Complete Chloroplast Genome of Chionographis japonica (Willd.) Maxim. (Melanthiaceae): Comparative Genomics and Evaluation of Universal Primers for Liliales

The complete chloroplast genome of Chionographis japonica (Willd.) Maxim. (Melanthiaceae, Liliales) was mapped using polymerase chain reaction and the Sanger method. The circular double-stranded DNA was a typical quadripartite structure consisting of two inverted repeated regions (27,397 bp), a small single copy region (18,205 bp), and a large single-copy region (81,646 bp), with a total length of 154,645 bp. The genome consisted of 137 coding genes, including 91 protein-coding genes, 38 distinct tRNA, and 8 rRNA genes. The ycf15 and ycf68 genes had several internal stop codons interpreted as pseudogenes. The inverted repeat (IR) region expanded to part of the rps3 gene in the junction between large single-copy and IRA regions in C. japonica. We designed 785 primers, of which 481 were used to map the entire chloroplast genome of C. japonica. Primers were compared with the complete chloroplast sequence of Smilax china (Smilacaceae) to identify primers that could be used for other Liliales members and whole chloroplast genome sequencing. Of the primers used for C. japonica, 398 could be used with other smaller species within the order.     

A restriction map of the ribosomal RNA genes and the short single-copy DNA sequence of the pearl millet chloroplast genome

The chloroplast rDNA genes of pearl millet (Pennisetum americanum) have been cloned and physically mapped. The chloroplast genome of the pearl millet contains two identical rRNA genes located on DNA sequences that are inverted with respect to one another and separated by 12 kb of single-copy DNA. The rRNA genes were positioned on a restriction endonuclease map by using as hybridization probes specific cloned rDNA sequences from the chloroplast DNA of the alga Euglena gracilis. The 16S and 23S rRNA genes were shown to be approx. 2 kb from one another, and the 5S RNA gene is immediately adjacent to the 23S tRNA gene.     

Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA

Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.     

A physical map of the ribosomal DNA repeat unit of Aspergillus nidulans

The ribosomal DNA repeat unit of Aspergillus nidulans has been cloned in pBR322 and a restriction map constructed. The genes coding for the 17S, 5.8S and 25S rRNAs are found in blocks separated by a 1.7 kb spacer region, with the 5.8S RNA gene lying between the genes for the two larger RNAs. The total length of the repeat unit is 7.7 kb. The 5S rRNA is not present in the repeat unit.     

The complete structure of the cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) chloroplast genome: Its composition and comparative analysis

The complete nucleotide sequence of the cucumber (C. sativus L. var. Borszczagowski) chloroplast genome has been determined. The genome is composed of 155,293 bp containing a pair of inverted repeats of 25,191 bp, which are separated by two single-copy regions, a small 18,222-bp one and a large 86,688-bp one. The chloroplast genome of cucumber contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species), and 37 tRNA genes (30 tRNA species), with 18 of them located in the inverted repeat region. Of these genes, 16 contain one intron, and two genes and one ycf contain 2 introns. Twenty-one small inversions that form stem-loop structures, ranging from 18 to 49 bp, have been identified. Eight of them show similarity to those of other species, while eight seem to be cucumber specific. Detailed comparisons of ycf2 and ycf15, and the overall structure to other chloroplast genomes were performed.     

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.     

Transfer RNA gene mapping studies on cyanelle DNA from Cyanophora paradoxa

Summary The 4S RNA of cyanelles from Cyanophora paradoxa strain LB 555 UTEX was fractionated by two-dimensional gel electrophoresis. Individual tRNA species were identified by aminoacylation, labeled in vitro and hybridized to restriction endonuclease fragments of cyanelle DNA. Hybridization experiments, using individual tRNA species, have revealed the location of two tRNA genes, coding for tRNA^Ala and tRNA^Ile, in each of the two spacer segments separating the 16S and 23S rRNA genes on the two inverted repeats (10 kbp each) and three tRNA genes in the small single-copy region (17 kbp) separating the two inverted repeats. A minimum of 14 tRNA genes in the large single-copy region (88.5 kbp) has also been found.Heterologous hybridization studies, using cyanelle tRNAs and chloroplast DNA from spinach, broad bean, or maize, indicate a high degree of homology between some tRNAs from cyanelles and chloroplasts.Although cyanelles are often condisered as having evolved from endosymbiotic cyanobacteria, the organization of tRNA genes on cyanelle DNA and the results of heterologous hybridization studies show that cyanelles are related to higher plant chloroplasts.     

Mapping of transfer RNA genes on tobacco chloroplast DNA

Tobacco chloroplast tRNAs have been purified by two-dimensional polyacrylamide gel electrophoresis, identified by aminoacylation, labelled at their 3-end and hybridized to tobacco chloroplast DNA restriction fragments, in order to establish a tRNA gene map. These hybridization studies have revealed the localization of at least seven genes in each inverted repeat region, a minimum of 22 tRNA genes in the large single copy region and one tRNA gene in the small single copy region. Comparison of the tobacco chloroplast tRNA gene map to that of maize shows many similarities, but also some differences suggesting that DNA sequence rearrangements have occurred in the chloroplast genome during evolution.     

