Biomedical subjects

Atsuhiko Shinmyo

Publications and source records attributed to Atsuhiko Shinmyo.

At least 19 recordsLinked to original sources

PubMed2006

Cell cycle regulated D3-type cyclins form active complexes with plant-specific B-type cyclin-dependent kinase in vitro.

Tobacco (Nicotiana tabacum L.) cv Bright Yellow-2 (BY-2) cells are the most highly synchronizable plant cell culture, and previously we used them to analyze cell cycle regulation of cyclin-dependent kinases (CDKs) containing the cyclin binding motifs PSTAIRE (CDKA) and PPTA/TLRE (CDKB). Here we describe the analysis of tobacco CycD3 cyclins whose transcripts predominantly accumulate during G2 to M phase, which represents a unique feature of this type of cyclin D in plants. Although protein levels of CycD3s fluctuate with different patterns during the cell cycle, kinase assays revealed that the CycD3-associated kinases phosphorylate histone H1 and the tobacco retinoblastoma related protein (NtRBR1) with two peaks at the G1/S and G2/M boundaries. In vitro pull-down assays revealed that cell cycle-regulated CycD3s bind to CDKA, but more weakly than does CycD3;3, and that they also bind to CDKB and the CDK inhibitor NtKIS1a. Mutations in the cyclin box of the CycD3s showed that two amino acids are required for binding with CDKA and NtKIS1a, but no diminished interaction was observed with CDKB. A reconstituted kinase assay was adapted for use with bacterially produced GST-CycD3s, and kinase activity could be activated by incubation of extracts from exponentially growing BY-2 cells. Such activated complexes contained CDKA and CDKB, and the reconstituted GST-CycD3 mutants, retaining binding ability to CDKB, showed kinase activity, suggesting that these cell cycle-regulated CycD3s form active complexes with both A- and B-type CDKs in vitro.

Amino Acid Motifs↗

PubMed2006

Arabidopsis CDKA;1, a cdc2 homologue, controls proliferation of generative cells in male gametogenesis.

The protein kinase cdc2 is conserved throughout eukaryotes and acts as a key regulator of the cell cycle. In plants, A-type cyclin-dependent kinase (CDKA), a homologue of cdc2, has a role throughout the cell cycle. Here we show that a loss-of-function mutation in CDKA;1, encoding the only Arabidopsis CDKA, results in lethality of the male gametophyte. Heterozygous plants produced mature siliques containing about 50% aborted seeds, and segregation distortion was observed in paternal inheritance. Microspores normally undergo an asymmetric cell division, pollen mitosis I (PMI), to produce bicellular pollen grains. The larger vegetative cell does not divide, but the smaller generative cell undergoes mitosis, PMII, to form the two sperm cells, thereby generating tricellular pollen grains. The cdka-1 mutant, however, produces mature bicellular pollen grains, consisting of a single sperm-like cell and a vegetative cell, due to failure of PMII. The mutant sperm-like cell is fertile, and preferentially fuses with the egg cell to initiate embryogenesis. As the central cell nucleus remains unfertilized, however, double fertilization does not occur. In heterozygous plants, the embryo is arrested at the globular stage, most likely because of loss of endosperm development, whereas it is arrested at the one- or two-cell stage in presumptive homozygous plants. Thus, CDKA;1 is essential for cell division of the generative cell in male gametogenesis.

Amino Acid Sequence↗

PubMed2006

Efficient translation destabilizes transcripts in chloroplasts of Chlamydomonas reinhardtii.

