PubMed Health⌕ Search

Biomedical subjects

K Bister

Publications and source records attributed to K Bister.

At least 19 recordsLinked to original sources
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

Myc protein structure: localization of DNA-binding and protein dimerization domains.

Wildtype and mutant v-Myc proteins were overexpressed in Escherichia coli using the T7 RNA polymerase system, and the in vitro DNA-binding activities of partially or highly purified proteins were analysed by native DNA-cellulose chromatography. For the construction of the expression plasmids, cloned proviral DNA from wildtype MC29 or from its spontaneous deletion mutant Q10C was used, the latter lacking internal v-myc sequences. Both the wildtype (p59) and the mutant (p42) recombinant protein contain at their amino termini 12 amino acids encoded by the vector, followed by 11 gag amino acids and 9 amino acids encoded by v-myc sequences derived from noncoding c-myc sequences. In addition, p59 contains 416 amino acids encoded by v-myc sequences derived from the complete chicken c-myc coding region, whereas p42 lacks 120 amino acids from the central region of the Myc protein including the highly acidic domain. Two additional proteins were engineered which contain the first 309 (p53) or the last 107 (p16) amino acids, respectively, of the Myc protein sequence in addition to vector-encoded amino acids. The p16 protein represents the carboxyl terminus of the Myc protein sequence containing both a muscle determination gene (MyoD1) homology region, including a basic motif and an amphipathic helix-loop-helix motif, and a leucine heptad repeat. All proteins, except p53 which lacks the carboxyl-terminal Myc protein sequences, bound to native DNA-cellulose and were eluted with 200-500 mM NaCl. Based on the DNA-binding activities of recombinant or spontaneous mutant v-Myc proteins extracted from bacterial or from transformed avian cells, we conclude that the DNA-binding domain of avian Myc proteins is confined within the last 86 carboxyl-terminal amino acids. The same region is also shown to be necessary and sufficient for Myc protein dimerization. This 86-amino acid region essentially encompasses a putative basic DNA contact surface and a tandem array of two presumed protein dimerization motifs, helix-loop-helix and leucine repeat.

Amino Acid Sequence↗
PubMed1988

Structural analysis of normal and transforming mil(raf) proteins: effect of 5'-truncation on phosphorylation in vivo or in vitro.

The phosphorylation sites of the cellular proto-oncogene product p71/73c-mil(raf) from quail and from human cells were analyzed by two-dimensional peptide mapping and compared to the sites phosphorylated in proteins encoded by three transforming alleles of c-mil(raf). These alleles all were 5'-truncated resulting from either retroviral transduction (v-mil, v-raf) or promoter insertion mutagenesis (LTR-c-raf). The normal cellular proteins each were phosphorylated in vivo on three major sites, two of which were identical in the two protein species. MH2 p100gag-mil, murine sarcoma virus 3611 p75gag-raf, and LTR-c-raf p45-50 delta c-raf were phosphorylated in vivo on several sites. One site was shared between these transforming proteins and was also conserved in both avian and human p71/73c-mil(raf). All normal and transforming mil(raf) proteins were phosphorylated on serine in vivo while p100gag-mil and p75gag-raf occasionally also contained low levels of phosphothreonine. No specific phosphorylation of p71/73c-mil(raf) was detected in vitro under conditions that readily revealed presumed autophosphorylation of p100gag-mil, p75gag-raf, and p45-50 delta c-raf. However, the in vitro phosphorylated sites of these proteins were different to each other and to the sites phosphorylated in vivo. In contrast to the predominant threonine phosphorylation of the two viral proteins, only phosphoserine could be detected in p45-50 delta c-raf phosphorylated in vitro.

Amino Acid Sequence↗
PubMed1988

Primary structure of the chicken c-mil protein:identification of domains shared with or absent from the retroviral v-mil protein.

