Nucleus Drosophila embryo

The very beginning of the first mitotic cell cycle of the mouse embryo seems to be controlled by the mechanisms characteristic for both meiotic and mitotic cell cycles. Active MAP kinase, its substrate p90rsk and the CSF activity itself could influence the cellular processes within the one-cell embryo. Indeed, we have observed that despite the entry into the interphase (as judged by the low activity of MPF) some proteins are actively phosphorylated as during the meiotic M phase (e.g. 35 kDa complex Howlett et al 1986, Szollosi et al 1993), the nuclei and the microtubule interphase network start to form only 1.5 hours after activation (Szollosi et al 1993). This delay in the phenomena characteristic for the interphase could be linked to the mixed meiotic/mitotic character of this early period. This delay probably allows the correct transformation of the sperm nucleus into the male pronucleus. In species like Xenopus or Drosophila the transitional period between the meiotic and the mitotic cell cycle control is probably much shorter since it is proportional to duration of the short first cell cycle of these rapidly cleaving embryos. Mammalian embryos are perhaps the most suitable to study this transition because of the exceptionally long first embryonic cell cycle. 

Unless otherwise noted, all procedures should be performed at 4°C or on ice. Once prepared. Drosophila nuclei should be handled identically, regardless of source. The nucleus-enriched pellet should be resuspended in 20 mM Tris-HCl, pH 7.5,5 mM MgCla (Buffer B). For nuclear resuspension, one volume of Buffer B relative to the initial volume of starting material should be used. For example, if nuclei are purified from 10 ml of embryos, 10 ml of Buffer B should be used if nuclei are purified from 1 ml of tissue culture cells, 1 ml of Buffer B should be used. Complete resuspension should be assured by Dounce homogenization (two-four strokes, loose pestle) and both DNase I and RNase A should be added to final concentrations of 10 and 8 /i.g/ml, respectively. Typically, RNase A is added first, the sample is mixed on a vortex mixer, and then the DNase I is added. After addition of DNase I, vortex mixing is avoided because DNase I is very sensitive to inactivation by oxidation. Rather, the sample is mixed by gentle agitation. 

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