Summary
The mutant allele rad9-192 renders Schizosaccharomyces pombe cells sensitive to ionizing radiation and UV light. We have isolated from a S. pombe genomic DNA library a unique recombinant plasmid that is capable of restoring wild-type levels of radioresistance to a rad9 192-containing cell population. Plasmid integration studies using the cloned DNA, coupled with mating and tetrad analyses, indicate that this isolated DNA contains the wild-type rad9 gene. We inactivated the repair function of the cloned fragment by a single insertion of the S. pombe ura4 gene. This nonfunctional fragment was used to create a viable disruption mutant, thus demonstrating that the rad9 gene does not encode an essential cellular function. In addition, the rad9-192 mutant population is as radiosensitive as the disruption mutant, indicating that rad9 gene function is severely if not totally inhibited by the molecular defect responsible for the rad9-192 phenotype. DNA sequence analysis of rad9 reveals an open reading frame of 1,278 bp, interrupted by three introns 53 bp, 57 bp, and 56 by long, respectively, and ending in the termination codon TAG. This gene is capable of encoding a protein of 426 amino acids, with a corresponding calculated molecular weight of 47,464 daltons. No significant homology was detected between the rad9 gene or its deduced protein sequence and sequences previously entered into DNA and protein sequence data banks.
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Allshire RC, Cranston G, Gosden JR, Moule JC, Hastie ND, Fantes PA (1989) A fission yeast chromosome can replicate autonomously in mouse cells. Cell 50:391–403
Bennetzen JL, Hall BD (1982) Codon selection in yeast. J Biol Chem 257:3026–3031
Fabre F (1970) UV sensitivity of the wild type and different UVS mutants of Schizosaccharomyces pombe. Influence of growth stage and DNA content of the cells. Mutat Res 10:415–426
Feinberg AP, Vogelstein B (1983) A technique for labeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13
Friedberg EC (1988) Deoxyribonucleic acid repair in the yeast Saccharomyces cerevisiae. Microbiol Rev 52: 70–102
Gentner NE (1977) Evidence for a second “prereplicative G2” pathway mechanism specific for γ-induced damage in wild type Schizosaccharomyces pombe. Mol Gen Genet 154:129–133
Gentner NE (1981) Both caffeine-induced lethality and the negative liquid holding effect, in UV- or γ-irradiated wild-type Schizosaccharomyces pombe, are consequences of interference with a recombinational repair process. Mol Gen Genet 181:283–287
Gentner NE, Werner MM (1976) Effect of protein synthesis inhibition on recovery of UV and γ-irradiated Schizosaccharomyces pombe from repair inhibition by caffeine. Mol Gen Genet 145:1–5
Gentner NE, Werner MM, Hannan MA, Nasim A (1978) Contribution of a caffeine-sensitive recombinational repair pathway to survival and mutagenesis in UV-irradiated Schizosaccharomyces pombe. Mol Gen Genet 167:43–49
Gutz H, Heslot H, Leupold U, Loprieno N (1974) Schizosaccharomyces pombe. In: King RC (ed) Handbook of genetics, Vol 1. Plenum, New York, pp 395–446
Hanahan D (1985) Techniques for transformation in E. coli. In: Glover DM (ed) DNA cloning, Vol 1. IRL Press, Washington, DC, pp 109–135
Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359
Higgins DR, Prakash S, Reynolds P, Polakowska R, Weber S, Prakash L (1983) Isolation and characterization of the rad3 gene of Saccharomyces cerevisiae and inviability of deletion mutants. Proc Natl Acad Sci USA 80:5680–5684
Hindley I, Phear GA (1984) Sequence of the cell division cycle gene CDC2 from Schizosaccharomyces pombe: Patterns of splicing and homology to protein kinases. Gene 31:129–134
Hoffman CS, Winston F (1987) A ten-minute preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272
Innis MA, Gelfand DH (1990) Optimization of PCRs. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols, A guide to methods and applications. Academic Press, San Diego, pp 3–12
