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Biomedical subjects

R Kettmann

Publications and source records attributed to R Kettmann.

At least 127 records · Page 7Linked to original sources

Transfection of BLV containing DNA into NIH 3T3 cells.

Cell DNA isolated from bovine leukosis virus (BLV) productive cell clones was transfected into the NIH3T3 cells. DNA from some cell clones was able to transform NIH3T3 cells. The transformed cells were cloned, and in 4 cell clones out of 33 bovine leukosis virus specific sequences were detected by hybridization with labeled BLV probe. According to the restriction analysis the BLV sequences were incomplete, they were rearranged, deleted, or both. The DNA from NIH3T3 transformants with BLV sequences was able to transform in the second round transfection experiments NIH3T3 cells again, but in these transformants BLV specific sequences were not detected. Cell DNA from sheep tumors induced by BLV was able to transform the NIH3T3 cells too, but BLV specific sequences were not present in the transformants. It appears that BLV specific sequences are not required for NIH3T3 cell transformation.

Animals↗

Bovine leukaemia virus and enzootic bovine leukosis.

Infection of bovines with bovine leukaemia virus (BLV) manifests itself in either of two ways: 30-70% of carriers develop persistent lymphocytosis (PL), with the viral genome integrated at a large number of different sites in the DNA of the affected B-lymphocytes, without causing any chromosomal abnormalities. Only 0,1-10% of carriers develop lymphoid tumours, which also consist of B-lymphocytes. In contrast to PL, however, they are of mono- or oligoclonal origin in terms of the integration site, which is characteristic for each tumour. All cells contain one or more copies of the viral genome, chromosomal aberrations are common and if deletions are present they are invariably found in the 5'-half of the virus DNA sequence. In both types of affected cells transcription is repressed in vivo, but transient virus production can be induced in vitro and detected by means of syncytia induction or haemagglutination. In vivo production of virus in some unknown cell is suggested by the presence of high antibody titres in infected animals, especially against the envelope glycoprotein gp51. This can be detected by various techniques such as immunodiffusion, radioimmune assay or ELISA. Monoclonal antibodies against gp51 have revealed 8 epitopes, 3 of which are recognized by neutralizing antibodies and one by a cytolytic antibody. The BLV genome, about 9 kb in size, have been cloned, and some of the information obtained on its molecular structure and function is discussed. It codes for at least 4 non-glycosylated and 2 glycoproteins. Of special interest is the recently discovered serological relationship between some of the non-glycosylated proteins and those of the human T-cell leukaemia virus. The functional role of BLV in leukaemogenesis is largely unknown. The presence of the viral genome seems to be necessary for the maintenance of the transformed state, but not its continuous expression nor an LTR-mediated promotion of transcription of cellular genes. No oncogene is carried by the virus. Although bovine leukosis is not of major economic importance, its eradication is desirable and feasible in countries with a relatively low incidence, by means of testing and elimination. For endemic situations vaccination would be preferable, and distinct possibilities exist for the development of gp51 based vaccines.

Animals↗

The nucleotide sequence of the env gene and post-env region of bovine leukemia virus.

The env gene of a bovine leukemia virus (BLV) tumor-derived proviral DNA clone has been located by comparison of the translated DNA sequence with amino acid sequence data on purified gp60 and p30env (A. M. Schultz, T. D. Copeland, and S. Oroszlan (1984) Virology 135, 417-427). There is a continuous open reading frame from the N terminus of gp60 for 1446 nucleotides; gp60 is predicted to contain 268 amino acids and p30env, 214. The predicted p30env shows structural features typical of type C viral transmembrane proteins. It is also clearly related to that of the human T-cell leukemia virus (HTLV), as predicted from the DNA sequence of Seiki et al. (M. Seiki, S. Hattori, Y. Hirayama, and M. Yoshida (1983) Proc. Natl. Acad. Sci. USA 80, 3618-3622) The two proteins show 36% identities in their amino acid sequence, in an alignment requiring six gaps. More distant relatedness is also seen between BLV p30env and both murine leukemia virus p15E and Rous sarcoma virus gp36. The gp60s of BLV and HTLV are more distantly related than their p30envs, but their homology is nonetheless statistically significant. Between the presumptive terminator of the env gene and the beginning of the 3'-long terminal repeat is a region of 1817 base pairs of unknown function. Just as in the HTLV post-envelope sequence, there are at least two reading frames which are open for a significant fraction of this region. In neither the tumor-derived clone nor a clone from a virus-producing cell line, however, is there a continuous open reading frame throughout the region. Comparison of the BLV and HTLV sequences within the post-envelope region revealed a very limited but possibly significant similarity.

Amino Acid Sequence↗

Experimental infection of sheep and goat with bovine leukemia virus: localization of proviral information on the target cells.

Bovine leukemia virus (BLV) proviral integration was studied in the DNA from circulating leucocytes or tumor cells of sheep and goats experimentally infected with BLV. Southern blot analysis of infected cell DNA for BLV proviral sequences indicate that: (1) the provirus may be found as unintegrated molecules in the circulating leucocytes of infected sheep; (2) the provirus is integrated at many sites in the genome of the leucocytes of infected goats and occasionally in infected sheep; (3) the provirus is present at only a few sites in the DNA of sheep or goat tumor cell clones. A second case of goat lymphosarcoma is also reported.

