Constructs for Production of a Probe for Monitoring of Chs3 Expression in Saccharomyces Cerevisiae

Abstract

Chitin and β-1,3-glucan are known to play an important role in determination of fungal cell wall rigidity in Saccharomyces cerevisiae 1 . Three chitin synthases participate in yeast chitin synthesis. ChS1p functions as a reparation enzyme after cell division 2 , ChS2p is responsible for the synthesis of primary septum 3,4 and ChS3p synthesizes chitin in the lateral wall and in the ring at the base of future septum 4 . All of the enzymes are supposed to be cell cycle regulated and expression of CHS3 was described to be strongly reduced in mpk1 mutant 5 . The Mpk1p MAP-kinase is a component of the cell wall integrity and proliferation pathway, known as PKC1-pathway as well. This pathway participates in the regulation of cell cycle progress 6, 7 and in hypotonic shock signalling 8 . Further, chitin synthesis was earlier described to increase markedly in yeast cells exposed to fluorescent brighteners Calcofluor white and Rylux BSU 9,10 . Chitin synthase 3 gene should be the main target of stimulation in vivo in both Calcofluor white 11 and Rylux BSU 10, 12 . Therefore, CHS3 expression is supposed to be increased in case of plasma membrane stretch. This stretch can be caused by hypotonic shock or weakening of cell wall if harmed by fluorescent brighteners. Kamada et al. 13 have found activation of Mpk1p-kinase in response not only to hypotonic shock, but in response to mild heat shock (37 °C) and in response to chlorpromazine, which inserts into the cytoplasmic leaflet of the plasma membrane lipid bilayer and thus induces an inward membrane stretch. However, we have not found any increase in cell wall chitin content in yeast cells treated with chlorpromazine and have found an decrease in cell wall chitin content in yeast cells grown at 37 °C (unpublished results). On the other hand, cell wall chitin content was increased in cells grown in hypotonic environment. To clarify the role of CHS3 expression in response to different environmental conditions, we decided to construct a RNA-probe, which would allow us to monitor the expression of CHS3. MATERIAL AND METHODSProbe design. Sequences of CHS3 and ACT1 (as control) were obtained from Saccharomyces Genome Database maintained at Stanford University, School of Medicine. These sequences were searched for recognition sites of restriction endonucleases using the freeware Digest. Sequence segments circumscribed by recognition sites of enzymes suitable for cloning were selected and primers were designed to amplify these regions of interest. Freeware Primer 0.5 was used for primer design, annealing temperature was set to be 56 °C. CHS3 and ACT1 sequences with marked primer sequences and restriction sites are summarised in Fig. 1 and Fig. 2. µl of TE buffer (TrisCl 100 mM, pH 7.5, EDTA 1mM, pH 8.0) were added, mixed shortly by vortexing and centrifuged at 12 000 g at laboratory temperature for 5 min. Supernatant was transferred into a clean tube, 4 µl of Proteinase K (10 mg/ml) were added, mixed and incubated at 37 °C for 60 min. Then, 68 µl of 5 M NaCl and 54 µl of 10% [w/v] CT...