Small epitope‐linker modules for PCR‐based C‐terminal tagging in Saccharomyces cerevisiae

PCR‐mediated gene modification is a powerful approach to the functional analysis of genes in Saccharomyces cerevisiae. One application of this method is epitope‐tagging of a gene to analyse the corresponding protein by immunological methods. However, the number of epitope tags available in a convenient format is still low, and interference with protein function by the epitope, particularly if it is large, is not uncommon. To address these limitations and broaden the utility of the method, we constructed a set of convenient template plasmids designed for PCR‐based C‐terminal tagging with 10 different, relatively short peptide sequences that are recognized by commercially available monoclonal antibodies. The encoded tags are FLAG, 3 × FLAG, T7, His‐tag, Strep‐tag II, S‐tag, Myc, HSV, VSV‐G and V5. The same pair of primers can be used to construct tagged alleles of a gene of interest with any of the 10 tags. In addition, a six‐glycine linker sequence is inserted upstream of these tags to minimize the influence of the tag on the target protein and maximize its accessibility for antibody binding. Three marker genes, HIS3MX6, kanMX6 and hphMX4, are available for each epitope. We demonstrate the utility of the new tags for both immunoblotting and one‐step affinity purification of the regulatory particle of the 26S proteasome. The set of plasmids has been deposited in the non‐profit plasmid repository Addgene ( http://www.addgene.org ). Copyright © 2009 John Wiley & Sons, Ltd.     

Improved tools for efficient mapping of fission yeast genes: identification of microtubule nucleation modifier mod22‐1 as an allele of chromatin‐ remodelling factor gene swr1

Fission yeast genes identified in genetic screens are usually cloned by transformation of mutants with plasmid libraries. However, for some genes this can be difficult, and positional cloning approaches are required. The mutation swi5‐39 reduces recombination frequency in homozygous crosses and has been used as a tool in mapping gene position (Schmidt, 1993 ). However, strain construction in swi5‐39‐based mapping is significantly more laborious than is desirable. Here we describe a set of strains designed to make swi5‐based mapping more efficient and more powerful. The first improvement is the use of a swi5Δ strain marked with kanamycin (G418) resistance, which greatly facilitates identification of swi5 mutants. The second improvement, which follows directly from the first, is the introduction of a large number of auxotrophic markers into mapping strains, increasing the likelihood of finding close linkage between a marker and the mutation of interest. We combine these new mapping strains with a rec12Δ‐based approach for initial mapping of a mutation to an individual chromosome. Together, the two methods allow an approximate determination of map position in only a small number of crosses. We used these to determine that mod22‐1, a modifier of microtubule nucleation phenotypes, encodes a truncation allele of Swr1, a chromatin‐remodelling factor involved in nucleosomal deposition of H2A.Z histone variant Pht1. Expression microarray analysis of mod22‐1, swr1Δ and pht1Δ cells suggests that the modifier phenotype of mod22‐1 mutants may be due to small changes in expression of one or more genes involved in tubulin function. Copyright © 2009 John Wiley & Sons, Ltd.