The complete sequence of the rice (Oryza sativa L.) mitochondrial genome: frequent DNA sequence acquisition and loss during the evolution of flowering plants.

The entire mitochondrial genome of rice (Oryza sativa L.), a monocot plant, has been sequenced. It was found to comprise 490,520 bp, with an average G+C content of 43.8%. Three rRNA genes, 17 tRNA genes and five pseudo tRNA sequences were identified. In addition, eleven ribosomal protein genes and two pseudo ribosomal protein genes were found, which are homologous to 13 of the 16 genes for ribosomal proteins in the mitochondrial genome of the liverwort (Marchantia polymorpha). A greater degree of variation in terms of presence/absence and integrity of genes was observed among the ribosomal protein genes and tRNA genes of rice, Arabidopsis and sugar beet. Transcription and post-transcriptional modification (RNA editing) in the rice mitochondrial sequence were also examined. In all, 491 Cs in the genomic DNA were converted to Ts in cDNA. The frequency of RNA editing differed markedly depending upon the ORF considered. Sequences derived from plastid and nuclear genomes make up 6.3% and 13.4% of the mitochondrial genome, respectively. The degree of conservation of plastid sequences in the mitochondrial genome ranged from 61% to 100%, suggesting that sequence migration has occurred very frequently. Three plastid DNA fragments that were incorporated into the mitochondrial genome were subsequently transferred to the nuclear genome. Nineteen fragments that were similar to transposon or retrotransposon sequences, but different from those found in the mitochondrial genomes of dicots, were identified. The results indicate frequent and independent DNA sequence flow to and from the mitochondrial genome during the evolution of flowering plants, and this may account for the range of genetic variation observed between the mitochondrial genomes of higher plants.

The gene for alternative oxidase-2 (AOX2) from Arabidopsis thaliana consists of five exons unlike other AOX genes and is transcribed at an early stage during germination.

We investigated the expressions of genes for alternative oxidase (AOX1a, AOX1b, AOX1c and AOX2) and genes for cytochrome c oxidase (COX5b and COX6b) during germination of Arabidopsis thaliana, and examined oxygen uptakes of the alternative respiration and the cytochrome respiration in imbibed Arabidopsis seeds. A Northern blot analysis showed that AOX2 mRNA has already accumulated in dry seeds and subsequently decreased, whereas accumulation ofAOX1a mRNA was less abundant from 0 hours to 48 hours after imbibition and then increased. The increase of the capacity of the alternative pathway appeared to be dependent on the expressions of both AOX2 and AOX1a. On the other hand, steady-state mRNA levels of COX5b and COX6b were gradually increased during germination, and the capacity of the cytochrome pathway was correlated with the increase of expressions of the COX genes. Antimycin A, the respiratory inhibitor, strongly increased the expression of AOX1a but had no effect on the expression of AOX2. A 5'RACE analysis showed that AOX2 consists of five exons, which is different from the case of most AOX genes identified so far. Analysis of subcellular localization of AOX2 using green fluorescent protein indicated that the AOX2 protein is imported into the mitochondria.

Characterization and expression of the genes for cytochrome c oxidase subunit VIb (COX6b) from rice and Arabidopsis thaliana.

Many of the subunits of cytochrome c oxidase (COX) in the mitochondria of higher plants are encoded by nuclear genes. These genes are less characterized compared to mitochondrial-encoded genes. We previously isolated a cDNA encoding COX6b (designated OsCOX6b1 in this study) from the rice nuclear genome and analyzed its expression. The deduced protein had an extended N-terminus compared with human and yeast COX6b proteins. In this study, we identified another COX6b gene (OsCOX6b2) in rice and revealed that it was actually expressed. The deduced protein of this gene did not have an extended N-terminus and had about the same size as the human and yeast proteins. Genomic Southern hybridization analysis revealed that there was at least one OsCOX6b-homologus sequences in the rice genome other than OsCOX6b1 and OsCOX6b2. Furthermore, we identified three COX6b genes in a dicotyledonous plant, Arabidopsis thaliana. One of these genes (AtCOX6b1) was relatively long, with a length similar to that of OsCOX6b1, and the other two (AtCOX6b2 and AtCOX6b3) were shorter, with lengths similar to the length of OsCOX6b2. Genomic Southern hybridization analysis indicated there were no additional COX6b genes in the Arabidopsis genome. The coding regions of OsCOX6b1 and AtCOX6b1 were separated by four introns and those of OsCOX6b2, AtCOX6b2 and AtCOX6b3 were separated by three introns. A Northern hybridization analysis showed that OsCOX6b1, AtCOX6b1 and AtCOX6b3 were expressed in all organs examined, although with some differences in the amount of expression among the organs. OsCOX6b2 and AtCOX6b2 were strongly expressed in roots but most of the transcripts of AtCOX6b2 were degraded. The evolution of COX6b genes from rice and Arabidopsis is discussed.

