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

一种新载体系统的开发和应用，用于制备人类 cDNA 文库的整个目录。

基本信息

批准号：
02557098
负责人：
NOJIMA Hiroshi
金额：
$ 8.64万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

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.

我们已经开发了一个新的向量系统（一系列PCD3向量），该系统是冈马 - 伯格矢量（PCD2矢量）的版本。 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为了有效的cDNA文库制备和DNA千目标测序。原始矢量系统的实用性是通过与s.pombe突变体的互补克隆作为宿主进行检查的；我们已经克隆并分析了3个补充CDC2和CDC13的新型人类基因。 DNA测序表明它们编码RNA结合蛋白。他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。我们还克隆了互补Cdc10突变体的8种新型基因。 DNA测序揭示了两个克隆用两个Ankyrin基序编码蛋白质，并具有与Cdc10基因产物以及SW14/SW16（S。s.cerevisiae）基因产物的同源域的蛋白质。发现一个克隆本身是CDC18。一个克隆（REP1）编码在前DNA合成中需要的锌指蛋白。一个克隆（HAC1）是AFT/CREB的同源物。三个克隆的DNA序列仍有待确定。