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The v-Myb DNA-binding domain differs from that of c-Myb mainly by deletion of the first of three repeats. This truncation correlates with efficient oncogenic transformation and a decrease in DNA-binding activity. Here we demonstrate that the D-type cyclins, cyclin D1 and D2 in particular, specifically inhibit transcription when activated through the v-Myb DNA-binding domain, but not the c-Myb DNA-binding domain. Analysis of a cyclin D1 mutant and a dominant-negative CDK4 mutant implied that this repression is independent of complex formation with a CDK partner. Association of cyclin D1 and D2 with the Myb DNA-binding domain could be demonstrated. Increased levels of cyclin D1 and D2 resulted in a stabilization of the Myb proteins, but not in an alteration in binding of the Myb proteins to DNA. These results highlight an unexpected role for cyclin D as a CDK-independent repressor of transcriptional activation by v-Myb but not c-Myb. This differential effect of D-type cyclins on v-Myb and c-Myb might help to explain the mechanism underlying the oncogenic activity of v-Myb, which appears to be a stronger transcriptional activator following the TPA-induced differentiation of transformed monoblasts when cyclin D1 and D2 are down-regulated.

The nuclear protein v-Myb, encoded by the avian myeloblastosis virus (AMV), can induce acute monoblastic leukemia in vivo and transform chicken myelomonocytic cells in culture. The N terminus of v-Myb functions as the DNA-binding domain, and multiple central and C-terminal regions of this protein have been reported to function in transcriptional activation of model reporter genes. We showed previously that a C-terminal domain (amino acids 296 to 371) is required for transcriptional activation and transformation of primary chicken myelomonocytic cells. In this study, we have now analyzed a series of C-terminal mutants of v-Myb to further investigate this domain. A strong correlation was observed between transcriptional activation and leukemic transformation by this series of mutants. Furthermore, deletion analyses demonstrate that the C-terminal 41 amino acids of v=MybAMV (amino acids 331 to 371 of the Myb portion) are nonessential whereas further deletion of amino acids 321 to 330 (EFAETLQLID) results in a nonfunctional protein. Hence, we defined a 10-amino-acid subregion (the "FAETL" motif) required for transcriptional activation and oncogenic transformation by v-Myb Amv. The FAETL region is part of a putative leucine zipper structure and lies near a cluster of phosphorylation sites. Our analysis of mutants with substitutions of the zipper leucines or multiple adjacent phosphorylation sites demonstrates that the function of the FAETL motif is not dependent on an intact leucine zipper structure or adjacent phosphorylation sites. The study of GAL4-Myb fusions suggests that this region is important in maintaining a fully functional conformation of v-Myb. The putative leucine zipper structure has previously been proposed to exert inhibitory effects on c-Myb because its mutation caused increased transcriptional transactivation and transformation. Interestingly, our results show that this region is essential for the functions of v-Myb without requiring a heptad leucine repeat.

Oncogenic activation of c-Myb in both avian and murine systems often involves N-terminal truncation. In particular, the first of three DNA-binding repeats in c-Myb has been largely deleted during the genesis of the v-myb oncogenes of avian myeloblastosis virus and E26 avian leukemia virus. This finding suggests that the first DNA-binding repeat may have an important role in cell growth control. We demonstrate that truncation of the first DNA-binding repeat of c-Myb is sufficient for myeloid transformation in culture, but deletion of the N-terminal phosphorylation site and adjacent acidic region is not. Truncation of the first repeat decreases the ability of a Myb-VP16 fusion protein to trans activate the promoter of a Myb-inducible gene (mim-1) involved in differentiation. Moreover, truncation of the first repeat decreases the ability of the Myb protein to bind DNA both in vivo and in vitro. These results suggest that N-terminal mutants of c-Myb may transform by regulating only a subset of those genes normally regulated by c-Myb.

