The first step of aminoacylation at the atomic level in histidyl-tRNA synthetase

The crystal structure of an enzyme–substrate complex with histidyl-tRNA synthetase from Escherichia coli, ATP, and the amino acid analog histidinol is described and compared with the previously obtained enzyme–product complex with histidyl-adenylate. An active site arginine, Arg-259, unique to all histidyl-tRNA synthetases, plays the role of the catalytic magnesium ion seen in seryl-tRNA synthetase. When Arg-259 is substituted with histidine, the apparent second order rate constant (kcat/Km) for the pyrophosphate exchange reaction and the aminoacylation reaction decreases 1,000-fold and 500-fold, respectively. Crystals soaked with MnCl2 reveal the existence of two metal binding sites between β- and γ-phosphates; these sites appear to stabilize the conformation of the pyrophosphate. The use of both conserved metal ions and arginine in phosphoryl transfer provides evidence of significant early functional divergence of class II aminoacyl-tRNA synthetases.

A first trial of retrospective collaboration for positional cloning in complex inheritance: Assay of the cytokine region on chromosome 5 by the Consortium on Asthma Genetics (COAG)

The central problem of complex inheritance is to map oligogenes for disease susceptibility, integrating linkage and association over samples that differ in several ways. Combination of evidence over multiple samples with 1,037 families supports loci contributing to asthma susceptibility in the cytokine region on 5q [maximum logarithm of odds (lod) = 2.61 near IL-4], but no evidence for atopy. The principal problems with retrospective collaboration on linkage appear to have been solved, providing far more information than a single study. A multipoint lod table evaluated at commonly agreed reference loci is required for both collaboration and metaanalysis, but variations in ascertainment, pedigree structure, phenotype definition, and marker selection are tolerated. These methods are invariant with statistical methods that increase the power of lods and are applicable to all diseases, motivating collaboration rather than competition. In contrast to linkage, positional cloning by allelic association has yet to be extended to multiple samples, a prerequisite for efficient combination with linkage and the greatest current challenge to genetic epidemiology.

Anti-β2 glycoprotein I (β2GPI) autoantibodies recognize an epitope on the first domain of β2GPI

Anticardiolipin (aCL) autoantibodies are associated with thrombosis, recurrent fetal loss, and thrombocytopenia. Only aCL found in autoimmune disease require the participation of the phospholipid binding plasma protein β2 glycoprotein I (β2GPI) for antibody binding and now are called anti-β2GPI. The antigenic specificity of aCL affinity purified from 11 patients with high titers was evaluated in an effort to better understand the pathophysiology associated with aCL. Seven different recombinant domain-deleted mutants of human β2GPI, and full length human β2GPI (wild-type), were used in competition assays to inhibit the autoantibodies from binding to immobilized wild-type β2GPI. Only those domain-deleted mutants that contained domain 1 inhibited the binding to immobilized wild-type β2GPI from all of the patients. The domain-deleted mutants that contained domain 1 inhibited all aCL in a similar but not identical pattern, suggesting that these aCL recognize a similar, but distinguishable, epitope(s) present on domain 1.

The First Step of Gibberellin Biosynthesis in Pumpkin Is Catalyzed by at Least Two Copalyl Diphosphate Synthases Encoded by Differentially Regulated Genes

The first step in gibberellin biosynthesis is catalyzed by copalyl diphosphate synthase (CPS) and ent-kaurene synthase. We have cloned from pumpkin (Cucurbita maxima L.) two cDNAs, CmCPS1 and CmCPS2, that each encode a CPS. Both recombinant fusion CmCPS proteins were active in vitro. CPS are translocated into plastids and processed by cleavage of transit peptides. For CmCPS1 and CmCPS2, the putative transit peptides cannot exceed the first 99 and 107 amino acids, respectively, because longer N-terminal deletions abolished activity. Levels of both CmCPS transcripts were strictly regulated in an organ-specific and developmental manner. Both transcripts were almost undetectable in leaves and were abundant in petioles. CmCPS1 transcript levels were high in young cotyledons and low in roots. In contrast, CmCPS2 transcripts were undetectable in cotyledons but present at significant levels in roots. In hypocotyls, apices, and petioles, CmCPS1 transcript levels decreased with age much more rapidly than those of CmCPS2. We speculate that CmCPS1 expression is correlated with the early stages of organ development, whereas CmCPS2 expression is correlated with subsequent growth. In contrast, C. maxima ent-kaurene synthase transcripts were detected in every organ at almost constant levels. Thus, ent-kaurene biosynthesis may be regulated through control of CPS expression.