Abstract
The efficiency of gene targeting within different segments of genes in yeast was estimated by transforming yeast cells with double-stranded integrative plasmids, bearing functional gene domains [promoter (P), ORF (O) and terminator (T)] derived from the common genetic markers HIS3, LEU2 , TRP1 and URA3. Transformation experiments with circular plasmids carrying a single gene domain demonstrated that the 5′ and 3′ flanking DNA regions (P and T) of the HIS3 and URA3 genes are preferred as sites for plasmid integration by several fold over the corresponding ORFs. Moreover, when plasmids bearing combinations of two or three regions were linearized to target them to a specific site of integration, three of the ORFs were found to be less preferred as sites for plasmid integration than their corresponding flanking regions. Surprisingly, in up to 50% of the transformants obtained with plasmids that had been linearized within coding sequences, the DNA actually integrated into neighbouring regions. Almost the same frequencies of ORF mis-targeting were obtained with plasmid vectors containing only two functional domains (“PO” or “OT”) of the gene URA3, demonstrating that this event is not the consequence of competition between homologous DNA regions distal to the ORF. Therefore, we suggest that coding sequences could be considered to be “cold spots” for plasmid integration in yeast.
Similar content being viewed by others
References
Adams DE, West SC (1996) Bypass of DNA heterologies during RuvAB-mediated three- and four-strand branch migration. J Mol Biol 263:582–596
Alani E, Padmore R, Kleckner N (1990) Analysis of wild-type and rad50 mutants of yeast suggests an intimate relationship between meiotic chromosome synapsis and recombination. Cell 61:419–436
Bartsch S, Kang LE, Symington LS (2000) RAD51 is required for the repair of plasmid double-stranded DNA gaps from either plasmid or chromosomal templates. Mol Cell Biol 20:1194–1205
Baudat F, Nicolas A (1997) Clustering of meiotic double-strand breaks on yeast chromosome III. Proc Natl Acad Sci USA 13:5213–5218
Bruschi CV, Esposito MS (1983) Enhancement of spontaneous mitotic recombination by the meiotic mutant spo11-1 in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 80:7566–7570
Bullard SA, Kim S, Galbraith AM, Malone RE (1996) Double strand breaks at the HIS2 recombination hot-spot in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 12:13054–13059
Clikeman JA, Wheeler SL, Nickoloff JA (2001) Efficient incorporation of large (>2 kb) heterologies into heteroduplex DNA: Pms1/Msh2-dependent and independent large loop mismatch repair in Saccharomyces cerevisiae. Genetics 157:1481–1491
Dardalhon M, DeMassy B, Nicolas A, Averbeck D (1998) Mitotic recombination and localized DNA double-strand breaks are induced after 8-methoxypsoralen and UVA irradiation in Saccharomyces cerevisiae. Curr Genet 34:30–42
Davidson JF, Schiestl RH (2000) Mis-targeting of multiple gene disruption constructs containing hisG. Curr Genet 38:188–190
DeMassy B, Rocco V, Nicolas A (1995) The nucleotide mapping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae. EMBO J 15:4589–4598
Fan Q, Xu F, Petes TD (1995) Meiosis-specific double-strand DNA breaks at the HIS4 recombination hot-spot in the yeast Saccharomyces cerevisiae: control in cis and trans. Mol Cell Biol 15:1679–1688
Gerton JL, DeRisi J, Shroff R, Lichten M, Brown PO, Petes TD (2000) Global mapping of meiotic recombination hotspots and coldspots in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci USA 97:11383–11390
Gietz RD, Woods RA (1994) High efficiency transformation with lithium acetate. In: Johnston JR (ed) Molecular genetics of yeast: a practical approach. IRL Press, Oxford, pp 121–134
Gietz RD, St. Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425–1430
Güldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Huxley C, Green ED, Dunham I (1990) Rapid assessment of S. cerevisiae mating type by PCR. Trends Genet 6:236
Inbar O, Kupiec M (1999) Homology search and choice of homologous partner during mitotic recombination. Mol Cell Biol 19:4134–4142
Inbar O, Liefshitz B, Bitan G, Kupiec M (2000) The relationship between homology length and crossing over during the repair of a broken chromosome. J Biol Chem 275:30833–30838
Keil RL, Roeder GS (1984) Cis -acting, recombination-stimulating activity of a fragment of the ribosomal DNA of S. cerevisiae. Cell 39:377–386
Koehler CM, Merchant S, Oppliger W, Schmid K, Jarosch E, Dolfini L, Junne T, Schatz G, Tokatlidis K (1998) Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins. EMBO J 17:6477–6486
Koren P, Svetec IK, Mitrikeski PT, Zgaga Z (2000) Influence of homology size and polymorphism on plasmid integration in the yeast CYC1 DNA region. Curr Genet 37:292–297
Lucau-Danila A, Wysocki R, Rognati T, Foury F (2000) Systematic disruption of 456 ORFs in the yeast Saccharomyces cerevisiae. Yeast 16:547–552
Mortimer RK, Johnston JR (1986) Genealogy of principal strains of the Yeast Genetic Stock Center. Genetics 113:35–43
Nasar F, Jankowski C, Nag DK (2000) Long palindromic sequences induce double-strand breaks during meiosis in yeast. Mol Cell Biol 20:3449–3458.