The v-myb oncogene and its cellular homolog c-myb encode sequence-specific DNA-binding proteins which regulate transcription from promoters containing Myb-binding sites in animal cells. We have developed a Saccharomyces cerevisiae system to assay transcriptional activation by v-Myb and c-Myb. In yeast strains containing integrated reporter genes, activation was strictly dependent upon both the Myb DNA-binding domain and the Myb recognition element. BAS1, an endogenous Myb-related yeast protein, was not required for transactivation by animal Myb proteins and by itself had no detectable effect on a Myb reporter gene. Deletion analyses demonstrated that a domain of v-Myb C terminal to the previously mapped Myb transcriptional activation domain was required for transactivation in animal cells but not in S. cerevisiae. The same domain is also required for the efficient transformation of myeloid cells by v-Myb. In contrast to results in animal cells, in S. cerevisiae the full-length c-Myb was a much stronger transactivator than a protein bearing the oncogenic N- and C-terminal truncations of v-Myb. These results imply that negative regulation of c-Myb by its own termini requires an additional animal cell protein or small molecule that is not present in S. cerevisiae.

The v-myb oncogene of avian myeloblastosis virus (AMV) differs from its normal cellular counterpart by a truncation at both its amino and carboxyl termini and by a substitution of 11 amino acid residues. We had previously shown that v-myb-containing AMV, in the presence of basic fibroblast growth factor, transformed chicken neuroretina (CNR) cells. To understand the mechanism of c-myb activation, we have tested whether avian retroviruses that express the full-length c-Myb are also active on CNR cells. We have found that c-Myb, like v-Myb, strongly increases the basic fibroblast growth factor response of CNR cells and that these c-myb-expressing cells are able to grow in soft agar in the presence of the growth factor. We have also found that, in contrast to normal or v-myb-expressing AMV-transformed CNR cells, c-Myb-transformed cells express mim-1, a granulocyte-specific gene. However, normal v-Myb- and c-Myb-expressing CNR cells all express the pax-QNR gene, a newly described paired and homeobox-containing gene specifically expressed in the neuroretina. We conclude that, in contrast to what has been described for hematopoietic cells, overexpression of c-Myb is sufficient to activate gene expression and to induce an abnormal behavior of CNR cells.

The v-myb- and v-ets-containing E26 retrovirus induces the proliferation of chicken neuroretina (CNR) cells in minimal medium. Proliferation of E26 CNR cells is strongly stimulated by basic fibroblast growth factor (bFGF). The v-myb-containing avian myeloblastosis virus also induces the proliferation of infected CNR cells stimulated by bFGF. Both E26 CNR and avian myeloblastosis virus CNR cells are able to form colonies in soft agar in the presence of bFGF. This suggests that the v-myb product, a nuclear sequence-specific DNA-binding protein which activates gene expression in transient transfection assays, plays a role in the proliferative response of the infected CNR cells. To determine the structure-function relationships of P135gag-myb-ets and p48v-myb, we have used deletion mutants expressed in retroviral vectors and have analyzed their effect on CNR cell proliferation as well as their effect on the CNR cell response to bFGF. We show that v-ets is not required for bFGF stimulation but increases the proliferation of CNR cells in minimal medium. In the v-myb mutants, the gag sequences derived from the helper virus increase the potency of the myb gene. The carboxyl-terminal domain required for the growth and transformation of myeloid cells and needed for maximal trans-activation in transient DNA transfection assays in fibroblasts was not required for the growth and bFGF response of CNR cells. In contrast, the domain encompassing amino acids 240 to 301 (containing part of the transcriptional activation domain of v-myb) was absolutely required for the response of CNR cells to bFGF and could be functionally replaced by the carboxyl-terminal transcriptional activation domain of the VP16 protein of herpes simplex virus.

A biologically active myeloblastosis-associated virus (MAV) provirus was cloned from a bacteriophage recombinant library constructed from leukemic chicken myeloblast DNA. The restriction endonuclease map of this clone was consistent with that of a type 1 MAV (MAV-1). Interference assays of virus recovered from cultured chicken embryo fibroblasts after DNA transfection established that the provirus was infectious and confirmed that it belonged to avian retrovirus subgroup A (type 1). Antipeptide antibodies raised against the env-encoded carboxyl terminus of p48myb, the transforming protein of avian myeloblastosis virus, specifically immunoprecipitated the gp37env from quail cells transfected with MAV-1 proviral DNA but not from cells infected with MAV-2. This suggests that MAV-1 rather than MAV-2 is the progenitor helper virus from which avian myeloblastosis virus arose by the transduction of cellular proto-oncogene sequences.

