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Expression of the protooncogene A-myb is restricted to the developing CNS, adult testes, breasts in late pregnancy, and germinal centers of secondary B cell follicles. The functional relevance of A-myb expression at three of these sites has been demonstrated previously via the generation and analysis of A-myb-deficient mice, which display behavioral abnormalities, male sterility, and perturbed breast development during pregnancy. In contrast, here we show that the germinal center response driven by T cell-dependent Ag immunization and the associated processes of Ab V gene somatic hypermutation, affinity maturation, and heavy chain class switching are overtly normal in A-myb-deficient mice. Nonetheless, these mice display mild splenic white pulp hypoplasia and blunted primary serum Ab responses, suggesting that although A-myb is not directly involved in the regulation of the memory B cell response, it may play a role in enhancing peripheral B cell survival or proliferative capacity.

Cytoplasmic Janus protein tyrosine kinases (JAKs) are crucial components of diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. Evidence to date, indicates that JAK kinase function may integrate components of diverse signaling cascades. While it is likely that activation of STAT proteins may be an important function attributed to the JAK kinases, it is certainly not the only function performed by this key family of cytoplasmic tyrosine kinases. Emerging evidence indicates that phosphorylation of cytokine and growth factor receptors may be the primary functional attribute of JAK kinases. The JAK-triggered receptor phosphorylation can potentially be a rate-limiting event for a successful culmination of downstream signaling events. In support of this hypothesis, it has been found that JAK kinase function is required for optimal activation of the Src-kinase cascade, the Ras-MAP kinase pathway, the PI3K-AKT pathway and STAT signaling following the interaction of cytokine/interferon receptors with their ligands. Aberrations in JAK kinase activity, that may lead to derailment of one or more of the above mentioned pathways could disrupt normal cellular responses and result in disease states. Thus, over-activation of JAK kinases has been implicated in tumorigenesis. In contrast, loss of JAK kinase function has been found to result in disease states such as severe-combined immunodeficiency. In summary, optimal JAK kinase activity is a critical determinant of normal transmission of cytokine and growth factor signals.

Diabetes mellitus ensues as a consequence of the body's inability to respond normally to high blood glucose levels. The onset of diabetes is due to several pathological changes, which are a reflection of either the inability of the pancreatic beta cells to secrete sufficient insulin to combat the hyperglycemia or a state of insulin resistance in target tissues. However, the significance of changes in beta cell mass and decreased beta cell proliferation or growth in progression of diabetes has been under-appreciated. Beta cells, like all other cells of our body are under the regulatory checks and balances enforced by changes in cell cycle progression. However, very little is known regarding the key components of the cell cycle machinery regulating cell cycle control of beta cells. Knowledge of key elements involved in cell cycle regulation of beta cells will go a long way in improving our understanding of the replication capacity and developmental biology of beta cells. This information is essential for us to design new approaches that can be used to correct beta cell deficiency in diabetes. This review focuses on the current knowledge of factors important for proliferation of beta cells and proposes a cell cycle model for regeneration of the beta cell population lost or reduced in diabetes.

To ascertain the role of cyclin-dependent kinase 4 (Cdk4) in vivo, we have targeted the mouse Cdk4 locus by homologous recombination to generate two strains of mice, one that lacks Cdk4 expression and one that expresses a Cdk4 molecule with an activating mutation. Embryonic fibroblasts proliferate normally in the absence of Cdk4 but have a delayed S phase on re-entry into the cell cycle. Moreover, mice devoid of Cdk4 are viable, but small in size and infertile. These mice also develop insulin-deficient diabetes due to a reduction in beta-islet pancreatic cells. In contrast, mice expressing a mutant Cdk4 that cannot bind the cell-cycle inhibitor P16INK4a display pancreatic hyperplasia due to abnormal proliferation of beta-islet cells. These results establish Cdk4 as an essential regulator of specific cell types.

