Euglena gracilis chloroplast ribosomal RNA transcription units. Nucleotide sequence polymorphism in 5 S rRNA genes and 5 S rRNAs

The three tandemly repeated ribosomal RNA operons from the chloroplast genome of Euglena gracilis Klebs, Pringsheim Strain Z each contain a 5 S rRNA gene distal to the 23 S rRNA gene (Gray, P.W., and Hallick, R.B. (1979) Biochemistry 18, 1820-1825). We have cloned two distinct 5 S rRNA genes, and determined the DNA sequence of the genes, their 5'- and 3'-flanking sequences, and the 3'-end of the adjacent 23 S rRNA genes. The two genes exhibit sequence polymorphism at five bases within the "procaryotic loop" coding region, as well as internal restriction endonuclease site heterogeneity. These restriction endonuclease site polymorphisms are evident in chloroplast DNA, and not just the cloned examples of 5 S genes. Chloroplast 5 S rRNA was isolated, end labeled, and sequenced by partial enzymatic degradation. The same polymorphisms found in 5 S rDNA are present in 5 S rRNA. Therefore, both types of 5 S rRNA genes are transcribed and are present in chloroplast ribosomes.     

Phylogeny of elasmobranchs based on LSU and SSU ribosomal RNA genes

The dominant view of the phylogeny of living elasmobranchs, based on morphological characters, is that batoids (skates and rays) are derived sharks, joined with saw sharks, and angel sharks in the clade Hypnosqualea [S. Shirai, Squalean Phylogeny: A New Framework of 'Squaloid' Sharks and Related Taxa, Hokkaido University Press, Sapporo, 1992]. By contrast, a recent molecular-phylogenetic study based on mitochondrial genes for 12S and 16S rRNA and tRNA valine [C.J. Douady et al., Mol. Phylogenet. Evol., 26 (2003) 215-221] supported the older view that batoids and sharks are separate lineages. Here, we tested these two different views using combined, nuclear large-subunit and small-subunit rRNA gene sequences ( approximately 5.3kb) from 22 elasmobranchs, two chimeras, and two bony fishes. We used maximum likelihood, maximum parsimony, minimum evolution, and Bayesian inference for tree reconstruction, and found the large-subunit rRNA gene to contain far more signal than the small-subunit gene for resolving this mostly Mesozoic radiation. Our findings matched those of in separating batoids from sharks and in statistically rejecting Hypnosqualea. The angel shark (Squatina) was the sister group to squaliforms (dogfish sharks), and our findings are consistent with the idea that "orbitostylic" sharks form a monophyletic group (squaliforms+the hexanchiform Chlamydoselachus+Squatina+Pristiophorus). In the galeomorph sharks, however, lamniforms grouped with orectolobiforms, opposing the widely accepted 'lamniform+carcharhiniform' grouping. A tree based on the mitochondrial gene for cytochrome b also supported a separation of sharks and batoids, in contrast to Hypnosqualea. Among elasmobranchs, variation in the evolutionary rates of the nuclear rRNA genes was higher than that of cytochrome b genes, mainly due to the relatively rapid evolution of rRNA in some carcharhiniforms. In conclusion, several different molecular studies now refute the Hypnosqualea hypothesis of elasmobranch interrelationships.     

Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi‐copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxonomic specificity and sensitivity of qPCR assays targeting the rodA gene (rodA984) and two regions of the multi‐copy 23S ribosomal RNA gene (EC23S and EC23S857). Experimental analyses of 28 culture collection strains representing E. coli and 21 related non‐target species indicated that the uidA405 and rodA984 assays were both 100% specific for E. coli while the EC23S assay was only 29% specific. The EC23S857 assay was only 95% specific due to detection of E. fergusonii. The uidA405, rodA984, EC23S and EC23S857 assays were 85%, 85%, 100% and 86% sensitive, respectively, in detecting 175 presumptive E. coli culture isolates from fresh, marine and waste water samples. In analyses of DNA extracts from 32 fresh, marine and waste water samples, the rodA984, EC23S and EC23S857 assays detected mean densities of target sequences at ratios of approximately 1 : 1, 243 : 1 and 6 : 1 compared with the mean densities detected by the uidA405 assay. Conclusions: The EC23S assay was less specific for E. coli, whereas the rodA984 and EC23S857 assay taxonomic specificities and sensitivities were similar to those of the uidA405 gene assay. Significance and Impact: The EC23S857 assay has a lower limit of detection for E. coli cells than the uidA405 and rodA984 assays due to its multi‐copy gene target and therefore provides greater analytical sensitivity in monitoring for these faecal pollution indicators in environmental waters by qPCR methods.     

The nucleolus and transcription of ribosomal genes

Ribosome biogenesis is a highly dynamic, steady-state nucleolar process that involves synthesis and maturation of rRNA, its transient interactions with non-ribosomal proteins and RNPs and assembly with ribosomal proteins. In the few years of the 21st century, an exciting progress in the molecular understanding of rRNA and ribosome biogenesis has taken place. In this review, we discuss the recent results on the regulation of rRNA synthesis in relation to the functional organization of the nucleolus, and put an emphasis on the situation encountered in mammalian somatic cells.     

Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma.