[Clinical efficacy of leuprolide acetate and combined treatment with estramustine for advanced prostate cancer].

BACKGROUND/PURPOSE: Twenty-two institutes have organized Keio University Prostate Cancer Study Group to study clinical efficacy and safety of Leuprolide acetate (Leuplin) for the treatment of advanced prostate cancer (clinical stage D1 and D2). Cotreatment of Leuplin and Estramustine phosphate disodium (Estracyt) has been performed to investigate its clinical efficacy. MATERIALS AND METHODS: One hundred and two cases of advanced prostate cancer were treated either with Leuplin alone (group I), Leuplin and Estracyt (group II) or Estracyt alone (group III). After 12 weeks treatment, clinical effects against subjective symptoms (pain, voiding difficulty, performance status and body weight), serum testosterone level, tumor size and serum PSA level were examined to investigate short-term effect of each treatment. The treatment had been continued for 24 months and the treatment effects including progression free survival and overall survival were analyzed. RESULTS: Clinical efficacy after 12 weeks treatment were examined among 97 cases (group I; 35 cases, group II; 36 cases, group III; 26 cases). The background of those patients in each group was statistically equal. Treatment effects against subjective symptoms and serum testosterone level statistically revealed no significant difference among 3 groups. Treatment effects against primary tumor, bone metastatic lesion, lymphnode metastatic lesion and serum PSA level were investigated and anti-tumor effect was characterized by total efficacy rate (complete remission rate plus partial remission rate) of each treatment group. Treatment efficacy rates for each lesion and PSA demonstrated no statistical difference among 3 treatment groups. Total efficacy rate of group I, II and III were 88.2%, 84.0% and 78.3%, respectively, which statistically revealed no significant difference. Total efficacy rate of each group after completing 24 months treatment was; group I 80.0%, group II 55.6% and group III 83.3%, which statistically showed no significant difference among 3 treatment groups. The median day for progression free survival of group I, II and III were 661, 731 and 517, respectively. The overall survival rate of group I, II and III after completing 24 months treatment were 77.5%, 83.0% and 72.4%, respectively. Both progression free survival rates and overall survival rates revealed no significant difference among 3 groups. Side effects during 24 months treatment were seen in 8.6% of group I, 47.2% of group II and 26.9% of group III, and these occurrence rates were significantly different among the groups (p = 0.0013). CONCLUSION: Although number of the cases had not been able to continue the treatment for their side effects, the statistical characterization demonstrated that cotreatment of Leuplin and Estracyt had no greater treatment effect than monotreatment of each drug.

Expression of a gene encoding mitochondrial aldehyde dehydrogenase in rice increases under submerged conditions.

It is known that alcoholic fermentation is important for survival of plants under anaerobic conditions. Acetaldehyde, one of the intermediates of alcoholic fermentation, is not only reduced by alcohol dehydrogenase but also can be oxidized by aldehyde dehydrogenase (ALDH). To determine whether ALDH plays a role in anaerobic metabolism in rice (Oryza sativa L. cv Nipponbare), we characterized a cDNA clone encoding mitochondrial ALDH from rice (Aldh2a). Analysis of sub-cellular localization of ALDH2a protein using green fluorescent protein and an in vitro ALDH assay using protein extracts from Escherichia coli cells that overexpressed ALDH2a indicated that ALDH2a functions in the oxidation of acetaldehyde in mitochondria. A Southern-blot analysis indicated that mitochondrial ALDH is encoded by at least two genes in rice. We found that the Aldh2a mRNA was present at high levels in leaves of dark-grown seedlings, mature leaf sheaths, and panicles. It is interesting that expression of the rice Aldh2a gene, unlike the expression of the tobacco (Nicotiana tabacum) Aldh2a gene, was induced in rice seedlings by submergence. Experiments with ruthenium red, which is a blocker of Ca(2+) fluxes in rice as well as maize (Zea mays), suggest that the induction of expression of Adh1 and Pdc1 by low oxygen stress is regulated by elevation of the cytosolic Ca(2+) level. However, the induction of Aldh2a gene expression may not be controlled by the cytosolic Ca(2+) level elevation. A possible involvement of ALDH2a in the submergence tolerance of rice is discussed.

