A simple Cre-loxP method for chromosomal N-terminal tagging of essential and non-essential Schizosaccharomyces pombe genes.
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.
Werler
Petra J H
PJ
Genome Damage and Stability Centre, School of Biological Sciences, University of Sussex, Falmer, Sussex BN1 9RQ, UK. a.m.carr@sussex.ac.uk
Hartsuiker
Edgar
E
Carr
Antony M
AM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics; D015825:Chromosomes, Fungal / Q000235:genetics*; D020043:Genes, Essential / Q000235:genetics*; D005800:Genes, Fungal / Q000235:genetics*; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D018797:Cell Cycle Proteins; C093953:NMT1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D014764:Viral Proteins; C080926:cdc22 protein, S pombe; D012264:Ribonucleotide Reductases; C080927:SUC22 protein, S pombe; C045073:Cre recombinase; D019426:Integrases