Chloroplast transfer RNAs and tRNA genes of wheat

Fractionation (by two-dimensional polyacrylamide gel electrophoresis) of total tRNA from wheat chloroplasts yields about 33 RNA spots. Of these, 30 have been identified by aminoacylation as containing tRNAs specific for 17 amino acids. Hybridization of labeled individual tRNAs to cloned chloroplast DNA fragments has revealed the location of at least nine pairs of tRNA genes in the segments of the inverted repeat, at least twelve tRNA genes in the large single copy region and one tRNA gene in the small single copy region. A comparison of this wheat chloroplast tRNA gene map to that of maize and of other higher plants suggests that gene rearrangements have occurred during evolution, even within cereal chloroplast DNA. These rearrangements have taken place within the inverted repeat, within the large single copy region and between the inverted repeat and the large single copy region.     

Construction of the physical map of the chloroplast DNA of Phaseolus vulgaris and localization of ribosomal and transfer RNA genes

Construction of a physical map of the chloroplast DNA from Phaseolus vulgaris showed that this circular molecule is segmentally organized into four regions. Unlike other chloroplast DNAs which have analogous organization, two single-copy regions that separate two inverted repeats have been demonstrated to exist in both relative orientations, giving rise to two populations of DNA molecules.Hybridization studies using individual rRNA and tRNA species revealed the location of a set of rRNA genes and at least seven tRNA genes in each inverted repeat region, a minimum of 17 tRNA genes in the large single-copy region and one tRNA gene in the small single-copy region. The tRNA genes code for 24 tRNA species corresponding to 16 amino acids. Comparison of this gene map with those of other chloroplast DNAs suggests that DNA sequence rearrangements, involving some tRNA genes, have occurred.     

Transfer RNA genes of Euglena gracilis chloroplast DNA

Summary Transfer RNA genes have been mapped to at least nine different loci on the physical map of the Euglena gracilis chloroplast genome. One of these loci in the ribosomal RNA operons is present three times per genome. The DNA sequences of six of the nine different loci, containing 21 different tRNA genes, have been determined. Genes corresponding to the amino acids Ala, Arg, Asn, Cys, Gln, Gly (2), Glu, His, Ile, Leu (2), Met (2), Phe, Ser, Thr, Trp, Tyr, Val, and one unassigned species have been identified. All genes except one are found in clusters of 2–6 genes. None of the known genes contains introns, nor codes for the 3-CCA terminus. In addition to these genes, two pseudo tRNA genes are present in the rDNA leader region.     

Structures of tobacco chloroplast genes for tRNAIle (CAU), tRNALeu (CAA), tRNACys (GCA), tRNASer (UGA) and tRNAThr (GGU): a compilation of tRNA genes from tobacco chloroplasts

Summary The location and nucleotide sequences of tobacco chloroplast genes for tRNA^Ile (CAU), tRNA^Leu (CAA), tRNA^Cys (GCA), tRNA^Ser (UGA) and tRNA^Thr (GGU) (trnI-CAU, trnL-CAA, trnC-GCA, trnS-UGA and trnT-GGU, respectively) have been determined. The trnI and trnL are located in the inverted repeat region. The trnC, trnS and trnT are present in the large single copy region. These five tRNA genes together with the 25 different tRNA genes previously published have been compiled and compared. These 30 tRNA genes corresponding to 20 amino acids are most likely to be all of the tRNA genes encoded in tobacco chloroplast genome.This paper is dedicated to Professor Morio Ikehara on the occasion of his retirement from Osaka University in March 1986.     