The complete primary structure of the protein product of the proto-oncogene c-mil was deduced from the nucleotide sequence of chicken c-mil cDNA clones. The c-mil protein contains 647 amino acid residues and has a calculated molecular weight of 73,132. Based on sequence comparisons with proteins of known or presumed biochemical function, two domains were recognized on the c-mil protein. In the carboxyl-terminal half of the protein, a 250-amino acid segment displays significant homology to the protein kinase domains of the src oncogene protein or of protein kinase C. In the amino-terminal half, a cysteine-rich segment (Cys-X2-Cys-X9-Cys-X2-Cys-X7-Cys-X7-Cys) of the c-mil protein shares significant homology with two similar repetitive domains of protein kinase C. Of the two structural and presumably functional domains of the c-mil protein, only the kinase domain is contained within the carboxyl-terminal 379-amino acid polypeptide encoded by the transduced v-mil allele of avian oncogenic retrovirus MH2. Hence, truncation of the 5' coding region in the course of the transduction and the resulting lack of the authentic amino-terminal domain in the protein product of the transduced allele may be a critical event in changing mil function from physiologic to oncogenic.

Amino Acid Sequence↗
PubMed1987

Structure of mutant and wild-type MC29 v-myc alleles and biochemical properties of their protein products.

Proviral DNAs of three fibroblast-transforming MC29 deletion mutants (MC29-10A, MC29-10C, MC29-10H) with defects in hemopoietic cell transformation and tumor induction were molecularly cloned and their deletions were defined by nucleotide sequence analysis. The MC29-10C and MC29-10H v-myc alleles have identical internal deletions overlapping with a smaller one in the MC29-10A v-myc allele, and MC29-10H has an additional internal deletion in the partial gag complement. All deletions are in frame, and the deduced sequences of the mutant gag-myc hybrid proteins lack 56 (MC29-10A) or 120 (MC29-10C, MC29-10H) myc-specific and 44 gag-specific (MC29-10H) amino acid residues. The deleted v-myc nucleotide sequences correspond to the 3' end of exon 2 and the 5' end of exon 3 of the cellular c-myc gene including a region that encodes a high number of acidic amino acid residues. Based on these structural analyses, biochemical properties of mutant and wild-type gag-myc hybrid proteins were compared. Tryptic digests of all three mutant proteins lack a large myc-specific peptide that is present in digests of the wild-type protein and is extensively phosphorylated at serine and threonine residues. Concordantly, the sequence analyses predict that such a large tryptic peptide with putative phosphorylation sites at serine and threonine residues is present in the wild-type gag-myc protein but absent in all three mutant proteins due to the v-myc deletions. Chromatography of wild-type and mutant gag-myc proteins on DNA-cellulose revealed that their in vitro DNA affinities are indistinguishable from each other. Correspondingly, the sequence analyses predict that the carboxyl-terminal region rich in basic amino acid residues and with putative DNA affinity is conserved in wild-type and mutant gag-myc proteins. We conclude that the internal v-myc protein sequences defined by the deletions are necessary for hemopoietic cell transformation and complete phosphorylation, but dispensable for fibroblast transformation and in vitro DNA binding.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence of the CMII v-myc allele.

The entire nucleotide sequence of the transduced v-myc allele in the genome of avian oncogenic retrovirus CMII was determined. The CMII v-myc and the chicken c-myc alleles differ in their shared coding sequences by a single nucleotide substitution causing a glutamic acid/alanine exchange in the predicted sequences of the corresponding protein products. This mutation has not been found in the transduced v-myc alleles of avian oncogenic retroviruses MC29, MH2, and OK10. We conclude that no specific, if any, missense mutation of the c-myc coding sequence is necessary for oncogenic activation upon transduction of the cellular gene.

Alleles↗
PubMed1986

v-mil induces autocrine growth and enhanced tumorigenicity in v-myc-transformed avian macrophages.

MH2, an avian retrovirus containing the v-myc and v-mil oncogenes, rapidly transforms chick hematopoietic cells in vitro. The transformed cells belong to the macrophage lineage and proliferate in the absence of exogenous growth factors. Here we analyze a series of MH2 deletion mutants and show that these two oncogenes together establish an autocrine growth system in which v-myc stimulates cell proliferation, while v-mil induces the production of chicken myelomonocytic growth factor (cMGF). We also demonstrate that these two oncogenes cooperate in vivo. MH2 efficiently induces monocytic leukemias and liver tumors, while deletion mutants lacking either a functional v-mil or v-myc do not.

Animals↗
PubMed1986

Interaction between Raf and Myc oncogenes in transformation in vivo and in vitro.

