Structure of cDNA clones for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from a fern (Asplenium cataractarum rosenstock,aspleniaceae), and its comparison with those of various seed plants

We isolated the small subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO SSu) from a fern,Asplenium cataractarum and determined its 34 N-terminal amino acid sequence. We obtained a cDNA clone that contains the entire coding region of the SSu from the same fern species, using synthetic oligonucleotide probes derived from the above amino acid sequence. It contains a 525 bp open reading frame capable of coding for a polypeptide with 174 amino acids, 31 bp 5′-and 206 bp 3′-noncoding regions. It was also elucidated that the precursor to the SSu contains a transit peptide of 53 amino acid residues and a mature protein of 121 residues. We compared the deduced amino acid sequence of the fern SSu with those of 11 other vascular plant species (including gymnosperms, monocots and dicots). As low as 55% homology was observed between those of a fern and seed plants. Constancy of the amino acid substitution rate in RuBisCO SSu was supported by our relative rate test. Amino acid substitution rate per year per site for RuBisCO SSu was calculated to be 0.81×10⁻⁹ assuming that the separation between pteridophytes and seed plants arose 380 million years ago.     

Conservation of fine-scale DNA marker order in the genomes of rice and the Triticeae.

DNA markers distribute over large chromosomal regions exhibit conservation of order (collinearity) in different cereal species, but it is not known whether this is maintained on a finer scale, i.e. < or = 2 cM. To address this, sets of two or more genetically linked DNA markers were localised to yeast artificial chromosomes containing rice DNA inserts. Linkage analysis of these DNA markers in barley revealed complete correspondence with their genetic order in rice, the distance between linked sequences on rice chromosomes being < 1.6 cM or < or = 1 + 10(6) bp (1 Mb). Thus, DNA markers separated in this range are collinear in rice, barley and, by inference, other members of the Triticeae. These results are discussed with respect to the use of rice as a key system for the isolation of cereal genes.     

Inferences of evolutionary history of a widely distributed mangrove species,Bruguiera gymnorrhiza,in the Indo‐West Pacific region

Inference of genetic structure and demographic history is fundamental issue in evolutionary biology. We examined the levels and patterns of genetic variation of a widespread mangrove species in the Indo‐West Pacific region, Bruguiera gymnorrhiza, using ten nuclear gene regions. Genetic variation of individual populations covering its distribution range was low, but as the entire species it was comparable to other plant species. Genetic differentiation among the investigated populations was high. They could be divided into two genetic clusters: the West and East clusters of the Malay Peninsula. Our results indicated that these two genetic clusters derived from their ancestral population whose effective size of which was much larger compared to the two extant clusters. The point estimate of speciation time between B. gymnorrhiza and Bruguiera sexangula was two times older than that of divergence time between the two clusters. Migration from the West cluster to the East cluster was much higher than the opposite direction but both estimated migration rates were low. The past Sundaland and/or the present Malay Peninsula are likely to prevent gene flow between the West and East clusters and function as a geographical or land barrier.     

Genome-wide searching of single-nucleotide polymorphisms among eight distantly and closely related rice cultivars (Oryza sativa L.) and a wild accession (Oryza rufipogon Griff.).

We searched the genomes of eight rice cultivars (Oryza sativa L. ssp. japonica and ssp. indica) and a wild rice accession (Oryza rufipogon Griffith) for nucleotide polymorphisms, and identified 7805 polymorphic loci, including single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels), in predicted intergenic regions. Polymorphisms are useful as DNA markers for genetic analysis or positional cloning with segregating populations of crosses. Pairwise comparison between cultivars and a neighbor-joining tree calculated from SNPs agreed very well with relationships between rice strains predicted from pedigree data or calculated with other DNA markers such as p-SINE1 and simple sequence repeats (SSRs), suggesting that whole-genome SNP information can be used for analysis of evolutionary relationships. Using multiple SNPs to identify alleles, we drew a map to illustrate the alleles shared among the eight cultivars and the accession. The map revealed that most of the genome is mono- or di-allelic among japonica cultivars, whereas alleles well conserved among modern japonica paddy rice cultivars were often shared with indica cultivars or wild rice, suggesting that the genome structure of modern cultivars is composed of chromosomal segments from various genetic backgrounds. Use of allele-sharing analysis and association analysis were also tested and are discussed.     