Interaction of IL-3 with its receptor is known to activate STAT-3 via phosphorylation of Tyrosine 701, which facilitates its dimerization and translocation to the nucleus, leading to the transcription of its target genes. In this communication, we have investigated the nature of tyrosine kinases that mediate STAT-3 phosphorylation during IL-3-mediated activation of myeloid cell proliferation. Our results show that interaction of IL-3 with its receptor leads to the activation of c-Src kinase activity, which in turn facilitates the binding of c-Src to STAT-3. This association leads to the phosphorylation of STAT-3, allowing this transcription factor to translocate to the nucleus. Expression of a dominant negative mutant of src (AMSrc) in these cells results in a block to IL-3 mediated phosphorylation of STAT-3, and its ability to bind to DNA. On the other hand, expression of a dominant negative mutant of JAK2 (JAK2KE) had no effect on IL-3-mediated activation of STAT-3. Our results also show that AMSrc does not affect the phosphorylation of JAK2, suggesting that JAK and STAT phosphorylation events are mediated by two independent pathways. Inhibition of c-Src activation by AMSrc, which leads to a block to STAT-3 activation, results in a dramatic inhibition of cell proliferation mediated by IL-3. However, expression of AMSrc does not activate apoptotic pathways. In contrast, expression of JAK2KE results in accelerated apoptosis of 32Dcl3 cells grown in the absence of IL-3 with concomitant down-regulation of Erk-2 kinase activity. These results suggest that Src family kinases mediate the phosphorylation of STATs and play a critical role in signal transduction pathways associated with myeloid cell proliferation while JAK kinases mediate the activation of Erk-2 pathway which appears to provide antiapoptotic signals. Thus the activation of JAKs and STATs appear to be two independent but related events, which dictate two separate biological outcomes, the combination of which results in proliferation and survival of myeloid precursor cells.

The myb gene family consists of three members, named A-, B-, and c-myb. All three members of this family encode nuclear proteins that bind DNA in a sequence-specific manner and function as regulators of transcription. In this report, we have examined the biochemical and biological activities of murine B-myb and compared these properties with those of murine c-myb. In transient transactivation assays, murine B-myb exhibited transactivation potential comparable to that of c-myb. An analysis of deletion mutants of B-myb and c-myb showed that while the C-terminal domain of c-Myb acts as a negative regulator of transcriptional transactivation, the C-terminal domain of B-Myb functions as a positive enhancer of transactivation. To compare the biological activities of c-myb and B-myb, the two genes were overexpressed in 32Dcl3 cells, which are known to undergo terminal differentiation into granulocytes in the presence of granulocyte colony-stimulating factor (G-CSF). We observed that c-myb blocked the G-CSF-induced terminal differentiation of 32Dcl3 cells, resulting in their continued proliferation in the presence of G-CSF. In contrast, ectopic overexpression of B-myb blocked the ability of 32D cells to proliferate in the presence of G-CSF and accelerated the G-CSF-induced granulocytic differentiation of these cells. Similar studies with B-myb-c-myb chimeras showed that only chimeras that contained the C-terminal domain of B-Myb were able to accelerate the G-CSF-induced terminal differentiation of 32Dcl3 cells. These studies show that c-myb and B-myb do not exhibit identical biological activities and that the carboxyl-terminal regulatory domain of B-Myb plays a critical role in its biological function.

The myb gene family consists of three members, named A-, B-, and c-myb, which encode nuclear proteins that bind to DNA and function as regulators of transcription. Our results show that murine A-myb is a poor transactivator of transcription compared with murine c-myb. Deletion of the COOH-terminal domain of A-Myb, or co-expression with Ets-2 resulted in increased transactivation potential. While ectopic overexpression of c-myb in 32Dcl3 cells results in a block to the ability of these cells to undergo terminal differentiation resulting in indefinite growth in granulocyte-colony-stimulating factor (G-CSF), similar overexpression of A-myb results in growth arrest and concomitant terminal differentiation of 32D cells into granulocytes. Co-expression of A-myb and ets-2 in these cells results in the restoration of the proliferative activity of the cells in G-CSF, but fails to induce a block to G-CSF-induced terminal differentiation. However, overexpression of the COOH-terminal deletion mutant of A-myb results in a block to G-CSF-induced differentiation of 32D cells, suggesting that the distinctive biological phenotypes produced by A-myb and c-myb genes are mediated by their COOH-terminal domains.

