The recombinant DNA laboratory will serve as a central resource in the program for obtaining information and materials related to new growth factors which will be purified from a variety of tissue culture systems of normal cells. The types of projects that will be served have a common element in that there is an unknown factor or factors either produced by cells or contained in crude extracts of tissues such as brain, which have a profound effect on the growth properties of those cells under study. As these new growth factors are purified the recombinant DNA laboratory will prepare complementary DNA libraries to total poly(A+)-RNA isolated from the cell lines under study in this program project. It will be fairly straightforward to prepare cloned cDNA libraries with 10/6 to 10/7 clones per mug of poly(A+)-RNA. This should be sufficient to find the cDNA to a given messenger RNA which is in a concentration as low as 1 part in 10/7 to the total messenger RNA. Once a cDNA to a new growth factor is identified, the complete nucleotide sequence of the messenger RNA can be determined. The cDNA libraries will mainly be prepared in the lambda vector lambda gt 11 or 10. Two of these vectors will allow high level expression of fused proteins coded for by these messenger RNAs. These various clones can then be screened by using antibody to the new factor, or as amino acid information becomes available on the new factors, specific oligonucleotide probes can be synthesized to screen and isolate the appropriate clones from the cDNA libraries. Finally, and most importantly, the cDNAs to the new growth factors will be used to analyze the expression of these genes in normal and abnormal tissues and cells in vitro which then may shed light on the role of these factors in normal abnormal cellular physiology both in vitro and in vivo.
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