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Biomedical subjects

H M Temin

Publications and source records attributed to H M Temin.

At least 109 records · Page 6Linked to original sources
PubMed1983

High-efficiency ligation and recombination of DNA fragments by vertebrate cells.

DNA-mediated gene transfer (transfection) is used to introduce specific genes into vertebrate cells. Events soon after transfection were quantitatively analyzed by determining the infectivity of the DNA from an avian retrovirus and of mixtures of subgenomic fragments of this DNA. The limiting step of transfection with two DNA molecules is the uptake by a single cell of both DNA's in a biologically active state. Transfected cells mediate ligation and recombination of physically unlinked DNA's at nearly 100 percent efficiency.

Animals↗
PubMed1983

Cellular DNA surrounding integration sites of an avian retrovirus.

The size of the direct repeats of cellular DNA next to a spleen necrosis virus (SNV) provirus from infected rat cells and the nature of the cellular DNA surrounding SNV integration sites in chicken DNA were studied. A five-base pair repeat, ATTTT, was observed at the SNV-rat cell junctions. Five of ten SNV proviruses from chicken cells were flanked by unique DNA and five others were flanked by repetitive DNA. No large rearrangements of cellular DNA at SNV integration sites were observed. A clone of uninfected chicken DNA containing the presumptive unoccupied integration site for one SNV provirus was obtained and part of it was sequenced. A small substitution, approximately 60 nucleotides, was observed at the site of virus DNA insertion. Its significance is not known.

Animals↗
PubMed1983

Structure and expression of c-rel, the cellular homolog to the oncogene of reticuloendotheliosis virus strain T.

The cellular homolog of the onc sequences in the avian retrovirus reticuloendotheliosis virus strain T (v-rel) was studied by molecular cloning and nucleic acid hybridization. In contrast to v-rel sequences, which are 1.4 kilobase pairs long, the cellular homolog, c-rel, from line 15B chickens is at least 25 kilobase pairs long, with multiple apparent introns. A 4.0-kilobase polyadenylic acid-containing RNA transcript is the primary species of c-rel RNA present in uninfected chicken cells. Sequences at the 3' end of this c-rel RNA are not present in v-rel.

Animals↗
PubMed1983

Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors.

We wished to construct cell lines that supply the gene products of gag, pol, and env for the growth of replication-defective reticuloendotheliosis retrovirus vectors without production of the helper virus. To do this, first we located by S1 mapping the donor and acceptor splice sites of reticuloendotheliosis virus strain A. The donor splice site is ca. 850 base pairs from the 5' end of proviral DNA. It is close to or overlaps the encapsidation sequences for viral RNA. The splice acceptor site is ca. 5.6 kilobase pairs from the 5' end of proviral DNA. Therefore, the encapsidation sequences and the donor splice site were removed from viral DNA to give expression of the gag and pol genes without virus production. The promoter in the long terminal repeat was fused to a site near the first ATG codon of the env gene, thereby deleting the encapsidation sequences and the gag and pol genes to give expression of the env gene without virus production. The permissive canine cell line D17 was transfected with the two modified viral DNAs. Two cell clones that contain both modified viral DNAs support the production of replication-defective spleen necrosis virus-thymidine kinase recombinant retrovirus vectors without the production of helper virus. To prevent recombination, the vector contains deletions that overlap with deletions in the integrated helper virus DNAs. This helper cell-vector system will be useful to derive infectious recombinant virus stocks of high titer (over 10(5) thymidine kinase transforming units per ml) which are able to infect avian, rat, and dog cells without the aid of helper virus.

Animals↗
PubMed1983

The organization of c-rel in chicken and turkey DNAS.

The process of transduction of a proto-oncogene involves the loss of intervening sequences, the loss of 3' mRNA terminal sequences, and possible base changes. It is not possible to know how similar the c-rel sequences we studied are to sequences from which v-rel arose. If one of the alleles we studied is identical to progenitor sequences of v-rel, then base changes must have occurred during the process of transduction. Alternatively, there may exist an allele of c-rel which did not need to undergo base changes to give rise to v-rel. The polymorphism of c-rel within a turkey population and between chicken and turkey shows clear evidence in c-rel coding sequences have not been detected within a turkey population. There is an EcoRI site in c-rel from turkey which is not in v-rel. There are unequal length substitutions in c-rel within the turkey population and between chicken and turkey. Unequal length substitutions can be explained if there has been a combination of two events, for example, insertion or deletion with extensive base changes, or two insertions or deletions. Two events occurring in a short evolutionary time span might occur if c-rel is diverging at a relatively fast rate. c-rel is much less conserved across species lines, in nucleic acid hybridization to v-rel (Chen et al. 1981; Wong and Lai 1981) than other proto-oncogenes, consistent with it diverging at a relatively fast rate. Since the evolutionary changes that have been seen for c-rel are in noncoding sequences, it is most likely that v-rel arose by transduction of a somatic mutation generated variant of c-rel or by transduction which involves sequence changes or later mutations.

Animals↗
PubMed1982

Substitution of 5' helper virus sequences into non-rel portion of reticuloendotheliosis virus strain T suppresses transformation of chicken spleen cells.