Neitz M, Carbon J (1987) Characterization of a centromere-linked recombination hot-spot in Saccharomyces cerevisiae. Mol Cell Biol 7:3871–3879
Nikawa J, Kawabata M (1998) PCR- and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruptions in Saccharomyces cerevisiae. Nucleic Acids Res 26:860–861
Ninkovic M, Alacevic M, Fabre F, Zgaga Z (1994) Efficient UV stimulation of yeast integrative transformation requires damage on both plasmid strands. Mol Gen Genet 243:308–314
Orr-Weaver TL, Szostak JW (1983) Multiple, tandem plasmid integration in Saccharomyces cerevisiae. Mol Cell Biol 3:747–749
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
Plessis A, Dujon B (1993) Multiple tandem integrations of transforming DNA sequences in yeast chromosomes suggest a mechanism for integrative transformation by homologous recombination. Gene 134:41–50
Puig O, Rutz B, Luukkonen BG, Kandels-Lewis S, Bragado-Nilsson E, Seraphin B (1998) New constructs and strategies for efficient PCR-based gene manipulations in yeast. Yeast 14:1139–1146
Saffran WA, Smith ED, Chan SK (1991) Induction of multiple plasmid recombination in Saccharomyces cerevisiae by psoralen reaction and double strand breaks. Nucleic Acids Res 19:5681–5687
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, New York
Saxe D, Datta A, Jinks-Robertson S (2000) Stimulation of mitotic recombination by high levels of RNA polymerase II transcription in yeast. Mol Cell Biol 20:5404–5414
Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, New York
Storici F, Coglievina M, Bruschi CV (1999) A 2-microm DNA-based marker recycling system for multiple gene disruption in the yeast Saccharomyces cerevisiae. Yeast 15:271–278
Stotz A, Linder P (1990) The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors. Gene 95:91–98
Sun H, Treco D, Schultes NP, Szostak JW (1989) Double-strand breaks at an initiation site for meiotic gene conversion. Nature 338:87–90
Wach A, Brachat A, Poehlmann R, Phillippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808
White MA, Dominska M, Petes TD (1993) Transcription factors are required for the meiotic recombination hotspot at the HIS4 locus of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 90:6621–6625
Acknowledgements
We wish to thank Valentina Tosato for her help in the sequencing of the plasmid constructs, and Zoran Zgaga for suggestions and comments. Kresimir Gjuracic was a Post-doctoral Fellow supported by ICGEB. This work has been carried out in compliance with the current laws governing genetic experimentation in Italy.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by A. Aguilera
Rights and permissions
About this article
Cite this article
Gjuracic, K., Pivetta, E. & Bruschi, C.V. Targeted DNA integration within different functional gene domains in yeast reveals ORF sequences as recombinational cold-spots. Mol Genet Genomics 271, 437–446 (2004). https://doi.org/10.1007/s00438-004-0994-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-004-0994-8