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.     

Intermolecular recombination of the c-myb proto-oncogene coding sequences in chicken: clues for a mechanism

The physical separation of c-myb coding exons on two different chromosomes, both in chicken and human, indicated that intermolecular recombination events might occur in higher eucaryotes. We present evidence in this paper suggesting that the expression of "antisens" mRNA species could be involved in the regulation of the intermolecular recombination process.     

Alternative splicing within the chicken c-ets-1 locus: implications for transduction within the E26 retrovirus of the c-ets proto-oncogene.

Two overlapping c-ets-1 cDNA clones were isolated which contained the alpha and beta genomic sequences homologous to the 5' end of v-ets not detected in the previously described c-ets RNA species or proteins. Nucleotide sequencing demonstrated that these cDNAs corresponded to the splicing of alpha and beta to a common set of 3' exons (a through F) already found in the p54c-ets-1 mRNA. They contained an open reading frame of 1,455 nucleotides which could encode a polypeptide of 485 amino acids with a predicted molecular mass of 53 kilodaltons. However, when expressed in COS-1 cells, the cDNAs directed the synthesis of a protein with an apparent molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 68 kilodaltons, p68c-ets-1, comigrating with a protein expressed at low levels in normal chicken spleen cells. These two proteins were shown to be identical by partial digestion with protease V8. Northern (RNA) blot hybridization analysis with the p68c-ets-1 -specific sequence and RNase protection experiments showed that the corresponding mRNA was expressed in normal chicken spleen and not in normal chicken thymus or in various T lymphoid cell lines. Thus, two closely related proteins, having distinct amino-terminal parts, are generated within the same locus by alternative addition of different 5' exons, alpha and beta or I54, respectively, onto a common set of 3' exons (a to F). Finally, we demonstrate that an aberrant splicing event between a cryptic splice donor site in c-myb exon E6 and the normal splice acceptor site of c-ets-1 exon alpha involved in the genesis of the E26 myb-ets sequence.     

Activation of the c-myb locus is insufficient for the rapid induction of disseminated avian B-cell lymphoma.

We have previously reported that infection of 9- to 13-day-old chicken embryos with RAV-1 results in rapid development of a novel B-cell lymphoma in which proviral insertion has activated expression of the c-myb gene (E. Pizer and E. H. Humphries, J. Virol. 63:1630-1640, 1989). The biological properties of these B-cell lymphomas are distinct from those associated with the B-cell lymphomas that develop following avian leukosis virus proviral insertion within the c-myc locus. In an extension of this study, more than 200 chickens, infected as 10- to 11-day-old embryos, were examined for development of lymphomas that possess disrupted c-myb loci. Fourteen percent developed disseminated B-cell lymphoma. In the majority of these tumors, the RAV-1 provirus had inserted between the first and second exons that code for p75c-myb. However, insertions between the second and third exons and between the third and fourth exons were also detected. In situ analysis of myb protein expression in tumor tissue revealed morphological features suggesting that the tumor originates in the bursa. Within the bursa, the lymphoma appeared to spread from follicle to follicle without compromising the structural integrity of the organ. Tumor masses in liver demonstrated heterogeneous levels of myb protein suggestive of biologically distinct subpopulations. In contrast to the morbidity data, immunohistological analysis of bursae from 4- to 6-week-old chickens at risk of developing lymphomas bearing altered c-myb loci revealed lesions expressing elevated levels of myb in 16 of 19 birds. The activated myb lymphoma displayed very poor capacity to proliferate outside its original host. Only 1 of 33 in vivo transfers of tumor to recipient hosts established a transplantable tumor. None of the primary tumor tissue nor the transplantable tumor exhibited the capacity for in vitro proliferation. Similar experimental manipulation has yielded in vitro lines established from avian B-cell lymphomas expressing elevated levels of c-myc or v-rel. The dependence on embryonic infection for development of activated-myb lymphoma suggests a requirement for a specific target cell in which c-myb is activated by proviral insertion. It is likely, moreover, that continued tumor development requires elevated expression of myb proteins within a specific cell population in a restricted stage of differentiation.     

Transduction of c-myb into avian myeloblastosis virus: locating points of recombination within the cellular gene.