The Myb gene family currently consists of three members, named A-, B- and c-myb. These genes encode nuclear proteins that bind DNA in a sequence-specific manner and function as regulators of transcription. In adult male mice, A-myb is expressed predominantly in male germ cells. In female mice, A-myb is expressed in breast ductal epithelium, mainly during pregnancy-induced ductal branching and alveolar development. We report here that mice homozygous for a germline mutation in A-myb develop to term but show defects in growth after birth and male infertility due to a block in spermatogenesis. Morphological examination of the testes of A-myb-/- males revealed that the germ cells enter meiotic prophase and arrest at pachytene. In adult homozygous null A-myb female mice, the breast epithelial compartment showed underdevelopment of breast tissue following pregnancy and the female mice were unable to nurse their newborn pups. These results demonstrate that A-myb plays a critical role in spermatogenesis and mammary gland development.

In this study, we have isolated the genomic DNA clone encoding the murine JAK3 gene and determined its sequence. Partial genomic clones of the JAK1 and JAK2 genes encoding the tyrosine kinase domain were also isolated and compared with JAK3. The genomic structure of JAK3 consists of 23 exons. The exon/intron boundaries and the distribution of coding sequences within the exons of each of the three JAK genes were found to follow a similar pattern suggesting that various members of the JAK family have originated from a single primordial gene by duplication and the structure has been closely maintained through evolution. Using in situ hybridization and FISH analysis, we have mapped the JAK3 gene to human chromosome 19p13.1-p13.2.

The myb gene has been shown to be an important regulator of hematopoietic cell proliferation, differentiation and apoptosis. Activation of the myb gene into an oncogenic form has involved structural alterations to the coding sequences. Thus, the v-myb gene encoded by the Avian Myeloblastosis Virus, is truncated at both the 5' and 3' ends. Additionally, tumor cells containing rearrangements in the myb locus, such as the ABPL tumors or NFS60 tumor cell line have recently been shown to display a heterogeneity of structure. In this study, we examined the growth and differentiation properties of clonal cell lines derived from 32Dcl3 which harbor myb transgenes; derived from v-myb, and the ABPL-1, ABPL-2, ABPL-4 and NFS-60 cell lines. Retroviral vectors containing the appropriate myb cDNAs were produced, transfected into packaging cell lines, and the viruses were used to generate the 32D derivative cell clones. Abrogation of IL-3 dependence was never observed in any cell line. Expression of c-myb, ABPL-1-myb and ABPL-2-myb isoforms in 32D cells resulted in a block to their ability to terminally differentiate into granulocytes at the pro-myelocytic stage. However, expression of ABPL-4-myb or NFS60-myb in these cells failed to result in a similar effect. These cells differentiated into granulocytes in the presence of G-CSF, albeit more slowly than control 32Dcl3 cells. We also examined the ability of various Myb-isoforms to transactivate transcription of reporter genes containing Myb-binding elements in their promoter/enhancer sequences, to determine whether the phenotypic effects produced by these various isoforms correlate with their ability to transactivate transcription. Our results show that while v-myb and c-myb transactivated transcription equally well, the NFS60-myb exhibited the highest levels of transcriptional transactivation. The ABPL-1, ABPL-2 and ABPL-4-myb isoforms showed very low levels of transcriptional transactivation potential with the same reporter genes. These results suggest that the ability of various Myb-isoforms to transactivate transcription does not by itself correlate with their ability to induce a block to G-CSF-induced terminal differentiation of myeloid precursor cells.

The ABPL tumor cell lines represent a group of myeloid cell lines which contain an altered myb locus due to viral insertional mutagenesis within the third exon of c-myb. Immunoprecipitation analysis of the proteins produced in three ABPL lines revealed an interesting anomaly. Despite the invariant position of the virus integration event, the three ABPL tumor cell lines we examined (ABPL-1, ABPL-2 and ABPL-4) produced three different sized proteins. In this report, we examined the molecular basis for this protein size heterogeneity. Molecular cloning and sequence analysis of the cDNAs derived from the myb transcripts show that ABPL-1 tumor produces a tripartate mRNA containing sequences derived from the viral gag and env genes fused to the myb coding region. This results in the synthesis of a 74 kd protein. In the ABPL-2 tumor line, a gag-myb fusion protein is produced which is of 68 kd. In ABPL-4 cell line a gag-myb fusion protein is produced which contains an internal deletion of coding sequences derived from exons 13 and 14. This deletion results in the synthesis of a 59 kd protein in ABPL-4 tumor cell line. These observations were further confirmed by RNase protection assays which demonstrate the presence of aberrantly spliced mRNAs in ABPL-1 and ABPL-4 tumor cells but not in cells containing an undisrupted c-myb locus. In vitro translation and immuno-precipitation analysis of the cRNAs derived from the ABPL-1, ABPL-2 and ABPL-4 cDNAs show the synthesis of protein products that were identical to Myb proteins produced by these tumors in vivo. These results suggest that integration of Mo-MuLV within the c-myb locus not only results in deletions of the 5' end of the transcript but splicing aberrations within the encoded mRNA, which results in the synthesis of a heterogeneous array of proteins, not seen in normal hematopoietic cells.

