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

Abstract

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 swi5Delta 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 rec12Delta-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, swr1Delta and pht1Delta 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.

Figure 1

Diagram showing the positions of markers for the six different mapping-strain chromosomes, drawn to scale with the genome sequence. Black circles, centromeres; grey-dashed regions, ribosomal DNA repeats at either end of chromosome III, which can vary in length (Pasero and Marilley, 1993)

Figure 2

Allelism of mod22 and swr1, a gene encoding a chromatin-remodelling factor. (A) Schematic of fission yeast Swr1 protein, showing position of the nonsense mutation in mod22-1. Shaded areas indicate two regions of high identity (>75%) to budding yeast Swr1p. (B) Cell-shape phenotypes in the indicated mutants, showing synthetic effect of combining either mod22-1 or swr1Δ with gfh1Δ. (C) Cell-shape phenotypes in the indicated mutants, showing that swr1ΔC (a de novo-created truncation-equivalent of mod22-1), pht1Δ (deletion of fission yeast H2A.Z) and swc2Δ (deletion of a component of the SWR1–SWR-C complex) all have similar phenotypes to mod22-1 and swr1Δ. D. Rescue of the cell-shape phenotype of gfh1Δ mod22-1 double mutant by plasmid-based expression of wild-type Swr1

Figure 3

Venn diagrams showing numbers of genes changed in expression in mod22-1, swr1Δ and pht1Δ mutants, either unique to each mutant, common to pairs of mutants or common to all three mutants. (A) Genes with reduced expression in the mutants. (B) Genes with increased expression in the mutants