Sequence studies on the soybean chloroplast 16S–23S rDNA spacer region

The sequence of the ribosomal spacer region of soybean chloroplast DNA including the 3 end of the 16S rRNA gene, the tRNA^Ala and tRNA^Ile genes (but not their introns), the three intergenic regions and the 5 end of the 23S rRNA gene, has been determined. This sequence has been compared to corresponding regions of other angiosperm chloroplast DNAs. Secondary structure models are proposed for the entirety of the intergenic regions a, b and c and for the flanking rRNA regions. A model for a common secondary structure of the ribosomal spacer intergenic regions from chloroplasts of higher plants is proposed, which is supported by comparative evidence.     

Organization of ribosomal RNA genes in Mycoplasma capricolum

Summary DNA segments carrying rRNA genes of Mycoplasma capricolum have been cloned and characterized by restriction endonuclease mapping, DNA-RNA hybridization and nucleotide sequencing. The M. capricolum genome has two sets of rRNA gene clusters, where the arrangement is in the order of (5)16S-23S-5S(3). The spacer region between 16S and 23S rDNA is extremely rich in AT and does not carry any tRNA genes. Present address: Division of Hematology and Immunology of Internal Medicine, Kanazawa Medical University, Uchinada-Cho, Kahoku-Gun Ishikawa Pref. 920-02, Japan     

Complex structure of the ribosomal DNA spacer of Cucumis sativus (cucumber)

Summary The nuclear 18 S, 5.8 S and 25 S ribosomal RNA genes (rDNA) of Cucumis sativus (cucumber) occur in at least four different repeat types of 10.2, 10.5, 11.5, and 12.5 kb in length. The intergenic spacer of these repeats has been cloned and characterized with respect to sequence organization. The spacer structure is very unusual compared to those of other eukaryotes. Duplicated regions of 197 bp and 311 bp containing part of the 3 end of the 25 S rRNA coding region and approximately 470 bp of 25 S rRNA flanking sequences occur in the intergenic spacer. The data from sequence analysis suggest that these duplications originate from recombination events in which DNA sequences of the original rDNA spacer were paired with sequences of the 25 S rRNA coding region. The duplicated 3ends of the 25 S rRNA are separated from each other mostly by a tandemly repeated 30 bp element showing a high GC-content of 87.5%. In addition, another tandemly repeated sequence of 90 bp was found downstream of the 3flanking sequences of the 25 S rRNA coding region. These results suggest that rRNA coding sequences can be involved in the generation of rDNA spacer sequences by unequal crossing over.     

An aberrant plastid ribosomal RNA gene cluster in the root parasite Conopholis americana

The plastid ribisomal RNA (rRNA) operon of the achlorophyllous root parasite Conopholis americana was completely sequenced. Full-length rRNA genes are retained in the gene cluster, but significant divergence has occurred in the 16S, 23S and 5S genes. Both the 16S–23S intergenic spacer and the 4.5S–5S intergenic spacer have suffered substantial deletions, including the two tRNA genes typically found in prokaryotic and plastid 16S–23S spacers.     

Restriction site and length polymorphism of the rDNA unit in the cultivated basidiomycetePleurotus cornucopiae

In the ribosomal DNA unit ofPleurotus cornucopiae, the rDNA coding regions are in the order 5, 5S-18S-5.8S-25S, 3, with the 5 location of the 5S gene differing from its 3 location found in other basidiomycetes. The most discriminating probe used to study the rDNA polymorphism consisted of a fragment that included the 5S, 18S and part of the 5.8S and 25S genes flanking three intergenic sequences. A high degree of rDNA polymorphism was observed in the sevenP. cornucopiae dikaryons studied. For the first time within a basidiomycete species, the restrictions maps distinguished two types of rDNA units (I and II). In each rDNA type, length variations in the external intergenic sequence IGS 1 located between the 25S and 5S genes allowed characterization of two different rDNA units in type I and four rDNA units in type II. This suggested that theP. cornucopiae rDNA units were derived from two kinds of ancestors (type I and II) by insertion or deletion events (100–700 bp) in the IGS 1. In four dikaryotic strains, two rDNA units of the same type (I or II) differing only by the IGS 1 length, were found in a similar number of copies, and presented a meiotic segregation in homokaryotic progeny. In one progeny, some homokaryotic strains possessed two different rDNA units: one with a high copy number and another with a lower one, showing that two different rDNA units could coexist in a single nucleus.     