We recently reported the cloning and sequencing of the mouse A-myb proto-oncogene cDNA and the abundant expression of this mRNA primarily in the testis of adult mice. The A-myb mRNA is detectable by in situ hybridization specifically in the spermatogenic cells, and is downregulated during terminal differentiation. A low level of expression is observed in a few other tissues, including ovary, spleen and brain. We have extended those studies by examining A-myb and B-myb expression during testis development in the mouse. The A-myb and B-myb genes are both expressed in a cell- and stage-specific manner during testis development. The B-myb mRNA is expressed most highly in gonocytes of the fetal testis and in spermatogonia and early spermatocytes in the adult. B-myb expression decreases at day 18 post partum, coincident with the initial appearance of late pachytene spermatocytes. B-myb expression was also detectable in some interstitial cells of the fetal and adult testis. The A-myb mRNA was not detectable by in situ hybridization in fetal day 15.5 gonocytes but was detectable at a low abundance by RT-PCR in fetal and newborn mice. A-myb mRNA expression increased at post-natal day 10, when primary spermatocytes first appear. In the adult, the A-myb mRNA was expressed highly in a sub-population of spermatogonia and in primary spermatocytes, but was not detectable in spermatids. This expression of A-myb is consistent with the meiotic arrest that is observed in A-myb-deficient male mice. We conclude that B-myb may play a critical role in controlling the proliferation or differentiation of gonocytes and spermatogonia and possibly the somatic lineages as well, whereas A-myb is required for progression through the first meiotic prophase. These distinct roles for B-myb and A-myb during spermatogenesis may reflect distinct transactivation potentials of the two proteins. Further studies to determine the functions of A-myb and B-myb in the developing testis should improve our understanding of the molecular events associated with spermatogenesis and differentiation of the Sertoli and other somatic cell types of the testis.

The myb gene family consists of three members, A-myb, B-myb and c-myb. The proteins encoded by these genes bind DNA in a sequence-specific manner and regulate transcription of target genes. In this communication, we report the nucleotide sequence of murine A-myb cDNA. This gene encodes a protein of 751 amino acids with an estimated molecular weight of 83 kDa. cDNA sequence analysis of multiple independent cDNA clones reveals the presence of alternatively spliced mRNAs that encode smaller proteins. Northern blot analysis of adult mouse tissue RNAs reveals A-myb expression predominantly in the testis, with very low levels of expression in the ovaries, spleen and brain. In situ hybridization analysis of adult mouse testis shows that this gene is expressed at high levels in type A spermatogonia (stem cells), and preleptotene and pachytene spermatocytes, with concomitant down-regulation of expression upon terminal differentiation of these cells into mature spermatozoa. This pattern of expression suggests that A-myb is involved in the control of proliferation and differentiation of spermatogonia. This potential function of A-myb in spermatogenesis is reminiscent of the role of c-myb in hematopoiesis.

