Biomedical subjects

K H Klempnauer

Publications and source records attributed to K H Klempnauer.

17 recordsLinked to original sources

PubMed1992

Functional antagonism between members of the myb family: B-myb inhibits v-myb-induced gene activation.

The oncogene v-myb and its cellular progenitor c-myb encode nuclear, DNA binding phosphoproteins that control the expression of certain target genes in immature hematopoietic cells. Here, we report the isolation of a myb-related chicken gene, chicken B-myb. We show that expression of B-myb, unlike that of c-myb, is not restricted to hematopoietic cells, suggesting that B-myb functions in a broader spectrum of cell types than c-myb. We have identified the authentic chicken B-myb protein as a nuclear protein of approximately 110 kDa. We show that the B-myb protein specifically recognizes v-myb binding sites in vitro and that binding is mediated by an N-terminally located DNA binding domain. Although B-myb protein recognizes myb binding sites, B-myb fails to transactivate several myb-responsive gene constructs as well as the endogenous myb-responsive gene mim-1. Instead, we find that B-myb represses v-myb- and c-myb-mediated activation of the mim-1 gene, most likely by competing with other myb proteins for binding sites. Our results raise the possibility that B-myb is an inhibitory member of the myb family.

Amino Acid Sequence↗

PubMed1991

Sequence-specific DNA binding by Myc proteins.

Myc proteins have a tripartite carboxyl-terminal domain containing specific amino acid sequence motifs: a basic motif, a helix-loop-helix motif, and a leucine heptad repeat. Similar sequence motifs have been identified in several eukaryotic transcription factors and were shown to facilitate protein-DNA and protein-protein interactions. By using recombinant v-Myc proteins obtained by bacterial expression of full-length or partially deleted avian v-myc alleles, the functional relevance of these sequence motifs for Myc protein oligomerization and for DNA binding was investigated. All recombinant v-Myc proteins that have retained the carboxyl-terminal domain dimerize and specifically bind to double-stranded DNA containing the palindromic core sequence CACGTG. This and a closely related DNA sequence element have been defined previously as part of the binding sites for human transcription factors USF and TFE3, which specifically bind to the adenovirus major late promoter or the muE3 motif within the immunoglobulin heavy-chain enhancer, respectively. It is shown that a 61-amino-acid peptide sequence containing only the bipartite basic motif/helix-loop-helix domain of Myc is necessary and sufficient for dimerization and sequence-specific DNA binding of v-Myc recombinant proteins.

Base Sequence↗

PubMed1991

Estrogen-dependent alterations in differentiation state of myeloid cells caused by a v-myb/estrogen receptor fusion protein.

The oncogene v-myb and its cellular progenitor c-myb encode nuclear, DNA binding phosphoproteins that are thought to regulate the expression of myb-responsive genes during myeloid differentiation. To identify such myb-regulated genes, and to explore the mechanisms by which v-myb affects their expression, we have established a conditional expression system for v-myb. We have converted the v-myb protein to an estrogen-inducible transactivator by fusing the protein to the hormone binding domain of the human estrogen receptor. Expression of the chimeric protein in a chicken macrophage cell-line causes estrogen-dependent, reversible changes in the differentiation state as well as alterations in the gene expression program of the cells. We have used this estrogen-dependent v-myb expression system to identify a novel v-myb regulated gene.

Amino Acid Sequence↗

PubMed1990

Characterization of the v-myb DNA binding domain.

The transforming protein encoded by the v-myb oncogene is a sequence-specific DNA-binding protein that is thought to be involved in the regulation of gene expression. The N-terminal region of the v-myb protein is composed of two highly conserved tandem repeat sequences of unknown function. It has been speculated that the N-terminal v-myb repeats might be crucial for DNA-binding, since N-terminal deletions destroy the DNA-binding activity of the v-myb protein. Here, we have studied the v-myb DNA-binding domain in more detail. Our results show that the N-terminal region of the v-myb protein is sufficient for specific DNA-binding. Dissection of this region suggests that both repeats are required for DNA-binding, but that both repeats play different roles in v-myb protein DNA interaction. We also show that the myb repeats of a drosophila melanogaster homolog of c-myb function as sequence-specific DNA-binding domain. Our results support the view that specific sequence-recognition, mediated by the conserved myb repeats, is a general feature of myb-related proteins.

