A new efficient gene disruption cassette for repeated use in budding yeast

The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre-lox P recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.

[1]  P. Philippsen,et al.  New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae , 1994, Yeast.

[2]  B. André,et al.  An overview of membrane transport proteins in Saccharomyces cerevisiae , 1995, Yeast.

[3]  P Manivasakam,et al.  Micro-homology mediated PCR targeting in Saccharomyces cerevisiae. , 1995, Nucleic acids research.

[4]  B. Sauer,et al.  Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. , 1987, Molecular and cellular biology.

[5]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[6]  E. Jacobs,et al.  A method for performing precise alterations in the yeast genome using a recycable selectable marker. , 1995, Nucleic acids research.

[7]  B. Bainbridge,et al.  Genetics , 1981, Experientia.

[8]  R. C. Dickson,et al.  Direct selection of Saccharomyces cerevisiae resistant to the antibiotic G418 following transformation with a DNA vector carrying the kanamycin-resistance gene of Tn903. , 1983, Gene.

[9]  O. Ozier-Kalogeropoulos,et al.  A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. , 1993, Nucleic acids research.

[10]  R. Schiestl,et al.  Improved method for high efficiency transformation of intact yeast cells. , 1992, Nucleic acids research.

[11]  Gerald R. Fink,et al.  Methods in Yeast Genetics: Laboratory Manual , 1981 .

[12]  Nancy Kleckner,et al.  A Method for Gene Disruption That Allows Repeated Use of URA3 Selection in the Construction of Multiply Disrupted Yeast Strains , 1987, Genetics.

[13]  S. Oliver.  From DNA sequence to biological function , 1996, Nature.

[14]  R. Müller,et al.  Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. , 1994, Nucleic acids research.

[15]  A. Jiménez,et al.  Expression of a transposable antibiotic resistance element in Saccharomyces , 1980, Nature.

[16]  J W Szostak,et al.  Yeast transformation: a model system for the study of recombination. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Ruth Lyck,et al.  The sequence of a 24 152 bp segment from the left arm of chromosome XIV from Saccharomyces cerevisiae between the BNI1 and the POL2 genes , 1996, Yeast.

[18]  R. Schiestl,et al.  Applications of high efficiency lithium acetate transformation of intact yeast cells using single‐stranded nucleic acids as carrier , 1991, Yeast.

[19]  M. Ciriacy,et al.  Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on qlycolytic flux , 1995, Molecular microbiology.

[20]  M. Fornerod,et al.  Cre-mediated site-specific translocation between nonhomologous mouse chromosomes. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[21]  A. Teunissen,et al.  The dominant flocculation genes of Saccharomyces cerevisiae constitute a new subtelomeric gene family , 1995, Yeast.

[22]  B. Sauer.  Recycling selectable markers in yeast. , 1994, BioTechniques.