THE TOPOISOMERASE-I GENE FROM USTILAGO-MAYDIS - SEQUENCE, DISRUPTION AND MUTANT PHENOTYPE

The Ustilago maydis genomic TOPI gene encoding DNA topoisomerase I was cloned by amplifying a gene fragment using the polymerase chain reaction, and using this fragment to search a genomic DNA library by hybridization. The predicted peptide sequence exhibited 30 - 40% identity to other eukaryotic TOPI genes, yet differed in several features. First, an unusually long acidic region was identified near the amino terminus (28/29 residues are acidic), which resembles other nucleolar peptide motifs. Second, an atypical carboxy-terminal 'tail', absent in other TOPI genes, followed the active site tyrosine residue. A top1 gene disruption mutant was constructed by replacing the genomic TOPI gene, with a top1::HygR null allele. This mutant lost the abundant topoisomerase I activity evident in wild-type U.maydis, and displayed a subtle coloration phenotype evident during cell senescence.