Living organisms respond to phosphate limitation by expressing various genes whose products maintain an appropriate range of phosphate concentrations within each cell. We identified a two-component system, which consists of histidine kinase Hik7 and its cognate response regulator Rre29, that was the dominant sensory system that controled gene expression in response to phosphate limitation in the cyanobacterium Synechocystis sp. PCC 6803. The response regulators PhoB in E. coli and PhoP in B. subtilis bind specifically to conserved sequences, known as Pho boxes. In order to investigate the possibility that Synechocystis might also have a Pho box, we examined whether Rre29 can bind to the upstream flanking regions of the phosphate limitation-inducible genes. We found that Rre29 bound to the upstream flanking regions of several phosphate limitation-inducible genes at repetitive PyTTAAPyPy(T/A)-like sequences, suggesting that this repetitive sequence might be the Pho box in Synechocystis .
EMSを用いて作出したシロイヌナズナのpcb2 (pale-green and chlorophyll b reduced 2) 変異株は、野性株に比べて淡い体色が特徴で、クロロフィル含量が約1/3に減少しており、特にクロロフィルbが減少していた。葉緑体の形状と数は野生株と大きく変わらなかったが、葉肉細胞の電子顕微鏡観察を行ったところ、グラナスタックが極度に減少していることがわかった。その原因遺伝子をマッピングしたところ、第5染色体の上腕の約190kbの範囲にあることがわかった。この領域には49個の遺伝子がコードされている。細胞内局在予測プログラムを利用して、葉緑体への局在が強く示唆される遺伝子に注目して野生株と変異株の塩基配列を比較したところ、そのうちの1つの遺伝子で一塩基置換の変異が見つかった。この遺伝子はイントロンを持たず、コードされるタンパク質は417のアミノ酸で構成され、N末端に葉緑体移行シグナルを持っており、変異は29番目のグルタミンコドンを終止コドンに変える、ナンセンス変異であった。データベースを検索したところ、イネや光合成細菌から相同性の高い配列が見つかったが、いずれも機能はまだ分かっていないようである。 この遺伝子をpcb2変異の原因遺伝子と仮定して解析を進めている。
C. merolaeの葉緑体ゲノムには、4種の転写因子 (Ycf27-30)が保持されており、これらは環境の変化に応答して、主要な光合成遺伝子群の転写制御を行っていることが予想される。私達は、これらの転写因子が認識しているシグナルや標的遺伝子群の同定は、複雑な光合成系の転写制御機構の最も単純なモデルを提供することになると考えて研究を進めている。本大会では、1）Ycf30によるルビスコ遺伝子群の転写制御機構と、2）集光装置の形成に働く転写因子の検討について報告する。1）精製したYcf30タンパク質は、ルビスコ遺伝子オペロンの上流域に結合した。転写開始点決定の結果、ルビスコ遺伝子オペロンは一箇所の転写開始点からの転写が活性化されていることが強く示唆された。2）C. merolaeを強光下で培養すると、通常条件で培養したものに比べて黄緑色を呈したが、弱光条件へ移すと通常の青緑色へと変わった。これは、強光から弱光へシフトにより、集光装置の形成が起きたものと考えられた。そこで、強光条件から弱光条件へ移して3時間後までの転写産物量を調べたところ、cpcA遺伝子とycf29遺伝子の転写産物量の増加がみられた。この結果は、Ycf29が集光装置の形成に関わる転写因子である可能性を示唆した。現在、Ycf29と集光装置に対応した遺伝子群の発現制御機構の関連について検討中である。
A growing body of evidence indicates that a RecA-mediated recombination system exists in chloroplast. However very little is known about mechanism for plastome homologous recombination. Using E. coli producing recombinant protein, we examined the enzymatic properties of chloroplast-targeted RecA homologue, recA-AT, encoded on the Arabidopsis nuclear genome (see Cerutti et al 1992). Although predicted mature recA-AT showed ATPase and DNA-binding activities, it had little strand exchange activity. More detailed characterization revealed that enzymatic properties of the recA-AT were distinct from those of authentic E. coli RecA: the effect of DNA on ATPase activity, the strength of DNA-binding activity and the migration pattern of DNA-protein complex in gel retardation assays. These differences between the recA-AT and the RecA raise a possibility that biochemical mechanism of the plastome homologous recombination is different from that of RecA-mediated homologous recombination in E. coli. Supported by METI/NEDO partly.
