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Biomedical subjects

J M Coffin

Publications and source records attributed to J M Coffin.

At least 91 records · Page 5Linked to original sources
PubMed1986

Differential transcription from the long terminal repeats of integrated avian leukosis virus DNA.

In avian leukosis virus-induced lymphoma and erythroblastosis, the expression of the proto-oncogenes c-myc and c-erbB is activated by downstream or readthrough transcripts initiated within integrated proviral DNA. To determine the relative abundance of viral RNAs extending into the downstream cellular sequences independently of the effects that may be exerted by specific sites of proviral integration, we examined the RNA of infected avian fibroblasts. Using a nuclease protection strategy to detect downstream, readthrough, and normal viral RNAs and to distinguish them from each other, we found that transcripts initiated within the 3' long terminal repeat, i.e., downstream transcripts, were undetectable in infected fibroblasts and could not have amounted to more than 1 to 2% of the total viral RNA. However, readthrough RNAs, which are transcripts initiated within the 5' long terminal repeat and extended beyond the viral polyadenylation site into the downstream cellular DNA, were present at relatively high levels, making up approximately 15% of the total viral RNA.

Animals↗
PubMed1985

Sequences in the gag-pol-5'env region of avian leukosis viruses confer the ability to induce osteopetrosis.

DNA sequences encoding the genomes of three subgroup E avian leukosis viruses have been molecularly cloned. Virus recovered from one of these cloned DNAs (pRAV-0) caused no osteopetrosis while virus recovered from the second (lambda NY203) caused late onset osteopetrosis and virus recovered from the third (lambda NTRE-2) caused intermediate onset osteopetrosis. Restriction endonuclease fragments of the cloned viral DNAs were used to construct recombinant viruses that could be used to test for the role of gag-pol-5'env and 3'env-LTR sequences in the induction of osteopetrosis. The results of the pathogenicity tests indicate that gag-pol-5'env sequences confer the ability to induce osteopetrosis while 3'env-LTR sequences influence the time of onset and the severity of osteopetrosis.

Animals↗
PubMed1985

Molecular basis of host range variation in avian retroviruses.

Previous genetic analysis has localized the region of the Rous sarcoma virus (RSV) env gene responsible for host range specificity to that encoding the middle one-third of gp85. To better understand the host range determinants, the relevant regions of the genomes of infectious molecular clones of the transformation-defective Prague strain of RSV, subgroup B (Pr-RSV-B) and Rous-associated virus 0 (RAV-0) (subgroup E) were sequenced and compared with the sequence of Pr-RSV-C. This comparative analysis identified two variable regions of low amino acid sequence homology flanked by highly conserved amino acid sequences. The first variable region (hr1) begins at base 5654 in the Pr-RSV-C sequence and encodes 32 amino acids. The second variable region (hr2) begins at base 5846 and encodes 27 amino acids. To test the role of the variable regions in host range specificity, we determined the sequence of this region of the env gene of NTRE-4, a recombinant virus between Pr-RSV-B and RAV-0 which exhibits an extended host range. This analysis revealed that the recombinant subgroup-encoding region of NTRE-4 is composed of 200 bases of RAV-0 sequence, including hr2, flanked by sequences which are otherwise of Pr-RSV-B origin. This study indicates that hr1 and hr2 are the domains of gp85 responsible for host range determination in avian retroviruses.

Amino Acid Sequence↗
PubMed1985

Evolutionary variants of Rous sarcoma virus: large deletion mutants do not result from homologous recombination.

Large deletion (LD) mutants of Prague strain Rous sarcoma virus, subgroup B (PrB), derived by serial undiluted passage through chicken (C/E) cells, were isolated and characterized. Individual LD viruses were initially isolated by cloning in soft agar of infected, chemically transformed quail (QT6) cells. Two regions of the PrB genome were deleted in the formation of the LD virus. This resulted in the junction of gag sequences in p12 to env sequences in gp37, and in the loss of the src gene. DNA restriction analysis of biologically active lambda Charon 27-LD recombinant clones indicated that individual LD viruses contained similar but not identical deletion endpoints. Two LD isolates, LD25 and LD85, were further subcloned into pBR322, and the deletion junctions were examined by DNA sequencing. Although the gag-env deletion endpoints were identical in the two subclones, heterogeneity was observed across the src deletion in that both mutants analyzed had the same 5' endpoint but slightly different 3' endpoints. In all cases, only a single homologous base (always an A residue) was found at the deletion endpoint. S1 nuclease analysis of the RNA from a number of QT6-LD clones gave similar results, indicating that the LD population was composed of viruses with similar but not identical deletion endpoints. Such viruses may have been generated from errors during reverse transcription of the virion RNA with subsequent selection assuring their dominance in the population.

