Rice mitochondrial genome contains a rearranged chloroplast gene cluster

Summary We have previously reported the isolation and partial sequence analysis of a rice mitochondrial DNA fragment (6.9 kb) which contains a transferred copy of a chloroplast gene cluster coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL), and subunits of ATPase (atpB and atpE), methionine tRNA (trnM) and valine tRNA (trnV). We have now completely sequenced this 6.9 kb fragment and found it to also contain a sequence homologous to the chloroplast gene coding for the ribosomal protein L2 (rpl2), beginning at a site 430 bp downstream from the termination codon of rbcL. In the chloroplast genome, two copies of rpl2 are located at distances of 20 kb and 40 kb, respectively, from rbcL. We have sequenced these two copies of rice chloroplast rpl2 and found their sequences to be identical. In addition, a 151 bp sequence located upstream of the chloroplast rpl2 coding region is also found in the 3 noncoding region of chloroplast rbcL and other as yet undefined locations in the rice chloroplast genome. Hybridization analysis revealed that this 151 bp repeat sequence identified in rice is also present in several copies in 11 other plant species we have examined. Findings from these studies suggest that the translocation of rpl2 to the rbcL gene cluster found in the rice mitochondrial genome might have occurred through homologous recombination between the 151 bp repeat sequence present in both rpl2 and rbcL.     

The barley chloroplast DNA atpBE, trnM2, and trnV1 loci.

The nucleotide sequence of a barley chloroplast DNA 3.7 kb SmaI-HindIII fragment is presented. This fragment contains atpBE, the genes for the beta and epsilon subunits of ATPase; trnM2, the gene for tRNA2met; and trnV1, the gene for tRNA1va1. The atpE-trnM2 interval is 126 bp and trnM2 is transcribed towards atpBE. The trnM2-trnV1 interval is 203 bp and trnV1 is transcribed away from trnM2. The trnV1 locus has a 597 bp intervening sequence. the organization and sequences of these genes are compared to the analogous genes from maize and tobacco chloroplast DNA. Using the latter comparisons the nature of sequence divergence between chloroplast DNAs is discussed.     

Positioning of protein-coding genes on the soybean chloroplast genome

Seven major plastid protein encoding genes were positioned on the soybean chloroplast DNA by heterologous hybridization. These include the genes for the alpha, beta and epsilon subunits of the CF₁ component of ATP synthase (atpA, atpB and atpE respectively), for subunit III of the CF₀ component of ATP synthase (atpH), for the cytochrome f (cytF), for the ‘32 Kd’ thylakoid protein (psbA), and for the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL), all of which map in the large single copy region. The atpB, atpE and rbcL genes are located in the region adjacent to one of the segments of the inverted repeat. The genetic organization of the soybean chloroplast DNA is compared to that of other plastid genomes.     

Phylogenetics of asterids based on 3 coding and 3 non-coding chloroplast DNA markers and the utility of non-coding DNA at higher taxonomic levels

Asterids comprise 1/4-1/3 of all flowering plants and are classified in 10 orders and >100 families. The phylogeny of asterids is here explored with jackknife parsimony analysis of chloroplast DNA from 132 genera representing 103 families and all higher groups of asterids. Six different markers were used, three of the markers represent protein coding genes, rbcL, ndhF, and matK, and three other represent non-coding DNA; a region including trnL exons and the intron and intergenic spacers between trnT (UGU) to trnF (GAA); another region including trnV exons and intron, trnM and intergenic spacers between trnV (UAC) and atpE, and the rps16 intron. The three non-coding markers proved almost equally useful as the three coding genes in phylogenetic reconstruction at the high level of orders and families in asterids, and in relation to the number of aligned positions the non-coding markers were even more effective. Basal interrelationships among Cornales, Ericales, lamiids (new name replacing euasterids I), and campanulids (new name replacing euasterids II) are resolved with strong support. Family interrelationships are fully or almost fully resolved with medium to strong support in Cornales, Garryales, Gentianales, Solanales, Aquifoliales, Apiales, and Dipsacales. Within the three large orders Ericales, Lamiales, and Asterales, family interrelationships remain partly unclear. The analysis has contributed to reclassification of several families, e.g., Tetrameristaceae, Ebenaceae, Styracaceae, Montiniaceae, Orobanchaceae, and Scrophulariaceae (by inclusion of Pellicieraceae, Lissocarpaceae, Halesiaceae, Kaliphoraceae, Cyclocheilaceae, and Myoporaceae+Buddlejaceae, respectively), and to the placement of families that were unplaced in the APG-system, e.g., Sladeniaceae, Pentaphylacaceae, Plocospermataceae, Cardiopteridaceae, and Adoxaceae (in Ericales, Ericales, Lamiales, Aquifoliales, and Dipsacales, respectively), and Paracryphiaceae among campanulids. Several families of euasterids remain unclassified to order.     