The Janus Kinases (JAK) JAK1, JAK2, and TYK2 are protein tyrosine kinases which play a pivotal role in the signal transduction process mediated by cytokines. These kinases appear to transduce signals via their substrates which modulate programs of gene expression specific to the respective signals. It is becoming increasingly evident that certain cytokines such as Granulocyte Colony Stimulating Factor (GCSF) can transmit signals for both cellular proliferation and differentiation. It is at present unclear whether both of these signals are transmitted by the same JAK kinase or whether an entire family of such kinases are involved in this process. To determine if additional members of JAK kinase family exist, we designed a polymerase chain reaction based strategy which resulted in the identification of a new member of the JAK kinase family. This new kinase, which we have named JAK3 is encoded by a 4.3 kb mRNA transcript. Nucleotide sequence analysis of a full length cDNA derived from this mRNA revealed that it encodes an open reading frame of 3897 bp. The protein encoded by this mRNA contains the double catalytic domain characteristic of the JAK family kinases. The most striking difference between JAK3 and the other JAK kinases is the presence of two stretches of additional amino acid sequence of 147 and 28 residues which span between amino acid positions 322 to 469 and 632 to 660 respectively. Expression studies indicate that JAK3 is expressed at very low levels in immature hematopoietic cells, but its expression is dramatically up-regulated during terminal differentiation of these cells. These results suggest that JAK3 plays an important role in the differentiation of hematopoietic cells.

The myb oncogene encodes a DNA-binding transcriptional transactivator which can become a hematopoietic cell-transforming protein following the deletion of amino acid sequences from either its amino or carboxyl terminus. Although a number of hematopoietic tumors express terminally deleted variants of Myb, the involvement of truncated Myb in nonhematopoietic tumors has not been adequately investigated. To assess the full spectrum of Myb's oncogenic capability, a replication-competent retroviral vector (RCAMV) was used to express a full-length protein (C-Myb), an amino-terminally truncated protein (VCC- or delta N-Myb), a carboxyl-terminally truncated protein (T-Myb), or a doubly truncated protein (VCT-Myb) in vivo. These viruses were injected intravenously into 10-day chicken embryos, and the infected chicks were monitored for tumors. Approximately 4 to 8 weeks after hatching, the majority (30 of 39 [77%]) of animals infected with the T-Myb retrovirus (without 214 carboxyl-terminal residues) developed nodular muscle tumors which could be identified by both morphologic and immunohistochemical criteria as fibrosarcomas. Identically appearing tumors could also be found in the kidney of some T-Myb-infected animals. The T-Myb-induced fibrosarcomas expressed the appropriately sized T-Myb protein, contained an unaltered proviral T-myb gene, and showed clonal proviral integration sites. In comparison, no sarcomas were observed in any of the animals infected with the amino-terminally truncated (VCC- and delta N-Myb) or doubly truncated (VCT-Myb) viruses. A loss of carboxyl-terminal but not amino-terminal sequences can thus convert Myb into a potent in vivo transforming protein for nonhematopoietic mesenchymal cells. In comparison, a truncation of either or both ends of the protein can activate Myb into a hematopoietic cell-transforming protein.

The nuclear oncogene v-myb and its cellular counterpart c-myb code for proteins that bind to DNA in a sequence specific manner and act as regulators of transcription. The Myb protein contains DNA binding and trans-regulatory domains which are important for its function. The DNA binding domain of Myb protein has been shown to contain three imperfectly conserved repeats of 50-52 amino acids that constitute the amino terminal end. In this communication, we show that Myb protein induces conformational change in DNA after protein-DNA complex formation. Circular permutation assays indicate that Myb protein induces DNA bending at the site of binding. Phasing analysis confirm the DNA bending and allowed the detection of relative orientation of bend. Myb proteins which comprise only DNA-binding domains either with three repeats or two repeats also bend DNA in the same orientation as the larger proteins with both DNA-binding and transactivating domains. However, the transactivating region seems to influence the magnitude of bend angle. We used molecular modeling to analyse the structure of Myb-DNA complex formation resulting in the bending of DNA. Data presented here show that Myb protein, like other transcriptional regulators, bends DNA upon binding allowing the interaction of regulatory elements.

