The topoisomerase I gene from Ustilago maydis: sequence, disruption and mutant phenotype.
The Ustilago maydis genomic TOP1 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 TOP1 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 TOP1 genes, followed the active site tyrosine residue. A top1 gene disruption mutant was constructed by replacing the genomic TOP1 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.
Gerhold
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Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107.
Thiyagarajan
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M
Kmiec
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EB
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004271:DNA, Fungal / Q000032:analysis; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D018628:Gene Dosage; D005800:Genes, Fungal / Q000235:genetics*; D015698:Genomic Library; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014588:Ustilago / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D004271:DNA, Fungal; D005656:Fungal Proteins; D004264:DNA Topoisomerases, Type I