Gao C Y, Bassnett S, Zelenka P S
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Dev Biol. 1995 May;169(1):185-94. doi: 10.1006/dbio.1995.1136.
Terminal differentiation of lens fiber cells is marked by chromatin condensation, abrupt dissolution of the nuclear lamina, vesicularization of the nuclear membrane, and complete degradation of the nucleus and other organelles. Since these events resemble the chromosomal condensation and nuclear envelope breakdown associated with mitosis, we investigated whether a similar biochemical mechanism might be involved by testing for the presence of cyclin B/p34cdc2 complexes and p34cdc2-associated histone kinase activity in differentiating lens fiber cells. A coupled reverse transcription/polymerase chain reaction using RNA from E7, E15, or E20 embryonic chicken lens fibers amplified a cyclin B product of the expected size, whose identity was confirmed by sequencing. In situ hybridization showed that cyclin B mRNA was present in nucleated lens fiber cells at E19. Immunoblotting of proteins isolated from E6 or E15 lens fibers by p13-agarose affinity chromatography with anti-cyclin B antibody detected the 45-kDa cyclin B protein, while immunoblotting with anti-PSTAIRE antibody detected a single, 34-kDa band, identified as p34cdc2. The p13-affinity purified fraction from E6 or E15 lens fibers showed histone H1 kinase activity in vitro. Immunocytochemistry of E6 lenses with anti-chicken cyclin B antiserum showed positive staining in the nuclei of postmitotic annular pad and fiber cells. These results demonstrate that cyclinB/p34cdc2 complexes and p34cdc2-associated histone kinase activity are present in postmitotic, differentiating lens fiber cells and support the possibility that phosphorylation of specific nuclear substrates by p34cdc2 may play a role in the denucleation of lens fiber cells.
晶状体纤维细胞的终末分化以染色质浓缩、核纤层突然溶解、核膜囊泡化以及细胞核和其他细胞器完全降解为特征。由于这些事件类似于与有丝分裂相关的染色体浓缩和核膜破裂，我们通过检测分化的晶状体纤维细胞中细胞周期蛋白B/p34cdc2复合物和p34cdc2相关组蛋白激酶活性的存在，来研究是否涉及类似的生化机制。使用来自E7、E15或E20胚胎鸡晶状体纤维的RNA进行的逆转录/聚合酶链反应扩增出了预期大小的细胞周期蛋白B产物，其身份通过测序得以确认。原位杂交显示，在E19时，有核晶状体纤维细胞中存在细胞周期蛋白B mRNA。用抗细胞周期蛋白B抗体通过p13-琼脂糖亲和层析从E6或E15晶状体纤维中分离的蛋白质进行免疫印迹，检测到45 kDa的细胞周期蛋白B蛋白，而用抗PSTAIRE抗体进行免疫印迹检测到一条单一的34 kDa条带，鉴定为p34cdc2。来自E6或E15晶状体纤维的p13亲和纯化级分在体外显示出组蛋白H1激酶活性。用抗鸡细胞周期蛋白B抗血清对E6晶状体进行免疫细胞化学分析，结果显示在有丝分裂后的环形垫和纤维细胞的细胞核中呈阳性染色。这些结果表明，细胞周期蛋白B/p34cdc2复合物和p34cdc2相关组蛋白激酶活性存在于有丝分裂后的、正在分化的晶状体纤维细胞中，并支持p34cdc2对特定核底物的磷酸化可能在晶状体纤维细胞核的去除中起作用的可能性。
