9424202 Maruyama Isolation of cDNA clones of interest is a crucial step toward understanding biological phenomena at the molecular level. cDNA clones are isolated from libraries by the technique of colony or plaque hybridization with labeled probes such as oligonucleoodes or antibodies, using membrane filters onto which proteins or DNAs of bacterial colonies or phage plaques have been transfered. However, screening of cDNA libraries using membrane filters is a laborious, time-consuming process, and is limited to the number of clones being searched. We have developed novel cloning vehicles, (foo, that are derived from bacteriophage ( and is capable of expressing foreign gene products as a fusion protein on the surface of the phage particle. The (foo vectors have multiple cloning sites for the insertion of a foreign DNA fragment and color selection for the recombinants. Foreign proteins are fused to the C-terminus of a truncated phage tail protein, pV, by a peptide linker. Conditional chain termination allows the assembly and fusion of a multisubunit protein. The (foo clones express functionally active proteins on the phage particle surface and can be purified by affinity chromatography with various probes immobilized on solid supports. The objective of this project is to determine experimental conditions for the general use of the (foo vector for construction of cDNA libraries and screening of cDNA clones with in vitro affinity selection techniques. (a) We will make the cDNA libraries with the (foo vectors from mRNA of the nematode Caenorhabditis elegans as a model organism. The following two approaches will be undertaken for the construction of the libraries: (1) The full-length cDNA libraries will be made from C. elegans mRNA with oligo(dt) as a primer for cDNA synthesis. and (2) the "domain library" will be made from cDNAs synthesized with random hexanucleotides. The two libraries will compensate each other for covering the entire mRNA population. (b) Using the (foo libraries, the follo wing affinity selection schemes will be examined for the isolation of cDNA clones: (1) antigen-antibody interactions, and (2) protein-protein interactions. In the first scheme, the proteins such as caltractin, centromere proteins, and/or neurofilament proteins, will be the targets to which we will use specific antibodies as probes immobilized on a microtiter plate. The second scheme aims to isolate cDNA clones encoding proteins that bind to the C2 domains of protein kinase C and Unc-13 proteins as well as to the SH3 domain of dynamin. During the selection process, we will determine optimum conditions for the amplification of the libraries by changing E. coli host strains and inoculum size of the library phages. After purification of the cDNA clones, the products will be expressed in bacteria, and specific binding will be confirmed by in vitro binding assay. (c) Toward understanding biological function of the clones, the isolated cDNA will be further analyzed by DNA sequencing, Northern and Southern blot hybridization. Full-length cDNA will be isolated by screening other cDNA libraries and PCR amplification of the cDNA ends. A genomic map position of the clones will also be determined by DNA hybridization to membrane filters blotted with the isolated genomic DNA in the genomic mapping project. The establishment of experimental conditions for the construction and screening of the (foo libraries shall dramatically improve technology of cDNA library screening. The system will allow one lo search 1013 phages or more by simple affinity selection using probes immobilized on solid supports such as a microtiter plate, paramagnetic beads, or agarose beads. Therefore, one can expect to isolate rare clones that have previously been diffficult or impossible to isolate by the conventional methods. One can also search the (foo libraries with many different probes including antibodies, DNA, inhibitors, ligands, and protein domains. The selection scheme is a simple and economical alternative to the convention al filter screening. Application of the (foo system is not limited to screening of cDNA libraries. It can be useful for the selection of a particular mutant gene by affinity chromatography after random mutagenesis of the gene. In the genome mapping projects, many genes encoding DNA-binding proteins may be efficiently identified by screening (foo libraries with isolated genomic DNAs as probes. %%% The development of an improved way of selecting for rare cloned genes will be of great use to researchers trying to identify and isolate important genes and to industrial scientists who are trying to isolate genes from any organism for biotechnology applications. ***
