TY - JOUR

T1 - A simple Cre-loxP method for chromosomal N-terminal tagging of essential and non-essential Schizosaccharomyces pombe genes

AU - Werler, Petra J H

AU - Hartsuiker, Edgar

AU - Carr, Antony M

PY - 2003/1/30

Y1 - 2003/1/30

N2 - To facilitate the N-terminal tagging of essential genes at their genomic locus and under control of their own promoters we have developed a series of novel polymerase chain reaction templates. Initially, a 1.8 kb DNA fragment is integrated upstream of the ATG of the gene of interest. This fragment encodes the tag, a loxP site, a selectable marker, an exogenous nmt1 promoter and a second loxP site. In a single homologous integration event, the gene of interest is placed under control of the thiamine regulated nmt1 promoter, allowing identification of potential integrants on the basis of phenotype. Subsequently, this integrant strain is transformed with a plasmid expressing the Cre recombinase. This results in excision of the marker and nmt1 promoter and leaves sequences encoding an in-frame tag at the N-terminus of the gene of interest under the control of its native promoter. We have created TAP-cdc22, TAP-suc22 and TAP-rad50 strains using this N-tagging system, and developed a range of vectors for introducing TAP-, (His)10HA-, (His)6Myc- and EGFP.

AB - To facilitate the N-terminal tagging of essential genes at their genomic locus and under control of their own promoters we have developed a series of novel polymerase chain reaction templates. Initially, a 1.8 kb DNA fragment is integrated upstream of the ATG of the gene of interest. This fragment encodes the tag, a loxP site, a selectable marker, an exogenous nmt1 promoter and a second loxP site. In a single homologous integration event, the gene of interest is placed under control of the thiamine regulated nmt1 promoter, allowing identification of potential integrants on the basis of phenotype. Subsequently, this integrant strain is transformed with a plasmid expressing the Cre recombinase. This results in excision of the marker and nmt1 promoter and leaves sequences encoding an in-frame tag at the N-terminus of the gene of interest under the control of its native promoter. We have created TAP-cdc22, TAP-suc22 and TAP-rad50 strains using this N-tagging system, and developed a range of vectors for introducing TAP-, (His)10HA-, (His)6Myc- and EGFP.

KW - Amino Acid Sequence

KW - Base Sequence

KW - Binding Sites

KW - Blotting, Western

KW - Cell Cycle Proteins

KW - Chromosomes, Fungal

KW - Genes, Essential

KW - Genes, Fungal

KW - Integrases

KW - Molecular Sequence Data

KW - Plasmids

KW - Recombination, Genetic

KW - Ribonucleotide Reductases

KW - Schizosaccharomyces

KW - Schizosaccharomyces pombe Proteins

KW - Transformation, Genetic

KW - Viral Proteins

KW - Journal Article

U2 - 10.1016/S0378-1119(03)00402-5

DO - 10.1016/S0378-1119(03)00402-5

M3 - Article

C2 - 12568722

VL - 304

SP - 133

EP - 141

JO - Gene

JF - Gene

SN - 0378-1119

ER -