3611 MSV, a raf-oncogene-transducing murine retrovirus, induces fibrosarcomas and erythroid hyperplasia in newborn mice after a latency of 4-8 wk. In contrast, new recombinant murine retroviruses carrying the myc oncogene (J-3, J-5 construct viruses) do not induce tumors before greater than 9 wk. A combination of both oncogenes in an infectious murine retrovirus (J-2) induces hematopoietic neoplasms in addition to less prominent fibrosarcomas and pancreatic adenocarcinoma 1-3 wk after inoculation. The hematologic neoplasms consist of immunoblastic lymphomas of T and B cell lineage and erythroblastosis. If animals were inoculated with a variant of the J-3 virus, which induces altered foci in cultures of NIH 3T3 cells, carcinoma developed in the pancreas with a 2-6 mo latency. In parallel to the synergistic action of both oncogenes on hematopoietic cells in vivo, we find that raf-oncogene-induced transformation of bone marrow cells in culture is enhanced by the addition of myc, which by itself does not transform these cells when grown in standard media. We conclude that concomitant expression of raf and myc oncogenes in hematopoietic and epithelial cells alters their respective transforming activities. The contribution of v-myc in this synergism was examined by use of a series of recombinant murine retroviruses capable of expressing the avian v-myc to study the effect of altered myc expression on hematopoietic/lymphoid cells. With either interleukin 3- or interleukin 2-dependent cell lines, introduction of the recombinant viruses abrogated the requirement for IL 3 or IL 2 for growth, and associated with this was the suppression of c-myc expression. The findings suggest that myc is a component in the signal transduction pathway for IL 3 and IL 2 and support an autoregulatory mechanism of c-myc expression. In contrast to v-myc, expression of v-raf in primary lymphoid/hematopoietic cells has an immortalizing function without abrogating the requirement for IL 3 for growth. This suggests that v-raf and v-myc affect different components of growth regulation, as, for example, commitment (v-myc) and cell cycle progression (v-raf).

Animals↗
PubMed1986

Structure and transforming function of transduced mutant alleles of the chicken c-myc gene.

A small retroviral vector carrying an oncogenic myc allele was isolated as a spontaneous variant (MH2E21) of avian oncovirus MH2. The MH2E21 genome, measuring only 2.3 kilobases, can be replicated like larger retroviral genomes and hence contains all cis-acting sequence elements essential for encapsidation and reverse transcription of retroviral RNA or for integration and transcription of proviral DNA. The MH2E21 genome contains 5' and 3' noncoding retroviral vector elements and a coding region comprising the first six codons of the viral gag gene and 417 v-myc codons. The gag-myc junction corresponds precisely to the presumed splice junction on subgenomic MH2 v-myc mRNA, the possible origin of MH2E21. Among the v-myc codons, the first 5 are derived from the noncoding 5' terminus of the second c-myc exon, and 412 codons correspond to the c-myc coding region. The predicted sequence of the MH2E21 protein product differs from that of the chicken c-myc protein by 11 additional amino-terminal residues and by 25 amino acid substitutions and a deletion of 4 residues within the shared domains. To investigate the functional significance of these structural changes, the MH2E21 genome was modified in vitro. The gag translational initiation codon was inactivated by oligonucleotide-directed mutagenesis. Furthermore, all but two of the missense mutations were reverted, and the deleted sequences were restored by replacing most of the MH2E21 v-myc allele by the corresponding segment of the CMII v-myc allele which is isogenic to c-myc in that region. The remaining two mutations have not been found in the v-myc alleles of avian oncoviruses MC29, CMII, and OK10. Like MH2 and MH2E21, modified MH2E21 (MH2E21m1c1) transforms avian embryo cells. Like c-myc, it encodes a 416-amino-acid protein initiated at the myc translational initiation codon. We conclude that neither major structural changes, such as in-frame fusion with virion genes or internal deletions, nor specific, if any, missense mutations of the c-myc coding region are necessary for activation of the basic oncogenic function of transduced myc alleles.

Alleles↗
PubMed1986

Protein product of proto-oncogene c-mil.