Variations in chloroplast proteins and nucleotide sequences of three chloroplast genes in Triticum and Aegilops.

Two alloplasmic wheat lines having the same common wheat nucleus but the cytoplasms of Aegilops crassa and Ae. columnaris together with the corresponding normal line (control) were used in the two-dimensional gel electrophoresis of soluble and thylakoid membrane proteins of the chloroplast. Three chloroplast polypeptides: the Rubisco large subunit, the beta subunit of ATP synthase, and an unidentified 31 kDa protein, differed in the common wheat and two Aegilops cytoplasms. Three chloroplast genes, atpB, atpE and trnM, that respectively encode the beta and epsilon subunits of ATP synthase and tRNA(met), were sequenced. The atpB gene differed by two synonymous base substitutions, whereas the other two genes were identical in the two Aegilops cytoplasms. From the predicted amino acid sequences, the beta subunits of the ATP synthase in the Aegilops cytoplasms were assumed to have three amino acid substitutions: Ala by Val, Asp- by Ala, and Gln by Lys+, in contrast to the cytoplasm of common wheat. This accounts for the difference in pI values found for the common wheat and Aegilops cytoplasms. The two base substitutions for the atpE genes of common wheat and the Aegilops cytoplasms were synonymous. The differences detected in the genes encoding the two subunits of ATP synthase do not appear to be ascribable to the differences in phenotypic effects for the common wheat and Aegilops cytoplasms. The base substitution rate of the atpB-atpE-trnM gene cluster was similar to that of the rbcL gene. From the rate for the atpB gene alone, evolutionary divergence of the wheat-Aegilops complex is assumed to have begun ca. 3.0 x 10(6) years ago, as compared to ca. 8.0 x 10(6) years ago for the divergence of the wheat-Aegilops complex and barley.     

水稻叶绿体DNA克隆片段的分析和rbcL基因在重组质粒上的定位

杂交灿稻(珍汕97B)的叶绿体DNA克隆到pBR 322载体上后,从克隆库中筛选出含核酮糖1.5-二磷酸羧化酶/加氧酶大亚基基因(rbcL)的重组子(19.3kb),用10种限制性内切酶分析了这个重组质粒并制作了完整的物理图谱,rbcL基因被定位在这个物理图谱上。     

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.     

Identification of EcoRV fragments spanning the N alpha-tubulin gene of Physarum

The N alpha-tubulin gene of Physarum polycephalum has an EcoRV site at codons 252/253. EcoRV digestion of physarum DNA generated two EcoRV fragments per gene copy comprising both coding and flanking sequences. Hybridisation probes which included coding sequences upstream from the central EcoRV site cross-hybridised with another alpha-tubulin gene. Probes derived from either 5'- or 3'-flanking regions were gene-specific. These probes identified two EcoRV fragments in the haploid strain CLdAXE viz 5.4 kb (5'-fragment) and 6.2 kb (3'-fragment). The same two fragments were identified in EcoRV digests of DNA of the diploid strain M3CVIII, and a second form of the gene was also identified comprising two fragments viz 5.0 kb (5'-end) and 5.5 kb (3'-end). Both forms gave rise to an identical 4.65 kb HindIII fragment as judged by restriction mapping.     

