Mamoru Sugita

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.

Regulation of gene expression in chloroplasts of higher plants

Chloroplasts contain their own genetic system which has a number of prokaryotic as well as some eukaryotic features. Most chloroplast genes of higher plants are organized in clusters and are cotranscribed as polycistronic pre-RNAs which are generally processes into many shorter overlapping RNA species, each of which accumulates of steady-state RNA levels. This indicates that posttranscriptional RNA processing of primary transcripts is an important step in the control of chloroplast gene expression. Chloroplast RNA processing steps include RNA cleavage/trimming, RNA splicing, ENA editing and RNA stabilization. Several chloroplast genes are interrupted by introns and therefore require processing for gene function. In tobacco chrloroplasts, 18 genes contain introns, six for tRNA genes and 12 for protein-encoding genes. A number of specific proteins and RNA factors are believed to be involved in splicing and maturation of pre-RNAs in chrloroplasts. Processing enzymes and RNA-binding proteins which could be involved in posttranscriptional steps have been identified in the last several years. Our current knowledge of the regulation of gene expression in chloroplasts of higher plants is overviewed and further studies on this matter are also considered.

A Pentatricopeptide Repeat Protein Is a Site Recognition Factor in Chloroplast RNA Editing

In higher plants, RNA editing is a post-transcriptional process that converts C to U in organelle mRNAs. We have previously shown that an Arabidopsis thaliana crr4 mutant is defective with respect to RNA editing for creating the translational initial codon of the plastid ndhD gene (the ndhD-1 site). CRR4 contains 11 pentatricopeptide repeat motifs but does not contain any domains that are likely to be involved in the editing activity. The green fluorescent protein fused to the putative transit peptide of CRR4 targeted the plastid. The recombinant CRR4 expressed in Escherichia coli specifically bound to the 25 nucleotides of the upstream and the 10 nucleotides of the downstream sequences surrounding the editing site of ndhD-1. The target C nucleotide of this editing is not essential for the binding of CRR4. Taken together with the genetic evidence, we conclude that the pentatricopeptide repeat protein CRR4 is a sequence-specific RNA-binding protein that acts as a site recognition factor in plastid RNA editing.

Pentatricopeptide Repeat Proteins with the DYW Motif Have Distinct Molecular Functions in RNA Editing and RNA Cleavage in Arabidopsis Chloroplasts

The plant-specific DYW subclass of pentatricopeptide repeat proteins has been postulated to be involved in RNA editing of organelle transcripts. We discovered that the DYW proteins CHLORORESPIRATORY REDUCTION22 (CRR22) and CRR28 are required for editing of multiple plastid transcripts but that their DYW motifs are dispensable for editing activity in vivo. Replacement of the DYW motifs of CRR22 and CRR28 by that of CRR2, which has been shown to be capable of endonucleolytic cleavage, blocks the editing activity of both proteins. In return, the DYW motifs of neither CRR22 nor CRR28 can functionally replace that of CRR2. We propose that different DYW family members have acquired distinct functions in the divergent processes of RNA maturation, including RNA cleavage and RNA editing.

A KaiC-associating SasA–RpaA two-component regulatory system as a major circadian timing mediator in cyanobacteria

KaiA, KaiB, and KaiC clock proteins from cyanobacteria and ATP are sufficient to reconstitute the KaiC phosphorylation rhythm in vitro, whereas almost all gene promoters are under the control of the circadian clock. The mechanism by which the KaiC phosphorylation cycle drives global transcription rhythms is unknown. Here, we report that RpaA, a potential DNA-binding protein that acts as a cognate response regulator of the KaiC-interacting kinase SasA, mediates between KaiC phosphorylation and global transcription rhythms. Circadian transcription was severely attenuated in sasA (Synechococcus adaptive sensor A)- and rpaA (regulator of phycobilisome-associated)-mutant cells, and the phosphotransfer activity from SasA to RpaA changed dramatically depending on the circadian state of a coexisting Kai protein complex in vitro. We propose a model in which the SasA–RpaA two-component system mediates time signals from the enzymatic oscillator to drive genome-wide transcription rhythms in cyanobacteria. Moreover, our results indicate the presence of secondary output pathways from the clock to transcription control, suggesting that multiple pathways ensure a genome-wide circadian system.

