A FRET-based analysis of SNPs without fluorescent probes

Fluorescence resonance energy transfer (FRET) is a simple procedure for detecting specific DNA sequences, and is therefore used in many fields. However, the cost is relatively high, because FRET-based methods usually require fluorescent probes. We have designed a cost-effective way of using FRET, and developed a novel approach for the genotyping of single nucleotide polymorphisms (SNPs) and allele frequency estimation. The key feature of this method is that it uses a DNA-binding fluorogenic molecule, SYBR Green I, as an energy donor for FRET. In this method, single base extension is performed with dideoxynucleotides labeled with an orange dye and a red dye in the presence of SYBR Green I. The dyes incorporated into the extended products accept energy from SYBR Green I and emit fluorescence. We have validated the method with ten SNPs, which were successfully discriminated by end-point measurements of orange and red fluorescence intensity in a microplate fluorescence reader. Using a mixture of homozygous samples, we also confirmed the potential of this method for estimation of allele frequency. Application of this strategy to large-scale studies will reduce the time and cost of genotyping a vast number of SNPs.     

Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery

Proper patterns of genome-wide DNA methylation, mediated by DNA methyltransferases DNMT1, -3A and -3B, are essential for embryonic development and genomic stability in mammalian cells. The de novo DNA methyltransferase DNMT3B is of particular interest because it is frequently overexpressed in tumor cells and is mutated in immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. In order to gain a better understanding of DNMT3B, in terms of the targeting of its methylation activity and its role in genome stability, we biochemically purified endogenous DNMT3B from HeLa cells. DNMT3B co-purifies and interacts, both in vivo and in vitro, with several components of the condensin complex (hCAP-C, hCAP-E and hCAP-G) and KIF4A. Condensin mediates genome-wide chromosome condensation at the onset of mitosis and is critical for proper segregation of sister chromatids. KIF4A is proposed to be a motor protein carrying DNA as cargo. DNMT3B also interacts with histone deacetylase 1 (HDAC1), the co-repressor SIN3A and the ATP-dependent chromatin remodeling enzyme hSNF2H. Further more, DNMT3B co-localizes with condensin and KIF4A on condensed chromosomes throughout mitosis. These studies therefore reveal the first direct link between the machineries regulating DNA methylation and mitotic chromosome condensation in mammalian cells.     

Phylogeography of the component species of broad-leaved evergreen forests in Japan,based on chloroplast DNA variation

In order to elucidate the past distribution and colonization routes of broad-leaved evergreen (lucidophyllous) forests, we investigated the intraspecific phylogeographic patterns of lucidophyllous forests in Japan and surrounding areas. We selected 6 component species with a similar geographic distributions growing in Castanopsis-dominant forests. We defined possible important refugia during the glacial periods as the regions rich in rare haplotypes (with a frequency of 5% or less), or as regions rich in the number of common haplotypes (with a frequency of more than 5%). We then located the sites of refuge by comparing the intraspecific phylogeographic patterns among 6 component species of lucidophyllous forests with respect to these two parameters (i.e., haplotype uniqueness and the number of haplotypes). The following results were obtained during the course of this study: (1) rare haplotypes were distributed among islands around the main islands of Japan; (2) rare subtypes and the greatest numbers of common haplotypes were observed in Kyushu, a finding which agreed with fossilized pollen data demonstrative of the past existence of refugia in southern Kyushu; and (3) rare haplotypes were found on the Muroto Peninsula, and the second greatest numbers of common haplotypes were observed on the Kii Peninsula, a finding which suggested the existence of additional important refugia along the Pacific coast of Japan during the glacial ages.     

Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos

Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.     

Crystallization and MAD data collection of high-molecular weight cytochrome c from Desulfovibrio vulgaris Miyazaki F

Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized. X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a=60.42, b=84.29 and c=144.16 A and contains one molecule per asymmetric unit.     

