Chloroplast group III twintron excision utilizing multiple 5''- and 3''-splice sites.

The chloroplast genes of Euglena gracilis contain more than 60 group II and 47 group III introns. Some Euglena chloroplast genes also contain twintrons, introns-within-introns. Two types of twintrons have previously been described, a group II twintron and a mixed group II/group III twintron. We report that four introns, three within the RNA polymerase subunit gene rpoC1 and one within ribosomal protein gene rpl16, with mean lengths twice typical group III introns, are a new type of twintron. The group III twintrons are composed of group III introns within other group III introns. The splicing of the twintrons was analyzed by PCR amplification, cloning and sequencing of cDNAs, and Northern hybridization. Excision of each group III twintron occurs by a two-step, sequential splicing pathway. Removal of the internal introns precedes excision of the external introns. Splicing of internal introns in three of the four group III twintrons involves multiple 5'- and/or 3'-splice sites. With two of the twintrons the proximal 5'-splice site can be spliced to an internal 3'-splice site, yielding alternative 'pseudo' fully spliced mRNAs. Excised group III introns of the rpl16 twintron are not linear RNA molecules but either lariat or circular RNAs, probably a lariat. The origins of alternative splicing and a possible evolutionary relationship between group II, group III and nuclear pre-mRNA introns are discussed.     

The complete nucleotide sequence of the coffee (Coffea arabica L.) chloroplast genome: organization and implications for biotechnology and phylogenetic relationships amongst angiosperms

The chloroplast genome sequence of Coffea arabica L., the first sequenced member of the fourth largest family of angiosperms, Rubiaceae, is reported. The genome is 155 189 bp in length, including a pair of inverted repeats of 25 943 bp. Of the 130 genes present, 112 are distinct and 18 are duplicated in the inverted repeat. The coding region comprises 79 protein genes, 29 transfer RNA genes, four ribosomal RNA genes and 18 genes containing introns (three with three exons). Repeat analysis revealed five direct and three inverted repeats of 30 bp or longer with a sequence identity of 90% or more. Comparisons of the coffee chloroplast genome with sequenced genomes of the closely related family Solanaceae indicated that coffee has a portion of rps19 duplicated in the inverted repeat and an intact copy of infA . Furthermore, whole-genome comparisons identified large indels (> 500 bp) in several intergenic spacer regions and introns in the Solanaceae, including trnE (UUC)– trnT (GGU) spacer, ycf4 – cemA spacer, trnI (GAU) intron and rrn5 – trnR (ACG) spacer. Phylogenetic analyses based on the DNA sequences of 61 protein-coding genes for 35 taxa, performed using both maximum parsimony and maximum likelihood methods, strongly supported the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids, asterids, eurosids II, and euasterids I and II. Coffea (Rubiaceae, Gentianales) is only the second order sampled from the euasterid I clade. The availability of the complete chloroplast genome of coffee provides regulatory and intergenic spacer sequences for utilization in chloroplast genetic engineering to improve this important crop.     

Euglena gracilis chloroplast ribosomal protein operon: a new chloroplast gene for ribosomal protein L5 and description of a novel organelle intron category designated group III

We describe the structure (3840 bp) of a novel Euglena gracilis chloroplast ribosomal protein operon that encodes the five genes rpl16-rpl14-rpl5-rps8-rpl36. The gene organization resembles the spc and the 3'-end of the S10 ribosomal protein operons of E. coli. The rpl5 is a new chloroplast gene not previously reported for any chloroplast genome to date and also not described as a nuclear-encoded, chloroplast protein gene. The operon contains at least 7 introns. We present evidence from primer extension analysis of chloroplast RNA for the correct in vivo splicing of five of the introns. Two of the introns within the rps8 gene flank an 8 bp exon, the smallest exon yet characterized in a chloroplast gene. Three introns resemble the classical group II introns of organelle genomes. The remaining 4 introns appear to be unique to the Euglena chloroplast DNA. They are uniform in size (95-109 nt), share common features with each other and are distinct from both group I and group II introns. We designate this new intron category as 'group III'.     

