A novel gene encoding an enzyme that degrades a polysaccharide from the sheath of Sphaerotilus natans

A gene encoding an enzyme that is able to depolymerize the basic polysaccharide prepared from the sheath of Sphaerotilus natans was identified in a sheath-degrading bacterium, Paenibacillus koleovorans. The gene was constructed from 2217 bp coding for 738 amino acids, including the signal sequence of 34 amino acids. No closely related protein or gene was indicated by a homology search. The gene was expressed in Escherichia coli as a glutathione S-transferase fusion protein. The fusion protein depolymerized the sheath polysaccharide into an oligosaccharide, introducing an unsaturated sugar residue, suggesting that the gene codes for a polysaccharide lyase acting on a basic polysaccharide.     

Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs

We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.     

Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the Na+ transporter AtHKT1

Sodium (Na+) is toxic to most plants, but the molecular mechanisms of plant Na+ uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na+ transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na+ levels and are more salt resistant than wild-type in short-term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na+ levels, and athkt1 and athkt1/sos3 shoots are Na+-hypersensitive in long-term growth assays. Thus wild-type AtHKT1 controls root/shoot Na+ distribution and counteracts salt stress in leaves by reducing leaf Na+ accumulation.     

Gene Rearrangements and Evolution of tRNA Pseudogenes in the Mitochondrial Genome of the Parrotfish (Teleostei: Perciformes: Scaridae)

Genomic size of animal mitochondrial DNA is usually minimized over time. Thus, when regional duplications occur, they are followed by a rapid elimination of redundant material. In contrast to this general view, we report here long-sustained tRNA pseudogenes in the mitochondrial genome (mitogenome) of teleost fishes of the family Scaridae (parrotfishes). During the course of a molecular phylogenetic study of the suborder Labroidei, we determined the complete nucleotide sequence of the mitogenome for a parrotfish, Chlorurus sordidus, and found a gene rearrangement accompanied by a tRNA pseudogene. In the typical gene order of vertebrates, a tRNA-gene cluster between ND1 and ND2 genes includes tRNA^Ile (I), tRNA^Gln (Q), and tRNA^Met (M) genes in this order (IQM). However, in the mitogenome of the parrotfish, the tRNA^Met gene was inserted between the tRNA^Ile and the tRNA^Gln genes, and the tRNA^Gln gene was followed by a putative tRNA^Met pseudogene (_M). Such a tRNA gene rearrangement including a pseudogene (IMQ_M) was found in all of the 10 examined species, representing 7 of the 10 currently recognized scarid genera. All sister groups examined (20 species of Labridae and a single species of Odacidae) had the typical gene order of vertebrate mitogenomes. Phylogenetic analysis of the tRNA^Met genes and the resulting pseudogenes demonstrated that the ancestral tRNA^Met gene was duplicated in a common ancestor of the parrotfish. Based on the fossil record, these results indicate that the pseudogenes have survived at least 14 million years. Most of the vertebrate mitochondrial gene rearrangements involving the IQM region have held the tRNA^Met gene just upstream of the ND2 gene, and even in a few exceptional cases, including the present ones, the tRNA pseudogenes have been found in that position. In addition, most of these tRNA^Met pseudogenes maintained clover-leaf secondary structures, with the remainder sustaining the clover-leaf structure in the top half (TC and acceptor arms). Considering their potential secondary structures (holding top halves of the clover-leaf structures), locations within mitogenomes (flanking the 5 ends of the ND2 genes) and stabilities over time (survived at least 14 Myr), it is likely that the tRNA pseudogenes retain function as punctuation marks for mitochondrial ND2 mRNA processing.This article contains online supplementary material.Reviewing Editor: Dr. Axel Meyer     

Organization of the Mitochondrial Genome of Antarctic Krill <Emphasis Type="Italic">Euphausia superba</Emphasis> (Crustacea: Malacostraca)

