Cotranscription, deduced primary structure, and expression of the chloroplast-encoded rbcL and rbcS genes of the marine diatom Cylindrotheca sp. strain N1.

The primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined. Unlike higher plants and green algae, the genes encoding the large and the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase are chloroplast-encoded and closely associated (Hwang and Tabita, 1989). The rbcL and rbcS genes in strain N1 are cotranscribed and are separated by an intergenic region of 46 nucleotide base pairs. Ribosome binding sites and a potential promoter sequence were highly homologous to previously determined chloroplast sequences. Comparison of the deduced primary structure of the diatom large and small subunits indicated significant homology to previously determined sequences from bacteria; there was much less homology to large and small subunits from cyanobacteria, green algae, and higher plants. Although high levels of recombinant diatom large subunits could be expressed in Escherichia coli, the protein synthesized was primarily insoluble and incapable of forming an active hexadecameric enzyme. Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphate carboxylase/oxygenase proteins, despite the distinctive procaryotic gene arrangement and sequence homology.     

High-throughput pyrosequencing of the chloroplast genome of a highly neutral-lipid-producing marine pennate diatom, Fistulifera sp. strain JPCC DA0580

The chloroplast genome of the highly neutral-lipid-producing marine pennate diatom Fistulifera sp. strain JPCC DA0580 was fully sequenced using high-throughput pyrosequencing. The general features and gene content were compared with three other complete diatom chloroplast genomes. The chloroplast genome is 134,918 bp with an inverted repeat of 13,330 bp and is slightly larger than the other diatom chloroplast genomes due to several low gene-density regions lacking similarity to the other diatom chloroplast genomes. Protein-coding genes were nearly identical to those from Phaeodactylum tricornutum. On the other hand, we found unique sequence variations in genes of photosystem II which differ from the consensus in other diatom chloroplasts. Furthermore, five functional unknown ORFs and a putative serine recombinase gene, serC2, are located in the low gene-density regions. SerC2 was also identified in the plasmids of another pennate diatom, Cylindrotheca fusiformis, and in the plastid genome of the diatom endosymbiont of Kryptoperidinium foliaceum. Exogenous plasmids might have been incorporated into the chloroplast genome of Fistulifera sp. by lateral gene transfer. Chloroplast genome sequencing analysis of this novel diatom provides many important insights into diatom evolution.     

CHARACTERIZATION OF A cDNA ENCODING GLUTAMINE SYNTHETASE FROM THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)

A cDNA-encoding glutamine synthetase (GS) was isolated from the marine diatom Skeletonema costatum (Greville) Cleve by PCR amplification. Nucleic acid and deduced amino acid sequences of the diatom GS were greater than 50% identical to GS from green algae and vascular plants, and phylogenetic analysis established the diatom GS as a member of the GSII gene family. The presence of an N-terminus signal sequence, identified on the basis of sequence similarity with other chloroplast-localized proteins from diatoms, suggests that the encoded GS isoenzyme is localized to the chloroplast. The GS mRNA was present in log-phase cells grown with either nitrate or ammonium as the sole added nitrogen source. Results from Southern blot analysis of genomic DNA suggested that the cDNA isolated in this study was either a member of a small, highly conserved gene family or that there was allelic variation within the region examined. Phylogenetic analyses further indicated that genes encoding GS from the diatom and two species of green algae diverged prior to the gene duplication, to the isoenzymes in vascular plants, supporting the hypothesis that GS isoenzymes in diatoms, green algae, and vascular plants arose through independent evolutionary events.     

Chloroplast ATPase genes in the diatom Odontella sinensis reflect cyanobacterial characters in structure and arrangement.

We have cloned and sequenced a 5200 base restriction fragment and an overlapping 3100 base fragment of the large single copy region of the chloroplast genome of the diatom Odontella sinensis, which hybridized to several ATPase gene probes. These fragments contain six closely linked reading frames that were identified as atpI, atpH, atpG, atpF, atpD, and atpA, coding for subunits IV, III, II, I, delta, and alpha, respectively. Remarkably, the genes atpG and atpD, which are nucleus-encoded in chlorophyll a + b plants, are present in the Odontella chloroplast gene cluster. They map at the same positions as in cyanobacteria. The genes atpD and atpF overlap by four base-pairs as in certain photosynthetic and heterotrophic eubacteria. Upstream from the atpA gene cluster an open reading frame coding for 251 amino acid residues was found, which shows sequence similarity to ATP-binding subunits of periplasmic prokaryotic and eukaryotic transport systems. No similar reading frame is present in the land plant chloroplast genomes analysed so far. Sequences and arrangement of the genes are discussed with respect to the peculiar evolution of the chlorophyll a + c-containing chromophytic plastids.     

