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Biomedical subjects

R Kettmann

Publications and source records attributed to R Kettmann.

At least 55 records · Page 3Linked to original sources
PubMed1996

The CREB, ATF-1, and ATF-2 transcription factors from bovine leukemia virus-infected B lymphocytes activate viral expression.

Efficient transcription and replication of the bovine leukemia virus (BLV) genome require both the viral long terminal repeat (LTR) and the virus-coded transcriptional activator Tax, which functions through a 21-bp sequence (Tax-responsive element [TxRE]) which is repeated three times within the LTR. Since Tax does not bind directly to DNA, host cell transcription factors play a central role in BLV expression. Electrophoretic mobility shift assays with nuclear extracts prepared with infected bovine B lymphocytes revealed five TxRE-specific complexes (C1, C2, C3, C4, and C5). Here, by using a UV-induced indirect labeling technique (UV cross-linking) in conjunction with mobility shift assays, eight major polypeptides of 31, 33, 42, 46, 51, 57, 87, and 119 kDa were identified within these five complexes. Immunoprecipitation experiments identified the 57- and 119-kDa proteins as cyclic AMP response element-binding (CREB) proteins, the 46- and 51-kDa proteins as activating transcription factor-1 (ATF-1), and the 87-kDa as protein ATF-2. All of these proteins (except the ATF-1 protein of 51 kDa) belong to the complex C1, which is the major complex identified in freshly isolated BLV-infected lymphocytes from cattle with persistent lymphocytosis. In transient-cotransfection experiments, these three transcription factors were able to activate LTR-directed gene expression in the presence of protein kinase A or Ca2+/calmodulin-dependent protein kinase IV. CREB protein, ATF-1, and ATF-2 thus appear to be the major transcription factors involved in the early stages of viral expression.

Activating Transcription Factor 1↗
PubMed1996

Cellular pathways involved in the ex vivo expression of bovine leukemia virus.

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. The virus adopts a strategy based on the lack of viral expression in vivo; only very rare BLV-infected B lymphocytes express viral information. When the cells are isolated from animals in persistent lymphocytosis and cultivated ex vivo, a tremendous increase in viral expression occurs. To gain insight into this mechanism, we employed a general approach using chemicals that interfere specifically with cellular pathways involved in signal transduction from the cell membrane to the nucleus. Our data demonstrate that BLV expression is not correlated with the activity of protein kinase A (PKA) and is even inhibited by cyclic AMP (cAMP). The cAMP/PKA pathway is thus apparently not involved in ex vivo viral expression. In contrast, PKC appears to play a key role in this process. Phorbol myristate acetate can directly activate viral expression in B cells (in the absence of T cells). Furthermore, calphostin C, a highly specific inhibitor of PKC, partly decreases ex vivo BLV expression. Our data further demonstrate that calmodulin and calcineurin, a calmodulin-dependent phosphatase, play a key role in the induction of viral expression. The involvement of this calmodulin-dependent pathway could explain the induction of expression that cannot be assigned to PKC. Furthermore, it appears that the activation of viral expression requires a calmodulin but not a PKA-dependent pathway. These data highlight major differences between transient transfection and ex vivo experiments. Finally, despite their homologies, BLV and human T-cell leukemia virus appear to use different signal transduction pathways to induce viral expression.

Animals↗
PubMed1995

Mutations in the p53 tumor-suppressor gene are frequently associated with bovine leukemia virus-induced leukemogenesis in cattle but not in sheep.

To investigate the mechanisms of bovine leukemia virus (BLV)-induced leukemogenesis, we have examined the alterations of the p53 tumor-suppressor gene in sheep and in cattle. The sequences of the open reading frames as well as the intron/exon junctions of the ovine and bovine p53 genes were determined. Pathological samples were screened for the presence of p53 mutations using a single-strand conformational polymorphism assay. Five of ten BLV-induced bovine tumors harbored p53 mutations. In contrast, only one of seven samples corresponding to circulating leukocytes from cattle in persistent lymphocytosis showed an alteration of the p53 gene. Surprisingly, no p53 mutation was found among the 10 BLV-induced sheep tumors analyzed. Altogether, these data indicate that p53 mutations are linked to BLV-induced leukemogenesis in cattle at the transition to the lymphomic stage. These results also enlighten different molecular mechanisms involved in sheep and in cattle during BLV-induced pathogenesis.

