The two genes for the P700 chlorophyll a apoproteins on the Euglena gracilis chloroplast genome contain multiple introns

Summary We have determined the complete nucleotide sequence of the two genes encoding the P₇₀₀ chlorophyll a apoproteins of the photosystem I reaction center of the Euglena gracilis chloroplast genome. The two genes are separated by 77 bp, are of the same polarity, and span a region which is greater than 9.0 kbp. The psaA gene (751 codons) is interrupted by three introns and the psaB gene (734 codons) by six introns. The introns range in size from 361 to 590 bp, whereas the exons range in size from 42 to 1,194 bp. The introns are extremely AT rich with a pronounced base bias of T > A > G > C in the RNA-like strand. Like other interrupted protein genes in the Euglena chloroplast genome, the psaA and psaB introns are similar to mitochondrial group II introns in having the splice junction consensus sequence, 5 GTGCGNTTCG ..... INTRON ..... TTAATTTTAT 3 and conserved secondary structural features. Except for the placement of the first intron, the intron-exon organization of these two highly homologous genes is not conserved. The other introns fall at or near putative surface domains of the predicted gene products. The psaA and psaB gene products are 74% homologous to one another and 93% and 95% homologous, respectively, to the psaA and psaB gene products of higher plant chloroplasts. The predicted secondary structure derived from the primary amino acid sequence has 11 potential membrane-spanning domains. Abbreviations and notations: Gene names follow the convention of Hallick and Bottomley (1983: psaA, psaB, genes for the P₇₀₀ apoprotein; psbE an psbF, genes foe the subunits of cytochrome b ₅₅₉; orfN, open reading frame of N condons     

Organisation of the chloroplast genome of kiwifruit (Actinidia deliciosa)

Summary A restriction map of the chloroplast genome has been determined for kiwifruit, Actinidia deliciosa. Single and multiple enzyme digests of kiwifruit chloroplast DNA were hybridised to a set of Brassica chloroplast probes, and the kiwifruit bands aligned with the known Brassica map. The chloroplast DNA of kiwifruit is typical of the majority of angiosperm chloroplast genomes; it is 160 kb in size, contains a 15–34 kb inverted repeat, and its gene content and gene order are similar to those of the Brassica chloroplast genome.     

The chloroplast trnP-trnW-petG gene cluster in the mitochondrial genomes of Beta vulgaris,B. trigyna and B. webbiana: evolutionary aspects

The chloroplast trnP-trnW-petG gene cluster has been identified in the mitochondrial DNA (mtDNA) of sugar beet (Beta vulgaris). The chloroplast-derived trnW gene is transcribed in the mitochondria; the other two genes, however, do not seem to be transcribed. This gene cluster is also present in the mitochondrial genomes of two wild Beta species, B. trigyna and B. webbiana. Sugar beet and the two wild relatives share 100% sequence identity in the coding regions of both the mitochondrial trnP and trnW genes. On the other hand, the petG genes from the wild Beta mtDNAs were found to be disrupted either by a 5-bp duplication (B. trigyna) or by a deletion of the 5 region (B. webbiana). A data-base search revealed that a conserved sequence of 60 bp is present in the trnP-trnW intergenic region of the mitochondrial genomes of the three Beta species as well as in other higher plants, including wheat and maize, and that the conserved sequence is absent from the chloroplast counterpart. Our results thus favour the hypothesis of a monophyletic origin of the trnP-trnW-petG cluster found in the plant mitochondrial genomes examined.     

Restriction site and genetic map of Cucurbita pepo chloroplast DNA

Summary A detailed restriction map of squash chloroplast DNA (cpDNA) was constructed with five restriction endonuclease, SalI, PvuII, BglI, SacII, and PstI. The cleavage sites were mapped by sequential digestion of cpDNA using low-gelling temperature agarose. The restriction map shows that squash cpDNA is an approximately 153 kilobase (kb) circle with a large inverted repeat sequence of 23.3 kb, separated by a large (83.7 kb) and a small (22.7 kb) single copy region. Genes for a number of chloroplast polypeptides were localized on the map by hybridizing the cpDNA restriction fragments to heterologous gene-specific probes from tobacco, pea, tomato, maize, and spinach chloroplasts. The gene locations and organization of squash cpDNA are highly conserved and similar to chloroplast genomes of tomato, pepper, and Ginkgo.Abbreviations cpDNA chloroplast DNA - kb kilobases - IR inverted repeat. Gene names follow the nomenclature recommendation of Hallick and Bottomley (1983)     

