Fig. 4

Quantification of the abundance of polyproline proteins involved possibly in tolerance to acetic acid. a GFP-tagged strategy for determination the abundance of polyproline proteins. Three pairs of primers were designed to amplify integrated DNA fragment to allow in-frame fusion of the GFP tag at the C-terminal coding region of the gene (X represents each gene name of interest). X-1/X-2 and X-5/X-6 were designed to amplify upstream and downstream homologous arms (about 400 bp) for recombination in chromosome, X-3/-X-4 were designed to amplify the GFP-marker cassette. X-2 and X-3, X-4 and X-5 shared complementary sequences, respectively. Then the gene-specific oligonucleotide fragment was obtained by overlap PCR of the three segments. b The relative abundance of polyproline proteins under condition without acetic acid. c The relative abundance of polyproline proteins in the presence of 87 mM acetic acid. d Comparison of mRNA level of UME6 and protein level of Ume6p. Yeast cells were cultured under conditions with or without 87 mM acetic acid (pH 4.2) for 4 h. The relative transcription level of UME6 or protein level of Ume6p in YS58-UME6G-V under non-stressed condition was defined as a value of 1, respectively. Data are presented as the means of the results of three independent experiments. Error bars represent standard deviations (*P < 0.05, **P < 0.01)