The coupling of cell growth to the cell cycle.

The development of a complex multicellular organism requires a coordination of growth and cell division under the control of patterning mechanisms. Studies in yeast have pioneered our understanding of the relationship between growth and cell division. In recent years, many of the pathways that regulate growth in multicellular eukaryotes have been identified. This work has revealed interesting and unexpected relationships between mechanisms that regulate growth and the cell cycle machinery.

Uncoupling cell fate determination from patterned cell division in the Drosophila eye.

Cell proliferation and cell fate specification are under strict spatiotemporal control in the developing Drosophila eye. Cells excluded from five-cell preclusters synchronously enter a single additional cell cycle, the second mitotic wave, after which the remaining cells are sequentially recruited. When the second mitotic wave was blocked with the human cyclin-dependent kinase inhibitor p21CIP1/WAF1, each cell type was still specified. Hence, cell fate determination is regulated independently of the division pattern of precursor cells. However, the second mitotic wave is needed to generate appropriate numbers of each cell type. Moreover, p21 can arrest precursor cell proliferation and allow appropriate fate choice in vivo.

Rescue of a Drosophila NF1 mutant phenotype by protein kinase A.

The neurofibromatosis type 1 (NF1) tumor suppressor protein is thought to restrict cell proliferation by functioning as a Ras-specific guanosine triphosphatase-activating protein. However, Drosophila homozygous for null mutations of an NF1 homolog showed no obvious signs of perturbed Ras1-mediated signaling. Loss of NF1 resulted in a reduction in size of larvae, pupae, and adults. This size defect was not modified by manipulating Ras1 signaling but was restored by expression of activated adenosine 3', 5'-monophosphate-dependent protein kinase (PKA). Thus, NF1 and PKA appear to interact in a pathway that controls the overall growth of Drosophila.

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Of flies and men--studying human disease in Drosophila.

During the past year, the Drosophila genome has been sequenced. More than 60% of genes implicated in human disease have Drosophila orthologues. Developments in RNA-mediated interference and homologous recombination have made 'reverse genetics' feasible in Drosophila. Conventional Drosophila genetics is being used increasingly to place human disease genes of unknown function in the context of functional pathways.

A bcr-v-abl oncogene induces lymphomas in transgenic mice.

In chronic myeloid leukemia and some cases of acute lymphoblastic leukemia, a 9;22 chromosome translocation has fused most of the c-abl oncogene to a gene designated bcr. To explore in vivo the biological effects of the chimeric gene, we introduced a facsimile of the translocation product, a bcr-v-abl gene, into the mouse germ line under the control of the immunoglobulin heavy-chain enhancer or a retroviral long terminal repeat. Some transgenic mice bearing either construct developed clonal lymphoid tumors. T lymphomas predominated, but some pre-B lymphomas developed. The transgenes were expressed in the tumors but not detectably in the lymphoid tissues of nontumorous transgenic animals, implying that transcription is activated by a low-frequency somatic event. These results demonstrate that bcr-v-abl is tumorigenic in vivo and provide a new animal model for lymphomagenesis.

Expression of the cyclin-dependent kinase inhibitor Dacapo is regulated by cyclin E.

The Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs) has been implicated in mediating cell cycle arrest prior to terminal differentiation. In many instances, increased expression of CKIs immediately precedes mitotic arrest. However, the mechanism that activates CKI expression in cells that are about to stop dividing has remained elusive. Here we have addressed this issue by investigating the expression pattern of dacapo, a Cip/Kip CKI in Drosophila. We show that the accumulation of dacapo RNA and protein requires Cyclin E and that increased expression of Cyclin E can induce dacapo expression. We also show that the oscillation of the Cyclin E and Dacapo proteins are tightly coupled during ovarian endocycles. Our results argue for a mechanism where Cyclin E/Cdk activity induces Dacapo expression but only within certain windows that are permissive for dacapo expression.

cDNA sequence for human bcr, the gene that translocates to the abl oncogene in chronic myeloid leukaemia.