Mitochondrial citrate synthase was overexpressed in Eucalyptus plant and its effects on organic acid excretion and low phosphorus tolerance were examined. The transformants and control plants were grown with a hydropoinc culture solution containing AlPO4 as a sole phosphorus source. CS activity in roots of the transformants showed 1.4- to 2.5-fold increase in comparison with that in the control. Symptom of phosphorus deficiency was ameliorated in the transformants as judged form their phosphorus content. One transgenic line showed slightly higher (1.6-fold) citrate excretion than the control. This line was then grown with an acid soil containing insoluble phosphate and its growth tended to be slightly greater than that of the control. Therefore, it is suggested that overexpression of mitochondrial citrate synthase improves the growth performance of Eucalyptus plant under low phosphorus stress due to the high ability of phosphate-acquisition caused by the enhanced citrate excretion.
To investigate the whole genetic system of legumes, we initiated large-scale sequencing of the genome of a model legume, Lotus japonicus. Using the information on ESTs and cDNA markers of legume plants, genomic clones corresponding to the multiple seed points of the genome have been chosen as initial targets. As the accumulation of the seed sequences has progressed, clone selection by walking has also initiated. Currently, a total of 1,550 clones have been selected. One hundred and twenty seven of them are in the library phase, 952 are in the finishing phase and 412 have been annotated. A total of 3,975 protein coding genes were identified in 42.2 Mbp. Mapping of the seed clones has also been performed by generating PCR-based markers from sequence information, and 1,140 clones have been located on the linkage map. The sequence data, gene information and mapping information are available through the WWW at http://www.kazusa.or.jp/lotus/.
Full-length cDNAs are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. Using the biotinylated CAP trapper method, we have constructed full-length cDNA libraries from Arabidopsis plants and isolated 224,641 RIKEN Arabidopsis full-length (RAFL) cDNA clones. They were clustered into 18,127 nonredundant cDNA groups, about 70% of predicted genes1). We have determined full-length cDNA sequences of 13,181 RAFL cDNA clones2). We have also used the RAFL cDNAs for the microarray analysis3), 4) of expression profiles and the structure analysis of Arabidopsis genes. In this meeting, overview of our RAFL cDNA project will be presented. 1) Seki et al. (2002) Science 296:141. 2) Yamada et al. (2003) Science 302:842. 3) Seki et al. (2003) Topics Curr. Genet. 4:271. 4) Seki et al. (2004) J. Exp. Bot. (in press).
Here we report the complete nucleotide sequence of the chloroplast genome of Populus alba. This is the first report on angiosperm in terms of tree chloroplast genomes. The genome as a circular DNA is composed of 156,505 bp, in which a pair of 27,660 bp-inverted repeat are contained. The inverted repeat region disrupts the others into the large (85,017 bp) and the small (16,168 bp) single copy regions. The genome contains genes which are identified to have similarity to the chloroplast genes previously reported. Those are 85 potential protein-coding genes, 4 ribosomal RNA genes and 37 tRNA genes. The chloroplast genome of P. alba lacks rpl32 & rps16 though it resembles that of tobacco in overall structure. In addition to the structural organization, the expression pattern of each gene in the chloroplast genome of the P. alba will be discussed. This work was partly supported by METI.
The complete nucleotide sequence of the chloroplast genome of Lactuca sativa (lettuce) has been determined. The chloroplast was isolated from 1-month-old seedlings by two stepwise percoll density gradient centrifugation. The Chloroplast DNA was extracted with CTAB and randomly cloned for DNA sequencing. Sequencing of the 5,000 individual clones by shotgun method covered the whole lettuce chloroplast genome. The chloroplast genome of lettuce is a circular double-stranded DNA of 152,765 bp. Two inverted repeat regions are separated by a large single copy (LSC) region of 84,103 bp and a small single copy (SSC) region of 18,595 bp. We have identified 121 genes including 80 potential-protein coding genes, 4 rRNA genes and 37 tRNA genes based on previously reported chloroplast genes. The gene content of total 80 potential-protein encoding genes in lettuce (compositae) chloroplast genome is identical with the chloroplast DNA of the different species tobacco (solanaceae). Supported by KEIHANNA/MEXT.