PubMed Health⌕ Search

Biomedical subjects

Junichi Obokata

Publications and source records attributed to Junichi Obokata.

14 recordsLinked to original sources
PubMed2006

Remote control of photosynthetic genes by the mitochondrial respiratory chain.

In this paper, we study whether mitochondrial respiration has an impact on the biogenesis of photosynthetic apparatus in the unicellular alga, Chlamydomonas reinhardtii. When respiration was activated by acetate in the dark, mRNAs of nuclear-encoded photosynthetic genes were induced. This induction did not occur in the cells treated with respiration inhibitors or in respiration mutants. An uncoupler of oxidative phosphorylation did not inhibit this mRNA induction; rather, it enhanced it in response to the increase in respiratory electron transport (RET). Plant and algal mitochondria have two RET pathways: the cytochrome pathway and the alternative pathway. Inhibitors of the former pathway inhibited mRNA induction, but inhibitors of the latter enhanced it. Taken together, these indicate that photosynthetic gene mRNAs are induced in response to activation of the cytochrome pathway. This RET-responsive induction is analogous to the photosynthetic electron transport (PET)-responsive induction of photosynthetic gene mRNAs (Matsuo and Obokata, Plant Cell Physiol. 43, 1189). PET-responsive induction occurred in photo-autotrophic and mixotrophic conditions, while RET-responsive induction occurred in mixotrophic and dark heterotrophic conditions. These results indicate that the regulatory system of photosynthetic genes changes between chloroplastic PET-dependent type and mitochondrial RET-dependent type in response to shifts in the dominant energy source between photosynthesis and respiration.

Animals↗
PubMed2006

In vitro selection of translational regulatory elements.

Untranslated regions (UTRs) of mRNAs carry various kinds of translational regulatory elements; however, our knowledge of them is still limited. We created an in vitro selection system that allows us to make a systematic enrichment of the sequences that alter translation efficiency (SESTRE) in any given mRNA and translation system. This method consists of the introduction of random nucleotide sequences into the UTRs of given mRNAs, followed by translation, size fractionation of the polyribosomes, and reverse transcription and PCR amplification (RT-PCR), with repeated cycles of these steps to enrich highly or poorly translatable mRNAs. With this experimental method, we examined how and where translational enhancer motifs emerge on mRNAs using the in vitro translation systems of wheat germ extract. The results indicate that the translational enhancers differentially emerge in response to the presence or absence of the 5' cap. Interestingly, the translational enhancers that activate cap-independent translation evolved more readily in the 3' UTR than in the 5' UTR in wheat germ extract. This SESTRE method should be a powerful tool with which to improve the translational efficiency of given mRNAs in given translation systems and to investigate the structure-function relationship of eukaryotic mRNAs underlying translational control.

3' Untranslated Regions↗
PubMed2005

The rice nuclear genome continuously integrates, shuffles, and eliminates the chloroplast genome to cause chloroplast-nuclear DNA flux.

Plastid DNA fragments are often found in the plant nuclear genome, and DNA transfer from plastids to the nucleus is ongoing. However, successful gene transfer is rare. What happens to compensate for this? To address this question, we analyzed nuclear-localized plastid DNA (nupDNA) fragments throughout the rice (Oryza sativa ssp japonica) genome, with respect to their age, size, structure, and integration sites on chromosomes. The divergence of nupDNA sequences from the sequence of the present plastid genome strongly suggests that plastid DNA has been transferred repeatedly to the nucleus in rice. Age distribution profiles of the nupDNA population, together with the size and structural characteristics of each fragment, revealed that once plastid DNAs are integrated into the nuclear genome, they are rapidly fragmented and vigorously shuffled, and surprisingly, 80% of them are eliminated from the nuclear genome within a million years. Large nupDNA fragments preferentially localize to the pericentromeric region of the chromosomes, where integration and elimination frequencies are markedly higher. These data indicate that the plant nuclear genome is in equilibrium between frequent integration and rapid elimination of the chloroplast genome and that the pericentromeric regions play a significant role in facilitating the chloroplast-nuclear DNA flux.

Active Transport, Cell Nucleus↗
PubMed2004

In vitro RNA editing systems from higher plant chloroplasts.

RNA editing in higher plant chloroplasts involves C-to-U conversion at ~30 specific sites. In vitro systems supporting accurate editing have been developed from tobacco and pea chloroplasts. mRNA substrates labeled with 32P at C residues to be edited provide sensitive detection of editing activity in vitro. The present systems allow the rapid identification of cis-elements using mutated mRNA substrates and trans-acting factors by ultraviolet crosslinking.

