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. 1991 Jan;127(1):139–149. doi: 10.1093/genetics/127.1.139

Mapping Point Mutations in the Drosophila Rosy Locus Using Denaturing Gradient Gel Blots

M Gray 1, A Charpentier 1, K Walsh 1, P Wu 1, W Bender 1
PMCID: PMC1204299  PMID: 1901817

Abstract

Mutations within the rosy locus of Drosophila were mapped using blots of genomic DNA fragments separated on denaturing gradient gels. DNA sequence differences between otherwise identical small rosy DNA fragments were detected among the mutants as mobility shifts on the blots. Mutations were mapped to within a few hundred base pairs of rosy sequence in 100 of 130 mutants tested--a 77% detection rate. The sequence changes in 43 rosy mutations are presented; all but six of these were single base changes. Thirty-four of 36 sequenced mutations induced by the alkylating agents N-ethyl-N-nitrosourea and ethyl methanesulfonate were transitions. All of the mutations mapped in the rosy transcription unit. Twenty-three of the 43 sequenced mutations change the predicted rosy gene polypeptide sequence; the remainder would interrupt protein translation (17), or disrupt mRNA processing (3).

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bender W., Spierer P., Hogness D. S. Chromosomal walking and jumping to isolate DNA from the Ace and rosy loci and the bithorax complex in Drosophila melanogaster. J Mol Biol. 1983 Jul 25;168(1):17–33. doi: 10.1016/s0022-2836(83)80320-9. [DOI] [PubMed] [Google Scholar]
  2. Birnstiel M. L., Busslinger M., Strub K. Transcription termination and 3' processing: the end is in site! Cell. 1985 Jun;41(2):349–359. doi: 10.1016/s0092-8674(85)80007-6. [DOI] [PubMed] [Google Scholar]
  3. Chovnick A., Gelbart W., McCarron M. Organization of the Rosy locus in Drosophila melanogaster. Cell. 1977 May;11(1):1–10. doi: 10.1016/0092-8674(77)90312-9. [DOI] [PubMed] [Google Scholar]
  4. Coté B., Bender W., Curtis D., Chovnick A. Molecular mapping of the rosy locus in Drosophila melanogaster. Genetics. 1986 Apr;112(4):769–783. doi: 10.1093/genetics/112.4.769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Curtis D., Clark S. H., Chovnick A., Bender W. Molecular analysis of recombination events in Drosophila. Genetics. 1989 Jul;122(3):653–661. doi: 10.1093/genetics/122.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Fischer S. G., Lerman L. S. Two-dimensional electrophoretic separation of restriction enzyme fragments of DNA. Methods Enzymol. 1979;68:183–191. doi: 10.1016/0076-6879(79)68013-8. [DOI] [PubMed] [Google Scholar]
  9. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  11. Hu N., Messing J. The making of strand-specific M13 probes. Gene. 1982 Mar;17(3):271–277. doi: 10.1016/0378-1119(82)90143-3. [DOI] [PubMed] [Google Scholar]
  12. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Keith T. P., Riley M. A., Kreitman M., Lewontin R. C., Curtis D., Chambers G. Sequence of the structural gene for xanthine dehydrogenase (rosy locus) in Drosophila melanogaster. Genetics. 1987 May;116(1):67–73. doi: 10.1093/genetics/116.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kelley M. R., Kidd S., Deutsch W. A., Young M. W. Mutations altering the structure of epidermal growth factor-like coding sequences at the Drosophila Notch locus. Cell. 1987 Nov 20;51(4):539–548. doi: 10.1016/0092-8674(87)90123-1. [DOI] [PubMed] [Google Scholar]
  15. Kessler C., Neumaier P. S., Wolf W. Recognition sequences of restriction endonucleases and methylases--a review. Gene. 1985;33(1):1–102. doi: 10.1016/0378-1119(85)90119-2. [DOI] [PubMed] [Google Scholar]
  16. Lee C. S., Curtis D., McCarron M., Love C., Gray M., Bender W., Chovnick A. Mutations affecting expression of the rosy locus in Drosophila melanogaster. Genetics. 1987 May;116(1):55–66. doi: 10.1093/genetics/116.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maniatis T., Jeffrey A., van deSande H. Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry. 1975 Aug 26;14(17):3787–3794. doi: 10.1021/bi00688a010. [DOI] [PubMed] [Google Scholar]
  18. Martin P. F., Place A. R., Pentz E., Sofer W. UGA nonsense mutation in the alcohol dehydrogenase gene of Drosophila melanogaster. J Mol Biol. 1985 Jul 20;184(2):221–229. doi: 10.1016/0022-2836(85)90375-4. [DOI] [PubMed] [Google Scholar]
  19. Myers R. M., Lerman L. S., Maniatis T. A general method for saturation mutagenesis of cloned DNA fragments. Science. 1985 Jul 19;229(4710):242–247. doi: 10.1126/science.2990046. [DOI] [PubMed] [Google Scholar]
  20. Myers R. M., Tilly K., Maniatis T. Fine structure genetic analysis of a beta-globin promoter. Science. 1986 May 2;232(4750):613–618. doi: 10.1126/science.3457470. [DOI] [PubMed] [Google Scholar]
  21. Nicholls R. D., Hill A. V., Clegg J. B., Higgs D. R. Direct cloning of specific genomic DNA sequences in plasmid libraries following fragment enrichment. Nucleic Acids Res. 1985 Nov 11;13(21):7569–7578. doi: 10.1093/nar/13.21.7569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pastink A., Vreeken C., Nivard M. J., Searles L. L., Vogel E. W. Sequence analysis of N-ethyl-N-nitrosourea-induced vermilion mutations in Drosophila melanogaster. Genetics. 1989 Sep;123(1):123–129. doi: 10.1093/genetics/123.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pastink A., Vreeken C., Schalet A. P., Eeken J. C. DNA sequence analysis of X-ray-induced deletions at the white locus of Drosophila melanogaster. Mutat Res. 1988 Jan;207(1):23–28. doi: 10.1016/0165-7992(88)90006-1. [DOI] [PubMed] [Google Scholar]
  24. Reardon J. T., Liljestrand-Golden C. A., Dusenbery R. L., Smith P. D. Molecular Analysis of Diepoxybutane-Induced Mutations at the rosy Locus of Drosophila melanogaster. Genetics. 1987 Feb;115(2):323–331. doi: 10.1093/genetics/115.2.323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yu Q., Jacquier A. C., Citri Y., Hamblen M., Hall J. C., Rosbash M. Molecular mapping of point mutations in the period gene that stop or speed up biological clocks in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1987 Feb;84(3):784–788. doi: 10.1073/pnas.84.3.784. [DOI] [PMC free article] [PubMed] [Google Scholar]

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