Characterization of the nuclear gene encoding chloroplast ribosomal protein S13 from Arabidopsis thaliana

We have characterised a cDNA clone and a nuclear gene encoding the chloroplast 30 s ribosomal protein S13 from Arabidopsis thaliana. The identification is based on the high similarity of the predicted amino-acid sequence with eubacterial S13 protein sequences, and immunodetection of a 14.5-kDa chloroplast ribosomal polypeptide using antibodies raised against the polypeptide produced from part of the cDNA expressed in bacteria. The predicted amino-acid sequence contains an N-terminal extension which has several features characteristic of chloroplast transit peptides. Experiments suggest there is a single copy of this gene in A. thaliana and multiple copies in Brassica species. The origin of the mitochondrial S13 polypeptide in crucifers is also discussed.     

Polymorphism of the mitochondrial L-RNA gene of the basidiomycete Coprinus cinereus

Summary Two unexpectedly small mitochondrial (mt) genomes of Coprinus cinereus, P and S, were compared with the H and J genomes we have described previously. H and J are 42 kb in size and differ in having alternative 1.23 kb insertions in or adjacent to the co-1 gene. P and S DNAs lacked both insertions and had an identical 4.4 kb deletion between the co-1 and L-RNA gene. P DNA contained a 700 by insertion and S DNA a 300 by deletion within a sequence coding the L-RNA gene. This was shown by Southern blot analysis using probes containing the 5 or the 3 exon sequences of the L-RNA gene of Neurospora crassa. These hybridisations showed also that the L-RNA gene and co-1 gene in the C. cinereus mt genome are oppositely orientated and must be transcribed from different DNA strands. No DNA homology was detected using probes containing intron sequences from the L-RNA genes of Saccharomyces cerevisiae or N. crassa. There was no evidence of respiratory deficiency in P and S strains and transfer of nuclei by dikaryon formation made it possible to recombine H nuclei with P and S mitochondria, S nuclei with H and P mitochondria and P nuclei with H mitochondria with no apparent detrimental effect on growth. We conclude that P and S mtDNAs represent naturally occurring variants of the C. cinereus mt genome.     

Chloroplast DNA from Adiantum capillus-veneris L., a fern species (Adiantaceae); clone bank,physical map and unusual gene localization in comparison with angiosperm chloroplast DNA

Summary We cloned all of Adiantum capillus-veneris chloroplast DNA PstI fragments longer than 1.0 kb, which cover 98% of the genome. These cloned fragments were used to construct a physical map for five restriction enzymes. The genome of A. capillus-veneris is approximately 153 kb long and contains a 24 kb inverted repeat. Mapping of 12 chloroplast DNA genes and heterologous hybridization, involving A. capillus-veneris chloroplast DNA and angiosperm chloroplast DNA probes, demonstrated that chloroplast DNA of A. capillus-veneris has a different gene order from typical angiosperm cpDNA (e.g., tobacco) in the inverted repeat region and the flanking segment of the large single copy region.     

The trans-spliced intron 1 in the psaA gene of the Chlamydomonas chloroplast: a comparative analysis

In the secondary structure model that has been proposed for the trans-spliced intron 1 in the Chlamydomonas reinhardtii psaA gene, a third RNA species (tscA RNA) interacts with the 5 and 3 intron parts flanking the exons to reconstitute a composite structure with several features of group-II introns. To test the validity of this model, we undertook the sequencing and modelling of equivalent introns in the psaA gene from other unicellular green algae belonging to the highly diversified genus Chlamydomonas. Our comparative analysis supports the model reported for the C. reinhardtii psaA intron 1, and also indicates that the 5 end of the tscA RNA and the region downstream from the psaA exon 1 cannot be folded into a structure typical of domain I as described for most group-II introns. It is possible that a fourth RNA species, yet to be discovered, provides the parts of domain I which are apparently missing.     

