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Biomedical subjects

R Kettmann

Publications and source records attributed to R Kettmann.

At least 145 records · Page 8Linked to original sources

Restriction endonuclease mapping of linear unintegrated proviral DNA of bovine leukemia virus.

A detailed restriction map was deduced for the genome of the exogenous bovine leukemia virus. The cleavage sites for nine restriction enzymes were mapped. The unintegrated linear viral DNA intermediate that is produced by infection of permissive cells with bovine leukemia virus was isolated. The linear viral DNA had a unique restriction map, indicating that it is not a set of random circular permutations of the RNA genome. From hybridization with a 3'-enriched probe, the DNA restriction map was aligned relative to the 5'-to-3' orientation of the viral RNA. Restriction enzyme analysis of integrated bovine leukemia virus information present in animals with enzootic bovine leukosis provided evidence for the existence of genetic variants of the virus.

Chromosome Mapping↗

Experiments with cloned complete tumor-derived bovine leukemia virus information prove that the virus is totally exogenous to its target animal species.

Taking advantage of the existence of a unique SacI restriction site in the long terminal repeats of the integrated bovine leukemia virus proviral DNA isolated from a bovine tumor, the total viral information (about 9.2 kilobases) was cloned in the lambdoid vector lambda WES. lambda B. Use of this cloned bovine leukemia virus DNA allowed us, for the first time, to definitely rule out the existence of any endogenous bovine leukemia virus sequence in the bovine, ovine, caprine, murine, feline, chicken, or human genomes. These data prove the absence of acquired cellular information in the provirus that has given rise to a tumor.

Animals↗

Goat lymphosarcoma from bovine leukemia virus.

A goat given inoculations of sheep lymphocytes from cultures that produced bovine leukemia virus (BLV) died 8 years later with lymphosarcoma. The tumors were located in various lymph nodes, the mesentery, omentum, body wall, and retrobulbar tissues. The BLV had been cultured from lymphocytes during the first year after the goat's infection, and persisting BLV antibodies could be demonstrated when the animal was 7.5 years old. BLV provirus was identified by molecular hybridization in the DNA of the goat tumors at the above five locations. The tumors were similar to those found in lymphosarcoma of the adult bovine type (BLV associated). Normal goat liver, normal calf thymus, and calf-type lymphosarcoma (not BLV associated) served as negative controls. Our serologic, histologic, and molecular hybridization studies are evidence that the lymphosarcoma was induced BLV.

Animals↗

Genomic integration of bovine leukemia provirus: comparison of persistent lymphocytosis with lymph node tumor form of enzootic.

Integration of bovine leukemia proviral DNA in the genome of infected cells was investigated in cattle affected by either the persistent lymphocytosis or the lymph node tumor form of enzootic bovine leukosis. In persistent lymphocytosis, proviral DNA was found to be integrated at a large number of genomic sites in one-fourth to one-third of circulating leukocytes. In the lymph node tumor form, in contrast, proviral DNA was found to be integrated at one or very few sites in the genomes of a larger fraction of both circulating leukocytes and lymph node tumor cells.

Animals↗

BLV proviral DNA in the genome of the target lymphocyte.

Integration of bovine leukemia proviral DNA in the genome of infected cells was investigated in cattle affected by either the persistent lymphocytosis or the lymph node tumor form of enzootic bovine leukosis. In persistent lymphocytosis, proviral DNA was found to be integrated at a large number of genomic sites in one-fourth to one-third of circulating leucocytes. In the lymph node tumor form, in contrast, proviral DNA was found to be integrated at one or very few sites in the genomes of a larger fraction of both circulating leucocytes and lymph node tumor cells.

Animals↗

Integration of bovine leukemia virus DNA in the bovine genome.

DNA preparations from circulating leukocytes, lymph node tumors, and spleens of three bovine leukemia virus-infected cattle were fractionated by Cs2SO4/3,6-bis(acetatomercurimethyl)dioxane density gradient centrifugation. Bovine leukemia virus proviral sequences were found in large GC-rich fragments having a buoyant density in CsCl close to 1.708 g/cm3. Provirus integration, therefore, does not take place at random locations in the host genome, but in a specific class of DNA segments. Hybridization of cDNA synthesized on viral RNA to EcoRI and Xba I restriction fragments of the DNA from infected cells showed that: (i) only one copy of proviral DNA is integrated per haploid genome; (ii) different restriction patterns were found in the proviral DNAs present in the genomes of different animals, providing evidence for the existence of several strains or mutants; and (iii) different integration sites for the proviral DNA were found in the genome of different animals and of different infected cells in the same animal. The latter finding strongly suggests a polyclonal origin of bovine leukemia virus-infected cells.