DNA sequence,structure, and phylogenetic relationship of the small subunit rRNA coding region of mitochondrial DNA fromPodospora anserina

Summary DNA sequence analysis and the localization of the 5 and 3 termini by S1 mapping have shown that the mitochondrial (mt) small subunit rRNA coding region fromPodospora anserina is 1980 bp in length. The analogous coding region for mt rRNA is 1962 bp in maize, 1686 bp inSaccharomyces cerevisiae, and 956 bp in mammals, whereas its counterpart inEscherichia coli is 1542 bp. TheP. anserina mt 16S-like rRNA is 400 bases longer than that fromE. coli, but can be folded into a similar secondary structure. The additional bases appear to be clustered at specific locations, including extensions at the 5 and 3 termini. Comparison with secondary structure diagrams of 16S-like RNAs from several organisms allowed us to specify highly conserved and variable regions of this gene. Phylogenetic tree construction indicated that this gene is grouped with other mitochondrial genes, but most closely, as expected, with the fungal mitochondrial genes.     

Structure and rearrangements of rRNA genes in chloroplast DNA in two strains of Euglena gracilis

Summary The organisation of the rRNA genes in the chloroplast genomes of two strains of Euglena gracilis were analyzed and compared. It was previously shown that the bacillaris strain contains three complete rrn (rRNA) operons (7) and that the Z-S strain contains one operon (21). Using heteroduplex analysis it was found that the bacillaris strain contains, apart from the three complete rrn operons, an extra 16S rRNA gene, an extra partial 23S rRNA gene sequence and an inverted duplication of a stretch within the 5S–16S spacer. In addition a short (<100 bp) inverted repeat sequence (13) which forms a stem/loop structure in single-stranded cpDNA was located between the 3-end of the extra 16S rRNA gene and the partial 23 S rRNA sequence.The Z-S strain differs from the bacillaris strain by a deletion of two units of the complete rrn operons. The region upstream of the single complete rrn operon, including the inverted repeats, the partial 23S and the extra 16S rRNA sequences is identical with the bacillaris strain.The only non-homology found in heteroduplexes between the SalI fragments of B of the two strains is the deletion-insertion loop which represents the two rrn operons. A small deletion loop was found occasionally in hetero-and in homoduplexes of both strands in the region of variable size. Apart from the deletion/insertion of two rrn operons the two genomes appear to be colinear as can be seen from partial denaturation mapping. The organisation of the rRNA genes of the two strains is compared with those of the Z strain and the bacillaris-ATCC strain.     

Phylogenetic relationship of the green alga Nanochlorum eukaryotum deduced from its chloroplast rRNA sequences

The marine green coccoidal alga Nanochlorum eukaryotum (N.e.) is of small size with an average diameter of 1.5 m. It is characterized by primitive-appearing biochemical and morphological properties, which are considerably different from those of other green algae. Thus, it has been proposed that N.e. may be an early developed algal form. To prove this hypothesis, DNA of N.e. was isolated by a phenol extraction procedure, and the chloroplast DNA separated by preparative CsCl density-gradient centrifugation. The kinetic complexity of the nuclear and of the chloroplast DNA was evaluated by reassociation kinetics to 3 × 10⁷ by and 9 × 10⁴ bp, respectively. Several chloroplast genes, including the rRNA genes, were cloned on distinct fragments. The order of the rRNA genes corresponds to the common prokaryotic pattern. The 16S rRNA gene comprises 1,548 bases and is separated from the 23S rRNA gene with its 2,920 bases by a short spacer of 460 bases, which also includes the tRNA^Ile and tRNA^Ala genes. The 5S rRNA gene has not been found; it must start further than 500 bases downstream from the 3-end of the 23S rRNA gene. From the chloroplast rRNA sequences, we have deduced secondary structures of the 16S and 23S rRNAs, which are in agreement with standard models. The rRNA sequences were aligned with corresponding chloroplast sequences; phylogenetic relationships were calculated by several methods. From these calculations, we conclude that N.e. is most closely related to Chlorella vulgaris. Therefore, N.e. does not represent an early developed algal species; the primitive-appearing morphological and biochemical characteristics of N.e. must rather be explained by secondary losses. Correspondence to: D. Weinblum     

Nucleotide sequence of cytoplasmic 5S ribosomal RNA fromEuglena gracilis

Summary The nucleotide sequence of cytoplasmic 5S ribosomal RNA fromEuglena gracilis has been determined to be: G- _A ^C -G-U-A-C-G-G-C-C-A-U-A-C-U-A-C-C-G-G-G-A-A-U-A-C-A-C-C-U-G-A-A-C-C-C-G--U-C-G-A-U-U-U-C-A-G-A-A-G-U-U-A-A-G-C-C-G-G-G-U-U-A-G-G-C-C-C-A-G-U-U-A-G-U-A-C-U-G-A-G-U-G-G-G-C-G-A-C-C-A-C-U-U-G-G-G-A-A-C-A-C-U-G-G-G-U-G-C-U-G-U-A-C-G-C-U-Up. This RNA is 119 nucleotides long and the sequence of a probable tRNA-binding site is GAUU (position 41–44 from the 5-terminus), which is the same as that of a trypanosoma species,Crithidia fasci-culata. TheEuglena 5S rRNA has a pseudouridine residue at position 38 and 3-terminus is phosphorylated. The 5S rRNA sequence ofEuglena resembles those of several other protozoa and higher animals rather than plants.On leave from Department of Zoology, Hiroshima University, Hiroshima, Japan     

