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The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMRalpha) and a common signal-transducing beta-subunit (hbetac) that is shared with the interleukin-3 and -5 receptors. We have previously identified a constitutively active extracellular point mutant of hbetac, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287). This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMRalpha (mGMRalpha) subunit, since introduction of mGMRalpha, but not hGMRalpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence. Experiments utilizing mouse/human chimeric GMRalpha subunits indicated that the species specificity lies in the extracellular domain of GMRalpha. Importantly, the requirement for mGMRalpha correlated with the ability of I374N (but not wild-type hbetac) to constitutively associate with mGMRalpha. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMRalpha surface expression. Taken together, these findings suggest a critical role for association with GMRalpha in the constitutive activity of I374N.

Murine bone marrow cells infected with a novel recombinant retrovirus, MPZen(GM‐CSF), were engrafted into lethally irradiated recipients. The transplanted animals developed extremely high circulating levels of GM‐CSF (up to 3 x 10(5) units/ml), and greatly elevated peripheral nucleated cell counts (up to 110 x 10(6) per ml). Their haemopoietic tissues contained GM‐CSF proviral DNA and produced substantial levels of GM‐CSF. The mice died within 4 weeks of transplantation with extensive neutrophil and macrophage infiltration of the spleen, lung, liver and peritoneal cavity and significant infiltration of both heart and skeletal muscle by neutrophils, macrophages and eosinophils. The thymus and lymph nodes were deficient in lymphoid cells. No disease occurred when infected cells from haemopoietic tissues of the primary transplanted animals were injected into normal or sub‐lethally irradiated mice. Dysregulated GM‐CSF expression by haemopoietic cells thus produces a fatal albeit non‐neoplastic myeloproliferative syndrome.

Murine retroviruses which encode c‐myb proteins that have either complete or truncated carboxy (C) termini were used to infect haemopoietic cells from murine fetal liver. Using an agar colony assay, we could show that infection with the virus encoding the truncated protein resulted in the persistence of colony‐forming cells well beyond the short period for which such cells are present in uninfected cultures. The resultant colonies failed to give rise to cell lines; however, clonal cell lines occasionally emerged from the original infected liquid cultures. The virus which encoded a myb protein with a complete C‐terminus was virtually inactive in the colony assay; surprisingly, however, this virus could enhance proliferation in liquid cultures and has led to the generation of at least one cell line. In addition to demonstrating ‘activation’ of c‐myb by C‐terminal truncation, our results imply that an unaltered c‐myb protein can also contribute to cellular transformation and that a second event is required to establish myb‐transformed cells as a permanent cell line.

To date, cellular transformation in vitro by the myb oncogene has been described for avian haemopoietic cells only. In order to exploit the well‐characterized murine haemopoietic system to study transformation by myb, we have infected fetal liver cells with retroviral vectors carrying cDNAs that encode either complete or carboxy‐terminally truncated c‐myb proteins. We describe four cell lines which, despite our ability to efficiently infect haemopoietic target cells, were generated at low frequency. This was due, as least in part, to the requirement for a rearrangement within the vector that allowed expression of myb sequences. Three of the lines express a truncated myb protein while the fourth apparently expresses a normal c‐myb protein, and thus constitutes an exception to the general association of truncation with transformation by myb. All four cell lines resemble immature cells of the myelomonocytic lineage and are dependent on colony‐stimulating factors (CSFs) for their growth in vitro. One representative line could be converted to CSF‐independence by infection with either Abelson murine leukaemia virus or a recombinant granulocyte‐macrophage‐CSF‐encoding retrovirus; unlike the parental line, the resultant sublines were highly tumorigenic when injected into syngeneic mice.

Single ice crystals were grown in air at low pressure (0.3 Torr) and 760 Torr at ȡ30°C and various constant supersaturations α. As a result, it is considered that the {0001} and {101¯0} faces of many ice crystals growing in air at ȡ30°C grow by a nucleation mechanism except for low supersaturations α < about 2%. In addition, the stability of the {0001} and {101¯0} faces of ice crystals growing in air at ȡ30°C and 760 Torr was investigated.

