Phosphorus deficiency-induced root elongation and its QTL in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A significant level of root elongation was induced in rice (Oryza sativa) grown under phosphorus-deficient conditions. The root elongation clearly varied among a total of 62 varieties screened under two different phosphorus levels. Two contrasting varieties, Gimbozu, with a low elongating response and Kasalath, with a high elongating response, were chosen and crossed to produce a hybrid population for QTL analyses. QTLs for the phosphorus deficiency-induced root elongation were detected by two linkage maps, i.e., one with 82 F₃ families constructed by 97 simple sequence repeat (SSR) and sequence-tag site markers and another with 97 F₈ lines by 790 amplified fragment length polymorphism and SSR markers. A single QTL for the elongation response was detected on chromosome 6, with a LOD score of 4.5 in both maps and explained about 20% of total phenotypic variance. In addition, this QTL itself, or a region tightly linked with it, partly explained an ability to reduce accumulation of excess iron in the shoots. The identified QTL will be useful to improve rice varieties against a complex nutritional disorder caused by phosphorus deficiency and iron toxicity.Electronic Supplementary Material Supplementary material is available for this article at     

Identification of AFLP markers linked to a resistance gene against pine needle gall midge in Japanese black pine

Bulked segregant and AFLP analyses of two mapping populations (R17 × S6 and R17 × S1) were used to identify markers linked to Rpgm, the only known gene responsible for resistance to pine needle gall midge in Pinus thunbergii Parl. Rpgm was found to be bracketed by ACCC/CCTTT ₁₉₀ on one side at a distance of 6.6 cM and ACGT/CCCGC ₂₅₀ at 15.3 cM on the other side. The segregation of these markers was analyzed in two other families in order to determine their phase and transferability. One of the two additional resistant parents carried ACCC/CCTTT ₁₉₀ in the homozygous state while the marker was in coupling (plus marker allele linked with an R allele) in a resistant parent, R17. The marker ACGT/CCCGC ₂₅₀ was in a repulsion phase in R17 and was not detected in the other two resistant pine trees. Out of four AFLP markers identified, only ACGT/CCAAT ₂₉₀ was transferable in all resistant trees tested, although its phase was opposite for different trees. These results indicate that in applying those markers to select resistant trees, the phase state of the markers in each resistant tree with respect to Rpgm needs to be considered.Communicated by D.B. Neale     

BAC system: A novel method for manipulation of herpesvirus genomes based on bacterial genetics

Although methods for reverse genetics of herpesviruses have been established in early 1980s, the steps are laborious and time-consuming. In 1997, Dr. Koszinwski's group reported a novel approach for the construction of herpesvirus mutants, based on cloning the viral genome as a bacterial artificial chromosome (BAC) in E. coli. This technique allows the maintenance of viral genomes as plasmid in E. coli and the reconstitution of viral progeny by transfection of the BAC plasmid into eukaryotic cells. Any genetics modification of the viral genome in E. coli using bacterial genetics is possible, thereby facilitating the introduction of mutagenesis into herpesvirus genome. This 'BAC system' has opened new avenues for reverse and forward genetics of herpesviruses in basic research and in vector development for human therapy. Here we describe the principle of the 'BAC system' in herpesvirus researches.     

A quantitative, non-radioactive single-nucleotide insertion assay for analysis of DNA replication fidelity by using an automated DNA sequencer

A method to determine the steady-state kinetic parameters of single-nucleotide insertion in replication was developed using an automated DNA sequencer. The insertion of nucleoside 5'-triphosphates into a 6-carboxyfluorescein-labeled primer by DNA polymerase was quantified from the band pattern on a gel using GeneScan software. The parameters determined by this method were consistent with those obtained by the conventional radioisotope-labeling method. This non-radioactive, fluorescent-based method is rapid and can handle a large number of samples to assess cognate or non-cognate base pair formation between natural or unnatural bases in replication.     

Flower color modulations of Torenia hybrida by downregulation of chalcone synthase genes with RNA interference

Suppression of biosynthetic genes involved in flower color formation is an important approach for obtaining target flower colors. Here we report that flower color of the garden plant Torenia hybrida was successfully modulated by RNA interference (RNAi) against a gene of chalcone synthase (CHS), a key enzyme for anthocyanin and flavonoid biosynthesis. By using each of the coding region and the 3'-untranslated region of the CHS mRNA as an RNAi target, exhaustive and gene-specific gene silencing were successfully induced, and the original blue flower color was modulated to white and pale colors, respectively. Our results indicate that RNAi is quite useful for modulations of flower colors of commercially important garden plants.     