We determined the nearly complete DNA sequence of the mitochondrial genome of Antarctic krill Euphausia superba (Crustacea: Malacostraca), one of the most ecologically and commercially important zooplankters in Antarctic waters. All of the genome sequences were purified by gene amplification using long polymerase chain reaction (PCR), and the products were subsequently used as templates for either direct sequencing using a primer-walking strategy or nested PCR with crustacea-versatile primers. Although we were unable to determine a portion of the genome owing to technical difficulties, the sequenced position, 14,606 bp long, contained all of the 13 protein-coding genes, 19 of the 22 transfer RNA genes, and the large subunit as well as a portion of the small subunit ribosomal RNA genes. Gene rearrangement was observed for 3 transfer RNA genes (tRNA^Cys, tRNA^Tyr, and tRNA^Trp) and the 2 leucine tRNA genes.     

Molecular phylogeny and possible scenario of ponyfish (Perciformes:Leiognathidae) evolution

The family Leiognathidae, commonly known as ponyfish or slip mouth, comprises three genera, each being characterized mainly by mouth morphology. To date, however, neither the phylogenetic relationships within the family nor monophyly of the genera has been tested. The phylogenetic relationships among 14 species of Leiognathidae, inferred from two protein coding mitochondrial genes (ND4 and 5), indicated monophyly of the studied species form genera Gazza and Secutor, and paraphyly of the genus Leiognathus, with L. equulus occupying a basal branch of the family. The relationships allowed phylogenetic analyses of mouthpart structures and light organ systems. The results suggested that the morphology of the upwardly and forwardly protractile mouth types (latter with canine-like teeth) are phylogenetically informative, and the downwardly protractile mouth type being ancestral in the family. The results also suggested that internal sexual dimorphism of the light organ system was present in the common ancestor of a sister clade to L. equulus, whereas external sexual dimorphism seems to have evolved subsequently in two monophyletic subgroups.     

Positions of disulfide bonds in rye (Secale cereale) seed chitinase-a

The positions of disulfide bonds of rye seed chitinase-a (RSC-a) were identified by the isolation of disulfide-containing peptides produced with enzymatic and/or chemical cleavages of RSC-a, followed by sequencing them. An unequivocal assignment of disulfide bonds in this enzyme was as follows: Cys3-Cysl8, Cys12-Cys24, Cys15-Cys42, Cys17-Cys31, and Cys35-Cys39 in the chitin-binding domain (CB domain), Cys82-Cys144, Cys156-Cys164, and Cys282-Cys295 in the catalytic domain (Cat domain), and Cys263 was a free form.     

The construction of an in vitro three-dimensional hematopoietic microenvironment for mouse bone marrow cells employing porous carriers

Spatial development of mouse bone marrow cellsemploying porous carriers was investigated in order todesign a bioreactor with a three-dimensionalhematopoietic microenvironment. Three types of porouscarriers were used for examining the spatialdevelopment of anchorage-dependent primary stromalcells as feeder cells. Stromal cells were found tospread well at a high density on a polyester nonwovendisc carrier (Fibra cel (FC)) under a scanningelectron microscope, while cells on porous cellulosebeads (Microcube (MC), 500 m pore diameter)spread at a low density; cells on another type ofcellulose porous beads (CPB, 100 m pore diameter)were globular. Mouse bone marrow cells wereinoculated to dishes containing three types of porouscarriers which shared more than 30% of the bottomsurface in a dish. The concentration of stromal cellsin the well containing FC was lower than that on theother two carriers. However, the weekly output oftotal hematopoietic cell (suspension cells) increasedbetween day 21 and 28 in the culture using FC while itdecreased monotonously in the cultures by use of theother two carriers. The proportion of progenitorcells (BFU-E, CFU-GM) in the total hematopoietic cellpopulation, after showing an initial decrease,increased after 1 week in the culture using FC whilethe proportion decreased monotonously to zero in thecultures using MC and CPB.