Rapid screening method of Saccharomyces cerevisiae mutants using calcofluor white and aniline blue. 2021

Francine Perrine-Walker, and Jennifer Payne
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC, 3086, Australia. marie.perrine-walker@sydney.edu.au.

Fungal cell walls are composed of polysaccharide scaffold that changes in response to environment. The structure and biosynthesis of the wall are unique to fungi, with plant and mammalian immune systems evolved to recognize wall components. Additionally, the enzymes that assemble fungal cell wall components are excellent targets for antifungal chemotherapies and fungicides. Understanding changes in the cell wall are important for fundamental understanding of cell wall dynamics and for drug development. Here we describe a screening technique to monitor the gross morphological changes of two key cell wall polysaccharides of chitin and β-1,3-glucan combined with polymerase chain reaction (PCR) genotyping. Changes in chitin and β-1,3-glucan were detected microscopically by using the dyes calcofluor white and aniline blue. Combining PCR and fluorescence microscopy, as a quick and easy screening technique, confirmed both the phenotype and genotype of the wild-type, h chitin synthase mutants (chs1Δ and chs3Δ) and one β-1,3-glucan synthase mutant fks2Δ from Saccharomyces cerevisiae knockout library. This combined screening method highlighted that the fks1Δ strain obtained commercially was in fact not FKS1 deletion strain, and instead had both wild-type genotype and phenotype. A new β-1,3-glucan synthase knockout fks1::URA3 strain was created. Fluorescence microscopy confirmed its phenotype revealing that the chitin and the new β-1,3-glucan profiles were elevated in the mother cells and in the emerging buds respectively in the fks1Δ cell walls. This combination of PCR with fluorescence microscopy is a quick and easy screening method to determine and verify morphological changes in the S. cerevisiae cell wall.

UI MeSH Term Description Entries
D008565 Membrane Proteins Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors. Cell Membrane Protein,Cell Membrane Proteins,Cell Surface Protein,Cell Surface Proteins,Integral Membrane Proteins,Membrane-Associated Protein,Surface Protein,Surface Proteins,Integral Membrane Protein,Membrane Protein,Membrane-Associated Proteins,Membrane Associated Protein,Membrane Associated Proteins,Membrane Protein, Cell,Membrane Protein, Integral,Membrane Proteins, Integral,Protein, Cell Membrane,Protein, Cell Surface,Protein, Integral Membrane,Protein, Membrane,Protein, Membrane-Associated,Protein, Surface,Proteins, Cell Membrane,Proteins, Cell Surface,Proteins, Integral Membrane,Proteins, Membrane,Proteins, Membrane-Associated,Proteins, Surface,Surface Protein, Cell
D002473 Cell Wall The outermost layer of a cell in most PLANTS; BACTERIA; FUNGI; and ALGAE. The cell wall is usually a rigid structure that lies external to the CELL MEMBRANE, and provides a protective barrier against physical or chemical agents. Cell Walls,Wall, Cell,Walls, Cell
D002686 Chitin A linear polysaccharide of beta-1->4 linked units of ACETYLGLUCOSAMINE. It is the second most abundant biopolymer on earth, found especially in INSECTS and FUNGI. When deacetylated it is called CHITOSAN.
D005936 Glucans Polysaccharides composed of repeating glucose units. They can consist of branched or unbranched chains in any linkages. Glucan,Polyglucose,Polyglucoses,Glucan (BO),Glucose Polymer,Polycose,Polymer, Glucose
D005964 Glucosyltransferases Enzymes that catalyze the transfer of glucose from a nucleoside diphosphate glucose to an acceptor molecule which is frequently another carbohydrate. EC 2.4.1.-. Glucosyltransferase
D000814 Aniline Compounds Compounds that include the aminobenzene structure. Phenylamine,Phenylamines,Anilines,Compounds, Aniline
D001557 Benzenesulfonates Organic salts and esters of benzenesulfonic acid.
D012441 Saccharomyces cerevisiae A species of the genus SACCHAROMYCES, family Saccharomycetaceae, order Saccharomycetales, known as "baker's" or "brewer's" yeast. The dried form is used as a dietary supplement. Baker's Yeast,Brewer's Yeast,Candida robusta,S. cerevisiae,Saccharomyces capensis,Saccharomyces italicus,Saccharomyces oviformis,Saccharomyces uvarum var. melibiosus,Yeast, Baker's,Yeast, Brewer's,Baker Yeast,S cerevisiae,Baker's Yeasts,Yeast, Baker
D054714 Echinocandins Cyclic hexapeptides of proline-ornithine-threonine-proline-threonine-serine. The cyclization with a single non-peptide bond can lead them to be incorrectly called DEPSIPEPTIDES, but the echinocandins lack ester links. Antifungal activity is via inhibition of 1,3-beta-glucan synthase production of BETA-GLUCANS. Aculeacin,Echinocandin,Pneumocandin,Aculeacins,Mulundocandins,Pneumocandins
D029701 Saccharomyces cerevisiae Proteins Proteins obtained from the species SACCHAROMYCES CEREVISIAE. The function of specific proteins from this organism are the subject of intense scientific interest and have been used to derive basic understanding of the functioning similar proteins in higher eukaryotes. Baker's Yeast Proteins,S cerevisiae Proteins

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