Amino Acid Sequence↗
PubMed1995

Lack of LTR and ENV genetic variation during bovine leukemia virus-induced leukemogenesis.

Genetic variation of the Bovine Leukemia Virus (BLV) appears to be limited in vitro and during the latent phase of the disease. However, cells in tumors often harbor deleted proviruses that are defective for expression. In order to gain insight into the involvement of viral genetic variation during pathogenesis, the BLV LTR and the env proviral sequences were analyzed in tumor tissues. A sheep (M230) was injected with the cloned BLV provirus 344 and became persistently infected with circulating lymphocytes reaching 345,000/mm3. After 11 months, this infected sheep developed leukemia-lymphoma. DNA was extracted from peripheral blood leukocytes at the time of tumor development and the LTR and the env gene were amplified, using the polymerase chain reaction procedure, cloned, and sequenced. Twenty independent LTR and twenty env clones were analyzed. It appeared that the in vivo mutation rate in the env gene was 0.043% (eight mutations including seven transitions out of 18,300 bp). Five point mutations (all transitions) were identified in the LTR, corresponding to 0.041% modifications (four mutations out of 9740 bp). These mutation rate values (0.043 and 0.041) were close to those due to the Taq DNA polymerase errors (0.030%). Altogether, these data demonstrate the lack of genetic variation in the LTR and the env gene during this case of BLV-induced pathogenesis in vivo. They confirm that the defectiveness of some BLV proviruses in vivo, thus, is not a mandatory step in the leukemogenic process.

Animals↗
PubMed1995

The YXXL signalling motifs of the bovine leukemia virus transmembrane protein are required for in vivo infection and maintenance of high viral loads.

The bovine leukemia virus (BLV) transmembrane protein (gp30) contains three YXXL motifs at its carboxyterminal end. Two of these motifs have been implicated in vitro in signal transduction pathways from the external to the intracellular compartment. In order to analyze the biological relevance of these motifs in vivo, recombinant BLV proviruses were constructed. A mutation of the tyrosine residue of the second YXXL motif completely destroyed the infectious potential of the virus in sheep. In contrast, the tyrosine of the first motif appeared to be dispensable for infectivity. However, the propagation of the recombinant virus within the animal was greatly impaired (as demonstrated by PCR and enzyme-linked immunosorbent assay). These recombinant BLVs thus exhibit an attenuated phenotype. Altogether, our data demonstrate the importance of the YXXL motifs of the BLV transmembrane protein for in vivo infection and viral propagation.

Animals↗
PubMed1995

Chromosomal localization of the BLV receptor candidate gene in cattle, sheep, and goat.

The BLV receptor candidate gene has been localized precisely on cattle, goat, and sheep chromosomes using nonradioactive in situ hybridization and simultaneous fluorescent R-banding. The probe, a whole plasmid containing a 2.3-kb bovine cDNA fragment encoding part of this gene, was nick-translated in the presence of biotin-11-dUTP. It hybridized on band q15 of cattle chromosome 7, goat chromosome 7, and sheep chromosome 5, thus confirming the homoeology based on banding patterns among the chromosomes of these three species.

Animals↗
PubMed1995

Nucleotide sequence of the ovine P53 tumor-suppressor cDNA and its genomic organization.

A 2155 bp cDNA clone corresponding to the ovine p53 tumor suppressor gene has been isolated from a cDNA library made from the BLV-transformed YR2 cell line RNA. After the sequencing analysis, it appeared that this clone contains the entire p53 coding region (including 126 bp upstream of the ATG initiation codon). The nucleotide sequence shows a high degree of homology with the human (73%), murine (74%) and rat (66%) cDNAs. The encoded ovine p53 protein is 382 amino acids long with an apparent molecular weight of 50 kDa and shares 79% and 72% amino acid homology with the human and the mouse p53 respectively. Furthermore, the homology is not equally distributed along the molecule but is mainly located within five highly conserved regions. As its mouse and human counterparts, the ovine p53 contains a high proportion of proline residues, an acidic N-terminal domain and a basic C-terminal domain. We also report the structure of the ovine p53 gene that is similar to those already defined from other species.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence of the bovine P53 tumor-suppressor cDNA.

