Common deletion polymorphisms in the human genome

The locations and properties of common deletion variants in the human genome are largely unknown. We describe a systematic method for using dense SNP genotype data to discover deletions and its application to data from the International HapMap Consortium to characterize and catalogue segregating deletion variants across the human genome. We identified 541 deletion variants (94% novel) ranging from 1 kb to 745 kb in size; 278 of these variants were observed in multiple, unrelated individuals, 120 in the homozygous state. The coding exons of ten expressed genes were found to be commonly deleted, including multiple genes with roles in sex steroid metabolism, olfaction and drug response. These common deletion polymorphisms typically represent ancestral mutations that are in linkage disequilibrium with nearby SNPs, meaning that their association to disease can often be evaluated in the course of SNP-based whole-genome association studies.     

Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607

Several hundred million tons of toxic mercurials are dispersed in the biosphere. Microbes can detoxify organo-mercurials and mercury salts through sequential action of two enzymes, organomercury lyase and mercuric ion reductase (MerA). The latter, a homodimer with homology to the FAD-dependent disulphide oxidoreductases, catalyses the reaction NADPH + Hg(II)----NADP+ + H+ + Hg(0), one of the very rare enzymic reactions with metal substrates. Human glutathione reductase serves as a reference molecule for FAD-dependent disulphide reductases and between its primary structure and that of MerA from Tn501 (Pseudomonas), Tn21 (Shigella), p1258 (Staphylococcus) and Bacillus, 25-30% of the residues have been conserved. All MerAs have a C-terminal extension about 15 residues long but have very varied N termini. Although the enzyme from Streptomyces lividans has no addition, from Pseudomonas aeruginosa Tn501 and Bacillus sp. strain RC607 it has one and two copies respectively of a domain of 80-85 residues, highly homologous to MerP, the periplasmic component of proteins encoded by the mer operon. These domains can be proteolytically cleaved off without changing the catalytic efficiency. We report here the crystal structure of MerA from the Gram-positive bacterium Bacillus sp. strain RC607. Analysis of its complexes with nicotinamide dinucleotide substrates and the inhibitor Cd(II) reveals how limited structural changes enable an enzyme to accept as substrate what used to be a dangerous inhibitor. Knowledge of the mode of mercury ligation is a prerequisite for understanding this unique detoxification mechanism.     

Loss of ACTN3 gene function alters mouse muscle metabolism and shows evidence of positive selection in humans

More than a billion humans worldwide are predicted to be completely deficient in the fast skeletal muscle fiber protein alpha-actinin-3 owing to homozygosity for a premature stop codon polymorphism, R577X, in the ACTN3 gene. The R577X polymorphism is associated with elite athlete status and human muscle performance, suggesting that alpha-actinin-3 deficiency influences the function of fast muscle fibers. Here we show that loss of alpha-actinin-3 expression in a knockout mouse model results in a shift in muscle metabolism toward the more efficient aerobic pathway and an increase in intrinsic endurance performance. In addition, we demonstrate that the genomic region surrounding the 577X null allele shows low levels of genetic variation and recombination in individuals of European and East Asian descent, consistent with strong, recent positive selection. We propose that the 577X allele has been positively selected in some human populations owing to its effect on skeletal muscle metabolism.     

Podocalyxin enhances the adherence of cells to platelets

Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Δ) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin αVβ3/αVβ5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid. Received 14 August 2007; received after revision 12 September 2007; accepted 13 September 2007     

Chromosomal localization of a unique gene by non-autoradiographic in situ hybridization

During the past few years, several methods have been developed for the detection of specific nucleic acid sequences by in situ hybridization using non-radioactive labels such as fluorochromes, cytochemically detectable enzymes and electron-dense markers. These methods are preferable to autoradiography in terms of speed of performance and topological resolution. Their limited sensitivity, however, has so far restricted their use to the detection of repeated sequences. Here we report single gene detection with a procedure using 2-acetylaminofluorene (AAF)-modified probes, immunoperoxidase cytochemistry and reflection-contrast microscopy. We confirmed the autoradiographic data on the localization of the human thyroglobulin (Tg) gene to the distal end of the long arm of chromosome 8. A mixture of cosmid cHT2-derived subclones of the 3' part of the Tg gene, 22.3 kilobase pairs (kbp) in total, was used as a hybridization probe. This procedure can be used to map other unique sequences, if genomic clones are available from which clones with an appropriate amount of inserts can be isolated.