Cyanelle DNA from Cyanophora paradoxa

Summary Cyanelles which have been found in few eukaryotic organisms are photosynthetically active organelles which strikingly resemble cyanobacteria. The complexity of the cyanelle genome in Cyanophora paradoxa (127 Kbp) is too low to consider them as independent organisms in a symbiotic relationship. In order to correlate cyanelle genome and gene structure with those of plastid chromosomes of other plants, a circular map of the cyanelle DNA from Cyanophora paradoxa (strain LB555 UTEX) has been constructed using the restriction endonucleases SalI (generating 6 DNA fragments), BamHI (6), SalI (5), XhoI (9), and BglII (19).Besides the rRNA genes (16S, 23S, 5S), genes for 14 proteins have been located on this circular map. Among those are components of several multienzyme complexes involved in photosynthetic electron transport, as well as the large subunit of ribulose-1,5-bisphosphate carboxylase and two ribosomal proteins. All the probes used, were derived from a collection of spinach chloroplast DNA clones. Hybridization experiments showed signals to DNA fragments primarily from the large single-copy region of cyanelle DNA. The arrangement of genes on cyanelle DNA is different from that on spinach chloroplast DNA. However, genes which have been shown to be cotranscribed in spinach chloroplasts are also clustered on cyanelle DNA.Abbreviations Kbp 10³ base pairs - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase holoenzyme     

Chloroplast DNA rearrangements are more frequent when a large inverted repeat sequence is lost

We examined the arrangement of sequences common to seven angiosperm chloroplast genomes. The chloroplast DNAs of spinach, petunia and cucumber are essentially colinear. They share with the corn chloroplast genome a large inversion of approximately 50 kb relative to the genomes of three legumes--mung bean, pea and broad bean. There is one additional rearrangement, a second, smaller inversion within the 50 kb inversion, which is specific to the corn genome. These two changes are the only detectable rearrangements that have occurred during the evolution of the species examined (corn, spinach, petunia, cucumber and mung bean) whose chloroplast genomes contain a large inverted repeat sequence of 22-25 kb. In contrast, we find extensive sequence rearrangements in comparing the pea and broad bean genomes, both of which have deleted one entire segment of the inverted repeat, and also in comparing each of these to the mung bean genome. Thus there is a relatively stable arrangement of sequences in those genomes with the inverted repeat and a much more dynamic arrangement in those that have lost it. We discuss several explanations for this correlation, including the possibility that the inverted repeat may play a direct role in maintaining a conserved arrangement of chloroplast DNA sequences.     

The chloroplast genome arrangement ofLobelia thuliniana (Lobeliaceae): Expansion of the inverted repeat in an ancestor of theCampanulales

A clone-bank ofSac I restriction fragments was constructed from the chloroplast DNA (cpDNA) ofLobelia thuliniana E. B. Knox (Lobeliaceae). These cloned fragments and a set of 106 clones spanning the tobacco chloroplast genome were used as probes to determine the cpDNA restriction fragment arrangement forSac I and six other restriction enzymes (BamH I,EcoR V,Hind III,Nci I,Pst I, andXho I) and the chloroplast genome arrangement ofL. thuliniana relative to tobacco, which has been fully sequenced and is collinear with the hypothesized ancestral genome arrangement of angiosperms. The results confirm and refine our previous understanding of the chloroplast genome arrangement in the large single-copy region (LSC) and reveal (1) a roughly 11 kilobase (kb) expansion of the inverted repeat (IR) into the small single-copy region (SSC) and (2) apparent sequence divergence of the DNA segment inL. thuliniana that corresponds to ORF1901 in tobacco. The expansion of the IR into the SSC is present in all other examined members ofLobeliaceae, Cyphiaceae, andCampanulaceae, which indicates that the IR expansion was an early event in the cpDNA evolution of theCampanulales. The IR expansion into the SSC was not present inSphenoclea, which additionally supports exclusion of this genus from theCampanulaceae.     