The product of the v-myb oncogene of avian myeloblastosis virus is a nuclear protein with an associated DNA-binding activity. We demonstrated that the highly conserved amino-terminal domain of p48v-myb is required for its associated DNA-binding activity. This activity is not required for the nuclear localization of p48v-myb. Furthermore, the associated DNA-binding activity and nuclear localization of p48v-myb together are not sufficient for transformation.

Interleukin 2 (IL-2), a product of activated T lymphocytes but not of T-cell-deficient spleen cells of congenitally athymic (nu/nu) mice, is shown to be strongly mitogenic for spleen cells from both nu/nu and nu/+ mice in the absence of serum. This response does not depend on the presence of additional mitogen or antigen as has been previously reported and is dose dependent with respect to IL-2. Plots of the logarithm of cell number versus logarithm of response indicate that IL-2-induced mitogenesis of nu/nu spleen cells is a one-cell event (slope = 1.28 +/- 0.21, mean +/- SD), whereas the response of nu/+ spleen cells to concanavalin A is a three-cell event (slope = 3.18 +/- 0.16). The nu/nu spleen cells proliferating in response to IL-2 are at least 80% T lymphocytes by the third day of culture, as demontrated by lysis with monoclonal anti-Thy-1 antibody and complement. These results suggest that one of the major roles of the thymus may be to induce IL-2 production in a subpopulation of lymphocytes that is then persistent.

The defective acute leukemia viruses avian myeloblastosis virus (AMV) and E26 virus each contain an inserted cellular sequence related to the same highly conserved cellular gene, proto-amv. The oncogenes of these two retroviruses differ from this cellular proto-oncogene in gene structure, transcript structure, and gene product. The product of the AMV oncogene (myb) is a 48,000 Mr protein, p48myb, encoded by a transduced segment (amv) of proto-amv flanked by short helper-virus-derived terminal sequences. The E26 virus oncogene product is a 135,000 Mr protein, p135gag-amve-ets, encoded by significant portions of a viral structural gene (gag), sequences related to proto-amv (amve), and additional E26-specific sequences (ets) transduced from cellular proto-ets. Both p48myb and p135gag-amve-ets transforming proteins are located in the nucleus of cells transformed by these viruses. A protein of 110,000 Mr which is specifically immunoprecipitated by antisera to amv peptides and may be the product of the normal cellular gene (proto-amv) has been located in the cytoplasm of cells that express proto-amv mRNA.

Spleen cells from conventional BALB/c or athymic mice with streptozotocin (SZ)-induced hyperglycemia failed to raise blood sugar levels when injected into athymic or thymus-sufficient recipients. Passive transfer efforts were unsuccessful despite variations in donor-recipient pairs with respect to age, thymic function, or time after sensitization of donor mice. Athymic mice develop hyperglycemia following SZ but fail to mount an inflammatory lymphocyte infiltration. In contrast, the heterozygotes show a marked cellular response, which seems to follow the onset of hyperglycemia. The injection of spleen cells from thymus-sufficient mice to athymic recipients confers immunologic competence on the latter as tested by antibody formation to sheep erythrocytes.

The v-myb oncogene of avian myeloblastosis virus transforms myeloid cells exclusively, both in vivo and in vitro. The c-myb proto-oncogene from which v-myb arose is expressed at relatively high levels in immature hematopoietic cells of the lymphoid, erythroid, and myeloid lineages but not in myeloblasts transformed by v-myb. This finding suggested that the nuclear v-myb gene product p48v-myb might act directly to inhibit the normal expression of the c-myb gene. I have therefore used a selectable avian retroviral vector to express p48v-myb in avian erythroblasts which normally express high levels of the c-myb gene product p75c-myb. The results demonstrate that p48v-myb and p75c-myb can be coexpressed in the nuclei of cloned cells. Therefore, p48v-myb does not invariably prevent the expression of p75c-myb.