Using antipeptide antibodies with specificity for the carboxyl termini of v-raf and v-mil protein products, two proteins with apparent molecular weights of approximately 71,000/73,000 and 215,000 were detected in immunoprecipitates from normal uninfected chicken cells. The 71,000/73,000-molecular-weight protein was identified as the product of the c-mil proto-oncogene by the close structural relationship of its 42,000-molecular-weight carboxyl-terminal domain to the v-mil-encoded domain of the hybrid protein p100gag-mil specified by the avian retrovirus MH2. The amino-terminal domain of the cellular protein is encoded by 5' c-mil sequences that have not been transduced into the genome of MH2. The c-mil protein (p71/73c-mil) was found to be phosphorylated in vivo, and homologous proteins were detected at variable levels in a variety of vertebrate cells, including human cells.

Animals↗
PubMed1985

Molecular and biological properties of MH2D12, a spontaneous mil deletion mutant of avian oncovirus MH2.

Avian oncogenic retrovirus MH2 carries two cell-derived oncogenes, v-mil and v-myc. From an infectious stock of MH2 a spontaneous deletion mutant, MH2D12, that has lost most of the v-mil gene but has retained a complete and functional v-myc gene, has been isolated. Nonproducer quail embryo cells transformed by MH2D12 in the absence of helper virus contain two virus-specific proteins: a gag-related protein of 53,000 Da (p53gag), and a v-myc gene product of 59,000/61,000 Da (p59/61v-myc) indistinguishable from the v-myc protein encoded by MH2. MH2D12 viral RNA contains all T1-oligonucleotides specific for the MH2 v-myc gene but none of those characteristic for the v-mil gene. The genetic structure of molecularly cloned proviral DNA of MH2D12 was revealed by restriction mapping, blot hybridization, heteroduplex analysis, and nucleotide sequencing. The MH2D12 provirus is homologous to the MH2 genome but has suffered a deletion of 1271 nucleotides from the central region encompassing the 3' end of delta gag and all of v-mil except the very 3' 31 nucleotides directly adjacent to the v-myc gene. A nine-nucleotide overlap of homology to gag or mil at the delta gag/delta mil junction suggests that recombination between homologous sequence elements of the delta gag and v-mil domains of MH2 was involved in the genesis of MH2D12. The nucleotide sequence analysis predicts that the carboxyterminal 17 amino acids of p53gag are encoded by the residual v-mil sequences and by intron-derived v-myc sequences. Transformation of quail embryo cells by MH2D12 can be assayed by focus and colony formation of transformed cells. This indicates that the v-mil gene is not essential for these activities. However, size and morphology of foci and colonies, and cellular morphology of cultured MH2D12-transformed cell lines can easily be distinguished from those observed in cell transformation by MH2 and resemble more those seen in cell transformation by viruses containing the myc oncogene only.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence analysis of the chicken gene c-mil, the progenitor of the retroviral oncogene v-mil.

The nucleotide sequence of the chicken gene c-mil was determined within and around all regions homologous to the oncogene v-mil of avian retrovirus MH2. The regions of homology to the previously determined v-mil sequence, ranging in size from 28 to 177 base pairs (bp), are distributed over 14 kilobase pairs (kbp) of the chicken genome and are organized in 11 exons. All exon-intron boundaries of c-mil, except the 5' boundary of exon 1 and the 3' boundary of exon 11, were unambiguously defined by the identification of consensus splice donor and acceptor sites precisely at positions where homology to v-mil ceases or resumes. The homology to v-mil starts within the coding sequence of exon 1 and ends within the 3' untranslated region of exon 11, 12 nucleotides downstream from the nonsense codon terminating the large open reading frame shared between c-mil and v-mil. The c-mil and v-mil sequences differ at only 7 out of 1153 nucleotide positions, and the predicted sequences of v-mil and c-mil proteins differ by one conservative and four nonconservative substitutions among 379 amino acid residues. Hence, the carboxy-terminal domains of the MH2 gag-mil hybrid protein and of the putative c-mil protein are very similar. However, the amino-terminal domain of the cellular protein is possibly encoded by additional 5' c-mil sequences not present in the transduced v-mil oncogene, while that of the MH2 hybrid protein is encoded by viral gag sequences. The sequence analysis also revealed that c-mil and c-myc derived sequences are immediately adjacent on the MH2 genome carrying both the v-mil and the v-myc oncogene. Hence, transduction of c-mil into MH2 involved recombination, at the 3' site, with either the c-myc locus or a previously transduced v-myc gene, and, at the 5' site, with gag sequences of the transducing virus. At both sites, no significant homologies were found between the sequence elements involved in the recombination.