We previously reported that high level of reporter gene transcript does not confer high amount of reporter protein accumulation in Chlamydomonas reinhardtii chloroplast transformants. Here, to further clarify the correlation between the level of transcript and protein accumulation, we generated the beta-glucuronidase (GUS) reporter gene (uidA) constructs with different potential for translation efficiency of the GUS protein by incorporating different 5' and 3'-untranslated regions of chloroplast genes into each construct. The relationship between mRNA stability and translation efficiency of the GUS reporter gene in each construct were then studied in C. reinhardtii stable chloroplast transformants. We found that sequences of the two nucleotides immediately upstream of the initial codon were important for translation efficiency and that transformants showing high GUS activity accumulated lower level of uidA transcripts than the transformants with low GUS activity. Moreover, accumulation and half-lives of these chimeric-uidA transcripts were increased to the same level in the presence of translation inhibitor. The accumulation and/or half-lives of several endogenous chloroplast transcripts were also increased by such inhibitor. Collectively, our results indicate that efficient translation destabilizes transcripts in chloroplasts of C. reinhardtii, and that there is an apparent negative correlation between protein accumulation and mRNA stability.

Animals↗

PubMed2006

Improvement on production of (R)-4-chloro-3-hydroxybutyrate and (S)-3-hydroxy-gamma-butyrolactone with recombinant Escherichia coli cells.

(R)-4-Chloro-3-hydroxybutyrate (CHB) and (S)-3-hydroxy-gamma-butyrolactone (HL) are used for the synthesis of biologically and pharmacologically important compounds. Enterobacter sp. DS-S-75 was found to have the unique activity to convert (S)-CHB in the racemate to (S)-HL through asymmetric dechlorination, hydrolysis, and lactonization. As a result, the remaining (R)-CHB and formed (S)-HL could be obtained in a one-pot reaction. We purified the CHB degrading enzyme which catalyzing these reactions and isolated the coding gene from the strain DS-S-75 in order to improve the productivity of these compounds using the transformant. Interestingly, the purified enzyme showed not only dechlorinating, but also hydrolyzing activities on CHB and the similar carboxylic esters, it was then designated CHB hydrolase, and appears to be a novel enzyme. The gene had 1101 bp encoding 367 amino acids including a signal peptide composed of 25 residues. The deduced amino acid sequence contained a conserved region generally found in esterases and lipases, but did not have significant similarity. When asymmetric degradation of racemic methyl CHB (CHBM) was performed using a culture broth of Escherichia coli DH5alpha transformed with the isolated gene, the reaction time was shortened 20-fold over that of the strain DS-S-75, and the maximum concentration of the substrate could be increased from 8% to 15% (w/v). Moreover, both of the obtained residual (R)-CHBM and the formed (S)-HL had high optical purities (>99% e.e.).

4-Butyrolactone↗

PubMed2006

High-efficiency secretory production of peroxidase C1a using vesicular transport engineering in transgenic tobacco.

Horseradish peroxidase isozyme C1a (HRP C1a) is widely used as a reporter enzyme in a variety of detection procedures such as enzyme-linked immunosorbent assay (ELISA) and western blotting. We previously isolated the gene encoding HRP C1a and showed that HRP C1a is at first translated as a preproprotein containing propeptides at its N- and C-termini (N-terminal secretion signal peptide and C-terminal propeptide; CTPP). The signal peptide (sp) is necessary for endoplasmic reticulum (ER) translocation and the CTPP acts as a vacuolar sorting determinant. Furthermore, HRP C1a was secreted into the culture medium from cells expressing the HRP C1a gene without the CTPP region. We optimized the secretory production system of HRP C1a in tobacco plants. To determine a suitable signal peptide for high-efficient secretion of proteins, three types of sp derived from HRP C1a (C1Psp), beta-D-glucan exohydrolase (GEsp) and 38 kDa peroxidase (38Psp) were compared. GE and 38P are secretory proteins highly accumulated in the culture medium of BY2 cells. The secretion efficiency was increased by 34% and 53% when GEsp and 38Psp was used, respectively. Next, we used a translational enhancer, the 5'-untranslated region of Nicotiana tabacum alcohol dehydrogenase gene (NtADH 5'-UTR). The production of HRP C1a was increased by placing NtADH 5'UTR in front of the ORF in BY2 cells. These results indicate that the localization and expression level of recombinant proteins can be controlled by the use of propeptides and 5'UTR, respectively. Finally, high-efficiency secretory production of the HRP C1a was also achieved in transgenic tobacco.