ORGANIZATION OF THE CHLOROPLAST GENOME OF THE FRESHWATER CENTRIC DIATOM CYCLOTELLA MENEGHINIANA1

We constructed a complete physical map and a partial gene map of the chloroplast genome of Cyclotella meneghiniana Kützing clone 1020-1a (Bacillariophyceae). The 128-kb circular molecule contains a 17-kb inverted repeat, which divides the genome into single copy regions of65 kb and 29 kb. This is the largest genome and inverted repeat found in any diatom examined to date. In addition to the 16S and 23S ribosomal RNA genes, the inverted repeat contains both the ndhD gene (as yet unexamined in other diatoms) and the psbA gene (located similarly in one of two other examined diatoms). The Cyclotella chloroplast genome exists as two equimolar populations of inversion isomers that differ in the relative orientation of their single copy sequences. This inversion heterogeneity presumably results from intramolecular recombination within the inverted repeat. For the first time, we map the ndhD, psaC, rpofi, rpoCl, and rpoC2 genes to the chloroplast genome of a chlorophyll c-containing alga. While the Cyclotella chloroplast genome retains some prokaryotic and land plant gene clusters and operons, it contains a highly rearranged gene order in the large and small single copy regions compared to all other examined diatom, algal, and land plant chloroplast genomes.     

Tripartite mitochondrial genome of spinach: physical structure, mitochondrial gene mapping, and locations of transposed chloroplast DNA sequences.

A complete physical map of the spinach mitochondrial genome has been established. The entire sequence content of 327 kilobase pairs (kb) is postulated to occur as a single circular molecule. Two directly repeated elements of approximately 6 kb, located on this "master chromosome", are proposed to participate in an intragenomic recombination event that reversibly generates two "subgenomic" circles of 93 kb and 234 kb. The positions of protein and ribosomal RNA-encoding genes, determined by heterologous filter hybridizations, are scattered throughout the genome, with duplicate 26S rRNA genes located partially or entirely within the 6 kb repeat elements. Filter hybridizations between spinach mitochondrial DNA and cloned segments of spinach chloroplast DNA reveal at least twelve dispersed regions of inter-organellar sequence homology.     

The chloroplast genome from a lycophyte (microphyllophyte), <Emphasis Type="Italic">Selaginella uncinata</Emphasis>, has a unique inversion,transpositions and many gene losses

We determined the complete nucleotide sequence of the chloroplast genome of Selaginella uncinata, a lycophyte belonging to the basal lineage of the vascular plants. The circular double-stranded DNA is 144,170 bp, with an inverted repeat of 25,578 bp separated by a large single copy region (LSC) of 77,706 bp and a small single copy region (SSC) of 40,886 bp. We assigned 81 protein-coding genes including four pseudogenes, four rRNA genes and only 12 tRNA genes. Four genes, rps15, rps16, rpl32 and ycf10, found in most chloroplast genomes in land plants were not present in S. uncinata. While gene order and arrangement of the chloroplast genome of another lycophyte, Hupertzia lucidula, are almost the same as those of bryophytes, those of S. uncinata differ considerably from the typical structure of bryophytes with respect to the presence of a unique 20 kb inversion within the LSC, transposition of two segments from the LSC to the SSC and many gene losses. Thus, the organization of the S. uncinata chloroplast genome provides a new insight into the evolution of lycophytes, which were separated from euphyllophytes approximately 400 million years ago. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Physical map of chloroplast DNA in sugi,Cryptomeria japonica

Summary To investigate the evolution of conifer species, we constructed a physical map of the chloroplast DNA of sugi, Cryptomeria japonica, with four restriction endonucleases, PstI, SalI, SacI and XhoI. The chloroplast genome of C. japonica was found to be a circular molecule with a total size of approximately 133 kb. This molecule lacked an inverted repeat. Twenty genes were localized on the physical map of C. japonica cpDNA by Southern hybridization. The chloroplast genome structure of C. japonica showed considerable rearrangements of the standard genome type found in vascular plants and differed markedly from that of tobacco. The difference was explicable by one deletion and five inversions. The chloroplast genome of C. japonica differed too from that of the genus Pinus which also lacks one of the inverted repeats. The results indicate that the conifer group originated monophyletically from an ancient lineage, and diverged independently after loss of an inverted repeat structure.     