Base Sequence↗
PubMed2003

A site-specific factor interacts directly with its cognate RNA editing site in chloroplast transcripts.

RNA editing involves a variety of genetic systems and occurs by different mechanisms. In higher plant chloroplasts, specific sites of some transcripts are subject to C-to-U conversion. We have previously shown that site-specific trans-acting factors for psbE and petB mRNA editing bind corresponding cis-elements, which are located 5 nucleotides upstream from the editing site. Here we report that, by using mRNAs labeled either at the center of the upstream cis-element or at the editing site, the site-specific factors can be cross-linked with nucleotides at both positions. Mutations of nucleotides in the proximal region of the editing site revealed a correlation between editing activity and cross-linking efficiency of factors with the editing site, even though cross-linking with the upstream cis-element was unaffected. These observations suggest that the site-specific factor binds stably to the upstream ciselement, whereas it interacts weakly with the editing site. This finding raises the intriguing possibility that the site-specific factor is involved in both site-determination and C-to-U conversion in chloroplast RNA editing.

Base Sequence↗
PubMed2003

Identification of RNA editing sites in chloroplast transcripts from the maternal and paternal progenitors of tobacco (Nicotiana tabacum): comparative analysis shows the involvement of distinct trans-factors for ndhB editing.

RNA editing alters genomic nucleotide sequences at the transcript level. In higher plant chloroplasts, C-to-U conversion is known to occur at around 30 specific sites. The tobacco cultivar Nicotiana tabacum is an amphidiploid derived from ancestors of N. sylvestris (maternal) and N. tomentosiformis (paternal). The chloroplast genome of N. tabacum is believed to originate from an ancestor of N. sylvestris. To study the evolution of RNA editing in higher plant chloroplasts, editing sites in the two likely progenitors have first been identified based on those found in N. tabacum. Altogether 34, 33, and 32 editing sites have been found in the chloroplast transcripts from N. tabacum, N. sylvestris, and N. tomentosiformis, respectively. Thirty-one sites are conserved among the three species, whereas remarkable differences are observed in the editing of ndhB and ndhD transcripts. Sites 7 and 8 in ndhB mRNAs are separated only by five nt, and both are edited in N. tabacum and N. sylvestris. However, site 8 is not edited in N. tomentosiformis, indicating that distinct trans-factors are involved in the two editing events. The first site in ndhD mRNAs is edited to produce an AUG start codon in N. sylvestris as well as in N. tabacum but not in N. tomentosiformis, suggesting that a distinct mechanism operates for the translational initiation of N. tomentosiformis ndhD mRNAs. Four to six sites are edited partially in green leaves. Some of these sites may represent evolutionary intermediates in the process of losing editing events.

Cell Lineage↗
PubMed2003

A tRNA(Leu)-like sequence located immediately upstream of an Arabidopsis clock-regulated gene is transcriptionally active: efficient transcription by an RNA polymerase III-dependent in vitro transcription system.

A tRNA(Leu)-like sequence is located within a probable enhancer region of the RNA polymerase II-dependent gene encoding an RNA-binding protein, Atgrp7, in Arabidopsis (Mol. Gen. Genet. 261 (1999) 811). To examine whether this sequence is transcribed, we used our in vitro transcription system from tobacco cell nuclei. In vitro assays demonstrated that this tRNA-like sequence is transcribed by RNA polymerase III and its transcript is processed into tRNA-size molecules. Transcription starts at the CAA motif, a transcription initiation site for many plant tRNA genes. Mutation analyses indicated that transcription of this sequence depends on promoter elements typical for plant tRNA genes. We therefore concluded that this is a transcriptionally active tRNA(Leu)(AAG) gene. Mutation of a basic promoter element of the tRNA gene exerted no influence on the transcription of the downstream protein-coding gene, suggesting that no apparent interference occurs between the two adjacent genes.

5' Flanking Region↗
PubMed2003

Array-based analysis on tobacco plastid transcripts: preparation of a genomic microarray containing all genes and all intergenic regions.

The plastid genome of higher plants includes about 120 genes. We adopted genomic array technologies to the tobacco plastid genome. A microarray was constructed, consisting of 220 DNA fragments that cover the whole genome sequence. Each DNA fragment corresponds to a single known gene or an intergenic region. We evaluated reliability of this microarray by comparing the plastid RNA level in light- or dark-grown tobacco seedlings. The transcripts encoding photosynthetic subunits increased significantly in light-grown tissues as expected. Furthermore, we found unexpected signals in several intergenic regions, suggesting the existence of novel transcripts in tobacco plastids.