5' Untranslated Regions↗

PubMed2005

Arabidopsis KRPs have distinct inhibitory activity toward cyclin D2-associated kinases, including plant-specific B-type cyclin-dependent kinase.

Arabidopsis contains seven Kip-related protein (KRP) genes encoding CDK (cyclin-dependent kinase) inhibitors (CKIs), which shares a restricted similarity with mammalian p27Kip1. Here, we analyze the characteristics of the KRPs. Although KRP1-KRP7 interact with active cyclin D2 (CYCD2)/CDKA and CYCD2/CDKB complexes to a similar extent, they inhibit kinase activity to a different extent. Our results suggest that inhibitory activity is related to the binding ability between KRP proteins and cyclin/CDK complexes, but secondary and tertiary structure may be also involved. These data provide the first evidence that KRPs inhibit kinase activity associated with plant-specific CDKB.

Arabidopsis↗

PubMed2005

Expression of randomly integrated single complete copy transgenes does not vary in Arabidopsis thaliana.

The high variability of transgene expression is frequently observed in independent transgenic lines. Variability of transgene expression has been attributed to several factors, including differences in chromosome position, repeat sequences and copy number. The eukaryotic genome, with a heterogeneous chromatin structure, is not homogeneous for transcriptional activity. Chromatin structure at the site of integration can affect transgene expression; this phenomenon is called the position effect. In this study, we investigated whether position effects confer variability of transgene expression in Arabidopsis thaliana. We analyzed the expression of randomly integrated single 'complete' (intact, non-truncated, non-rearranged) copy transgenes in A. thaliana. Ten independent lines containing single complete copies of the transgene located at different chromosome positions showed very similar levels of transgene expression, and variability of transgene expression was not observed. This result indicates that position effects may not generally be a major cause of variability of transgene expression in A. thaliana.

Arabidopsis↗

PubMed2005

Transcriptional activation of tobacco E2F is repressed by co-transfection with the retinoblastoma-related protein: cyclin D expression overcomes this repressor activity.

Evidence is emerging that the E2F family of transcription factors plays an important role in the regulation of gene expression at the G1/S transition in plants. Here, we show that in the tobacco proliferating cell nuclear antigen (PCNA), whose transcript is specifically expressed at G1/S phase, the two E2F binding sites are synergistically responsible for transcriptional activation at G1/S phase in synchronized tobacco BY-2 cells transformed with promoter constructs fused to a reporter gene. In addition, we have isolated the tobacco DP cDNA (NtDP) and showed that significant activation of the reporter gene was observed in transient expression assays by concomitantly transfecting with plasmids expressing NtE2F and NtDP. This transcriptional activation was repressed by co-transfection with a plasmid expressing NtRBR1; in vitro pull-down assays also revealed that NtRBR1 binds directly to NtE2F, thereby potentially blocking the transcriptional activation of NtE2F. Importantly, this repressor activity was cancelled when NtRBR1 was further co-transfected with a plasmid expressing cyclin D but not with cyclin A or cyclin B. These results are discussed with respect to the repression activity of NtRBR1 on the NtE2F/NtDP complex.

Amino Acid Sequence↗

PubMed2005

A nuclear-encoded sigma factor, Arabidopsis SIG6, recognizes sigma-70 type chloroplast promoters and regulates early chloroplast development in cotyledons.