Restriction endonuclease cleavage site map of safflower (Carthamus tinctorius L.) chloroplast DNA

Summary The restriction endonucleases SalI, PstI, KpnI and HindIII have been used to construct a physical map of safflower (Carthamus tinctorius L.) chloroplast DNA. This was accomplished by hybridizing Southern blots of single and double digested chloroplast DNA with ³²P-dCTP nick-translated SalI, KpnI and HindIII probes which were individually isolated from agarose gels. The chloroplast DNA was found to be circular and to contain approximately 151 kbp. In common with many other higher plant chloroplast DNAs a sequence of about 25 kbp is repeated in an inverted orientation. The small and large single copy regions separating the two repeated segments contain about 20 kbp and 81 kbp, respectively. The rRNA structural genes were also mapped by Southern blot hybridization and are co-linear with several other plant species.     

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.     

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.     

Recombination of Chlamydomonas chloroplast DNA occurs more frequently in the large inverted repeat sequence than in the single-copy regions

Summary It is well documented that chloroplast DNA (cpDNA) recombination occurs at a relatively high frequency during sexual reproduction of unicellular green algae from the Chlamydomonas genus. Like the cpDNAs of most land plants, those of Chlamydomonas species are divided into two single-copy regions by a large inverted repeat sequence, part of which encodes the chloroplast rRNA genes. In the present study, we scored the inheritance of polymorphic loci spanning the entire chloroplast genome in hybrids recovered from reciprocal interspecific and F₁ crosses between Chlamydomonas eugametes and C. moewusii, and from these data, estimated the density of recombination junctions within each region of recombinant cpDNAs. Our results indicate that recombination junctions occur at highly variable frequencies across the three main domains of the chloroplast genome. The large inverted repeat sequence was found to exhibit at least a five-fold higher density of recombination junctions compared to one of the singlecopy regions, whereas junctions in the latter region were five-fold more abundant relative to those in the other single-copy region. This marked difference in the densities of recombination junctions implies that the extent of genetic linkage between two given chloroplast loci will depend not only on their physical distance, but also on their locations within the genome.     

Identification of the genus Geobacillus using genus-specific primers, based on the 16S-23S rRNA gene internal transcribed spacer

The aim of this study was to develop an easy and accurate technique for the identification of the genus Geobacillus. For this purpose, Geobacillus genus-specific primers GEOBAC (GEOBAC-F and GEOBAC-R) based on the 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences have been designed. In total, 52 sequences from three species of the genus Geobacillus (Geobacillus stearothermophilus, Geobacillus kaustophilus and Geobacillus lituanicus) were examined for the design of these primers. Analysis of the sequences revealed three highly conservative regions common to these species: 5' and 3' end regions of 16S-23S rRNA gene ITSs and box A. Some sequences possessed two additional conservative regions - genes of tRNA(Ile) and tRNA(Ala). These particular sequences were chosen for the construction of the primers. The designed primers targeted the gene of tRNA(Ile) and the 3' end region of ITSs. This technique was validated with both the reference strains of the genus Geobacillus and the thermophilic aerobic endospore-forming environmental isolates. Different Geobacillus species could be grouped according to the number and size of GEOBAC-PCR products and identified on the basis of the AluI and TaqI restriction analysis of these products.     

Sequence analysis of the junctions between a large inverted repeat and single-copy regions in tobacco chloroplast DNA

Summary Tobacco chloroplast DNA contains a large inverted repeat sequence of 26 kilobase pairs (kbp). The inverted repeat is separated by 20 kbp small single-copy and 90 kbp large single-copy regions. We have cloned four DNA fragments containing each junction between the inverted repeat and the single-copy regions. The sequence analysis revealed the exact edges of the inverted repeat. A putative coding region for a ribosomal protein CS19 was found 4 base pairs (bp) away from the inverted repeat on the left margin of the large single-copy region. A sequence AGGAG, which is complementary to the 3 terminal sequence of tobacco chloroplast 16S rRNA, was found within the inverted repeat. A tRNA^His gene was found 5 bp away from the inverted repeat on the right-hand margin of the large single-copy region.     

Application of random amplified polymorphic DNA (RAPD) assays in identifying conserved regions of actinomycete genomes

Abstract Various arbitrary primers as well as pUC18/19 'reverse' sequencing primers were used for random amplified polymorphic DNA assays. Use of a modified reverse primer led to amplification of one major approx. 1100-bp band from the chromosomal DNA of all actinomycetes tested; however, the band was not found when DNAs from other bacteria were used in comparable experiments. Hybridization experiments showed that these bands all contained similar genomic regions. Subsequent sequencing of four of these fragments showed they each contained the sequence of the 3' end of the 23S rRNA gene, the intergenic region and the start of the 5S rRNA gene.     

Nicotiana chloroplast genes for components of the photosynthetic apparatus

In order to understand more fully chloroplast genetic systems, we have determined the complete nucleotide sequence (155, 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA. It contains two copies of an identical 25,339 bp inverted repeat, which are separated by 86, 684 bp and 18,482 bp single-copy regions. The genes for 4 different rRNAs, 30 different tRNAs, 44 different proteins and 9 other predicted protein-coding genes have been located. Fifteen different genes contain introns.Twenty-two genes for components of the photosynthetic apparatus have so far been identified. Most of the genes (except the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) code for thylakoid membrane proteins. Twenty of them are located in the large single-copy region and one gene for a 9-kd polypeptide of photosystem I is located in the small single-copy region. The gene for the 32-kd protein of photosystem II as well as the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase have strong promoters and are transcribed monocistronically while the other genes are transcribed polycistronically. We have found that the predicted amino acid sequences of six DNA sequences resemble those of components of the respiratory-chain NADH dehydrogenase from human mitochondria. As these six sequences are highly transcribed in tobacco chloroplasts, they are probably genes for components of a chloroplast NADH dehydrogenase. These observations suggest the existence of a respiratory-chain in the chloroplast of higher plants.