The bovine p53 open reading frame was cloned and characterized using a reverse transcription-polymerase chain reaction amplification (RT-PCR) method. After sequencing, it appeared that this cDNA is able to code for a 386 amino acids protein with high degree of homology with the ovine p53. Furthermore, the homology is not equally distributed along the molecule since the highest divergence is located within the exon 4. The bovine p53 shares 93% and 91% homology with the ovine p53 at the DNA and protein level respectively.

Amino Acid Sequence↗
PubMed1994

Structure of the gene encoding pig phosphoglucose isomerase.

Genomic clones encoding pig phosphoglucose isomerase (PGI) have been isolated and partially sequenced. The gene (Pgi) contains 18 exons, 17 introns and spans about 32 kb. This structure is partially conserved between plant and animal. A major transcription start point (tsp) has been identified 74 nucleotides (nt) upstream from the AUG. The nt sequence around the tsp is very G+C rich; a 5'-ATAAA sequence, as well as four putative Sp1-binding sites, are present. In the 3'-flanking region, an AATAAA signal has been identified. Extending from the 5'-flanking region to the first intron, a 0.6-kb CpG island has been identified. The Pgi structural gene contains several DNA repetitive elements in its non-coding regions.

Alternative Splicing↗
PubMed1994

Attenuation of bovine leukemia virus by deletion of R3 and G4 open reading frames.

Complex oncoviruses contain, in addition to the classical retroviral genes (gag, pol, and env), a region (X) located between the envelope sequences and the 3' long terminal repeat. The X region contains two genes, tax and rex, whose protein products are involved in transcriptional and posttranscriptional regulation of viral expression. In addition to these activators, the bovine leukemia virus (BLV) and the human T-cell leukemia virus (HTLV) contain alternative open reading frames (R3 and G4 for BLV; p30, p13, and p12 for HTLV). As a virus/animal model for HTLV-induced leukemogenesis, BLV provirus can be injected intradermally into sheep, where it induced B-lymphocyte transformation. Deletion of the R3 and G4 sequences from an infectious and tumorigenic BLV provirus greatly impaired the in vivo propagation of the viruses as demonstrated by DNA polymerase chain reaction, RNA blots, structural-protein ELISA, and immunofluorescence analysis. Our results show that the alternative open reading frames are required for maintaining high virus loads during the course of persistent infection in vivo. Thus, R3 and G4 are candidates for antiviral drug development. Furthermore, viruses with a deletion in these sequences should be tested as live attenuated vaccines.

Animals↗
PubMed1994

Isolation of the missing 5'-end of the encoding region of the bovine leukemia virus cell receptor gene.

The missing 5'-end of the encoding region of the bovine leukemia virus (BLV) cell receptor gene (BLVRcp1/5') was isolated from a lambda gt11 cDNA library using the 32P-labeled EcoRI-SamI fragment corresponding to the 5'-end of a 2.3 kbp cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor gene (BLVRcp1). The nucleotide and amino acid sequence analysis of the BLVRcp1/5' cDNA revealed that the 1058 bp EcoRI fragment at its 5'-end contained a new 114 amino acid long sequence, and at its 3'-end contained a completely identical 88 amino acid overlapping region with the 5'-end of the BLVRcp1 cDNA. The combined sequences of both cDNAs represent the whole encoding region of the BLV cell receptor gene. The longest open reading frame of the BLV cell receptor gene encodes a protein containing 843 amino acids with a calculated molecular mass of 94.2 kDa which concurs with experimentally detected native BLV receptor protein. Search for homology has shown that about 250 bp of the BLV cell receptor gene is highly homologous to Venter's tag sequences of an unidentified gene from the human brain library.

Amino Acid Sequence↗
PubMed1994

Expression of interleukin 6 receptors and interleukin 6 mRNA by bovine leukaemia virus-induced tumour cells.

