Mechanisms of Initiation of DNA replication after fertilization in Starfish eggs

Cdc 6是Mcm（mini-chromosome maintenance）蛋白的核苷酸依赖性装载因子。我们克隆了海星Cdc 6的cDNA，并通过免疫荧光显微镜技术制备了能识别染色质上Cdc 6蛋白的抗体。Cdc 6蛋白在海星未成熟卵母细胞中的表达水平很低。减数分裂恢复后，Cdc 6增加了5-10倍的初始水平。使用吗啉代双链体（M0）敲低Cdc 6。在卵母细胞成熟过程中，MO对Cdc 6的表达有90%以上的抑制作用。令人惊讶的是，Cdc 6敲低的卵母细胞、卵和胚胎中染色质上的Cdc 6与注射对照（β-珠蛋白）吗啉代的对照卵中的水平相同。此外，Ced 6敲除的胚胎至少发育了原肠胚。这些结果表明，细胞需要非常少量的Cdc 6（正常水平的1/5-1110）来复制DNA。

Cdc6 is a member of nucleotide-dependent loading factors of Mcm (mini-chromosome maintenance) proteins. We have cloned starfish Cdc6 cDNA and raised its antibody that recognized Cdc6 protein on chromatin by immuno fluorescent microscopy. Very low level of Cdc6 protein was identified in immature oocytes of starfish. After resumption of meiosis Cdc6 increased 5-10 times of initial level. Using morpholino oligonucleoties(M0) to knockdown Cdc6. Microinjection of MO inhibited more than 90% of expression of Cdc6 during oocyte, maturation. Surprisingly, Cdc6 on the chromatin in the Cdc6 knockdown oocytes, eggs and embryos was same level as in the control eggs that were injected with control (beta-globin) morpholino. Furthermore, the Ced6 knocked down embryos developed at least gastrula. These results suggest that cells require very small amount of Cdc6 (1/5-1110 of normal level) to replicate DNA.