Avian Sarcoma Viruses↗
PubMed1985

Truncated gag-related proteins are produced by large deletion mutants of Rous sarcoma virus and form virus particles.

Large deletion (LD) mutants of Prague strain Rous sarcoma virus subgroup B (PrB), derived by serial undiluted passage through chicken (C/E) cells, contain two deletions relative to wild-type virus. One of these joins gag sequences in the p12 coding region to env sequences in region encoding gp37; the other deletion spans the src region. Analysis of the viral proteins of QT6 cell clones containing only LD proviruses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major truncated gag-related phosphoprotein of 60,000 to 66,000 daltons (P63LD). P63LD was stable, but could be cleaved in vitro to the predicted products by p15gag. A second gag-related LD protein of about 68,000 to 74,000 molecular weight (P70LD) was also found which often reacted with an anti-gp37 serum. P70LD was unstable and may represent a short-lived gag-gp37 fusion protein. Finally, immunoprecipitation indicated that particles containing P63LD were shed from QT6-LD clones. Thin section preparations of these clones viewed in an electron microscope showed enveloped budding particles of "immature" morphology. Thus, the synthesis and release of particles from infected cells does not require cleavage of the gag precursor, nor does it require the presence of p15 or (most of) p12.

Animals↗
PubMed1985

Sequences outside of the long terminal repeat determine the lymphomogenic potential of Rous-associated virus type 1.

Recombinant avian leukosis viruses have been constructed from the molecularly cloned DNAs of Rous-associated virus type 1 (RAV-1) and Rous-associated virus type 0(RAV-0). Virus encoded by the cloned RAV-1 DNA induced a high incidence of B-cell lymphoma and a moderate incidence of a variety of other neoplasms. Virus encoded by the cloned RAV-0 DNA did not cause disease. Virus recovered from DNA constructions that encoded the gag, pol, and 5' env sequences of RAV-0 and the 3' env and long terminal repeat sequences of RAV-1 did not cause a high incidence of lymphoma. Rather, these constructed viruses induced a low incidence of a variety of neoplasms. Virus recovered from reconstructed pRAV-1 DNA had the same disease potential as did virus recovered from the parental pRAV-1 DNA. These results indicate that the long terminal repeat sequences of RAV-1 do not confer the potential to induce a high incidence of B-cell lymphoma.

Animals↗
PubMed1985

Bacterial beta-galactosidase as a marker of Rous sarcoma virus gene expression and replication.

We have developed a convenient and sensitive assay of eucaryotic gene expression which uses the Escherichia coli lacZ gene product, beta-galactosidase, as a nonselectable marker. This system has been applied to the analysis of Rous sarcoma virus replication and gene expression. Avian cells were transfected with plasmids encoding in-frame gene fusions of the N-terminal portion of the gag gene to a 'lacZ gene, which requires both transcriptional and translational initiation signals; these were supplied by the virus long terminal repeat and leader region. Readily detectable quantities of beta-galactosidase were synthesized in transfected cells; it was demonstrated that the levels of enzyme activity induced in such cultures increased linearly with the input DNA concentration and also correlated with mRNA levels. By using a Rous sarcoma virus-derived vector containing the src gene and a related virus as a helper, it was shown that lac sequences were compatible with all phases of the virus life cycle. gag-lacZ fusion proteins were immunoprecipitable from cultures which stably expressed lacZ as well as src. Virus rescued from stably transfected cultures resulted in continued lac and src expression in recipient cells. One particular construction was efficiently transmitted as virus, although it lacked sequences thought to be important for encapsidation of RNA into virions. The data presented here demonstrate the use of lacZ as a marker of retrovirus gene expression and replication.

Avian Sarcoma Viruses↗
PubMed1984

Role of recombinant ecotropic and polytropic viruses in the development of spontaneous thymic lymphomas in HRS/J mice.