A region of calpastatin domain L that reprimes cardiac L-type Ca2+ channels

Calpastatin, an endogenous inhibitor of calpain, is composed of domain L and four repetitive homologous domains 1-4. Domains 1-4 inhibit calpain, whereas domain L partially reprimes L-type Ca2+ channels for voltage-gated activation. In the present study, the effects on Ca2+ channel activity of four isoforms and a series of fragments of calpastatin domain L were investigated in guinea-pig ventricular myocytes with the patch-clamp method. With one exception, all the isoforms and fragment peptides that contained amino acid residues 54-64 of domain L reprimed the Ca2+ channels to comparable levels (9-15% of control activity) to those observed previously with a full-length form of calpastatin. These results suggest that the region containing amino acid residues 54-64 (EGKPKEHTEPK) is responsible for the Ca2+ channel repriming function of calpastatin domain L.     

Transformation of pBR322-Derived Plasmids in Phytopathogenic Pseudomonas avenae and Enhanced Transformation in Its Proline-Auxotrophic Mutant

Efficient transformation of pBR322 and its derived plasmids, which have been widely used as cloning vectors in Escherichia coli, was observed in Pseudomonas avenae (K1), the pathogen of leaf blight disease in cereals. Moreover, there was a 10- to 50-fold transformation efficiency (1.3–3.0 × 10⁶/μg DNA) in the proline-auxotrophic mutant (Pr47), whose virulence to rice seedlings decreased. Similar enhancement of the frequency of transfer by mobilization of RSF1010, a broad host range plasmid, was observed in the recipient Pr47 strain in mating with donor Pseudomonas syringae. The plasmids harbored in these strains were maintained very stably after subcultures. Thus, a highly efficient transformation system with pBR322-derived plasmids used as a vector and Pseudomonas as a host bacterium was developed. Received: 13 July 1996 / Accepted: 26 August 1996     

Further characterization of the PW peptide family that inhibits neuron differentiation in <Emphasis Type="Italic">Hydra</Emphasis>

From an evolutionary point of view, Hydra has one of the most primitive nervous systems among metazoans. Two different groups of peptides that affect neuron differentiation were identified in a systematic screening of peptide signaling molecules in Hydra. Within the first group of peptides, a neuropeptide, Hym-355, was previously shown to positively regulate neuron differentiation. The second group of peptides encompasses the PW family of peptides that negatively regulate neuron differentiation. In this study, we identified the gene encoding PW peptide preprohormone. Moreover, we made the antibody that specifically recognizes LPW. In situ hybridization and immunohistochemical analyses showed that the PW peptides and the gene encoding them were expressed in ectodermal epithelial cells throughout the body except for the basal disk. The PW peptides are produced by epithelial cells and are therefore termed “epitheliopeptides.” Together with Hym-355, the PW family peptides mediate communication between neurons and epithelial cells and thereby maintain a specific density of neurons in Hydra. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Toshio Takahashi, Osamu Koizumi equally contributed to this study.     

Calpastatin domain L is a partial agonist of the calmodulin-binding site for channel activation in Cav1.2 Ca2+ channels

Cav1.2 Ca(2+) channel activity diminishes in inside-out patches (run-down). Previously, we have found that with ATP, calpastatin domain L (CSL) and calmodulin (CaM) recover channel activity from the run-down in guinea pig cardiac myocytes. Because the potency of the CSL repriming effect was smaller than that of CaM, we hypothesized that CSL might act as a partial agonist of CaM in the channel-repriming effect. To examine this hypothesis, we investigated the effect of the competitions between CSL and CaM on channel activity and on binding in the channel. We found that CSL suppressed the channel-activating effect of CaM in a reversible and concentration-dependent manner. The channel-inactivating effect of CaM seen at high concentrations of CaM, however, did not seem to be affected by CSL. In the GST pull-down assay, CSL suppressed binding of CaM to GST fusion peptides derived from C-terminal regions in a competitive manner. The inhibition of CaM binding by CSL was observed with the IQ peptide but not the PreIQ peptide, which is the CaM-binding domain in the C terminus. The results are consistent with the hypothesis that CSL competes with CaM as a partial agonist for the site in the IQ domain in the C-terminal region of the Cav1.2 channel, which may be involved in activation of the channel.     

Specific removal of endotoxin from protein solutions by immobilized histidine

A method for reducing endotoxin contamination in various solutions by immobilized histidine is described. Immobilized histidine is a porous adsorbent suitable for the adsorption of endotoxin with a high affinity over a wide range of pH and temperature and at low ionic strength (gamma/2 less than or equal to 0.1). When a purified endotoxin originating from Escherichia coli UKT-B was studied, the apparent dissociation constant between endotoxin and the adsorbent was 7.3 X 10(-13) M. The adsorbent was able to remove various kinds of endotoxin originating from gram-negative bacteria; the concentration of endotoxin was reduced from 1000 to less than 0.01 ng/ml in water. It is shown that the adsorbent specifically adsorbs endotoxin provided that the adsorption conditions are properly selected. Some examples of the specific removal of endotoxin from high-molecular-weight physiologically active substances such as tumor necrosis factor and lysozyme are shown.