The chloroplast genome of Nicotiana sylvestris and Nicotiana tomentosiformis: complete sequencing confirms that the Nicotiana sylvestris progenitor is the maternal genome donor of Nicotiana tabacum.

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.

Comparative analysis of RNA editing sites in higher plant chloroplasts.

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.

The genomics of land plant chloroplasts: Gene content and alteration of genomic information by RNA editing.

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.

A graphic tool for circular genome maps.

A program has been developed for drawing map of circular DNA such as organelle or plasmid genome. Total size of the genome, gene names and positions, and other details, if required, should be prepared in a simple format text file then the program process it to a PostScript(R) (PS) file with which you can print a image of the map on suitable device(s). The final touch on the map can be given through editing the PS file.

RNA editing sites in tobacco chloroplast transcripts: editing as a possible regulator of chloroplast RNA polymerase activity.

Genetic information in chloroplast DNA is sometimes altered at the transcript level by a process known as RNA editing. Sequence analysis of amplified cDNAs for 69 potential editing sites revealed 13 real editing sites in transcripts of 11 tobacco chloroplast genes. Together with those reported previously, these bring the total of edited sites observed in tobacco chloroplast transcripts to 31 (all involve C to U conversion). Alignment of sequences around the 31 editing sites revealed no obvious consensus, apart from an apparent bias for U or C at position -1 and A at position +2. Editing in tobacco rpoA mRNA restores the conserved leucine residue which is known to be important for transcriptional activation of the alpha subunit of E. coli RNA polymerase. Editing of this site is partial and the extent of editing depends on developmental conditions, suggesting that editing is, at least in part, involved in the regulation of chloroplast-encoded RNA polymerase activity.

Complete nucleotide sequence of the chloroplast genome from the green alga Chlorella vulgaris: the existence of genes possibly involved in chloroplast division.

The complete nucleotide sequence of the chloroplast genome (150,613 bp) from the unicellular green alga Chlorella vulgaris C-27 has been determined. The genome contains no large inverted repeat and has one copy of rRNA gene cluster consisting of 16S, 23S, and 5S rRNA genes. It contains 31 tRNA genes, of which the tRNALeu(GAG) gene has not been found in land plant chloroplast DNAs analyzed so far. Sixty-nine protein genes and eight ORFs conserved with those found in land plant chloroplasts have also been found. The most striking is the existence of two adjacent genes homologous to bacterial genes involved in cell division, minD and minE, which are arranged in the same order in Escherichia coli. This finding suggests that the mechanism of chloroplast division is similar to bacterial division. Other than minD and minE homologues, genes encoding ribosomal proteins L5, L12, L19, and S9 (rpl5, rpl12, rpl19, and rps9); a chlorophyll biosynthesis Mg chelating subunit (chlI); and elongation factor EF-Tu (tufA), which have not been reported from land plant chloroplast DNAs, are present in this genome. However, many of the new chloroplast genes recently found in red and brown algae have not been found in C. vulgaris. Furthermore, this algal species possesses two long ORFs related to ycf1 and ycf2 that are exclusively found in land plants. These observations suggest that C. vulgaris is closer to land plants than to red and brown algae.

Creation of a novel protein-coding region at the RNA level in black pine chloroplasts: the pattern of RNA editing in the gymnosperm chloroplast is different from that in angiosperms.

The phenomenon of RNA editing has been found to occur in chloroplasts of several angiosperm plants. Comparative analysis of the entire nucleotide sequence of a gymnosperm [Pinus thunbergii (black pine)] chloroplast genome allowed us to predict several potential editing sites in its transcripts. Forty-nine such sites from 14 genes/ORFs were analyzed by sequencing both cDNAs from the transcripts and the corresponding chloroplast DNA regions, and 26 RNA editing sites were identified in the transcripts from 12 genes/ORFs, indicating that chloroplast RNA editing is not restricted to angiosperms but occurs in the gymnosperm, too. All the RNA editing events are C-to-U conversions; however, many new codon substitutions and creation of stop codons that have not so far been reported in angiosperm chloroplasts were observed. The most striking is that two editing events result in the creation of an initiation and a stop codon within a single transcript, leading to the formation of a new reading frame of 33 codons. The predicted product is highly homologous to that deduced from the ycf7 gene (ORF31), which is conserved in the chloroplast genomes of many other plant species.

Occurrence of silent RNA editing in chloroplasts: its species specificity and the influence of environmental and developmental conditions.

