Biochemical screening of stable dinucleosomes using DNA fragments from a dinucleosome DNA library

The dinucleosome is an informative unit for analysis of the higher-order chromatin structure. DNA fragments forming stable dinucleosomes were screened from a dinucleosome DNA library after the reconstitution of nucleosomes in vitro and digestion with micrococcal nuclease. Reconstituted dinucleosomes showed a diversity of sensitivity to micrococcal nuclease, suggesting that the biochemical stability of a dinucleosome depends, in part, on the DNA fragments. The DNA fragments after the screening were classified into three groups represented by clones bf10, af14 and af32 according to the sensitivity to micrococcal nuclease. Mapping of the nucleosome boundaries by Southern blotting of the DNA after restriction digestion and by primer extension analysis showed that each nucleosome position of clone af32 was fixed. Analysis of reconstituted dinucleosomes using mutant DNA fragments of clone af32 revealed a unique property characteristic of a key nucleosome, given that the replacement of a DNA fragment corresponding to the right nucleosome position resulted in marked sensitivity to micrococcal nuclease, whereas the replacement of the other nucleosome fragment had almost no effect on sensitivity as compared to the original af32 construct. The mutant construct in which the right nucleosome was removed showed multiple nucleosome phases, suggesting that the right nucleosome stabilized first each mononucleosome and then the dinucleosome. An oligonucleotide bending assay revealed that the DNA fragment in the right nucleosome included curved DNA, suggesting that the positioning activity of the nucleosome was attributed to its DNA structure. These results suggest that information for forming stable dinucleosome is embedded in the genomic DNA and that a further characterization of the key nucleosome is useful for understanding the building up of the chromatin structure.     

Rice plant development: from zygote to spikelet

Rice is becoming a model plant in monocotyledons and a model cereal crop. For better understanding of the rice plant, it is essential to elucidate the developmental programs of the life cycle. To date, several attempts have been made in rice to categorize the developmental processes of some organs into substages. These studies are based exclusively on the morphological and anatomical viewpoints. Recent advancement in genetics and molecular biology has given us new aspects of developmental processes. In this review, we first describe the phasic development of the rice plant, and then describe in detail the developmental courses of major organs, leaf, root and spikelet, and specific organs/tissues. Also, for the facility of future studies, we propose a staging system for each organ.     

Update of mouse microsatellite database of Japan (MMDBJ).

We updated a database of microsatellite marker polymorphisms found in inbred strains of the mouse, most of which were derived from the wild stocks of four Mus musculus subspecies, M. m. domesticus, M. m. musculus, M. m.castaneus and M. m. molossinus. The major aim of constructing this database was to establish the genetic status of these inbred strains as resources for linkage analysis and positional cloning. The inbred strains incorporated in our database are A/J, C57BL/6J, CBA/J, DBA/2J, SM/J, SWR/J, 129Sv/J, MSM/Ms, JF1/Ms, CAST/Ei, NC/Nga, BLG2/Ms, NJL/Ms, PGN2/Ms, SK/CamEi and SWN/Ms, which have not or have only been poorly incorporated in the Whitehead Institute/MIT (WI/MIT) microsatellite database. The number of polymorphic microsatellite loci incorporated in our database is over 1,000 in all strains, and the URL site for our database is located at http:// www.shigen.nig.ac.jp /mouse/mmdbj/mouse.html.     

An efficient synthesis of methyl tetra-o-hexyl gentiooctaoside, an octaosyl analogue of ANP receptor antagonist HS-142-1

Methyl

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, a 3-O hexyl analogue of the octaosyl component of fungal lipooligosaccharide HS-142-1, was stereo- and regioselectively synthesized as a potent antagonist for the tetrameric atrial natriuretic peptide (ANP) receptors.     

Virulence-defective strains of Salmonella enterica serovar Typhi as candidates for education at level 2 facilities

The use of biosafety level 3 pathogens is an essential element of education and training at medical schools. We previously reported on invasion-defective strains of Salmonella enterica serovar Typhi, GTC 3P408 (DeltainvA, DeltasipB) and GTC 3P409 (DeltainvA, DeltasipB, and DeltaviaB), as candidates for use in educational programs. Vi negative strains of S. enterica serovar Typhi became extremely sensitive to complement attack but showed increased invasiveness. Therefore, this study was conducted to construct two virulencedefective strains, GTC 3P460 (DeltainvA, DeltasipB, and DeltarpoS) and GTC 3P461 (DeltainvA, DeltasipB, DeltaviaB, and DeltarpoS), of S. enterica serovar Typhi by deleting rpoS from the GTC 3P409 and GTC 3P408 strains. Stress tests demonstrated that GTC 3P460 and GTC 3P461 are sensitive to conditions of starvation, acid stress and oxidative stress. These results suggest that these virulence-defective strains have difficulty surviving in the gastric environment and in macrophages, characteristics that make them ideal candidates for education at level 2 facilities. Colony morphology and conventional biochemical features of these strains are identical to the parent strain S. enterica serovar Typhi GIFU 10007.     