Bovine leukaemia virus (BLV) is the aetiologic agent of bovine leucosis. The virus induces malignancies of the B-cell lineage (leukaemia/lymphoma). The role played by interleukin 6 (IL-6) in the BLV-induced leukemogenesis process was evaluated. Six cell lines derived from BLV-induced tumours were tested for the expression of IL-6 receptors. Two cell lines (LB155 and YR2) display 250-300 receptor per cell (kd = 1.7 10(-10) M and 1.4 10(-10) M, respectively) whereas the other four (LB159, LB167, YR1 and M51) do not display detectable amounts of receptors. Very low (if any) expression of IL-6 receptors has been found in the case of the B lymphocytes of animals in persistent lymphocytosis (PL). Despite the presence of IL-6 receptors on the surface of LB155 and YR2 cells, no influence of exogenous IL-6 on their growth has been observed. Northern analyses indicated the presence of IL-6 transcripts only in the case of mRNA isolated from LB155 cells. Since this cell line also expresses receptors for the cytokine, an autocrine loop may exist in these cells. Experiments in which bovine and bovine epithelial cell lines were transfected with a plasmid containing the bovine IL-6 promoter controlling the expression of the reporter cat gene failed to indicate any influence of the viral transactivator p34tax on the activity of this promoter. We conclude that IL-6 receptors and IL-6 mRNA can be found in some BLV-induced tumours, but this does not correlate with viral expression in BLV-induced leukaemia/lymphoma.

Animals↗
PubMed1994

Involvement of the cyclic AMP-responsive element binding protein in bovine leukemia virus expression in vivo.

The TAR element (Tax-responsive element; also called TxRE) is a major determinant of the regulation of bovine leukemia virus (BLV) expression. In order to gain insight into the mechanisms of viral expression, complexes formed between proteins and the TAR enhancer DNA were analyzed by gel retardation assays. We report here that nuclear lysates from ex vivo-isolated B lymphocytes contain proteins that specifically bind to TAR. An antibody directed toward the cyclic AMP-responsive element binding (CREB) protein supershifted a complex (C1) present only in BLV-infected B lymphocytes. The CREB protein thus appears to be a major transcription factor involved in BLV expression in vivo.

Animals↗
PubMed1994

Nucleotide sequence of ovine thioredoxin cDNA.

We report the cloning of an ovine thioredoxin cDNA. The clone was isolated from a bovine leukemia virus-infected cell line (FLK) cDNA library cloned in the lambda gt11 vector. The clone encodes the full length thioredoxin protein made of 105 amino acids with 92 and 83% identity to published sequences of human and mouse thioredoxin, respectively.

Amino Acid Sequence↗
PubMed1993

Developmental and circadian pattern of rubisco activase mRNA accumulation in apple plants.

An apple cDNA encoding the precursor of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase has been characterized. Using this cDNA as a probe, leaf-specific and light-regulated accumulation of corresponding transcripts was detected. Rubisco activase transcripts also turned out to accumulate at growing levels during apple leaf development, to reach a maximum in fully expanded leaves. In contrast, chlorophyll a/b-binding protein (Cab) and rubisco large subunit mRNA levels reach a maximum earlier in the course of leaf development. Moreover, the accumulation of rubisco activase messengers appeared to follow an oscillating circadian rhythm qualitatively similar to that observed for Cab mRNA levels.

Amino Acid Sequence↗
PubMed1993

Cloning and characterization of the tandemly arranged bovine lymphotoxin and tumour necrosis factor-alpha genes.

The screening of a bovine genomic library with a human tumour necrosis factor-alpha (TNF-alpha) cDNA probe resulted in the isolation of a 7.2 kb DNA fragment containing the entire bovine TNF-alpha gene. Analysis of this genomic clone showed that it also contains the bovine lymphotoxin (LT, TNF-beta) gene. Comparison to published sequences of human, murine, ovine and rabbit counterparts allowed us to delineate the coding sequences, the promoters and the enhancers of these two genes. Sequences involved in the regulation of translation and in the mRNA stability were found in the 3' untranslated regions.

Amino Acid Sequence↗
PubMed1993

A calcium/calmodulin-binding serine/threonine protein kinase homologous to the mammalian type II calcium/calmodulin-dependent protein kinase is expressed in plant cells.

cDNA fragments corresponding to an apple (Malus domestica [L.] Borkh) calmodulin-binding polypeptide have been isolated and characterized. The protein encoded by this messenger contains a serine/threonine protein kinase catalytic domain followed by a calcium/calmodulin-binding regulatory domain, both exhibiting significant sequence similarities to the corresponding regions of the mammalian calcium/calmodulin-dependent protein kinase II subunits. These results confirm a potential regulatory role for calmodulin in phosphorylation-mediated signal transduction events.

Amino Acid Sequence↗