Molecular and cellular characterizations of a cDNA clone encoding a novel isozyme of aldehyde dehydrogenase from rice.

Aldehyde dehydrogenases (ALDHs) are a group of enzymes catalyzing the conversion of aldehydes to the corresponding acids. In mammals and yeasts, at least two isozymes of ALDH are known to be involved in ethanol metabolism (cytosolic ALDH1 and mitochondrial ALDH2). Although mitochondrial ALDH isozymes have previously been identified in several plants, such as maize and tobacco, it is unclear whether cytosolic ALDH isozymes also exist in plants. In this study, we identified and characterized a cDNA clone encoding aldehyde dehydrogenase (ALDH1a) from rice (Oryza sativa L. cv. Nipponbare). The open reading frame of this clone did not contain a typical mitochondrial targeting signal. Analysis of the subcellular localization of ALDH1a using green fluorescent protein (GFP) suggested that ALDH1a is a cytosolic enzyme rather than a mitochondrial enzyme. A genomic Southern hybridization indicated that sequences homologous to the ALDH1a gene are present in at least two regions of the rice genome. Amplification by RT-PCR showed that ALDH1a is expressed strongly in roots, but not in leaves, of rice seedlings, suggesting that ALDH1a functions in roots.

Transcript levels of the nuclear-encoded respiratory genes in rice decrease by oxygen deprivation: evidence for involvement of calcium in expression of the alternative oxidase 1a gene.

We investigated the effect of oxygen on the expressions of respiratory genes encoded in the nuclear and mitochondrial genomes of rice (Oryza sativa L.). Hypoxic treatment decreased the transcript levels of nuclear-encoded, but not mitochondrial-encoded respiratory genes. The effects of ruthenium red (an inhibitor of Ca(2+) fluxes from organelles) and/or CaCl(2) on plants under hypoxic conditions suggested that Ca(2+) is a physiological transducer of a low-oxygen signaling pathway for expression of the alternative oxidase 1a gene (AOX1a), but not for expressions of genes involved in the cytochrome respiratory pathway, in rice.

Antibody-mediated insulin resistance treated by cessation of insulin administration.

A 45-year-old Japanese man was referred to our hospital because of hyperglycemia despite the administration of as much as 120 U/day of human insulin. He had no history of injecting animal insulin. Free insulin was below 5 microU/ml, but a high titer of total insulin (about 3,000 microU/ml) was observed, suggesting the presence of antibodies against human insulin. Scatchard analysis showed an increased insulin binding capacity in the plasma characterized by a higher affinity for insulin. He was successfully treated by cessation of insulin administration. A Scatchard analysis series showed that a reduction in the insulin binding capacity of antibodies paralleled the improvement in glycemic control.

Characterization of two cDNA clones encoding isozymes of the F(1)F(0)-ATPase inhibitor protein of rice mitochondria.

Two cDNA clones encoding F(1)F(0)-ATPase inhibitor proteins, which are loosely associated with the F(1) part of the mitochondrial F(1)F(0)-ATPase, were characterized from rice (Oryza sativa L. cv. Nipponbare). A Northern hybridization showed that the two genes (designated as IF(1)-1 and IF(1)-2) are transcribed in all the organs examined. However, the steady-state mRNA levels varied among organs. A comparison of the deduced amino acid sequences of the two IF(1) genes and the amino acid sequence of the mature IF(1) protein from potato revealed that IF(1)-1 and IF(1)-2 have N-terminal extensions with features that are characteristic of a mitochondrial targeting signal. To determine the subcellular localization of the gene products, the IF(1)-1 or IF(1)-2 proteins were fused in frame to the green fluorescent protein (GFP) or the fused GFP-beta-glucuronidase, and expressed transiently in onion or dayflower epidermal cells. Localized fluorescence was detected in mitochondria, confirming that the two IF(1) proteins are targeted to mitochondria.

