A clone containing the DNA repair gene uvs-2 of Neurospora crassa was identified from a Neurospora genomic DNA library using the sib-selection method. Transformants were screened for resistance to methyl methane sulfonate (MMS). A DNA fragment that complements the uvs-2 mutation was subcloned by monitoring its ability to transform the uvs-2 mutant to MMS resistance. Deletion analysis of the cloned DNA indicated that the size of the uvs-2 gene is approximately 1.6 kb. The identity of the uvs-2 gene was verified by restriction fragment length polymorphism (RFLP) mapping. The sensitivity of the transformant to three different mutagens was similar to that of the wild-type strain. Nucleotide sequences of genomic DNA and cDNA of the uvs-2 gene indicated that it has an open reading frame (ORF) of 1572 bp with a 69 bp intron in the middle of the sequence. Two transcription initiation sites located around 73 bp and 290 bp upstream of the translation initiation codon were identified by primer extension experiments. Northern analysis revealed that the nature transcript of the uvs-2 gene was about 1.8 kb long. The uvs-2 gene ORF is deduced to encode a polypeptide of 501 amino acids with a molecular mass of 54 kDa. The proposed polypeptide has 25% identity to the RAD18 polypeptide of Saccharomyces cerevisiae and contains two unique zinc finger motifs for nucleic acid binding. Similarities between the phenotypes of the rad18 and uvs-2 mutants suggest that the uvs-2 gene encodes a protein which is involved in postreplication repair, rather than excision repair.

• PMID: 8097557
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Comparative Study | Journal Article | Research Support, Non-U.S. Gov't

Authors

Tomita H, Soshi T, Inoue H

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