The molecular biology core laboratory will serve as a central resource in the program for obtaining information at the gene level related to various proteins under investigation. The proteins under study in the individual projects cover a wide range of activities, and they have a significant effect on the growth properties of the cells under study. The recombinant DNA laboratory will prepare complementary DNA libraries to total poly(A+)- RNA. Libraries with 10,000,000 to 100,000,000 recombinant clones per ug of poly (A+)RNA will be prepared. This should be sufficient to find the cDNA to a given messenger RNA which is in a concentration as low as 1 part in 100,000,000 to the total messenger RNA. Once a particular cDNA has been identified, the complete nucleotide sequence of the messenger RNA can be determined. The cDNA libraries will mainly be prepared in the lambda vectors lambda gt11 and lambda ZAP (Stratagene). These vectors will allow high level expression of fused proteins coded for by the cDNAs. The various clones can be screened by using antibodies and specific oligonucleotide probes. The cDNAs will also be used to analyze the expression of these genes in vitro. This may shed light on the role of the proteins under investigation both in vitro and in vivo. State-of-art DNA sequencing and cloning procedures will be used.
Showing the most recent 10 out of 109 publications