Characterization of the complete chloroplast genome of Hevea brasiliensis reveals genome rearrangement, RNA editing sites and phylogenetic relationships

Rubber tree (Hevea brasiliensis) is an economical plant and widely grown for natural rubber production. However, genomic research of rubber tree has lagged behind other species in the Euphorbiaceae family. We report the complete chloroplast genome sequence of rubber tree as being 161,191 bp in length including a pair of inverted repeats of 26,810 bp separated by a small single copy region of 18,362 bp and a large single copy region of 89,209 bp. The chloroplast genome contains 112 unique genes, 16 of which are duplicated in the inverted repeat. Of the 112 unique genes, 78 are predicted protein-coding genes, 4 are ribosomal RNA genes and 30 are tRNA genes. Relative to other plant chloroplast genomes, we observed a unique rearrangement in the rubber tree chloroplast genome: a 30-kb inversion between the trnE(UUC)-trnS(GCU) and the trnT(GGU)-trnR(UCU). A comparison between the rubber tree chloroplast genes and cDNA sequences revealed 51 RNA editing sites in which most (48 sites) were located in 26 protein coding genes and the other 3 sites were in introns. Phylogenetic analysis based on chloroplast genes demonstrated a close relationship between Hevea and Manihot in Euphorbiaceae and provided a strong support for a monophyletic group of the eurosid I.     

A mixed group II/group III twintron in the Euglena gracilis chloroplast ribosomal protein S3 gene: evidence for intron insertion during gene evolution.

The splicing of a 409 nucleotide intron from the Euglena gracilis chloroplast ribosomal protein S3 gene (rps3) was examined by cDNA cloning and sequencing, and northern hybridization. Based on the characterization of a partially spliced pre-mRNA, the intron was characterized as a 'mixed' twintron, composed of a 311 nucleotide group II intron internal to a 98 nucleotide group III intron. Twintron excision is via a 2-step sequential splicing pathway, with removal of the internal group II intron preceding excision of the external group III intron. Based on secondary structural analysis of the twintron, we propose that group III introns may represent highly degenerate versions of group II introns. The existence of twintrons is interpreted as evidence that group II introns were inserted during the evolution of Euglena chloroplast genes from a common ancestor with eubacteria, archaebacteria, cyanobacteria, and other chloroplasts.     

Complete structure of the chloroplast genome of Arabidopsis thaliana.

The complete nucleotide sequence of the chloroplast genome of Arabidopsis thaliana has been determined. The genome as a circular DNA composed of 154,478 bp containing a pair of inverted repeats of 26,264 bp, which are separated by small and large single copy regions of 17,780 bp and 84,170 bp, respectively. A total of 87 potential protein-coding genes including 8 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acid species were assigned to the genome on the basis of similarity to the chloroplast genes previously reported for other species. The translated amino acid sequences from respective potential protein-coding genes showed 63.9% to 100% sequence similarity to those of the corresponding genes in the chloroplast genome of Nicotiana tabacum, indicating the occurrence of significant diversity in the chloroplast genes between two dicot plants. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

Complete chloroplast genome sequences from Korean ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants.

The nucleotide sequence of Korean ginseng (Panax schinseng Nees) chloroplast genome has been completed (AY582139). The circular double-stranded DNA, which consists of 156,318 bp, contains a pair of inverted repeat regions (IRa and IRb) with 26,071 bp each, which are separated by small and large single copy regions of 86,106 bp and 18,070 bp, respectively. The inverted repeat region is further extended into a large single copy region which includes the 5' parts of the rpsl9 gene. Four short inversions associated with short palindromic sequences that form stem-loop structures were also observed in the chloroplast genome of P. schinseng compared to that of Nicotiana tabacum. The genome content and the relative positions of 114 genes (75 peptide-encoding genes, 30 tRNA genes, 4 rRNA genes, and 5 conserved open reading frames [ycfs]), however, are identical with the chloroplast DNA of N. tabacum. Sixteen genes contain one intron while two genes have two introns. Of these introns, only one (trnL-UAA) belongs to the self-splicing group I; all remaining introns have the characteristics of six domains belonging to group II. Eighteen simple sequence repeats have been identified from the chloroplast genome of Korean ginseng. Several of these SSR loci show infra-specific variations. A detailed comparison of 17 known completed chloroplast genomes from the vascular plants allowed the identification of evolutionary modes of coding segments and intron sequences, as well as the evaluation of the phylogenetic utilities of chloroplast genes. Furthermore, through the detailed comparisons of several chloroplast genomes, evolutionary hotspots predominated by the inversion end points, indel mutation events, and high frequencies of base substitutions were identified. Large-sized indels were often associated with direct repeats at the end of the sequences facilitating intra-molecular recombination.     

