Direct cloning of full-length mouse mitochondrial DNA using a Bacillus subtilis genome vector

The complete mouse mitochondrial genome (16.3 kb) was directly cloned into a Bacillus subtilis genome (BGM) vector. Two DNA segments of 2.06 and 2.14 kb that flank the internal 12 kb of the mitochondrial DNA (mtDNA) were subcloned into an Escherichia coli plasmid. Subsequent integration of the plasmid at the cloning locus of the BGM vector yielded a derivative specific for the targeted cloning of the internal 12-kb mtDNA region. The BGM vector took up mtDNA purified from mouse liver and integrated it by homologous recombination at the two preinstalled mtDNA-flanking sequences. The complete cloned mtDNA in the BGM vector was converted to a covalently closed circular (ccc) plasmid form via gene conversion in B. subtilis. The mtDNA carried on this plasmid was then isolated and transferred to E. coli. DNA sequence fidelity and stability through the BGM vector-mediated cloning process were confirmed.     

Molecular characterization of methicillin-resistant Staphylococcus aureus in hospitals in Niigata, Japan: divergence and transmission

The major methicillin-resistant Staphylococcus aureus(MRSA) distributed among hospitals in Japan is New York/Japan clone [multilocus sequence type 5 (ST5), agr type 2 and methicillin resistance locus type (SCC mec) II] which possesses both the toxic shock syndrome toxin 1 gene (tst) and staphylococcal enterotoxin C gene (sec). In this study, we collected 245 MRSA strains from four hospitals during 2001 to 2005 in Niigata, Japan, and analyzed tst and sec genes and SCC mec type among them. A total of 13 strains were further examined for their genotypes, virulence gene patterns and drug resistance. Among the 245 strains four tst sec genes patterns were observed; tst(+) sec(+) strains represented a majority of 86.5% and 9.4% were tst(-) sec(-). SCCmec typing revealed that 91.4% had type II, 4.1% type IV and 4.1% type I. Multilocus sequence typing (MLST) revealed that 10 of the 13 typed strains belonged to clonal complex 5 (7 had ST5 while 3 were single locus variants of ST5) with similar characteristics to the New York/Japan clone and possessed multi-drug resistance with high virulence gene content. The remaining 3 strains were ST8 (n=2) and ST91 (n=1). The ST91 strain had SCC mec IV and seemed to originate in the community, while ST8 strains exhibited SCC mec type I, which is distinct from community type IV. The data suggest that MRSA in hospitals in Niigata now mainly includes the New York/Japan clone (undergoing genomic divergence and clonal expansion) and other minor types (e.g. ST8) as well as the community type.     

Common reed produces starch granules at the shoot base in response to salt stress

Common reed (Phragmites australis) is a well known salt-tolerant plant and it is suggested that reeds recover Na(+) in the xylem sap of the shoot base (basal part of the shoot), store it temporarily in the shoot base, release it into the phloem sap, and then retranslocate it to the roots. To investigate whether Na(+) is retained in the shoot base of reeds, confocal laser scanning microscope (CLSM) observations were conducted using an intracellular Na(+)-specific fluorescent probe. The CLSM observations revealed that reeds produced a large number of the starch granules at the shoot base when salt-stressed, and that the fluorescence indicating the location of intracellular free Na(+) was observed in the same position as the starch granules. The Na content of starch granules was considerably greater than that of the shoot base, whereas the potassium (K) contents of the granules was only slightly greater than that of the shoot base. Reeds produced Na(+)-binding starch granules in the parenchyma cells of the shoot base when salt-stressed; these starch granules may decrease intracellular free Na(+). It is proposed that the site-specific production of Na(+)-binding starch granules constitutes a novel salt tolerance mechanism.     

Relationship between Human Evolution and Neurally Mediated Syncope Disclosed by the Polymorphic Sites of the Adrenergic Receptor Gene α2B-AR

The objective of this study was to clarify the effects of disease on neurally mediated syncope (NMS) during an acute stress reaction. We analyzed the mechanism of the molecular interaction and the polymorphisms of the alpha-2 adrenoreceptor (α2B-AR) gene as the potential psychiatric cause of incentive stress. We focused on the following three genotypes of the repeat polymorphism site at Glu 301–303 in the α2B-AR gene: Glu12/12, Glu12/9, and Glu9/9. On the basis of our clinical research, NMS is likely to occur in people with the Glu12/9 heterotype. To verify this, we assessed this relationship with the interaction of Gi protein and adenylate cyclase by in silico analysis of the Glu12/9 heterotype. By measuring the difference in the dissociation time of the Gi-α subunit twice, we found that the Glu12/9 heterotype suppressed the action of adenylate cyclase longer than the Glu homotypes. As this difference in the Glu repeat number effect is thought to be one of the causes of NMS, we investigated the evolutionary significance of the Glu repeat number. Glu8 was originally repeated in simians, while the Glu12 repeats occurred over time during the evolution of bipedalism in humans. Taken with the Glu12 numbers, NMS would likely become a defensive measure to prevent significant blood flow to the human brain.