A flavokinase preparation from Bacillus subtilis is described which catalyzes the phosphorylation of reduced, but not oxidized, riboflavin. The enzyme is distinguished from other known flavokinases also in having an unusually low Km for the flavin substrate, 50 to 100 nM. ATP is the obligatory phosphate donor; one ATP is utilized for each FMNH2 formed. Mg2+ or Zn2+ is required for the reaction; Co2+ and Mn2+ will substitute, but less effectively. The same enzyme preparation catalyzes the synthesis of FADH2 from FMNH2 and ATP, but not the synthesis of FAD from FMN and ATP. FADH2 is also formed from reduced riboflavin, presumably by sequential flavokinase and FAD synthetase action. Zn2+ cannot replace Mg2+ in FADH2 formation. The reverse reaction, formation of FMN from FAD, occurs only with reduced FAD, giving rise to FMNH2, and is dependent on the presence of inorganic pyrophosphate. The enzyme thus appears to be an FADH2 pyrophosphorylase. The two enzymatic activities, flavokinase and FADH2 pyrophosphorylase, although not separated during the purification procedure, are distinguished by differences in metal ion specificity, in concentration dependence for ATP (apparent Km for ATP = 300 microM for FADH2 synthesis and 6.5 microM for flavokinase), and in the inhibitory effects of riboflavin analogues.

We demonstrated that molecular clones of the v-myb oncogene of avian myeloblastosis virus (AMV) can direct the synthesis of p48v-myb both in avian and mammalian cells which are not targets for transformation by AMV. To accomplish this, we constructed dominantly selectable avian leukosis virus derivatives which efficiently coexpress the protein products of the Tn5 neo gene and the v-myb oncogene. The use of chemically transformed QT6 quail cells for proviral DNA transfection or retroviral infection, followed by G418 selection, allowed the generation of cell lines which continuously produce both undeleted infectious neo-myb viral stocks and p48v-myb. The presence of a simian virus 40 origin of replication in the proviral plasmids also permitted high-level transient expression of p48v-myb in simian COS cells without intervening cycles of potentially mutagenic retroviral replication. These experiments establish that the previously reported DNA sequence of v-myb does in fact encode p48v-myb, the transforming protein of AMV.

The v-myb oncogene of avian myeloblastosis virus induces acute myeloblastic leukemia in chickens and transforms avian myeloid cells in vitro. The protein product of this oncogene, p48v-myb, is partially encoded by the retroviral gag and env genes. We demonstrated that the env-encoded carboxyl terminus of p48v-myb is not required for transformation. Our results showed, in addition, that a coding region of c-myb which is not essential for transformation was transduced by avian myeloblastosis virus.

The expression of p80c-myb was examined during the activation of resting human T lymphocytes. Before activation, no detectable p80c-myb was present. Synthesis of p80c-myb was observed only after initiation of the S phase of the cell cycle.

The genome of the replication-defective avian myeloblastosis virus (AMV) contains an inserted cellular sequence (amv) that is part of the oncogene responsible for acute myeloblastic leukemia in chickens infected with AMV. Three antisera raised against distinct synthetic peptides predicted from the long open reading frame of amv specifically precipitated the same 48-kilodalton protein (p48amv) from leukemic myeloblasts but not from normal hematopoietic tissue, fibroblasts, or from fibroblasts infected with the AMV helper virus, MAV-1 (myeloblastosis-associated virus type 1). p48amv is not glycosylated or phosphorylated and does not appear to act as a protein kinase in vitro. The same three antisera that recognized p48amv also specifically precipitated a common 110-kilodalton protein from normal uninfected hematopoietic tissue. This normal cellular homologue of the AMV leukemogenic protein, p110proto-amv, was not present in normal fibroblasts, MAV-1 infected fibroblasts, or, interestingly, in some leukemic myeloblasts. We conclude that p48amv is the leukemogenic product of an altered, transduced, partial protooncogene. Short helper-virus sequences provide its carboxyl terminus and also may provide the amino terminus.

Antibodies directed against a bacterial fusion protein that contains the domain encoded by the highly evolutionarily conserved 5' one-third of the v-myb oncogene of avian myeloblastosis virus (AMV) detect the protein products of various members of the myb gene family. Immunoprecipitation or immunoblot analyses with these antibodies yielded the following information. First, the products of the v-myb oncogenes of AMV (p48v-myb) and of E26 virus (p135gag-myb-ets) contain this highly conserved amino acid sequence, as previously hypothesized. Second, p75c-myb, the product of the chicken c-myb protooncogene, also contains this protein domain. Third, these antibodies have identified the products of the human, murine, and Drosophila c-myb genes, which were all found to be nuclear proteins of Mr 75,000-80,000. The human c-myb protein product is present in immature cells of the erythroid, myeloid, and lymphoid lineages.