Complete structure of the chloroplast genome of a legume, Lotus japonicus.

The nucleotide sequence of the entire chloroplast genome (150,519 bp) of a legume, Lotus japonicus, has been determined. The circular double-stranded DNA contains a pair of inverted repeats of 25,156 bp which are separated by a small and a large single copy region of 18,271 bp and 81,936 bp, respectively. A total of 84 predicted protein-coding genes including 7 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids species were assigned on the genome based on similarity to genes previously identified in other chloroplasts. All the predicted genes were conserved among dicot plants except that rpl22, a gene encoding chloroplast ribosomal protein CL22, was missing in L. japonicus. Inversion of a 51-kb segment spanning rbcL to rpsl6 (positions 5161-56,176) in the large single copy region was observed in the chloroplast genome of L. japonicus. The sequence data and gene information are available on our World Wide Web database at http://www.kazusa.or.jp/en/plant/database.html.     

Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.     

Complete Chloroplast Genome of Tanaecium tetragonolobum: The First Bignoniaceae Plastome

Bignoniaceae is a Pantropical plant family that is especially abundant in the Neotropics. Members of the Bignoniaceae are diverse in many ecosystems and represent key components of the Tropical flora. Despite the ecological importance of the Bignoniaceae and all the efforts to reconstruct the phylogeny of this group, whole chloroplast genome information has not yet been reported for any members of the family. Here, we report the complete chloroplast genome sequence of Tanaecium tetragonolobum (Jacq.) L.G. Lohmann, which was reconstructed using de novo and referenced-based assembly of single-end reads generated by shotgun sequencing of total genomic DNA in an Illumina platform. The gene order and organization of the chloroplast genome of T. tetragonolobum exhibits the general structure of flowering plants, and is similar to other Lamiales chloroplast genomes. The chloroplast genome of T. tetragonolobum is a circular molecule of 153,776 base pairs (bp) with a quadripartite structure containing two single copy regions, a large single copy region (LSC, 84,612 bp) and a small single copy region (SSC, 17,586 bp) separated by inverted repeat regions (IRs, 25,789 bp). In addition, the chloroplast genome of T. tetragonolobum has 38.3% GC content and includes 121 genes, of which 86 are protein-coding, 31 are transfer RNA, and four are ribosomal RNA. The chloroplast genome of T. tetragonolobum presents a total of 47 tandem repeats and 347 simple sequence repeats (SSRs) with mononucleotides being the most common and di-, tri-, tetra-, and hexanucleotides occurring with less frequency. The results obtained here were compared to other chloroplast genomes of Lamiales available to date, providing new insight into the evolution of chloroplast genomes within Lamiales. Overall, the evolutionary rates of genes in Lamiales are lineage-, locus-, and region-specific, indicating that the evolutionary pattern of nucleotide substitution in chloroplast genomes of flowering plants is complex. The discovery of tandem repeats within T. tetragonolobum and the presence of divergent regions between chloroplast genomes of Lamiales provides the basis for the development of markers at various taxonomic levels. The newly developed markers have the potential to greatly improve the resolution of molecular phylogenies.     

Complete nucleotide sequence of the cotton (Gossypium barbadense L.) chloroplast genome with a comparative analysis of sequences among 9 dicot plants

