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Objective The outcome of trial of voiding without catheter in patients treated combination therapy with dutasteride and alpha1-adrenergic receptor blocker for acute urinary retention caused by benign prostatic hyperplasia was not reported. We evaluated the clinical efficacy of combination therapy with dutasteride in patients with unsuccessful trial without catheter after treatment with an alpha1-adrenergic receptor blocker monotherapy for acute urinary retention caused by benign prostatic hyperplasia. Patients and methods Patients with acute urinary retention due to prostatic hyperplasia were catheterized and treated alpha1-adrenergic receptor blocker monotherapy. After two weeks later, patients were put on trial without catheter. 52 patients who were unsuccessful trial without catheter administered combination therapy with dutasteride and alpha1-adrenergic receptor blocker. We use criteria that voiding urine volume over 100 ml and post-void residual urine volume below 100 ml in deciding whether catheter should be removed. Results 33 (63.5%) men did not require re-catheterization within 7 months after combination therapy. The successful rate of Performance Status (PS) 0-1 group was significantly superior to that of PS 2-4 group. Conclusions PS 0-1 men catheterized for AUR can void more successfully after catheter removal than PS 2-4 men if treated with combination therapy.

We previously developed an in vitro translation system derived from tobacco chloroplasts. Here, we report a significantly improved in vitro translation system. By modifying preparation procedures for chloroplast extracts and reaction conditions, we achieved 100-fold higher translation activity than the previous system. The new system does not require the supplement of Escherichia coli tRNAs due to the omission of micrococcal nuclease treatment, thus the tRNA population reflects the intrinsic tRNA population in tobacco chloroplasts. The rate of translation initiation from a variety of chloroplast mRNAs may be measured by monitoring the fluorescence intensity of synthesized green fluorescent protein, which is a non-radioactive detection method. Incorporation of an amino acid linked to a fluorescent dye also allows detection of the translation products in vitro. Using our new system, we found that mRNAs carrying unprocessed or processed atpH and rbcL 5'-UTRs were efficiently translated at similar rates, whereas translation of mRNAs with processed atpB and psbB 5'-UTRs was more efficient than those with unprocessed 5'-UTRs. These results suggest that the role of 5'-UTR processing in the regulation of chloroplast gene expression differs between mRNAs. The new in vitro translation system will be a powerful tool to investigate the mechanism of chloroplast mRNA translation.

Little is known about the biochemical mechanism of translation in cyanobacteria though substantial studies have been made on photosynthesis, nitrogen fixation, circadian rhythm, and genome structure. To analyze the mechanism of cyanobacterial translation, we have developed an in vitro translation system from Synechococcus cells using a psbAI-lacZ fusion mRNA as a model template. This in vitro system supports accurate translation from the authentic initiation site of a variety of Synechococcus mRNAs. In Synechococcus cells, rbcL and rbcS encoding the large and small subunits, respectively, of ribulose-1,5-bisphosphate carboxylase/oxygenase are co-transcribed as a dicistronic mRNA, and the downstream rbcS mRNA possesses two possible initiation codons separated by three nucleotides. Using this in vitro system and mutated mRNAs, we demonstrated that translation starts exclusively from the upstream AUG codon. Although there are Shine-Dalgarno-like sequences in positions similar to those of the functional Shine-Dalgarno elements in Escherichia coli, mutation analysis indicated that these sequences are not required for translation. Assays with deletions within the 5'-untranslated region showed that a pyrimidine-rich sequence in the -46 to -15 region is necessary for efficient translation. Synechococcus cells contain two ribosomal protein S1 homologues of 38 and 33 kDa in size. UV cross-linking and immunoprecipitation experiments suggested that the 38-kDa S1 is involved in efficient translation via associating with the pyrimidine-rich sequence. The present in vitro translation system will be a powerful tool to analyze the basic mechanism of translation in cyanobacteria.

Codon usage in chloroplasts is different from that in prokaryotic and eukaryotic nuclear genomes. However, no experimental approach has been made to analyse the translation efficiency of individual codons in chloroplasts. We devised an in vitro assay for translation efficiencies using synthetic mRNAs, and measured the translation efficiencies of five synonymous codon groups in tobacco chloroplasts. Among four alanine codons (GCN, where N is U, C, A or G), GCU was the most efficient for translation, whereas the chloroplast genome lacks tRNA genes corresponding to GCU. Phenylalanine and tyrosine are each encoded by two codons (UUU/C and UAU/C, respectively). Phenylalanine UUC and tyrosine UAC were translated more than twice as efficiently than UUU and UAU, respectively, contrary to their codon usage, whereas translation efficiencies of synonymous codons for alanine, aspartic acid and asparagine were parallel to their codon usage. These observations indicate that translation efficiencies of individual codons are not always correlated with codon usage in vitro in chloroplasts. This raises an important issue for foreign gene expression in chloroplasts.

RNA editing is found in various transcripts from land plant chloroplasts. In tobacco chloroplasts, C-to-U conversion occurs at 36 specific sites including two sites identified in this work. Our RNA editing assay system using chloroplast extracts facilitated biochemical analyses of editing reactions but required mRNAs labeled with (32)P at specific sites. Here, we have improved the in vitro system using fluorescence-labeled chain terminators, ddGTP and ddATP, and have measured the editing activity at 19 sites in ndh transcripts. Editing activities varied from site to site. It has been reported that one editing site in ndhA mRNAs is present in spinach but absent in tobacco, but a corresponding editing capacity had been found in vivo in tobacco using biolistic transformation. We confirmed biochemically the existence of this activity in tobacco extracts. Using the non-radioactive assay, we examined sequences essential for editing within a 50-nt mRNA region encompassing an editing site. Editing of the ndhB-2 site requires a short sequence in front of the editing site, while that of the ndhF mRNA requires two separate regions, a sequence surrounding the editing site and a 5' distal sequence. These results suggest that distinct editing mechanisms are present in chloroplasts.

