Drosophila embryonic cell-cycle mutants.

Nearly all cell division mutants in Drosophila were recovered in late larval/pupal lethal screens, with less than 10 embryonic lethal mutants identified, because larval development occurs without a requirement for cell division. Only cells in the nervous system and the imaginal cells that generate the adult body divide during larval stages, with larval tissues growing by increasing ploidy rather than cell number. Thus, most mutants perturbing mitosis or the cell cycle do not manifest a phenotype until the adult body differentiates in late larval and pupal stages. To identify cell-cycle components whose maternal pools are depleted in embryogenesis or that have specific functions in embryogenesis, we screened for mutants defective in cell division during embryogenesis. Five new alleles of Cyclin E were recovered, ranging from a missense mutation that is viable to stop codons causing embryonic lethality. These permitted us to investigate the requirements for Cyclin E function in neuroblast cell fate determination, a role previously shown for a null Cyclin E allele. The mutations causing truncation of the protein affect cell fate of the NB6-4 neuroblast, whereas the weak missense mutation has no effect. We identified mutations in the pavarotti (pav) and tumbleweed (tum) genes needed for cytokinesis by a phenotype of large and multinucleate cells in the embryonic epidermis and nervous system. Other mutations affecting the centromere protein CAL1 and the kinetochore protein Spc105R caused mitotic defects in the nervous system.

Drosophila follicle cell amplicons as models for metazoan DNA replication: a cyclinE mutant exhibits increased replication fork elongation.

Gene clusters amplified in the ovarian follicle cells of Drosophila serve as powerful models for metazoan DNA replication. In response to developmental signals, specific genomic regions undergo amplification by repeated firing of replication origins and bidirectional movement of replication forks for approximately 50 kb in each direction. Previous work focused on initiation of amplification, defining replication origins, establishing the role of the prereplication complex and origin recognition complex (ORC), and uncovering regulatory functions for the Myb, E2F1, and Rb transcription factors. Here, we exploit follicle cell amplification to investigate the control of DNA replication fork progression and termination, poorly understood processes in metazoans. We identified a mutant in which, during gene amplification, the replication forks move twice as far from the origin compared with wild type. This phenotype is the result of an amino acid substitution mutation in the cyclinE gene, cyclinE(1f36). The rate of oogenesis is normal in cyclinE(1f36)/cyclinE(Pz8) mutant ovaries, indicating that increased replication fork progression is due to increased replication fork speed, possibly from increased processivity. The increased amplification domains observed in the mutant imply that there are not replication fork barriers preventing replication forks from progressing beyond the normal 100-kb amplified region. These results reveal a previously unrecognized role for CyclinE in controlling replication fork movement.

Death of CD4(+) T-cell lines caused by human immunodeficiency virus type 1 does not depend on caspases or apoptosis.

A critical aspect of AIDS pathogenesis that remains unclear is the mechanism by which human immunodeficiency virus type 1 (HIV-1) induces death in CD4(+) T lymphocytes. A better understanding of the death process occurring in infected cells may provide valuable insight into the viral component responsible for cytopathicity. This would aid the design of preventive treatments against the rapid decline of CD4(+) T cells that results in AIDS. Previously, apoptotic cell death has been reported in HIV-1 infections in cultured T cells, and it has been suggested that this could affect both infected and uninfected cells. To evaluate the mechanism of this effect, we have studied HIV-1-induced cell death extensively by infecting several T-cell lines and assessing the level of apoptosis by using various biochemical and flow cytometric assays. Contrary to the prevailing view that apoptosis plays a prominent role in HIV-1-mediated T-cell death, we found that Jurkat and H9 cells dying from HIV-1 infection fail to exhibit the collective hallmarks of apoptosis. Among the parameters investigated, Annexin V display, caspase activity and cleavage of caspase substrates, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) signal, and APO2.7 display were detected at low to negligible levels. Neither peptide caspase inhibitors nor the antiapoptotic proteins Bcl-x(L) or v-FLIP could prevent cell death in HIV-1-infected cultures. Furthermore, Jurkat cell lines deficient in RIP, caspase-8, or FADD were as susceptible as wild-type Jurkat cells to HIV-1 cytopathicity. These results suggest that the primary mode of cytopathicity by laboratory-adapted molecular clones of HIV-1 in cultured cell lines is not via apoptosis. Rather, cell death occurs most likely via a necrotic or lytic form of death independent of caspase activation in directly infected cells.