Recently, the complete chloroplast genome sequences of many important crop plants were determined, and this can be considered a major step forward toward exploiting the usefulness of chloroplast genetic engineering technology. Economically, cotton is one of the most important crop plants for many countries. To further our understanding of this important crop, we determined the complete nucleotide sequence of the chloroplast genome from cotton (Gossypium barbadense L.). The chloroplast genome of cotton is 160,317 base pairs (bp) in length, and is composed of a large single copy (LSC) of 88,841 bp, a small single copy (SSC) of 20,294 bp, and two identical inverted repeat (IR) regions of 25,591 bp each. The genome contains 114 unique genes, of which 17 genes are duplicated in the IRs. In addition, many open reading frames (ORFs) and hypothetical chloroplast reading frames (ycfs) with unknown functions were deduced. Compared to the chloroplast genomes from 8 other dicot plants, the cotton chloroplast genome showed a high degree of similarity of the overall structure, gene organization, and gene content. Furthermore, the sequences of the genes showed high degrees of identity at the DNA and amino acid levels. The cotton chloroplast genome was somewhat longer than the chloroplast genomes of most of the other dicot plants compared here. However, this elongation of the cotton chloroplast genome was found to be due mainly to expansions of the intergenic regions and introns (non-coding DNA). Moreover, these expansions occurred predominantly in the LSC and SSC regions.     

A comparative analysis of the Lactuca and Helianthus (Asteraceae) plastid genomes: identification of divergent regions and categorization of shared repeats

We have sequenced two complete chloroplast genomes in the Asteraceae, Helianthus annuus (sunflower), and Lactuca sativa (lettuce), which belong to the distantly related subfamilies, Asteroideae and Cichorioideae, respectively. The Helianthus chloroplast genome is 151?104 bp and the Lactuca genome is 152?772 bp long, which is within the usual size range for chloroplast genomes in flowering plants. When compared to tobacco, both genomes have two inversions: a large 22.8-kb inversion and a smaller 3.3-kb inversion nested within it. Pairwise sequence divergence across all genes, introns, and spacers in Helianthus and Lactuca has resulted in the discovery of new, fast-evolving DNA sequences for use in species-level phylogenetics, such as the trnY-rpoB, trnL-rpl32, and ndhC-trnV spacers. Analysis and categorization of shared repeats resulted in seven classes useful for future repeat studies: double tandem repeats, three or more tandem repeats, direct repeats dispersed in the genome, repeats found in reverse complement orientation, hairpin loops, runs of A's or T's in excess of 12 bp, and gene or tRNA similarity. Results from BLAST searches of our genomic sequence against expressed sequence tag (EST) databases for both genomes produced eight likely RNA edited sites (C → U changes). These detailed analyses in Asteraceae contribute to a broader understanding of plastid evolution across flowering plants.     

Sequence and comparative analysis of the maize NB mitochondrial genome

The NB mitochondrial genome found in most fertile varieties of commercial maize (Zea mays subsp. mays) was sequenced. The 569,630-bp genome maps as a circle containing 58 identified genes encoding 33 known proteins, 3 ribosomal RNAs, and 21 tRNAs that recognize 14 amino acids. Among the 22 group II introns identified, 7 are trans-spliced. There are 121 open reading frames (ORFs) of at least 300 bp, only 3 of which exist in the mitochondrial genome of rice (Oryza sativa). In total, the identified mitochondrial genes, pseudogenes, ORFs, and cis-spliced introns extend over 127,555 bp (22.39%) of the genome. Integrated plastid DNA accounts for an additional 25,281 bp (4.44%) of the mitochondrial DNA, and phylogenetic analyses raise the possibility that copy correction with DNA from the plastid is an ongoing process. Although the genome contains six pairs of large repeats that cover 17.35% of the genome, small repeats (20-500 bp) account for only 5.59%, and transposable element sequences are extremely rare. MultiPip alignments show that maize mitochondrial DNA has little sequence similarity with other plant mitochondrial genomes, including that of rice, outside of the known functional genes. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly three-fourths of the maize NB mitochondrial genome is still of unknown origin and function.     

Complete mitochondrial genome of Oxuderces dentatus (Perciformes, Gobioidei)

The complete mitochondrial genome (mitogenome) of Oxuderces dentatus was determined first. The genome was 17,116?bp in length and consisted of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and 2 main non-coding regions [the control region (CR) and the origin of the light strand replication], the gene composition and order of which was similar to most other vertebrates. The overall base composition of the heavy strand was T 27.9%, C 26.8%, A 30.2%, and G 15.1%, with a slight A+T bias of 58.1%. In addition to the discrete and conserved sequence blocks, unusual long tandem repeat unit (three 150-bp tandem repeat units and an incomplete copy of 146?bp) was also detected within CR. This mitogenome sequence data would play an important role in population genetics and phylogenetic analysis of the Gobioidei.     

