Multiple site-directed mutagenesis of more than 10 sites simultaneously and in a single round.

Site-directed mutagenesis is a powerful tool to explore the structure-function relationship of proteins, but most traditional methods rely on the mutation of only one site at a time and efficiencies drop drastically when more than three sites are targeted simultaneously. Many applications in functional proteomics and genetic engineering, including codon optimization for heterologous expression, generation of cysteine-less proteins, and alanine-scanning mutagenesis, would greatly benefit from a multiple-site mutagenesis method with high efficiency. Here we describe the development of a simple and rapid method for site-directed mutagenesis of more than 10 sites simultaneously with up to 100% efficiency. The method uses two terminal tailed primers with a unique 25-nucleotide tail each that are simultaneously annealed to template DNA together with the set of mutagenic primers in between. Following synthesis of the mutant strand by primer extension and ligation with T4 DNA polymerase and ligase, the unique mutant strand-specific tails of the terminal primers are used as anchors to specifically amplify the mutant strand by high-fidelity polymerase chain reaction. We have employed this novel method to mutate simultaneously all 9 and 11 CUG leucine codons of the Hyg and Neo resistance genes, respectively, to the Candida albicans-friendly UUG leucine codon at 100% efficiency.

High-affinity myo-inositol transport in Candida albicans: substrate specificity and pharmacology.

Inositol is considered a growth factor in yeast cells and it plays an important role in Candida as an essential precursor for phospholipomannan, a glycophosphatidylinositol (GPI)-anchored glycolipid on the cell surface of Candida which is involved in the pathogenicity of this opportunistic fungus and which binds to and stimulates human macrophages. In addition, inositol plays an essential role in the phosphatidylinositol signal transduction pathway, which controls many cell cycle events. Here, high-affinity myo-inositol uptake in Candida albicans has been characterized, with an apparent K(m) value of 240 +/- 15 microM, which appears to be active and energy-dependent as revealed by inhibition with azide and protonophores (FCCP, dinitrophenol). Candida myo-inositol transport was sodium-independent but proton-coupled with an apparent K(m) value of 11.0 +/- 1.1 nM for H(+), equal pH 7.96 +/- 0.05, suggesting that the C. albicans myo-inositol-H(+) transporter is fully activated at physiological pH. C. albicans inositol transport was not affected by cytochalasin B, phloretin or phlorizin, an inhibitor of mammalian sodium-dependent inositol transport. Furthermore, myo-inositol transport showed high substrate specificity for inositol and was not significantly affected by hexose or pentose sugars as competitors, despite their structural similarity. Transport kinetics in the presence of eight different inositol isomers as competitors revealed that proton bonds between the C-2, C-3 and C-4 hydroxyl groups of myo-inositol and the transporter protein play a critical role for substrate recognition and binding. It is concluded that C. albicans myo-inositol-H(+) transport differs kinetically and pharmacologically from the human sodium-dependent myo-inositol transport system and constitutes an attractive target for delivery of cytotoxic inositol analogues in this pathogenic fungus.