The aim of this proposal is to use PCR/recombination-directed library construction to create libraries that will facilitate genome-wide analysis of yeast. The proposal can be divided into three specific aims: (i) A set of unique primers (called """"""""adaptamers"""""""" in this proposal) to every intergenic region on chromosome V will be designed to permit many possible gene fusion, gene disruption and gene insertion strategies. Each adaptamer will have a tag that will allow the fusion of any adjacent sequence by PCR. Using various combinations of adaptamers, we will construct two arrayed libraries. (ii) The first library will contain the fusion of promoters from every open reading frame (ORF) on chromosome V to the green fluorescent protein (GFP) gene cloned into a circular autonomously replicating plasmid. Introduction of this library into various strains will permit a """"""""readout"""""""" of gene expression for each promoter under different conditions (e.g., various carbon sources, differing osmolarity, after irradiation, etc.) or in different genetic backgrounds (e.g., various deletions such as transcription factors, rad genes, etc.). The library will be transferred from strain to strain by using a kar1 mutation that blocks nuclear fusion but permits the transfer of plasmids or chromosomes between nuclei. (iii) The second library will contain a gene disruption of each chromosome V ORF in a vector specifically designed to permit the liberation of the disrupting fragment after transfer into the strain of choice. Once again, a kar1 mutant strain will be used as the host strain. In addition, unique restriction sites (I-SceI) and an inducible copy of the I-SceI enzyme will be included in this plasmid. Upon transfer into the recipient nucleus, the I-SceI enzyme will be induced, releasing the disruption fragment and thereby stimulating its integration into the recipient genome. Many of the methods outlined here are not specific for yeast and can be applied to any sequenced genome. For example, a two-hybrid library for any genome can be constructed in yeast using adaptamers. Therefore, a library can be designed for sequenced bacterial genomes or even human cDNAs. In addition, this technology will serve as a testing ground for these other organisms.