Nucleotide sequence of the single ribosomal RNA operon of pea chloroplast DNA

The nucleotide sequence of an 8 kbp region of pea ( Pisum sativum L.) chloroplast DNA containing the rRNA operon and putative promoter sites has been determined and compared to the corresponding sequences from maize, tobacco and the liverwort Marchantia polymorpha . The chloroplast DNA species of all vascular plants investigated, with the exception of a few legumes including pea, and of Marchantia contain an inverted repeat with an rRNA operon. The pea rRNA operon is the first sequenced rRNA operon from a plant with only one copy of the rRNA genes per molecule of chloroplast DNA. The organization of the operon is the same as for maize, tobacco and Marchantia . i.e. tRNA-Val gene/16S rRNA gene/spacer with intron-containing genes for tRNA-Ile and tRNA-Ala/23S rRNA gene/4.5S rRNA gene/5S rRNA gene. Current evidence suggests that the tRNA-Val gene may not be contranscribed with the other genes. For pea 16S, 23S, 4.5S and 5S rRNA have 1488, 2813, 105 and 121 nucleotides, respectively. The homologies of the entire operon (the tRNA-Val gene - 5S rRNA region) to those from tobacco, maize and Marchantia are 88, 82 and 79%, respectively. The corresponding homologies for tobacco/maize, tobacco/ Marchantia and maize/ Marchantia have similar values. The 16S and 23S rRNA genes from pea are more than 90% homologous to those from the 3 other species. We conclude that the fact that pea only has one set of rRNA genes per molecule of chloroplast DNA is apparently not correlated with any significant difference between the pea operon and the rRNA operons from tobacco, maize and Marchantia .     

Transfer RNA genes ofZea mays chloroplast DNA

A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA₂ ^Ile and tRNA^Ala map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAs^Leu and the three for tRNAs^Ser, as well as the two isoaccepting species for tRNA^Asn, tRNA^Gly, tRNAs^Ile, tRNAs^Met, tRNAs^Thr, are shown to be encoded at different loci. Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were³²P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases.     

Nucleotide sequences of transfer RNA genes in the Pisum sativum chloroplast DNA

Summary Eight transfer RNA (tRNA) genes which were previously mapped to five regions of the Pisum sativum (pea) chloroplast DNA (ctDNA) have been sequenced. They have been identified as tRNA^Val(GAC), tRNA^Asn(GUU), tRNA^Arg(ACG), tRNA^Leu(CAA), tRNA^Tyr(GUA), tRNA^Glu(UUC), tRNA^His(GUG), and tRNA^Arg(UCU) by their anticodons and by their similarity to other previously identified tRNA genes from the chloroplast DNAs of higher plants or from E. gracilis. In addition,two other tRNA genes, tRNA^Gly (UCC) and tRNA^Ile(GAU), have been partially sequenced. The tRNA genes are compared to other known chloroplast tRNA genes from higher plants and are found to be 90–100% homologous. In addition there are similarities in the overall arrangement of the individual genes between different plants. The 5 flanking regions and the internal sequences of tRNA genes have been studied for conserved regions and consensus sequences. Two unusual features have been found: there is an apparent intron in the D-loop of the tRNA^Gly(UCC), and the tRNA^Glu(UUC) contains GATTC in its T-loop.     

Localization of tRNA genes on the Petunia hybrida 3704 mitochondrial genome

22 tRNA genes corresponding to 17 tRNA species were localized on the master circle of Petunia hybrida mitochondrial (mt) DNA. Genes for trnN, trnM, trnS-GGA, trnW and trnH are of the chloroplast-like type and presumably originate from promiscuous chloroplast (cp) DNA sequences inserted into the petunia mitochondrial genome. A comparison of the mt tRNAs or tRNA genes population present in two monocotyledonous plants (wheat and maize) and two dicotyledonous plants (petunia and potato) show slight differences in the genetic origin of individual tRNAs. The organization of the petunia mt tRNA genes as well as the number of tRNA gene copies, compared to other plant species, is discussed.     