Identification of cDNA encoding cytochrome c oxidase subunit 5c (COX5c) from rice: comparison of its expression with nuclear-encoded and mitochondrial-encoded COX genes.

Little is presently known about the nuclear-encoded genes for cytochrome c oxidase (COX) in higher plants. In rice, only the nuclear-encoded COX5b gene has been reported. To understand the relationship between the expression of nuclear-encoded and mitochondrial-encoded COX genes in rice, we first characterized a cDNA encoding one of the other nuclear COX genes, COX5c, which encodes 63 amino acids. The deduced amino acid sequence of COX5c from rice was highly homologous to that from sweet potato. Genomic Southern hybridization indicated that the rice COX5c subunit is encoded by a single copy of the COX5c gene. Furthermore, we compared the expression patterns of the nuclear-encoded COX5c and COX5b genes with the expression pattern of the mitochondrial-encoded COX1 gene among several organs by Northern blot analysis. The results suggested that regulatory systems of expression between the nuclear-encoded and the mitochondrial-encoded COX genes are different among different organs in rice.

A novel plant nuclear gene encoding chloroplast ribosomal protein S9 has a transit peptide related to that of rice chloroplast ribosomal protein L12.

We have cloned a novel nuclear gene for a ribosomal protein of rice and Arabidopsis that is like the bacterial ribosomal protein S9. To determine the subcellular localization of the gene product, we fused the N-terminal region and green fluorescent protein and expressed it transiently in rice seedlings. Localized fluorescence was detectable only in chloroplasts, indicating that this nuclear gene encodes chloroplast ribosomal protein S9. The N-terminal region of rice ribosomal protein S9 was found to have a high sequence similarity to the transit peptide region of the rice chloroplast ribosomal protein L12, suggesting that these transit peptides have a common lineage.

AtUCP2: a novel isoform of the mitochondrial uncoupling protein of Arabidopsis thaliana.

Mitochondrial uncoupling proteins (UCPs) play a central role in adaptive thermogenesis in mammals. The UCPs dissipate the proton gradient formed through respiration without ATP synthesis, and the freed energy is readily converted to heat, helping the animals to maintain their body temperature in cold environments. Recently, it was found that UCPs also function in plant mitochondria. Subsequently, a cDNA clone encoding a UCP in potato was isolated. Whereas the UCP gene constitutes a multigene family in mammals, only a single cDNA sequence has been reported so far for the potato UCP. Moreover, it has been recently suggested that Arabidopsis has only a single nuclear gene for UCP. Here we report the existence of another UCP gene in the Arabidopsis genome, showing for the first time the occurrence of a multigene family for the protein in higher plants. A cDNA analysis of this gene showed that the novel isoform possesses all typical features reported for known UCPs. However, the new gene, unlike the other gene in Arabidopsis and the gene in potato, did not appear to respond to low temperature.

Transcription of plastid-derived tRNA genes in rice mitochondria.

Previous investigations located nine of the genes for rice tRNAs on plastid (pt)-derived sequences in mitochondrial DNA. In the present study, we examined whether these genes were also transcribed in rice mitochondria. Northern-blot hybridization revealed that seven of these genes (trnC, trnF, trnH, trnM, trnN, trnS and trnW) are transcribed and are precisely processed to mature tRNAs. One of the other two genes (trnP) is transcribed but cannot be processed efficiently, while the other (trnR), which has 100% identity to the original plastid tRNAArg gene, is not transcribed in rice mitochondria. These results suggest that seven of the nine pt-derived tRNAs may be utilized for the biosynthesis of protein in plant mitochondria.

A chloroplast derived trnH gene is expressed in the mitochondrial genome of gramineous plants.

