Efficient isolation and mapping of rad genes of the fungus Coprinus cinereus using chromosome-specific libraries.

Abstract

We have constructed cosmid libraries from electrophoretically separated chromosomes of the basidiomycete Coprinus cinereus. These libraries greatly facilitate the isolation of genes by complementation of mutant phenotypes and are particularly useful for map-based cloning strategies. From a library constructed from two co-migrating C.cinereus chromosomes, we isolated a clone that complements the C.cinereus rad9-1 mutation. Examination of this clone showed that it complements both the repair and meiotic defects of this mutant. Restriction fragment length polymorphism mapping using a portion of this clone showed that it maps to the rad9 locus. In addition, a single copy of transforming DNA is sufficient to complement the rad9-1 defects. Thus, we believe we have cloned the rad9 gene itself. We also used a chromosome-specific library and backcrossed isolates to rapidly identify a cosmid clone which is tightly linked to the rad11 locus and is therefore a suitable starting point for a chromosome walk. These rapid methods of gene mapping and isolation should be applicable to any organism with separable chromosomes.