Heterologous modules for efficient and versatile PCR‐based gene targeting in Schizosaccharomyces pombe

Abstract

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was > or = 50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.

Role of the α‐glucanase Agn2p in ascus‐wall endolysis following sporulation in fission yeast

“…Diploid strains ND080 (h − /h + ade6-M210 /ade6-M216 ) and ND088 (h − /h + agn2 ::kanMX4 /agn2 ::kanMX4 ade6-M210 /ade6-M216 ) were constructed by conjugating strains FYC11 (h − ade6-M210 ) with FYC15 (h + ade6-M216 ) and strains ND044 (h − agn2 ::kanMX4 ade6-M210 ) with ND047 (h + agn2 ::kanMX4 ade6-M216 ), respectively, and by selecting for adenine prototrophy using the complementing chromosomal alleles ade6-M210 and ade6-M216. For overexpression of agn2 from its chromosomal locus, a kanMX6-Pnmt1 cassette was integrated in front of the agn2 ORF of wild-type strain 972 (h − ) by using a PCR-mediated strategy (Bähler et al, 1998) with minor modifications (Dekker et al, 2004), using primers ND161 (5 -TTGTATAGCGGCAGTCATCGGCTAGACCTT-TGAAAGCGGACAGCTTGCATTTCCACTAAC-CAACTTAAATATCACTTAGAATTCGAGCTC-GTTTAAACTG) and ND162 (5 -AGCATAATTG-TACGTAAGACCCATCATAAAATGGGCTACA-ACGGCTTTATTCGGTAAAGCTGTTGTTAAC-GACGCCATGTTTAACAAAGCGACTATAAG). Using several mating steps together with diploid selection, diploid strain ND227 (h − /h + agn2::Pnmt1-kanMX6 /agn2 ::kanMX4 ade6-M210 /ade6-M216 ) was generated.…”

Two F‐box/WD‐repeat proteins Pop1 and Pop2 form hetero‐ and homo‐complexes together with cullin‐1 in the fission yeast SCF (Skp1‐Cullin‐1‐F‐box) ubiquitin ligase

“…To examine the loss-of-function phenotypes of pcu1 þ , an entire ORF of the pcu1 þ gene was deleted by use of a PCR-generated fragment containing the ura4 þ marker (Bähler et al 1998). Tetrad manipulation of a heterozygous diploid (IO100 , Table 1) showed that pcu1 þ is essential for cell viability.…”

Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast

“…The Mcs6-TAP and Mcs2-TAP strains were generated as described previously [36,37]. The sequence of the primers is available upon request.…”