Chloroplast genes encoding subunits of the H+-ATPase complex of Chlamydomonas reinhardtii are rearranged compared to higher plants: sequence of the atpE gene and location of the atpF and atpI genes

Summary The chloroplast gene for the epsilon subunit (atpE) of the CF₁/CF₀ ATPase in the green alga Chlamydomonas reinhardtii has been localized and sequenced. In contrast to higher plants, the atpE gene does not lie at the 3 end of the beta subunit (atpB) gene in the chloroplast genome of C. reinhardtii, but is located at a position 92 kb away in the other single copy region. The uninterrupted open reading frame for the atpE gene is 423 bp, and the epsilon subunit exhibits 43% derived amino acid homology to that from spinach. Codon usage for the atpE gene follows the restricted pattern seen in other C. reinhardtii chloroplast genes.The genes for the CF₀ subunits I (atpF) and IV (atpI) of the ATPase complex have also been mapped on the chloroplast genome of C. reinhardtii. The six chloroplast ATPase genes in C. reinhardtii are dispersed individually between the two single copy regions of the chloroplast genome, an organization strikingly different from the highly conserved arrangement in two operon-like units seen in chloroplast genomes of higher plants.Abbreviations bp base pairs - CF₁ chloroplast coupling factor 1 - CF₀ chloroplast coupling factor 0 - F₁ coupling factor 1 - F₀ coupling factor 0 - kb kilobase pairs     

Molecular and genetic analysis of the chloroplast ATPase of chlamydomonas

Summary We have carried out a molecular and genetic analysis of the chloroplast ATPase in Chlamydomonas reinhardtii. Recombination and complementation studies on 16 independently isolated chloroplast mutations affecting this complex demonstrated that they represent alleles in five distinct chloroplast genes. One of these five, the ac-u-c locus, has been positioned on the physical map of the chloroplast DNA by deletion mutations. The use of cloned spinach chloroplast ATPase genes in heterologous hybridizations to Chlamydomonas chloroplast DNA has allowed us to localize three or possibly four of the ATPase genes on the physical map. The beta and probably the epsilon subunit genes of Chlamydomonas CF₁ lie within the same region of chloroplast DNA as the ac-u-c locus, while the alpha and proteolipid subunit genes appear to map adjacent to one another approximately 20 kbp away. Unlike the arrangement in higher plants, these two pairs of genes are separated from each other by an inverted repeat.     

Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa.

A rapid and simple method for constructing restriction maps of large DNAs (100-200 kb) is presented. The utility of this method is illustrated by mapping the Sal I, Sac I, and Hpa I sites of the 152 kb Atriplex triangularis chloroplast genome, and the Sal I and Pvu II sites of the 155 kb Cucumis sativa chloroplast genome. These two chloroplast DNAs are very similar in organization; both feature the near-universal chloroplast DNA inverted repeat sequence of 22-25 kb. The positions of four different genes have been localized on these chloroplast DNAs. In both genomes the 16S and 23S ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat, while genes for the large subunit of ribulose-1,5-bisphosphate carboxylase and a 32 kilodalton photosystem II polypeptide are separated by 55 kb of DNA within the large single copy region. The physical and genetic organization of these DNAs is compared to that of spinach chloroplast DNA.     

