Cloning by synteny: identifying C. briggsae homologues of C. elegans genes.

Phylogenetic comparisons of gene and protein sequences between related species are often used to identify evolutionarily conserved elements that are important for gene expression, function, or regulation. However, homologoues may sometimes be difficult to identify by conventional low stringency hybridisation techniques, if they have undergone substantial sequence divergence. A new approach, cloning by synteny, is described that was used to identify the C. briggsae homologue of the C. elegans sex-determining gene tra-2. We show that four genes tra-2, ppp-1, art-1, and sod-1 are organised in a syntenic cluster and suggest that extensive conservation of gene linkage may exist between C. briggsae and C. elegans. We have also constructed a C. briggsae cDNA library to facilitate characterisation of these genes. Given the rapid progress in the physical mapping and sequencing of the C. elegans genome, cloning by synteny may provide the fastest method for identifying C. briggsae gene homologues, especially for genes encoding novel proteins.     

Sequence specificity of the deoxyribonuclease activity of 1,10-phenanthroline-copper ion

A statistical analysis of a data set composed of over 1600 scission events of DNA produced by the 2:1 1,10-phenanthroline-copper complex (OP-Cu) has demonstrated that the nucleotide 5' to the site of phosphodiester bond scission is a primary influence in the kinetics of cleavage at any sequence position. The scission was less affected by the 3' neighbor. For each of the sixteen possible dinucleotides, a kinetic parameter can be computed reflecting scission at the 3' nucleotide. When used to predict the scission pattern of a DNA sequence not part of the present data set, correlation coefficients of about 0.6 between predicted and observed patterns were obtained.     

Human galectin-3 is a novel chemoattractant for monocytes and macrophages

Galectin-3 is a beta-galactoside-binding protein implicated in diverse biological processes. We found that galectin-3 induced human monocyte migration in vitro in a dose-dependent manner, and it was chemotactic at high concentrations (1.0 microM) but chemokinetic at low concentrations (10-100 nM). Galectin-3-induced monocyte migration was inhibited by its specific mAb and was blocked by lactose and a C-terminal domain fragment of the protein, indicating that both the N-terminal and C-terminal domains of galectin-3 are involved in this activity. Pertussis toxin (PTX) almost completely blocked monocyte migration induced by high concentrations of galectin-3. Galectin-3 caused a Ca2+ influx in monocytes at high, but not low, concentrations, and both lactose and PTX inhibited this response. There was no cross-desensitization between galectin-3 and any of the monocyte-reactive chemokines examined, including monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, and stromal cell-derived factor-1alpha. Cultured human macrophages and alveolar macrophages also migrated toward galectin-3, but not monocyte chemotactic protein-1. Finally, galectin-3 was found to cause monocyte accumulation in vivo in mouse air pouches. These results indicate that galectin-3 is a novel chemoattractant for monocytes and macrophages and suggest that the effect is mediated at least in part through a PTX-sensitive (G protein-coupled) pathway.     

The 2.0 A crystal structure of Thermus thermophilus methionyl-tRNA synthetase reveals two RNA-binding modules

Background: The 20 aminoacyl-tRNA synthetases are divided into two classes, I and II. The 10 class I synthetases are considered to have in common the catalytic domain structure based on the Rossmann fold, which is totally different from the class II catalytic domain structure. The class I synthetases are further divided into three subclasses, a, b and c, according to sequence homology. No conserved structural features for tRNA recognition by class I synthetases have been established. Results: We determined the crystal structure of the class Ia methionyl-tRNA synthetase (MetRS) at 2.0 A resolution, using MetRS from an extreme thermophile, Thermus thermophilus HB8. The T. thermophilus MetRS structure is in full agreement with the biochemical and genetic data from Escherichia coli MetRS. The conserved 'anticodon-binding' residues are spatially clustered on an alpha-helix-bundle domain. The Rossmann-fold and anticodon-binding domains are connected by a beta-alpha-alpha-beta-alpha topology ('SC fold') domain that contains the class I specific KMSKS motif. Conclusions: The alpha-helix-bundle domain identified in the MetRS structure is the signature of the class Ia enzymes, as it was also identified in the class Ia structures of the isoleucyl- and arginyl-tRNA synthetases. The beta-alpha-alpha-beta-alpha topology domain, which can now be identified in all known structures of the class Ia and Ib synthetases, is likely to dock with the inner side of the L-shaped tRNA, thereby positioning the anticodon stem.     

Effects of antioxidants on X-ray- or hyperthermia-induced apoptosis in human lymphoma U937 cells

Hydroxyl radicals (.OH) and superoxide anion radicals (O2.-) are known to play cardinal roles in cell killing and various types of cell damage. In order to elucidate the mechanism of the involvement of both free radicals on apoptosis, the correlation between anti-apoptotic effects and free radical scavenging abilities of anti-oxidants was studied. As an indicator of anti-apoptotic effects, C1/2 (antioxidant concentration to inhibit DNA fragmentation by 50%) was evaluated in human lymphoma cell line U937 cells 6 hr after X-ray (10 Gy) or hyperthermia (44 degrees C, 30 min) treatment. Rate constants of the reactions between antioxidants and .OH or O2.- were calculated as the scavenging ability of the antioxidants with graded concentration estimated by EPR spectroscopy. No apparent correlation between C1/2 obtained in apoptosis induced by X-rays or hyperthermia and the rate constants of antioxidants for .OH or O2.- was observed. On the other hand, the partition coefficients in 1-octanol/water of the antioxidants, an indicator of hydrophobicity, revealed a correlation with the C1/2 of the agents with hyperthermia, but not with X-ray irradiation. These results indicate that the prevention of apoptosis by an antioxidant is not simply associated with its scavenging ability for .OH or O2.-. The hydrophobicity of the antioxidant, among other possible factors, is involved in the inhibition of hyperthermia- induced apoptosis.     