Eubacterial-type multi-subunit plastid RNA polymerase (PEP) is responsible for the principal transcription activity in chloroplasts. PEP is composed of plastid-encoded core subunits and one of multiple nuclear-encoded sigma factors that confer promoter specificity on PEP. Thus, the replacement of sigma factors associated with PEP has been assumed to be a major mechanism for the switching of transcription patterns during chloroplast development. The null mutant (sig6-1) of plastid sigma factor gene AtSIG6 exhibited a cotyledon-specific pale green phenotype. Light-dependent chloroplast development was significantly delayed in the sig6-1 mutant. Genetic complementation of the mutant phenotype by the AtSIG6 cDNA demonstrated that AtSIG6 plays a key role in light-dependent chloroplast development. Northern and array-based global analyses for plastid transcripts revealed that the transcript levels of most PEP-dependent genes were greatly reduced in the sig6-1 mutant, but that the accumulation of nuclear-encoded RNA polymerase (NEP)-dependent transcripts generally increased. As the PEP alpha subunit and PEP-dependent trnV accumulated at normal levels in the sig6-1 mutant, the AtSIG6 knockout mutant probably retained functional PEP, and the transcriptional defects are likely to have been directly caused by AtSIG6 deficiency. Most of the AtSIG6-dependent genes are preceded by sigma70-type promoters comprised of conserved -35/-10 elements. Thus, AtSIG6 may act as a major general sigma factor in chloroplasts during early plant development. On the other hand, the mutant phenotype was restored in older seedlings. Arabidopsis probably contains another late general sigma factor, the promoter specificity of which widely overlaps with that of AtSIG6.

Arabidopsis↗

PubMed2004

Yeast plasma membrane Ena1p ATPase alters alkali-cation homeostasis and confers increased salt tolerance in tobacco cultured cells.

In plants, the plasma membrane Na(+)/H(+) antiporter is the only key enzyme that extrudes cytosolic Na(+) and contributes to salt tolerance. But in fungi, the plasma membrane Na(+)/H(+) antiporter and Na(+)-ATPase are known to be key enzymes for salt tolerance. Saccharomyces cerevisiae Ena1p ATPase encoded by the ENA1/PMR2A gene is primarily responsible for Na(+) and Li(+) efflux across the plasma membrane during salt stress and for K(+) efflux at high pH and high K(+). To test if the yeast ATPase would improve salt tolerance in plants, we expressed a triple hemagglutinin (HA)-tagged Ena1p (Ena1p-3HA) in cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells. The Ena1p-3HA proteins were correctly localized to the plasma membrane of transgenic BY2 cells and conferred increased NaCl and LiCl tolerance to the cells. Under moderate salt stress conditions, the Ena1p-3HA-expressing BY2 clones accumulated lower levels of Na(+) and Li(+) than nonexpressing BY2 clones. Moreover, the Ena1p-3HA expressing BY2 clones accumulated lower levels of K(+) than nonexpressing cells under no-stress conditions. These results suggest that the yeast Ena1p can also function as an alkali-cation (Na(+), Li(+), and K(+)) ATPase and alter alkali-cation homeostasis in plant cells. We conclude that, even with K(+)-ATPase activity, Na(+)-ATPase activity of the yeast Ena1p confers increased salt tolerance to plant cells during salt stress.

Adenosine Triphosphatases↗

PubMed2004

Efficient construction of cDNA microarrays utilizing normalized cDNA libraries of Arabidopsis thaliana.

We have demonstrated the utility of normalization for efficient cDNA microarray preparation using Arabidopsis as a model. Nonredundant cDNAs including 5722 species were efficiently collected from random 7914 expressed sequence tags (ESTs) in four normalized cDNA libraries. The prepared microarrays were successfully used to monitor gene expression. These methodologies should be applicable to the study of other species in plant biotechnology.

Journal Article↗

PubMed2004

The 5'-untranslated region of the tobacco alcohol dehydrogenase gene functions as an effective translational enhancer in plant.