The chloroplast genome of Carthamus tinctorius

Summary A physical map of safflower (Carthamus tinctorius L.) chloroplast DNA has been generated using Sall, Pstl, Kpnl and HindIII restriction endonucleases. Southern blots to single and double digests by these enzymes were hybridized with ³²P-dCTP nick-translated Kpnl probes, which were individually isolated from agarose gels. The plastid genome was found to be circular (151 kbp), to contain a repeated sequence of about 25 kbp, and to have small and large single copy regions of approximately 20 and 81 kbp, respectively. Heterologous probes from spinach and Euglena containing psbA, rbcL, atpA or rrnA structural genes were also hybridized with such single and double restriction enzyme digests and mapped on this circular chlorpolast genome. The genetic map was found to be co-linear with that of spinach and many other higher plants.     

Chloroplast genome organization of bromegrass,Bromus inermis Leyss

Summary A physical map of the Bromus inermis chloroplast genome was constructed using heterologous probes of barley and wheat chloroplast DNA (cpDNA) to locate restriction sites. The map was aligned from data obtained from filter hybridization experiments on single and double enzyme digests. Cleavage sites for the enzymes PstI, SalI, KpnI, XhoI and PvuII were mapped. The chloroplast genome of B. inermis is similar in physical organization to that of other grasses. The circular cpDNA molecule of B. inermis has the typical small (12.8 kbp) and large (81.3 kbp) single-copy regions separated by a pair of inverted repeat (21 kbp) regions. The cpDNA molecule of B. inermis is collinear in sequence to that of wheat, rye, barley and oats. No structural rearrangements or major deletions were observed, indicating that the cpDNA of Bromus is a useful tool in phylogenetic studies.     

Extensive Rearrangements in the Chloroplast Genome of <Emphasis Type="Italic">Trachelium caeruleum</Emphasis> Are Associated with Repeats and tRNA Genes

Chloroplast genome organization, gene order, and content are highly conserved among land plants. We sequenced the chloroplast genome of Trachelium caeruleum L. (Campanulaceae), a member of an angiosperm family known for highly rearranged genomes. The total genome size is 162,321 bp, with an inverted repeat (IR) of 27,273 bp, large single-copy (LSC) region of 100,114 bp, and small single-copy (SSC) region of 7,661 bp. The genome encodes 112 different genes, with 17 duplicated in the IR, a tRNA gene (trnI-cau) duplicated once in the LSC region, and a protein-coding gene (psbJ) with two duplicate copies, for a total of 132 putatively intact genes. ndhK may be a pseudogene with internal stop codons, and clpP, ycf1, and ycf2 are so highly diverged that they also may be pseudogenes. ycf15, rpl23, infA, and accD are truncated and likely nonfunctional. The most conspicuous feature of the Trachelium genome is the presence of 18 internally unrearranged blocks of genes inverted or relocated within the genome relative to the ancestral gene order of angiosperm chloroplast genomes. Recombination between repeats or tRNA genes has been suggested as a mechanism of chloroplast genome rearrangements. The Trachelium chloroplast genome shares with Pelargonium and Jasminum both a higher number of repeats and larger repeated sequences in comparison to eight other angiosperm chloroplast genomes, and these are concentrated near rearrangement endpoints. Genes for tRNAs occur at many but not all inversion endpoints, so some combination of repeats and tRNA genes may have mediated these rearrangements.     

PSIIP680 ChlaAP基因在水稻叶绿体基因组中的定位

本实验总结出一套水稻叶绿体DNA的提取方法,并获得清晰的叶绿体DNA限制性内切酶图谱。Southern杂交结果表明,菠菜PSIIP680ChlaAP基因探针与水稻叶绿体DNA的Pst-1,Pst-14,Pvu-2和Sal-1片段的部分顺序有较高的同源性。根据Hirai和赵衍的水稻叶绿体基因组物理图,可以确定该基因位于紧靠RuBPCaseLS基因,距反向重复区约26kb处。高等植物叶绿体基因组中这种基因排列方式还未见报道。     

The first report describes features of the chloroplast genome of Withania frutescens

