Abstract
Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprene biosynthetic pathway. The Saccharomyces cerevisiae gene for IPP isomerase, IDI1, was recently isolated and characterized (Anderson, M. S., Muehlbacher, M., Street, I. P., Proffitt, J., and Poulter, C. D. (1989) J. Biol. Chem. 264, 19169-19175), and the wild-type gene, IDI1, was disrupted with a LEU2 marker to create a diploid yeast strain heterozygous for the idi1::leu2 disruption, which revealed that IDI1 was an essential single-copy gene (Mayer, M.P., Hahn, F. M., Stillman, D. J., and Poulter, C. D. (1992) Yeast 8, 743-748). We now report the isolation of a cDNA clone from Schizosaccharomyces pombe by a plasmid shuffle-mediated complementation of the LEU2 disrupted yeast gene. The S. pombe clone encoded a 26,864-dalton polypeptide of 227 amino acids with a high degree of similarity to the S. cerevisiae IDI1 enzyme. S. pombe IPP isomerase contained the essential Cys and Glu catalytic residues identified in yeast isomerase (Street, I. P., Coffman, H. R., Baker, J., and Poulter, C. (1994) Biochemistry 33, 4212-4217) but was significantly smaller than the S. cerevisiae enzyme. The plasmid shuffle technique is an excellent procedure for screening expression libraries for IPP isomerase activity by complementation of the idi1 mutation.
Highlights
Xuan et al [19] reported a human cDNA clone with an open reading frame that encoded a protein with a high degree of similarity to yeast Isopentenyl diphosphate (IPP) isomerase, including the essential Cys and Glu residues
We previously described construction of S. cerevisiae strain FH2-5b [15] and report its use to isolate a cDNA clone for IPP isomerase from Schizosaccharomyces pambe by a plasmid shuffle technique and synthesis of the S. pambe enzyme in Escherichia coli
The conversion ofIPP to DMAPP catalyzed by IPP isomerase is an essential step in the biosynthesis of all isoprenoids
Summary
Materials-[1- '4ClIPP was purchased from Amersham Corp. DE52 ion exchange resin was from Whatman, and phenyl-Sepharose was purchased from Pharmacia Biotech Inc. Tran sform ation of S. cerevisiae Strain FH2-5b-S. cerevisiae strain FH2-5b was grown in YEPD medium to OD6DD - 0.7 5 a nd tra nsform ed with 10 JJ.g of DNA from the S. pombe libra ry by the modifi ed li thium acetate procedUJ·e of Elbe [30]. Cl-AA Selection l Counterselection-Pl ates containing S. cerevisiae transformed with the S. pombe cDNA library were washed with 5 ml of medium containing Cl-AA [24]. Pombe IPP Isomerase Gene-Hind lJI di gests were perform ed on f our plasmids identifi ed as co nta ining DNA able to compl ement di s rupted S. cerevisiae IDI1. The DNA was electrophoresed on a O.S% agarose gel, excised, purified by GeneClean , and ligated into the NdeI a nd Sall sites of the E. coli expression vector pARC306N.
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