A DNA fragment hybridizing to a nif probe in Rhodobacter capsulatus is homologous to a 16S rRNA gene

We have sequenced the Rhodobacter capsulatus nifH and nifD genes. The nifH gene, which codes for the dinitrogenase reductase protein, is 894 bp long and codes for a polypeptide of predicted Mr 32,412. The nifD gene, which codes for the alpha subunit of dinitrogenase, is 1,500 bp long and codes for a protein of predicted Mr 56,113. A 776-bp BglII-XhoI fragment containing only nif sequences was used as a hybridization probe against R. capsulatus genomic DNA. Two HindIII fragments, 11.8 kb and 4.7 kb in length, hybridize to this probe. Both fragments have been cloned from a cosmid library. The 11.8-kb fragment contains the nifH, D and K genes, as previously demonstrated (Scolnik and Haselkorn, 1984). In this paper we present evidence that suggests that the 4.7-kb HindIII fragment contains a gene coding for 16S rRNA, and that although homology between nif and this fragment can be observed in filter hybridization experiments, a second copy of the nif structural genes seems not to be present in this region.     

The Nature of Nucleotide Sequence Divergence between Barley and Maize Chloroplast DNA

Analysis of a 2175-base pair (bp) SmaI-HindIII fragment of barley chloroplast DNA revealed that rbcL (the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase) and atpB (the gene for the beta subunit of ATPase) are transcribed divergently and are separated by an untranscribed region of 155-166 bp. The rbcL mRNA has a 320-residue untranslated leader region, whereas the atpB mRNA has a 296- to 309-residue leader region. The sequence of these regions, together with the initial 113 bp of the atpB-coding region and the initial 1279 bp of the rbcL-coding region, is compared with the analogous maize chloroplast DNA sequences. Two classes of nucleotide differences are present, substitutions and insertions/deletions. Nucleotide substitutions show a 1.9-fold bias toward transitions in the rbcL-coding region and a 1.5-fold bias toward transitions in the noncoding region. The level of nucleotide substitutions between the barley and maize sequences is about 0.065/bp. Seventy-one percent of the substitutions in the rbcL-coding region are at the third codon position, and 95% of these are synonymous changes. Insertion/deletion events, which are confined to the noncoding regions, are not randomly distributed in these regions and are often associated with short repeated sequences. The extent of change for the noncoding regions (about 0.093 events/bp) is less than the extent of change at the third codon positions in the rbcL-coding region (about 0.135 events/bp), including insertion/delection events. Limited sequence analysis of the analogous DNA from a wild line ( Hordeum spontaneum) and a primitive Iranian barley (H. vulgare) suggested a low rate of chloroplast DNA evolution. Compared to spinach chloroplast DNA, the barley rbcL-atpB untranslated region is extremely diverged, with only the putative rbcL promoters and ribosome-binding site being extensively conserved.     

Genetic determinant of pyocin R2 in Pseudomonas aeruginosa PAO. II. Physical characterization of pyocin R2 genes using R-prime plasmids constructed from R68.45

The chromosome segment which contains the genes responsible for production of pyocin R2 in P. aeruginosa PAO was defined physically using R-prime plasmids constructed in vivo from R68.45. The previous conclusion from genetic mapping that the cluster of pyocin R2 genes is located in between trpC and trpE genes was confirmed by deletion mapping of various R prime plasmids bearing the trpC gene. The pyocin R2 gene cluster was further localized on two contiguous HindIII fragments of 16 kb and 8.0 kb. PML14 strain, in which R-type pyocin genes were completely deleted, had only one 11 kb HindIII fragment instead. Heteroduplexes between this 11 kb fragment with the two HindIII fragments of PAO revealed that the cluster of pyocin R2 genes was an insertion 13 kb long.     

Genes encoding the beta and epsilon subunits of the proton-translocating ATPase from Anabaena sp. strain PCC 7120.

The genes encoding the beta (atpB) and epsilon (atpE) subunits of the ATPase from the cyanobacterium Anabaena sp. strain PCC 7120 were cloned, and their sequences were determined. atpB and atpE are each single-copy genes in the Anabaena genome. The two genes are separated by a 96-base-pair intergenic spacer and transcribed as a single mRNA of 2.3 kilobases that initiates approximately 200 base pairs upstream of the atpB coding region. The predicted translation product of atpB has 81 and 68% amino acid identity with the corresponding proteins from spinach chloroplasts and Escherichia coli, respectively. The atpE gene product is less conserved, with 41 and 33% amino acid identity with the corresponding proteins from spinach chloroplasts and E. coli, respectively. The organization of the Anabaena atpB and atpE genes relative to adjacent genes differs from that of both E. coli and chloroplasts.