Structure of the rec12 gene and constructs used. (A) Intron/exon structure. Genomic DNA (D) and total RNA (R) obtained from meiotic cultures of strain WSP0020 were subject to PCR and RT-PCR, respectively, using primers flanking the putative introns (a-d). Products were resolved on a 2% agarose gel stained with EtBr. (B) Primary sequence of Rec12 protein based upon the DNA sequence of a complementing cDNA clone. Residues of Rec12 with at least 50% identity (black boxes) or 50% conservation (gray boxes) relative to other eukaryotic Spo11 family members are indicated. Also shown are the positions of the active site tyrosine (*) and residues conserved among other Spo11 family members, but not conserved in S. pombe (open boxes). Alignments evaluated proteins from S. pombe (P40384), Neurospora crassa (Q9P6Y7), Coprinus cinereus (Q9P4D2), Homo sapiens (Q9NQM7), Mus musculus (Q9QZS1), Arabidopsis thaliana (AAL01152), Drosophila melanogaster (O77205), Caenorhabditis elegans (Q22236), and Saccharomyces cerevisiae (P23179). (C) Structure of constructs. Gene targeting of the endogenous rec12+ locus was used to introduce a null allele (rec12-D15::ura4+) lacking the complete coding region and a point mutation allele (rec12-Y98F) encoding a protein in which the active site tyrosine at position 98 was replaced with phenylalanine. Placing the rec12+ coding region and the rec12+ cDNA into the various pREP plasmids  allowed for a wide range of regulated gene expression.