Genomic organization, sequence analysis and expression of all five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato

SummaryWe have cloned and sequenced all five members of the gene family for the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato, Lycopersicon esculentum cv. VFNT LA 1221 cherry line. Two of the five genes, designated Rbcs-1 and Rbcs-2, are present as single genes at individual loci. Three genes, designated Rbcs-3A, Rbcs-3B and Rbcs-3C, are organized in a tandem array within 10 kb at a third independent locus. The Rbcs-2 gene contains three introns; all the other members of the tomato gene family contain two introns. The coding sequence of Rbcs-1 differs by 14.0% from that of Rbcs-2 and by 13.3% from that of Rbcs-3 genes. Rbcs-2 shows 10.4% divergence from Rbcs-3. The exon and intron sequences of Rbcs-3A are identical to those of Rbcs-3C, and differ by 1.9% from those of Rbcs-3B. Nucleotide sequence analysis suggests that the five rbcS genes encode four different precursors, and three different mature polypeptides. S1 nuclease mapping of the 5′ end of rbcS mRNAs revealed that the mRNA leader sequences vary in length from 8 to 75 nucleotides. Northern analysis using gene-specific oligonucleotide probes from the 3′ non-coding region of each gene reveals a four to five-fold difference among the five genes in maximal steady-state mRNA levels in leaves.

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 ) .

Cyanobacterial daily life with Kai-based circadian and diurnal genome-wide transcriptional control in Synechococcus elongatus

In the unicellular cyanobacterium Synechococcus elongatus PCC 7942, essentially all promoter activities are under the control of the circadian clock under continuous light (LL) conditions. Here, we used high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild-type, kaiABC-null, and kaiC-overexpressor strains under LL and continuous dark (DD) conditions. In the wild-type strains, >30% of transcripts oscillated significantly in a circadian fashion, peaking at subjective dawn and dusk. Such circadian control was severely attenuated in kaiABC-null strains. Although it has been proposed that KaiC globally represses gene expression, our analysis revealed that dawn-expressed genes were up-regulated by kaiC-overexpression so that the clock was arrested at subjective dawn. Transfer of cells to DD conditions from LL immediately suppressed expression of most of the genes, while the clock kept even time in the absence of transcriptional feedback. Thus, the Synechococcus genome seems to be primarily regulated by light/dark cycles and is dramatically modified by the protein-based circadian oscillator.

Suppression mechanism of mitochondrial ORF79 accumulation by Rf1 protein in BT‐type cytoplasmic male sterile rice

SUMMARY In BT-type cytoplasmic male sterile rice (Oryza sativa L.) with Chinsurah Boro II cytoplasm, cytoplasmic male sterility (CMS) is caused by an accumulation of the cytotoxic peptide ORF79. The ORF79 protein is expressed from a dicistronic gene atp6-orf79, which exists in addition to the normal atp6 gene in the BT-type mitochondrial genome. The CMS is restored by a PPR (pentatricopeptide-repeat) gene, Rf1, via RNA processing. However, it has not yet been elucidated how the accumulation of ORF79 is reduced by the action of the Rf1 protein. Here, we report that the level of processed orf79 transcripts in the restorer line was reduced to 50% of the unprocessed atp6-orf79 transcripts in the CMS line. Ninety percent of the processed orf79 transcripts, which remained after degradation, were not associated with the ribosome for translation. Our data suggests that the processing of atp6-orf79 transcripts diminishes the expression of orf79 by the translational reduction and degradation of the processed orf79 transcripts.

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.