Cloning and analysis of the genes involved in the regulation of cell cycle and growth by complementation cloning.

我们利用两种不同的研究系统进行我们的研究项目，分别如下：(1)使用正常大鼠肾脏细胞：NRK细胞是有用的，因为它们可以通过添加EGF和转化生长因子-β进行可逆转化。我们已经建立了几个对EGF和转化生长因子-β不敏感的突变株。利用这些突变体，我们研究了EGF和几个癌基因的信号通路以及锚定依赖性。(2)利用Pombe细胞分裂周期(CDC)突变体cdc2、cdc13和cdc10：我们克隆并分析了3个补充cdc2和cdc13的人类新基因。DNA测序显示，它们编码RNA结合蛋白。它们似乎通过稳定和诱导cdc13基因产物的翻译来补充这些突变体。我们还克隆了8种与CDC 10突变体互补的新基因。DNA测序表明，这两个克隆编码的蛋白具有两个锚定基序，并且与cdc10基因产物和SWI4/SW16(酿酒酵母)基因产物具有同源结构域。其中一个克隆被发现是cdc18本身。一个克隆(REP1)编码锌指蛋白，这是减数分裂前DNA合成所必需的。其中一个克隆(HAC1)与ATF/CREB同源。其余三个克隆的DNA序列仍有待确定。

We have pursued our research project by utilizing two kinds of research systems as described separately as follows;(1) Using NRK(normal rat kidney) cells: NRK cells are useful because they are transformed reversibly by the addition of EGF and TGF-beta. We have established several mutant strains which are insensitive to the effect of EGF and TGF-beta. Usingthese mutants, we have investigated the signal pathway of EGF and several oncogenes and anchorage dependency.(2) Using S.pombe cell division cycle (cdc) mutants, cdc2, cdc13 and cdc10: We have cloned and analysed 3 novel human genes that complement both cdc2 and cdc13. DNA sequencing revealed that they encode RNA binding proteins. They seem to complement these mutants by stabilizing and inducing translation of cdc13 gene product. We also cloned 8 kinds of novel genes that complement cdc 10 mutant. DNA sequencing unveiled that two clones encode proteins with two ankyrin motifs and with homologous domains to cdc10 gene product and those of SWI4/SW16 (S.cerevisiae) gene products. One clone was found to be cdc18 itself. One clone (rep1) encode a zinc-finger protein which is required in premeiotic DNA synthesis. One clone (HAC1) was a homologue of ATF/CREB. DNA sequence of the rest of three clones remain to be determined.