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Mutations at abnormal spindle result in abnormally long and wavy microtubules in the meiotic spindles of males. Some of these spindles have a single pole and take the form of unopposed hemi-spindles. Unfertilised eggs produced by homozygous asp females may have either no nuclei, or a small number of large nuclei, consistent with there also being an effect upon female meiosis. Such eggs also display free centrosomes and independent arrays of microtubules. Embryos that have this phenotype are also present among the progeny of fertilised homozygous asp females, together with embryos that undergo varying degrees of aberrant morphogenesis, developing a variety of abnormal cuticle patterns. This latter category shows asynchronous mitoses prior to cellularisation, and has abnormal arrays of spindle microtubules. Such embryos can develop large areas that are either devoid of or have a reduced number of nuclei, in which there are centrosomes that have dissociated from the mitotic spindles. Neuroblasts in the brains of homozygous asp larvae display a high mitotic index, and have condensed chromosomes aligned as if blocked at metaphase. Immunostaining reveals that many cells contain a single centrosome connected to the metaphase chromosomes by microtubules in a hemi-spindle-like structure.

We have studied the mutant phenotypes brought about during early embryogenesis by mutation in the gammaTub37C gene, one of the two isoforms of gamma-tubulin that have been identified in Drosophila. We have focused our attention on fs(2)TW1(1) and fs(2)TW1(RU34), a null and a hypomorph allele of this gene, whose sequences we report in this work. We have found that the abnormal meiotic figures observed in mutant stage 14 oocytes are not observed in laid oocytes or fertilised embryos, suggesting that these abnormal meiotic figures are not terminally arrested. We have also concluded that both null and hypomorph alleles lead to a total arrest of nuclear proliferation during early embryogenesis. This is in contrast to their effect on female meiosis-I where hypomorph alleles display a much weaker phenotype. Finally, we have observed that null and hypomorph alleles lead to some distinct phenotypes. Unfertilised laid oocytes and fertilised embryos deficient for gammaTub37C do not contain polar bodies and have a few bipolar microtubule arrays. In contrast, oocytes and embryos from weaker alleles do not have these microtubule arrays, but do contain polar bodies, or polar-body-like structures. These results indicate that gammaTub37C is essential for nuclear proliferation in the early Drosophila embryo.

To determine the molecular composition of the centrosome of a higher eukaryote, we carried out a systematic nano-electrospray tandem or MALDI mass spectrometry analysis of the polypeptides present in highly enriched preparations of immunoisolated Drosophila centrosomes. One of the proteins identified is Hsp83, a member of the highly conserved Hsp90 family including chaperones known to maintain the activity of many proteins but suspected to have other essential, unidentified functions. We have found that a fraction of the total Hsp90 pool is localized at the centrosome throughout the cell cycle at different stages of development in Drosophila and vertebrates. This association between Hsp90 and the centrosome can be observed in purified centrosomes and after treatment with microtubule depolymerizing drugs, two criteria normally used to define core centrosomal components. Disruption of Hsp90 function by mutations in the Drosophila gene or treatment of mammalian cells with the Hsp90 inhibitor geldanamycin, results in abnormal centrosome separation and maturation, aberrant spindles and impaired chromosome segregation. This suggests that another role of Hsp90 might be to ensure proper centrosome function.

We report that in Drosophila, gamma-tubulin is required for the structure as well as the function of microtubule organizing centres (MTOCs). This conclusion is based on the identification and phenotypic characterization of a mutant allele of the gamma-tubulin gene located at region 23C of the polytene chromosome map. This mutation, which we have called gamma-tub23CPl, is caused by the insertion of a P-element within the 5' untranslated leader of the gamma-tubulin transcript. Northern and Western analysis show that gamma-tub23CPl is either a null or a very severe hypomorph as no gamma-tubulin mRNA or protein can be detected in mutant individuals. Visualization of DNA, MTOCs and microtubules by confocal laser scanning microscopy of cells from individuals homozygous for gamma-tub23CPl reveals a series of phenotypic abnormalities. Some of these are similar to those observed after disruption of gamma-tubulin function in other organisms, including mitotic arrest and a dramatic decrease in the number of microtubules, but, in addition, we have observed that mutation in this gene also results in highly abnormal MTOCs which show a variety of shapes and sizes which we never observed in wild type cells. These results show that gamma-tubulin is required for both structural and functional roles in the MTOCs.

Mutation in the gene merry-go-round (mgr) of Drosophila causes a variety of phenotypic traits in somatic and germinal tissues, such as polyploid cells, metaphasic arrest, postmeiotic cysts with 16 nuclei, and spermatids with four times the normal chromosome content. The most characteristic phenotype is the appearance of mitotic and meiotic figures where all chromosomes are arranged in a circle. Treatment with anti-mitotic drugs and the phenotype of double mutants mgr asp (asp being a mutation altering the spindle) show that these circular figures need a functional spindle for their formation. These abnormal figures are caused by monopolar spindles similar to those observed after different treatments in several organisms. All mutant traits indicate that mgr performs a function necessary for the correct behaviour of centrosomes, thus opening this organelle to genetic analysis.

We have used fluorescence in situ hybridisation to wild-type and rearranged mitotic chromosomes to map the Drosophila melanogaster dodecasatellite sequence. It is located at a unique site, within the pericentric heterochromatin of the right arm of the third chromosome, closely linked to the primary constriction. In polytene chromosomes, dodecasatellite is found as one or a few dots in the central region of the chromocentre. In untreated diploid cells, dodecasatellite sequences are found as one or two dots throughout the cell cycle. This distribution can be altered in a cell cycle-dependent manner in two ways. Firstly, in interphase cells, hypotonic shock promotes the decondensation of the genomic region containing this satellite, resulting in a string-like structure. Secondly, some of the precociously separated sister chromatids produced by colchicine treatment show dodecasatellite within the intervening space connecting the main dodecasatellite signals of each chromatid. The distribution of dodecasatellite seems to be rather constant between individuals of the same species, as indicated by the lack of any detectable variations in its pattern amongst individuals from six geographically distant strains of D. melanogaster. On the other hand, the distribution of dodecasatellite shows a remarkable degree of variation amongst closely related species of the melanogaster subgroup ranging from a non-detectable signal in Drosophila yakuba and Drosophila teissieri, to staining in the X, second and third chromososomes of Drosophila mauritiana.