The tobacco cultivar Nicotiana tabacum is a natural amphidiploid that is thought to be derived from ancestors of Nicotiana sylvestris and Nicotiana tomentosiformis. To compare these chloroplast genomes, DNA was prepared from isolated chloroplasts from green leaves of N. sylvestris and N. tomentosiformis, and subjected to whole-genome shotgun sequencing. The N. sylvestris chloroplast genome comprises of 155,941 bp and shows identical gene organization with that of N. tabacum, except one ORF. Detailed comparison revealed only seven different sites between N. tabacum and N. sylvestris; three in introns, two in spacer regions and two in coding regions. The chloroplast DNA of N. tomentosiformis is 155,745 bp long and possesses also identical gene organization with that of N. tabacum, except four ORFs and one pseudogene. However, 1,194 sites differ between these two species. Compared with N. tabacum, the nucleotide substitution in the inverted repeat was much lower than that in the single-copy region. The present work confirms that the chloroplast genome from N. tabacum was derived from an ancestor of N. sylvestris, and suggests that the rate of nucleotide substitution of the chloroplast genomes from N. tabacum and N. sylvestris is very low.

The NOx reduction activity on γ-alumina was significantly improved by discharging nonthermal plasma in a simulated oxidizing exhaust gas under conditions of a high space velocity. On the other hand, those on a Pt-loaded catalyst and Cu-ZSM-5 could not be improved. The discharging nonthermal plasma converted NO to NO2 and hydrocarbons to partial-oxidized hydrocarbons, such as aldehydes, under the oxidizing conditions. From the relationship between the NOx reduction activities and the properties of several alumina catalysts, it was found that the NOx reduction activity was correlated with the number of acid sites on the alumina based on the NH3 adsorption measurement. Therefore, it is concluded that the NOx reduction occurred at the acid sites of the alumina, and the discharging nonthermal plasma improves the NOx reduction activity by NO2 formation and partial-oxidation of hydrocarbons. These results suggest that a catalytic reaction assisted by the discharging nonthermal plasma could be a promising technology for NOx reduction in lean-burn and diesel exhaust gases even under the conditions of high space velocity. Copyright © 2006 Society of Chemical Industry

The complete nucleotide sequence of tobacco chloroplast DNA was first determined in 1986, and then its updated gene map was reported in 1998. During the course of sequencing the chloroplast DNA ofNicotiana sylvestris, the female progenitor of tobacco, we found some sequence errors and amended the 1998 version. The tobacco chloroplast DNA comprises 155,943 bp, 4 bp longer than the 1998 version.

Rice, one of the world's most important food plants, has important syntenic relationships with the other cereal species and is a model plant for the grasses. Here we present a map-based, finished quality sequence that covers 95% of the 389 Mb genome, including virtually all of the euchromatin and two complete centromeres. A total of 37,544 non-transposable-element-related protein-coding genes were identified, of which 71% had a putative homologue in Arabidopsis. In a reciprocal analysis, 90% of the Arabidopsis proteins had a putative homologue in the predicted rice proteome. Twenty-nine per cent of the 37,544 predicted genes appear in clustered gene families. The number and classes of transposable elements found in the rice genome are consistent with the expansion of syntenic regions in the maize and sorghum genomes. We find evidence for widespread and recurrent gene transfer from the organelles to the nuclear chromosomes. The map-based sequence has proven useful for the identification of genes underlying agronomic traits. The additional single-nucleotide polymorphisms and simple sequence repeats identified in our study should accelerate improvements in rice production.

The genes transcribed by RNA polymerase III (pol III) display a great diversity in terms of promoter structure and are placed in four groups accordingly. Type 3 subset of pol III genes has promoter elements which reside entirely upstream of the coding region of the gene whereas type 4 consists of genes with mixed promoters that enclose intra- and extragenic regulatory sequences. Plant 7SL RNA genes have been previously classified as type 3 of pol III genes requiring an upstream sequence element and a canonical TATA box for transcriptional activity in transfected plant protoplasts. We have identified two novel functional control regions within the coding region of an Arabidopsis 7SL RNA gene (At7SL-1) that resemble tRNA gene-specific A and B boxes with respect to sequence and position. Single and multiple nucleotide substitutions in either of these regions resulted in a pronounced reduction of transcription activity in tobacco nuclear extract that was not caused by a decreased stability as shown by decay kinetics of wild type and mutant RNA transcripts. These findings suggest that plant 7SL RNA genes should be actually placed in type 4 of pol III-transcribed genes. As a consequence of substantially different upstream promoters utilized by plant and human pol III, in vitro transcription of 7SL RNA genes in heterologous systems is severely impaired. A chimeric human 7SL RNA gene that contains the 5' flanking region up to position -300 of At7SL-1 is yet transcribed with a reduced efficiency in tobacco extract when compared with the plant wild-type gene, supporting the notion that internal regulatory elements contribute to full activity.

Innovative three-way catalysts containing ceria-zirconia solid solutions (CZ) with a ZrO 2 content ranging from 5 to 75 mol% were developed. Such catalysts have very high oxygen storage/release capacity (OSC). The OSC of'catalysts containing first-generation CZ was found to increase linearly with the amount of zirconia dissolved in the solid solution in the 0-20 mol%( ZrO 2 range. The thermal stability of the catalyst containing first-generation CZ was higher than that containing ceria. Second-generation CZ with greater OSC than first-generation CZ was developed by a homogeneous coprecipitation process, followed by heat-treatment below 1000 °C, enabling dissolution of zirconia into ceria up to 75 mol% zirconia as estimated by XRD. Third-generation CZ, CZ-doped alumina (ACZ), was developed by homogeneous coprecipitation based on the "diffusion barrier concept". The OSC of ACZ was 23 times larger than that of pure ceria. The NOx emission over the catalyst containing ACZ was reduced by about a factor of five compared with that containing pure ceria. Toyota Motor Corp. has put three-way catalysts containing first-, second-, and third-generation CZ into practical use globally.