Animals↗

Translation of bovine leukemia virus virion RNAs in heterologous protein-synthesizing systems.

Bovine leukemia virus 60 to 70S RNA was heat denatured, the polyadenylic acid-containing species were separated by velocity sedimentation, and several size classes were translated in a micrococcal nuclease-treated cell-free system from rabbit reticulocytes. The major RNA species sedimented at 38S and migrated as a single component of molecular weight 2.95 x 10(6) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The predominant polypeptides of the in vitro translation of bovine leukemia virus 38S RNA were products with molecular weights of 70,000 and 45,000; minor components with molecular weights of 145,000 and 18,000 were also observed. Two lines of evidence indicate that the 70,000- and 45,000-molecular weight polypeptides represent translation products of the gag gene of the bovine leukemia virus genome (Pr70gag and Pr45gag). First, they are specifically precipitated by a monospecific antiserum to the major internal protein, p24, and second, they are synthesized and correctly processed into virion proteins p24, p15, and p10 in Xenopus laevis oocytes microinjected with bovine leukemia virus 38S RNA. The 145,000-molecular weight polypeptide was immunoprecipitated by the anti-p24 serum and not by an antiserum to the major envelope glycoprotein, gp60. It contained all the tryptic peptides of Pr70gag and additional peptides unique to it, and thus represents in elongation product of Pr70gag in an adjacent gene, presumably the pol gene. The 18,000-molecular weight product was antigenically unrelated to p24 and gp60 and shared no peptides in common with Pr70gag, Pr45gag, or the 145,000-molecular weight polypeptide. It was maximally synthesized on a polyadenylic acid-containing virion 16 to 18S RNA, and we present evidence that this RNA is a 3' end-derived subgenomic fragment of the bovine leukemia virus genome rather than a contaminating cellular RNA.

Cell-Free System↗

Reverse transcription of turnip yellow mosaic virus RNA primed with calf-thymus DNA hydrolysate: characterization of the purified cDNA product.

Complementary DNA was transcribed from turnip yellow mosaic virus RNA, using the method of Taylor et al. (1). The purified cDNA thus obtained sedimented between 2 and 4 S and was a mostly uniform transcript of template RNA. It hybridized with a sharp transition to homologous TYMV-RNA (Crt 1/2 = 2.7 x 10(-2)), but showed a low level of hybridization (less than 5%) to the RNAs of two other tymoviruses, namely Andean potato latent virus and eggplant mosaic virus.

Avian Myeloblastosis Virus↗

Distribution of bovine leukemia virus proviral sequences in tissues of bovine, ovine and human origin.

Bovine leukemia virus (BLV) single-stranded cDNA was used to study the distribution of DNA sequences in tissues (normal or malignant) from bovine, ovine and human origin. After recycling against normal bovine DNA, BLV (3H) cDNA hybridized with bovine enzootic tumor DNA but did not hybridize with normal bovine DNA. These results indicate that BLV is an exogenous RNA oncogenic virus and confirm that enzootic bovine leukosis (EBL) is an infectious disease. Proviral BLV sequences were also detected in buffy coat cells of animals in persistent lymphocytosis (PL) and carrying antibodies to BLV but no tumors. In animals at the tumor stage of EBL, the proviral sequences were found in buffy coat cells, in solid tumors (lymphosarcomas) and in organs infiltrated with tumoral lymphoid cells but not in apparently normal organs. No hybridization above background was observed between BLV (3H) cDNA and DNAs extracted from buffy coat cells and tumors corresponding to sporadic forms of bovine leukosis and some human leukemias and sarcomas.

Animals↗

Differences in mouse mammary tumor viruses. Relationship to early and late occurring mammary tumors.

The murine model has been used extensively to study the various factors involved in the etiology of mammary carcinoma. Inbred mouse strains have been classically categorized into (i) high incidence stains with tumors occurring relatively early in the life of the animal, or (ii) low or moderate incidence strains with tumors occurring later on in life. We have radioactively labeled the RNA genome of the mouse mammary tumor virus (MMTV) from each of several mouse strains. We report here, using the technique of molecular hybridization, that the class of MMTVs responsible for the early occurring mammary tumors in high incidence strains can be clearly distinguished from the MMTVs associated with late occurring mammary tumors in low or moderate incidence strains; we also demonstrate that minor differences in MMTV genomes can also exist within these classes. Our findings show that MMTVs are transmitted via the germ line (as a germinal provirus) in some mouse strains, whereas in other strains, a non-germ line transmission is clearly demonstrated. Biochemical techniques can thus be used to track the mode of transmission of oncogenic viruses. The relationship of these findings to an understanding of the etiology of mammary carcinoma is discussed.

Animals↗