Coumailleau P, Billoud B, Sourrouille P, Moreau N, Angelier N
UA 1135 CNRS Groupe Gènes et Développement, Université Pierre et Marie Curie, Paris, France.
Dev Biol. 1995 Apr;168(2):247-58. doi: 10.1006/dbio.1995.1077.
In order to study expression of a 90-kDa heat-shock protein during amphibian oogenesis at physiological temperature, we isolated a Pleurodeles waltl hsc90 cDNA by screening an ovarian cDNA library with a chicken hsp90 cDNA probe. The cDNA thus obtained--named Pw90--shows a high homology level with the hsp90 gene in other species. RNase protection analysis led us to conclude that this sequence is part of the cognate gene hsc90 and is constitutively expressed in oocytes. Furthermore, results of quantitative Northern blot analysis, as well as in situ hybridizations on oocyte sections or lampbrush chromosome spreads, provide evidence for expression of hsc90 transcripts at every stage of oogenesis. Moreover, they point to the fact that an accumulation of transcripts occurs very early in oogenesis. Simultaneously, the expression of HSC90-related protein was analyzed on Western blots using a monoclonal antibody (AC88) and a polyclonal antibody (AP90Ct) raised against the Pleurodeles C-terminal part of HSC90. We provide evidence for a net accumulation of HSC90-related protein in oocytes. Immunolocalization shows that a nuclear transfer occurs in the course of oogenesis and leads to a concentration equilibrium between cytoplasm and nucleus in stage VI oocytes.
为了研究90 kDa热休克蛋白在两栖动物卵子发生过程中生理温度下的表达情况，我们用鸡hsp90 cDNA探针筛选卵巢cDNA文库，分离出了疣螈（Pleurodeles waltl）hsc90 cDNA。由此获得的cDNA——命名为Pw90——与其他物种的hsp90基因具有高度同源性。核糖核酸酶保护分析使我们得出结论，该序列是同源基因hsc90的一部分，且在卵母细胞中组成性表达。此外，定量Northern印迹分析结果以及对卵母细胞切片或灯刷染色体铺片的原位杂交结果，为hsc90转录本在卵子发生的各个阶段均有表达提供了证据。而且，这些结果表明转录本在卵子发生早期就开始积累。同时，我们使用针对疣螈HSC90 C末端部分产生的单克隆抗体（AC88）和多克隆抗体（AP90Ct），通过蛋白质免疫印迹法分析了HSC90相关蛋白的表达。我们提供了卵母细胞中HSC90相关蛋白净积累的证据。免疫定位显示，在卵子发生过程中发生了核转运，并导致VI期卵母细胞的细胞质和细胞核之间达到浓度平衡。