The genome of the highly oncogenic avian retrovirus reticuloendotheliosis virus strain T (REV-T) differs from that of the helper virus reticuloendotheliosis virus strain A by a substitution (rel and a large deletion. Further deletions, constructed in vitro, within the helper-virus-related sequences of REV-T have little effect on the ability of the virus to transform chicken spleen cells in vitro. However, deletions that extend into rel abolish transformation. Substitution of helper-virus-related sequences for the deleted region in the non-rel portion of REV-T also abolishes transformation. Viruses with revertant phenotype were obtained both spontaneously and by construction in vitro from these substituted recombinants. The revertant viruses have various mutations, including deletions and insertions, in the helper-virus-related sequences. Thus the additional helper-virus-related sequences suppress expression of transformation in cis, and the deletion in REV-T seems necessary for expression of the transforming properties of the virus.

Animals↗
PubMed1982

Encapsidation sequences for spleen necrosis virus, an avian retrovirus, are between the 5' long terminal repeat and the start of the gag gene.

The minimal cis-acting sequences outside the long terminal repeat (LTR) required for formation of an infectious retrovirus cloning vector were determined with recombinants of spleen necrosis virus (SNV) DNA and herpes simplex virus type 1 thymidine kinase gene. The 3' end of SNV DNA was removed to within 40 base pairs (bp) from the 3' LTR with only a 2-fold effect on the recovery of infectious recombinant virus. However, when the 5' end of SNV DNA was removed to within 100 bp from the 5' LTR, infectious recombinant virus was not recovered. Deletion mutants constructed around this latter region showed that nucleotides between 100 and 285 bp from the 5' LTR are necessary for encapsidation of genomic viral RNA. We call this region required for encapsidation E.

Animals↗
PubMed1982

Spontaneous changes in nucleotide sequence in proviruses of spleen necrosis virus, an avian retrovirus.

We determined the nucleotide sequence of about 1 kilobase of DNA 3' to the 5' long terminal repeat of three noninfectious ad one infectious proviral DNA clones of spleen necrosis virus, an avian retrovirus, to determine if the types of nucleic acid changes involved in retrovirus mutation shed light on special features of retrovirus replication. An open reading frame was found starting 411 base pairs from the end of the long terminal repeat. It contained sequences coding for the 36 amino acids at the amino terminus of the p30 of a related reticuloendotheliosis virus [Oroszlan, S., Barbacid, M., Copeland T., Aaronson, S. A. & Gilden, R. V. (1981) J. Virol. 39, 845-854]. Therefore, the open reading frame represents the 5' end of the gag gene. A mutation in one noninfectious provirus changed the initiation codon for the gag polypeptide; a mutation in another noninfectious provirus caused premature termination of gag polypeptide synthesis; and a nontandem duplication into gag resulting from a mistake in initial (+) strand DNA synthesis changed amino acids and the reading frame in a third noninfectious provirus. These mutations appear to be responsible for the lack of infectivity of these provirus clones and indicate a higher relative frequency of mutation in this region of the genome. In addition, all four clones have multiple other mutations. These mutations are mostly base pair substitutions and many are clustered for any one clone, reflecting certain special features of retrovirus replication.

Amino Acid Sequence↗
PubMed1982

Spontaneous variation and synthesis in the U3 region of the long terminal repeat of an avian retrovirus.

Recombinant DNA clones of a viral clone of spleen necrosis virus, an avian retrovirus, were found to have long terminal repeats of different sizes. The variation was in the U3 region of the long terminal repeats, and any one clone had U3 of the same size in both long terminal repeats. The U3 regions in the 5' and 3' long terminal repeat were shown both to be derived from the 3' long terminal repeat of parental virus DNA.

Alpharetrovirus↗
PubMed1982

Establishment of infection by spleen necrosis virus: inhibition in stationary cells and the role of secondary infection.

The relationship of two early events in the establishment of infection by avian retroviruses, the inhibition of viral DNA synthesis in stationary avian cells and the secondary infection which occurs after infection of replicating cells, was investigated. When neutralizing antibody to spleen necrosis virus was used to prevent secondary infection, the amount of unintegrated linear spleen necrosis virus DNA detected was much lower in infected stationary cells than in infected replicating cells. The amount of unintegrated linear spleen necrosis virus DNA in stationary cells was less than one copy per cell even at high multiplicities of infection. Viral DNA synthesis resumed after stimulation of the cells to replicate. The time of this viral DNA synthesis was closely correlated with renewed cellular DNA synthesis. In addition, blocking secondary infection of replicating cells prevented the rate of virus production from reaching the high levels usually associated with a normal productive infection by SNV. Virus production increased if secondary infection was allowed. However, this rise in virus production was not proportional to the amounts of viral DNA integrated after secondary infection.

Alpharetrovirus↗
PubMed1981

Formation of infectious progeny virus after insertion of herpes simplex thymidine kinase gene into DNA of an avian retrovirus.

We have prepared several infectious stocks of an avian retrovirus, spleen necrosis virus, containing the herpes simplex virus type 1 thymidine kinase (tk) gene. The viruses were produced after cotransfection of chicken cells with DNA from recombinants between cloned spleen necrosis virus and tk DNAs and DNA of cloned reticuloendotheliosis virus strain A. removal of sequences in the tk gene for the end of tk mRNA increased a thousand fold the yield of infectious recombinant virus. Infection of chicken or rat tk- cells with the recombinant virus transformed them to a tk+ phenotype.

Animals↗