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R Kettmann

Publications and source records attributed to R Kettmann.

At least 19 recordsLinked to original sources
PubMed1997

Knotted1-like homeobox genes are expressed during apple tree (Malus domestica [L.] Borkh) growth and development.

Apple (Malus domestica [L.] Borkh) cDNA clones encoding two distinct but very similar kn1-like homeobox gene class 1 homologues (KNAP1 and KNAP2) were isolated using a fragment amplified with degenerated primers as a probe. A fragment corresponding to the homeodomain region of KNAP1 was used to isolate a cDNA (KNAP3) belonging to the kn1-like homeobox gene class 2. These probes were used to detect corresponding gene copies in apple genomic DNA digests, together with other putative members of an apple kn1-like homeobox gene family. The kn1-like homeobox gene probes were also used to detect corresponding messenger accumulation levels in various organs of an apple tree. Transcripts corresponding to KNAP1 and KNAP2 genes appeared to be absent from leaves or floral organs, but they accumulate in tissue samples from elongated parts of the stem. In contrast, KNAP3 mRNAs accumulate at detectable levels in a wider range of both vegetative and reproductive organs.

Amino Acid Sequence↗
PubMed1997

The major homology region of bovine leukaemia virus p24gag is required for virus infectivity in vivo.

In order to gain insight into the role of the major homology region (MHR) in the infectious potential of bovine leukaemia virus (BLV), mutations were introduced into the capsid gene of an infectious molecular clone. A provirus that was designed to contain only a slightly modified version of the MHR (substitution of phenylalanine 147 with a tyrosine) was still infectious in vivo. Furthermore, the provirus loads were not significantly different from those obtained with a wild-type virus. A second mutant was designed to analyse a mild modification of the MHR at the level of arginine 150. The substitution of this residue with a lysine completely destroyed the infectious potential of the recombinant virus. Finally, a third mutant that was deleted in the MHR region was unable to infect the host. Thus it appears that the integrity of the MHR domain is essential for BLV infectivity in vivo.

Amino Acid Sequence↗
PubMed1997

Both wild-type and strongly attenuated bovine leukemia viruses protect peripheral blood mononuclear cells from apoptosis.

Bovine leukemia virus (BLV) and the human T-cell leukemia viruses belong to the same subfamily of oncoviruses. Although much attention has focused on the mechanisms of cell proliferation and transformation by these viruses, experiments on the apoptotic process have yielded conflicting data in in vitro cell culture. Experimental infection of sheep with BLV proviruses offers the opportunity to analyze apoptosis in vivo. Here, we show that BLV-infected peripheral mononuclear cells, cultivated ex vivo, are protected from spontaneous programmed cell death. Moreover, the virus is able to specifically interfere with the apoptotic program of infected B lymphocytes. Strongly attenuated mutant proviruses that harbor deletions in the G4 and/or R3 genes also decrease the global susceptibility to apoptosis at levels similar to those obtained with the wild-type virus. In addition, cell culture supernatants from wild-type and mutant viruses can prevent uninfected cells from undergoing programmed cell death. These observations demonstrate that the R3 and G4 genes are not required to maintain both direct and indirect protection against apoptosis. They also imply that the level of programmed cell death observed ex vivo is independent of the amounts of proviruses in the animals. The failure of these cells to undergo apoptosis might be related to the pathogenesis induced by BLV.

Animals↗
PubMed1997

Polyclonal bovine sera but not virus-neutralizing monoclonal antibodies block bovine leukemia virus (BLV) gp51 binding to recombinant BLV receptor BLVRcp1.

