Cytological characterisation of the mutant phenotypes produced during early embryogenesis by null and loss-of-function alleles of the gammaTub37C gene in Drosophila.

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.

Centrosomes and microtubule organisation during Drosophila development.

Are the microtubule-organising centers of the different cell types of a metazoan interchangeable? If not, what are the differences between them? Do they play any role in the differentiation processes to which these cells are subjected? Nearly one hundred years of centrosome research has established the essential role of this organelle as the main microtubule-organising center of animal cells. But only now are we starting to unveil the answers to the challenging questions which are raised when the centrosome is studied within the context of a pluricellular organism. In this review we present some of the many examples which illustrate how centrosomes and microtubule organisation changes through development in Drosophila and discuss some of its implications.

6DMAP inhibition of early cell cycle events and induction of mitotic abnormalities.

N-6 dimethylaminopurine (6DMAP) has been shown to induce aberrant mitosis in different cell types including Chinese hamster fibroblasts (CHEF/18). The mechanism of action and the cellular targets, however, are still not clear. We showed previously that in CHEF/18 cells this compound inhibits DNA synthesis with a kinetic of inhibition suggestive of an effect on early events of the cell cycle. In this paper we investigated which cellular targets were affected by 6DMAP and found that: (i) the compound inhibits phosphorylation of ribosomal protein S6 and activation of the 70 kDa S6 kinase (p70S6k) known to be activated by epidermal growth factor (EGF) in keeping with the notion that it is a protein kinase inhibitor; however the inhibition in vivo appears to be specific as MAP kinase phosphorylation is not inhibited; (ii) 6DMAP drastically affects cytoskeletal components leading to a rapid morphological change in most cells. These data, together with the findings that the dose range and the treatment time effective in inducing the micronuclei containing chromosomes were the same as for DNA synthesis inhibition, suggest that a disturbance in G1 of signal transduction pathways may contribute to abnormal mitosis.

Essential role for gamma-tubulin in the acentriolar female meiotic spindle of Drosophila.

Microtubule nucleation in vivo requires gamma-tubulin, a highly conserved component of microtubule-organizing centers. In Drosophila melanogaster there are two gamma-tubulin genes, gammaTUB23C and gammaTUB37C. Here we report the cytological and molecular characterization of the 37C isoform. By Western blotting, this protein can only be detected in ovaries and embryos. Antibodies against this isoform predominantly label the centrosomes in embryos from early cleavage divisions until cycle 15, but fail to reveal any particular localization of gamma-tubulin in the developing egg chambers. The loss of function of this gene results in female sterility and has no effect on viability or male fertility. Early stages of oogenesis are unaffected by mutations in this gene, as judged both by morphological criteria and by localization of reporter genes, but the female meiotic spindle is extremely disrupted. Nuclear proliferation within the eggs laid by mutant females is also impaired. We conclude that the expression of the 37C gamma-tubulin isoform of D. melanogaster is under strict developmental regulation and that the organization of the female meiotic spindle requires gamma-tubulin.

The induction of aneuploidy by alkylated purines: effects on early and late cell cycle events.

It has been shown that alkylated bases induce aneuploidy in mammalian cells in culture. The mechanism of action is not clear, however, data with 6-dimethyl amino purine (6DMAP) suggest that this analogue might act by affecting the cytoskeleton and protein kinases involved in cell cycle regulation (cdc2/p34). The aim of this work was to study the effect of O6methylguanine (O6meG), O6ethylguanine (O6etG) and 6DMAP on DNA synthesis induced by growth factors in two cell lines, 3T3 and CHEF/18 fibroblasts, which respond in opposite ways to substances affecting the cytoskeleton, colchicine and cholera toxin: DNA synthesis initiation is stimulated in 3T3 cells and inhibited in CHEF/18 cells by such compounds. Our results indicate that O6meG and O6etG behave like cholera toxin, in as much as they inhibit DNA synthesis induced by epidermal growth factor plus insulin in CHEF/18 cells, and stimulate it in 3T3 cells. 6DMAP behaves differently and inhibits DNA synthesis in both cell lines. The inhibition (or stimulation) was greater when alkylated bases were added before S phase started, suggesting that these compounds might affect early events of the cell cycle. In CHEF/18 cells the three alkylated bases were able to induce aberrant metaphases and ana-telophases with different efficiency (70-100%). The effect was not dependent on the G1-S block and it was reversible even after cell commitment to DNA synthesis.