Plastid DNA in the mitochondrial genome of Oenothera: Intra- and interorganellar rearrangements involving part of the plastid ribosomal cistron

Summary Part of the plastid rRNA cistron is present in the mitochondrial genome of Oenothera. This sequence of 2081 nucleotides contains the 3 half of the plastid 23 S rRNA, the adjacent intergenic region and the 4.5 S rRNA. Secondary intramitochondrial sequence rearrangements involve this region of plastid origin and the gene encoding the putative mitochondrial small ribosomal protein S13. Sequence comparison suggests that the interorganellar transfer event occurred a long time ago. The mitochondrial sequence contains regions more homologous to the plastid DNA from tobacco than from Oenothera itself in the regions analysed, suggesting faster sequence evolution in plastids than in mitochondria of Oenothera.     

Characterization of replication origins flanking the 23S rRNA gene in tobacco chloroplast DNA

Using 5 end-labeled nascent strands of tobacco chloroplast DNA (ctDNA) as a probe, replication displacement loop (D-loop) regions were identified. The strongest hybridization was observed with restriction fragments containing the rRNA genes from the inverted repeat region. Two-dimensional gel analysis of various digests of tobacco ctDNA suggested that a replication origin is located near each end of the 7.1 kb BamHI fragment containing part of the rRNA operon. Analysis of in vitro replication products indicated that templates from either of the origin regions supported replication, while the vector alone or ctDNA clones from other regions of the genome did not support in vitro replication. Sequences from both sides of the BamHI site in the rRNA spacer region were required for optimal in vitro DNA replication activity. Primer extension was used for the first time to identify the start site of DNA synthesis for the D-loop in the rRNA spacer region. The major 5 end of the D-loop was localized to the base of a stem-loop structure which contains the rRNA spacer BamHI site. Primer extension products were insensitive to both alkali and RNase treatment, suggesting that RNA primers had already been removed from the 5 end of nascent DNA. Location of an origin in the rRNA spacer region of ctDNA from tobacco, pea and Oenothera suggests that ctDNA replication origins may be conserved in higher plants.     

New species in the genus <Emphasis Type="Italic">Francisella</Emphasis> (Gammaproteobacteria; Francisellaceae); <Emphasis Type="Italic">Francisella piscicida</Emphasis> sp. nov. isolated from cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511^T = DSM 18777^T = LMG registration number not yet available).     

Sequence analysis of the rDNA spacer ofParacentrotus lividus and observations about pre-rRNA processing

We have isolated and sequenced one intergenic region and a small part of the flanking regions (18S and 26S rRNA coding regions) of the rRNA-encoding genes (rDNA) from the sea urchinParacentrotus lividus. This region is about 3.8 Kb long. Northern blot hybridizations and S1 mapping experiments demonstrated the presence of a partially processed 21S rRNA precursor which has the same 5 terminus as the 32S primary precursor, also in developmental stages characterized by a low rate of rRNA synthesis.Abbreviations bp base pair(s) - Kb Kilobase(s) or 1000 bp - nt nucleotide(s) - rDNA DNA encoding rRNA - rRNA ribosomal RNA - S sedimentation constant     

Sequence variation in the putative replication origins of the five genetically distinct basicEuoenothera plastid chromosomes (plastomes)

Approximately 4200 nucleotides of the 16S/23S rDNA spacer and the 5 region flanking therrn operon of the plastid chromosomes representing the five basic, phylogenetically relatedEuoenothera plastomes were sequenced and compared. The sequences that harbor the putative replication origins are almost identical except for a 785-bp intercistronic segment between the genes for the 16S rRNA andtrnI. Differences are mainly caused by insertions/deletions and duplications; the predicted potential for formation of quite extensive secondary structure differs among the plastomes. Unexpected intraplastome variation has also been noted. Furthermore, the sequence-based and published genetically deduced plastome pedigrees differ significantly.     