Characterization of genes encoding the light-harvesting proteins in diatoms: biogenesis of the fucoxanthin chlorophylla/c protein complex

In chromophytic algae the major light-harvesting complex is the fucoxanthin chlorophylla/c protein complex. Recently, we have cloned several highly related cDNA and genomic sequences encoding the fucoxanthin chlorophylla/c proteins from the diatomPhaeodactylum tricornutum. These genes are clustered on the nuclear genome. The sequences of the fucoxanthin chlorophylla/c proteins as deduced from the gene sequences have some similarity to the chlorophylla/b proteins associated with light-harvesting complexes of higher plants and green algae. Like the chlorophylla/b proteins of higher plants, the fucoxanthin chlorophylla/c proteins are synthesized as higher-molecular weight precursors in the cytoplasm of the cell and are transported into the plastids. However, the mode of transport into diatom plastids is very different from the mechanism involved in transporting proteins into the chloroplasts of higher plants and green algae. We focus here on the characteristics of the fucoxanthin chlorophylla/c proteins, the mode of transport of these proteins into plastids, the arrangement of the genes encoding these proteins, and efforts to utilize these genes to develop a DNA transformation system for diatoms.     

A novel protein in Photosystem II of a diatom Chaetoceros gracilis is one of the extrinsic proteins located on lumenal side and directly associates with PSII core components

The gene encoding a novel extrinsic protein (Psb31) found in Photosystem II (PSII) of a diatom, Chaetoceros gracilis, was cloned and sequenced. The deduced protein contained three characteristic leader sequences targeted for chloroplast endoplasmic reticulum membrane, chloroplast envelope membrane and thylakoid membrane, indicating that Psb31 is encoded in the nuclear genome and constitutes one of the extrinsic proteins located on the lumenal side. Homologous genes were found in a red alga and chromophytic algae but not in other organisms. Genes encoding the other four extrinsic proteins in C. gracilis PSII were also cloned and sequenced, and their leader sequences were characterized and compared. To search for the nearest neighbor relationship between Psb31 and the other PSII components, we crosslinked the PSII particles with the water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and found that Psb31 directly associates with PSII core components through electrostatic interaction, suggesting that the novel Psb31 protein is one of the extrinsic proteins constituting the functional oxygen-evolving complex of C. gracilis PSII.     

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in Euglena gracilis chloroplast DNA: location, polarity, cloning, and evidence for an intervening sequence

The gene for the large subunit (LS) of ribulose-1,5,-bisphosphate carboxylase of Euglena gracilis Z chloroplast DNA has been mapped by heterologous hybridization with DNA restriction fragments containing internal sequences from the Zea mays and Chlamydomonas reinhardii LS genes. The Euglena LS gene which has the same polarity as the Euglena rRNA genes has been located with respect to Pst I, Pvu I, and HindIII sites within the Eco RI fragment Eco A. The region of Euglena chloroplast DNA complementary to an 887 bp internal fragment from the Chlamydomonas chloroplast LS gene is interrupted by a 0.5-1.1 kbp non-complementary sequence. This is the first chloroplast protein gene located on the Euglena genome, and the first evidence for an intervening sequence within any chloroplast protein gene.     

杨树叶绿体3′rps12基因,rps7基因和ndhB基因片段的克隆及序列分析

通过烟草叶绿体相关序列设计引物 ,从杨树的叶绿体基因组中克隆出 2个相邻的DNA片段 .经分析发现 ,这 2个DNA片段包含核糖体蛋白 3′rps12、rps7基因和NADH脱氢酶第二亚基ndhB基因片段 .利用DNAMAN等软件 ,将扩增到的杨树叶绿体DNA片段与烟草、拟南芥、玉米和黑松的相关序列进行比较 ,证实所扩增的片段具有较高的保守性 ,尤其是在这 3个基因的编码区 ,同源性均在 90 %以上 .插入或缺失常发生在基因间隔区 ,同源性在 80 %左右 ;对其编码区所推导的氨基酸序列进行了比较 ,同源性均在 92 %以上 .首次克隆了杨树叶绿体的部分DNA序列 ,详细报道并分析了杨树 3′rps12、rps7基因和ndhB基因片段及其边界序列信息 .所报告的基因序列均已登录GenBank .     