The complete mitochondrial genome sequence of Brassica oleracea and analysis of coexisting mitotypes

The complete mitochondrial genome sequences of Brassica species have provided insight into inter- and intraspecific variation of plant mitochondrial genomes. However, the size of mitochondrial genome sequenced for Brassica oleracea hitherto does not match to its physical mapping data. This fact led us to investigate B. oleracea mitochondrial genome in detail. Here we report novel B. oleracea mitochondrial genome, derived from var. capitata, a cabbage cultivar ‘‘Fujiwase’’. The genome was assembled into a 219,952-bp circular sequence that is comparable to the mitochondrial genomes of other Brassica species (ca. 220–232 kb). This genome contained 34 protein-coding genes, 3 rRNA genes and 17 tRNA genes. Due to absence of a large repeat (140 kb), the mitochondrial genome of ‘‘Fujiwase’’ is clearly smaller than the previously reported mitochondrial genome of B. oleracea accession ‘‘08C717’’ (360 kb). In both mitotypes, all genes were identical, except cox2-2, which was present only in the Fujiwase type. At least two rearrangement events via large and small repeat sequences have contributed to the structural differences between the two mitotypes. PCR-based marker analysis revealed that the Fujiwase type is predominant, whereas the 08C717 type coexists at low frequency in all B. oleracea cultivars examined. Intraspecific variations in the mitochondrial genome in B. oleracea may occur because of heteroplasmy, coexistence of different mitotypes within an individual, and substoichiometric shifting. Our data indicate that the Fujiwase-type genome should be used as the representative genome of B. oleracea.     

Comparative analysis of the mitochondrial genomes of Chlamydomonas eugametos and Chlamydomonas moewusii

Summary We report the cloning and physical mapping of the mitochondrial genome of Chlamydomonas eugametos together with a comparison of the overall sequence structure of this DNA with the mitochondrial genome of Chlamydomonas moewusii, its closely related and interfertile relative. The C. eugametos mitochondrial DNA (mtDNA) has a 24 kb circular map and is thus 2 kb larger than the 22 kb circular mitochondrial genome of C. moewusii. Restriction mapping and heterologous fragment hybridization experiments indicate that the C. eugametos and C. moewusii mtDNAs are colinear. Nine cross-hybridizing restriction fragments common to the C. eugametos and C. moewusii mtDNAs, and spanning the entirety of these genomes, show length differences between homologous fragments which vary from 0.1 to 2.3 kb. A 600 bp subfragment of C. moewusii mtDNA, within one of these conserved fragments, showed no hybridization with the C. eugametos mtDNA. Of the 73 restriction sites identified in the C. eugametos and C. moewusii mtDNAs, five are specific to C. moewusii, eight are specific to C. eugametos and 30 are common to both species. Hybridization experiments with gene probes derived from protein-coding and ribosomal RNA-coding regions of wheat and Chlamydomonas reinhardtii mtDNAs support the view that the small and large subunit ribosomal RNA-coding regions of the C. eugametos and C. moewusii mtDNAs are interrupted and interspersed with each other and with protein-coding regions, as are the ribosomal RNA-coding regions of C. reinhardtii mtDNA; however, the specific arrangement of these coding elements in the C. eugametos and C. moewusii mtDNAs appears different from that of C. reinhardtii mtDNA.     

Optional elements in the chloroplast DNAs of Chlamydomonas eugametos and C. moewusii: unidirectional gene conversion and co-conversion of adjacent markers in high-viability crossses

Unlike most polymorphic markers in the Chlamydomonas eugametos and Chlamydomonas moewusii chloroplast DNAs (cpDNAs), the C. moewusii 6- and 21-kb extra sequences and the C. eugametos-specific CeLSU ⋅ 5 intron are transmitted to all of the few viable progeny in reciprocal crosses between the two green algae. To determine whether this unidirectional transmission pattern is due to gene conversion or to selection for F1 hybrid survival, we followed the inheritance of the parental alleles at the loci featuring these three deletions/additions and at several other polymorphic cpDNA loci in zygospore clones derived from high-viability crosses. The great majority of the zygospore clones examined inherited exclusively the long alleles from the mt ^– parent at the loci containing the three optional cpDNA elements, but as expected, they preferentially inherited the markers from the mt ⁺ parent at most other loci. Our results therefore indicate that all three optional cpDNA sequences propagate themselves very efficiently by gene conversion in crosses between strains differing by the presence of these elements. The co-conversion tracts associated with these sequences are longer (>3 kb) than those previously reported for mobile elements spreading by gene conversion. Our results also revealed that less efficient gene conversion events occurred at two other cpDNA loci. Received: 12 February / 14 May 1996     

