Structural features and transcript-editing analysis of sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) chloroplast genome

The complete nucleotide sequence of the chloroplast genome of sugarcane (Saccharum officinarum) was determined. It consists of 141,182 base-pairs (bp), containing a pair of inverted repeat regions (IR(A), IR(B)) of 22,794 bp each. The IR(A) and IR(B) sequences separate a small single copy region (12,546 bp) and a large single copy (83,048 bp) region. The gene content and relative arrangement of the 116 identified genes (82 peptide-encoding genes, four ribosomal RNA genes, 30 tRNA genes), with the 16 ycf genes, are highly similar to maize. Editing events, defined as C-to-U transitions in the mRNA sequences, were comparable with those observed in maize, rice and wheat. The conservation of gene organization and mRNA editing suggests a common ancestor for the sugarcane and maize plastomes. These data provide the basis for functional analysis of plastid genes and plastid metabolism within the Poaceae. The sugarcane chloroplast DNA sequence is available at GenBank under accession NC005878.     

Organization of ribosomal protein genes rpl23, rpl2, rps19, rpl22 and rps3 on the Euglena gracilis chloroplast genome

The nucleotide sequence (4,814 bp) was determined for a cluster of five ribosomal protein genes and their DNA flanking regions from the chloroplast genome of Euglena gracilis. The genes are organized as rpl23-150 bp spacer-rpl2-59 bp spacer-rps19-110 bp spacer-rpl22-630 bp spacer-rps3. The genes are all of the same polarity and reside 148 bp downstream from an operon for two genes of photosystem I and four genes of photosystem II. The Euglena ribosomal protein gene cluster resembles the S-10 ribosomal protein operon of Escherichia coli in gene organization and follows the exact linear order of the analogous genes in the tobacco and liverwort chloroplast genomes. The number and positions of introns in the Euglena ribosomal protein loci are different from their higher plant counterparts. The Euglena rpl23, rps19 and rps3 loci are unique in that they contain three, two and two introns, respectively, whereas rpl2 and rpl22 lack introns. The introns found in rpl23 (106, 99 and 103 bp), rps19 (103 and 97 bp) and rps3 intron 2 (102 bp) appear to represent either a new class of chloroplast intron found only in constitutively expressed genes, or possibly a degenerate version of Euglena chloroplast group II introns. They are deficient in bases C and G and extremely rich in base T, with a base composition of 53-76% T, 25-34% A, 3-10% G and 2-7% C in the mRNA-like strand. These six introns show minimal resemblance to group II chloroplast introns. They have a degenerate version of the group II intron conserved boundary sequences at their 5' and 3' ends. No conserved internal secondary structures are apparent. By contrast, rps3 intron 1 (409 bp) has a potential group II core secondary structure. The five genes, rpl23 (101 codons), rpl2 (278 codons), rps19 (95 codons), rpl22 (114 codons) and rps3 (220 codons) encode lysine-rich polypeptides with predicted molecular weights of 12,152, 31,029, 10,880, 12,819, and 25,238, respectively. The Euglena gene products are 18-50%, and 29-58% identical in primary structure to their E. coli and higher plant counterparts, respectively. Oligonucleotide sequences corresponding to Euglena chloroplast ribosome binding sites are not apparent in the intergenic regions. Inverted repeat sequences are found in the upstream flanking region of rpl23 and downstream from rps3.     