A new, spontaneously occurring diabetic syndrome has been observed in the aged males of an inbred strain of Wistar rats, WBN/Kob. The main clinical sign, glycosuria, was first detected at about 60 weeks of age, and thereafter some animals developed hyperlipidaemia and gradual emaciation. Prior to the onset of glucosuria, male rats showed impaired glucose tolerance after a glucose load at 21 weeks of age. The histopathologic lesions of the pancreas in the diabetic males consisted of multifocal fibrosis, decrease in number and size of islets and atrophy of exocrine tissue. Multifocal inflammatory foci of varying stages were the main pancreatic lesion in prediabetic male rats. This inflammatory change was detected even in 12-week-old rats and tended to occur around the islets. Therefore focal fibrosis and the decrease in the number and size of islets were considered to result from post-inflammatory scarring. The maturity-onset of this syndrome and the impaired glucose tolerance in younger animals suggested that diabetes mellitus of this rat strain is insulin-independent type II. However, the histological lesions of the pancreas were somewhat different from previous reports of both type I and II diabetes mellitus in man and animals.

A previous study using random mutagenesis identified an activating mutation in the common beta subunit (hbetac) of the human granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5 receptors in which an isoleucine residue (Ile374) in the extracellular region of hbetac is replaced by asparagine (Jenkins, B. J., D'Andrea, R., and Gonda, T. J. (1995) EMBO J 14, 4276-4287). To investigate the mechanism by which this mutation (I374N) acts, we employed site-directed mutagenesis to explore predictions based on a structural model of hbetac. We focused on possible interactions between Ile374 and other hydrophobic residues in its vicinity and found that replacement of two such residues, Leu356 and Trp358, with asparagine resulted in constitutive activation of hbetac. Hydrophilic substitutions at both of these positions and at position 374 resulted in the greatest degree of activation, as measured by the growth rate of factor-independent cells, while hydrophobic substitutions had lesser or no effects. Moreover, these "weak" substitutions appeared to synergize, since factor-independent cells expressing the double mutants I374F/W358F and I374F/L356A showed substantially higher growth rates than the single mutants. Taken together, these results suggest that Ile374 normally interacts with Leu356 and Trp358, and that disruption of these interactions results in a conformational change in hbetac that leads to constitutive activity. A model relating this notion to the predicted structure and to ligand- and alpha subunit-dependent activation of hbetac is proposed.

Two murine monocytic leukemia cell lines, WEHI-265 and WEHI-274, were found to carry a rearranged c-myb gene. The rearrangements are due to insertion of a deleted Moloney murine leukemia virus (Mo-MLV) provirus in the 5' region of the c-myb gene and thus are similar to rearrangements in the ABPL tumors (G. L. C. Shen-Ong, M. Potter, J. F. Mushinski, S. Lavu, and E. P. Reddy, Science 226:1077-1080, 1984). In each cell line, the retroviral insertion has induced high levels of two aberrant RNA species, which, as in the ABPL tumors (G. L. C. Shen-Ong, H. C. Morse, M. Potter, and J. F. Mushinski, Mol. Cell. Biol. 6:380-392, 1986), contain both viral (Mo-MLV) and cellular (myb) sequences. Both species lack the sequences encoding the amino terminus of the c-myb protein and thus could encode a protein which, like the v-myb gene products (and the predicted ABPL myb proteins), is truncated at the amino terminus. We have found that the larger (5.3 kilobase [kb]) and more abundant of the tumor-specific myb RNAs was predominantly nuclear, while the smaller species (3.9 kb) was cytoplasmic. Furthermore, our data imply that the 3.9-kb RNA was derived from the 5.3-kb RNA by an additional splice which utilized a cryptic splice acceptor site within the viral gag sequences. On the basis of subcellular distribution and predicted translational potential, we conclude that the 3.9-kb RNA is probably the mRNA which encodes a truncated myb protein. We also show that, due to different insertion points in W265 and W274, the W274 myb RNAs contained sequences from a c-myb exon upstream of the exons represented in the W265 (and ABPL) RNAs. The significance of our findings with regard to transformation by myb in these tumors is discussed.