The alcohol dehydrogenase gene (NtADH) was previously isolated from tobacco BY2 suspension cultured cell. Expression of this gene was dramatically increased only during the early stationary phase, and the 5'-untranslated region (5'-UTR) was hypothesized to be involved in the stimulatory effect at the post-transcriptional level. In this paper, we investigated whether the NtADH 5'-UTR possesses the ability to positively enhance gene expression at the translational level. For easily estimating translational efficiency, we used beta-glucuronidase (GUS) gene as a reporter and tobacco BY2 cell, Arabidopsis thaliana T87 cell, and rice Oryza sativa suspension cultured cells as host cells in a transient assay system. Compared with the control plasmid pBI221, insertion of the NtADH 5'-UTR enhanced GUS expression levels about 30- to 100-fold and 30- to 60-fold in transiently transformed BY2 and T87 cells, respectively. However, in transiently transformed O. sativa cells, expression was barely enhanced. In comparison with the 5'-UTR of tobacco mosaic virus (Omega sequence), a known translational enhancer, the NtADH 5'-UTR enhanced translation to a similar level. Meanwhile, the translational efficiency was affected by the sequence context around the AUG initiation codon at the translational initiation step. Moreover, this NtADH 5'-UTR also worked in stable tobacco transformants. Therefore, it is expected that this 5'-UTR will serve as a powerful tool for enhancing foreign gene expression.

Journal Article↗

PubMed2004

Systematic transient assays of promoter activities for leaf-specific genes identified by gene-expression profiling with cDNA microarrays in Arabidopsis thaliana.

We identified 85 genes highly expressed in leaves using an Arabidopsis cDNA microarray. A vector, pRAB5, was designed to allow cloning and assaying of the promoters. Fifty-one promoters from the selected genes were cloned and then assayed using a microprojectile bombardment and dual luciferase reporter assay system. This system allowed efficient systematic assays of promoter activity.

Journal Article↗

PubMed2004

Overexpression of NtHAL3 genes confers increased levels of proline biosynthesis and the enhancement of salt tolerance in cultured tobacco cells.

The Hal3 protein of Saccharomyces cerevisiae inhibits the activity of PPZ1 type-1 protein phosphatases and functions as a regulator of salt tolerance and cell cycle control. In plants, two HAL3 homologue genes in Arabidopsis thaliana, AtHAL3a and AtHAl3b, have been isolated and the function of AtHAL3a has been investigated through the use of transgenic plants. Expressions of both AtHAL3 genes are induced by salt stress. AtHAL3a overexpressing transgenic plants exhibit improved salt and sorbitol tolerance. In vitro studies have demonstrated that AtHAL3 protein possessed 4'-phosphopantothenoylcysteine decarboxylase activity. This result suggests that the molecular function of plant HAL3 genes is different from that of yeast HAL3. To understand the function of plant HAL3 genes in salt tolerance more clearly, three tobacco HAL3 genes, NtHAL3a, NtHAL3b, and NtHAL3c, from Nicotiana tabacum were identified. NtHAL3 genes were constitutively expressed in all organs and under all conditions of stress examined. Overexpression of NtHAL3a improved salt, osmotic, and lithium tolerance in cultured tobacco cells. NtHAL3 genes could complement the temperature-sensitive mutation in the E. coli dfp gene encoding 4'-phosphopantothenoyl-cysteine decarboxylase in the coenzyme A biosynthetic pathway. Cells overexpressing NtHAL3a had an increased intracellular ratio of proline. Taken together, these results suggest that NtHAL3 proteins are involved in the coenzyme A biosynthetic pathway in tobacco cells.

Amino Acid Sequence↗

PubMed2004

Enhanced resistance to blast fungus and bacterial blight in transgenic rice constitutively expressing OsSBP, a rice homologue of mammalian selenium-binding proteins.