Phormium yellow leaf (PYL) phytoplasma causes a lethal disease of the monocotyledon, New Zealand flax (Phormium tenax). The 16S rRNA genes of PYL phytoplasma were amplified from infected flax by PCR and cloned, and the nucleotide sequences were determined. DNA sequencing and Southern hybridization analysis of genomic DNA indicated the presence of two copies of the 16S rRNA gene. The two 16S rRNA genes exhibited sequence heterogeneity in 4 nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. A phylogenetic tree based on 16S rRNA gene sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas, which form the stolbur subgroup of the aster yellows group. This phylogenetic position of PYL phytoplasma was supported by 16S/23S spacer region sequence data.     

Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with an unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal core.

Microsporidia are eukaryotic parasites lacking mitochondria, the ribosomes of which present prokaryote-like features. In order to better understand the structural evolution of rRNA molecules in microsporidia, the 5S and rDNA genes were investigated in Encephalitozoon cuniculi . The genes are not in close proximity. Non-tandemly arranged rDNA units are on every one of the 11 chromosomes. Such a dispersion is also shown in two other Encephalitozoon species. Sequencing of the 5S rRNA coding region reveals a 120 nt long RNA which folds according to the eukaryotic consensus structural shape. In contrast, the LSU rRNA molecule is greatly reduced in length (2487 nt). This dramatic shortening is essentially due to truncation of divergent domains, most of them being removed. Most variable stems of the conserved core are also deleted, reducing the LSU rRNA to only those structural features preserved in all living cells. This suggests that the E.cuniculi LSU rRNA performs only the basic mechanisms of translation. LSU rRNA phylogenetic analysis with the BASEML program favours a relatively recent origin of the fast evolving microsporidian lineage. Therefore, the prokaryote-like ribosomal features, such as the absence of ITS2, may be derived rather than primitive characters.     

Linkage of two distinct AT-rich minisatellites at multiple loci in the genome of Theileria parva

Minisatellite tandem repeat elements are well known components of vertebrate genomes, but have not yet been extensively characterized in lower eukaryotes. We describe two unusual, AT-rich minisatellites of the protozoan parasite Theileria parva whose sequences are unrelated to the G/C-rich `chi minisatellite superfamily' of vertebrate and plant genomes. The T. parva tandem repeats, one with a conserved sequence T_2-5ACACA (6–17 copies), and the other with a 6-bp core sequence of either ACTATA or TATACT associated with additional variable sequences in repeats of 10–17 bp (3–7 copies), were closely linked at more than 20 sites in the T. parva genome, separated by 390, 510 and 660 bp at three loci analysed in detail. Such linkage is without precedent in minisatellites so far analysed in other organisms. The minisatellite loci were widely dispersed on 13 out of 33 genomic SfiI fragments, on all four T. parva chromosomes and did not exhibit a telomeric bias in their distribution. Analysis of flanking sequences revealed no obvious conserved sequences between the five loci, or other multicopy repeat sequences outside the minisatellite regions. The T_2-5 ACACA minisatellite was highly effective as a multilocus fingerprinting probe for discrimination of T. parva isolates. Analysis of two individual minisatellite loci revealed variation between the genomic DNAs of two T. parva isolates in the copy number of the constituent repeats within the array, similar to that typical of vertebrate minisatellites.     

Sequence analysis of 16S rRNA genes amplified from two ribosomal RNA gene clusters of Bifidobacterium bifidum

Two rRNA gene clusters were detected in the genome of Bifidobacterium bifidum KCTC 3202^T using Southern blot analysis. To analyse the sequences of the 16S rRNA genes from rrnA and rrnB, 16S rDNAs were amplified by PCR using DNA fragments purified from gel slices containing each of the rRNA gene clusters. The amplified 16S rDNAs from rrnA and rrnB were cloned into vectors and three clones of each gene sequenced. The resultant sequences were confirmed by direct sequencing of the 16S rDNAs from rrnA and rrnB. Sequence differences were not found between rrnA and rrnB in 1488 bp of the 16S rRNA genes.     

Conserved restriction sites within the ribosomal RNA genes of vertebrates

We have mapped the cleavage sites of four restriction enzymes which recognize six-base sequences within the nuclear ribosomal (rRNA) genes of twelve vertebrates, including several placental mammals (Homo sapiens, man; Bos taurus, cow; Equus caballus, horse; Sus scofra, pig; Ovis aries, sheep; Rattus rattus, rat), a marsupial (Didelphis marsupialis, opossum), a bird (Gallus domesticus, chicken), an amphibian (Xenopus laevis), a reptile (Alligator mississipiensis), a bony fish (Cynoscion nebulosus, sea trout), and a cartilagenous fish (Carcharhinus species, requiem shark). These animals represent a span of approx. 400 million years of evolutionary divergence. Our data identify restriction sites in the rRNA genes which are highly conserved among higher vertebrates and therefore are likely to be in functionally important regions. Additionally, the restriction enzyme sites identified will be useful in cloning and sequencing the rRNA genes in any vertebrate. Finally, the consistent size and conserved sequence homology suggests that these rRNA gene segments will be useful as internal controls in hybridization experiments involving other genomic regions in vertebrates.