A genome-wide gain-of-function analysis of rice genes using the FOX-hunting system

Funding Information: Acknowledgements This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Green Technology Project EF-1004). We are grateful to Dr. Takuji Sasaki for his encouragement throughout the project and his excellent advice on the improvement of this manuscript, and to Dr. Shoshi Kikuchi for providing useful information on rice FL-cDNAs. We thank Professors Kokichi Hinata, Atsushi Hirai, Hiroshi Kamada and Masashi Ugaki for their encouragement, critical comments and helpful suggestions, and Drs. Hisato Okuizumi and Hiroyuki Kawahigashi for their administrative support throughout the project. We also thank Mayumi Akagawa, Hiroko Abe, Keiko Mori, Etsuko Sugai, Yumiko Nakane, Ken-ichi Watanabe, Mayumi Takeya, and Kana Miyata for their technical assistance; the members of the Technical Support Section of the National Institute of Agrobiological Sciences for their help in the care of the FOX-rice plants; Haruko Onodera and Kazuko Ono for their technical assistance and advice on rice transformation; Inplanta Innovations Inc. for their technical help on the construction of theThe latest report has estimated the number of rice genes to be ∼32 000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13 980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12 000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene, confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.publishersversionPeer reviewe

Substance use and sexual behaviours of Japanese men who have sex with men: A nationwide internet survey conducted in Japan

BACKGROUND: Japanese men who have sex with men (MSM), especially those living in large metropolitan areas such as Tokyo and Osaka, are facing a growing HIV/AIDS epidemic. Although the Internet is used as a new venue for meeting sex partners, it can also serve as a useful research tool for investigating the risk behaviours of Japanese MSM. This Internet survey explored the extent of substance use and its association with sexual risk behaviours among Japanese MSM. METHODS: Between 28 February 2003 and 16 May 2003 MSM were recruited through 57 Japanese gay-oriented Web sites, gay magazines, and Internet mailing lists. Participants completed a structured questionnaire anonymously through the Internet. RESULTS: In total, 2,062 Japanese MSM completed the questionnaire. The average age of participants was 29.0 years and 70.5% identified as gay, 20.8% as bisexual, and 8.7% as other. Overall, 34.5% reported never using a substance, 45% reported ever using one type of substance (lifetime reported single substance users), and 19.6% had used more than 1 type of substance (lifetime reported multiple substance users) in their lifetimes. The substances most commonly used were amyl nitrite (63.2%), 5-methoxy-N, N-diisopropyltryptamine (5MEO-DIPT) (9.3%), and marijuana (5.7%). In the multivariate analysis, unprotected anal intercourse, having had 6 or more sexual partners, visiting a sex club/gay venue in the previous 6 months, a lower education level, and being 30 to 39 years of age were associated with both lifetime single and lifetime multiple substance use. Lifetime reported multiple substance use was also correlated with having a casual sex partner, having symptoms of depression, being diagnosed as HIV-positive, and greater HIV/AIDS-related knowledge. CONCLUSION: This is the first Internet-based research focused on the sexual and substance use behaviours of MSM in Asia. Our findings suggest a compelling need for prevention interventions to reduce HIV risk-related substance use behaviours among Japanese MSM. The results also suggest that the Internet is potentially a useful tool for collecting behavioural data and promoting prevention interventions among this population

Filament formation and robust strand exchange activities of the rice DMC1A and DMC1B proteins

The DMC1 protein, a meiosis-specific DNA recombinase, catalyzes strand exchange between homologous chromosomes. In rice, two Dmc1 genes, Dmc1A and Dmc1B, have been reported. Although the Oryza sativa DMC1A protein has been partially characterized, however the biochemical properties of the DMC1B protein have not been defined. In the present study, we expressed the Oryza sativa DMC1A and DMC1B proteins in bacteria and purified them. The purified DMC1A and DMC1B proteins formed helical filaments along single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and promoted robust strand exchange between ssDNA and dsDNA over five thousand base pairs in the presence of RPA, as a co-factor. The DMC1A and DMC1B proteins also promoted strand exchange in the absence of RPA with long DNA substrates containing several thousand base pairs. In contrast, the human DMC1 protein strictly required RPA to promote strand exchange with these long DNA substrates. The strand-exchange activity of the Oryza sativa DMC1A protein was much higher than that of the DMC1B protein. Consistently, the DNA-binding activity of the DMC1A protein was higher than that of the DMC1B protein. These biochemical differences between the DMC1A and DMC1B proteins may provide important insight into their functional differences during meiosis in rice

Inefficient double-strand DNA break repair is associated with increased fasciation in Arabidopsis BRCA2 mutants

BRCA2 is a breast tumour susceptibility factor with functions in maintaining genome stability through ensuring efficient double-strand DNA break (DSB) repair via homologous recombination. Although best known in vertebrates, fungi, and higher plants also possess BRCA2-like genes. To investigate the role of Arabidopsis BRCA2 genes in DNA repair in somatic cells, transposon insertion mutants of the AtBRCA2a and AtBRCA2b genes were identified and characterized. atbrca2a-1 and atbrca2b-1 mutant plants showed hypersensitivity to genotoxic stresses compared to wild-type plants. An atbrca2a-1/atbrca2b-1 double mutant showed an additive increase in sensitivity to genotoxic stresses compared to each single mutant. In addition, it was found that atbrca2 mutant plants displayed fasciation and abnormal phyllotaxy phenotypes with low incidence, and that the ratio of plants exhibiting these phenotypes is increased by γ-irradiation. Interestingly, these phenotypes were also induced by γ-irradiation in wild-type plants. Moreover, it was found that shoot apical meristems of the atbrca2a-1/atbrca2b-1 double mutant show altered cell cycle progression. These data suggest that inefficient DSB repair in the atbrca2a-1/atbrca2b-1 mutant leads to disorganization of the programmed cell cycle of apical meristems

The Current Status and Challenges for the Nursing Education with Information and Communication Technology

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Effects of Pyrolysis Temperature and Chemical Modification on the Adsorption of Cd and As(V) by Biochar Derived from Pteris vittata

Phytoremediation can be applied successfully to solve the serious worldwide issue of arsenic (As) and cadmium (Cd) pollution. However, the treatment of biomass containing toxic elements after remediation is a challenge. In this study, we investigated the effective use of biomass resources by converting the As hyperaccumulator P. vittata into biochar to adsorb toxic elements. Plant biomass containing As was calcined at 600, 800, and 1200 °C, and its surface structure and adsorption performances for As(V) and Cd were evaluated. Pyrolysis at 1200 °C increased the specific surface area of the biochar, but it did not significantly affect its adsorption capacity for toxic elements. The calcined biochar had very high adsorption capacities of 90% and 95% for As(V) and Cd, respectively, adsorbing 6000 mmol/g-biochar for As(V) and 4000 mmol/g-biochar for Cd. The As(V) adsorption rate was improved by FeCl3 treatment. However, the adsorption capacity for Cd was not significantly affected by the NaOH treatment. In conclusion, it was found that after phytoremediation using P. vittata biomass, it can be effectively used as an environmental purification material by conversion to biochar. Furthermore, chemical modification with FeCl3 improves the biochar’s adsorption performance