Tobacco is a valuable model system for investigating the origin of mitochondrial DNA (mtDNA) in amphidiploid plants and studying the genetic interaction between mitochondria and chloroplasts in the various functions of the plant cell. As a first step, we have determined the complete mtDNA sequence of Nicotiana tabacum. The mtDNA of N. tabacum can be assumed to be a master circle (MC) of 430,597 bp. Sequence comparison of a large number of clones revealed that there are four classes of boundaries derived from homologous recombination, which leads to a multipartite organization with two MCs and six subgenomic circles. The mtDNA of N. tabacum contains 36 protein-coding genes, three ribosomal RNA genes and 21 tRNA genes. Among the first class, we identified the genes rps1 and psirps14, which had previously been thought to be absent in tobacco mtDNA on the basis of Southern analysis. Tobacco mtDNA was compared with those of Arabidopsis thaliana, Beta vulgaris, Oryza sativa and Brassica napus. Since repeated sequences show no homology to each other among the five angiosperms, it can be supposed that these were independently acquired by each species during the evolution of angiosperms. The gene order and the sequences of intergenic spacers in mtDNA also differ widely among the five angiosperms, indicating multiple reorganizations of genome structure during the evolution of higher plants. Among the conserved genes, the same potential conserved nonanucleotide-motif-type promoter could only be postulated for rrn18-rrn5 in four of the dicotyledonous plants, suggesting that a coding sequence does not necessarily move with the promoter upon reorganization of the mitochondrial genome.

The three-way catalytic performance (TWC performance) and the NO x purification activity through storage-reduction (NSR activity) over a Pt/Ba/(Al 2 O 3 -CeO 2 -Fe 2 O 3 ) catalyst were investigated in comparison with a Pt/Ba/(Al 2 O 3 -CeO 2 ) catalyst. Two kinds of reducing agents, C 3 H 6 and CO, were used for the measurement of the NSR activity. There was no difference in the TWC performance and the NSR activity between these two fresh catalysts. After thermal aging at 1123 K, however, the aged Pt/Ba/(Al 2 O 3 -CeO 2 -Fe 2 O 3 ) catalyst was found to show a higher TWC performance and a higher NSR activity in the case of using CO as a reducing agent than did the aged Pt/Ba/(Al 2 O 3 -CeO 2 ) catalyst, although there was no difference in the NSR activity in the case of using C 3 H 6 as a reducing agent between these two aged catalysts. Oxygen storage-release capacity measurement showed that an Fe-compound on the aged Pt/Ba/(Al 2 O 3 -CeO 2 -Fe 2 O 3 ) catalyst acted to store oxygen more effectively than did CeO 2 . Reaction analysis of a simple binary gas system showed that the Fe-compound weakened the CO self-poisoning for the CO–O 2 reaction and the CO–NO x reaction under reducing conditions, probably by provision of oxygen to the CO from Fe 2 O 3 or/and Fe in a Pt–Fe alloy. The more effective storing of oxygen and the weaker CO self-poisoning for the CO–O 2 reaction and the CO–NO x reaction lead to a higher TWC performance after thermal aging. The weaker CO self-poisoning for the CO–NO x reaction also leads to a greater ability to reduce stored NO x on the catalysts by CO under reducing conditions, causing a higher NSR activity in the case of using CO as a reducing agent after thermal aging.

The CeO 2 -ZrO 2 solid solution is the most important oxygen storage/release material for use in automobile catalysts. In particular, the ceria-zirconia solid solution with a pyrochlore structure exhibits the highest oxygen storage capacity (OSC). In this study, CeO 2 -ZrO 2 solid solutions containing Y 2 O 3 were synthesized and their OSCs were measured to clarify the influence of an ordered arrangement of Ce and Zr ions on the OSC. As a result of this study, it is concluded that an ordered arrangement of Ce and Zr ions markedly improves the OSC of CeO 2 -ZrO 2 solid solutions.

As an important oxygen storage material, ceria-zirconia solid solution is widely used in automotive three-way catalysts. For the Ce-Zr solid solution being usually used with a noble metal, the evaluation of oxygen storage capacity (OSC) should be conducted with the Ce-Zr solid solution loaded with a noble metal. The measured OSC of noble-metal-loaded Ce-Zr solid solution consists of two parts: the stored oxygen within the solid solution itself and the adsorbed oxygen on the loaded noble metal particles. Therefore, it is very necessary to clarify the influence of metal loading on the measured OSC in order to ascertain the exact OSC of the solid solution itself. In this research, the authors attempted to clarify the above issues by characterizing the OSC performance of Ce-Zr solid solutions with platinum (Pt) loading from 0.0001-10 mass%. We found that increasing the amount of Pt loading increases the oxygen storage and release rates and that increasing the temperature also increases the oxygen storage rate, although the oxygen release rate is not affected by increasing the temperature. The apparent OSC reached saturation over a certain amount of Pt loading and temperature. The saturated OSC increased with increasing temperature.

Ceria-zirconia (Ce-Zr) solid solution is widely used as an oxygen storage material, which is a key component in the automotive three-way catalysts. The Ce-Zr solid solution is usually used with noble metals supported on their surface. Thus the evaluation of oxygen storage capacity (OSC) should be conducted on the Ce-Zr solid solution loaded with a noble metal. The measured OSC of noble-metal-loaded Ce-Zr solid solution consists of two parts; one is the stored oxygen within the solid solution itself, and the other is the adsorbed oxygen on the surface of the supported noble metal particles. Therefore, it is necessary to clarify the influence of the noble metal on the measured OSC, and this will also help to ascertain the exact OSC of the solid solution itself. In this research, the authors tried to clarify the above issues by characterizing the OSC performance on Ce-Zr solid solutions with Pt loading from 0.0001 to 10 wt%. We found that, increasing the Pt loading makes the oxygen storage and release rate higher. Temperature increase also makes the oxygen storage rate higher, however, the oxygen release rate is not affected by the temperature. The apparent OSC saturated above a certain level of Pt loading and temperature. The saturated OSC increased with increasing temperature.