Animals↗

PubMed1989

Activation of transcription by v-myb: evidence for two different mechanisms.

The retroviral oncogene v-myb encodes a nuclear, sequence-specific DNA-binding protein. To investigate the possibility that v-myb encodes a transcriptional regulator, we used a transient cotransfection assay to explore the potential of v-myb to influence the expression of other genes. We found that expression of a chicken lysozyme promoter/CAT gene construct was activated by v-myb in the presence of myb-specific binding sites. Action was not observed with a truncated v-myb protein lacking its DNA-binding domain. We also observed that expression of a hybrid human HSP70 promoter/CAT gene, lacking myb-specific binding sites, was activated by v-myb. However, in this case, the truncated v-myb protein, which lacked its DNA-binding domain, also activated HSP70/CAT expression, indicating that trans-activation of this gene construct was independent of the sequence-specific DNA-binding activity of the v-myb protein. These observations suggest that v-myb encodes a trans-activator and that activation of gene expression occurs by two different mechanisms, one of which involves specific binding of v-myb protein to the regulated gene.

Animals↗

PubMed1989

Association of v-myc protein with chromatin.

The subnuclear distribution of proteins encoded by v-myb and v-myc was analysed in a cell-line of AMV-transformed chicken myeloblasts superinfected by the myc-containing retrovirus MC29. p45v-myb and p110gag-myc, co-expressed in these cells, were released in similar fashion when nuclei were treated with salt or DNAase. Analysis of nucleoprotein complexes extracted from nuclease-treated nuclei shows that p45v-myb and p110gag-myc are associated with a chromatin fraction of enhanced nuclease sensitivity. v-myb and v-myc proteins thus share the same subnuclear location and apparently interact directly with the cellular DNA.

Animals↗

PubMed1988

Viral myb oncogene encodes a sequence-specific DNA-binding activity.

The retroviral oncogene v-myb and its cellular progenitor c-myb encode nuclear DNA-binding proteins. Myb genes have been identified in a broad range of species, including vertebrates, the fruit fly Drosophila melanogaster and the plant Zea mays. The localization of the DNA-binding domain of the v-MYB protein to the highly conserved amino-terminal region suggests that the MYB/DNA interaction is important for MYB function. We show here that v-MYB specifically recognizes the nucleotide sequence pyAACG/TG. So like other nuclear transforming proteins, v-MYB seems to be a member of the class of sequence-specific DNA-binding factors presumably involved in gene regulation.

Base Sequence↗

PubMed1988

Interaction of myb proteins with nuclear matrix in vitro.

Fractionation studies of isolated nuclei have shown that the proteins encoded by the retroviral oncogene v-myb and its cellular homologue c-myb are associated to a variable extent with the nuclear matrix, suggesting that the nuclear matrix might contain a cellular target for myb proteins. I have explored the possible existence of such a target by incubating soluble v-myb and c-myb protein with nuclear matrix prepared from a separate source. The results presented here suggest that nuclear matrices from various cells contain binding sites for myb proteins. Matrix-binding appears to be an intrinsic property of myb proteins. In addition to myb proteins I have demonstrated the existence of a small group of proteins possessing nuclear matrix binding activity. These findings suggest that the nuclear matrix serves as a target for a specific set of proteins, including the products of myb genes.

Animals↗

PubMed1987

The highly conserved amino-terminal region of the protein encoded by the v-myb oncogene functions as a DNA-binding domain.

