Exploitation of colinear relationships between the genomes of<Emphasis Type="Italic"> Lotus japonicus</Emphasis>,<Emphasis Type="Italic"> Pisum sativum</Emphasis> and<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>, for positional cloning of a legume symbiosis gene

The Lotus japonicus LjSYM2 gene, and the Pisum sativum orthologue PsSYM19, are required for the formation of nitrogen-fixing root nodules and arbuscular mycorrhiza. Here we describe the map-based cloning procedure leading to the isolation of both genes. Marker information from a classical AFLP marker-screen in Lotus was integrated with a comparative genomics approach, utilizing Arabidopsis genome sequence information and the pea genetic map. A network of gene-based markers linked in all three species was identified, suggesting local colinearity in the region around LjSYM2/PsSYM19. The closest AFLP marker was located just over 200 kb from the LjSYM2 gene, the marker SHMT, which was converted from a marker on the pea map, was only 7.9 kb away. The LjSYM2/PsSYM19 region corresponds to two duplicated segments of the Arabidopsis chromosomes AtII and AtIV. Lotus homologues of Arabidopsis genes within these segments were mapped to three clusters on LjI, LjII and LjVI, suggesting that during evolution the genomic segment surrounding LjSYM2 has been subjected to duplication events. However, one marker, AUX-1, was identified based on colinearity between Lotus and Arabidopsis that mapped in physical proximity of the LjSym2 gene.Communicated by J.S. Heslop-Harrison     

Fine genetic mapping of the <Emphasis Type="Italic">TuNI</Emphasis> locus causing systemic veinal necrosis by turnip mosaic virus infection in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In the pathosystem of turnip mosaic virus (TuMV) and Arabidopsis thaliana, two distinct symptoms (mosaic symptom and veinal necrosis) were observed that were dependent upon the combination of the TuMV isolate and the Arabidopsis ecotype. The Col-0 ecotype developed mosaic symptoms after infection with the TuMV isolate Azu while the Ler ecotype developed veinal necrosis after infection with the same TuMV isolate. The Ler phenotype is controlled by a single dominant gene TuNI (TuMV necrosis inducer) which is located on chromosome 1. The TuNI gene was precisely mapped to the ~105 kb interval between the two markers of mXF41 and mRF28 by using several types of DNA polymorphism markers. Within this region, which included largely duplicated sequences, a total of 19 putative genes were predicted and 15 of these were classified into five gene families. The genes belonging to the gene families At1g58480 and At1g58602 may function in response to infection by pathogens. The gene family At1g58480 encodes lipase-like proteins, which might be involved in the induction of defence responses that are mediated by salicylic acid. The gene family At1g58602 encodes the CC-NBS-LRR (CNL) proteins, which are known to function as one of the plant resistance (R) proteins against pathogens. In the present study, the possibility that TuNI might function as an R gene was discussed.Y.K. and T.I. contributed equally to this study     

migS, a cis-acting site that affects bipolar positioning of oriC on the Escherichia coli chromosome

During replication of the Escherichia coli chromosome, the replicated Ori domains migrate towards opposite cell poles, suggesting that a cis-acting site for bipolar migration is located in this region. To identify this cis-acting site, a series of mutants was constructed by splitting subchromosomes from the original chromosome. One mutant, containing a 720 kb subchromosome, was found to be defective in the bipolar positioning of oriC. The creation of deletion mutants allowed the identification of migS, a 25 bp sequence, as the cis-acting site for the bipolar positioning of oriC. When migS was located at the replication terminus, the chromosomal segment showed bipolar positioning. migS was able to rescue bipolar migration of plasmid DNA containing a mutation in the SopABC partitioning system. Interestingly, multiple copies of the migS sequence on a plasmid in trans inhibited the bipolar positioning of oriC. Taken together, these findings indicate that migS plays a crucial role in the bipolar positioning of oriC. In addition, real-time analysis of the dynamic morphological changes of nucleoids in wild-type and migS mutants suggests that bipolar positioning of the replicated oriC contributes to nucleoid organization.     

Development and neural organization of the tornaria larva of the Hawaiian hemichordate, Ptychodera flava

We report scanning and transmission electron microscopic studies of the early development of the Hawaiian acorn worm, Ptychodera flava. In addition, we provide an immunohistochemical identification of the larval nervous system. Development occurs and is constrained within the stout chorion and fertilization envelope that forms upon the release of the cortical granules in the cytoplasm of the egg. The blastula consists of tall columnar blastomeres encircling a small blastocoel. Typical gastrulation occurs and a definitive tornaria is formed compressed within the fertilization envelope. The young tornaria hatches at 44 hr and begins to expand. The major circumoral ciliary band that crosses the dorsal surface and passes preorally and postorally is well developed. In addition, we find a nascent telotroch, as well as a midventral ciliary band that is already clearly developed. The epithelium of tornaria is a mosaic of monociliated and multiciliated cells. Immunohistochemistry with a novel neural marker, monoclonal antibody 1E11, first detects nerve cells at the gastrula stage. In tornaria, 1E11 staining nerve cells occur throughout the length of the ciliary bands, in the apical organ, in a circle around the mouth, in the esophageal epithelium and in circumpylorus regions. Axon(s) and apical processes extend from the nerve cell bodies and run in tracks along the ciliary bands. Axons extending from the preoral and postoral bands extend into the oral field and form a network. The tornaria nervous system with ciliary bands and an apical organ is rather similar to the echinoderm bipinnaria larvae.     

