Development and application of a new vector system for the preparation of the whole catalog of a human cDNA library.

我们开发了一个新的矢量系统（一系列pcd3矢量），它是冈山-伯格矢量（pcD2矢量）的升级版。这些向量具有以下特征；(1)利用这些载体，将cDNA通过BstXI位点（具有5′-GGGG-3′粘端）插入，可从cDNA上游5′直接测序，适用于PCR。(2)引入f1起源和T3/T7 RNA聚合酶启动子，可以在cDNA文库之间进行有效的减法。(3)介绍了一种用于高效cDNA文库制备和DNA千碱基测序的多克隆位点。以S.pombe突变体为寄主，通过互补克隆验证了原载体体系的有效性。我们克隆并分析了3个新的人类基因，它们与cdc2和cdc13互补。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cdc10突变体互补的新基因。DNA测序结果显示，两个克隆编码的蛋白具有两个锚蛋白基序，并且与cdc10基因产物和SW14/SW16(S)具有同源结构域。酿酒酵母基因产物。一个克隆体被发现是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）是AFT/CREB的同源物。这三个克隆的DNA序列还有待确定。

We have developed an new vector system (a series of pcD3vectors), which is a version-up of Okayama-Berg vector (pcD2 vectors). These vectors have following features;(1) With these vectors, cDNA is inserted via BstXI site (with 5'-GGGG-3' sticky ends), which enables direct sequencing from the 5'upstream of cDNAs and is applicable to PCR.(2) Introduction of f1 origin and promotors of T3/T7 RNA polymerases enables efficient subtraction among cDNA libraries.(3) A multicloning site useful for efficient cDNA library preparation and DNA kilobase- sequencing is introduced.The usefulness of the original vector system was checked by complementation cloning with S.pombe mutant as host as follows;We have cloned and analyzed 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 cdc10 mutant. DNA sequencing unveiled that two clones encode proteins with two ankyrin motifs and with homologous domains to cdc10 gene product and those of SW14/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 AFT/CREB. DNA sequence of the three clones remain to be determined.