Hirohiko Hirochika

Silencing of Retrotransposons in Arabidopsis and Reactivation by the ddm1 Mutation

Gene silencing associated with repeated DNA sequences has been reported for many eukaryotes, including plants. However, its biological significance remains to be determined. One important function that has been proposed is the suppression of transposons. Here, we address transposon suppression by examining the behavior of the tobacco retrotransposon Tto1 and endogenous retrotransposons in Arabidopsis. After an initial increase in copy number because of active transposition in the Arabidopsis genome, Tto1 became silent. The amount of transcript was reduced, and the inactivated Tto1 became methylated. This silencing correlated with an increase in copy number. These phenomena mimic repeat-induced gene silencing. The homozygous ddm1 (for decrease in DNA methylation) mutation of Arabidopsis results in genomic DNA hypomethylation and the release of silencing in repeated genes. To investigate the role of DNA methylation and the gene-silencing machinery in the suppression of Tto1, we introduced the ddm1 mutation into an Arabidopsis line carrying inactivated Tto1 copies. In the homozygous ddm1 background, Tto1 became hypomethylated and transcriptionally and transpositionally active. In addition, one of the newly isolated endogenous Arabidopsis retrotransposon families, named Tar17, also became hypomethylated and transcriptionally active in the ddm1 mutant background. Our results suggest that the inactivation of retrotransposons and the silencing of repeated genes have mechanisms in common.

RiceXPro: a platform for monitoring gene expression in japonica rice grown under natural field conditions.

Elucidating the function of all predicted genes in rice remains as the ultimate goal in cereal genomics in order to ensure the development of improved varieties that will sustain an expanding world population. We constructed a gene expression database (RiceXPro, URL: http://ricexpro.dna.affrc.go.jp/) to provide an overview of the transcriptional changes throughout the growth of the rice plant in the field. RiceXPro contains two data sets corresponding to spatiotemporal gene expression profiles of various organs and tissues, and continuous gene expression profiles of leaf from transplanting to harvesting. A user-friendly web interface enables the extraction of specific gene expression profiles by keyword and chromosome search, and basic data analysis, thereby providing useful information as to the organ/tissue and developmental stage specificity of expression of a particular gene. Analysis tools such as t-test, calculation of fold change and degree of correlation facilitate the comparison of expression profiles between two random samples and the prediction of function of uncharacterized genes. As a repository of expression data encompassing growth in the field, this database can provide baseline information of genes that underlie various agronomically important traits in rice.

Autonomous transposition of the tobacco retrotransposon Tto1 in rice.

The complete nucleotide sequence of the tobacco retrotransposon Tto1, one of the few active retrotransposons of plants, was determined. The sequence analysis suggests that Tto1 carries all functions required for autonomous transposition through reverse transcription. Gene organization and the nature of the transcription product suggest that Tto1 uses a gene expression mechanism different from those employed by retroviruses and most retrotransposons to regulate Gag and Pol stoichiometry. Tto1 was introduced into rice to study its autonomous transposition in heterologous hosts. Transcription and transposition of Tto1 were observed in rice cells. To probe the autonomous transposition through reverse transcription, a modified Tto1 retrotransposon in which part of a reverse transcriptase gene was replaced with an intron-containing hygromycin resistance gene was constructed and introduced into rice cells. Loss of the intron was observed only when intact Tto1 was cotransfected. These results indicate that Tto1 can transpose autonomously through reverse transcription and that the host factors required for transposition are conserved among monocots (class Magnoliopsida; rice) and dicots (class Liliopsida; tobacco), which diverged approximately 200 million years ago. These findings are discussed in relation to the regulation and evolution of retrotransposons and the possible use of Tto1 as a molecular genetic tool.

