Mechanisms of Initiation of DNA replication after fertilization in Starfish eggs

Cdc6是Mcm（迷你染色体维持）蛋白的核苷酸依赖装载因子的成员。我们克隆了海星Cdc6 cDNA，并在免疫荧光显微镜下获得了在染色质上识别Cdc6蛋白的抗体。在海星未成熟卵母细胞中发现了极低水平的Cdc6蛋白。减数分裂恢复后，Cdc6增加5-10倍于初始水平。利用morpholino寡核（M0）敲除Cdc6。在卵母细胞成熟过程中，微量注射MO可抑制Cdc6 90%以上的表达。令人惊讶的是，Cdc6敲除的卵母细胞、卵子和胚胎中染色质上的Cdc6与注射了对照（β -珠蛋白）morpholino的对照卵子中的Cdc6水平相同。此外，敲除Ced6的胚胎至少发育出原肠胚。这些结果表明细胞需要非常少量的Cdc6（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.