Spindle assembly and mitosis without centrosomes in parthenogenetic Sciara embryos.

In Sciara, unfertilized embryos initiate parthenogenetic development without centrosomes. By comparing these embryos with normal fertilized embryos, spindle assembly and other microtubule-based events can be examined in the presence and absence of centrosomes. In both cases, functional mitotic spindles are formed that successfully proceed through anaphase and telophase, forming two daughter nuclei separated by a midbody. The spindles assembled without centrosomes are anastral, and it is likely that their microtubules are nucleated at or near the chromosomes. These spindles undergo anaphase B and successfully segregate sister chromosomes. However, without centrosomes the distance between the daughter nuclei in the next interphase is greatly reduced. This suggests that centrosomes are required to maintain nuclear spacing during the telophase to interphase transition. As in Drosophila, the initial embryonic divisions of Sciara are synchronous and syncytial. The nuclei in fertilized centrosome-bearing embryos maintain an even distribution as they divide and migrate to the cortex. In contrast, as division proceeds in embryos lacking centrosomes, nuclei collide and form large irregularly shaped nuclear clusters. These nuclei are not evenly distributed and never successfully migrate to the cortex. This phenotype is probably a direct result of a failure to form astral microtubules in parthenogenetic embryos lacking centrosomes. These results indicate that the primary function of centrosomes is to provide astral microtubules for proper nuclear spacing and migration during the syncytial divisions. Fertilized Sciara embryos produce a large population of centrosomes not associated with nuclei. These free centrosomes do not form spindles or migrate to the cortex and replicate at a significantly reduced rate. This suggests that the centrosome must maintain a proper association with the nucleus for migration and normal replication to occur.

Incomplete sister chromatid separation is the mechanism of programmed chromosome elimination during early Sciara coprophila embryogenesis.

Sex in Sciara coprophila is determined by maternally supplied factors that control the number of paternal X chromosomes eliminated during the syncytial embryonic divisions. Confocal microscopy and FISH demonstrate that the centromeres of the X chromosomes separate at anaphase and remain functional during the cycle in which the X chromosomes are eliminated. However, a region of the sister chromatids fails to separate and the X chromosomes remain at the metaphase plate. This indicates that failure of sister chromatid separation is the mechanism of chromosome elimination. Elimination of the X chromosomes requires the presence of a previously discovered Controlling Element that acts in cis during male meiosis. Using an X-autosome translocation, we demonstrate that the Controlling Element acts at-a-distance to prevent sister chromatid separation in the arm of an autosome. This indicates that the region in which sister chromatid separation fails is chromosome-independent. Although chromosome elimination occurs in all somatic nuclei and is independent of location of the nuclei within the embryo, the decision to eliminate is made at the level of the individual nucleus. Programmed X chromosome elimination occurs at different cycles in male and female embryos. These observations support a model in which elements on the X chromosome are titrating maternally supplied factors controlling the separation of sister X chromatids.

The ultraviolet-sensitizing function of plasmid R391 interferes with a late step of postreplication repair in Escherichia coli.

The conjugative plasmid R391 increases the UV radiation sensitivity of wild-type, uvrA, and lexA cells of Escherichia coli, but not recA strains. To investigate the UV-sensitizing function of R391, we examined the effect of R391 on the repair of DNA daughter-strand gaps and on the UV radiation sensitivities of various repair and/or recombination-deficient mutants. The presence of R391 did not significantly inhibit the repair of DNA daughter-strand gaps in uvrB cells. The presence of R391 increased the UV radiation sensitivity of uvrA, uvrA recF, uvrB, uvrB recF, uvrB recB, and uvrB ssb-113 cells to UV irradiation, but did not significantly increase the UV radiation sensitivity of uvrA ruvA and uvrA ruvC strains. Based on these results, we propose that the UV-sensitizing activity of R391 acts by inhibiting or interfering with the ruvABC-mediated postsynapsis step of recombinational repair.