Animals↗

Nucleotide sequence analysis of the long terminal repeat of integrated bovine leukemia provirus DNA and of adjacent viral and host sequences.

The nucleotide sequence of the 3' long terminal repeat and adjacent viral and host sequences was determined for a bovine leukemia provirus cloned from a bovine tumor. The long terminal repeat was found to comprise 535 nucleotides and to harbor at both ends an imperfect inverted repeat of 7 bases. Promoter-like sequences (Hogness box and CAT box), an mRNA capping site, and a core enhancer-related sequence were tentatively located. No kinship was detected between this bovine leukemia proviral fragment and other retroviral long terminal repeats, including that of human T-cell leukemia virus.

Animals↗

Different bovine leukemia virus-induced tumors harbor the provirus in different chromosomes.

DNAs from 25 independent hamster X bovine somatic cell hybrids were submitted to the Southern blot analysis with probes made of unique cellular fragments adjacent to single-copy proviruses from three different bovine tumors. It appeared that these cellular sequences, and thus the respective proviruses, belonged to three different chromosomes in the three different tumors examined.

Animals↗

Characterization of cellular sequences flanking an integrated bovine leukemia virus genome.

We cloned a 20.2-kilobase fragment, including the unique proviral integration site, from the tumor tissue DNA of a bovine leukemia virus-induced lymphoma (tumor 1351). This clone contained 5.5 kilobases of flanking sequences at either side of the full-length integrated viral genome. Hybridization analysis of both 3' and 5' cellular flanking regions provided no evidence for rearrangements at this locus in other tumors, RNA transcription from either flanking region, or homology to eight known onc genes.

Animals↗

Bovine leukemia virus, a distinguished member of the human T-lymphotropic virus family.

Bovine leukemia virus (BLV) has many structural and functional characteristics in common with the human T-lymphotropic viruses (HTLVs). The most striking feature of these retroviruses is the existence of a long open reading frame (LOR) located at the 3' side of the provirus between the right end of the 3' side of env gene and the left end of the long terminal repeat (LTR). It is believed that the LOR protein product is of critical importance in the induction process of the tumor phase of bovine leukemia. Prevention of BLV infection will be attempted by vaccination. To that aim, careful study of BLV envelope glycoprotein epitopes has shown that epitopes F, G, and H play a major role in biological activities of the virus. Their native structure depends upon glycosylation of the peptide backbone.

Animals↗

[Oncogenes].

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Animals↗

Chromosome integration domain for bovine leukemia provirus in tumors.

The 3'-end host-virus junction fragments from two bovine leukemia virus (BLV)-induced lymphoid tumors (tumors 15-4 and 1351), each containing a single provirus, were used as probes to detect large restriction fragments flanking these proviruses. The DNAs from 28 other independent BLV-induced tumors were checked by Southern analysis of their restriction fragments for possible rearrangement due to the insertion of a BLV provirus in the cellular sequences corresponding to those flanking the proviruses in tumors 15-4 and 1351. In no case did proviral integration occur in cellular sequences corresponding to those implicated in the tumors of origin. According to the statistical analysis performed, if a preferential domain for BLV integration exists, it has a size of 1,304 kilobases when the probability of not observing an integration event in the cellular fragments considered in tumors 15-4 and 1351 is 0.50.

Animals↗

Leukemogenesis by bovine leukemia virus: proviral DNA integration and lack of RNA expression of viral long terminal repeat and 3' proximate cellular sequences.

The DNA from 17 lymphoid tumors induced by bovine leukemia virus (BLV) was digested with the restriction endonuclease EcoRI. Filter hybridization analysis using radioactive probes specific for the BLV genome showed that all tumors contained at least one or a portion of one provirus. Digestion of these proviruses with Sac I demonstrated that deletions occurred in about 25% of the cases and involved sequences located in the 5' half of the provirus. No sequence homology was observed between the cloned proximate cellular sequences flanking two different proviruses at their 3' end and the corresponding sequences in 16 other tumor DNAs, thus showing that a wide range of genomic sites could accommodate BLV proviruses. Transcription of viral DNA including long terminal repeated sequences was not detected, strongly suggesting that viral gene expression is not required for maintenance of the tumor state. No expression of 3'-proximate cellular sequences was observed, indicating that no proximate downstream promotion took place in the cases examined.

Animals↗

Viral RNA content of bovine leukemia virus-infected cells.

A bovine leukemia virus (BLV)-producing cell line, fetal lamb kidney cells infected with BLV (FLK) contains one or a few copies of BLV proviral DNA in its genome. These cells contain 0.002% of viral RNA which sediments, in a sucrose gradient, at about 35S and between 18S and 28S. In cattle affected by enzootic bovine leukosis, tumor cells and circulating lymphocytes also contain one or a few copies of BLV proviral DNA integrated in their genome. However, in all cases tested (except one), no viral RNA was detected in these cells in conditions where one or two copies of viral genomic RNA per cell would have been easily detected.

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