We have identified three new C-to-U RNA editing sites, one in atpF and two in atpA transcripts from tobacco chloroplasts. Two of them lead to amino acid substitutions to restore the conserved amino acid found in the corresponding genes of other plants. However, one editing site in the atpA transcript was found to take place partially at the third base of a serine codon (CUC_ to CUU_), thus not leading to an amino acid substitution. This is the first report of silent editing in chloroplasts. The extent of silent editing depends on plastid stage and light conditions, while editing as another site (found 4 nt upstream from the silent editing site) takes place constitutively even in non-photosynthetic cultured cells and bleached white seedlings grown in the presence of spectinomycin and streptomycin. In pea and spinach, despite a conservation in sequence, no editing at the site corresponding to the silent site in tobacco was found. This observation suggests that the silent editing detected in this study is species-specific.

Loss of all ndh genes as determined by sequencing the entire chloroplast genome of the black pine Pinus thunbergii.

The complete nucleotide sequence (119,707 bp) of the black pine (Pinus thunbergii) chloroplast genome has been determined. It contains 4 rRNA genes and 32 tRNA genes. To our knowledge, the tRNAPro (GGG) gene has not been found in any other chloroplast genome analyzed. Sixty-one genes encoding proteins and 11 conserved open reading frames are also found. Extensive rearrangements are apparent in the chloroplast genome relative to those of other land plants. The most striking feature is the loss of all 11 functional genes (ndh genes) for subunits of a putative NADH dehydrogenase that are found in the chloroplast genomes of angiosperms and a bryophyte. Four ndh genes were completely lost and the other 7 genes remain as obvious pseudogenes. This unexpected finding raises the possibility that all ndh genes have been transferred to the nucleus or that an NADH dehydrogenase is not essential in black pine chloroplasts.

A new gene encoding tRNA(Pro) (GGG) is present in the chloroplast genome of black pine: a compilation of 32 tRNA genes from black pine chloroplasts.

The chloroplast genome of black pine (Pinus thunbergii), a gymnosperm, contains 32 different tRNA genes, 30 of which correspond to those previously identified in tobacco and rice chloroplast genomes. Two additional genes encode tRNA(Pro) (GGG) and tRNA(Arg) (CCG); the former is newly identified while the latter is present in liverwort, Physcomitrella patens and Angiopteris lygodiifolia, chloroplast genomes. Moreover, a partial copy of the split tRNA(Gly) (UCC) gene and full copies of tRNA(His) (GUG), tRNA(Thr) (GGU) and tRNA(Ser) (GCU) genes are present in the large single-copy region of the genome, suggesting extensive rearrangements of the chloroplast genome during evolution. No tRNA genes whose tRNA products can recognize codons CUU/C (Leu) and GCU/C (Ala) have been found. We propose that the 32 tRNAs are sufficient to read all the 61 sense codons in the black pine system using the "two-out-of-three" and the "U:N wobble" mechanisms.

Chloroplast DNA of black pine retains a residual inverted repeat lacking rRNA genes: nucleotide sequences of trnQ, trnK, psbA, trnI and trnH and the absence of rps16.

A physical map of black pine (Pinus thunbergii) chloroplast DNA (120 kb) was constructed and two separate portions of its nucleotide sequence were determined. One portion contains trnQ-UUG, ORF510, ORF83, trnK-UUU (ORF515 in the trnK intron), ORF22, psbA, trnI-CAU (on the opposing strand) and trnH-GUG, in that order. Sequence analysis of another portion revealed the presence of a 495 bp inverted repeat containing trnI-CAU and the 3' end of psbA but lacking rRNA genes. The position of trnI-CAU is unique because most chloroplast DNAs have no gene between psbA and trnH (trnI-CAU is usually located further downstream). Black pine chloroplast DNA lacks rps16, which has been found between trnQ and trnK in angiosperm chloroplast DNAs, but possesses ORF510 instead. This ORF is highly homologous to ORF513 found in the corresponding region of liverwort chloroplast DNA and ORF563 located downstream from trnT in Chlamydomonas moewusii chloroplast DNA. A possible pathway for the evolution of black pine chloroplast DNA is discussed.

Glutaric aciduria type II: autopsy study of a case with electron-transferring flavoprotein dehydrogenase deficiency.