Crystal structure of aspartic proteinase from Irpex lacteus in complex with inhibitor pepstatin

The crystal structure of Irpex lacteus aspartic proteinase (ILAP) in complex with pepstatin (a six amino acid residue peptide-like inhibitor) was determined at 1.3A resolution. ILAP is a pepsin-like enzyme, widely distributed in nature, with high milk-clotting activity relative to proteolytic activity. The overall structure was in good topological agreement with pepsin and other aspartic proteases. The structure and interaction pattern around the catalytic site were conserved, in agreement with the other aspartic proteinase/inhibitor complex structures reported previously. The high-resolution data also supported the transition state model, as proposed previously for the catalytic mechanism of aspartic proteinase. Unlike the other aspartic proteinases, ILAP was found to require hydrophobic residues either in the P(1) or P(1') site, and also in the P(4) and/or P(3) site(s) for secondary interactions. The inhibitor complex structure also revealed the substrate binding mechanism of ILAP at the P(3) and P(4) site of the substrate, where the inserted loop built up the unique hydrophobic pocket at the P(4) site.     

Effect of interaction between hepatitis C virus NS5A and NS5B on hepatitis C virus RNA replication with the hepatitis C virus replicon

Hepatitis C virus (HCV) NS5A has been reported to be important for the establishment of replication by adaptive mutations or localization, although its role in viral replication remains unclear. It was previously reported that NS5A interacts with NS5B via two regions of NS5A in the isolate JK-1 and modulates the activity of NS5B RdRp (Y. Shirota et al., J. Biol. Chem., 277:11149-11155, 2002), but the biological significance of this interaction has not been determined. In this study, we addressed the effect of this interaction on HCV RNA replication with an HCV replicon system derived from the isolate M1LE (H. Kishine et al., Biochem. Biophys. Res. Commun., 293:993-999, 2002). We constructed three internal deletion mutants, M1LE/5Adel-1 and M1LE/5Adel-2, each encoding NS5A which cannot bind NS5B, and M1LE/5Adel-3, encoding NS5A that can bind NS5B. After transfection into Huh-7 cells, M1LE/5Adel-3 was replication competent, but both M1LE/5Adel-1 and M1LE/5Adel-2 were not. Next we prepared 20 alanine-substituted clustered mutants within both NS5B-binding regions and examined the effect of these mutants on HCV RNA replication. Only 5 of the 20 mutants were replication competent. Subsequently, we introduced a point mutation, S225P, a deletion of S229, or S232I into NS5A and prepared cured Huh-7 cells that were cured of RNA replication by alpha interferon. Finally, with these point mutations and cured cells, we established a highly improved replicon system. In this system, only the same five mutants were replication competent. These results strongly suggest that the interaction between NS5A and NS5B is critical for HCV RNA replication in the HCV replicon system.     

Effect of multiple-load training on the force-velocity relationship

The effect of training with a combination of different loads (multiple-load training) on the force-velocity and force-power relationships was examined with training programs that included maximal isometric contraction (Fmax) and concentric contraction of the elbow flexor muscles. Twenty-one male college students were placed into 3 equal training groups (G(30 + 60), G(30 + 100), and G(30 + 60 + 100)) and performed multiple-load training 3 days per week for 8 weeks. The training load was a set fraction of the maximal isometric strength (% Fmax). The G(30 + 60) group performed 6 repetitions of elbow flexion at 30 and 60% Fmax. The G(30 + 100) group performed 6 repetitions at 30% Fmax and six 5-second Fmax loads. The G(30 + 60 + 100) group performed 4 repetitions at 30 and 60% Fmax and four 5-second Fmax loads. After training, Fmax and maximal velocity significantly increased (p < 0.05) in all 3 training groups. The increases in maximal power were significantly (p < 0.05) different between the G(30 + 60 + 100) group (52.9%) and the G(30 + 100) group (24.2%). These results suggest that multiple-load training programs with 4-6 repetitions are effective for improving muscle power and velocity of the elbow flexors.     

Molecular Characterization of Mn-superoxide Dismutase and Gene Expression Studies in Dietary Restricted <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> Rotifers

Superoxide dismutases (SODs) promote a conversion of harmful reactive oxygen species (ROS) to relatively moderate forms, resulting in the extension of lifespan in the nematode Caenorhabditis elegans under caloric restriction. The lifespan of the rotifer Brachionus plicatilis is also markedly extended by caloric restriction. We, therefore, cloned cDNA encoding SOD activated with Mn (Mn SOD) from B. plicatilis and examined its expression pattern in rotifers raised with energy restricted diet. The full length deduced amino acid sequence of the rotifer Mn SOD showed 61% identity with the C. elegans ortholog. Four amino acid residues that are essential to the binding of this enzyme to Mn were conserved in the rotifer Mn SOD. Subsequently we examined the mRNA expression patterns of Mn SOD using highly sensitive quantitative real-time PCR for various rotifer populations that are likely to differ in their lifespans in experiments on calorie restricted diets. The accumulated mRNA levels of Mn SOD were found to increase in supposedly long-lived rotifers. These results suggest that Mn SOD is possibly related to the aging of B. plicatilis.