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Jones RH, Moreno S, Nurse P, Jones NC (1988) Expression of the SV40 promoter in fission yeast: identification of an AP-1-like factor. Cell 53:659–667
Kaufer NF, Simanis V, Nurse P (1985) Fission yeast Schizosaccharomyces pombe correctly excises a mammalian RNA transcript intervening sequence. Nature 318:78–80
Kawasaki ES (1990) Amplification of RNA. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols, A guide to methods and applications. Academic Press, San Diego, pp 21–27
Kozak M (1989) The scanning model for translation: an update. J Cell Biol 108:229–241
Lee MG, Nurse P (1987) Complementation used to clone human homologue of the fission yeast cell cycle control gene cdc2. Nature 327:31–35
Lieberman HB, Riley R, Martel M (1989) Isolation and initial characterization of a Schizosaccharomyces pombe mutant exhibiting temperature-dependent radiation sensitivity due to a mutation in a previously unidentified rad locus. Mol Gen Genet 218:554–558
Mao J, Appel B, Schaak J, Sharp S, Yamada H, Soll D (1982) The 5S genes of Schizosaccharomyces pombe. Nucleic Acids Res 10:487–498
McCready SJ, Burkill H, Evans S, Cox BS (1989) The Saccharomyces cerevisae RAD2 gene complements a Schizosaccharomyces pombe repair mutation. Curr Genet 15:27–30
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Murray JM, Watts FZ (1990) Isolation of a Schizosaccharomyces pombe homologue to the rat ribosomal protein L7. Nucleic Acids Res 18:4590
Nasim A, Smith BP (1974) Dark repair inhibitors and pathways for repair of radiation damage in Schizosaccharomyces pombe. Mol Gen Genet 132:13–22
Naumovski L, Friedberg EC (1983) A DNA repair gene required for the incision of damaged DNA is essential for viability in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 80:4818–4821
Okazaki K, Okazaki N, Kume K, Jinno S, Tanaka K, Okayama H (1990) High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by transcomplementation of Schizosaccharomyces pombe. Nucleic Acids Res 18:6485–6489
Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: A model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358
Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448
Phipps J, Nasim A, Miller DR (1985) Recovery, repair and mutagenesis in Schizosaccharomyces pombe. Adv Genet 23:1–72
Reynolds P, Koken MHM, Hoeijmakers JHJ, Prakash S, Prakash L (1990) The rhp + gene of Schizosaccharomyces pombe: a structural and functional homologue of the RAD6 gene from a distantly related yeast Saccharomyces cerevisiae. EMBO J 9:1423–1430
Rubin JS (1988) Review. The molecular genetics of the incision step in the DNA excision repair process. Int J Radiat Biol 54:309–365
Russell P (1989) Gene cloning and expression in fission yeast. In: Nasim A, Young P, Johnson BF (eds) Molecular biology of the fission yeast. Academic Press, San Diego, pp 243–271
Russell PR, Hall BD (1983) The primary structure of the alcohol dehydrogenase gene from the fission yeast Schizosaccharomyces pombe. J Biol Chem 258:143–149
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schüpbach M (1971) The isolation and genetic classification of UV-sensitive mutants of Schizosaccharomyces pombe. Mutat Res 11:361–371
Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Sunnerhagen P, Seaton BL, Nasim A, Subramani S (1990) Cloning and analysis of a gene involved in DNA repair and recombination, the rad1 gene of Schizosaccharomyces pombe. Mol Cell Biol 10:3750–3760
Wright A, Maundrell K, Heyer W-D, Beach D, Nurse P (1986) Vectors for the construction of gene banks and the integration of cloned genes in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Plasmid 15:156–158
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Communicated by B.J. Kilbey
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Lieberman, H.B., Hopkins, K.M., Laverty, M. et al. Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis. Molec. Gen. Genet. 232, 367–376 (1992). https://doi.org/10.1007/BF00266239
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DOI: https://doi.org/10.1007/BF00266239