The c-myb gene products are thought to be regulators of cellular replication and of differentiation and heterogeneity may underlie their multiple functions. To investigate the possible existence of heterogeneity we have examined the chicken c-myb mRNAs by Northern blot analysis and polymerase chain reaction amplification of cDNAs (RT-PCR). Northern blot analysis with the c-myb cDNA clone pSG3, which contains the entire open reading frame (ORF) plus 500 base pairs of 3' untranslated sequences (Gerondakis & Bishop, 1986), and genomic probes revealed c-myb RNA species of 4.3 kb in addition to the major 4.0 kb species. The 4.3 kb c-myb RNA contained the alternatively spliced exon 9A which is highly conserved and has also been detected in a minor 4.3 kb alternatively spliced c-myb mRNA in murine and human cells. Sequencing of the avian exon 9A revealed 360 bp exon homologous to that found in murine and human mRNAs, which contains three highly conserved sequence regions shared by all three species. RT-PCR demonstrated usage of exon 9A in five hematopoietic tissues and revealed an additional splicing variant which used the 3' portion of exon 9A. Northern blot analysis using splice site-specific oligonucleotide probes spanning the two splice junctions between exon 9 and 9A revealed four additional c-myb RNAs of 4.4 kb, 2.2 kb, 2.0 kb and 1.4 kb.

In the generation of the acutely transforming avian retrovirus E26, both myb and ets genes have been transduced, leading to the production of a Gag-Myb-Ets fusion protein. This co-occurrence of v-myb and v-ets oncogenes suggests that the two might have a functional relationship. To look for such a relationship, we tested the transcriptional activation activity of Myb alone or with coexpressed Ets-1 or Ets-2. Using the promoter of the v-Myb-inducible mim-1 gene as a target, we found that full-length c-Myb gene products were poor activators of transcription, while an oncogenic (truncated) form of this protein was a strong trans-activator. However, coexpression of Ets-2 with full-length or truncated forms of Myb greatly increased trans-activation. Coexpression of Ets-1, Fos, Jun, or Myc with Myb did not increase trans-activation of the mim-1 promoter. The ability of Myb and Ets-2 to transactivate was cooperative, since Ets-2 alone gave little or no activation. Bacterially synthesized Ets-2 protein was found to bind specifically to the mim-1 promoter, suggesting that it may be a target for both Myb and Ets proteins. Thus, Myb and Ets proteins can cooperate in transcriptional activation, and their co-occurrence in the E26 virus may reflect a functional relationship between these two oncoproteins. Truncated forms of Myb may have a reduced need for cooperating factors such as Ets-2, and this might constitute an important mechanism associated with oncogenic activation.

The proto-oncogene c-myb encodes a nuclear transcription factor that binds to DNA in a sequence-specific manner and activates transcription of several viral and cellular genes. Expression of the c-myb gene is induced in mitogen- and/or antigen-stimulated T lymphocytes, which are also the preferential target cells of human T-lymphotropic virus type I (HTLV-I) in vivo and in vitro. We report here that Myb binds to the HTLV-I long terminal repeat (LTR) in four different regions in a sequence-specific manner. Electrophoretic mobility shift assay using labeled LTR fragments as well as labeled double-stranded oligonucleotides show that there are two high-affinity and two low-affinity Myb-binding sites present in the HTLV-I LTR. DNase I footprinting analysis and oligonucleotide competition experiments indicate that this binding is sequence specific. Cotransfection experiments in HeLa cells, using a Myb expression vector and chloramphenicol acetyltransferase reporter gene linked to the HTLV-I LTR, show that Myb activates HTLV-I LTR-mediated transcription by a factor of four-to sixfold. Thus, in HTLV-I-infected T cells, Myb protein binding to the HTLV-I LTR may constitute one of the signal that regulate HTLV-I transcription in vivo.

The c-myb protooncogene codes for a sequence-specific DNA-binding protein that appears to act as a transcriptional regulator and is highly conserved through evolution. The DNA-binding domain of Myb has been shown to contain three imperfectly conserved repeats of 52 amino acids that constitute the amino-terminal end. Within each repeat, there are three tryptophans that are separated by 18 or 19 amino acids and are flanked by basic amino acids. To determine the role of tryptophans and the flanking basic amino acids in the DNA-binding activity of Myb proteins, we have selectively mutagenized individual tryptophans as well as some of the amino acid residues that flank these tryptophans. Replacement of these tryptophans with glycine, proline, or arginine abolished the DNA-binding activity whereas replacement with other aromatic amino acids or leucine or alanine did not appreciably affect this activity. On the other hand the replacement of two amino acids, asparagine and lysine, that flank the last tryptophan with acidic amino acids completely abolished their DNA-binding activity. These results are consistent with a model we present in which the tryptophans form a hydrophobic scaffold that plays a crucial role in maintaining the helix-turn-helix structure of the DNA binding domain. Basic and polar amino acids adjacent to these tryptophans seem to participate directly in DNA binding.

