Journal: The Journal of Biological Chemistry
Article Title: Regulation of Inositol Metabolism Is Fine-tuned by Inositol Pyrophosphates in Saccharomyces cerevisiae * * ♦
Figure Lengend Snippet: bZIP and inositol pyrophosphate kinase (DINS) domains of Kcs1 are required for INO1 transcription. A, diagram of the bZIP and the DINS functional domains of Kcs1 indicating site mutations disrupting individual domains. B, serial 10-fold dilutions of kcs1 Δ cells carrying either empty vector (pURA3), mutated bZIP domain (p kcs1 L1L2→AA ), mutated DINS domain (p kcs1 SLL→AAA ), or WT KCS1 were spotted on I− or I+ plates. Plates were incubated at 30 °C for 3 days. The figure shown is representative of three experiments. C, cells harboring the empty vector (pEV), WT KCS1, or mutated KCS1 were cultured in I+ to the mid-logarithmic phase ( A 550 of 0.5), pelleted, washed with prewarmed I+ or I−, and resuspended in fresh prewarmed I+ or I−. After the shift, cells were grown for 2 h. INO1 mRNA was quantified using RT-qPCR as described under “Experimental Procedures.” The data shown in C are the average of three experiments ± S.D.
Article Snippet: RT-qPCRs were performed in a 20-μl volume using Brilliant III Ultra-Faster SYBR Green qPCR master mix (Agilent Technologies, Santa Clara, CA).
Techniques: Functional Assay, Plasmid Preparation, Incubation, Cell Culture, Quantitative RT-PCR