The hallmark of human chronic myeloid leukaemia is a 9;22 chromosome translocation that fuses most of the c-abl oncogene to the 5' portion of the breakpoint cluster region (bcr) gene, such that a hybrid bcr-abl mRNA and polypeptide are generated. To clarify further the nature of this translocation, we have analysed the structure of normal human bcr mRNA by isolating large cDNA clones that collectively span the entire coding region and extend 2.6 kb upstream of those previously described. The 3150-bp nucleotide sequence reported here includes 534 bp of a GC-rich 5' non-coding segment and indicates, in conjunction with published sequences, that the bcr polypeptide comprises 1271 amino acid residues. The predicted polypeptide is unrelated to serine or tyrosine kinases, or indeed to any previously published sequence; its structure provides no evidence of a transmembrane region. Since probes from throughout the 4.8-kb cloned region hybridized to both the 4.5 and 6.7 kb normal bcr transcripts, both RNAs contain most or all of that region.

Star is required for neuronal differentiation in the Drosophila retina and displays dosage-sensitive interactions with Ras1.

The photoreceptor cells R8, R2, and R5 are the first cells to initiate neuronal differentiation in the Drosophila eye imaginal disc. We have shown previously that these three cells require Star gene function for proper ommatidial assembly. Due to the embryonic lethality associated with mutations in Star, we have analyzed the consequence of loss of Star function in mosaic eye imaginal discs. Presumptive R8, R2, and R5 cells that lack Star function fail to differentiate neuronally and die a few hours later. Enhancer trap insertions reveal that Star expression in the eye disc is restricted to the developing R8, R2, and R5 cells. Taken together, these data suggest that Star is required for the reception of a signal and/or the execution of a developmental program that leads to the neuronal differentiation of R8, R2, and R5. Star is also required for the formation of wing veins and is expressed in developing veins, suggesting that at least partially overlapping pathways may operate during photoreceptor cell differentiation and wing vein formation. The role of Star in cell-cell signaling is supported by the observation of genetic interactions between Star and mutations that reduce signaling through both sevenless and the Drosophila EGF-receptor homologue, including Ras1 and Son of sevenless.

The Drosophila roughened mutation: activation of a rap homolog disrupts eye development and interferes with cell determination.

Roughened is a dominant mutation of D. melanogaster that disrupts eye development. The majority of the ommatidia in the adult eye lack a single photoreceptor cell, which is most commonly the R7 cell. The Roughened mutation disrupts the early stages of photoreceptor cell determination. Roughened is a dominant gain-of-function mutation that results from a single amino acid change (Phe157 to Leu) in the Drosophila Rap1 protein. Loss of function Rap1 mutations are lethal. Drosophila Rap1 protein is 88% identical to human rap1A/K-rev1 protein, a putative antagonist of ras action.

bcr-abl oncogene renders myeloid cell line factor independent: potential autocrine mechanism in chronic myeloid leukemia.

In chronic myeloid leukemia (CML), a chromosome translocation has fused the bcr gene to the c-abl oncogene, such that a chimeric bcr-abl polypeptide can be made. To explore the biological properties of bcr-abl and compare them with those of the Abelson virus (AMuLV) transforming gene (gag-v-abl), we have used either a synthetic bcr-v-abl gene that mimics the translocation product or, in some experiments, a bcr-c-abl cDNA. A new retroviral vector was used to introduce the genes into the factor-dependent myeloid line FDC-P1. Both bcr-abl and v-abl efficiently rendered the myeloid cells factor independent and tumorigenic. Their fully autonomous growth may be due to the myeloid growth factor interleukin-3 (IL-3) made in small amounts by the infected cells. Hence autocrine factor production may feature in CML development and Abelson virus transformation.

Conformation and segregation of nucleoids accompanying cell length extension after completion of a single round of DNA replication in germinated and outgrowing Bacillus subtilis spores.

When germinating spores of the temperature-sensitive DNA initiation mutant of Bacillus subtilis TsB134 are shifted to the restrictive temperature at a time such that just one or two rounds of replication are accomplished, the completed, nonreplicating nucleoids that form eventually adopt a doublet conformation. This conformation has now been observed after fixation by glutaraldehyde or osmium tetroxide, as well as by Formalin as found previously. The doublet was observed in media of different degrees of richness and under both light and electron microscopes. Electron micrographs of serial sections through the doublet were consistent with its formation by the gradual pulling apart of a single mass of DNA into two lobes. A systematic study was made of the effect of the time of shifting from the permissive to the restrictive temperature and of the restrictive temperature used on the number of nucleoids segregating within the outgrowing rod. It was established that the doublet nucleoid behaved as a single unit in replication control and segregation in both rich and poor media. Measurement of the relative position of the two segregating nucleoids within the outgrowing rod after completion of just one round of replication yielded quantitative information on the segregation and cell length extension processes. Segregation was accompanied by cell length extension at approximately equal rates on both sides of each nucleoid. Furthermore, the data were consistent with an exponential increase in such an extension with time over the early and major portion of the period studied, but it was not possible to rule out other models of length extension.

