Nucleotide sequence and linkage map position of the secX gene in maize chloroplast and evidence that it encodes a protein belonging to the 50S ribosomal subunit

The nucleotide sequence of the segment of maize chloroplast DNA lying between the map coordinate positions 32.59 and 32.98 Kb and containing the secX gene has been determined. The derived amino acid sequence of maize chloroplast secX is 95%, 87% and 62% identical to the corresponding derived amino acid sequences from two plant chloroplasts and Escherichia coli, respectively. It is also 70% identical to the experimentally determined amino acid sequence of a protein isolated from Bacillus stearothermophilus ribosomes. Separation of the 50S ribosomal subunit proteins of E. coli by reversed phase HPLC gave a peak which contained pure secX protein, as determined by N-terminal amino acid sequencing. Spinach chloroplast 50S subunit proteins separated by HPLC also gave a peak corresponding to pure secX protein. From these results we conclude that the secX gene in E. coli and in plant chloroplasts encodes a small (37-38 amino acid residues) ribosomal protein belonging to the 50S subunit. The same conclusion has been reached recently by A. Wada with respect to E. coli secX. In agreement with Wada, we name the secX protein L36. Its chloroplast gene is designated rpL36.     

Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli RecA.

The occurrence of homologous DNA recombination in chloroplasts is well documented, but little is known about the molecular mechanisms involved or their biological significance. The endosymbiotic origin of plastids and the recent finding of an Arabidopsis nuclear gene, encoding a chloroplast-localized protein homologous to Escherichia coli RecA, suggest that the plastid recombination system is related to its eubacterial counterpart. Therefore, we examined whether dominant negative mutants of the E. coli RecA protein can interfere with the activity of their putative homolog in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. Transformants expressing these mutant RecA proteins showed reduced survival rates when exposed to DNA-damaging agents, deficient repair of chloroplast DNA, and diminished plastid DNA recombination. These results strongly support the existence of a RecA-mediated recombination system in chloroplasts. We also found that the wild-type E. coli RecA protein enhances the frequency of plastid DNA recombination over 15-fold, although it has no effect on DNA repair or cell survival. Thus, chloroplast DNA recombination appears to be limited by the availability of enzymes involved in strand exchange rather than by the level of initiating DNA substrates. Our observations suggest that a primary biological role of the recombination system in plastids is in the repair of their DNA, most likely needed to cope with damage due to photooxidation and other environmental stresses. This hypothesis could explain the evolutionary conservation of DNA recombination in chloroplasts despite the predominantly uniparental inheritance of their genomes.     

Sequence of a putative promoter region for the rRNA genes of tobacco chloroplast DNA.

The nucleotide sequence of the segment of tobacco chloroplast DNA adjacent to and including the start of the 16S rRNA gene has been determined. The region just preceding this gene was found to contain a tRNAVal gene and promoter-type sequences similar to those which occur in E. coli were found before this tRNA gene. E. coli RNA polymerase can recognize these sequences and in vitro co-transcribes the tRNA and rRNA genes.     

Roles of novobiocin-sensitive topoisomerases in chloroplast DNA replication in Chlamydomonas reinhardtii.

We have examined DNA replication in Chlamydomonas reinhardtii chloroplasts in vivo when chloroplast type II topoisomerases are inactivated with sublethal doses of novobiocin. DNA replication is at first inhibited under these conditions. However, after a delay of several hours, chloroplast chromosomes initiate a novobiocin-insensitive mode of DNA replication. This replication starts preferentially near a hotspot of recombination in the large inverted repeats, instead of from the normal chloroplast origins, oriA and oriB. It replicates one, but not the other single-copy region of the chloroplast chromosome. We speculate that novobiocin-insensitive DNA replication in chloroplasts requires recombination in this preferred initiation region.     

The RNase E/G-type endoribonuclease of higher plants is located in the chloroplast and cleaves RNA similarly to the E. coli enzyme

RNase E is an endoribonuclease that has been studied primarily in Escherichia coli, where it is prominently involved in the processing and degradation of RNA. Homologs of bacterial RNase E are encoded in the nuclear genome of higher plants. RNA degradation in the chloroplast, an organelle that originated from a prokaryote similar to cyanobacteria, occurs via the polyadenylation-assisted degradation pathway. In E. coli, this process is probably initiated with the removal of 5'-end phosphates followed by endonucleolytic cleavage by RNase E. The plant homolog has been proposed to function in a similar way in the chloroplast. Here we show that RNase E of Arabidopsis is located in the soluble fraction of the chloroplast as a high molecular weight complex. In order to characterize its endonucleolytic activity, Arabidopsis RNase E was expressed in bacteria and analyzed. Similar to its E. coli counterpart, the endonucleolytic activity of the Arabidopsis enzyme depends on the number of phosphates at the 5' end, is inhibited by structured RNA, and preferentially cleaves A/U-rich sequences. The enzyme forms an oligomeric complex of approximately 680 kDa. The chloroplast localization and the similarity in the two enzymes' characteristics suggest that plant RNase E participates in the initial endonucleolytic cleavage of the polyadenylation-stimulated RNA degradation process in the chloroplast, perhaps in collaboration with the two other chloroplast endonucleases, RNase J and CSP41.     

Screening recombinant clones containing sequences homologous to Escherichia coli genes using single-stranded bacteriophage vector

Detection and isolation of Escherichia coli clones carrying vectors with foreign DNA sequences partially homologous to specific E. coli genes is difficult because denatured DNA in the host genome can hybridize with the probe. In this paper we present a procedure which simplifies this task by using bacteriophage M13 as the cloning vector. The procedure takes advantage of the secretory properties of the phage, as well as the property of nitrocellulose membrane to bind protein and single-stranded DNA but not double-stranded DNA. This procedure is shown to be effective in identifying E. coli clones containing sequences of Chlamydomonas reinhardtii chloroplast DNA that are homologous to the rpoC gene of E. coli. We suggest that this procedure can be used generally for rapid isolation of DNA sequences that are homologous to E. coli genes.     