Partial conservation of the 5′ ndhE-psaC-ndhD 3′ gene arrangement of chloroplasts in the cyanobacterium Synechocystis sp. PCC 6803: implications for NDH-D function in cyanobacteria and chloroplasts

The psaC gene, which encodes the 8.9 kDa iron-sulfur containing subunit of Photosystem I, has been sequenced from Synechocystis sp. PCC 6803 and shows greater similarity to reported plant sequences than other cyanobacterial psaC sequences. The deduced amino acid sequence of the protein encoded by the Synechocystis psaC gene is identical to the tobacco PSA-C sequence. In plants psaC is located in the small single-copy region of the chloroplast genome between two genes (designated ndhE and ndhD) with similarity to genes encoding subunits of the mitochondrial NADH Dehydrogenase Complex I. The 5 ndhE-psaC-ndhD3 gene arrangement of higher plants is only partially conserved in Synechocystis. An open reading frame (ORF) upstream of the Synechocystis psaC gene has 85% identity to the tobacco ndhE gene. Downstream of psaC there is a 273 bp ORF with 48% identity to the 5 portion of the tobacco ndhD gene (1527 bp). psaC, ndhE and the region of similarity to ndhD are present in a single copy in the Synechocystis genome. Part of the wheat ndhD gene was sequenced and used as a probe for the presence of the 3 portion of the ndhD gene. The wheat ndhD probe did not hybridize to Synechocystis or Anabaena sp. PCC 7120 genomic DNA, but did hybridize to Oenothera chloroplast DNA. These results indicate the complete ndhD gene is absent in two cyanobacteria, and raises the question of what role, if any, the ndhD gene product plays in the facultative heterotroph Synechocystis sp. PCC 6803.     

Rice chloroplast DNA: a physical map and the location of the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase and the 32 KD photosystem II reaction center protein

Summary By homogenizing rice leaves in liquid nitrogen, it was possible to isolate intact chloroplasts and, subsequently, pure rice chloroplast DNA from the purified chloroplasts. The DNA was digested by several restriction enzymes and fragments were fractionated by agarose gel electrophoresis. The sum of the fragment sizes generated by the restriction enzymes showed that the total length of the DNA is 130 kb. A circular physical map of fragments, generated by digestion with SalI, PstI, and PvuII, has been constructed. The circular DNA contains two inverted repeats of about 20 kb separated by a large, single copy region of about 75 kb and a short, single copy region of about 15 kb. The location of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase (Fraction I protein) and the 32 KD photosystem II reaction center gene were determined by using as probes tobacco chloroplast DNAs containing these genes. Rice chloroplast DNA differs from chloroplast DNAs of wheat and corn as well as from dicot chloroplast DNAs by having the 32 KD gene located 20 kb removed from the end of an inverted repeat instead of close to the end, as in other plants.     

Locations and sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC): the tRNAGly contains only two base-pairs in the D stem.

The nucleotide sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC) have been determined. None of these genes contains an intron. One unusual feature is that the tRNAGly contains only two base-pairs (A-U, G-U) in the D stem. These four tRNA genes were located in the known physical map of tobacco chloroplast DNA. Hybridization analysis to chloroplast tRNA revealed that all four tRNA genes are transcribed in vivo.     

Complete structure of the chloroplast genome of a legume, Lotus japonicus.

The nucleotide sequence of the entire chloroplast genome (150,519 bp) of a legume, Lotus japonicus, has been determined. The circular double-stranded DNA contains a pair of inverted repeats of 25,156 bp which are separated by a small and a large single copy region of 18,271 bp and 81,936 bp, respectively. A total of 84 predicted protein-coding genes including 7 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids species were assigned on the genome based on similarity to genes previously identified in other chloroplasts. All the predicted genes were conserved among dicot plants except that rpl22, a gene encoding chloroplast ribosomal protein CL22, was missing in L. japonicus. Inversion of a 51-kb segment spanning rbcL to rpsl6 (positions 5161-56,176) in the large single copy region was observed in the chloroplast genome of L. japonicus. The sequence data and gene information are available on our World Wide Web database at http://www.kazusa.or.jp/en/plant/database.html.     

