Cloning of open reading frames and promoters from the Saccharomyces cerevisiae genome: construction of genomic libraries of random small fragments

Abstract

We have developed a novel efficient method, carrier-facilitated insertion, to insert small (150–600 bp) DNA fragments into plasmid vectors. This method employs a carrier segment of vector DNA to circumvent the difficulties in ligating two fragments together to generate a recombinant circle efficiently. We have used carrier-facilitated insertion to construct three genomic libraries of random (DNase I-generated) fragments from the Saccharomyces cerevisiae genome. One of these was an expression library, and the other two were promoter-cloning libraries. 87–90% of the Escherichia coli colonies in each library contained recombinant plasmids, and less than 3 % of the recombinants contained more than one insert. Detection of open reading frames among the inserts in the expression library was accomplished by testing for β-galactosidase activity. This methodology, unencumbered by the intrinsic disproportionality of cDNA libraries, can be used to identify and clone DNA that codes for a specific antigenic determinant. When used in combination with a method to detect and isolate random constitutive, repressible and inducible yeast promoters, these libraries should permit a comprehensive analysis of the yeast genome and its expression.