We determined the distribution of 11 different transposable elements on Drosophila melanogaster mitotic chromosomes by using high-resolution fluorescent in situ hybridization (FISH) coupled with charge-coupled device camera analysis. Nine of these transposable elements (copia, gypsy, mdg-1, blood, Doc, I, F, G, and Bari-1) are preferentially clustered into one or more discrete heterochromatic regions in chromosomes of the Oregon-R laboratory stock. Moreover, FISH analysis of geographically distant strains revealed that the locations of these heterochromatic transposable element clusters are highly conserved. The P and hobo elements, which are likely to have invaded the D. melanogaster genome at the beginning of this century, are absent from Oregon-R heterochromatin but clearly exhibit heterochromatic clusters in certain natural populations. Together these data indicate that transposable elements are major structural components of Drosophila heterochromatin, and they change the current views on the role of transposable elements in host genome evolution.

We describe the phenotypic and molecular characterization of twinstar (tsr), an essential gene in Drosophila melanogaster. Two P-element induced alleles of tsr (tsr1 and tsr2) result in late larval or pupal lethality. Cytological examination of actively dividing tissues in these mutants reveals defects in cytokinesis in both mitotic (larval neuroblast) and meiotic (larval testis) cells. In addition, mutant spermatocytes show defects in aster migration and separation during prophase/prometaphase of both meiotic divisions. We have cloned the gene affected by these mutations and shown that it codes for a 17-kD protein in the cofilin/ADF family of small actin severing proteins. A cDNA for this gene has previously been described by Edwards et al. (1994). Northern analysis shows that the tsr gene is expressed throughout development, and that the tsr1 and tsr2 alleles are hypomorphs that accumulate decreased levels of tsr mRNA. These findings prompted us to examine actin behavior during male meiosis to visualize the effects of decreased twinstar protein activity on actin dynamics in vivo. Strikingly, both mutants exhibit abnormal accumulations of F-actin. Large actin aggregates are seen in association with centrosomes in mature primary spermatocytes. Later, during ana/telophase of both meiotic divisions, aberrantly large and misshaped structures appear at the site of contractile ring formation and fail to disassemble at the end of telophase, in contrast with wild-type. We discuss these results in terms of possible roles of the actin-based cytoskeleton in centrosome movement and in cytokinesis.

On the basis of the hypothesis that mutants in genes controlling essential cell cycle functions in Drosophila should survive up to the larval-pupal transition, 59 such 'late lethals' were screened for those mutants affecting cell division. Examination of mitosis in brain neuroblasts revealed that 30 of these lethals cause disruptions in mitotic chromosome behavior. These mutants identify genes whose wild-type functions are important for: (1) progression through different steps of interphase, (2) the maintenance of mitotic chromosome integrity, (3) chromosome condensation, (4) spindle formation and/or function, and (5) completion of cytokinesis or completion of chromosome segregation. The presence of mitotic defects in late lethal mutants is correlated tightly with the presence of defective imaginal discs. Thus, the phenotypes of late lethality and poorly developed imaginal discs are together almost diagnostic of mutations in essential cell-cycle functions. The terminal phenotypes exhibited by these Drosophila mitotic mutants are remarkably similar to those observed in mammalian cell-cycle mutants, suggesting that these diverse organisms use a common genetic logic to regulate and integrate the events of the cell cycle.

We have used double-stranded RNA-mediated interference (RNAi) to study Drosophila cytokinesis. We show that double-stranded RNAs for anillin, acGAP,pavarotti, rho1, pebble,spaghetti squash, syntaxin1A, andtwinstar all disrupt cytokinesis in S2 tissue culture cells, causing gene-specific phenotypes. Our phenotypic analyses identify genes required for different aspects of cytokinesis, such as central spindle formation, actin accumulation at the cell equator, contractile ring assembly or disassembly, and membrane behavior. Moreover, the cytological phenotypes elicited by RNAi reveal simultaneous disruption of multiple aspects of cytokinesis. These phenotypes suggest interactions between central spindle microtubules, the actin-based contractile ring, and the plasma membrane, and lead us to propose that the central spindle and the contractile ring are interdependent structures. Finally, our results indicate that RNAi in S2 cells is a highly efficient method to detect cytokinetic genes, and predict that genome-wide studies using this method will permit identification of the majority of genes involved inDrosophila mitotic cytokinesis.

Larval and pupal testes of Drosophila melanogaster were fixed with a methanol/acetone fixation procedure that results in good preservation of cell morphology; fixed cells viewed by phase-contrast optics exhibit most of the structural details that can be seen in live material. Fixed testis preparations were treated with anti-tubulin antibodies and Hoechst 33258 to selectively stain microtubules and DNA. The combined analysis of cell morphology, chromatin and microtubule organization allowed a fine cytological dissection of gonial cell multiplication, spermatocyte development, meiosis and the early stages of spermatid differentiation. We placed special emphasis on the spermatocyte growth phase and the meiotic divisions, providing a description of these processes that is much more detailed than those previously reported. In addition, by means of bromo-deoxyuridine incorporation experiments, we were able to demonstrate that premeiotic DNA synthesis occurs very early during spermatocyte growth.