Basic methods for fission yeast

Forsburg, Susan L.; Rhind, Nick

doi:10.1002/yea.1347

Cited by 482 publications

(506 citation statements)

References 25 publications

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“…The induced level is about 806 greater than the repressed level. pREP41 has a 66 lower induced level and a 156 lower repressed level than the wild-type promoter (Basi et al, 1993;Maundrell, 1993;Forsburg & Rhind, 2006). As expected, HRP-PNA staining showed that galactosylation of the cell surface of the uge1D strain was restored in an expression-dependent manner (Fig.…”

supporting

confidence: 70%

Characterization of two different types of UDP-glucose/-galactose4-epimerase involved in galactosylation in fission yeast

et al. 2010

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Schizosaccharomyces species are currently the only known organisms with two types of genes encoding UDP-glucose/-galactose 4-epimerase, uge1 + and gal10 + . A strain deleted for uge1 + exhibited a severe galactosylation defect and a decrease in activity and in UDP-galactose content when grown in glucose-rich medium (2 % glucose), indicating that Uge1p is a major UDPglucose/-galactose 4-epimerase under these growth conditions. In contrast, gal10 + was efficiently expressed and involved in galactosylation of cell-surface proteins in low-glucose medium (0.1 % glucose and 2 % glycerol), but not in galactose-containing medium. In a uge1Dgal10D strain, the galactosylation defect was suppressed and UDP-galactose content restored to wild-type levels in galactose-containing medium. Disruption of gal7 + , encoding galactose-1-phosphate uridylyltransferase, in the uge1Dgal10D strain reversed suppression of the galactosylation defect and reduced levels of UDP-galactose, indicating that galactose is transported from the medium to the cytosol and is converted into UDP-galactose via galactose 1-phosphate by Gal7p in Sch. pombe. INTRODUCTIONGlycoproteins in the fission yeast Schizosaccharomyces pombe contain a large amount of galactose in addition to both N-and O-linked mannan (Manners & Meyer, 1977;Moreno et al., 1985;Ikeda et al., 2009;Ohashi et al., 2009), indicating that Sch. pombe is equipped with mechanisms for glycoprotein galactosylation like animal cells. Research has demonstrated that the outer chain structure of galactomannan has an a-1,2-linked galactose or pyruvylated galactose attached to a poly-a-1,6-linked mannose backbone (Gemmill & Trimble, 1998), and that the pyruvylated galactose epitope bears remarkable structural resemblance to the N-acetylneuraminic acid-linked galactose epitope found in mammalian cells (Gemmill & Trimble, 1996). These galactose residues are enzymically modified by galactosyltransferases using UDP-galactose as substrate (Andreishcheva et al., 2004; Chappell et al., 1994;Yoko-o et al., 1998). UDP-galactose is transported to the Golgi lumen from the cytosol by the Golgi-localized UDPgalactose transporter (Gms1p) (Tabuchi et al., 1997;). In Sch. pombe, the precise manner in which UDP-galactose is synthesized in the cytosol has been unclear. In the cytosol, the key enzyme for UDP-galactose synthesis is UDP-galactose/-glucose 4-epimerase, which catalyses the interconversion of UDPgalactose and UDP-D-glucose. Interestingly, Schizosaccharomyces species have two types of UDP-glucose/-galactose 4-epimerase, while other organisms have only one. It is not known why two types of epimerase are needed and how they are regulated in Sch. pombe.In Saccharomyces cerevisiae, Gal10p is the sole UDPglucose/-galactose 4-epimerase, which consists of a UDPglucose/-galactose 4-epimerase domain and a galactose mutarotase domain (Majumdar et al., 2004;Scott & Timson, 2007;Thoden & Holden, 2005), and which catalyses mutarotation of a-galactose to b-galactose. Expression of the GAL10 gene is regulated by Gal4p, a DNA...

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confidence: 70%

Characterization of two different types of UDP-glucose/-galactose4-epimerase involved in galactosylation in fission yeast

et al. 2010

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“…YN001 (msy1ura4 + in FY15952), YN002 (msy2kanMX6 in FY15952), YN003 (msy1ura4 + msy2kanMX6 in FY15952) and YN004 (msy1 + -4HA in FY15952) were created in this study. Cells were cultured at 30 °C in rich medium (YES) or synthetic medium (EMM2) 37 . E. coli strain PB113 (∆mscs ∆msck) was used for the electrophysiological experiment 38 .…”

Section: Methodsmentioning

confidence: 99%

Organellar mechanosensitive channels in fission yeast regulate the hypo-osmotic shock response

2012

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A key molecule of sensing machineries essential for survival upon hypo-osmotic shock is the mechanosensitive channel. The bacterial mechanosensitive channel mscs functions directly for this purpose by releasing cytoplasmic solutes out of the cell, whereas plant mscs homologues are found to function in chloroplast organization. Here we show that the fission yeast mscs homologues, designated msy1 and msy2, participate in the hypo-osmotic shock response by a mechanism different from that operated by the bacterial mscs. upon hypoosmotic shock, msy2 -and msy1 -msy2 -cells display greater cell swelling than wild-type cells and undergo cell death. Cell swelling precedes an intracellular Ca 2 + increase, which was greater in msy1 -and msy1 -msy2 -cells than in wild-type cells. Fluorescent microscopy showed that msy1 and msy2 localize mainly to the endoplasmic reticulum. These observations suggest that organellar msy1 and msy2 regulate intracellular Ca 2 + and cell volume for survival upon hypo-osmotic shock.

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“…General methods to handle fission yeast cells were as described (Forsburg and Rhind, 2006). Highefficiency transformation of Sz.…”

Section: Genetic Methods and Transformation Of Sz Pombementioning

confidence: 99%

“…Complete medium (YE; 0.5% yeast extract, 2% glucose, 5 µg/ml adenine), minimal medium (SD), and minimal medium (MM) were used for the culture of Sz. pombe cells (reviewed by Forsburg and Rhind, 2006). Thiamine and leucine were added to the media at a final concentration of 15 µM and 50 µg/ml, respectively, when needed.…”

Section: Sz Pombe Strains and Mediamentioning

confidence: 99%

A series of promoters for constitutive expression of heterologous genes in fission yeast

Matsuyama

Shirai

Yoshida

2008

Yeast

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Inducible/repressible promoters are useful for the maintenance of toxic genes or timely expression. For ectopic expression of cloned genes in the fission yeast Schizosaccharomyces pombe, the thiamine-regulatable nmt1 promoter has been widely used, since the transcriptional activity of this promoter can be controlled by thiamine. However, this property sometimes limits a certain type of research, since the expression inevitably requires cells to be cultivated under the conditions that induce promoter activation. To allow constitutive expression of heterologous genes, we cloned three promoters of cam1 + , tif51 + and ef1a-c + . Construction of a series of vectors comprising these promoters and their introduction into the fission yeast cells demonstrated that the activity was different among these promoters but was not affected by cultured media commonly used in fission yeast. Therefore, a promoter with appropriate strength would be selectable from these promoters, depending on the genes to be expressed.

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Basic methods for fission yeast

Cited by 482 publications

References 25 publications

Characterization of two different types of UDP-glucose/-galactose4-epimerase involved in galactosylation in fission yeast

Characterization of two different types of UDP-glucose/-galactose4-epimerase involved in galactosylation in fission yeast

Organellar mechanosensitive channels in fission yeast regulate the hypo-osmotic shock response

A series of promoters for constitutive expression of heterologous genes in fission yeast

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