Variable copy number DNA sequences in rice

Summary We have cloned two types of variable copy number DNA sequences from the rice embryo genome. One of these sequences, which was cloned in pRB301, was amplified about 50-fold during callus formation and diminished in copy number to the embryonic level during regeneration. The other clone, named pRB401, showed the reciprocal pattern. The copy numbers of both sequences were changed even in the early developmental stage and eliminated from nuclear DNA along with growth of the plant. Sequencing analysis of the pRB301 insert revealed some open reading frames and direct repeat structures, but corresponding sequences were not identified in the EMBL and LASL DNA databases. Sequencing of the nuclear genomic fragment cloned in pRB401 revealed the presence of the 3rps12-rps7 region of rice chloroplast DNA. Our observations suggest that during callus formation (dedifferentiation), regeneration and the growth process the copy numbers of some DNA sequences are variable and that nuclear integrated chloroplast DNA acts as a variable copy number sequence in the rice genome. Based on data showing a common sequence in mitochondria and chloroplast DNA of maize (Stern and Lonsdale 1982) and that the rps12 gene of tobacco chloroplast DNA is a divided gene (Torazawa et al. 1986), it is suggested that the sequence on the inverted repeat structure of chloroplast DNA may have the character of a movable genetic element.     

Gene organization of chloroplast DNA from the broad bean Vicia faba

Summary Six protein genes have been mapped on broad bean chloroplast DNA by Southern hybridization using the tobacco chloroplast genes as probes. In broad bean chloroplast DNA, the genes for the and subunits of proton-translocating ATPase and the 32,000 dalton thylakoid membrane protein are located near the large subunit gene of ribulose-1,5-bisphosphate carboxylase/oxygenase. The gene for the subunit of proton-translocating ATPase is distantly located from the and genes. The gene for the ribosomal protein CS19 was found close to the ribosomal RNA operon. The gene organization of broad bean chloroplast DNA is therefore quite different from that of tobacco chloroplast DNA. The nucleotide sequence of the spacer region between the large subunit and the genes of the broad bean has been determined. Conserved sequences are found among the putative promoter regions of the chloroplast protein genes.     

Physical map and gene localization on sunflower (Helianthus annuus) chloroplast DNA: evidence for an inversion of a 23.5-kbp segment in the large single copy region

As a first step in the study of chloroplast genome variability in the genus Helianthus, a physical restriction map of sunflower (Helianthus annuus) chloroplast DNA (cpDNA) has been constructed using restriction endonucleases BamH I, Hind III, Pst I, Pvu II and Sac. I. Sunflower circular DNA contains an inverted repeat structure with the two copies (23 kbp each) separated by a large (86 kbp) and a small (20 kbp) single copy region. Its total length is therefore about 152 kbp. Sunflower cpDNA is essentially colinear with that of tobacco with the exception of an inversion of a 23.5-kbp segment in the large single copy region. Gene localization on the sunflower cpDNA and comparison of the gene map with that from tobacco chloroplasts have revealed that the endpoints of the inversion are located between the trnT and trnE genes on the one hand, and between the trnG and trnS genes on the other hand.Analysis of BamH I restriction fragment patterns of H. annuus, H. occidentalis ssp. plantagineus, H. grossesseratus, H. decapetalus, H. giganteus, H. maximiliani and H. tuberosus cpDNAs suggests that structural variations are present in the genus Helianthus.     

Divergence of chloroplast gene organization in three legumes: Pisum sativum,Vicia faba and Phaseolus vulgaris

Summary Isolated chloroplasts from Pisum sativum were found to contain at least 32 tRNA species. Hybridization of in vitro labeled, identified, chloroplast tRNAs to Pisum chloroplast DNA fragments revealed the locations of the tRNA genes on the circular chloroplast genome. Comparison of this gene map to the maps of Vicia faba and Phaseolus vulgaris showed that the chloroplast genomes of Pisum and Phaseolus are otherwise more closely related than either genome is to the chloroplast genome of Vicia. Furthermore, the results suggest how possible recombination events could be involved in the evolution of these three closely related, but divergent, chloroplast genomes.     

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.