Identification of mating-type dependent genes by non-radioactive, arbitrarily primed PCR in Schizophyllum commune.

Abstract

Using arbitrary primers for a PCR reaction on cDNA from different mating interactions, genes differentially expressed in a mating-type dependent manner have been identified. The cDNAs were prepared from mRNA extracted from liquid grown cultures of the fungus in order to prevent further steps in development and increase the possibility of identifying genes directly under control of the mating type system. A reaction using a monokaryotic strain was compared to a semicompatible mating reaction induced for B-regulated development and a reaction using a fully compatible mating. In the comparison of silver-stained, non-denaturing polyacrylamide gel separation of the PCR products bands differentially detected between the samples were cloned and subjected to further analysis. Bands were characterized which were present in both mating reactions but absent in the monokaryon while the third band was specifically absent in the fully compatible mating interaction. Sequence analysis allowed design of specific primers for verification of differential expression in a semi-quantitative PCR. Thus, use of radioactivity was not necessary for the fast and reproducible method identifying genes specifically regulated by mating reactions.