The biological and genetic characteristics of murine leukemia viruses (MuLV) derived from leukemic and normal HRS/J mice were studied. T1-oligonucleotide fingerprinting and mapping of viral RNAs from unpassaged isolates revealed the presence of complex mixtures of viral genomes. MuLV that were purified by endpoint dilution were genetically heterogeneous. Thus, endogenous retroviral sequences expressed in the tissues of HRS/J mice readily recombined with one another. Furthermore, the regular recovery of recombinant ecotropic MuLV suggested reciprocal in vivo complementation of a genetic defect(s) in each of the endogenous ecotropic proviruses Emv-1 and Emv-3. Some recombinant ecotropic viruses contained sequences in the p15E-U3 region that were not derived from Emv-1 and Emv-3 but were found in recombinant polytropic HRS/J viruses. Finally, comparison of the genetic structures of leukemogenic and nonleukemogenic MuLV of this strain implied that the oncogenic phenotype of these MuLV is encoded within env or the U3 region of the genome or both. Our results are consistent with a stepwise convergent evolution of recombinant MuLV in vivo in individual HRS/J mice. Ultimately, this process of selection results in formation of leukemogenic polytropic viruses.

Animals↗
PubMed1983

Genomes of endogenous and exogenous avian retroviruses.

The endogenous viruses of chickens are closely related to the exogenous avian leukosis viruses (ALV) yet as a group differ from these viruses in their host range, growth rate, and oncogenicity. The present study was undertaken to determine the patterns of relationship among the genomes of endogenous and exogenous ALVs. Complete or partial T1 oligonucleotide maps were prepared from the genomes of endogenous viruses that reside at eight distinct loci in chickens. Selected endogenous viruses and recombinants of endogenous or endogenous and exogenous viruses were characterized for host range and growth rate. From these data we could infer the following: (1) Endogenous viruses form a distinct lineage of ALVs with the most distinctive differences occurring in the portion of env that encodes host range and the U3 portion of the long terminal repeat; (2) The U3 sequences of endogenous ALVs determine the low growth rates of these viruses; and (3) Endogenous ALVs have distinctive oligonucleotide markers that allow them to be subclassified into distinct lineages. Our results suggest that endogenous viruses are derived from one another and not from exogenous field strains of ALV. This phenomenon may be related to the unique env encoded host range of endogenous ALVs, their unique U3 encoded growth rates, or perhaps their unique access, as residents of germ line DNA, to germ line cells.

Animals↗
PubMed1983

Thymic epithelial genotype influences the production of recombinant leukemogenic retroviruses in mice.

By using T1 oligonucleotide fingerprinting and mapping techniques, we analyzed the genomic structure of retroviruses produced by thymocytes and splenocytes of reciprocal bone marrow-and thymus-grafted chimeras. We found that the genetic factor(s) derived from NZB mice that suppresses the development of thymic leukemia in (AKR X NZB)F1 mice also prevents the formation of recombinant leukemogenic viruses and the expression of preleukemic changes in the (AKR X NZB)F1 thymocytes. The NZB mouse gene or genes appeared to exert this suppressive effect by acting on the thymic reticuloepithelial cells and not on the thymic lymphocytes of (AKR X NZB)F1 hybrids. Prospective studies with thymic epithelial grafts from young mice showed that the AKR thymic epithelium could mediate the formation and expression of leukemogenic recombinant viruses and preleukemic changes in thymocytes that lead to the development of thymic leukemia, whereas the (AKR X NZB)F1 thymic epithelium was deficient in this regard. Our results also confirmed a previous observation that during in vivo generation of recombinant leukemogenic viruses, the acquisition of polytropic virus-related sequences in the 3' portion of the p15E gene and the U3 region and in the 5' part of the gp70 gene can occur independently.

Animals↗
PubMed1983

Rous sarcoma virus contains sequences which permit expression of the gag gene in Escherichia coli.

Several aspects of Rous sarcoma virus gene expression, including transcription, translation, and protein processing, can occur within Escherichia coli containing cloned viral DNA. The viral long terminal repeat contains a bacterial promoter, and viral sequences at or near the authentic viral initiation codon permit the initiation of translation. These signals can direct the synthesis in E. coli of the viral gag gene precursor Pr76 or, when fused to a portion of the lacZ gene, a gag-beta-galactosidase fusion protein. Pr76 is processed into gag structural proteins in E. coli in a process which is dependent upon the gag product p15. These observations suggest that E. coli can be used for the introduction and analysis of mutations in sequences relevant to viral gene expression.

Animals↗
PubMed1982

At least two regions of the viral genome determine the oncogenic potential of avian leukosis viruses.

Recombinants of oncogenic and nononcogenic avian leukosis viruses were tested for their oncogenic potential in chickens. The results indicate that at least two regions of the viral genome determine the oncogenic potential of these viruses. The first region contains sequences that control viral mRNA synthesis. These sequences determine the potential of a virus to induce a low incidence of lymphomas, carcinomas, chondrosarcomas, fibrosarcomas, and osteopetrosis. The second region lies outside the sequences that control viral mRNAs synthesis. These sequences determine the ability of a virus to induce a high incidence of lymphomas or osteopetrosis.

