A large-scale sonication assay for cell wall mutant analysis in yeast

Abstract

The complete yeast genome contains a large number of genes of unknown biological function. Simple, rapid and reliable specific screens are valuable tools in exploring gene function via systematic phenotypic analysis of large mutant collections. This report provides a new approach for monitoring changes in cell wall strength, based on the deleterious effects caused by ultrasound on the yeast cell surface. Sonication can thus be used for the screening of mutants affected in the architecture or stability of the cell wall, since such mutants are expected to have an altered sensitivity to this treatment compared to that of a wild‐type. The experimental procedure, consisting in the quantification of damaged cells after a mild sonication treatment, by means of flow cytometry, can be applied on a large scale. The usefulness of the sonication assay as a primary screen for cell wall‐related mutants is evaluated on the collection of calcofluor white‐hypersensitive and ‐resistant mutants obtained by Lussier et al. (1997). A further phenotypic characterization of the sonication‐hypersensitive mutants within the calcofluor white collection is also presented. Copyright © 1999 John Wiley & Sons, Ltd.

Discoveries of the phosphatidate phosphatase genes in yeast published in the Journal of Biological Chemistry

“…The increase in phospholipid synthesis is associated with the induction of phospholipid synthesis gene expression (69,99), whereas the reduction in the synthesis of TAG is associated with a decrease in lipid droplet formation (100 -102). The imbalance of lipid homeostasis in cells lacking PAH1 results in a multitude of other phenotypes that include fatty acid-induced toxicity (100), hypersensitivity to oxidative stress (103), loss in cell wall strength (104,105), reduction in chronological life span (103), the inability to fuse vacuoles (106), the inability to degrade cellular components (e.g. autophagy) (107), and the inability to grow on nonfermentable carbon sources (57,68) and at elevated temperatures (57,68,69).…”

Improvement of FISH–FCM enumeration performance in filamentous yeast species in activated sludge by snailase partial digestion

“…To gain a better understanding of the content of specific yeast species in microbial community and its evolution in response to environmental conditions, it is often necessary and effective to use FISH–FCM to distinguish between the specific yeast species and bacteria or other fungal species and to further enumerate it (Zheng et al , ). However, FCM is an established cell‐analysis technique that permits rapid analysis and quantitative evaluation at single‐cell level, based on light‐scattering and fluorescent signals (Deere et al , ; Ruiz et al , ; Foladori et al , ). At that time, filamentous fungus or algae that form chains are often counted as single entities during the sorting step if they are not separated (Cellamare et al , ).…”

Protein kinase A phosphorylates the Nem1–Spo7 protein phosphatase complex that regulates the phosphorylation state of the phosphatidate phosphatase Pah1 in yeast

“…The increase in phospholipid synthesis correlates with the derepression of phospholipid synthesis gene expression (4,25), whereas the decrease in TAG synthesis correlates with a decrease in the formation of cytoplasmic lipid droplets (26 -28). The loss of Pah1 function gives rise to a multiple assortment of other phenotypes that include the susceptibility of cells to fatty acidinduced toxicity (26), defects in cell wall integrity (29,30), vacuole fusion and acidification (31,32), and the inability to grow on nonfermentable carbon sources (7,33) and at elevated temperatures (4,7,33). Cells lacking Pah1 are also hypersensitive to oxidative stress and exhibit an apoptosis phenotype (26) and a decrease in chronological life span (34).…”