Complete sequence and organization of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome

The nucleotide sequence of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome was completed (DQ119058). The circular double-stranded DNA, consisting of 155,527 bp, contained a pair of inverted repeat regions (IRa and IRb) of 25,187 bp each, which were separated by small and large single copy regions of 86,879 and 18,274 bp, respectively. The presence and relative positions of 113 genes (76 peptide-encoding genes, 30 tRNA genes, four rRNA genes, and three conserved open reading frames) were identified. The major portion (55.76%) of the C. sativus chloroplast genome consisted of gene-coding regions (49.13% protein coding and 6.63% RNA regions; 27.81% LSC, 9.46% SSC and 18.49% IR regions), while intergenic spacers (including 20 introns) made up 44.24%. The overall G-C content of C. sativus chloroplast genome was 36.95%. Sixteen genes contained one intron, while two genes had two introns. The expansion/contraction manner of IR at IRb/LSC and IR/SSC border in Cucumis was similar to that of Lotus and Arabidopsis, and the manner at IRa/LSC was similar to Lotus and Nicotiana. In total, 56 simple sequence repeats (more than 10 bases) were identified in the C. sativus chloroplast genome.     

Group II and group III introns of twintrons: potential relationships with nuclear pre-mRNA introns

Two new and important features of introns have emerged from analysis of the Euglena gracilis chloroplast genome. One is a new class of introns, designated group III, that may be the closest contemporaries to nuclear pre-mRNA introns. The second is introns that are interrupted by other introns termed twintrons.     

Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome.

Analysis of the mitochondrial DNA of a liverwort Marchantia polymorpha by electron microscopy and restriction endonuclease mapping indicated that the liverwort mitochondrial genome was a single circular molecule of about 184,400 base-pairs. We have determined the complete sequence of the liverwort mitochondrial DNA and detected 94 possible genes in the sequence of 186,608 base-pairs. These included genes for three species of ribosomal RNA, 29 genes for 27 species of transfer RNA and 30 open reading frames (ORFs) for functionally known proteins (16 ribosomal proteins, 3 subunits of H(+)-ATPase, 3 subunits of cytochrome c oxidase, apocytochrome b protein and 7 subunits of NADH ubiquinone oxidoreductase). Three ORFs showed similarity to ORFs of unknown function in the mitochondrial genomes of other organisms. Furthermore, 29 ORFs were predicted as possible genes by using the index of G + C content in first, second and third letters of codons (42.0 +/- 10.9%, 37.0 +/- 13.2% and 26.4 +/- 9.4%, respectively) obtained from the codon usages of identified liverwort genes. To date, 32 introns belonging to either group I or group II intron have been found in the coding regions of 17 genes including ribosomal RNA genes (rrn18 and rrn26), a transfer RNA gene (trnS) and a pseudogene (psi nad7). RNA editing was apparently lacking in liverwort mitochondria since the nucleotide sequences of the liverwort mitochondrial DNA were well-conserved at the DNA level.     

Evidence for the late origin of introns in chloroplast genes from an evolutionary analysis of the genus Euglena.

The origin of present day introns is a subject of spirited debate. Any intron evolution theory must account for not only nuclear spliceosomal introns but also their antecedents. The evolution of group II introns is fundamental to this debate, since group II introns are the proposed progenitors of nuclear spliceosomal introns and are found in ancient genes from modern organisms. We have studied the evolution of chloroplast introns and twintrons (introns within introns) in the genus Euglena. Our hypothesis is that Euglena chloroplast introns arose late in the evolution of this lineage and that twintrons were formed by the insertion of one or more introns into existing introns. In the present study we find that 22 out of 26 introns surveyed in six different photosynthesis-related genes from the plastid DNA of Euglena gracilis are not present in one or more basally branching Euglena spp. These results are supportive of a late origin for Euglena chloroplast group II introns. The psbT gene in Euglena viridis, a basally branching Euglena species, contains a single intron in the identical position to a psbT twintron from E.gracilis, a derived species. The E.viridis intron, when compared with 99 other Euglena group II introns, is most similar to the external intron of the E.gracilis psbT twintron. Based on these data, the addition of introns to the ancestral psbT intron in the common ancester of E.viridis and E.gracilis gave rise to the psbT twintron in E.gracilis.