Analysis of linear plasmids isolated from Streptomyces: Association of protein with the ends of the plasmid DNA

Abstract Linear DNA plasmids, designated pSLA1 and pSLA2, which were isolated from two strains of Streptomyces sp. producing lankacidin group antibiotics, were analyzed by using restriction endonucleases. Cleavage patterns of these plasmids were very similar, indicating that they are closely related. Cleavage maps of pSLA2 were constructed with BamHI, SalI, BglII, and EcoRI. A protein was associated with the restriction fragments of both ends of pSLA2 and this was not removed by sodium dodecyl sulfate-phenol treatment. pSLA2 was senstive to a 3′-exonuclease, exonuclease III, but was resistant to the 5′-exonuclease, λ-exonuclease. These results suggest that the protein is associated with the 5′ termini of pSLA2. The same terminal structure was also found on pSLA1. The approximate copy number of the plasmid was estimated by a brief method using agarose gel electrophoresis.

Cloning and expression of the hydrogenase gene from Clostridium butyricum in Escherichia coli

Hydrogenase gene from Clostridium butyricum was cloned in Escherichia coli HK16 (Hyd−) using pBR322 and PstI. The plasmid, pCBH1, containing hydrogenase gene was 7.3 MDa and pCBH1 had 5 PstI‐DNA fragments (3.9, 2.6, 0.7, 0.03–0.04, <0.02 MDa, respectively). The hydrogenase activity of HK16 (pCBH1) was about 3.1–3.5‐times as high as those of the present strains, such as C. butyricum and E. coli C600 (Hyd+).

Isolation and partial characterization of large plasmids in hydrogen-evolving bacterium Clostridium butyricum

SummaryThe hydrogen-evolving bacterium Clostridium butyricum IFO 3847 was found to carry three plasmids: pSSK1, pSSK2, and pSSK3. The molecular weights of pSSK1, 2 and 3, determined by agarose gel electrophoresis and electron microscopy, were about 51, 32, and 9.4 megadaltons respectively.The two larger plasmids were analysed by digestion with several restriction enzymes such as AvaII, BamHl, EcoRl, Pst1, and Sall. With each enzyme, 10–20 fragments were produced from pSSK1 and five to six fragments from pSSK2. Since in each digestion some fragments were common to both plasmids, the two plasmids pSSK1 and pSSK2 must be related to each other to a high degree.The other small plasmid pSSK3 was digested by restriction endonucleases, EcoR1, Pst1, and Sall at single sites, so that this plasmid might be a candidate as a vector for gene cloning in Clostridium species.

Linear Plasmids with Terminal Inverted Repeats Obtained from Streptomyces Rochei and Kluyveromyces Lactis

Linear plasmids with inverted terminal repeats of 614 bp were obtained from Streptomyces rochei which produced lankacidin. The 5 ends were blocked by the association of a terminal protein. A DNA model of racket frame-like structure is presented implying the juxtaposition of 2 double-stranded DNAs of the same sequence through the binding of cohesive proteins which recognize and bind to the DNA. Two linear plasmids with the inverted terminal repeats of 202 and 184 bp were obtained from a yeast, Kluyveromyces lactis. A killer toxin was produced from the shorter plasmid. The protein toxin inhibited the adenylatecyclase activity of the yeast membrane.

Identification of Photosynthesis-Related Genes in Rice Using FOX Hunting System

Photosynthesis is one of the most important determinants of crop productivity. Although many studies have been conducted to improve yield, very little progress has been made. In this study, we used rice FOX (Full length cDNA over-expressing) lines that rice full-length cDNAs were over-expressed in Arabidopsis to identify novel photosynthesis-related genes of rice. We have used imaging of chlorophyll fluorescence to screen candidates. To confirm over-expression of transformed cDNAs cause the observed phenotype, we generated transformants overexpressing each cDNA, resulting that eight lines showed the same phenotype as original lines. Isolated candidates could be classified into four categories depending on chlorophyll fluorescence parameters. We analyzed gene expression profile of three candidate lines showing similar photosynthetic characteristics using DNA microarray. We found commonly regulated genes among three candidates, indicating that candidates have both shared and independent influences on gene expression.