The expression of p80c-myb was examined during the activation of resting human T lymphocytes. Before activation, no detectable p80c-myb was present. Synthesis of p80c-myb was observed only after initiation of the S phase of the cell cycle.

Summary Two chicken lectins with similar sugar specificities are mitogenic for nonadherent, Thy-1 negative mouse spleen cells in serum-free cultures. This effect appears to be due to the sugar-binding properties of these lectins since asialofetuin and lactose, specific inhibitors of lectin activity, inhibit mitogenesis. Among other lectins with similar sugar specificity, purpurin, the lectin from Dictyostelium purpureum , is also mitogenic for these cells whereas peanut lectin is not. The mitogenic lectins work similarly with spleen cells from athymic (nu/nu) mice and their heterozygote (nu/+) littermates.

The protein product of the v-myb oncogene of avian myeloblastosis virus, p48v-myb, differs structurally in several ways from its normal cellular homolog, p75c-myb. We demonstrated that the 11 specific amino acid substitutions found in two independent molecular clones of this virus were not required for the transformation of myeloblasts by v-myb.

The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in vivo and transforms only myeloid cells in vitro. Its product, p48v-myb, is a nuclear protein of unknown function. To determine structure-function relationships for this protein, we constructed a series of deletion mutants of v-myb, expressed them in retroviral vectors, and studied their biochemical and biological properties. We used these mutants to identify two separate domains of p48v-myb which had distinct roles in its accumulation in the cell nucleus. We showed that the viral sequences which normally encode both termini of p48v-myb were dispensible for transformation. In contrast, both copies of the highly conserved v-myb amino-terminal repeat were required for transformation. We also identified a carboxyl-terminal domain of p48v-myb which was required for the growth of v-myb-transformed myeloblasts in soft agar but not for morphological transformation.

The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its protein product p48v-myb is a nuclear, sequence-specific, DNA-binding protein which activates gene expression in transient DNA transfection studies. To investigate the relationship between transformation and trans-activation by v-myb, we constructed 15 in-frame linker insertion mutants. The 12 mutants which transformed myeloid cells also trans-activated gene expression, whereas the 3 mutants which did not transform also did not trans-activate. This implies that trans-activation is required for transformation by v-myb. One of the transformation-defective mutants localized to the cell nucleus but failed to bind DNA. The other two transformation-defective mutants localized to the cell nucleus and bound DNA but nevertheless failed to trans-activate. These latter mutants define two distinct domains of p48v-myb which control trans-activation by DNA-bound protein, one within the amino-terminal DNA-binding domain itself and one in a carboxyl-terminal domain which is not required for DNA binding.

Cellular oncogenes comprise a small family of genes, highly conserved throughout vertebrate evolution, that code for proteins with diverse functions including DNA binding, protein kinase, and cellular growth factor activities. Cellular oncogenes are important in certain aspects of the proliferation and differentiation of normal cells. Under some circumstances these genes may also induce malignant transformation of normal cells. Various mechanisms may underlie their involvement in carcinogenesis. Incorporation of all, or part of, cellular oncogenes into RNA tumor viruses, mutations in gene structure, or translocation of cellular oncogenes from one chromosome to another may all be associated with the induction of malignant change in cells. In some of these situations altered oncogene products are made. Knowledge about the biology of oncogenes may lead to improved techniques for cancer detection and perhaps new approaches to cancer treatment.

The c-Myb protein is a transcriptional regulator initially identified by homology to the v-Myb oncoprotein, and has since been implicated in human cancer. The most highly conserved portion of the c-Myb protein is the DNA-binding domain which consists of three imperfect repeats. Many other proteins contain one or more Myb-related domains, including a number of proteins that do not bind directly to DNA. We performed a phylogenetic analysis of diverse classes of Myb-related domains and discovered a highly conserved patch of acidic residues common to all Myb-related domains. These acidic residues are positioned in the first of three alpha-helices within each of the three repeats that comprise the c-Myb DNA-binding domain. Interestingly, these conserved acidic residues are present on a surface of the protein which is distinct from that which binds to DNA. Alanine mutagenesis revealed that the acidic patch of the third c-Myb repeat is essential for transcriptional activity, but neither for nuclear localization nor DNA-binding. Instead, these acidic residues are required for efficient chromatin binding and interaction with the histone H4 N-terminal tail.