The gene for the 9 kd polypeptide,a possible apoprotein for the iron-sulfur centers A and B of the photosystem I complex,in tobacco chloroplast DNA

Summary The gene for the 9 kd polypeptide (a possible apoprotein for the iron-sulfur centers A and B) of photosystem I has been located in the small single-copy region of tobacco chloroplast DNA. This gene (psaC) was identified by comparing the N-terminal amino acid sequence of the spinach 9 kd polypeptide with the entire sequence of tobacco chloroplast genome. The gene organization is ndhE (101 codons) — 263 by spacer — psaC (S1 codons) — 94 by spacer - ndhD (509 codons). Northern blot hybridization revealed that psaC is transcribed in the chloroplasts. The deduced amino acid sequence and secondary structure are presented. The predicted polypeptide is rich in cysteine residues and contains a unique repeated sequence.     

Structure and expression of tomato mitochondrial genes coding for tRNACys (GCA), tRNAAsn (GUU) and tRNATyr (GUA): A native tRNACys gene is present in dicot plants but absent in monocot plants

Summary The nucleotide sequences of tRNA^Asn (GUU) and tRNA^Tyr (GUA) genes from tomato mitochondria and their flanking regions have been determined. The tomato mitochondrial tRNA^Asn gene is located 2.1 kb downstream from the tRNA^Cys gene reported previously (Izuchi and Sugita 1989) and shows a nearly complete identity with the corresponding chloroplast gene. The tRNA^Tyr gene, which shows only 73% homology with the corresponding chloroplast gene, has to be considered a native mitochondrial tRNA gene and is 535 bp from the chloroplast-like tRNA^Asn gene on the same strand. Northern hybridization analysis revealed that the three tRNA genes are transcribed in tomato mitochondria. Southern hybridization analysis of tomato, sugar beet, rice and wheat mitochondrial DNAs, with oligonucleotide probes for mitochondrial or chloroplast tRNA genes, demonstrated that the mitochondrial tRNA^Cys gene found in tomato is present in dicot plants but not in monocots. On the other hand, a chloroplast-like tRNA^Cys gene exists in monocot plants.     

Organization of the psbE,psbF, orf38, and orf42 gene loci on the Euglena gracilis chloroplast genome

Summary The genes for cytochrome b559, designated psbE and psbF, and two highly conserved open reading frames of 38 and 42 codons have been located and characterized on the chloroplast genome of Euglena gracilis. The organization of the genes is psbE — 8 by spacer —psbF — 110 by spacer — orf38 — 87 by spacer — orf42. All genes are of the same polarity. The psbE gene contains two introns of 350 and 326 bp. The psbF gene contains a single large intron of 1,042 bp. The orf38 and orf42 loci lack introns. The introns are extremely AT rich with a pronounced base composition bias of T > A > G > C in the mRNA-like strand and group II-like boundary sequences at their 3 and 5 ends having the consensus 5-GTGTG .. INTRON .. TTAATTTNAT-3. The psbE gene consists of 82 codons and encodes a polypeptide with a predicted molecular weight of 9,212. The psbF gene consists of 42 codons, which specify a polypeptide with a predicted molecular weight of 4,785. The highly conserved open reading frames of 38 and 42 codons code for polypeptides with predicted molecular weights of 4,405 and 4,426, respectively. The gene products of psbE psbF orf38 and orf42 are, respectively, 69.5%, 70% and 61.5% identical to those found in higher plants. The predicted secondary structure of the proteins from hydropathy plots is consistent with each containing a single membrane-spanning domain of at least 20 amino acids. Each of the genes is preceded by sequences which may serve as ribosome binding sites. All four genes are transcribed.Abbreviations and notations DEP diethyl pyrocarbonate; gene names follow the convention of Hallick and Bottomley (1983) - psaA, psaB genes for the P₇₀₀ apoproteins - psbE and psbF genes for the subunits of cytochrome b ₅₅₉ - orfN open reading frame of N codons     