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.

In vivo, we have already shown that only two of the 5S rDNA array blocks of the Arabidopsis thaliana genome produce the mature 5S rRNAs. Deletions and point mutations were introduced in an Arabidopsis 5S rDNA-transcribed region and its 5'- and 3'-flanks in order to analyse their effects on transcription activity. In vitro transcription revealed different transcription control regions. One control region essential for transcription initiation was identified in the 5'-flanking sequence. The major sequence determinants were a TATA-like motif (-28 to -23), a GC dinucleotide (-12 to -11), a 3-bp AT-rich region (-4 to -2) and a C residue at -1. They are important for both accurate transcription initiation and transcription efficiency. Transcription level was regulated by polymerase III (Pol III) re-initiation rate as in tRNA genes in which TATA-like motif is involved. Active 5S rDNA transcription additionally required an intragenic promoter composed of an A-box, an Intermediate Element (IE) and a C-box. Double-stranded oligonucleotides corresponding to different fragments of the transcribed region, used as competitors, revealed the main importance of internal promoter elements. A stretch of four T is sufficient for transcription termination. Transcription of Arabidopsis 5S rDNA requires 30 bp of 5'-flanking region, a promoter internal to the transcribed region, and a stretch of T for transcription termination.

Most prokaryotic mRNAs contain within the 5' untranslated region (UTR), a Shine-Dalgarno (SD) sequence, which is complementary to the 3' end of 16S rRNA and serves as a major determinant for correct translational initiation. The tobacco chloroplast rps2 mRNA possesses an SD-like sequence (GGAG) at a proper position (positions -8 to -5 from the start codon). Using an in vitro translation system from isolated tobacco chloroplasts, the role of this sequence in translation was examined. Unexpectedly, the mutation of the SD-like element resulted in a large increase in translation. Internal and external deletions within the 5' UTR revealed that the region from -20 to -5 was involved in the negative regulation of translation. Scanning mutagenesis assays confirmed the above result. Competition assays suggested the existence of a trans-acting factor(s) involved in translational regulation. In this study, we discuss a possible mechanism for the negative regulation of rps2 mRNA translation.

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.

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.

Genes encoding chlorophyll a/b-binding proteins of photosystem II (Lhcb) constitute a multigene family. Nine Lhcb1 genes have previously been isolated from the tobacco species, Nicotiana sylvestris, and the transcription initiation sites in vivo have been mapped. Reaction conditions from a previously developed in vitro transcription system from tobacco cultured cells were optimized for the Lhcb1 genes. Transcription initiation sites in vitro predominantly coincided with those found in vivo and were typically cytidines, a system unique to N. sylvestris Lhcb1 genes. CTC*A (C* for initiation site) was a consensus motif for the initiation region in vitro, as reported in vivo. Mutation analysis defined functionally that the TATA box is essential for transcription initiation and that the CTCA motif is a determinant of transcription initiation sites but not transcript levels. Polyadenylation sites were determined from in vivo transcripts, located 12-21 nt downstream from likely poly(A) signals. Four major 3'-ends of in vitro transcripts from Lhcb1*6 were detected, 40-300 nt downstream of the poly(A) site, suggestive of multiple, discrete transcription termination sites. These 3'-ends are mapped in or nearby T-rich sequences.

The presence of chloroplast DNA was established in 1963. With the development of recombinant DNA technologies, chloroplast DNA was selected as one of the first candidates for genome sequencing. The first physical map was reported for maize chloroplasts in 1976. As tobacco has been popular as an experimental system, tobacco chloroplast DNA has been extensively analyzed and the complete nucleotide sequence was established in 1986. This sequencing and the availability of tobacco chloroplast transformation techniques and of in vitro expression systems have formed the basis of an ongoing functional genomics program.

This paper reviews progress in the development of oxygen storage materials for automotive exhaust catalysts. The research was mainly conducted as a study and development exercise in the author's laboratory in Japan. Ceria-lanthana solid solutions (CL) and the first generation of ceriazirconia solid solutions (CZ) were developed as excellent oxygen storage materials for automotive catalysts in the 1980s. These materials consist of ceria doped with less than 20 mol% of La4+ or Zr4+. An increase in oxygen defects in CL and CZ under reductive conditions is responsible for an enhanced oxygen storage capability on the cerium atoms. An accurate measure of the oxygen storage capacity (OSC) per cerium is very important for theoretical and practical treatments of the catalyst. The term “partial OSC” was introduced to describe this capacity and to differentiate it from the usual definition of the OSC, known also as the “total OSC”. After the development of CL and CZ, a new technology was developed to dissolve more than 20 mol% of zirconia in the ceria, allowing second generation CZ and third generation CZ (known as ACZ, which is doped with alumina) to be successfully developed in the 1990s. The partial OSC of these materials increases with increasing amounts of zirconia dissolved in the ceria, and also with decreasing material particle size after an engine durability test. In the case of ACZ, alumina was added to CZ based on the “diffusion barrier concept”, in which a diffusion barrier layer inhibits the coagulation of CZ and A when the material is required for duty at high temperature in air. Furthermore, the relationship between the total or partial OSC and the structure of the ceriazirconia solid solutions is explained in this paper. For ceriazirconia solid solutions composed of equimolar CeO2 and ZrO2(Ce/Zr=1), the total or partial OSC of the κ-phase CeZrO4, in which the cerium and zirconium ions are regularly distributed, was about twice as large as that of a ceriazirconia solid solution with a relatively irregular distribution of cerium and zirconium ions, and about five times larger than that of a mixture of ceria powder and zirconia containing only a small amount of ceriazirconia solid solution. It corresponds to about 89% of the theoretical maximum value. For a ceriazirconia solid solution composed of non-equimolar CeO2 and ZrO2(Ce/Zr ≠ 1), the partial OSC of a ceria-κ-phase solid solution with a zirconia content of between 30 and 50mol% is much higher than that of a ceriazirconia solid solution of the same zirconia content. The partial OSC of a κ-phase and zirconia mixed oxide, which is formed by reducing the material at 1200 °C, reaches a value above 0.20 mol-O2/mol-Ce (about 80% of the theoretical maximum value of the partial OSC), when the zirconia content is between 50 and 80 mol%. The Toyota Motor Corp. has put automotive three-way catalysts containing the first, second and third generations of CZ into practical use on a global basis.

