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

点击翻译按钮获取中文摘要

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.