Genomic studies not only help researcher not only to identify genomic features in organisms, but also facilitate understanding of evolutionary relationships. Species in the Withania genus have medicinal benefits, and one of them is Withania frutescens, which is used to treat various diseases. This report investigates the nucleotides and genic features of chloroplast genome of Withania frutescens and trying to clarify the evolutionary relationship with Withania sp and family Solanaceae. We found that the total size of Withania frutescens chloroplast genome was 153.771 kb (the smallest chloroplast genome in genus Withania). A large single-copy region (91.285 kb), a small single-copy region (18.373 kb) form the genomic region, and are distinct from each other by a large inverted repeat (22.056 kb). 137 chloroplast genes are found including 4 rRNAs, 38 tRNAs and 83 protein-coding genes. The Withania frutescens chloroplast genome as well as four closest relatives was compared for features such as structure, nucleotide composition, simple sequence repeats (SSRs) and codon bias. Compared to other Withania species, Withania frutescens has unique characteristics. It has the smallest chloroplast genome of any Withania species, isoleucine is the major amino acid, and tryptophan is the minor, In addition, there are no ycf3 and ycf4 genes, fourth, there are only fifteen replicative genes, while in most other species there are more. Using fast minimum evolution and neighbor joining, we have reconstructed the trees to confirm the relationship with other Solanacaea species. The Withania frutescens chloroplast genome is submitted under accession no. ON153173.     

Mapping of genes on the chloroplast DNA of Spirodela oligorhiza

With the use of spinach chloroplast RNAs as probes, we have mapped the rRNA genes and a number of protein genes on the chloroplast DNA (cpDNA) of the duckweed Spirodela oligorhiz. For a more precise mapping of these genes we had to extend the previously determined [14] restriction endonuclease map of the duckweed cpDNA with the cleavage sites for the restriction endonucleases Sma I and Bgl I. The physical map indicates that duckweed cpDNA contains two inverted repeat regions (18 Md) separated by two single copy regions with a size of 19 Md and 67 Md, respectively.By hybridization with spinach chloroplast rRNAs it could be shown that each of the two repeat units contains one set of rRNA genes in the order: 16S rRNA gene — spacer — 23S rRNA gene — 5S rRNA gene.A spinach chloroplast mRNA preparation (14S RNA), which is predominantly translated into a 32 Kilodalton (Kd) protein [9], hybridized strongly to a DNA fragment in the large single copy region, immediately outside one of the inverted repeats. With another mRNA preparation (18S), which mainly directs the in vitro synthesis of a 55 Kd protein [9], hybridization was observed with two DNA regions, located between 211° and 233° and between 137° and 170°, respectively. Finally, with a spinach chloroplast genomic probe for the large subunit of ribulose 1,5-bisphosphate carboxylase [17], hybridization was found with a DNA fragment located between 137° and 158° on the map.     

Chloroplast Genome of Vigna aconififolia and Localization of Certain Photosynthesis-related Genes

The restriction analysis of chloroplast genome of Vigna aeonitifolia has revealed that it is about 150 kb in size, similar to V. radiata. The restriction pattern of chloroplast DNA (cpDNA) for Pst I is also the same from both the species, but restriction fragment length polymorphism is observed in cases of Kpn I and Sstl. These differences in the restriction patterns have arisen because of the occurrence of different restriction sites in the chloroplast genome of V. aconitifolia. A restriction map of cpDNA for V. aeonitifolia has been prepared on the basis of these observations. Furthermore, seven genes (psbA, psbB, psbC, psbD, psaA, psaB and rbcL) — coding for polypeptides of photosystems I and II as well as the large subunit of ribulose 1,5-bisphosphate carboxylaseloxygenase — have been localized on the Pst I — and Kpn I — generated restriction fragments of V. aconitifolia with the help of heterologous gene-specific probes and their relative position on the restriction map is presented. The gene organization supports the view that an inversion of about 50 kb has occurred in Vigna cpDNA as compared to other species.