The 18S and 5S ribosomal RNA genes in Oenothera mitochondria: Sequence rearrangments in the 18S and 5S rRNA genes of higher plants

Summary The 18S and 5S ribosomal RNA genes are separated by a 582-nucleotide-long spacer region in the Oenothera mitochondrial genome. The 5S rRNA gene is 7 bp shorter than the maize and 3 bp shorter than the wheat sequences due to a 4 bp deletion in a side arm of the secondary structure model. The 18S rRNA molecule can be folded analogously to the maize and wheat mitochondrial and Escherichia coli models for this rRNA. Most of the sequence variations between the wheat and Oenothera molecules are located in the variable domains identified for the wheat 18S rRNA.The comparison of the 18S rRNA from the mitochondria of Oenothera as a representative of dicotyledonous plants with that of the monocotyledons wheat and maize provides an indication of the rate of diversity in higher plant mitochondrial genes and gives direct evidence for sequence rearrangements within the 18 S rRNA genes.     

Sequence of the plastid rDNA spacer region of the brown alga Pylaiella littoralis (L.) Kjellm. Evolutionary significance

The DNA segment situated between the 16S and 23S rRNA genes belonging to the plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm. has been sequenced. This small region (322 bp) contains two unsplit tRNA genes separated by 3 bp. A comparison with similar regions from different plants shows that this region has evolved in two different ways according to the place of plants in evolution. In the primitive group, this region is reduced in size when compared to prokaryotes. In the other groups, it is considerably enlarged by insertion of repetitive sequences, open reading frames and introns.     

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.     

Site-specific circularisation at an intragenic sequence in Oenothera mitochondria

Summary The mitochondrial genome of Oenothera is divided between a number of circular molecules that are derived from master circles containing the total sequence complexity. The circularisation event which leads to the formation of one of the small molecules involves a decanucleotide within the region coding for the large ribosomal RNA. This decanucleotide, 5-GGAAGCAGCC-3, is repeated 7.5 kb further downstream. The repeat recombines with the intragenic sequence to form circle no. 3, which thus contains the 3 part of the large rRNA gene. The other theoretical result from such a circularisation with the 5 part of the gene cannot be detected in the mitochondrial genome, thus eliminating the reformation of the original sequence arrangement. Different models are discussed for this apparent non-reciprocal circularisation.     

The Arabidopsis nuclear DAL gene encodes a chloroplast protein which is required for the maturation of the plastid ribosomal RNAs and is essential for chloroplast differentiation

Altered pigmentation is an easily scored and sensitive monitor of plastid function. We analyzed in detail a yellow colored transposon-tagged mutant (dal1-2) that is allelic to the dal mutant previously identified (Babiychuk et al., 1997). Mesophyll cells of mutant plants possess abnormal nucleoids and more but smaller plastids than wild type cells. Plastid development in dal1-2 is not altered in the dark but is arrested at the early steps of thylakoid assembly. The amino acid sequence of the protein deduced from our cDNA clone is 21 amino acids longer than the previously published DAL sequence (Babiychuk et al., 1997) and allowed us to show that DAL codes for a chloroplast protein. The dal1-2 mutation has a global negative effect on plastid RNA accumulation and on expression of nuclear encoded photosynthetic genes. We show that the plastid RNA polymerases, the nuclear-encoded NEP and the plastid-encoded PEP, are functional in the mutant. Precursor 16S and 23S rRNA species specifically accumulate at a high level in the mutant but the 5-end and the long 3-end trailer are not modified. We suggest that the dal mutation is involved in plastid rRNA processing and consequently in translation and early chloroplast differentiation.     

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.     