An autopsy study of glutaric aciduria type II in a 62-day-old Japanese boy is presented. The diagnosis was made by analysis of organic acids in the urine. Immunoblot analysis of liver homogenate confirmed the diagnosis, revealing absence of electron-transferring flavoprotein dehydrogenase. The major findings were fatty changes of variable degree in many organs and tissues, the most severe being found in cardiac myocytes, hepatocytes, renal tubular epithelium, and skeletal muscle fibers. Other pertinent findings included multicystic and dysplastic kidney, pulmonary alveolar proteinosis, and spongiosis and gliosis of the spinal cord. The thymus was markedly depleted, and lymphocytes in the lymph nodes were mainly B cells. Although some of these changes may have been secondary to the sepsis and immunosuppression complicating 2 months of intensive care, the abnormal organic acid metabolism with severe acidosis may have been a significant contributing factor.

Changes in physicochemical properties of mitochondrial membranes during the formation process of megamitochondria induced by hydrazine.

Changes in some biochemical and physico-chemical properties of rat liver mitochondrial membranes during the formation process of megamitochondria induced by hydrazine were analyzed. Hepatic mitochondria obtained from rats placed on a 1% hydrazine diet for 3 days became slightly enlarged and sometimes elongated, while they became gigantic after 7 days of hydrazine intoxication. Changes were observed in mitochondria from rats treated with hydrazine for 3 days. Total amounts of phospholipids extracted from mitochondria and submitochondrial fractions were increased. Among phospholipid species, relative amounts of acidic phospholipids were increased. Contents of Ca2+ in mitochondria were increased. Differential scanning calorimetric analysis of mitochondria, especially that of the outer membrane fraction, showed that the thermotropic lipid phase transition temperatures were elevated accompanying the broadening of thermograms and the increase in transition enthalpy. Contents of water in mitochondria were increased significantly with the ratio of freezable water to unfreezable water unchanged. Among the changes observed was that the total amount of phospholipids (except for that of the outer membrane fraction) and the contents of water and Ca2+ nearly returned to normal in megamitochondria after 7 days of hydrazine intoxication. Relative amounts of phospholipids and thermotropic lipid phase transition temperatures of megamitochondria did not return to normal levels and yet changes were smaller than those obtained from 3 days of hydrazine intoxication. The fluidity of mitochondrial membranes was not affected by hydrazine treatment. These data would suggest that hydrazine-induced megamitochondrial formation is not due simply to the swelling of mitochondria, but might be due to the fusion of adjacent mitochondria by Ca2+-acidic phospholipid interactions, and once megamitochondria are formed the mitochondrial membranes are stabilized.

Delta pH-dependent and -independent uptake of [3H]dopamine by synaptic vesicles isolated from rat brain.

The dopamine (DA)-translocating mechanism of synaptic vesicles isolated from rat brain has been studied in the presence of an artificially imposed delta pH on the vesicle membranes (acidic inside with respect to the external medium) without the aid of ATP-Mg2+. Under the experimental conditions, [3H]DA uptake by the synaptic vesicles was driven by two different, i.e. delta pH-dependent and -independent processes. Both processes appeared to be carrier-mediated based on the inhibition by NEM (N-ethylmaleimide), an -SH reagent, and by nomifensine, a DA uptake blocker at nerve terminals. The DA carrier of the vesicles was similar to that of the nerve terminal plasma membrane with respect to their susceptibility to nomifensine. The delta pH-dependent uptake was transient and most of the incorporated DA was easily lost from the vesicles. On the other hand, the delta pH-independent uptake increased with time and the amine was retained in the vesicles. The initial rate of the delta pH-independent uptake was lower than that of the delta pH-dependent one but their extents were comparable with each other. These results indicate that rat brain synaptic vesicles have a DA uptake system requiring no ATP hydrolysis. A preparation of synaptic vesicles used here exhibited an inwardly directed proton translocation in the presence of ATP-Mg2+ when monitored by following changes in the fluorescence of ANS (8-anilino-1-naphthalene sulfonate). However, the time course of the delta pH-generation was not influenced by the addition of 1 mM DA.(ABSTRACT TRUNCATED AT 250 WORDS)

The Ca2+-dependent dissociation of [3H]dopamine bound to an acidic glycoprotein present in the synaptosomal cytoplasm of rat brain.