A large deletion in the plastid DNA of the holoparasitic flowering plant Cuscuta reflexa concerning two ribosomal proteins (rpl2, rpl23), one transfer RNA (trnI) and an ORF 2280 homologue

We have determined the nucleotide sequence of a 5.3-kb region of the plastid DNA (ptDNA) from the heterotrophic holoparasitic plant Cuscuta reflexa. The cloned area contains genes for the D1-protein (32-kDa protein; psbA), tRNA^His (trnH), ORF 740 (homologous to ORF 2280 from Nicotiana tabacum), ORF 77 (homologous to ORF 70), tRNA^Leu (trnL) and a hypothetical ORF 55 which has no homology to any known gene among higher plants. This 5.3-kb area is colinear with a 12.4-kb region of tobacco ptDNA and has therefore undergone several deletions totalling 7.1 kb. Most of the missing nucleotides belong to one large deletion in the ptDNA of C. reflexa of approximately 6.5 kb. This deletion involves two ribosomal protein genes, rpl2 and rpl23, as well as the transfer RNA for Isoleucin (trnI) and a region encoding 1540 amino-acid residues of an ORF 2280 homologue, as compared to tobacco chloroplast DNA. This is remarkable since the remaining genes, especially the psbA gene, are highly conserved in C. reflexa. Furthermore, we found that the expression of the psbA gene is in the same range as in the autotrophic Ipomoea purpurea which belongs to the same family as Cuscuta (Convolvulaceae). Here we hypothesize a total loss of rpl2 and rpl23 in the entire genome of C. reflexa. The phylogenetic position of, and the evolutionary change of ptDNA from, Cuscuta are discussed.     

Organization of ribosomal protein genes rp123, rp12, rps19, rp122 and rps3 on the Euglena gracilis chloroplast genome

Summary The nucleotide sequence (4,814 bp) was determined for a cluster of five ribosomal protein genes and their DNA flanking regions from the chloroplast genome of Euglena gracilis. The genes are organized as rp123 — 150 by spacer — rpl2 — 59 by spacer —rps19 — 110 by spacer — rp122 — 630 by spacer — rps3. The genes are all of the same polarity and reside 148 bp downstream from an operon for two genes of photosystem I and four genes of photosystem II. The Euglena ribosomal protein gene cluster resembles the S-10 ribosomal protein operon of Escherichia coli in gene organization and follows the exact linear order of the analogous genes in the tobacco and liverwort chloroplast genomes. The number and positions of introns in the Euglena ribosomal protein loci are different from their higher plant counterparts. The Euglena rp123, rps19 and rps3 loci are unique in that they contain three, two and two introns, respectively, whereas rp12 and rp122 lack introns. The introns found in rpl23 (106, 99 and 103 bp), rps19 (103 and 97 bp) and rps3 intron 2 (102 bp) appear to represent either a new class of chloroplast intron found only in constitutively expressed genes, or possibly a degenerate version of Euglena chloroplast group II introns. They are deficient in bases C and G and extremely rich in base T, with a base composition of 53–76% T, 25–34% A, 3–10% G and 2–7% C in the mRNA-like strand. These six introns show minimal resemblance to group IT chloroplast introns. They have a degenerate version of the group II intron conserved boundary sequences at their 5 and 3 ends. No conserved internal secondary structures are apparent. By contrast, rps3 intron 1 (409 bp) has a potential group II core secondary structure. The five genes, rpl23 (101 codons), rpl2 (278 codons), rpsl9 (95 codons), rpl22 (114 codons) and rps3 (220 codons) encode lysine-rich polypeptides with predicted molecular weights of 12,152, 31,029, 10,880, 12,819, and 25,238, respectively. The Euglena gene products are 18–50%, and 29–58% identical in primary structure to their E. coli and higher plant counterparts, respectively. Oligonucleotide sequences corresponding to Euglena chloroplast ribosome binding sites are not apparent in the intergenic regions. Inverted repeat sequences are found in the upstream flanking region of rp123 and downstream from rps3. Abreviations: Gene names follow the convention of Hallick and Bottomley (1983): rp123, rpl2, rpl22 are, respectively, genes for the L23, L2, L22 polypeptides of the 50S ribosomal subunit; rps19 and rps3 are genes for the S19 and S3 polypeptides of the 30S ribosomal subunit     