The oncogene (v-myb) of avian myeloblastosis virus apparently arose by transduction of nucleotide sequences from a cellular gene (c-myb). In c-myb the nucleotide sequences that formed v-myb exist at seven distinct regions separated by nontransduced stretches of sequence that are flanked by eucaryotic splice signals. By contrast, the sequences at the outside boundaries of the transduced region of c-myb do not resemble splice sites. We mapped the nucleotide sequences that are homologous to the ends of v-myb with respect to the exons and introns of c-myb. The results indicate that the leftward recombination between c-myb and the transducing retrovirus occurred within an intron of the cellular gene, whereas the rightward recombination took place in an exon of c-myb. Transduction of c-myb sequences may therefore have involved a DNA rearrangement.     

Multiple domains for the chicken cellular sequences homologous to the v-ets oncogene of the E26 retrovirus.

We have investigated the structure of chicken genomic DNA homologous to v-ets, the second cell-derived oncogene of avian retrovirus E26. We isolated a c-ets locus spanning ca. 30.0 kilobase pairs (kbp) in the chicken genome with homologies to 1,202 nucleotides (nt) of v-ets (total length, 1,508 nt) distributed in six clusters along 18.0 kbp of the cloned DNA. The 5'-distal part of v-ets (224 nt) was homologous to chicken cellular sequences contained upstream within a single 16.0-kbp EcoRI fragment as two typical exons but not found transcribed into the major 7.5-kb c-ets (or 4.0-kb c-myb) RNA species. Between these two v-ets-related cellular sequences we found ca 40.0 kbp of v-ets-unrelated DNA. Finally, the most 3' region of homology to v-ets in the cloned DNA was shown to consist of a truncated exon lacking the nucleotides coding for the 16 carboxy-terminal amino acids of the viral protein but colinear to one of the two human c-ets loci, c-ets-2.     

Sequences of complete cDNAs encoding four variants of chicken skeletal muscle troponin T

cDNAs containing the complete coding sequences of four isoforms of troponin T derived from 1-week-old chick skeletal muscle have been isolated and sequenced. While the 5' and 3' untranslated regions and most of the coding sequence were identical for each, dramatic differences were observed in the NH2-terminal region corresponding to amino acid residues 10-37 of rabbit skeletal troponin T. These sequence differences correspond to the alternatively spliced but not mutually exclusive exons 4 to 8 of the rat skeletal muscle troponin T gene. In addition, we observe a sequence corresponding to an extra exon or exons (between 5 and 6) present in the chicken skeletal muscle gene and not previously detected in the rat skeletal or chicken cardiac genes. This sequence of 63 nucleotides consists of an almost perfect repeat of 30 and 33 nucleotides and has previously been shown to be represented as a protein variant in chicken skeletal muscle. A difference is also present in one cDNA clone corresponding to the alternatively spliced (mutually exclusive) exons 16 and 17 of the rat gene. In the protein, this corresponds to a region implicated in the interaction of troponin T with troponin C, tropomyosin, and perhaps troponin I and F-actin.     

Cloning and mapping of 5' exons from the gene encoding chicken beta nerve growth factor.

An NGF cDNA containing the 5' exons of the nerve growth factor (NGF) messenger was obtained from chicken heart mRNA using the anchored polymerase chain reaction technique. Alignment of the chicken with the corresponding murine and human sequences reveals interspecies similarities. A sequence corresponding to an exon found only in the NGF messenger, which is abundant in the submaxillary gland of the male mouse, is present in the chicken NGF cDNA. The first non-coding exons of the NGF gene are much less conserved between chicken and mouse or human than the region of the last exon encoding the mature protein. After the cloning of the chicken NGF gene from a cosmid library, the chicken NGF exons have been located within 20 kb of DNA. The chicken NGF gene is therefore shorter than its murine counterpart which spans more than 43 kb. Furthermore, the organization of the chicken and murine NGF genes markedly differs in their 5' portion.     

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.     

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.     

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5''-disrupted myb loci.

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.     

Analysis of the human apolipoprotein B gene; complete structure of the B-74 region

In this paper we describe the nucleotide sequence of the B-74 region of human apolipoprotein B-100 mRNA. This region comprises the 3'-proximal three-quarters of the mRNA and contains 10,089 nucleotides (nt), 9786 of which are coding. Combining our data with the published sequence of the 5'-proximal one-quarter (i.e., the B-26 region [Protter et al., Proc. Natl. Acad. Sci. USA 83 (1986) 5678-5682] assigns 14,059 nt to the apoB-100 mRNA. The coding sequence spans 13,548 nt or 4516 amino acids (leader peptide excluded). The B-74 part of the apoB gene is built up of five exons separated by small introns, and is dominated by an unusually large exon of 7.5 kb. The derivation of two (EcoRI and XbaI) restriction fragment length polymorphisms occurring in the coding region is discussed.