Fig. 4

Construction of a MIP-GFP reporter enzyme. The complete INO1 gene, located on a 4.2 kb Hind lll- Kpn l restriction enzyme fragment, was cloned into a high copy number plasmid vector, pDK 396. A [51, 52], The plasmid was used to construct an INO1: GFP fusion gene, encoding a fluorescent MIP-GFP reporter enzyme. The INO1 gene encodes MIP in S. cerevisiae [51, 52]. A GFP gene [53], with Csp45I restriction site adapters, was inserted into a Csp45I restriction enzyme site (shown in bold type) located at the 3 prime end of INO1. DNA sequencing and bioinformatics analyses confirmed the correct reading frame, GFP is underlined. B while polymerase chain reaction cloning, using gene specific primers [54] and Southern blotting [55] confirmed the correct genomic location of the fusion gene after its integration into the yeast genome, using homologous recombination as described by Huh et al.[56], replacing resident genomic copy of the INO1 gene, (C). Western blotting studies of MIP-GFP and WT-MIP organelles, found that MIP-GFP in the plasma membrane is less sensitive to inositol feedback inhibition than Wt-MIP (D)