The retroviral oncogene v-myb encodes a 45,000 Mr nuclear protein (p45v-myb) that is predominantly associated with the chromatin of transformed cells. It has previously been shown that p45v-myb, when released from chromatin by salt-treatment, binds to DNA. To analyse the biochemical properties of p45v-myb in more detail we have expressed the v-myb coding region in Escherichia coli. Our results demonstrate that bacterially expressed myb protein has an intrinsic DNA-binding activity. Using two alternative strategies, (i) inhibition of DNA-binding by monoclonal antibodies and (ii) analysis of DNA-binding activities of partially deleted forms of the bacterial myb protein, we show that the DNA-binding domain is located in the amino-terminal region of the v-myb protein. This region has been highly conserved between myb genes of different species. Our results are therefore consistent with the hypothesis that DNA-binding is an important aspect of myb protein function.

Amino Acid Sequence↗

PubMed1987

Drosophila and vertebrate myb proteins share two conserved regions, one of which functions as a DNA-binding domain.

We report the nucleotide sequence of a cDNA clone of the Drosophila melanogaster homologue of c-myb, a member of the class of vertebrate transforming genes encoding nuclear proteins. We predict the mol. wt of the Drosophila myb (D-myb) protein to be 74,000. The D-myb protein contains two clusters of sequences homologous to vertebrate myb proteins, surrounded by sequences lacking homology. These results extend previous evidence for the existence of a D. melanogaster homologue of c-myb and identify two highly conserved and therefore presumably functionally important domains of c-myb proteins. DNA-binding experiments indicate that the NH2-proximal of the two homology regions functions as a DNA-binding domain. Based on the absence of the COOH-proximal homology region in truncated oncogenic derivatives of c-myb it is likely that this homology region encodes a function whose loss is involved in activating the oncogenic potential of c-myb.

Amino Acid Sequence↗

PubMed1986

Identification and characterization of the protein encoded by the human c-myb proto-oncogene.

We have identified the product of the human c-myb proto-oncogene as a 80,000-Mr protein, p80c-myb, by using polyclonal and monoclonal antibodies raised against a bacterially synthesized polypeptide from the amino terminus of the viral myb protein. p80c-myb shares at least two distinct antigenic sites with the amino terminal region of the v-myb protein. p80c-myb is found only in hematopoietic cells or in cells that contain amplified c-myb genes. Like the chicken myb proteins, p80c-myb is a nuclear DNA-binding protein that is predominantly associated with chromatin and exhibits a short half-life of approximately 1 hour.

Animals↗

PubMed1986

Subnuclear localization of proteins encoded by the oncogene v-myb and its cellular homolog c-myb.

The retroviral transforming gene v-myb encodes a 45,000-Mr nuclear transforming protein (p45v-myb). p45v-myb is a truncated and mutated version of a 75,000-Mr protein encoded by the chicken c-myb gene (p75c-myb). Like its viral counterpart, p75c-myb is located in the cell nucleus. As a first step in identifying nuclear targets involved in cellular transformation by v-myb and in c-myb function, we determined the subnuclear locations of p45v-myb and p75c-myb. Approximately 80 to 90% of the total p45v-myb and p75c-myb present in nuclei was released from nuclei at low salt concentrations, exhibited DNA-binding activity, and was attached to nucleoprotein particles when released from the nuclei after digestion with nuclease. A minor portion of approximately 10 to 20% of the total p45v-myb and p75c-myb remained tightly associated with the nuclei even in the presence of 2 M NaCl. These observations suggest that both proteins are associated with two nuclear substructures tentatively identified as the chromatin and the nuclear matrix. The function of myb proteins may therefore depend on interactions with several nuclear targets.

Animals↗

PubMed1986

Coordinate regulation of myelomonocytic phenotype by v-myb and v-myc.