Genetic variations at urotensin II and urotensin II receptor genes and risk of type 2 diabetes mellitus in Japanese

Urotensin II is among the most potent vasoactive hormones known and the urotensin II (UTS2) gene is localized to 1p36-p32, one of the regions reported to show possible linkage with type 2 diabetes in Japanese. When we surveyed genetic polymorphisms in the UTS2 and urotensin II receptor (GPR14) gene, we identified two SNPs with amino acid substitutions (designated T21M and S89N and an SNP in the promotor region (-605G>A) of the UTS2 gene, and two SNPs in the non-coding region of the GPR14 gene. We then studied these three SNPs in the UTS2 gene and two SNPs in the GPR14 gene in 152 Japanese subjects with type 2 diabetes mellitus and two control Japanese populations. The allele frequency of 89N was significantly higher in type 2 diabetic patients than in both elderly normal subjects (P = 0.0018) and subjects with normal glucose tolerance (P = 0.0011), whereas the allele frequency of T21M and -605G>A in the UTS2 gene and those of two SNPs in the GPR14 gene were essentially identical in these three groups. Furthermore, in the subjects with normal glucose tolerance, 89N was associated with significantly higher insulin levels on oral glucose tolerance test, suggesting reduced insulin sensitivity in subjects with 89N. These results strongly suggest that subjects with S89N in the UTS2 gene are more insulin-resistant and thus more susceptible to type 2 diabetes mellitus development.     

An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's disease

Various types of stress, such as disruption of calcium homeostasis, inhibition of protein glycosylation and reduction of disulfide bonds, result in accumulation of misfolded proteins in the endoplasmic reticulum (ER). The initial cellular response involves removal of such proteins by the ER, but excessive and/or long-term stress results in apoptosis. In this study, we used a randomized ribozyme library and ER stress-mediated apoptosis (tunicamycin-induced apoptosis) in SK-N-SH human neuroblastoma cells as a selective phenotype to identify factors involved in this process. We identified a double-stranded RNA-dependent protein kinase (PKR) as one of the participants in this process. The level of nuclear PKR was elevated, but the level of cytoplasmic PKR barely changed in tunicamycin-treated SK-N-SH cells. Furthermore, tunicamycin also raised levels of phosphorylated PKR in the nucleus. We also detected the accumulation of phosphorylated PKR in the nuclei of autopsied brain tissues in Alzheimer's disease. Thus, PKR might play a role in ER stress-induced apoptosis and in Alzheimer's disease.     

Design, synthesis and structure-activity relationship studies of novel indazole analogues as DNA gyrase inhibitors with Gram-positive antibacterial activity

In this study, we report the design, synthesis and structure-activity relationships of novel indazole derivatives as DNA gyrase inhibitors with Gram-positive antibacterial activity. Our results show that selected compounds from this series exhibit potent antibacterial activity against Gram-positive bacteria including multi-drug resistant strains that is methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE).     

Novel biodegradable polyphosphate cross-linker for making biocompatible hydrogel

To obtain a novel biodegradable cross-linker, polymerizable polyphosphate (PIOP) was synthesized by ring-opening polymerization of 2-i-propyl-2-oxo-1,3,2-dioxaphospholane with 2-(2-oxo-1,3,2-dioxaphosphoroyloxyethyl methacrylate) (OPEMA). The number averaged molecular weight of the PIOP was 1.2 x 10(4), and the number of OPEMA units in one PIOP molecule was 2.2. Nonenzymatic degradation of the PIOP was evaluated in various pH aqueous media. The degree of hydrolysis was dependent on the pH; that is, it increased with an increase in the pH of the medium. At pH 11.0, the PIOP completely degraded in only 6 days. The poly[2-methacryloyloxyethyl phosphorylcholine (MPC)] cross-linked with the PIOP was prepared by radical polymerization. This polymer could form hydrogel, and the free water fraction in the hydrogel was high. The enzymatic activity of trypsin in contact with the hydrogel was similar to that in buffer solution. There is no adverse effect caused by the hydrogel to reduce the function of the trypsin. The cytotoxicity of poly(MPC) and degraded PIOP was evaluated using v79 cells, and it was not observed in either case. In conclusion, PIOP is a hydrolyzable polymer, which can be used as a cross-linker, and novel hydrogels having biodegradability and biocompatibility were prepared from poly(MPC) cross-linked with the PIOP.     

Requirement for Tec kinases in chemokine-induced migration and activation of Cdc42 and Rac

Cell polarization and migration in response to chemokines is essential for proper development of the immune system and activation of immune responses. Recent studies of chemokine signaling have revealed a critical role for PI3-Kinase, which is required for polarized membrane association of pleckstrin homology (PH) domain-containing proteins and activation of Rho family GTPases that are essential for cell polarization and actin reorganization. Additional data argue that tyrosine kinases are also important for chemokine-induced Rac activation. However, how and which kinases participate in these pathways remain unclear. We demonstrate here that the Tec kinases Itk and Rlk play an important role in chemokine signaling in T lymphocytes. Chemokine stimulation induced transient membrane association of Itk and phosphorylation of both Itk and Rlk, and purified T cells from Rlk(-/-)Itk(-/-) mice exhibited defective migration to multiple chemokines in vitro and decreased homing to lymph nodes upon transfer to wt mice. Expression of a dominant-negative Itk impaired SDF-1alpha-induced migration, cell polarization, and activation of Rac and Cdc42. Thus, Tec kinases are critical components of signaling pathways required for actin polarization downstream from both antigen and chemokine receptors in T cells.