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Fig. 4. (A) Chitinase activity in culture supernatants. Wild-type (YPA24, black circles) and mutant cells (YPA207, white circles) cells were grown overnight in YEPD medium at 28°C and then diluted into fresh YEPD medium that was transferred to 38°C. At the indicated times, aliquots were taken, cells were removed by centrifugation and chitinase activity was measured using the fluorogenic substrate 4-methylumbelliferyl-ß-D-N,N',N''-triacetylchitotrioside. Activity is expressed in nmoles 4-methylumbelliferone (4-MU) released per minute and per millilitre of culture. (B) Expression of CTS1 in wild-type and mutant cells. RNA was purified from wild-type (strain TD28) and swm1 (strain LS40) cells at the indicated times (hours) after transfer to 38°C. RNA blots were hybridized sequentially with radioactively labelled probes for CTS1 and ACT1. (C) Analysis of CTS1 mRNA stability. RNA was purified from swm1 cells (strain LS40) transformed with vector (left, swm1) or pSU50 (right, swm1+P_TPI-CTS1) containing the CTS1 ORF under the control of the TPI promoter (P_TPI-CTS1 allele) at the indicated times (hours) after transfer to 38°C. RNA blots were hybridized with radioactively labelled probes for CTS1 and ACT1. Arrows indicate the position of the transcript corresponding to the heterologous promoter (upper) or the chromosomal locus (lower). (D) Expression of genes involved in cell separation in swm1 cells. RNA was purified from wild-type (strain TD28) and swm1 cells (strain LS40) at the indicated times (hours) after transfer to 38°C. RNA blots were hybridized sequentially with radioactively labelled probes for CTS1, DSE1, DSE2, SCW11, EGT2 and ACE2. The ACT1 gene was used to test for equal RNA loading in all lanes.

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