A new method to measure the amount of oxygen storage in/release from catalysts on a millisecond scale per two CO pulses (MS-OSC) has been developed. The MS-OSC measurement is based on the principle of the fast detection of reaction products on catalyst surfaces just after CO and O 2 injections using pulsed valves by time-of-flight mass spectrometry. Each catalyst was slurry-coated on planar cordierite substrate. MS-OSC was measured under high-vacuum (10 −7 Pa) conditions, which allowed the MS-OSC to be directly measured as the amount of CO 2 without diffusion effects. We applied the MS-OSC to the following three catalysts: Pt/CeO 2 –ZrO 2 mixture, Pt/CeO 2 –ZrO 2 solid solution, and Pt/CeO 2 –ZrO 2 –Y 2 O 3 (Pt/CZY) solid solution. Pt/CZY was reported to improve the transient catalytic activity on a millisecond scale, which could not be explained by the result of conventional thermogravimetric OSC measurements on a second scale. From a comparison of the three catalysts, the MS-OSC of Pt/CZY was found to be the highest. This result is supported by a previously reported engine test result (H. Tanaka, I. Tan, K. Yamada, and M. Yamamoto, ASATA 98ATE018, 1998).

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.

Nine Lhcb1 genes encoding the light-harvesting chlorophyll a/b-binding proteins of photosystem II were isolated and characterized from Nicotiana sylvestris. Their nucleotide sequences are highly similar. Lhcb1 transcripts are accumulated in leaves and stems but not in roots and non-green cultured cells. RNase protection assay revealed that no transcripts were detected from the gene, Lhcb1*2, in Nicotiana tabacum. This finding raises the possibility that the amphidiploid tobacco cultivar (N. tabacum) lost one gene from the female progenitor (N. sylvestris) during evolution. Transcriptional initiation sites were mapped and found to be mostly cytidine residues, which is unique to the N. sylvestris Lhcb1 genes. Four of the nine genes have single start sites and the remaining genes possess multiple initiation sites. The TATA-like sequences of nine Lhcb1 genes can be classified into two groups; one that possesses a TTTATA sequence and the other that has a sequence diverged from it. The genes with single initiation sites belong to the first group. A consensus motif for the initiation region is CTC*A (C* for initiation site), which differs from those of other plant genes or mammalian genes.

RNA polymerase III-driven cassettes for the expression of antisense RNAs and ribozymes have recently attracted much attention because (1) pol III genes are transcribed abundantly in all kinds of tissues and (2) the transcripts are very stable by virtue of their small and compact size. We have designed two types of pol III-based expression vehicles. Antisense RNA sequences targeted against conserved structural elements or domains in the RNAs of potato spindle tuber viroid, hop latent viroid and potato virus S were either embedded in the anticodon region of a Nicotiana tRNATyr gene or near the 3′ end of an Arabidopsis 7SL RNA gene. Both classes of chimeric genes were transcribed in vitro in a homologous plant extract. Our studies clearly revealed that the modified tRNA and 7SL RNA genes, carrying insertions of up to 90 and 120 bp, respectively, were expressed efficiently in the tobacco nuclear extract, resulting in high levels of stable chimeric transcripts. 7SL RNA (also termed SRP RNA) represents the RNA component of the signal recognition particle. This is the first report of demonstrating the employment of 7SL RNA genes as potential cassettes for the expression of antisense RNA and ribozyme sequences and might be helpful in future experiments to control their localization in specific sub-cellular compartments.

Three types of CeO2–ZrO2 (Ce:Zr=1:1 molar ratio) compounds with different oxygen storage/release capacities (OSCs) were characterized by means of the Ce K-edge and Zr K-edge X-ray absorption fine structure (XAFS). In order to investigate the relationship between the OSC and local structure, the quantitative EXAFS curve-fitting analysis was applied. By enhancing the homogeneity of the Ce and Zr atoms in the CeO2–ZrO2 solid solution, the OSC performance increased. Especially, the atomically homogeneous Ce0.5Zr0.5O2 solid solution exhibited the highest OSC among these CeO2–ZrO2 samples. Additionally, the local oxygen environment around Ce and Zr was remarkably modified by enhancing the homogeneity of the CeO2–ZrO2 solid solution. It was postulated that the enhancement of the homogeneity of the CeO2–ZrO2 solid solution and the modification of the oxygen environment would be the source for the OSC improvement.

The function of oxygen storage capacity (OSC) is very important to improve a performance for an automobile exhaust catalyst. CeO2-ZrO2 solid solution has an excellent OSC, and is available for a catalytic promoter. In this paper, OSC of the CO reduced CeO2-ZrO2 solid solution having various composition loaded with Pt was estimated by thermo gravimeter, and the phases and the crystalline structure of the reduced CeO2-ZrO2 solid solution were studied by X-ray diffraction method. The maximum total OSC-value of the reduced material occurs at the composition of 50mol% CeO2-50mol% ZrO2. The maximun value is about 753μmol O2/g-solid solution, which is about twice the value (416μmol O2/g-solid solution) of the attritionmilled CeO2-ZrO2 solid solution, and 5 times higher than that (150μmol O2/g-solid solution) of the material synthesized by conventional impregnation. The reduced material consists of Ce2Zr2O8 and CeO2 solid solution in the composition range from 30 to 50mol% ZrO2. The reduced material consists of the mixture, Ce2Zr2O8 and ZrO2 in the composition range from 50mol% ZrO2 to 100mol% ZrO2. The presence of Zr4+ near Ce4+ makes the expansion easier enhancing further valence change, because Zr4+ is very small in volume. Regular distribution of Ce and Zr ions would make it possible to place Zr4+ in the nearest position to Ce ion. This would be the reason why the partial OSC of Ce2Zr2O8 is much higher than the complete solid solution.