Activation of the c-abl protooncogene occurs in Abelson murine leukemia virus, in Hardy-Zuckerman 2 feline sarcoma virus, and during the chromosomal translocations that generate BCR-ABL gene fusion products. To study the molecular mechanism involved in the c-abl activation, we have created a series of modifications in murine c-abl and assayed these constructs for oncogenic activity using the NIH 3T3 cell transformation assay. Our results show that amino-terminal deletions are sufficient for oncogenic activation of c-abl and high levels of oncogenic activities were generated by a deletion of 114 codons from the 5' end that deleted the SH3 region. A deletion of 53 codons from the 5' end (inclusive of deletions seen in Hardy-Zuckerman 2 feline sarcoma virus and BCR-ABL gene products) that retains the SH3 region of c-abl resulted in the generation of low levels of transforming activity. This transforming potential was substantially increased with the introduction of a G----A point mutation in codon 832 that is present in v-abl. The point mutation was found to affect the secondary structure and the tyrosine kinase activity of the mutant gene products.

Among a series of myeloid leukemia cell lines, one (NFS-60) was found to have a rearrangement of the c-myb locus. The rearrangement involved the integration of a retrovirus into the region of the gene corresponding to the sixth exon of the avian c-myb locus. The insertion is associated with the production of a truncated RNA and the introduction of a terminator codon at the juncture of the long terminal repeat and the c-myb locus. The properties of the NSF-60 cells were compared with those of other myeloid cell lines, and the known sequence of differentiation induced by interleukin 3. Similar to other myeloid cell lines, the NFS-60 cells do not terminally differentiate in response to interleukin 3, granulocyte/macrophage, or granulocyte colony-stimulating factor suggesting that the cells are transformed with regard to their ability to differentiate. The NFS-60 cells are totally dependent on interleukin 3 for growth and maintenance of viability in vitro but also proliferate in response to granulocyte colony-stimulating factor. The properties of the cells support the concept that the c-myb protooncogene is involved in the control of normal differentiation of hematopoietic cells.

Activation of the abl gene and its involvement in human leukemia is one of the most thoroughly characterized examples of the structural alterations of chromosomes associated with the conversion of a normal cell into a cancer cell. The abl oncogene as first identified on the Abelson murine leukemia virus (A-MuLV). Activation of the viral oncogene is associated, in part, with the truncation of the gene at its 5' end. As in studies with other retroviruses, results with A-MuLV presaged the mechanism of activation by abl in naturally occurring human malignancies. Thus, chronic myelogenous leukemia (CML) is consistently associated with a translocation of a piece of chromosome 9 onto chromosome 22 creating what is known as the Philadelphia chromosome (Ph1). The result of this translocation is the truncation of the 5' end of the cellular abl gene, which is located at the breakpoint of chromosome 9. The function of the abl gene product is poorly understood but is thought to participate in an, as yet, undefined pathway of growth control signals, which originate outside the cell, and traverse through the cell into its nucleus. The loss of the gene product's N-terminal amino acid sequences brought about by the truncation of the 5' portion of the gene is consistent with the hypothesis that the protein's growth-controlling activity is deregulated by the structural alterations which occur in the cancer cells. The abl gene and CML serve as a paradigm of the mechanism of activation of proto-oncogenes by chromosomal alterations. The case of CML and the Ph1 chromosome illustrates the findings we might expect as other chromosomal abnormalities are characterized at the molecular level.

Nonrandom involvement of chromosome 6 in cutaneous malignant melanoma have been noted by several investigators. Recently an alteration in the c-myb locus (6q22-23) has been identified by Southern analysis in the WM983A cell line which was derived from a primary melanoma of the vertical growth phase. In the present study, the nature of this rearrangement in the WM983A cell line has been further characterized by molecular cloning and nucleotide sequence analysis of the break-point region in the c-myb locus. The results of this investigation demonstrate that the rearrangement in the 6q22-23 region results in deletion of the 3'-end of the c-myb locus with the concomitant translocation of a portion of chromosome 12 to chromosome 6.