The interaction of [3H]dopamine with acidic proteins present in the synaptosomal cytoplasm of rat brain was studied from the point of view of Ca2+-dependent release of dopamine as a neurotransmitter. Some of the proteins were glycoproteins and showed various affinities for [3H]dopamine. [3H]Dopamine bound to an acidic protein with a molecular weight of about 45,000 daltons became reversibly unbound in the presence of Ca2+, while that bound to other proteins did not. The amount of [3H]dopamine bound to the 45,000-dalton glycoprotein was 48.7 pmol/mg protein, and the Ca2+-concentration required to release half of the bound dopamine was found to be 0.2 to 0.25 mM under the experimental conditions. However, Ca2+ did not bind to any component of the acidic proteins and it is likely that the cation dissociates the bound dopamine by forming a complex with it. No calmodulin (-like protein) was detectable among the acidic proteins. Possible roles of the glycoproteins are discussed in relation to a Ca2+-dependent mechanism of neurotransmitter release.

Newly synthesized L-[14C]dopamine failed to accumulate immediately in synaptic vesicles within isolated rat brain synaptosomal cytoplasm.

It was examined whether or not enzymically synthesized L-dopamine could be immediately incorporated and stored in synaptic vesicles within a synaptosomal cytoplasm fraction isolated from the rat brain. The synaptosomal cytoplasm was isotonically prepared by disrupting the synaptosomes in 0.32 M sucrose using an N2-gas pressure homogenizer. Synaptic vesicles present in the isolated synaptosomal cytoplasm appeared to be osmotically sensitive (i.e., not leaky vesicles) under iso-osmotic conditions. DOPA decarboxylase [EC 4.1.1.28] present in the soluble cytosol region synthesized L-[14C]dopamine from added L-[14C]DOPA. However, the newly synthesized L-[14C]dopamine was not immediately incorporated in the synaptic vesicles within the synaptosomal cytoplasm during the reaction period of 20 min at 30 degrees C. Plain synaptic vesicles prepared by the method of Kadota and Kadota (10) also failed to take up the newly synthesized dopamine under the same conditions. It was found that soluble protein(s) in the synaptosomal cytoplasm was able to bind as much added L-[3H]dopamine as 35 pmol per mg of protein, and the binding ability seemed to inhibit the amine uptake by synaptic vesicles within the synaptosomal cytoplasm. The [3H]dopamine bound to the protein(s) became unbound in the presence of Ca2+ (5 mM). This effect of Ca2+ may have an important role in the releasing process of neurotransmitters.

Studies on the uptake of [3H]dopamine by synaptic vesicle fraction isolated from bovine brain.

1. A synaptic vesicle fraction isolated from bovine caudatolenticular nuclei showed enzymic activities of tyrosine hydroxylase [EC 1.14.16.2] and dopamine beta-hydroxylase [EC 1.14.17.1]. Tyrosine hydroxylase, whose subcellular localization is uncertain, appeared to be associated with the synaptic vesicles. 2. The vesicle fraction took up [3H]dopamine increasingly with time without the aid of ATP (6.3 pmol of [3H]dopamine per mg of vesicle proteins, or 3-4% of the added dopamine, at 30 min). In the presence of ATP, a transient accumulation of the amine was observed, reaching the highest level at 7-10 min (5.6 pmol dopamine/mg protein), and then a rapid release of the amine took place, obeying first-order kinetics with respect to the amine concentration. The amine uptake was strongly inhibited with NEM, regardless of the presence or absence of ATP. 3. The vesicle fraction also exhibited a weak ability to translocate protons inward and ATP-dependently, as monitored by an increase in the fluorescence intensity of ANS. The fluorescence enhancement persisted for at least 30 min and this time-dependent change was not consistent with that of the transient accumulation of [3H]dopamine mentioned above. Since the present vesicle preparation contained a small amount of mitochondrial ATPase (18-20% of the total activity), the proton translocating ability could be attributable to contaminating submitochondrial particles. 4. Therefore these results made it impossible to conclude that the transient uptake of [3H]dopamine by the synaptic vesicles was coupled to ATP hydrolysis.

Adenosine triphosphate-dependent calcium uptake of synaptic vesicle fraction is largely due to contaminating microsomes.

Ca2+ uptake by synaptic vesicle fractions isolated from bovine caudatolenticular nuclei and from rat brain was studied. The purified vesicle fractions from both materials took up very little Ca2+ even in the presence of ATP and Mg2+, but the crude fractions took up Ca2+ actively, showing the maximum uptake around pH 7.0. Since the crude fractions were contaminated by microsomes, which are known to accumulate Ca2+ actively (Yoshida, H., Kadota, K., & Fujisawa, H. (1966) Nature 212, 291--292; Otsuka, M., Ohtsuki, I., & Ebashi, S. (1965) J. Biochem. 58, 188-190), the active uptake of Ca2+ appeared to be largely, if not wholly, due to microsomal contamination.