An interactive gene network for securin‐separase, condensin, cohesin, Dis1/Mtc1 and histones constructed by mass transformation

Abstract

The small genome of fission yeast Schizosaccharomyces pombe contains 4824 predicted genes and gene disruption suggests that ∼ ∼ ∼ ∼ 850 are essential for viability. To obtain information on interactions among genes required for chromosome segregation, an approach called Strategy B was taken using mass transformation of the 1015 temperature-sensitive (ts) mutants that were made by random mutagenesis and transformed by plasmids carrying the genes for securin, separase, condensin, cohesin, kinetochore microtubule-binding proteins Dis1/Mtc1 or histones. Mutant strains whose phenotypes were either suppressed or inhibited by plasmids were selected. Each plasmid interacted positively or negatively with the average 14 strains. Identification of the mutant gene products by cloning revealed many hitherto unknown interactions. The interactive networks of segregation therefore may consist of genes with a variety of functions. For example, separase/Cut1 interacts with Cdc48/p97/VCP, which stabilizes securin and separase. Surprisingly, S. pombe cdc48 mutants displayed the mitotic phenotype highly similar to separase/ cut1 mutants. This approach also provides a novel way of mutant isolation, resulting in two histone H2B strains and a cohesion mutant with a new phenotype.

Yeast Sterol Regulatory Element-binding Protein (SREBP) Cleavage Requires Cdc48 and Dsc5, a Ubiquitin Regulatory X Domain-containing Subunit of the Golgi Dsc E3 Ligase

“…ϩ is an essential gene, and complete loss-of-function mutants would be inviable (29). The success of our unbiased approach makes it an attractive platform for identifying important functional residues of Cdc48.…”

“…3A and Table 1). cdc48 is an essential gene, possibly explaining the absence of nonsense mutations (29). Reconstitution of the individual cdc48 mutations in an otherwise wild-type background conferred a CoCl 2 -sensitive phenotype to the strains (data not shown).…”

“…Given the central role of Cdc48 in the ubiquitin-proteasome system and the role of the Dsc E3 ubiquitin ligase complex in Sre1 processing, we considered cdc48 ϩ a strong candidate for dsc6 ϩ . Linkage tests using tetrad analysis between an existing cdc48-353 temperature-sensitive mutant and dsc6-1 demonstrated that dsc6-1 was an allele of cdc48 (29). Next, we sequenced the cdc48 locus for each dsc6 mutant and found that each allele harbored a missense mutation in cdc48 (Fig.…”

Mapping epigenetic mutations in fission yeast using whole-genome next-generation sequencing

“…In a genetic screen for transcriptional silencing defects, we assayed a subset of a large collection of mutagenized S. pombe strains that were temperature sensitive (ts) for growth. In each case, the growth defect could be suppressed by one or more plasmids recovered from a genomic library that had been systematically transformed into each strain (Matsumura et al 2003;Hayashi et al 2004;Yuasa et al 2004). Twenty-seven ts strains were screened by reverse transcription polymerase chain reaction (RT-PCR) for repeat transcript accumulation at the nonpermissive temperature of 36°C.…”

Cut1/separase-dependent roles of multiple phosphorylation of fission yeast cohesin subunit Rad21 in post-replicative damage repair and mitosis

“…A novel mis4 temperature-sensitive mutant, mis4-450, was isolated as previously described. 42 Immunochemistry. The protein extracts were prepared by using glass beads in the KB buffer (25 mM Tris-HCl at pH 7.5, 15 mM EDTA, 60 mM b-glycerophosphate, 0.1% NP-40, 10% glycerol, 1 mM phenylmethylsulphonyl fluoride, 2 mg/ml pepstatin A and 1 mg/ml aprotinin), or by the TCA precipitation method.…”