Both avian myeloblastosis virus (by the action of v-myb) and avian myelocytomatosis virus MC29 (by the action of v-myc) transform cells of the myelomonocytic lineage. Whereas avian myeloblastosis virus elicits a relatively immature phenotype, cells transformed by MC29 resemble mature macrophages. When cells previously transformed by v-myb were superinfected with MC29, their phenotype was rapidly altered to that of a more mature cell. These superinfected cells expressed both v-myb (at a level similar to that found before superinfection) and v-myc. It therefore appears that the expression of v-myc can elicit certain properties of a more differentiated phenotype. In addition, unlike cells transformed by v-myb alone, the cells expressing both v-myb and v-myc could not be induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate to differentiate to fully mature macrophages. Cells with a morphology similar to that of the superinfected cells were elicited by simultaneously infecting yolk sac macrophages with avian myeloblastosis virus and MC29. Such cells expressed both v-myb and v-myc. These results indicate that expression of v-myb and v-myc in infected cells coordinately regulates myelomonocytic phenotype and that the two viral oncogenes vary in their ability to interfere with tumor promoter-induced differentiation. Our findings also sustain previous suggestions that the oncogenes v-myb and v-myc may not transform target cells by simply blocking differentiation.

Animals↗

PubMed1986

Rearrangement at the 5' end of amplified c-myc in human COLO 320 cells is associated with abnormal transcription.

The proto-oncogene c-myc is amplified in sublines of human COLO 320 cells carrying either homogeneously staining chromosomal regions or double minutes. COLO 320 cells carrying homogeneously staining chromosomal regions have 15 to 20 copies of an apparently normal c-myc allele and 1 to 2 copies of an abnormal c-myc allele lacking exon 1 and express high levels of a normal c-myc mRNA 2.5 kilobases in size. COLO 320 cells carrying double minutes have about 25 copies each of the normal allele and the abnormal allele but express preferentially an abnormal c-myc mRNA 2.2 kilobases in size. Nucleotide sequence analyses revealed that the break point of rearrangement resulting in the loss of exon 1 in the abnormal allele lies within a region frequently rearranged in human and murine B-cell tumors.

Alleles↗

PubMed1985

Transformation-defective mutant of avian myeloblastosis virus that is temperature sensitive for production of transforming protein p45v-myb.

We have characterized a mutant of avian myeloblastosis virus (strain GA907/7) that shows a reduced capacity to transform myelomonocytic cells at the nonpermissive temperature. Myeloblasts transformed by this mutant suffer a substantial decrease in the amount of the transforming protein p45v-myb when shifted from the permissive to the nonpermissive temperature. We presume that the 5- to 10-fold decrease in the amount of p45v-myb causes the loss of the transformed phenotype. The decrease is due to a reduction in the level of v-myb mRNA. Mutant GA907/7 thus provides genetic evidence that p45v-myb is the transforming protein of avian myeloblastosis virus and apparently represents an unusual defect in the production or stability of mRNA.

Animals↗

PubMed1978

The structure of chromatin replicated in vitro.

Nuclei from concanavalin-A-activated lymphocytes were used to study the replication of chromatin in vitro. Micrococcal nuclease was employed to obtain information about the structure of the replicated chromatin. The nuclease digestion products were examined by sucrose gradient sedimentation and by gel electrophoresis. Experiments are presented which indicate that DNA replicated in vitro is organized into chromatin whose structure is similar to that of bulk chromatin. This conclusion is based on the following observations: (a) DNA replicated in vitro is associated with typical chromatin subunits (nucleosomes) even after short replication times, when the newly replicated DNA consists almost entirely of Okazaki fragments; (b) the length of internucleosomal spacer DNA in part of the replicated chromatin corresponds to that in bulk chromatin. Evidence which suggests that the structure of nucleosomes is transiently altered in the vicinity of the replication fork is presented.

Animals↗

PubMed1978

Association of DNA polymerase with nucleosomes from mammalian cell chromatin.

More than half of the DNA polymerase beta in mouse ascites cell chromatin was found to be associated with monomeric nucleosomal particles (produced by micrococcal nuclease treatment of chromatin). Almost all nuclear DNA polymerase activity in lymphocytes was found to be associated with nucleosomes. The nucleosome-associated enzyme was mainly DNA polymerase beta in chromatin from resting and mainly DNA polymerase alpha in chromatin from concanavalin-A-stimulated lymphocytes.

Animals↗