We reported previously that the mitochondrial sequence that contains the chloroplast-derived trnH gene has been highly conserved in the region around one terminus of the junction between chloroplast-derived and mitochondrion-specific sequences in most of the gramineous plants analyzed [15]. The results of RT-PCR, northern hybridization, in vitro capping and ribonuclease protection experiments show that the chloroplast-derived trnH gene is transcribed from a putative promoter that is located in the mitochondrion-specific sequence. Gene expression in this region seems to be correlated with the conservation of the sequence at the junction between the chloroplast-derived fragment and the mitochondrion-specific sequence.

[Establishment of two renal cancer cell lines].

Two renal cell carcinoma (RCC) cell lines, JMSU2 and JMSU3, derived from the primary sites of mixed cell type and spindle cell type RCC, respectively, have been established and maintained for 31 and 22 months. Karyotypic analysis revealed human karyotypes with modal numbers of 84 and 55, respectively. Consistent chromosomal abnormalities were 1p+, 3p-, 6q- or 8p- in the JMSU2 cells and 1p-, inv (5p + q-) or loss of sex chromosome in the JMSU3 cells. Electron microscopy revealed abundant glycogen granules, lipid droplets and microvilli. The JMSU3 cells transplanted to nude mice produced tumors with a spindle cell pattern similar to that of the original tumor. High concentrations of cytokines, such as interleukin-6 (145,000 pg/ml), interleukin-8 (35,300 pg/ml) and granulocyte-colony stimulating factor (6,340 pg/ml), were detected in the culture supernatant of the JMSU3 cells. Interleukin-1 beta (IL-1 beta) dose-dependently inhibited the proliferation of the JMSU2 and JMSU3 cells in culture. Tumor cytotoxic factor/hepatocyte growth factor (TCF/HGF) dose-dependently enhanced JMSU3 cell proliferation, but suppressed JMSU2 cell proliferation. These findings suggest that IL-1 beta and TCF/HGF have regulatory roles in the proliferation of RCC.

Characterization of the gene family for alternative oxidase from Arabidopsis thaliana.

We investigated the copy number of the gene for alternative oxidase (AOX) of Arabidopsis thaliana by amplification by PCR and Southern hybridization. These studies indicated that there are at least four copies of the AOX gene in Arabidopsis. We isolated genomic clones containing individual copies (designated as AOX1a, AOX1b, AOX1c and AOX2) of the AOX genes. Interestingly, two of the AOX genes (AOX1a and AOX1b) were located in tandem in a ca. 5 kb region on one of the chromosomes of Arabidopsis. Comparison between genomic and cDNA sequences of the four AOX genes showed that all AOX genes are divided by three introns and the positions of the introns in AOX1a, AOX1b, AOX1c and AOX2 are the same. We examined whether expression of Arabidopsis AOX genes, like the tobacco AOX1a gene, is enhanced by treatment with antimycin A, an inhibitor of complex III in the mitochondrial respiratory chain. We found that, in young plants, the amount of Arabidopsis AOX1a mRNA was dramatically increased by addition of antimycin A, while the transcription of the other three genes (AOX1b, AOX1c and AOX2) did not respond to antimycin A. Amplification by RT-PCR showed that AOX1a and AOX1c were expressed in all organs examined (flowers and buds, stems, rosette, and roots of 8-week old plants). In contrast, transcripts of AOX1b were detected only in the flowers and buds, and transcripts of AOX2 were detected mainly in stems, rosette and roots. These results suggested that transcriptions of the four genes for alternative oxidase of Arabidopsis are differentially regulated.

Maintenance of chloroplast-derived sequences in the mitochondrial DNA of Gramineae.

Evidence for the transfer of DNA from the chloroplast to the mitochondrion has been reported in many higher plants and, in most cases, the transferred chloroplast genes do not have the ability to encode functional products as a consequence of base substitutions and/or multiple rearrangements. We reported previously that the sequence of one end of a chloroplast-derived (ct-derived) fragment of DNA that contained the rps19 and trnH genes has been maintained in most gramineous plants and that its presence seems to be correlated with gene expression in this region. In the present study, we have investigated whether or not the ct-derived sequences in mitochondrial DNA (mtDNA) from some gramineous plants and species of Oryza are conserved, and whether or not such conservation is related to gene expression in these regions. We identified two junctions between ct-derived and mitochondrial sequences that were conserved among some gramineous plants. Around these regions, we found a ct-derived gene for tRNA and the promoter of a mitochondrial gene on the ct-derived sequences, respectively, and these regions were transcribed through the junctions. This result indicates that the junctions and/or regions that are transcribed and functional in mitochondria have been strongly conserved and maintained during their evolution. In Oryza, some junctions between ct-derived and mitochondrial sequences were conserved and other junctions were not. These variations seem to have been caused by deletions and/or rearrangements, and appear to be specific to the type of genome. In the case of Oryza, the timing of deletions and/or rearrangements of ct-derived sequences is likely to have coincided with the divergence of the various genome types.