The oxidation behavior of reduced CeO2 and CeO2-ZrO2 solid solution (CZS) was studied by measuring the weight and temperature change during oxidization, in the near-room-temperature range. A flaming oxidation at close to room temperature was observed in some of the reduced CZS. This oxidation takes place in the CZS reduced at much lower temperatures, 800°C and 1000°C, compared with CeO2, for which the temperature of 1400°C or so is required for the occurrence of the flaming oxidation. The oxidation was not observed in the CZS reduced at a temperature higher than 1200°C. The material, which exhibited the flaming oxidation, possessed a high oxygen storage capacity (OSC) and a large specific surface area. The dissolution of ZrO2 into CeO2 results in an increase of OSC and in the retardation of grain growth. These two effects lead reduced CZS to a flaming oxidation at close to room temperature.

What is an industrial researcher expecting from the use of synchrotron radiation at SPring-8? It seems that there are two answers. One expectation is that he is able to explain convincingly to his customers a distinctive advantage of the developed product in a visible manner, and the other is that he investigates essential quality of materials by returning to the starting point when he is at a loss what to do in the process of his RD thus he can gain the opportunity of making a technical breakthrough in his R&D. This is because SPring-8 provides the most powerful synchrotron radiation currently available. As a result, a researcher can understand the chemical-bonding states of the material structure at the atomic and electronic levels by utilizing new analytical tools such as XAFS (X-ray Absorption Fine Structure) and XRD (X-ray Diffraction), etc.

The chloroplast psbB operon includes five genes encoding photosystem II and cytochrome b 6 /f complex components. The psbN gene is located on the opposite strand. PsbN is localized in the thylakoid and is present even in the dark, although its level increases upon illumination and then decreases. However, the translation mechanism of the psbN mRNA remains unclear. Using an in vitro translation system from tobacco chloroplasts and a green fluorescent protein as a reporter protein, we show that translation occurs from a tobacco primary psbN 5′-UTR of 47 nucleotides (nt). Unlike many other chloroplast 5′-UTRs, the psbN 5′-UTR has two processing sites, at −39 and −24 upstream from the initiation site. Processing at −39 enhanced the translation rate fivefold. In contrast, processing at −24 did not affect the translation rate. These observations suggest that the two distinct processing events regulate, at least in part, the level of PsbN during development. The psbN 5′-UTR has no Shine–Dalgarno (SD)-like sequence. In vitro translation assays with excess amounts of the psbN 5′-UTR or with deleted psbN 5′-UTR sequences demonstrated that protein factors are required for translation and that their binding site is an 18 nt sequence in the 5′-UTR. Mobility shift assays using 10 other chloroplast 5′-UTRs suggested that common or similar proteins are involved in translation of a set of mRNAs lacking SD-like sequences.

Objective: To investigate the risk of bladder cancer with pioglitazone, the data of diabetic patients treated at Teikyo Chiba Medical Center were analyzed retrospectively. Methods: From February 2009 to October 2011, 720 patients were treated with pioglitazone, and 742 patients were not treated with pioglitazone at Teikyo University Chiba Medical Center. The numbers of newly diagnosed bladder cancers from February 2009 to December 2013 in these patients and the occurrence of bladder cancer among type 2 diabetes mellitus patients were identified. The duration of diabetes, sex, age, smoking, and medication for diabetes were obtained from the medical records. The occurrence of bladder cancer was defined as incident cases after the initiation of pioglitazone. Statistical significance was analyzed by Fisher’s exact test. Results: About 2% of diabetic patients had bladder cancer. The incidence of bladder cancer was not different between the pioglitazone group (15/720, 2.0%) and the no pioglitazone group (14/742, 1.9%). No oral diabetes medication was related to the risk of bladder cancer. However, use of insulin was significantly related to bladder cancer risk (hazard ratio [HR]: 2.83; 95% confidence interval [CI]: 1.15–6.84; p=0.0246), and sex was also significantly related to bladder cancer (HR: 6.001; 95% CI: 1.397–43.06; p=0.0137). The duration of diabetes and smoking were not associated with bladder cancer risk. Conclusion: In this study, use of pioglitazone was not related to bladder cancer risk, while use of insulin was related

Transcription is an important control point in the transposable element mobilization process. To better understand the regulation of the plant SINE (Short Interspersed Elements) S1, its promoter sequence was studied using an in vitro pol III transcription system derived from tobacco cells. We show that the internal S1 promoter can be functional although upstream external sequences were found to enhance this basal level of transcription. For one putative 'master' locus (na7), three CAA triplets (in positions -12, -7 and -2) and two overlapping TATA motifs (in positions -54 to -43) were important to stimulate transcription. For this locus, two transcription initiation regions were characterized, one centered on position + 1 (first nucleotide of the S1 element) and one centered on position - 19 independently of the internal motifs. The CAA triplets only influence transcription in + 1 and work in association with the internal motifs. We show that methylation can inhibit transcription at the na7 locus. We also observe that S1 RNA is cleaved in a smaller Poly (A) minus product by a process analogous to the maturation of mammalian SINEs.