Restriction and gene maps of plastid DNA from Capsicum annuum

Summary Chloroplasts and chromoplasts were isolated from green and red fruits, respectively, of the bell pepper, Capsicum annuum var. Emerald giant. A comparison of the restriction patterns of DNAs isolated from these plastids was made using single and double digests by SacI, PvuII, PstI, and SalI and found to be indistinguishable. It is inferred therefore that the conversion of chloroplasts to chromoplasts in Capsicum annuum does not involve any large rearrangements of the plastid chromosome. A restriction map of Capsicum annuum plastid DNA was constructed using the same restriction enzymes in single digests and in all possible pair combinations. Overlapping restriction fragments were identified by digesting each product of a single digest with each of the other three enzymes. The resulting restriction map is similar to that of chloroplast DNA from other members of the Solanaceae with respect to most restriction sites. The genome size corresponds to 143 kbp. The locations of 24 genes, coding for ribosomal RNAs and for proteins of Photosystem I (PSI), Photosystem II (PSII), ATP synthase, cytochromes, the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (E.C. 4.1.1.29) (RuBPC), and ribosomal proteins were determined by probing Southern blots of Capsicum chloroplast DNA with probes of genes from spinach and tobacco. The gene locations are completely conserved with respect to those of other members of the Solanaceae and the majority of higher land plants.Abbreviations and notations atpA, atpB, atpE, and atpF genes for the , , , and I subunit, respectively, of ATP synthase - cpDNA chloroplast DNA - petA, petB, petD genes for cytochrome f, cytochrome b ₆, and subunit IV of cytochrome b ₆/f complex, respectively - psaA, psaB, psaC genes for the P₇₀₀ apoproteins - psbA gene for Q_B - psbB and psbC genes for the 51-kDa and 44-kDa proteins, respectively, of PSII - psbD gene for the Q_B-like polypeptide of PSII - psbE gene for cytochrome b ₅₅₉ - rbcL gene for the large subunit of RuBPC - rpl2 gene for ribosomal protein L2 - rpoA gene for the subunit of RNA polymerase - rps11, rps12 and rps19 genes for ribosomal proteins S11, S12, and S19, respectively - rps19 open reading frame for a protein with N terminus similar to that of S19 - RuBPC ribulose-1,5-bisphosphate carboxylase-oxygenase (E.C. 4.1.1.29) - trnH gene for histidine transfer RNA - URF39 and URF509 unidentified reading frames for polypeptides of 39 and 509 amino acids, respectively Gene names follow the convention of Hallick and Bottomley (1983)     

A mutation hotspot in the chloroplast genome of a conifer (Douglas-fir: Pseudotsuga) is caused by variability in the number of direct repeats derived from a partially duplicated tRNA gene

We determined the DNA sequence of a 2.7-kb cpDNA XbaI fragment from douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. RFLPs revealed by the 2.7-kb XbaI clone were observed to vary up to 1 kb among species within the genus Pseudotsuga and up to 200 bp among trees of P. menziesii. The polymerase chain reaction (PCR) allowed the locus of polymorphism to be identified, and the variable region was then sequenced in a second Douglas-fir tree, a single tree of a related species, Japanese Douglas-fir (P. japonica), and in a species lacking a mutation hotspot in the region, Pinus radiata (Monterey pine). The locus of polymorphism is characterized by hundreds of base pairs of imperfect, tandem direct repeats flanked by a partially duplicated and an intact trnY-GUA gene. The duplication is direct in orientation and consists of 43 bp of the 3 end of trnY and 25 bp of its 3 flanking sequence. Tandem repeats show high sequence similarity to a 27-bp region of the trnY gene that overlaps one end of the duplication. The two trees of Douglas-fir sequenced differed by a single tandem repeat unit, whereas these trees differed from the Japanese Douglas-fir sequenced by approximately 34 repeat units. Repetitive DNA in the Pseudotsuga cpDNA hotspot was most likely generated at the time of the partial trnY gene duplication and these sequences expanded by slipped-strand mispairing and unequal crossingover.     

