E/DLS), was moved from its normal location near the 5' end of the retroviral genome to a location near the 3' end of the genome. We characterized four pairs of retroviral vectors: (i) with both E/DLSs at the 5' ends of the genomes, (ii) with both E/DLSs at the 3' ends of the genomes, and (iii) two with one E/DLS at the 5' end of the genome and one at the 3' end of the genome. We found that moving the E/DLS to the 3' end of the genome resulted in at most an approximately factor of 5 reduction in virus titer in a single cycle of retroviral replication. Furthermore, we found no changes that were attributable to the alteration of the position of the E/DLS in the minus-strand DNA primer transfers or the plus-strand DNA primer transfers, the rate of homologous recombination, or the number of internal template switches in recombinant proviruses. These results indicate that any alignment or conformation necessary for retroviral replication or recombination is not the result of the position of the E/DLS.

Animals↗
PubMed1992

Unusually high frequency of reconstitution of long terminal repeats in U3-minus retrovirus vectors by DNA recombination or gene conversion.

Recently, we described a retrovirus vector system with which to study formation of cDNA genes (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 6:2328-2334, 1988; Mol. Cell. Biol. 8:64-72, 1990; J. Virol. 64:886-889, 1990). For these studies, retrovirus vectors were constructed in which the U3 region of the 3' long terminal repeat (LTR) was deleted. After one round of retrovirus replication, such vectors formed a provirus with two U3-minus LTRs. However, the insertion of some additional sequences into such vectors promoted vector rearrangements with an efficiency greater than 95%. Such rearranged vectors behaved like vectors with two wild-type LTRs. Proviruses derived from such vectors were investigated by Southern blot analysis, polymerase chain reaction, and DNA sequencing. We found that the U3 region was reconstituted, resulting in vectors with LTRs like wild-type virus. The sequences that reconstituted the U3 region of the vector LTR were derived from LTR sequences present in the helper cell. Since no retroviral protein coding sequences were detected in infected target cells, recombination of vector sequences with coencapsidated helper cell sequences during reverse transcription seems very unlikely. Thus, it appears that the recombination (or gene conversion) events leading to a vector with reconstituted LTRs occurred at the DNA level. The high frequency of this recombination (or gene conversion) was dependent on internal vector sequences.

Animals↗
PubMed1992

5-Azacytidine and RNA secondary structure increase the retrovirus mutation rate.

A broad spectrum of mutations occurs at a high rate during a single round of retrovirus replication (V.K. Pathak and H. M. Temin, Proc. Natl. Acad. Sci. USA 87:6019-6023, 1990). We have now determined that this high rate of spontaneous mutation can be further increased by 5-azacytidine (AZC) treatment or by regions of potential RNA secondary structure. We found a 13-fold increase in the mutation rate after AZC treatment of retrovirus-producing cells and target cells. The AZC-induced substitutions were located at the same target sites as previously identified spontaneous substitutions. The concordance of the AZC-induced and spontaneous substitutions indicates the presence of reverse transcription "pause sites," where the growing point is error prone. An analysis of nucleotides that neighbored substitutions revealed that transversions occur primarily by transient template misalignment, whereas transitions occur primarily by misincorporation. We also introduced a 34-bp potential stem-loop structure as an in-frame insertion within a lacZ alpha gene that was inserted in the long terminal repeat (LTR) U3 region and determined whether this potential secondary structure increased the rate of retrovirus mutations. We found a threefold increase in the retrovirus mutation rate. Fifty-seven of 96 mutations were deletions associated with the potential stem-loop. We also determined that these deletion mutations occurred primarily during minus-strand DNA synthesis by comparing the frequencies of mutations in recovered provirus plasmids containing both LTRs and in provirus plasmids containing only one LTR.

Animals↗
PubMed1992

Effect of gamma radiation on retroviral recombination.

To elucidate the mechanism(s) of retroviral recombination, we exposed virions to gamma radiation prior to infecting target cells. By using previously described spleen necrosis virus-based vectors containing multiple markers, recombinant proviruses were studied after a single round of retrovirus replication. The current models of retroviral recombination predict that breaking virion RNA should promote minus-strand recombination (forced copy-choice model), decrease or not affect plus-strand recombination (strand displacement/assimilation model), and shift plus-strand recombination towards the 3' end of the genome. However, we found that while gamma irradiation of virions reduced the amount of recoverable viral RNA, it did not primarily cause breaks. Thus, the frequency of selected recombinants was not significantly altered with greater doses of radiation. In spite of this, the irradiation did decrease the number of recombinants with only one internal template switch. As a result, the average number of additional internal template switches in the recombinant proviruses increased from 0.7 to 1.4 as infectivity decreased to 6%. The unselected internal template switches tended to be 5' of the selected crossover even in the recombinants from irradiated viruses, inconsistent with a plus-strand recombination mechanism.

Base Sequence↗
PubMed1992

Multiple sequence elements are involved in RNA 3' end formation in spleen necrosis virus.

The function of the poly(A) signal in spleen necrosis virus (SNV) is dependent upon the distance between the cap site and the poly(A) site, while the function of the SV40 late poly(A) signal is independent of the distance. Deletions in the SNV poly(A) sequence do not alter the distance-dependent function. SNV/SV40 chimeric poly(A) signals show intermediate behavior between the SNV and SV40 poly(A) signals. These results indicate that multiple sequence elements are involved in the functions of either the SNV or SV40 poly(A) signals. This intermediate behavior is also observed with poly(A) signals from the mouse alpha-globin and herpes simplex virus thymidine kinase genes.

