EFFICIENT CLONING OF ASCOMYCETE MATING-TYPE GENES BY PCR AMPLIFICATION OF THE CONSERVED MAT HMG BOX

Cloning of mating type (MAT) genes from ascomycetes has been hampered by low conservation among them, One of the pair of MAT genes, represen ted by MAT-2 of Cochliobolus heterostrophus (a loculoascomycete) and m t a of Neurospora crassa (a pyrenomycete), encodes a protein with a co nserved DNA binding motif called the high mobility group (HMG) box. PC R with primer pairs corresponding to the borders of the C, heterostrop hus and the N. crassa HMG boxes generated an similar to 0,3-kb product from genomic DNAs of MAT-2 and mt a strains, respectively, but not fr om MAT-1 and mt A strains, The C, heterostrophus primers amplified sim ilar to 0,3-kb products from DNA of most loculoascomycete genera teste d but not from DNA of pyrenomycete genera; this specificity was revers ed with the N, crassa primers, The validity of the PCR procedure was d ocumented by near sequence identity between the C, heterostrophus MAT- 2 HMG box and PCR products from several Cochliobolus spp, and by coseg regation of the PCR product with mating type in progeny of Setosphaeri a turcica and of Cryphonectria parasitica, Regions of the MAT locus fl anking the HMG box were readily cloned using the TAIL-PCR procedure wi th a combination of random and specific primers. (C) 1997 Academic Pre ss.