DNA, Plant↗
PubMed2002

Photosynthesis nuclear genes generally lack TATA-boxes: a tobacco photosystem I gene responds to light through an initiator.

The promoter architecture of the nuclear-encoded photosystem I genes was studied using a tobacco gene, psaDb, as a model case. Linker scanning mutations revealed that the psaDb promoter does not have a TATA box. Instead, pyrimidine-rich Initiator (Inr) elements that overlap the transcription start sites are essential for light-responsive transcription of this gene. When the psaDb promoter was mutated to have a TATA-box but no Inr, light-responsive transcription was impaired, indicating that the regulatory system of this gene prefers Inr to a TATA box. As very little is known about plant TATA-less promoters, we subsequently examined whether this promoter architecture is unique to psaDb. Computer analysis of 232 plant promoters revealed surprising features; the majority of photosynthesis nuclear genes lacked TATA boxes, although the frequency of the TATA-less promoters in non-photosynthesis genes was less than 10%. These results strongly suggest that TATA-independent transcription mechanisms play important roles in the regulated expression of photosynthesis nuclear genes.

5' Flanking Region↗
PubMed2002

Polyribosome loading of spinach mRNAs for photosystem I subunits is controlled by photosynthetic electron transport.

In light-, but not in dark-grown spinach seedlings, the mRNAs for the nuclear-encoded photosystem I subunits D, F and L are associated with polyribosomes and this association is prevented by the application of 3-(3',4'-dichlorophenyl)-1,1'-dimethyl urea (DCMU), an inhibitor of the photosynthetic electron transport. To identify the cis-elements which are responsible for this regulation, we generated a series of chimeric PsaD constructs and tested them in transgenic tobacco. The spinach PsaD 5'-untranslated region is sufficient to confer light- and photosynthesis-dependent polyribosome association onto the uidA reporter gene, while the tobacco PsaD 5'-untranslated region directs constitutive polyribosome association. These results are discussed with regard to signals from photosynthetic electron flow which control processes in the cytoplasm.

Base Sequence↗
PubMed2002

Dual roles of photosynthetic electron transport in photosystem I biogenesis: light induction of mRNAs and chromatic regulation at post-mRNA level.

Light regulation of photosystem I (PSI) biogenesis was studied in a unicellular green alga, Chlamydomonas reinhardtii. When Chlamydomonas cells were transferred from darkness to the light, mRNAs for both nuclear- and chloroplast-encoded PSI subunits were induced in concert. This light induction was inhibited by photosynthetic electron transport (PET) inhibitors, 3-(3,4 dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6 isopropyl-p-benzoquinone, but not by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone. This indicated that PET plays a pivotal role in the light induction of PSI subunit mRNAs, but that photophosphorylation is not necessary. When we irradiated the Chlamydomonas cells with PSI-light (695 nm) or PSII-light (644 nm), which makes the plastoquinone pool oxidative and reductive, respectively, PSII-light caused the accumulation of PSI proteins more abundantly than did PSI-light. However, there was no difference for the PSI subunit mRNA levels between these light sources. From these results, we conclude that PET plays dual roles in the regulation of PSI biogenesis in Chlamydomonas: when cells are illuminated, PET first induces the PSI subunit mRNAs irrespective of the redox state of the intersystem electron carriers, and then their redox state fine-tunes PSI biogenesis at translational and/or post-translational steps to fulfil the chromatic adaptation.

Animals↗
PubMed2002

Recognition of RNA editing sites is directed by unique proteins in chloroplasts: biochemical identification of cis-acting elements and trans-acting factors involved in RNA editing in tobacco and pea chloroplasts.

RNA editing in higher-plant chloroplasts involves C-to-U conversions at specific sites. Although in vivo analyses have been performed, little is known about the biochemical aspects of chloroplast editing reactions. Here we improved our original in vitro system and devised a procedure for preparing active chloroplast extracts not only from tobacco plants but also from pea plants. Using our tobacco in vitro system, cis-acting elements were defined for psbE and petB mRNAs. Distinct proteins were found to bind specifically to each cis-element, a 56-kDa protein to the psbE site and a 70-kDa species to the petB site. Pea chloroplasts lack the corresponding editing site in psbE since T is already present in the DNA. Parallel in vitro analyses with tobacco and pea extracts revealed that the pea plant has no editing activity for psbE mRNAs and lacks the 56-kDa protein, whereas petB mRNAs are edited and the 70-kDa protein is also present. Therefore, coevolution of an editing site and its cognate trans-factor was demonstrated biochemically in psbE mRNA editing between tobacco and pea plants.

5' Untranslated Regions↗