Human bronchial smooth muscle cell proliferation via thromboxane A2 receptor

Thromboxane A2 receptor (TP) mediates bronchial smooth muscle cell (BSMC) contraction, airway hyperresponsiveness, and airway inflammation in patients with asthma. In the present study, a pathogenic role of TP activation in airway remodeling was examined using primary cultures of human BSMC. A TP agonist, I-BOP, concentration-dependently enhanced not only bromodeoxyuridine (BrdU) uptake but also cell proliferation of BSMC. A TP-selective antagonist, AA-2414, blocked the effects of I-BOP on both BrdU uptake and cell proliferation. I-BOP-induced BrdU uptake was significantly blocked by two non-selective tyrosine kinase inhibitors, genistein and herbimycin A, or a Src family tyrosine kinase inhibitor, PP2, but not by an inhibitor of epidermal growth factor (EGF) receptor-associated tyrosine kinase, AG1478. In conclusion, TP receptor activation causes DNA synthesis and cell proliferation of human BSMC by activating tyrosine kinases including Src, but not by EGF receptor transactivation.     

Taurine inhibits apoptosis by preventing formation of the Apaf-1/caspase-9 apoptosome

Cardiomyocyte apoptosis contributes to cell death during myocardial infarction. One of the factors that regulate the degree of apoptosis during ischemia is the amino acid taurine. To study the mechanism underlying the beneficial effect of taurine, we examined the interaction between taurine and mitochondria-mediated apoptosis using a simulated ischemia model with cultured rat neonatal cardiomyocytes sealed in closed flasks. Exposure to medium containing 20 mM taurine reduced the degree of apoptosis following periods of ischemia varying from 24 to 72 h. In the untreated group, simulated ischemia for 24 h led to mitochondrial depolarization accompanied by cytochrome c release. The apoptotic cascade was also activated, as evidenced by the activation of caspase-9 and -3. Taurine treatment had no effect on mitochondrial membrane potential and cytochrome c release; however, it inhibited ischemia-induced cleavage of caspase-9 and -3. Taurine loading also suppressed the formation of the Apaf-1/caspase-9 apoptosome and the interaction of caspase-9 with Apaf-1. These findings demonstrate that taurine effectively prevents myocardial ischemia-induced apoptosis by inhibiting the assembly of the Apaf-1/caspase-9 apoptosome. ischemia; cultured cardiomyocytes     

Expression patterns of dachshund during head development of Gryllus bimaculatus (cricket)

We report that Gryllus bimaculatus dachshund (Gbdac), a cricket homologue of Drosophila dachshund (Dmdac), is expressed in the developing eye and brain. During brain development, Gbdac was first expressed in the medial head region, corresponding to a part of developing protocephalic region, and expressed in the primordial and adult Kenyon cells. During eye development, Gbdac was first expressed in the lateral head region, becoming to the eye primordium and a part of the deutocerebrum. Then, Gbdac was expressed in the posterior region of the eye primordium, prior to the formation of compound eyes. The expression domain shifted to the anterior domain concomitantly with the movement of morphogenetic furrows. Gbdac was also expressed in the developing optic lobes during differentiation of the retina. These expression patterns were compared with those of Dmdac. We found that although developmental processes of the Gryllus eye and brain differ from those of the Drosophila ones, the expression patterns of Gbdac are essentially similar to those of the Dmdac.     

Three-dimensional solution structure of an archaeal FKBP with a dual function of peptidyl prolyl cis-trans isomerase and chaperone-like activities

Here we report the solution structure of an archaeal FK506-binding protein (FKBP) from a thermophilic archaeum, Methanococcus thermolithotrophicus (MtFKBP17), which has peptidyl prolyl cis-trans isomerase (PPIase) and chaperone-like activities, to reveal the structural basis for the dual function. In addition to a typical PPIase domain, a newly identified domain is formed in the flap loop by a 48-residue insert that is required for the chaperone-like activity. The new domain, called IF domain (the Insert in the Flap), is a novel-folding motif and exposes a hydrophobic surface, which we consider to play an important role in the chaperone-like activity.