The ribosomal RNA genes from chloroplasts of mustard (Sinapis alba L.): mapping and sequencing of the leader region

Summary The genes coding for rRNAs from mustard chloroplasts were mapped within the inverted repeat regions of intact ctDNA and on ctDNA fragments cloned in pBR322. R-loop analysis and restriction endonuclease mapping show that the genes for 16S rRNA map at distances of 17 kb from the junctions of the repeat regions with the large unique region. The genes for 23S rRNA are located at distances of 2.8 kb from the junctions with the small unique region. Genes for 4.5S and 5S rRNA are located in close proximity to the 23S rRNA genes towards the small unique region. DNA sequencing of portions of the 5 terminal third from the mustard 16S rRNA gene shows 96–99% homology with the corresponding regions of the maize, tobacco and spinach chloroplast genes. Sequencing of the region proximal to the 16S rRNA gene reveals the presence of a tRNA^Val gene in nearly the same position and with identical sequence as in maize, tobacco and spinach. Somewhat less but still strong homology is also observed for the tDNA ^Val/16S rDNA intercistronic regions and for the regions upstream of the tRNA^Val gene. However, due to many small and also a few larger deletions and insertions in the leader region, common reading frames coding for homologous peptides larger than 44 amino acids can not be detected; it is therefore unlikely that this region contains a protein coding gene.     

The NADH-dehydrogenase subunit 5 gene in Oenothera mitochondria contains two introns and is co-transcribed with the 5 S rRNA gene

Summary The gene encoding subunit 5 of the NADH dehydrogenase complex (ND 5) has been identified in Oenothera mitochondria from a cDNA clone. The coding region is interrupted by a type II intron of 850 nucleotides and a second intervening sequence of 357 nucleotides. Genomic sequence rearrangement within the first intron creates a nontranscribed partial copy of the gene. The intact ND 5 gene is transcribed in a complex pattern with mRNAs including the 5 S rRNA sequence. Excision of the two introns appears to proceed slowly in vivo since the steady state mitochondrial RNA contains significant proportions of unprocessed precursor molecules.     

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.     

DNA sequence and secondary structures of the large subunit rRNA coding regions and its two class I introns of mitochondrial DNA fromPodospora anserina

Summary DNA sequence analysis has shown that the gene coding for the mitochondrial (mt) large subunit ribosomal RNA (rRNA) fromPodospora anserina is interrupted by two class I introns. The coding region for the large subunit rRNA itself is 3715 bp and the two introns are 1544 (r1) and 2404 (r2) bp in length. Secondary structure models for the large subunit rRNA were constructed and compared with the equivalent structure fromEscherichia coli 23S rRNA. The two structures were remarkably similar despite an 800-base difference in length. The additional bases in theP. anserina rRNA appear to be mostly in unstructured regions in the 3 part of the RNA. Secondary structure models for the two introns show striking similarities with each other as well as with the intron models from the equivalent introns inSaccharomyces cerevisiae, Neurospora crassa, andAspergillus nidulans. The long open reading frames in each intron are different from each other, however, and the nucleotide sequence similarity diverges as it proceeds away from the core structure. Each intron is located within regions of the large subunit rRNA gene that are highly conserved in both sequence and structure. Computer analysis showed that the open reading frame for intron r1 contained a common maturase-like polypeptide. The open reading frames of intron r2 apeared to be chimeric, displaying high sequence similarity with the open reading frames in the r1 and ATPase 6 introns ofN. crassa.     

A point mutation in the core subunit gene of yeast mitochondrial RNA polymerase is suppressed by a high level of specificity factor MTF1

Analysis of a transcribed region in the mitochondrial genome of Oenothera revealed an open reading frame (ORF) of 577 codons (orf577) that is also conserved in carrot, here encoding a protein of 579 amino acids (orf579). RNA editing alters the mRNA sequence of orf577 in Oenothera with 46 C to U transitions, many of which improve sequence similarity with the homologous Marchantia gene orf509. The deduced polypeptides show significant similarity with the ccll-encoded protein involved in cytochrome c biogenesis in the photosynthetic bacterium Rhodobacter capsulatus. A highly conserved domain is also found in plastid ORFs, suggesting that these bacterial, chloroplast and mitochondrial genes encode polypeptides with analogous functions in assembly and maturation of cytochromes c.     

Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae

Summary An 8.6-kb fragment was isolated from an EcoRI digest of Candida albicans ATCC 10261 genomic DNA which conferred the property of autonomous replication in Saccharomyces cervisiae on the otherwise non-replicative plasmid pMK155 (5.6 kb). The DNA responsible for the replicative function was subcloned as a 1.2-kb fragment onto a non-replicative plasmid (pRC3915) containing the C. albicans URA3 and LEU2 genes to form plasmid pRC3920. This plasmid was capable of autonomous replication in both S. cerevisiae and C. albicans and transformed S. cerevisiae AH22 (leu2 ^–) to Leu⁺ at a frequency of 2.15 × 10³ transformants per pg DNA, and transformed C. albicans SGY-243 (ura3) to Ura⁺ at a frequency of 1.91 × 10³ transformants per g DNA. Sequence analysis of the cloned DNA revealed the presence of two identical regions of eleven base pairs (5TTTTATGTTTT3) which agreed with the consensus of autonomously replicating sequence (ARS) cores functional in S. cerevisiae. In addition there were two 10/11 and numerous 9/11 matches to the core consensus. The two 11/11 matches to the consensus, CaARS1 and CaARS2, were located on opposite strands in a non-coding AT-rich region and were separated by 107 bp. Also present on the C. albicans DNA, 538 by from the ARS cores, was a gene for 5S rRNA which showed sequence homology with several other yeast 5S rRNA genes. A sub-fragment (494 bp) containing the 5S rRNA gene (but not the region containing the ARS cores) hybridized to genomic DNAs from a number of yeast species, including S. cerevisiae, C. tropicalis, C. pseudotropicalis, C. parapsilosis, C. kruseii, C. (Torulopsis) glabrata and Neurospora crassa. The 709-bp ARS element (but not the 5S rRNA gene) was necessary for high-frequency transformation and autonomous plasmid replication in both S. cerevisiae and C. albicans.EMBL/GenBank database accession number: X16634 (5S rRNA)     

An unlinked 5 S ribosomal RNA gene in the archaebacterium, Thermococcus celer

Summary We have determined the nucleotide sequence of an unlinked 5 S rRNA gene region from a thermophilic archaebacterium, Thermococcus celer. This 5 S rRNA gene is flanked by a single tRNA^Asp sequence and appears to be transcribed as part of a very short operon consisting of only two gene sequences. Comparative studies indicate features in the 5 and 3 flanking sequences, which bear similarity with promoter and termination signals in eubacteria, but also reflect unusual features found in at least some archaebacteria. The evolution of this unlinked operon and the unusual features are discussed.     

Unusual characteristics of Codium fragile chloroplast DNA revealed by physical and gene mapping

Summary A complete physical map of the Codium fragile chloroplast genome was constructed and the locations of a number of chloroplast genes were determined. Several features of this circular genome are unusual. At 89 kb in size, it is the smallest chloroplast genome known. Unlike most chloroplast genomes it lacks any large repeat elements. The 8 kb spacer region between the 16 S and 23 S rRNA genes is the largest such spacer characterized to date in chloroplast DNA. This spacer region is also unusual in that it contains the rps12 gene or at least a portion thereof. Three regions polymorphic for size are present in the Codium chloroplast genome. The psbA and psbC genes map closely to one of these regions, another region is in the spacer between the 16 S and 23 S rRNA genes and the third is very close to or possibly within the 16 S rRNA gene. The gene order in the Codium genome bears no marked resemblance to either the consensus vascular plant order or to that of any green algal or bryophyte genome. Present address: Department of Biology, Texas A&M University, College Station, TX 77843; USA     

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     

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.     

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.     

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.     

Unusual evolutionary conservation of 5S rRNA pseudogenes inAspergillus nidulans: Similarity of the DNA sequence associated with the pseudogenes with the mouse immunoglobulin switch region

Summary AllAspergillus nidulans 5S rRNA pseudogenes known so far are the result of integration of an approx. 0.2-kbp-long DNA sequence into the 5S rRNA genes. This sequence, called block C, is present in at least five copies in theA. nidulans genome and seems to be associated either with 5S rRNA genes or pseudogenes. In contrast to the 78% sequence conservation of the C-block in pseudogenes, the truncated 5 halves of the pseudogenes are very highly conserved (96.9–100%). We postulate that the 5S rRNA pseudogenes are still a subject of concerted evolution. The C-block sequence shows similarity to the switch region of the mouse heavy chain immunoglobulin gene. A characteristic motif GGGTGAG is repeated several times in both sequences; the sequence conservation is 63%.