Amino Acid Sequence↗
PubMed1985

Molecular cloning of proviral DNA and structural analysis of the transduced myc oncogene of avian oncovirus CMII.

Molecularly cloned proviral DNA of avian oncogenic retrovirus CMII was isolated by screening a genomic library of a CMII-transformed quail cell line with a myc-specific probe. On a 10.4-kilobase EcoRI fragment, the cloned DNA contained 4.4 kilobases of CMII proviral sequences extending from the 5' long terminal repeat to the EcoRI site within the partial (delta) complement of the env gene. The gene order of CMII proviral DNA is 5'-delta gag-v-myc-delta pol-delta env-3'. All three structural genes are partially deleted: the gag gene at the 3' end, the env gene at the 5' end, and the pol gene at both ends. The delta gag (0.83 kilobases)-v-myc (1.50 kilobases) sequences encode the p90gag-myc transforming protein of CMII. In comparison with the p110gag-myc protein of acute leukemia virus MC29, p90gag-myc lacks amino acids corresponding to additional 516 bases of gag sequences and 12 bases of 5' v-myc sequences present in the MC29 genome. Nucleotide sequence analysis of CMII proviral DNA at the delta gag-v-myc and the v-myc-delta pol junctions revealed significant homologies between avian retroviral structural genes and the cellular oncogene c-myc precisely at the positions corresponding to the gene junctions in CMII. Furthermore, the delta gag-v-myc junction in CMII corresponds to sequence elements in gag and C-myc that are possible splicing signals. The data suggest that transduction of cellular oncogenes may involve RNA splicing and recombination with homologous sequences on retroviral vectors. Different sequence elements of both the retroviral vectors and the c-myc gene recombined during genesis of highly oncogenic retroviruses CMII, MC29, or MH2.

Animals↗
PubMed1985

Rapid induction of hemopoietic neoplasms in newborn mice by a raf(mil)/myc recombinant murine retrovirus.

3611 MSV, a raf oncogene-transducing murine retrovirus, induced fibrosarcomas in newborn mice after a latency of 4 to 8 weeks. In contrast, newly constructed recombinant murine retroviruses carrying the myc oncogene did not induce tumors before greater than or equal to 9 weeks. A combination of both oncogenes in an infectious murine retrovirus induced hematopoietic neoplasms in addition to less prominent fibrosarcomas and pancreatic acinar dysplasia 1 to 3 weeks after inoculation. The hematological neoplasms consisted of immunoblastic lymphomas of T- and B-lineage cells and erythroblastosis. Cell lines from these tumors could be readily established in culture in regular medium, whereas culture of cells from raf oncogene-induced tumors required the addition of interleukin 3. In parallel to the synergistic action of both oncogenes on hematopoietic cells in vivo, we found that raf oncogene-induced transformation of fibroblast cell lines in culture was enhanced by the addition of myc, which by itself did not morphologically transform these permanent cell lines. We conclude that concomitant expression of raf and myc oncogenes in hematopoietic cells and fibroblastic cell lines enhances their respective transforming activities.

Animals↗
PubMed1984

Immunological analysis of v-myc gene products using antibodies against a myc-specific synthetic peptide.

Rabbit antisera were prepared against a synthetic peptide corresponding to the carboxyterminal amino acid sequences of the transforming protein p110gag-myc of avian oncovirus MC29. Analysis of immunoprecipitates formed with these sera (anti-mycC) demonstrated that the gag-myc hybrid proteins encoded by the avian leukemia viruses MC29, CMII, and OK10 were recognized by the anti-peptide sera, but not the gag or pol precursor proteins of helper viruses or the MH2-encoded p100gag-mil protein. In cells transformed by OK10 or MH2, putative v-myc proteins of 60K (OK10) and 59/61K (MH2) were also precipitated by anti-mycC. In addition, the anti-peptide sera reacted specifically with the gag-myc proteins encoded by three partially transformation-defective mutants of MC29, td10A, td10C, and td10H, and by MC29 variant HBI.

Amino Acid Sequence↗