The rice Oryza sativa selenium-binding protein homologue (OsSBP) gene encodes a homologue of mammalian selenium-binding proteins, and it has been isolated as one of the genes induced by treating a plant with a cerebroside elicitor from rice blast fungus. The possible role of OsSBP in plant defense was evaluated by using a transgenic approach. Plants overexpressing OsSBP showed enhanced resistance to a virulent strain of rice blast fungus as well as to rice bacterial blight. The expression of defense-related genes and the accumulation of phytoalexin after infection by rice blast fungus were accelerated in the OsSBP overexpressors. A higher level of H(2)O(2) accumulation and reduced activity of such scavenging enzymes as ascorbate peroxidase and catalase were seen when the OsSBP-overexpressing plants were treated with the protein phosphatase 1 inhibitor, calyculin A. These results suggest that the upregulation of OsSBP expression conferred enhanced tolerance to different pathogens, possibly by increasing plant sensitivity to endogenous defense responses. Additionally, the OsSBP protein might have a role in modulating the defense mechanism to biotic stress in rice.

Animals↗

PubMed2003

Introduction of the archaebacterial geranylgeranyl pyrophosphate synthase gene into Chlamydomonas reinhardtii chloroplast.

Geranylgeranyl pyrophosphate (GGPP) synthase gene (gds) derived from a thermophilic Archae Sulfolobus acidocaldarius, was introduced into a unicellular green alga Chlamydomonas reinhardtii chloroplast. Heat treatment abolished the prenyltransferase activity of the wild strain, but the activity of the transforment remained. The transformant accumulated gds gene mRNA and translation product.

Journal Article↗

PubMed2003

5' Untranslated region of the HSP18.2 gene contributes to efficient translation in plant cells.

Maximizing the amount of protein translated per unit mRNA is an important goal in establishing expression systems. The 5' untranslated region (5'-UTR) of mRNA is known to play an important role in determining the rate of translation. The full length 5'-UTR from the Arabidopsis thaliana heat shock protein (HSP) gene,HSPI8.2 gene, was inserted into the cloning site between the cauliflower mosaic virus 35S RNA promoter and beta-glucuronidase (GUS) gene in the pBI221 plasmid. When this construct was transfected into Nicotiana tabacum (tobacco) BY-2 protoplasts, the level of protein was about 10-fold higher than that of unmodified pB1221. The accumulation of each transcript was the same level. We also demonstrated that the 5'-UTR of the HSP18.2 gene enhances the rate of translation in stable transgenic BY-2 clones and Arabidopsis T87 protoplasts. The 5'-UTRs of the other Arabidopsis HSP genes -HSP17.4, HSP81-1,HSP81-2, andHSP81-3 - also conferred efficient translation. These 5'-UTRs ofHSP genes may be of use in increasing the expression of foreign proteins. In combination with a strong promoter, it can be used in the development of efficient protein production systems.

Journal Article↗

PubMed2003

Ectopic expression of a horseradish peroxidase enhances growth rate and increases oxidative stress resistance in hybrid aspen.

We previously demonstrated that overexpression of the horseradish (Armoracia rusticana) peroxidase prxC1a gene stimulated the growth rate of tobacco (Nicotiana tabacum) plants. Here, the cauliflower mosaic virus 35S::prxC1a construct was introduced into hybrid aspen (Populus sieboldii x Populus grandidentata). The growth rate of these transformed hybrid aspen plants was substantially increased under greenhouse conditions. The average stem length of transformed plants was 25% greater than that of control plants. There was no other obvious phenotypic difference between the transformed and control plants. Fast-growing transformed hybrid aspen showed high levels of expression of prxC1a and had elevated peroxidase activities toward guaiacol and ascorbate. However, there was no increase of the endogenous class I ascorbate peroxidase activities in the transformed plants by separate assay and activity staining of native polyacrylamide gel electrophoresis. Furthermore, calli derived from the transformed hybrid aspen grew faster than those from control plants and were resistant to the oxidative stress imposed by hydrogen peroxide. Therefore, enhanced peroxidase activity affects plant growth rate and oxidative stress resistance.

Gene Expression Regulation, Plant↗