The molecular mechanisms that modulate c-myb mRNA levels in hematopoietic cells appear to involve premature termination of transcription in the first intron of the gene. We have examined the DNA-protein interactions within the first intron of the c-myb gene and identified a 1.0-kilobase region that could be responsible for its transcriptional regulation. Using the mobility-shift assay, we show a direct correlation between the extent of sequence-specific protein binding to intron 1 DNA fragments, and c-myb mRNA levels in different cell types. During dimethyl sulfoxide-induced differentiation of mouse erythroleukemic cells, there was a dramatic decrease in these nuclear factors that correlated with the decrease in the levels of c-myb mRNA. Nucleotide sequence analysis and DNase I footprinting revealed the presence of putative regulatory elements that are implicated in the binding of these nuclear factors. We propose that binding of nuclear factors to the site of transcriptional pause could play an important role in the regulation of c-myb transcription.

Using a panel of anti-myb antibodies, we have examined the c-myb proteins present in the NFS-60 and ABPL-1, ABPL-2, and ABPL-4 tumor cell lines, all of which have an altered myb locus due to viral insertional mutagenesis. As predicted from previous DNA and RNA analysis, NFS-60 cells produce myb protein with a truncated C-terminus and a normal N-terminus. The three ABPL tumor cell lines studied here were previously shown to have undergone similar rearrangements towards the 5' end of myb locus and were expected to synthesize identical myb RNAs and proteins. These cell lines were found to produce myb proteins with N-terminal modifications. Surprisingly, however, the three cell lines were found to synthesize a heterogenous array of myb proteins of different sizes. These observations suggest that oncogenic activation of myb in ABPL tumors may involve additional changes in the coding region in addition to those occurring at the N-terminus.

The c-abl gene codes for a protein-tyrosine kinase and is expressed in most examined murine cell types as two distinct mRNA species of 5.5 kilobases (kb) and 6.5 kb. In mouse testis, an additional species of 4.0 kb is expressed in very high levels. To study the interrelationship between various c-abl transcripts and to compare their sequence with the v-abl transcript, we prepared c-abl-specific cDNA clones from mouse testis and determined the complete nucleotide sequence of the 4.0-kb cDNA that appears to be the reverse transcript of the testis-specific mRNA. In addition, we have determined the 3' sequence of an additional clone derived from the larger mRNA species that is expressed in somatic as well as germ-line cells. These cDNA sequences have been compared with the v-abl sequences to understand the mechanism of activation of this oncogene. The results demonstrate that (i) testis-specific c-abl mRNAs arise as a result of 3' truncation, and (ii) the v-abl gene has arisen from its cellular homologue as a result of an extensive deletional/mutational process.

The murine c-myb gene has been recently shown to code for two protein products of 75kd and 89kd. The 89kd protein appears to be generated from an alternatively spliced mRNA which contains an additional stretch of 363 bases between exons 9 and 10. In this communication, we have examined whether similar alternatively spliced mRNAs of c-myb occur in human cells. Human c-myb exon 9A has been identified and sequenced in a cDNA clone (ML5) generated from the acute myeloid leukemic cell line ML-2. This alternatively spliced exon of c-myb has been found to contain the same number of nucleotides (363bp) as the corresponding mouse exon. Between murine and human exon 9A sequences, 81% sequence homology was found at the DNA level, while the homology at the predicted amino acid level was found to be 73%. A stretch of 14 amino acid residues at the junction of exons 9A and 10 have been found to be conserved between Drosophila and human sequences indicating that this region might perform an essential biological function which was deemed necessary through evolution.

The c-myb gene is the normal cellular homolog of v-myb, the oncogenic component of Avian Myeloblastosis Virus (AMV). The c-myb gene has previously been shown to code for a single protein species of about 75 kd. However, accumulating evidence indicates that this gene could code for multiple mRNAs as a result of differential splicing. This was first detected in the ABPL-2 tumor line and was later shown to occur in normal cells. To test if indeed these differentially spliced mRNAs code for a new protein species that went hitherto undetected, a series of antibodies directed against specific domains of the myb protein, including the putative sequences that could be generated from the alternatively spliced mRNAs were generated. Immunoprecipitation analysis using these antibodies show that both normal and tumor cell lines synthesize at least two myb proteins, one of 75 kd and the other of 89 kd.