Transcript levels of tandem-arranged alternative oxidase genes in rice are increased by low temperature.

We identified two genes for alternative oxidase (AOX) from rice. One AOX gene (designated AOX1a) is located approx. 1.9 kb downstream of another AOX gene (designated AOX1b). Comparison of the genomic and cDNA sequences of the two AOX genes showed that the AOX1a gene is interrupted by three introns, as are AOX genes of other plants. On the other hand, two introns are inserted in the AOX1b gene. The predicted AOX1a and AOX1b precursor proteins consist of 332 and 335 amino acid residues, respectively. A genomic Southern hybridization analysis indicated that rice has several AOX genes other than the two tandem-arranged AOX genes. Steady-state mRNA levels of both of the genes for AOX1a and AOX1b were increased under low temperature (4 degrees C). However, no difference in the pattern of induction of transcription between the genes for AOX1a and AOX1b was observed.

A chloroplast-derived sequence is utilized as a source of promoter sequences for the gene for subunit 9 of NADH dehydrogenase (nad9) in rice mitochondria.

The chloroplast-derived sequence trnS-rps4/ 3'trnL-trnF-ndhJ-ndhK (4066 bases in length) is present in a region that starts 355 bases upstream of the gene for subunit 9 of NADH dehydrogenase (nad9) in the mitochondrial genome of rice. Northern blot hybridization revealed that three large transcripts of 3.05, 1.62 and 1.05 kb hybridized to strand-specific probes for both the nad9 gene and the chloroplast-derived sequence, indicating that the nad9 gene was transcribed together with the chloroplast-derived sequence. From the results of in vitro capping and ribonuclease protection experiments, as well as primer extension analysis, we identified at least seven sites for the initiation of transcription of nad9 in the chloroplast-derived sequence. All of the initiation sites for transcription of the nad9 gene were located in sequences homologous to chloroplast DNA. Two of seven initiation sites were flanked by a sequence homologous to the consensus promoter motif that includes the CRTA motif (where R is A or G) of the rice mitochondrion. However, the sequences surrounding the other five sites showed only limited similarity to the conserved sequence. It is suggested that all the promoters of the rice nad9 gene exist in a sequence that was transferred from the chloroplast during evolution. Thus, the chloroplast-derived sequence has a novel, significant function in the mitochondrial genome of this higher plant.

The gene for a subunit of an ABC-type heme transporter is transcribed together with the gene for subunit 6 of NADH dehydrogenase in rice mitochondria.

We previously identified a chloroplast-derived (ct-derived) sequence of 32 base pairs (bp) in rice mitochondrial DNA that includes a part (30 bp; psitrnI) of a gene for isoleucine tRNA (CAU) of the chloroplast. Analyzing the ct-derived psitrnI, we found that an open reading frame (orf240), which was homologous to the gene for a subunit of an ATP-binding cassette-type (ABC-type) heme transporter, namely helC, of Rhodobacter capsulatus, and a gene for subunit 6 of NADH dehydrogenase (nad6) were located upstream of and downstream from the ct-derived psitrnI, respectively. Northern-blot hybridization and analysis by reverse transcription-polymerase chain reaction (RT-PCR) revealed that both orf240 and nad6 were co-transcribed in rice mitochondria. An analysis of PCR-amplified fragments of the region of orf240/nad6 from the DNA of some Gramineae suggests that the arrangement of orf240/nad6 was generated in the mitochondrial genome of the genus Oryza during evolution after its divergence from the other Gramineae. Most of the transcripts of orf240 are edited, with a change from cytidine to uridine, at 35 positions. Editing of the RNA changes 33 amino-acid residues among the 240 encoded amino-acid residues, suggesting that the orf240 gene is functional in rice mitochondria.