CHARACTERIZATION OF DIATOM (BACILLARIOPHYCEAE) NITRATE REDUCTASE GENES AND THEIR DETECTION IN MARINE PHYTOPLANKTON COMMUNITIES1

The complete assimilatory nitrate reductase (NR) gene from the pennate diatom Phaeodactylum triconutum Bohlin was sequenced from cDNA and compared with NR sequences from fungi, green algae, vascular plants, and the recently sequenced genome of the centric diatom Thalassiosira pseudonana Hasle and Heimdal CCMP1335. In all the major eukaryotic nitrate reductase (Euk‐NR) functional domains, diatom NR gene sequences are generally 50%–60% identical to plant and alga sequences at the amino acid level. In the less conserved N‐terminal, hinge 1, and hinge 2 regions, homology to other NR sequences is weak, generally<30%. Two PCR primer sets capable of amplifying Euk‐NR from plants, algae, and diatoms were designed. One primer set was used to amplify a 750‐base pair (bp) NR fragment from the cDNA of five additional diatom strains. The PCR amplicon spans part of the well‐conserved dimer interface region, the more variable hinge 1 region, and part of the conserved cytochrome b heme binding region. The second primer set, targeted to the dimer region, was used to amplify an approximately 400‐bp fragment of the NR gene from DNA samples collected in Monterey Bay, California and in central New Jersey inner continental shelf (LEO‐15 site) waters. Only diatom‐like NR sequences were recovered from Monterey Bay samples, whereas LEO‐15 samples yielded NR sequences from a range of photosynthetic eukaryotes. The prospect of using DNA‐ and RNA‐based methods to target the NR genes of diatoms specifically is a promising approach for future physiological and ecological experiments.     

Complementation of the nuclear antisense rbcS-induced photosynthesis deficiency by introducing an rbcS gene into the tobacco plastid genome

The small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a nuclear gene-encoded protein that is imported into chloroplasts where it assembles with the large subunit (LS) after removal of the transit peptide to form Rubisco. We have explored the possibility that the severe deficiency in photosynthesis exhibited in nuclear transgenic tobacco (line alpha5) expressing antisense rbcS coding DNA that results in low SS and Rubisco protein content [Rodermel et al. (1988) Cell 55: 673] could be complemented by introducing a copy of the rbcS gene into its plastid genome through chloroplast transformation. Two independent lines of transplastomic plants were generated, in which the tobacco rbcS coding sequence, either with or without the transit sequence, was site-specifically integrated into the plastid genome. We found that compared with the antisense plants, expression of the plastid rbcS gene in the transplastomic plants resulted in very high mRNA abundance but no increased accumulation of the SS and Rubisco protein or improvement in plant growth and photosynthesis. Therefore, there is a limitation in efficient translation of the rbcS mRNA in the plastid or an incorrect processing and modification of the plastid-synthesized SS protein that might cause its rapid degradation.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase gene expression and diversity of Lake Erie planktonic microorganisms.

Carbon dioxide fixation is carried out primarily through the Calvin-Benson-Bassham reductive pentose phosphate cycle, in which ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme. The primary structure of the large subunit of form I RubisCO is well conserved; however, four distinct types, A, B, C, and D, may be distinguished, with types A and B and types C and D more closely related to one another. To better understand the environmental regulation of RubisCO in Lake Erie phytoplanktonic microorganisms, we have isolated total RNA and DNA from four Lake Erie sampling sites. Probes prepared from RubisCO large-subunit genes (rbcL) of the freshwater cyanobacterium Synechococcus sp. strain PCC6301 (representative of type IB) and the diatom Cylindrotheca sp. strain N1 (representative of type ID) were hybridized to the isolated RNA and DNA. To quantitate rbcL gene expression for each sample, the amount of gene expression per gene dose (i.e., the amount of mRNA divided by the amount of target DNA) was determined. With a limited number of sampling sites, it appeared that type ID (diatom) rbcL gene expression per gene dose decreased as the sampling sites shifted toward open water. By contrast, a similar trend was not observed for cyanobacterial (type IB) rbcL gene expression per gene dose. Complementary DNA specific for rbcL was synthesized from Lake Erie RNA samples and used as a template for PCR amplification of portions of various rbcL genes. Thus far, a total of 21 clones of rbcL genes derived from mRNA have been obtained and completely sequenced from the Ballast Island site. For surface water samples, deduced amino acid sequences of five of six clones appeared to be representative of green algae. In contrast, six of nine sequenced rbcL clones from 10-m-deep samples were of chromophytic and rhodophytic lineages. At 5 m deep, the active CO2-fixing planktonic organisms represented a diverse group, including organisms related to Chlorella ellipsoidea, Cylindrotheca sp. strain N1, and Olisthodiscus luteus. Although many more samplings at diverse sites must be accomplished, the discovery of distinctly different sequences of rbcL mRNA at different water depths suggests that there is a stratification of active CO2-fixing organisms in western Lake Erie.     