In order to identify factors that influence expression by retroviral vectors in hemopoietic cells, we have compared viral RNA levels in cells infected with several different recombinant viruses. All of the vectors tested carry the neomycin resistance gene and provide for the insertion of a second gene which, in these studies, comprised sequences from the myc or myb oncogenes or the gene encoding granulocyte-macrophage colony-stimulating factor. The vectors utilize two different strategies for the coexpression of the two genes: alternate splicing and the use of a separate internal promoter. We found that expression in hemopoietic cells could be increased by substituting sequences from the myeloproliferative sarcoma virus long terminal repeat for those of the Moloney murine leukemia virus long terminal repeat. However, none of the vectors examined was able to express a second gene at levels equivalent to those achieved by the parental vectors carrying only the neomycin resistance gene. The reasons for this varied with the different vectors and included inefficient splicing and/or a reduction in the level of unspliced transcripts upon insertion of a second gene. Although the basis of the latter phenomenon is not clear, it is probably related to the position--near the 5' long terminal repeat--at which the second gene was inserted, since insertion of the same genes near the 3' end of another vector had no effect on viral RNA levels. In an attempt to circumvent some of these problems, we constructed a vector that employs an internal beta-actin promoter. Although this vector could express granulocyte-macrophage colony-stimulating factor sequences in a responsive hemopoietic cell line, the level of granulocyte-macrophage colony-stimulating factor produced was disappointingly low. The results from these studies suggest approaches to the design of improved vectors for effective expression of genes in hemopoietic cells.

We isolated and characterized molecular clones containing the chicken cellular homolog (c-myb) of the avian myeloblastosis virus oncogene (v-myb). Mapping of the c-myb clones using restriction endonucleases and hybridization to radiolabeled v-myb probes revealed that the sequences homologous to v-myb are contained within four separate regions, which have since been shown by nucleotide sequencing (Klempnauer et al., Cell 31:453-463, 1982) to carry seven exons. Analysis of c-myb transcripts showed the presence of several large precursor RNAs in addition to the 4.0-kilobase cytoplasmic mRNA. We also determined the approximate positions of the c-myb sequences that are present in the 4.0-kilobase c-myb mRNA but not present in v-myb. Some of these sequences are found in a separate region 5' to the v-myb-related sequences, whereas the remainder appear to be located immediately 3' to the v-myb-related sequences. The data presented here, in conjunction with nucleotide sequence analysis (Klempnauer et al., Cell 31:453-463, 1982), indicate that the c-myb gene contains at least eight exons which span a total of about 16 kilobase pairs. The presence of exon sequences in c-myb outside the regions of homology with v-myb raises the possibility that the v-myb and c-myb gene products may differ significantly.

The c-myb protooncogene encodes a sequence-specific DNA-binding protein (c-Myb) that induces transcriptional activation or repression. We have identified three functional domains of the mouse c-Myb protein that are responsible for DNA binding, transcriptional activation, and negative regulation, respectively. In addition to the DNA-binding domain, which is located near the N terminus, an adjacent region (the transcriptional activation domain) containing about 80 amino acids was found to be essential for transcriptional activation. Deletion of a region spanning about 175 amino acids of the C-proximal portion increased transcriptional activation markedly, revealing that this domain normally represses activation. Differences between the transcriptional activation and repression functions of c-Myb and v-Myb are discussed in the light of these functional domains. Our results suggest that transcriptional activation may be involved in transformation by myb gene products.