Major differences between the rrnA operons of two strains of Agrobacterium vitis

The sequence of the rrnA operon and its flanking regions was determined for the Agrobacterium vitis type strain NCPPB3554. Compared to the earlier obtained rrnA sequence of A. vitis strain S4, several important differences were noted: the sequences diverged at the 5′-flanking region, within the 16S–23S intergenic region, and within the 23S rRNA sequence. The B8 stem-loop structure at the 5′-end of the 23S rRNA of strain NCPPB3554 was 142 nt shorter than that of strain S4. These findings have important consequences for the use of ribosomal RNA gene sequences in phylogenetic comparisons. Received: 16 February 1996 / Accepted: 26 April 1996     

Structural features of the plastid ribosomal RNA operons of two red algae: Antithamnion sp. and Cyanidium caldarium

The nucleotide sequences of the plastid 16S rDNA of the multicellular red alga Antithamnion sp. and the 16S rDNA/23S rDNA intergenic spacers of the plastid DNAs of the unicellular red alga Cyanidium caldarium and of Antithamnion sp. were determined. Sequence comparisons support the idea of a polyphyletic origin of the red algal and the higher-plant chloroplasts. Both spacer regions include the unsplit tRNA^Ile (GAU) and tRNA^Ala (UGC) genes and so the plastids of both algae form a homogeneous group with those of chromophytic algae and Cyanophora paradoxa characterized by small-sized rDNA spacers in contrast to green algae and higher plants. Nevertheless, remarkable sequence differences within the rRNA and the tRNA genes give the plastids of Cyanidium caldarium a rather isolated position.     

Cloning and characterization of genes coding for ribosomal RNA inCampylobacter jejuni

The DNA fragments coding for ribosomal RNA inCampylobacter jejuni have been cloned from a genomic library ofC. jejuni constructed inEscherichia coli. Clones carrying DNA Sequences for rRNA were identified by hybridization of 5-end-labeled rRNA fromC. jejuni to colony blots of transformants from this gene library. Cloned DNA sequences homologous to each of 5S, 16S, and 23S rRNA were idenfified by hybridization of labeled plasmid DNA to Northern blots of rRNA. The gene coding for 23S rRNA was found to be located on a 5.5kb HindIII fragment, while the 5S and 16S rRNA genes were on HindIII fragments of 1.65 and 1.7 kb, respecitively. The DNA fragment containing the 16S rRNA gene was characterized by restriction endonuclease mapping, and the location of the 16S rRNA gene on this fragment was determined by hybridization of 5-end-labeled rRNA to restriction fragments and also by DNA sequence determination. It appears that the major portion of the coding region for 16S rRNA is located on the 1.7-kb HindIII fragment, while a small portion is carried on an adjacent HindIII fragment of 7.5 kb. Cloned rRNA genes fromC. jejuni were used to study the organization of the rDNA inC. jejuni and other members of the genùsCampylobacter.     

Unusual intragenomic and interspecific variability of <Emphasis Type="Italic">Geobacillus</Emphasis> 16S-23S rRNA internal transcribed spacers

The aim of this study was to evaluate the inter-and intraspecific as well as intragenomic variability of Geobacillus 16S–23S rRNA internal transcribed spacers without tRNA genes and to compare these sequences with sequences bearing tRNA genes. In this study the structural analysis was performed in a unique way because the length and the sequence of the structural blocks were adjusted to fit the structure of 16S–23S rRNA internal transcribed spacers of five different Geobacillus species. Our study demonstrated the mosaic-like structure of 16S–23S rRNA internal transcribed spacers in Geobacillus. Some characteristics of these spacers of geobacilli were not previously reported for other bacteria: unusually short conserved sequence in the 5′ end region, some identical conserved blocks in both 5′ and 3′ regions of 16S–23S rRNA internal transcribed spacers, the same sequence blocks in both 16S–23S and 23S–5S rRNA intergenic spacers. Our study demonstrated quite uniform arrangement of the sequence blocks in Geobacillus thermodenitrificans. This species diverged early in the phylogenetic tree of the genus Geobacillus. For the phylogenetically recent species Geobacillus kaustophilus and Geobacillus lituanicus the low inter-and intraspecific, but high intragenomic variability, as a consequence of recent phylogenetic events, was established.     

Nucleotide sequence of the rrnB 16S ribosomal RNA gene from Mycoplasma capricolum

Summary The nucleotide sequences of the rrnB 16S ribosomal RNA gene and its 5-and 3-flanking regions from Mycoplasma capricolum have been determined. The coding sequence is 1521 base pairs long, being 21 base pairs shorter than that of the Scherichia coli 16S rRNA gene. The 16S rRNA sequence of M. capricolum reveals 74% and 76% identity with that of E. coli and Anacystis nidulans, respectively. The secondary structure model constructed from the M. capricolum 16S rRNA.gene sequence resembles that proposed for E. coli 16S rRNA. A large stem structure can be constructed between the 5- and 3-flanking sequences of the 16S rRNA gene. The flanking regions are extremely rich in AT.