Loss of the rpl32 gene from the chloroplast genome and subsequent acquisition of a preexisting transit peptide within the nuclear gene in Populus

Gene transfer events from organelle genomes (mitochondria and chloroplasts in plants) to the nuclear genome are important processes in the evolution of the eukaryotic cell. It is highly likely that the gene transfer event is still an ongoing process in higher plant mitochondria and chloroplasts. The number and order of genes encoded in the chloroplast genome of higher plants are highly conserved. Recently, several exceptional cases of gene loss from the chloroplast genome have been discovered as the number of complete chloroplast genome sequences has increased. The Populus chloroplast genome has lost the rpl32 gene, while the corresponding the chloroplast rpl32 (cp rpl32) gene has been identified in the nuclear genome. Nuclear genes transferred from the chloroplast genome need to gain a sequence that encodes a transit peptide. Here, we revealed that the nuclear cp rpl32 gene has acquired the exon sequence, which is highly homologous to a transit peptide derived from the chloroplast Cu-Zn superoxide dismutase (cp sod-1) gene. The cp rpl32 gene has acquired the sequence that encodes not only for the transit peptide, but also for the conserved N-terminal portion of the mature SOD protein from the cp sod-1 gene, suggesting the occurrence of DNA sequence duplication. Unlike cp SOD-1, cp RPL32 did not show biased localization in the chloroplasts. This difference may be caused by mutations accumulated in the sequence of the SOD domain on the cp rpl32 gene. We provide new insight into the fate of the inherent sequence derived from a transit peptide.     

Chloroplast genome characterization in the red alga Griffithsia pacifica

Summary It has been suggested that cyanobacteria served as the ancestors for rhodophytic algae whose chloroplasts contain chlorophyll a and phycobilins, and that a rhodophyte served as the plastid source for chromophytic plants that contain chlorophylls a and c. Although organellar DNA has been used to assess phylogenetic relatedness among terrestrial plants and green algae whose chloroplasts contain chlorophylls a and b, few data are presently available on the molecular profile of plastid DNA in chromophytes or rhodophytes.In this study the chloroplast genome of the rhodophytic, filamentous alga Griffithsia pacifica has been characterized. DNA was purified from isolated chloroplasts using protease k treatment and sodium dodecyl sulfate lysis followed by density centrifugation in Hoescht-33258 dye-CsCl gradients. Single and double restriction enzyme digests demonstrate that the DNA prepared from purified chloroplasts has a genome size of about 178 kilobase pairs (kb). A restriction map of this chloroplast genome demonstrates that it is circular and, unlike the chloroplast DNA (cpDNA) in most other plants, contains only a single ribosomal DNA operon. DNA was also purified from the mitochondria that co-isolated with chloroplasts. Mitochondrial DNA consists of molecules that range in size from 27 to 350 kb based on restriction endonuclease digestion and electron microscopic analysis.     

COLINEARITY OF CHLOROPLAST GENOMES IN DIVERGENT ECOTYYES OF THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYTA)1

The chloroplast genomes of three isolates of the marine diatom Skeletonema costatum (Grev.) Cleve were mapped and found to be 131 ± 2 kb with inverted repeats (IRs) of approximately 20 kb. In contrast to higher plants, the psbA gene mapped to the IR, and rbcS mapped to the same fragment as the rbcL gene in the large single-copy region. The maps of the three isolates were colinear and revealed as many as 20 site mutations out of a total of 47 sites. The number of site mutations among isolates was consistent with previous data on their genetic diversity and physiology. Comparisons of gene order among our maps and those of three other diatom species showed that closely related genera retained similar gene orders but that more distantly related taxa exhibited extensive rearrangements. We conclude that simple restriction fragment analysis of chloroplast DNA is useful in comparative studies of diatom populations and species but that other analytical methods are more appropriate for phylogenetic studies at higher levels.     

Cloning and characterization of chloroplast ribosomal protein-encoding genes, rpl16 and rps3, of the marine macro-algae, Gracilaria tenuistipitata

In Gracilaria tenuistipitata, a highly differentiated multicellular member of the marine red algae, Rhodophyta, chloroplast (cp) DNA can be separated as a satellite band from the nuclear DNA in a CsCl gradient. Using a heterologous probe from Chlamydomonas, the ribosomal protein-encoding gene, rpl16, was located on a 4.5-kb EcoRI fragment of cp DNA. The fragment was cloned and a 1365-bp region around rpl16 was sequenced. The gene order around rpl16, 5′ rpl22-rps3-rpl16, is identical to that detected in the chloroplast DNA of liverwort, tobacco and maize. Both the nucleotide sequence and the amino-acid sequence of rpl16 are more conserved than that of rps3. The rpl16 gene contains no intron, a feature which shows more similarity to the unicellular green algae, Chlamydomonas, than the other land plants. Sequences that may form a stable stem-loop structure were detected within the coding sequence of rpl16.     

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.