Nucleotide sequence of cDNA clones of the murine myb proto-oncogene.

We have isolated cDNA clones of murine c-myb mRNA which contain approximately 2.8 kb of the 3.9-kb mRNA sequence. Nucleotide sequencing has shown that these clones extend both 5' and 3' to sequences homologous to the v-myb oncogenes of avian myeloblastosis virus and avian leukemia virus E26. The sequence contains an open reading frame of 1944 nucleotides, and could encode a protein which is both highly homologous, and of similar size (71 kd), to the chicken c-myb protein. Examination of the deduced amino acid sequence of the murine c-myb protein revealed the presence of a 3-fold tandem repeat of 52 residues near the N terminus of the protein, and has enabled prediction of some of the likely structural features of the protein. These include a high alpha-helix content, a basic region toward the N terminus of the protein and an overall globular configuration. The arrangement of genomic c-myb sequences, detected using the cDNA clones as probes, was compared with the reported structure of rearranged c-myb in certain tumour cells. This comparison suggested that the rearranged c-myb gene may encode a protein which, like the v-myb protein, lacks the N-terminal region of c-myb.     

Studies of c-myb gene regulation in MRL-lpr/lpr mice. Identification of a 5' c-myb nuclear protein binding site and high levels of binding factors in nuclear extracts of lpr/lpr lymph node cells

Lymph node T cells of MRL-lpr/lpr mice are characterized by the production of very large amounts of c-myb mRNA. To study the control of c-myb expression, a search was made for sites on the 5' c-myb gene which could bind regulatory proteins. DNase I digestion of nuclear chromatin uncovered four DNase I hypersensitive sites in the first intron of the c-myb gene, and a single site approximately 300 bp 5' to the initiation codon. Lambda exonuclease digestion of a 5'-myb fragment in the presence of nuclear extracts from either MRL-lpr/lpr PLN or EL-4 thymoma revealed stop sites approximately 300 bp 5' (-271 to -322) to the ATG initiation codon. DNase I footprint analysis demonstrated a guanine-cytosine enriched region of potential binding sites (-274 to -319) in the region of the stop sites and a fifth potential binding site closer to the initiation codon (-163 to -168). Specific gel shift bands were detected by a 5'-myb fragment (-346 to -155) with extracts from a number of different lymphoid cell lines and the appropriate specific and non-specific competitor DNA. The DNA giving rise to these gel shift bands encompassed the region defined by the stop site and footprinting studies. To determine whether or not the protein binding to the 5' c-myb gene at -274 to -319 was associated with increased c-myb mRNA, we studied nuclear extracts of several cell lines and compared the amount of binding to the amount of c-myb mRNA found on Northern analyses. Among the cell lines, there was a correlation between c-myb expression and the amount of the 5'-myb DNA binding protein. In addition, MRL-lpr/lpr lymph node cells had high c-myb expression and large amounts of the 5'-myb binding protein. This result suggests that the binding may play some role in the c-myb expression. Moreover, the most immature cell lines had the greatest amount of the binding factor, suggesting that its regulatory effect on c-myb expression might be important in early differentiation events.     

Nucleotide sequence of chicken myb proto-oncogene promoter region: detection of an evolutionarily conserved element.

The nucleotide sequence of the chicken myb proto-oncogene putative promoter region was determined and compared with the corresponding sequence of the mouse c-myb gene (1). 118 bp upstream from the initiation codon suggested by Gerondakis and Bishop (2) for the chicken c-myb protein, a 124-bp-long conserved element was found (92% identity in chicken and mouse sequences). Sequences homologous to this element were detected on Southern blots of restricted genomic DNAs from mouse, man, lizard, frog, and carp. No hybridization was observed with Drosophila, yeast, or Escherichia coli DNA. In human DNA, sequences homologous to this element were located at the 5' end of the c-myb gene, i.e. in the same position as in the chicken and mouse genes. Several lines of evidence suggest that the element is not a coding exon of a gene overlapping the c-myb gene. It may be of importance that one of the DNase I-sensitive sites and several c-myb mRNA cap sites localized recently in the mouse c-myb gene (3,4) lie within this region. It is suggested that this evolutionarily conserved element is involved in the regulation of myb proto-oncogene expression in vertebrates.     

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.     

Identification of c-myb (chicken), c-myb (mouse) and v-myb (AMV) protein products by immunoprecipitation with antibodies directed against a synthetic peptide

A synthetic nonadecapeptide (IL 19) derived from a sequence of v-myb was covalently bound to haemocyanin and used for immunization. Anti-IL 19 serum immunoprecipitated a 75 kDa protein in the lysate of metabolically labelled chicken and murine thymus cells. Presaturation of the serum with IL 19 abolished this immunoprecipitation, thus indicating that the product of c-myb in both chicken and murine thymuses is the 75 kDa protein (p75c-myb). Anti IL 19 serum also precipitated p48v-myb in the lysate of nonproducer myeloblasts.