Cleavage of an inaccessible site by the maxizyme with two independent binding arms: an alternative approach to the recruitment of RNA helicases

To overcome obstacles to target site selection, we recently created a novel hybrid ribozyme that could access any chosen site by the recruitment of intracellular RNA helicases [Warashina et al. (2001) Proc. Natl. Acad. Sci. USA 98, 5572-5577; Kawasaki et al. (2002) Nat. Biotech. 20, 376-380]. We also demonstrated previously that pol III-driven maxizymes with two substrate-binding arms that were directed against two different sites within a target mRNA formed very active heterodimers in vivo [Kuwabara, et al. (2000) Trends Biotechnol. 18, 462-468; Tanabe et al. (2001) Nature 406, 473-474]. Despite the complicated dimerization process, all the maxizymes that we tested in cultured cells had greater catalytic activity than the parental ribozymes. To investigate the action of maxizymes in cells, we designed a specific maxizyme with two substrate-binding arms that was directed against endogenously expressed LTR-luciferase chimeric mRNA, where LTR refers to the long terminal repeat of HIV-1. One substrate-binding arm of the maxizyme was designed to bind to a site within HIV-1 TAR RNA that is known to form a stable stem structure that normally prevents binding of a ribozyme. The other substrate-binding arm was directed against a relatively accessible site within the luciferase gene. As expected, the conventional ribozyme failed to cleave the TAR region in vivo because of the latter's stable secondary structure. However, to our surprise, the maxizyme cleaved the TAR region within the stem with high efficiency in vivo. The enhanced cleavage in vivo by the maxizyme might have resulted from an entropically favorable, intramolecular, second binding process that occurred during the breathing of the stem structure of the target mRNA. Importantly, our data suggest that this maxizyme technology might be used as an alternative approach to the recruitment of RNA helicases in cleaving sites previously found to be inaccessible.     

Mutation screening of the muscarinic m2 and m3 receptor genes in asthmatics,outgrow subjects,and normal controls

Muscarinic receptors are important in the development of airway hyperresponsiveness. In some patients with asthma and in animal models of hyperreactivity, functional abnormalities in these receptors are suggested to contribute to disease. Here, we have screened for single nucleotide polymorphisms in the coding region of human muscarinic m2 and m3 receptor genes using direct fluorescence sequencing. DNA samples from 102 current asthmatics and 58 who had outgrown asthma ("outgrow" patients) were compared with 70 random non-asthmatic controls. A mutation characterized by a single base substitution (A1050G, Ser350Ser) was identified in the muscarinic m2 receptor gene. This polymorphism was common and was represented in all three groups studied. In contrast, in the m3 receptor coding region examined, we found a very rare nucleotide variant (C261T, Ile87Ile), identified in only one of the 230 samples genotyped. Therefore, neither A1050G in the m2 receptor nor C261T in the m3 receptor is likely to be functionally significant for airway hyperresponsiveness in asthma. Our data suggest that both the m2 and m3 receptor genes are highly conserved, and no significant genetic mutations are related to their possible functional changes in human asthma.     

A novel bacterial pathogen, Microbacterium nematophilum, induces morphological change in the nematode C. elegans

The Dar (deformed anal region) phenotype, characterized by a distinctive swollen tail, was first detected in a variant strain of Caenorhabditis elegans which appeared spontaneously in 1986 during routine genetic crosses [1] and [2]. Dar isolates were initially analysed as morphological mutants, but we report here that two independent isolates carry an unusual bacterial infection different from those previously described [3], which is the cause of the Dar phenotype. The infectious agent is a new species of coryneform bacterium, named Microbacterium nematophilum n. sp., which fortuitously contaminated cultures of C. elegans. The bacteria adhere to the rectal and post-anal cuticle of susceptible nematodes, and induce substantial local swelling of the underlying hypodermal tissue. The swelling leads to constipation and slowed growth in the infected worms, but the infection is otherwise non-lethal. Certain mutants of C. elegans with altered surface antigenicity are resistant to infection. The induced deformation appears to be part of a survival strategy for the bacteria, as C. elegans are potentially their predators.     

Fe(III) oxides protect fermenter–methanogen syntrophy against interruption by elemental sulfur via stiffening of Fe(II) sulfides produced by sulfur respiration

Thermosipho globiformans (rod-shaped thermophilic fermenter) and Methanocaldococcus jannaschii (coccal hyperthermophilic hydrogenotrophic methanogen) established H₂-mediated syntrophy at 68 °C, forming exopolysaccharide-based aggregates. Electron microscopy showed that the syntrophic partners connected to each other directly or via intercellular bridges made from flagella, which facilitated transfer of H₂. Elemental sulfur (S⁰) interrupted syntrophy; polysulfides abiotically formed from S⁰ intercepted electrons that were otherwise transferred to H⁺ to produce H₂, resulting in the generation of sulfide (sulfur respiration). However, Fe(III) oxides significantly reduced the interruption by S⁰, accompanied by stiffening of Fe(II) sulfides produced by the reduction of Fe(III) oxides with the sulfur respiration-generated sulfide. Sea sand replacing Fe(III) oxides failed to generate stiffening or protect the syntrophy. Several experimental results indicated that the stiffening of Fe(II) sulfides shielded the liquid from S⁰, resulting in methane production in the liquid. Field-emission scanning electron microscopy showed that the stiffened Fe(II) sulfides formed a network of spiny structures in which the microorganisms were buried. The individual fermenter rods likely produced Fe(II) sulfides on their surface and became local centers of a core of spiny structures, and the connection of these cores formed the network, which was macroscopically recognized as stiffening.