A deletion in a cis element of Foxe3 causes cataracts and microphthalmia in rct mice

The Rinshoken cataract (rct) mutation, which causes congenital cataracts, is a recessive mutation found in SJL/J mice. All mutants present with opacity in the lens by 2?months of age. The rct locus was mapped to a 1.6-Mb region in Chr 4 that contains the Foxe3 gene. This gene is responsible for cataracts in humans and mice, and it plays a crucial role in the development of the lens. Furthermore, mutation of Foxe3 causes various ocular defects. We sequenced the genomic region of Foxe3, including the coding exons and UTRs; however, no mutations were discovered in these regions. Because there were no differences in Foxe3 sequences between the rct/rct and wild-type mice, we inferred that a mutation was located in the regulatory regions of the Foxe3 gene. To test this possibility, we sequenced a 5' noncoding region that is highly conserved among vertebrates and is predicted to be the major enhancer of Foxe3. This analysis revealed a deletion of 22-bp located approximately 3.2-kb upstream of the start codon of Foxe3 in rct mice. Moreover, we demonstrated by RT-PCR and in situ hybridization that the rct mutant has reduced expression of Foxe3 in the lens during development. We therefore suggest that cataracts in rct mice are caused by reduced Foxe3 expression in the lens and that this decreased expression is a result of a deletion in a cis-acting regulatory element.     

Low-intensity pulsed ultrasound (LIPUS) induces RANKL, MCP-1, and MIP-1beta expression in osteoblasts through the angiotensin II type 1 receptor

Constant mechanical stress is essential for the maintenance of bone mass and strength, which is achieved through the cooperative functions of osteoblasts and osteoclasts. However, it has not been fully elucidated how these cell types mediate mechanical signals. Low-intensity pulsed ultrasound (LIPUS) therapy is a recently developed method for application of mechanical stress, and is used clinically to promote bone fracture healing. In the present study, we applied LIPUS to osteoblasts at different stages of maturation and analyzed their chemokine and cytokine expression. In comparison with their immature counterparts, mature osteoblasts expressed significantly higher levels of mRNAs for the receptor activator of nuclear factor kappa B ligand (RANKL), monocyte chemoattractant protein (MCP)-1, and macrophage-inflammatory protein (MIP)-1beta after a few hours of LIPUS treatment. Intriguingly, protein and mRNA expression of angiotensin II type 1 receptor (AT1), a known mechanoreceptor in cardiomyocytes, was detected in osteoblasts, and the level of expression increased significantly during cell maturation. Furthermore, LIPUS-induced extracellular signal-regulated kinase (ERK) phosphorylation and RANKL/chemokine expression was abrogated by a specific AT1 inhibitor. Thus, AT1 may play one of the essential roles in bone metabolism as a mechanoreceptor of osteoblasts.     

Scolecenchelys fuscogularis (Anguilliformes: Ophichthidae, Myrophinae), a new worm eel from Japan

A new species of worm eel (Ophichthidae, subfamily Myrophinae), Scolecenchelys fuscogularis, is described from two specimens collected at 90–147 m depth off the coast of Japan. The new species is characterized by its dorsal-fin origin, which is located posterior to a vertical through the anus, its high total number of vertebrae (146–149), and its uniserial dentition on jaws and vomer. The new species is similar to Scolecenchelys australis and Scolecenchelys tasmaniensis in having 148–152 total and 60–61 preanal vertebrae and its uniserial teeth, but can be distinguished from the latter two species as it has a larger head [8.5–8.8 % of total length (TL) vs. 7.8–8.3 %], a longer trunk (39 % TL vs. 34–35 %), and a shorter tail (52–53 % TL vs. 56–58 %). Although S. fuscogularis most resembles Scolecenchelys chilensis in having 146–159 total and 59–64 preanal vertebrae and uniserial teeth, as well as in the proportions of the head, trunk and tail, the new species differs from the latter in having a smaller head (8.5–8.8 % TL vs. 8.9–9.7 %), a more slender body (body depth 1.5–1.6 % TL vs. 2.3–2.9 %), a more posterior dorsal-fin origin (horizontal distance between the origin and a vertical through the anus 83 % of head length vs. 36–54 %), no groove on the ventral side of its snout, and a dark lower jaw with a patch of melanophores on the ventral side of its branchial basket.