Tatsuya Wakasugi

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single‐copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory‐chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ‘two out of three’ and ‘U:N wobble’ mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

Six chloroplast genes (ndhA-F) homologous to human mitochondrial genes encoding components of the respiratory chain NADH dehydrogenase are actively expressed: determination of the splice sites in ndhA and ndhB pre-mRNAs.

SummarySequences (ndhA-F) homologous to human mitochondrial genes for components of the respiratory chain NADH dehydrogenase have been found in the chloroplast DnA of tobacco. The ndhA, D, E and F sequences corresponding to the mitochondrial URF1, 4, 4L and 5 are located in the small single copy region, the ndhB sequence corresponding to URF2 in the inverted repeat and the ndhC sequence corresponding to URF3 in the large single copy region of the chloroplast DNA. Northern blot hybridization revealed that all six ndh sequences are actively expressed in the chloroplasts. The ndhA and ndhB sequences contain single introns and the splice sites of their pre-mRNAs were determined by reverse transcription analysis. These findings suggest that potential components of an NADH dehydrogenase are synthesized in the chloroplasts.

Comparative analysis of RNA editing sites in higher plant chloroplasts.

Abstract. 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 complete nucleotide sequence of the tobacco chloroplast genome

The c o m p l e t e n u c i e o t i d e sequence [155 ,844 bp) o f t o b a c c o ( N i c o t i a n a tabecum v a r . B r i g h t y e l l o w 4) c h l o r o p l a s t DNA [ S h i n o z a k i e t e l . 1986) is p r e s e n t e d . The c i r c u l a r DNA [see F ig . 1) i8 i nea r zed by c u t t i n g a t the j u n c t i o n JLA between IR A and LSC JLA is d e s i g n a t e d ze ro and numbered p r o c e e d i n g t owa rds LSC The DNA s t r a n d which codes f o r the l a r g e s u b u n i t of r i b u l o s e l , % b 8phospha te c a r b o x y l a s e is d e s i g n a t e d as A s t r a n d and the comp lemen ta ry s t r a n d as B s t r a n d . The B s t r a n d i8 shown he re . 6enes are boxed . The n o m e n c l a t u r e f o r genes f o l l o w s the p r o p o s a l s of H a l l i c k and B o t t o m l e y [1983, see Tab le 1) . A s t e r i s k s i n d i c a t e s p l i t genes and [C) deno tes genes l o c a t e d on the comp lemen ta ry s t r a n d ( t h e A s t r a n d ) .

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.

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.

SummaryA 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 by 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.

Updated Gene Map of Tobacco Chloroplast DNA

The choroplast DNA from tobacco (Nicotiana tabacum) has often served as areference for plastid genomes. It is now analyzed extensively due to advancesin transplastome techniques and in vitro technology. The complete nucleotidesequence and gene map was published in 1986 (Shinozaki et al., 1986a,b).Since then, 24 new genes have been identified and sequencing errors havebeen found. Genes found after 1986 include: one small RNA gene (sprA)and 23 protein-coding genes(psaC, psaI, psaJ, psbI, psbJ, psbK, psbL, psbM,psbN, psbT, petG, petL, ndhG, ndhH, ndhI, ndhJ, ndhK, rp132, rp136, rpoC2,matK, accD and clpP). Figure 1 shows the updated gene map which includes105 different genes and 9 ycfs.Some of the sequence errors were found by our group (e.g. Shimada etal., 1990), and the remaining errors were reported and suggested by othergroups (e.g. Olmstead et al., 1993). We have examined these errors by re-sequencing. All corrections (Table 1) were made to the original sequenceand the numbering system was changed accordingly. The updated gene list(Table 2) and sequence have been deposited with the EMBL Nucleotide Se-quence Database under the accession number Z00044. A printed sequence inwhich genes/ycfs/orfs (see Figure 1) are boxed is available upon request toM. Sugiura.

Cotranscription of the genes encoding two P700 chlorophyll a apoproteins with the gene for ribosomal protein CS14: determination of the transcriptional initiation site by in vitro capping

SummaryTranscription of the psaA operon in tobacco chloroplasts has been studied. This operon contains in linear sequence the genes encoding the P700 chlorophyll a A1 and A2 apoproteins (psaA and psaB) and the gene encoding the ribosomal CS14 protein (rps14). Northern blot hybridization revealed that a 5.2 kb transcript hybridizes to psaA, psaB, and rps14, but not to the fMet-tRNA (trnfM) gene which follows. Primer extension and in vitro capping assays indicated that the transcriptional initiation site is 194 by upstream of psaA. The 3′ end of the transcript was determined by S1 mapping to be 105 bp downstream of rps14. The transcript is calculated to be 5,207 nucleotides long.

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 at 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.

A physical map and clone bank of the black pine Pinus thunbergii) chloroplast genome.

Black pine chloroplast DNA is 119,707 bp long. The physical map is shown and the genes are listed. Plasmid clones covering the entire DNA sequence have been ordered and available upon request.