Loss of mt +-derived zygotic chloropiast DNA is associated with a lethal allele in Chlamydomonas monoica

Summary The Chlamydomonas monoica mutant allele mtl-1, is associated with the formation of nonviable zygospores following self-mating of the mutant strain. Furthermore, mtl-1 heterozygote populations show a 50% reduction in germination frequency and no transmission of a chloroplast antibiotic resistance marker carried by the mtl-1 parent. To determine whether the effects on zygospore viability and chloroplast gene transmission resulted from the direct involvement of the mtl-1 locus in the control of mt ⁺-directed uniparental inheritance of chloroplast DNA (cpDNA), we have used the DNA-specific fluorochrome DAPI to follow the fate of cpDNA during the maturation of zygotes. Throughout the first few hours after the initial fusion of gametes, the young zygotes show DAPI-fluorescent nucleoids distributed symmetrically around the region of nuclear fusion, and presumably located within both of the parental chloroplasts. Wild-type and mtl-1 mutant zygotes show similar early staining patterns. As the zygotes age, the staining patterns become asymmetric for the wildtype population, with all of the visible cytoplasmic nucleoids restricted to one side of the zygote. In contrast, mtl-1 homozygotes appear to lose cytoplasmic nucleoids from both sides of the zygote simultaneously and within 24 h are apparently devoid of cpDNA. By introducing a mutation which arrests cell fusion (and prevents plastid fusion), we can show that (1) the asymmetric nucleoid distribution in wildtype zygotes results from the loss of nucleoids from one gamete in each mating event, and (2) the additional loss of cpDNA in mtl-1 homozygotes does not require contact between parental plastids (thus the nuclease responsible for cpDNA degradation is not sequestered within the chloroplast of one gamete). We propose that the mtl-1 mutant strain is defective for a process which normally protects cpDNA of mt ⁺ origin.     

Cloning and characterization of the European seabass,Dicentrarchus labrax,mitochondrial genome

Mitochondrial DNA (mtDNA) from the European seabass, Dicentrarchus labrax, has been cloned and characterized. Its gene organization was deduced by a comparison of the sequenced termini of different subclones obtained from European seabass mtDNA to the completely-sequenced mtDNAs from carp and freshwater loach. The difference in genome size between the European seabass mtDNA (approximately 18 kb) and most of the other characterized fish mtDNAs (approximately 16.5 kb) is accounted for by the displacement-loop (D-loop). Comparisons have been performed between the derived amino-acid sequences of three sequenced genes, cytochrome c oxidase subunit 2 (COII), NADH dehydrogenase subunit 4L (ND4L) and ATP synthase subunit 8 (ATPase8), from D. labrax, and their counterparts in other fishes and Xenopus laevis.     

Structural analysis of length mutations in a hot-spot region of wheat chloroplast DNAs

Summary The hot-spot region related to length mutations in the chloroplast genome of the wheat group was precisely analyzed at the DNA sequence level. This region, located downstream from the rbcL gene, was highly enriched in A+T, and contained a number of direct and inverted repeats. Many deletions/insertions were observed in the region. In most deletions/insertions of multiple nucleotides, short repeated sequences were found at the mutation points. Furthermore, a pair of short repeated sequences was also observed at the border of the translocated gene. A sequence homologous with ORF512 of tobacco cpDNA was truncated in cpDNAs of the wheat group and found only in the mitochondrial DNA of Ae. crassa, suggesting the inter-organellar translocation of this sequence. Mechanisms that could generate structural alterations of the chloroplast genome in the wheat group are discussed.     

Unicircular structure of the Brassica hirta mitochondrial genome

Summary Restriction mapping studies reveal that the mitochondrial genome of white mustard (Brassica hirta) exists in the form of a single circular 208 kb chromosome. The B. hirta genome has only one copy of the two sequences which, in several related Brassica species, are duplicated and undergo intramolecular recombination. This first report of a plant mitochondrial DNA that does not exist in a multipartite structure indicates that high frequency intramolecular recombination is not an obligatory feature of plant mitochondrial genomes. Heterologous filter hybridizatios reveal that the mitochondrial genomes of B. hirta and B. campestris have diverged radically in sequence arrangement, as the result of approximately 10 large inversions. At the same time, however, the two genomes are similar in size, sequence content, and primary sequence.