The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reductase were mapped and cloned from Salmonela typhimurium and Escherichia coli. Two classes of oxidant-resistant ahp mutants which overexpress the two proteins were isolated. ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. ahp-2 was isolated in an oxyR deletion background and is oxyR independent. Transposons linked to ahp-l and ahp-2 or inserted in ahp mapped the genes to 13 min on the S. typhimurium chromosome, 59% linked to ent. Deletions of ahp obtained in both S. typhimurium and E. coli resulted in hypersensitivity to killing by cumene hydroperoxide (an alkyl hydroperoxide) and elimination of the proteins F52a and C22 from two-dimensional gels and immunoblots. ahp clones isolated from both S. typhimurium and E. coli complemented the cumene hydroperoxide sensitivity of the ahp deletion strains and restored expression of the F52a and C22 proteins. A cis-acting element required for oxyR-dependent, rpoH-independent heat shock induction of the F52a protein was present at the S. typhimurium but not the E. coli ahp locus.When Salmonella typhimurium and Escherichia coli cells are pretreated with low doses of hydrogen peroxide, they become transiently resistant to lethal doses of hydrogen peroxide (4,5). Coincident with this increased resistance is the induction of at least 30 proteins as seen by two-dimensional gel electrophoresis (4,14). The synthesis of some of the hydrogen peroxide-inducible proteins is also elevated by heat shock and treatment with nalidixic acid or ethanol, although each of these stresses also induces a group of unique proteins (14, 21). We have identified and characterized the oxyR gene that regulates the expression of nine of the hydrogen peroxide-inducible proteins and have isolated mutants in S. typhimurium (oxyRI) and E. coli (oxyR2) that are resistant to hydrogen peroxide and constitutively overexpress the nine proteins (4).Some of the stress proteins that are overexpressed in the oxyRi mutant have been identified, including catalase, manganese superoxide dismutase, and glutathione reductase (4). We found that two of the proteins overexpressed by the oxyRI-and the oxyR2 mutants, designated F52a and C22 on two-dimensional gels, make up a novel alkyl hydroperoxide reductase activity (4, 9a). The proteins were purified to homogeneity and characterized. The F52a flavoprotein, together with the smaller C22 protein, reduces lipid hydroperoxides and other alkyl hydroperoxides directly to their corresponding alcohols by using either NADH or NADPH as an electron donor (9a). We are interested in studying the role of the alkyl hydroperoxide reductase in defending against oxidative stress, since hydroperoxides have been shown to be mutagenic in * Corresponding author. (22) salts to 1 x 108 cells per ml. Diethylsulfate (0.1 ml) was added to the 10-ml culture, and the tube was vortexed vigorously for 20 s. After the culture was l...

Phosphorylation of the regulatory light chain is an important mechanism for the activation of myosin in non‐muscle cells. Unlike most myosin light chain kinases (MLCKs), MLCK‐A from Dictyostelium is not activated by Ca2+/calmodulin. Autophosphorylation increases activity, but only to a low level, suggesting that there is an additional activation mechanism. Here, we show that MLCK‐A is autophosphorylated on Thr289, which is C‐terminal to the catalytic domain. Phosphorylation of MLCK‐A increases in response to concanavalin A (conA) treatment of cells, which was previously shown to activate MLCK‐A. However, a mutant kinase with an alanine at position 289 (T289A) is also phosphorylated in vivo, indicating that there is an additional phosphorylated residue. Based on comparisons with other protein kinases, we tested whether phosphorylation of Thr166 drives activation of MLCK‐A. Our data indicate that phosphorylation of Thr289 occurs in vivo, but is not associated with conA‐induced activation, whereas phosphorylation of Thr166 by some as yet unidentified kinase is associated with activation. Replacement of Thrl66 with glutamate results in a 12‐fold increase in activity as compared with the wild‐type enzyme, supporting the idea that phosphorylation of Thr166 increases MLCK‐A activity.

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References linking genes to complex human traits, such as personality type or disease susceptibility, abound in the news media and popular culture. In his book The God Gene: How Faith is Hardwired into Our Genes, Dean Hamer argues that a variation in the VMAT2 gene plays a role in one's openness to spiritual experiences. In a nonmajors class, we read and discussed The God Gene and conducted on a small scale an extension of the study it describes. Students used polymerase chain reaction to replicate a portion of their VMAT2 genes, and they analyzed three polymorphic sites in the sequence of these products. Associations between particular VMAT2 alleles and scores on a personality test were assessed by t test. The course, of which this project was a major part, stimulated student learning; scores on a test covering basic genetic concepts, causation/correlation, and laboratory methodology improved after completion of the course. In a survey, students reported the laboratory project aided their learning, especially in the areas of statistics and the linking of genes to behaviors. They reported high levels of engagement with the project, citing in particular its personal nature as motivating their interest.