Patent ID: 6991900
Filing Date: 2006-01-31
Classification: C12N

Abstract:
1. A method for identifying an essential chromosomal gene in a haploid test organism, said method comprising: a) constructing a bacterial artificial chromosome (BAC)-carrying merodiploid test cell by transforming a wild-type haploid host cell whose genomic sequence is known, which is naturally capable of being transformed and undergoing DNA recombination, with a BAC carrying a known segment of DNA of the haploid test organism with about 80% to 100% sequence identity to a known segment of chromosomal DNA in the host cell, and wherein replication of the BAC in the test cell is sensitive to an environmental condition that selectively prevents replication of the BAC in the host cell; b) inserting randomly a bacterial transposon into the merodiploid test cell so as to disrupt function of a gene therein; c) culturing one or more of the BAC-carrying merodiploid test cells in a suitable culture medium while introducing the environmental condition so as to transform the merodiploid test cells into haploid test cells; d) identifying one or more of the haploid test cells that contain transposon-mutagenized DNA in an essential chromosomal gene therein, and e) obtaining the identity of the essential chromosomal gene in the test organism by locating a gene in the known segment of DNA of the haploid test organism that contains the transposon-mutagenized DNA.