Bovine leukemia virus (BLV), a transactivating lymphotropic retrovirus, is the etiologic agent of enzootic lymphosarcoma or leukemia in cattle. Sera from BLV-infected animals possess high BLV-neutralizing antibody titres. The availability of the recombinant BLV receptor candidate, BLVRcp1, allowed us to determine a mechanism of virus neutralization by polyclonal sera and monoclonal antibodies (MAbs). Bovine sera from animals naturally infected with BLV blocked gp51 binding to recombinant BLVRcp1. In contrast, virus-neutralizing MAbs specific for gp51 F, G, and H epitopes did not prevent gp51-receptor attachment. Furthermore, gp51 neutralization epitopes F, G, and H were accessible to antibodies following gp51 attachment to BLVRcp1. This finding implies that virus neutralization by MAbs to defined BLV gp51 epitopes can occur subsequent to virus engagement of the receptor while polyclonal sera can specifically block virus attachment to the receptor. In conclusion, these data suggest that cell infection by BLV is a multistep process requiring receptor binding (inhibited by polyclonal sera) followed by a second, postbinding event(s) at the cell membrane (inhibited by anti-gp51 MAbs).

3T3 Cells↗
PubMed1997

The cloning and sequencing of an ovine c-myc cDNA.

We report the cloning of an ovine c-myc cDNA. The clone was isolated from a bovine leukemia virus-infected cell line (YR2) cDNA library cloned in the lambda gt10 vector. The clone encodes the full length c-Myc protein made of 439 amino-acids with 93, 96, 92 and 93% similarity with human, feline, murine and rat c-Myc proteins, respectively.

Amino Acid Sequence↗
PubMed1996

Modulation of CD3-gamma gene expression after HIV type 1 infection of the WE17/10 T cell line is progressive and occurs in concert with decreased production of viral p24 antigen.

HIV-1 infection of WE17/10, an IL-2-dependent CD4+ human T cell line, abrogates T cell receptor (TCR)/CD3 expression due to a transcription level defect in the CD3-gamma chain gene. Kinetic examination of surface receptor density reveals that these complexes are progressively reduced early after HIV-1 infection as the cells transition from TCR/CD3hi-->TCR/CD3lo-->TCR/CD3-. The passage from TCR/CD3hi reversible TCR/CD3lo is characterized by a steady decrease in receptor density from 100 to 50% of control values with similar kinetic for all of the viral variants tested. This first phase in TCR/CD3 downmodulation was found to occur in concert with a decrease in viral p24 antigen production. The switch from TCR/CD3- is distinguished by the conversion of individual cells to the receptor negative phenotype. Although broad kinetic differences in this second phase were observed between viral variants, its onset was consistently accompanied by a further reduction in virus production. In some of the HIV-1-infected WE17/10 cell lines, surface receptor expression was spontaneously upregulated during the second phase of infection, reversing the progression from TCR/CD3(-)-->TCR/CD3lo-->TCR/CD3hi. Thus, in HIV-1-infected WE17/10 cells, changes in CD3-gamma gene transcription are accompanied by altered viral p24 antigen production and the resulting modulation of surface receptor expression can be summarized by the formula: TCR/CD3hi reversible TCR/CD3lo reversible TCR/CD3-.

Cell Line↗
PubMed1996

Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships.

We have isolated, sequenced, and characterized a human MN/CA9 gene. This gene is a novel member of the carbonic anhydrase (CA) family, which codes for widely distributed catalysts of the reversible conversion of carbon dioxide to carbonic acid. So far, MN/CA IX is the only tumor-associated CA isoenzyme. The entire genomic sequence of MN/CA9, including the 5'-flanking region, encompasses 10.9 kb. The coding sequence is divided into 11 exons, whose organization and relationships to predicted protein domains suggest that the gene arose by exon shuffling. Exon 1 encodes a signal peptide and a proteoglycan-related region. Exons 2-8 code for a CA domain with a highly conserved active site. The exon/intron pattern of the CA coding region is similar but not identical to other described animal kingdom alpha-CA genes. Exons 10 and 11 encode a transmembrane anchor and an intracytoplasmic tail, respectively. We have also determined the transcription initiation and termination sites by RNase protection assay and analyzed the 3. 5-kb region upstream of the MN/CA9 gene. Sequence of the proximate 5' end of the flanking region shows extensive homology to the long terminal repeats of HERV-K endogenous retroviruses. The putative MN/CA9 promoter immediately preceding the transcription start site does not possess a TATA box, but contains consensus sequences for the AP1, AP2, p53, and Inr transcription factors. This study will allow further investigations of the molecular events regulating expression of MN/CA IX as well as elucidation of its biological function.

Amino Acid Sequence↗
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↗