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

Using arbitrary primers for a PCR reaction on cDNA from different mating in teractions, genes differentially expressed in a mating-type dependent manne r have been identified. The cDNAs were prepared from mRNA extracted from li quid grown cultures of the fungus in order to prevent further steps in deve lopment and increase the possibility of identifying genes directly under co ntrol of the mating type system. A reaction using a monokaryotic strain was compared to a semicompatible mating reaction induced for B-regulated devel opment and a reaction using a fully compatible mating. In the comparison of silver-stained, non-denaturing polyacrylamide gel separation of the PCR pr oducts bands differentially detected between the samples were cloned and su bjected to further analysis. Bands were characterized which:were present in both mating reactions but absent in the monokaryon while the third band wa s specifically absent in the fully compatible mating interaction. Sequence analysis allowed design of specific primers for verification of differentia l expression in a semi-quantitative PCR. Thus, use of radioactivity was not necessary for the fast and reproducible method identifying genes specifica lly regulated by mating reactions.