Cloning and characterization of a receptor-class phosphotyrosine phosphatase gene expressed on central nervous system axons in Drosophila melanogaster.

We have cloned and characterized cDNAs coding for a receptor-class phosphotyrosine phosphatase gene from Drosophila melanogaster. The gene maps to the polytene chromosome bands 99A7-8. The cDNA clones code for a polypeptide of 1301 amino acids with a predicted molecular mass of 145 kDa. The extracellular domain includes two fibronectin-type III-like domains. The cytoplasmic region contains two tandemly repeated phosphotyrosine phosphatase-like domains. Residues shown crucial for catalytic activity are absent in the second domain. This Drosophila receptor-class phosphotyrosine phosphatase polypeptide is expressed on axons of the embryonic central nervous system.

bcr-abl, the hallmark of chronic myeloid leukaemia in man, induces multiple haemopoietic neoplasms in mice.

The chromosome translocation forming the hybrid bcr-abl gene is thought to be the initiating event in chronic myeloid leukaemia (CML) and some cases of acute lymphoblastic leukaemia. To assess the impact of bcr-abl upon haemopoiesis, lethally irradiated mice were reconstituted with bone marrow cells enriched for cycling stem cells and infected with a bcr-abl bearing retrovirus. The mice developed several fatal diseases with abnormal accumulations of macrophage, erythroid, mast and lymphoid cells, and marked strain differences in disease distribution and kinetics. Some mice exhibited more than one neoplastic cell type and, in some instances, these were clonally related, indicating that a progenitor or stem cell had been transformed. While classical CML was not observed, the macrophage tumours were accompanied by a mild CML-like syndrome, probably due to myeloid growth factor production by tumour cells. The erythroid and mast cell diseases were rarely transplantable, in contrast to the macrophage tumours and lymphomas, but all disease types displayed limited clonality. These results establish that bcr-abl confers a proliferative advantage on diverse haemopoietic cells but complete transformation probably involves additional genetic changes.

A lethal myeloproliferative syndrome in mice transplanted with bone marrow cells infected with a retrovirus expressing granulocyte-macrophage colony stimulating factor.

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.

A cyclin-dependent kinase inhibitor, Dacapo, is necessary for timely exit from the cell cycle during Drosophila embryogenesis.

In a screen for genes that interact with the Rap1 GTPase, we have identified a Drosophila gene, dacapo (dap), which is a member of the p21/p27 family of cdk inhibitors. Unlike mammalian cdk inhibitors studied to date, dap is essential for normal embryonic development. Dacapo inhibits cyclin-cdk activity in vitro. Overexpressing dap during eye development interferes with cell cycle progression and interacts genetically with the retinoblastoma homolog (Rbf) and cyclin E. dap expression in embryos parallels the exit of cells from the cell cycle. dap mutant embryos delay the normal cell cycle exit during development; many cells complete an additional cycle and subsequently become quiescent. Thus, dap functions during embryogenesis to achieve a precisely timed exit from the cell cycle.

Biological characterization of Drosophila Rapgap1, a GTPase activating protein for Rap1.

The activity of Ras family proteins is modulated in vivo by the function of GTPase activating proteins, which increase their intrinsic rate of GTP hydrolysis. We have isolated cDNAs encoding a GAP for the Drosophila Rap1 GTPase. Drosophila Rapgap1 encodes an 850-amino acid protein with a central region that displays substantial sequence similarity to human RapGAP. This domain, when expressed in Escherichia coli, potently stimulates Rap1 GTPase activity in vitro. Unlike Rap1, which is ubiquitously expressed, Rapgap1 expression is highly restricted. Rapgap1 is expressed at high levels in the developing photoreceptor cells and in the optic lobe. Rapgap1 mRNA is also localized in the pole plasm in an oskar-dependent manner. Although mutations that completely abolish Rapgap1 function display no obvious phenotypic abnormalities, overexpression of Rapgap1 induces a rough eye phenotype that is exacerbated by reducing Rap1 gene dosage. Thus, Rapgap1 can function as a negative regulator of Rap1-mediated signaling in vivo.