Animals↗
PubMed1982

Genetic alterations of RNA leukemia viruses associated with the development of spontaneous thymic leukemia in AKR/J mice.

T1-oligonucleotide fingerprinting and mapping were used to study the expression of RNA leukemia viruses in leukemic and preleukemic AKR/J mice, with techniques designed to minimize the loss or inadvertent selection of viruses in vitro before biochemical analysis. In leukemic animals, complex mixtures of ecotropic and mink-tropic viruses were expressed. Unique but similar polytropic virus-like genomes were present in each tumor isolate. In preleukemic mice, viral isolates from the thymus that were grown on NIH3T3 fibroblasts contained genomes with non-Akv polytropic virus-related oligonucleotides. This phenomenon was not evident in fingerprints of viruses from the spleen and bone marrow of the same animals. Remarkably, the non-Akv oligonucleotides located in the 3' portion of the P15E gene, the U3 noncoding region, and the 5' part of the gp70 gene were often expressed independently. Our results suggest the following. (i) Recombinant viruses can be detected in the thymuses of young preleukemic AKR mice and increase in relative abundance with age. (ii) During in vivo generation of the recombinant leukemogenic viruses, the selection of polytropic virus-related sequences in the 3' part of p15E and the U3 region and the 5' portion of gp70 occurs independently. (iii) Independent biological properties encoded in the gp70 and p15E regions of env of the recombinant viruses may mediate viral selection or leukemogenicity. (iv) The leukemogenic polytropic viruses of AKR/J mice arise via genetic recombination involving at least three endogenous viral sequences.

Animals↗
PubMed1982

Role of methylation in the induced and spontaneous expression of the avian endogenous virus ev-1: DNA structure and gene products.

The endogenous avian provirus ev-1 is widespread in white leghorn chickens. Although it has no major structural defects, ev-1 has not been associated with any phenotype and is ordinarily expressed at a very low level. In this report, we describe a chicken embryo (Number 1836) cell culture containing both ev-1 and ev-6 which spontaneously expressed the ev-1 provirus. This culture released a high level of noninfectious virions containing a full complement of virion structural (gag) proteins but devoid of reverse transcriptase activity or antigen. These virions contained 70S RNA closely related to the genome of Rous-associated virus type 0, but identifiable as the ev-1 genome by oligonucleotide mapping. A fraction of the RNA molecules in the 70S complex were unusual in that they were polyadenylated 100 to 200 nucleotides downstream of the usual polyadenylation site. Eight sibling embryo cultures did not share this unusual phenotype with 1836, indicating that it was not inherited. However, an identical phenotype was inducible in the sibling cultures by treatment with 5-azacytidine, an inhibitor of DNA methylation, and the induced expression was stable for more than 10 generations. Analysis of chromatin structure and DNA methylation of the ev-1 provirus in 1836 cells revealed the presence (in a fraction of the proviruses) of both DNase I hypersensitive sites in the long terminal repeats and in gag and a pattern of cleavage sites for methyl-sensitive restriction endonuclease not found in a nonexpressing sibling. These results lend strong support to the role of DNA methylation in the control of gene expression. Additionally, they explain the lack of phenotype associated with ev-1 as due to a combination of its low expression and defectiveness in pol and env.

Animals↗
PubMed1980

Expression of leukemogenic recombinant viruses associated with a recessive gene in HRS/J mice.

HRS/J inbred mice carry a mutant autosomal recessive gene (hr), which in homozygotes coincides with susceptibility to spontaneous thymic leukemia. Unlike their heterozygote (hr/+) littermates, hr/hr homozygotes express high levels of xenotropic virus during the preleukemic period, and viruses with a broadened host range (termed polytropic viruses) can be isolated from their preleukemic and leukemic tissues. Because hr/hr and hr/+ mice are otherwise genetically identical, the virological differences between them support the role of polytropic viruses in the generation of thymic leukemia. In the present report we show that the HRS/J polytropic viruses are env gene recombinants with unique oligonucleotide and peptide maps. These polytropic viruses appear to arise by recombination between ecotropic virus and an unidentified genome related, but not identical to, the endogenous xenotropic viruses. Moreover, polytropic viruses not only accelerate leukemogenesis in HRS/J mice, but also induce thymic leukemia in the low leukemia strain CBA/J. By contrast, cloned ecotropic and xenotropic viruses have no leukemogenic action.

Animals↗