Nuclear tRNA genes are transcribed by RNA polymerase III (Pol III) and pre-tRNAs are processed into mature tRNAs via complex processes in the nucleus. We have developed an in vitro Pol III-dependent transcription system derived from tobacco cultured cells, which supports efficiently not only transcription of a variety of plant tRNA genes but also 5'-and 3'-end processing, nucleotide modification and splicing of intron-containing pre-tRNAs. The structures of in vitro transcripts have been confirmed by primer extension analysis and by RNase T1 fingerprinting. The optimal Mg2+ concentration differed for each step so that each reaction can be controlled by adjusting the Mg2+ concentration. At 1 mm Mg2+, only transcription occurs so that pre-tRNAs accumulate. The splicing reaction can be initiated by raising Mg2+ ions (> 5 mm) and enhanced by adding 1 mm hexamminecobalt chloride. Using the optimized system for the Nicotiana intron-containing tRNATyr gene, the precise initiation and termination sites of transcription and the splice sites were determined. The presence of 1 mm NAD+ in the reaction mixture leads to the removal of the 2' phosphate at the splice junction of tRNATyr, demonstrating the activity of a 2'-phosphotransferase in the tobacco nuclear extract. Many modified nucleosides such as m2G, m22G, m1A, phi27 and phi35 are introduced in either of the studied transcripts. As shown in other systems, the conversion of U35 to phi requires an intron-containing substrate.

We have isolated and sequenced a nuclear gene and cDNA encoding bacteriophage T7-type single subunit RNA polymerase, NsRpoT-A, from Nicotiana sylvestris. NsRpoT-A consists of 19 exons and 18 introns; the first intron is 17 kb, the longest yet identified in a plant gene. Genomic Southern analysis indicated that N. sylvestris contains a small family of NsRpoT genes. Quantitative RT-PCR revealed that steady-state mRNA levels are highest in the leaves and lowest in the cotyledons. Phylogenetic analysis of NsRpoT-A and the RpoT proteins of other plant species suggested that NsRpoT-A is a mitochondrial protein. The TargetP program predicted localization of the NsRpoT-A gene product to the mitochondria. Using a transient expression assay and protoplasts from N. sylvestris mesophyll cells, we clearly demonstrated that the N-terminal sequence of NsRpoT-A targets the protein to the mitochondria. We therefore named this protein NsRpoTm.

Transcripts of land plant chloroplast genomes undergo C-to-U RNA editing. Systematic search disclosed 31 editing sites in tobacco, 27 in maize, and 21 in rice. Based on these identified sites, potential editing sites have been predicted in the transcripts from four angiosperm chloroplast genomes which have been completely sequenced. Most RNA editing events occur in internal codons, which result in amino-acid substitutions. The initiation codon AUG was found to be created from ACG by RNA editing in the transcripts from rpl2, psbL, and ndhD genes. Comparison of editing patterns raises a possibility that many editing sites were acquired in the evolution of angiosperms.

A numerical model for lean NOx catalytic reaction in consideration of hydrocarbon adsorption and desorption process, which is capable of predicting the performance of diesel lean NOx catalyst using diesel fuel as a supplemental reductant, has been developed. This model is based on our simplified kinetics for the adsorption and desorption of hydrocarbons, for the oxidation of hydrocarbons, and for the reduction of NOx by hydrocarbons including the desorbed one. The adsorption of diesel exhaust hydrocarbons was expressed by the Freundlich-type isotherm that has the adsorption equilibrium constant of exponential temperature dependence. The behavior of HC and NOx reactions and HC adsorption and desorption in diesel exhaust has been simulated successfully.

The NO x storage-reduction catalysis under oxidizing conditions in the presence of SO 2 has been investigated on Pt/Ba/Fe/Al 2 O 3 , Pt/Ba/Co/Al 2 O 3 , Pt/Ba/Ni/Al 2 O 3 , and Pt/Ba/Cu/Al 2 O 3 catalysts compared with Pt/Ba/Al 2 O 3 , Pt/Fe/Al 2 O 3 , Pt/Co/Al 2 O 3 , Pt/Ni/Al 2 O 3 , Pt/Cu/Al 2 O 3 and Pt/Al 2 O 3 catalysts. The NO x purification activity of Pt/Ba/Fe/Al 2 O 3 catalyst was the highest of all the catalysts investigated in this paper after an aging treatment. That of the aged Pt/Ba/Co/Al 2 O 3 and Pt/Ba/Ni/Al 2 O 3 catalysts was essentially the same as that of the aged Pt/Ba/Al 2 O 3 catalyst, while that of the aged Pt/Ba/Cu/Al 2 O 3 and Pt/Cu/Al 2 O 3 catalysts was substantially lower than the others. The Fe-compound on the aged Pt/Ba/Fe/Al 2 O 3 catalyst has played a role in decreasing the sulfur content on the catalyst after exposure to simulated reducing gas compared with the Pt/Ba/Al 2 O 3 catalyst without the Fe-compound. XRD and EDX show that the Fe-compound inhibits the growth in the size of BaSO 4 particles formed on the Pt/Ba/Fe/Al 2 O 3 catalyst under oxidizing conditions in the presence of SO 2 and promotes the decomposition of BaSO 4 and desorption of the sulfur compound under reducing conditions.

RNA editing in higher plant chloroplasts involves C→U conversion at ∼30 specific sites. An in vitro system supporting accurate editing has been developed from tobacco chloroplasts. Mutational analysis of substrate mRNAs derived from tobacco chloroplast psbL and ndhB mRNAs confirmed the participation of cis ‐acting elements that had previously been identified in vivo . Competition analysis revealed the existence of site‐specific trans ‐acting factors interacting with the corresponding upstream cis ‐elements. A chloroplast protein of 25 kDa was found to be specifically associated with the cis ‐element involved in psbL mRNA editing. Immunological analyses revealed that an additional factor, the chloroplast RNA‐binding protein cp31, is also required for RNA editing at multiple sites. This combination of site‐specific and common RNA‐binding proteins recognizes editing sites in chloroplasts.

