Abstract
Genetic rearrangements such as deletions or duplications of DNA sequences are rarely detected in the yeast Saccharomyces cerevisiae. We have developed a screening system using the URA2 gene coding for the bi-functional CPSase-ATCase (carbamyl phosphate synthetase — aspartate transcarbamylase) to select positively for these kinds of events. Nonsense mutations in the CPSase region cause a complete loss of the ATCase activity because of their strong polar effect. Thirty-seven ATCase+ revertants were isolated from a strain containing three nonsense mutations in the proximal CPSase region. Genetic and structural analysis of the URA2 locus in these strains allowed us to characterize two major classes of revertants. In the first, an entire copy of a Ty transposon was found to be inserted in the CPSase coding domain. This event, which represents a new form of Ty-mediated gene activation was further analysed by mapping the Ty integration site in 26 strains. In a second class of revertants, we observed chromosomal rearrangements and, in particular, duplication of the ATCase region and its integration in a new chromosomal environment in which this sequence becomes active.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bach ML (1984) Tyl-promoted expression of aspartate transcarbamylase in the yeast Saccharomyces cerevisiae. Mol Gen Genet 194:395–401
Boeke JD, Sandmeyer SB (1991) Yeast transposable elements. In: Broach JR, Pringle JF, Jones EW (eds) The molecular and cellular biology of the yeast Saccharomyces: genome dynamics, protein synthesis, and energetics, vol 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 193–262
Bradshaw VA, McEntee K (1989) DNA damage activates transcription and transposition of yeast Ty retrotransposons. Mol Gen Genet 218:465–474
Carle GF, Olson MV (1985) An electrophoretic karyotype for yeast. Proc Natl Acad Sci USA 82:3756–3760
Chu G, Vollrath GD, Davis RW (1986) Separation of large DNA molecules by contour-clamped homogenous electric fields. Science 234:1582–1585
Derr LK, Strathern JN, Garfinkel DJ (1991) RNA-mediated recombination in S. cerevisiae. Cell 67: 355–364
Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395
Exinger F, Lacroute F (1979) Genetic evidence for the creation of a reinitiation site by mutation inside the yeast URA2 gene. Mol Gen Genet 173:109–113
Lacroute F, Pierard A, Grenson M, Wiame JM (1965) The biosynthesis of carbamoylphosphate in Saccharomyces cerevisiae. J Gen Microbiol 40: 127–142
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Morawetz C, Hagen U (1990) Effect of irradiation and mutagenic chemicals on the generation of ADH2 and ADH4 constitutive mutants in yeast: the inducibility of Ty transposition by UV and ethyl methane sulfonate. Mutat Res 229: 69–77
Mortimer RK, Hawthorne DC (1966) Genetic mapping in Saccharomyces cerevisiae. Genetics 53: 165–173
Mortimer RK, Contopoulou CR, King JS (1992) Genetic and physical maps of Saccharomyces cerevisiae, edition 11. Yeast 8:817–902
Natsoulis G, Thomas W, Roghmann M-C, Winston F, Boeke JD (1989) Tyl transposition in Saccharomyces cerevisiae is nonrandom. Genetics 123: 269–279
Paquin CE, Williamson VM (1984) Temperature effects on the rate of Ty transposition. Science 226: 53–55
Potier S, Souciet JL, Lacroute F (1987) Correlation between restriction map, genetic map and catalytic functions in the gene complex URA2. Mol Gen Genet 209:283–289
Roeder GS, Farabaugh PJ, Chaleff DT, Fink GR (1980) The origins of gene instability in yeast. Science 209: 1375–1380
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Sathe GM, O'Brien S, McLauglin M, Watson F, Livi GP (1991) Use of polymerase chain reaction for rapid detection of gene insertions in whole yeast cells. Nucleic Acids Res 19:4775
Souciet JL, Nagy M, Le Göuar M, Lacroute F, Potier S (1989) Organization of the URA2 gene: identification of a defective dihydroorotase-like domain in the multifunctional carbamoylphosphate synthetase-aspartate transcarbamylase complex. Gene 79:59–70
Sutton PR, Liebman SW (1992) Rearrangements occurring adjacent to a single Ty1 yeast retrotransposon in the presence and absence of full-length Ty1 transcription. Genetics 131:833–850
Wiesenberger G, Link TA, von Ahsen U, Waldherr M, Schweyen RJ (1991) MRS3 and MRS4, two suppressors of mtRNA splicing defects in yeast, are new members of the mitochondrial carrier family. J Mol Biol 217:23–37
Wilke CM, Heidler SH, Brown N, Liebman SW (1989) Analysis of yeast retrotransposon Ty insertions in the CAN1 locus. Genetics 123:655–665
Winston F, Chumley F, Fink GR (1983) Eviction and transplacement of mutant genes in yeast. Methods Enzymol 101: 211–228
Author information
Authors and Affiliations
Additional information
Communicated by C. Hollenberg
Rights and permissions
About this article
Cite this article
Roelants, F., Potier, S., Souciet, J.L. et al. Reactivation of the ATCase domain of the URA2 gene complex: a positive selection method for Ty insertions and chromosomal rearrangements in Saccharomyces cerevisiae . Molec. Gen. Genet. 246, 767–773 (1995). https://doi.org/10.1007/BF00290725
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00290725