Detection by denaturing gradient gel electrophoresis of pncA mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis isolates from the United States-Mexico border region.

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint.

Detection of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis using denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with rifampin (RIF) resistance in the rpoB gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and is comparable to DNA sequencing in detecting DNA alterations. Specific mutations are often recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint.

Kinetic properties and metabolic contributions of yeast mitochondrial and cytosolic NADP+-specific isocitrate dehydrogenases.

To compare kinetic properties of homologous isozymes of NADP+-specific isocitrate dehydrogenase, histidine-tagged forms of yeast mitochondrial (IDP1) and cytosolic (IDP2) enzymes were expressed and purified. The isozymes were found to share similar apparent affinities for cofactors. However, with respect to isocitrate, IDP1 had an apparent Km value approximately 7-fold lower than that of IDP2, whereas, with respect to alpha-ketoglutarate, IDP2 had an apparent Km value approximately 10-fold lower than that of IDP1.

Multiple cellular consequences of isocitrate dehydrogenase isozyme dysfunction.

To probe the functions of multiple forms of isocitrate dehydrogenase in Saccharomyces cerevisiae, mutants lacking three of the isozymes were constructed and analyzed. Results show that, while the mitochondrial NAD+-dependent enzyme, IDH (composed of Idh1p and Idh2p subunits) is not the major contributor to total isocitrate dehydrogenase activity under any growth condition, loss of IDH produces the most dramatic growth phenotypes. These include reduced growth in the absence of glutamate, as well as an increase in expression of Idp2p (the cytosolic NADP+-dependent enzyme) under some growth conditions.

Modulation of citrate metabolism alters aluminum tolerance in yeast and transgenic canola overexpressing a mitochondrial citrate synthase.

Aluminum (Al) toxicity is a major constraint for crop production in acid soils, although crop cultivars vary in their tolerance to Al. We have investigated the potential role of citrate in mediating Al tolerance in Al-sensitive yeast (Saccharomyces cerevisiae; MMYO11) and canola (Brassica napus cv Westar). Yeast disruption mutants defective in genes encoding tricarboxylic acid cycle enzymes, both upstream (citrate synthase [CS]) and downstream (aconitase [ACO] and isocitrate dehydrogenase [IDH]) of citrate, showed altered levels of Al tolerance.

Global transcription analysis of Krebs tricarboxylic acid cycle mutants reveals an alternating pattern of gene expression and effects on hypoxic and oxidative genes.

To understand the many roles of the Krebs tricarboxylic acid (TCA) cycle in cell function, we used DNA microarrays to examine gene expression in response to TCA cycle dysfunction. mRNA was analyzed from yeast strains harboring defects in each of 15 genes that encode subunits of the eight TCA cycle enzymes. The expression of >400 genes changed at least threefold in response to TCA cycle dysfunction.