The formation of ceria-zirconia solid solution by the solid phase reaction of ceria and zirconia powders near room temperature during wet attrition milling was recently reported. The solid phase reaction occurred as a result of a large plastic deformation, breaking and mutual combining on the ceria particle as well as the solid solution. The size of the particle in which the solid phase reaction can occur was examined in this study. The reaction that forms ceria-zirconia solid solution from large ceria particles (4m2/g) begins when the average particle size reaches 28nm or smaller. The reaction does not occur in CeO2 powder with particle size larger than 30nm. The critical particle size of ceria powder to begin the reaction with zirconia is thought to be approximately 20nm, which was found asymptotically by two methods, X-ray diffraction and specific surface area measurements.

The catalytic activity of Pt catalyst loaded on γ-alumina was improved by Ba addition in simulated automotive exhaust gases. On the other hand, the result of Rh catalyst was the opposite. From the results of the partial reaction orders in C3H6–O2 reaction and TPR, it was concluded that the Ba addition to Pt catalyst suppressed the hydrocarbon chemisorption on the Pt catalyst and therefore allowed the catalytic reaction to proceed smoothly. On the other hand, Ba addition to Rh catalyst caused such a strong oxygen adsorption on Rh that rejected the hydrocarbon adsorption and suppressed the reaction.

The entire nucleotide sequence of the chloroplast genome has been determined from 12 land plants. The gene content and arrangement are relatively uniform from species to species, and the genome contains an average of 111 identified gene species (except Epifagus). Chloroplast genes can be classified into three main categories: Genes for the photosynthetic apparatus, those for the transcription/translation system, and those related to biosyntheses. The genes encoding components of the photosynthesis apparatus have been identified by protein chemical analyses from higher plants, Chlamydomonas and cyanobacteria, and then by chloroplast transformation techniques using tobacco and Chlamydomonas. The genes for subunits of RNA polymerases and of ribosomes were initially deduced similarity to those in E. coli, and later confirmed by protein analyses. Coding information is often modified at the level of transcripts by RNA editing (mostly C-U changes), resulting in amino acid substitutions and creation of novel reading frames. Perspectives of chloroplast genomics are discussed.

Capability of oxygen store and release of ceria-zirconia solid solution made by attrition mill process was measured by thermo-gravimeter with 1mass% of Pt. Ceria-zirconia solid solution made by conventional heating method of ceria powder with zirconium salt was also characterized for comparison. Capability of oxygen store and release of ceria-zirconia solid solution made by attrition mill process is about three times larger than that of conventionally made samples. And, the point, where zirconium content is 100% on the relation between capability of oxygen store and release and zirconia content in the ceria-zirconia solid solution, corresponds to the state in which the valence of all the cerium ion being contained in the solid solution is 3. This fact suggests that the mechanism, that the increase of capability of oxygen store and release by dissolving zirconia into ceria, is that zirconium ions, which are smaller than cerium ions of the valence 4, release the strain caused by the expansion due to a reduction of cerium ions.

The effect of alteration of 5' and 3' flanking sequences on the transcription of plant tRNA genes was analysed using an RNA polymerase III-dependent in vitro transcription system derived from nuclei of cultured tobacco cells. A TATA-like sequence and the CAA motif frequently observed upstream of plant tRNA genes, and the poly(T) stretch usually present downstream, were shown to be necessary for efficient re-initiation of transcription. The CAA motif was shown to be a transcription initiation site. Introduction of the CAA and TATA-like motifs into a gene naturally lacking them greatly enhanced transcription by promoting efficient re-initiation.

The surprisingly low temperature (-323K) synthesis of the ceria-zirconia solid solution upon milling ceria powder with zirconia mill and zirconia balls (in ethanol and/or water) was studied. Solid solutions up to 60mol%ZrO2 were obtained, whose formation was proved to result from solid phase reaction between ceria and zirconia powders, enhanced by contact stress (either shear or compressive one). Furthermore, the occurrence of large plastic deformation, breaking and mutual combining was found on the ceria powder before and after solid solution. The grain size of ceria-zirconia solid solution was ≤20nm, which would cause on easy rearrangement of low diffusion atoms of cerium and zirconium to form the solid solution at such a low temperature. In addition, the possible existence of a solid solution for which the migration of the constituents is much faster (or the stability of the solid at the composition is much higher) than that of other composition, was suggested around 50mol%CeO2-50mol%ZrO2.

Calcined sepiolite-supported Pt (Pt/CSp) catalysts indicate the high activity for complete oxidation of hydrocarbons (HC). The simultaneous oxidation of HC and SO 2 on the Fe-impregnated calcined sepiolite-supported Pt (Pt/Fe-CSp) catalysts was studied in comparison with that on Pt/CSp catalysts. There was a negligible difference in HC activity. Nevertheless, SO 2 oxidation activity on the Pt/Fe-CSp catalyst was suppressed more than that on the Pt/CSp catalyst. The calcination temperature of sepiolite affects the quantity of Fe additive. XRD and NMR show that the quantity of Fe additive depends on the distortion of the local structure of the calcined sepiolite. At the same time, the quantity of Fe additive affects the SO 2 oxidation activity. Fresh Pt/Fe-CSp catalyst absorbs SO 2 . The absorbed SO 2 reacted with Mg of CSp to form MgSO 4 on the catalyst. After this absorption was saturated, the selectivity of the oxidation was maintained. XPS and Mossbauer spectroscopy show that most of Fe on the Pt/Fe-CSp catalyst is Fe 3+ Pulsed chemisorption analysis of the redox cycle shows that Fe on the calcined sepiolite has a high oxygen storage capacity (OSC). This redox reaction suggests the mechanism of this selective oxidation reaction. The selective oxidation property is expected to be useful for a catalyst of purification for exhaust gas from a boiler or a combustion engine using a fuel which contains sulfur.

Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNATyr GΨ A and elongator tRNAMet CmAU contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNATyr. Here we have studied the expression of an Arabidopsis elongator tRNAMet gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNAMet precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNAMet to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3′ and 5′ splice sites and of a structured intron for pre-tRNAMet splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNAMet splicing and that a highly structured intron is indispensable for pre-tRNAMet splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNAMet gene, is efficiently processed and spliced in both plant extracts.