Base Sequence↗
PubMed1991

Characterization of large deletions occurring during a single round of retrovirus vector replication: novel deletion mechanism involving errors in strand transfer.

Retroviruses mutate at a high rate during replication. We used a spleen necrosis virus-based vector system and helper cell line to characterize mutations occurring during a single round of retrovirus replication. The vector used, JD216HyNeo, codes for two drug resistance genes, hygromycin resistance (hygro) and neomycin resistance (neo). The downstream neo gene is expressed only when a mutation alleviates a block to splicing which is located in the upstream hygro gene. The mutations allowing splicing were large deletions, ranging in size from about 500 to about 2,000 bp. Most of the mutant proviruses lacked the encapsidation sequence, as shown by our inability to rescue the mutant proviruses with wild-type reticuloendotheliosis virus strain A and confirmed by Southern blotting and direct DNA sequence analysis. We therefore concluded that most of the deletions arose during reverse transcription in the target cell, rather than during transcription in the host cell. The sequence data also indicated that the deletions occurred by at least three different mechanisms: (i) misalignment of the growing point; (ii) incorrect synthesis and termination in the primer-binding sequence during synthesis of the plus-strand strong-stop DNA; and (iii) incorrect synthesis and termination before the primer-binding sequence during synthesis of the plus-strand strong-stop DNA. The second mechanism also led to the incorporation of cellular sequences into the proviral genome, pointing to a potential novel mechanism by which retroviruses can acquire cellular genes.

Amino Acid Sequence↗
PubMed1991

Molecular mechanisms of transformation by the v-rel oncogene.

Our knowledge of the molecular mechanisms that underlie the diverse cellular phenotypes collectively called cancer has increased dramatically over the past 20 years. A significant contribution to our current understanding of cancer has come from research into the behavior of a unique group of viruses, the acutely transforming retroviruses. The acutely transforming retroviruses contain one, or occasionally two, genes that are responsible for the transforming properties of the viruses. These genes, called retroviral oncogenes, have been transduced from genes present in the normal cellular genome, called proto-oncogenes. The proto-oncogenes encode diverse proteins that are important for the regulation of normal cell growth and differentiation. One such proto-oncogene, the c-rel proto-oncogene, has recently been shown to encode a member of the Nuclear Factor-kappa B (NF-kappa B) transcription factor family. The structural and functional relationship between NF-kappa B and the c-rel protein provides a basis for understanding the molecular mechanism of neoplastic transformation by the v-rel protein.

Animals↗
PubMed1990

Retroviral recombination and reverse transcription.

Recombination occurs at a high rate in retroviral replication, and its observation requires a virion containing two different RNA molecules (heterodimeric particles). Analysis of retroviral recombinants formed after a single round of replication revealed that (i) the nonselected markers changed more frequently than expected from the rate of recombination of selected markers; (ii) the transfer of the initially synthesized minus strand strong stop DNA was either intramolecular or intermolecular; (iii) the transfer of the first synthesized plus strand strong stop DNA was always intramolecular; and (iv) there was a strong correlation between the type of transfer of the minus strand strong stop DNA and the number of template switches observed. These data suggest that retroviral recombination is ordered and occurs during the synthesis of both minus and plus strand DNA.

Biological Evolution↗
PubMed1990

Overview of biological effects of addition of DNA molecules to cells.

Injected DNA proceeds with certain probabilities through the following steps: degradation by serum nucleases, adsorption to cells, uptake into cells, ligation to other DNA, mutation, expression of unintegrated DNA, integration, expression of integrated DNA, and activation of or inactivation of cellular genes. The maximal probability per DNA molecule of each of these steps is estimated based on experimental results in cell culture with transfection of DNA and with infection by retroviruses. A maximum cumulative probability of having a harmful effect is calculated to be less than 10(-16) to 10(-19) per DNA molecule from a cell without activated proto-oncogenes or active viral oncogenes. The most frequent harmful effects considered are inactivation of a tumor suppressor gene and activation of a proto-oncogene. Such inactivation and activation in a cell that could give rise to cancer would increase the age-standardized incidence of cancer by a small amount. The amount of increase would differ among individuals depending upon their genotypes and their environments. Thus, the magnitude of the increase will depend upon the frequency of more sensitive individuals. The probability of an increased incidence of cancer as a possible effect of the vaccination should be compared to the number of DNA molecules to be injected per person and to the protective effects of a successful HBV vaccine.

Animals↗
PubMed1990

Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: substitutions, frameshifts, and hypermutations.

We determined the in vivo forward mutation rate in a single replication cycle for spleen necrosis virus (SNV). A method was developed to clone integrated proviruses of retroviral shuttle vectors by exploiting the tight binding of the lac operator to the lac repressor protein. The vectors contained the lacZ alpha gene as a reporter of mutations. Thirty-seven of the 16,867 proviruses recovered contained five classes of mutations, including substitutions and frameshifts. Runs of 9 and 10 identical base pairs and a direct repeat of 110 base pairs were mutational hotspots. In addition, two copies of a provirus contained 15 G-to-A substitutions. Such proviruses, which we name hypermutants, may arise through the action of an error-prone polymerase and could significantly contribute to the genetic variation in retroviral populations.

Animals↗