A pseudogene cluster in the leader region of the Euglena chloroplast 16S-23S rRNA genes.

The nucleotide sequence of a region (leader region) preceding the 5'-end of 16S-23S rRNA gene region of Euglena gracilis chloroplast DNA was compared with the homologous sequences that code for the 16S-23S rRNA operons of Euglena and E. coli. The leader region shows close homology in sequence to the 16S-23S rRNA gene region of Euglena (Orozco et al. (1980) J. Biol.Chem. 255, 10997-11003) as well as to the rrnD operon of E. coli, suggesting that it was derived from the 16S-23S rRNA gene region by gene duplication. It was shown that the leader region had accumulated nucleotide substitutions at an extremely rapid rate in its entirety, similar to the rate of tRNAIle pseudogene identified in the leader region. In addition, the leader region shows an unique base content which is quite distinct from those of 16S-23S rRNA gene regions of Euglena and E. coli, but again is similar to that of the tRNAIle pseudogene. The above two results strongly suggest that the leader region contains a pseudogene cluster which was derived from a gene cluster coding for the functional 16S-23S rRNA operon possibly by imperfect duplication during evolution of Euglena chloroplast DNA.     

A comparison of rice chloroplast genomes

Using high quality sequence reads extracted from our whole genome shotgun repository, we assembled two chloroplast genome sequences from two rice (Oryza sativa) varieties, one from 93-11 (a typical indica variety) and the other from PA64S (an indica-like variety with maternal origin of japonica), which are both parental varieties of the super-hybrid rice, LYP9. Based on the patterns of high sequence coverage, we partitioned chloroplast sequence variations into two classes, intravarietal and intersubspecific polymorphisms. Intravarietal polymorphisms refer to variations within 93-11 or PA64S. Intersubspecific polymorphisms were identified by comparing the major genotypes of the two subspecies represented by 93-11 and PA64S, respectively. Some of the minor genotypes occurring as intravarietal polymorphisms in one variety existed as major genotypes in the other subspecific variety, thus giving rise to intersubspecific polymorphisms. In our study, we found that the intersubspecific variations of 93-11 (indica) and PA64S (japonica) chloroplast genomes consisted of 72 single nucleotide polymorphisms and 27 insertions or deletions. The intersubspecific polymorphism rates between 93-11 and PA64S were 0.05% for single nucleotide polymorphisms and 0.02% for insertions or deletions, nearly 8 and 10 times lower than their respective nuclear genomes. Based on the total number of nucleotide substitutions between the two chloroplast genomes, we dated the divergence of indica and japonica chloroplast genomes as occurring approximately 86,000 to 200,000 years ago.     

Acyl carrier protein-derived sequence encoded by the chloroplast genome in the marine diatom Cylindrotheca sp. strain N1.

The chloroplast genome of chromophytic and rhodophytic algae differs from the plastid genome of plants and green algae in that it encodes the gene for the small subunit (rbcS) of ribulose 1,5-bisphosphate carboxylase/oxygenase. Hybridization studies indicated that there was a second region of chloroplast DNA from the marine diatom Cylindrotheca sp. strain N1 that strongly hybridized to a previously isolated Cylindrotheca fragment that contained the rbcS gene and flanking sequences. Subsequent determination of the oligonucleotide sequence of this second chloroplast DNA fragment, however, indicated that hybridization was due to identical sequences 3' to the previously cloned Cylindrotheca chloroplast rbcL rbcS genes. Sequences derived from the 5' end of the second chloroplast DNA fragment contained a short open reading frame of 80 amino acids which was found to be highly homologous to bacterial acyl carrier protein and nuclear-encoded acyl carrier protein from plants. Amino acid residues in the environment of Ser-36 of the Escherichia coli protein, which is bound to a 4'-phosphopantetheine moiety, are virtually identical in the Cylindrotheca deduced sequence and all other sources of this protein. Unlike plant acyl carrier-deduced amino acid sequences, there was no leader peptide sequence found for the presumptive Cylindrotheca protein, consistent with the location of this DNA fragment on the chloroplast genome of this organism. DNA encoding the putative acyl carrier protein gene and rbcS thus represent two genes that are chloroplast-encoded in the chromophytic marine diatom Cylindrotheca, a significant departure from the organization of such genes in plants.     

Detection and localization of a chloroplast-encoded HU-like protein that organizes chloroplast nucleoids

Chloroplast DNA (cpDNA) is packed into discrete structures called chloroplast nucleoids (cp-nucleoids). The structure of cpDNA is thought to be important for its maintenance and regulation. In bacteria and mitochondria, histone-like proteins (such as HU and Abf2, respectively) are abundant and play important roles in DNA organization. However, a primary structural protein has yet to be found in cp-nucleoids. Here, we identified an abundant DNA binding protein from isolated cp-nucleoids of the primitive red alga Cyanidioschyzon merolae. The purified protein had sequence homology with the bacterial histone-like protein HU, and it complemented HU-lacking Escherichia coli mutants. The protein, called HC (histone-like protein of chloroplast), was encoded by a single gene (CmhupA) in the C. merolae chloroplast genome. Using immunofluorescence and immunoelectron microscopy, we demonstrated that HC was distributed uniformly throughout the entire cp-nucleoid. The protein was expressed constitutively throughout the cell and the chloroplast division cycle, and it was able to condense DNA. These results indicate that HC, a bacteria-derived histone-like protein, primarily organizes cpDNA into the nucleoid.