The mitochondrial DNA of land plants: peculiarities in phylogenetic perspective

Land plants exhibit a significant evolutionary plasticity in their mitochondrial DNA (mtDNA), which contrasts with the more conservative evolution of their chloroplast genomes. Frequent genomic rearrangements, the incorporation of foreign DNA from the nuclear and chloroplast genomes, an ongoing transfer of genes to the nucleus in recent evolutionary times and the disruption of gene continuity in introns or exons are the hallmarks of plant mtDNA, at least in flowering plants. Peculiarities of gene expression, most notably RNA editing and trans-splicing, are significantly more pronounced in land plant mitochondria than in chloroplasts. At the same time, mtDNA is generally the most slowly evolving of the three plant cell genomes on the sequence level, with unique exceptions in only some plant lineages. The slow sequence evolution and a variable occurrence of introns in plant mtDNA provide an attractive reservoir of phylogenetic information to trace the phylogeny of older land plant clades, which is as yet not fully resolved. This review attempts to summarize the unique aspects of land plant mitochondrial evolution from a phylogenetic perspective.     

Editing site analysis in a gymnosperm mitochondrial genome reveals similarities with angiosperm mitochondrial genomes

Sequence analysis of organelle genomes and comprehensive analysis of C-to-U editing sites from flowering and non-flowering plants have provided extensive sequence information from diverse taxa. This study includes the first comprehensive analysis of RNA editing sites from a gymnosperm mitochondrial genome, and utilizes informatics analyses to determine conserved features in the RNA sequence context around editing sites. We have identified 565 editing sites in 21 full-length and 4 partial cDNAs of the 39 protein-coding genes identified from the mitochondrial genome of Cycas taitungensis. The information profiles and RNA sequence context of C-to-U editing sites in the Cycas genome exhibit similarity in the immediate flanking nucleotides. Relative entropy analyses indicate that similar regions in the 5′ flanking 20 nucleotides have information content compared to angiosperm mitochondrial genomes. These results suggest that evolutionary constraints exist on the nucleotide sequences immediately adjacent to C-to-U editing sites, and similar regions are utilized in editing site recognition.     

Identification of an RNA element for specific coordination of A‐to‐I RNA editing on HTR2C pre‐mRNA

Adenosine‐to‐Inosine (A‐to‐I) RNA editing is an intracellular mechanism in which inosine is specifically substituted against adenosine by the action of adenosine deaminases acting on RNA (ADARs). Serotonin 2C receptor (HTR2C) is encoded through combinatorial A‐to‐I RNA editing at recoding sites (A – E site) on its pre‐mRNA. Although the efficiency of RNA editing at particular sites is known to be critical for modulating the serotonin signaling, the mechanistic details of site‐specific editing on HTR2C pre‐mRNA are not fully understood. Toward complete understanding of this mechanism, we discovered an RNA element, which coordinates site‐specific RNA editing on HTR2C pre‐mRNA by an in vitro editing assay and secondary structural analysis of mutant HTR2C RNA fragments. Our results showed that HTR2C pre‐mRNA forms a characteristic structure, which was restricted by the internal loop and Watson–Crick base‐pair interaction on site E, for intrinsic editing. We suggest that the internal loop would contribute toward adjusting the relative distance and/or geometry between the editing sites and the scaffold for ADAR.     

Identification of an aspartate transfer RNA gene in maize mitochondrial DNA

Summary A gene for a transfer RNA (tRNA) specific for aspartic acid was identified in maize mitochondrial DNA. The nucleotide sequence and predicted secondary structure of this tRNA more closely resemble eubacterial and chloroplast aspartate tRNA genes than other mitocondrial aspartate tRNA genes. This gene is located on a 3,123 base pair EcoRl DNA fragment that also contains an elongator methionine tRNA gene. These two tRNA genes are separated by 726 nucleotides and are located on opposite strands of DNA.Paper No. 9755 of the Journal series of the North Carolina Agricultural Research Service, Raleigh