rRNA Sites Research Articles

An RNA Conformational Switch Regulates Pre-18S rRNA Cleavage

To produce mature ribosomal RNAs (rRNAs), polycistronic rRNA transcripts are cleaved in an ordered series of events. We have uncovered the molecular basis for the ordering of two essential cleavage steps at the 3′-end of 18S rRNA. Using in vitro and in vivo structure probing, RNA binding and cleavage experiments, and yeast genetics, we demonstrate that a conserved RNA sequence in the spacer region between the 18S and 5.8S rRNAs base-pairs with the decoding site of 18S rRNA in early assembly intermediates. Nucleolar cleavage at site A2 excises this sequence element, leading to a conformational switch in pre-18S rRNA, by which the ribosomal decoding site is formed. This conformational switch positions the nuclease Nob1 for cytoplasmic cleavage at the 3′-end of 18S rRNA and is required for the final maturation step of 18S rRNA in vivo and in vitro. More generally, our data show that the intrinsic ability of RNA to form stable structural switches is exploited to order and regulate RNA-dependent biological processes.

Protein S20 Binds Two 16S rRNA Sites as Assembly Is Initiated

Ribosomal protein S20 is a primary binding protein that bridges the 5′ domain and the 3′ minor domain of the 16S ribosomal RNA (rRNA) in the 30S ribosomal subunit. Using time-dependent dimethyl sulfate modification, we have determined that as it is bound to 16S rRNA, protein S20 causes rapid protection of bases A246, A274, A279, and A282 in the stem region of helix 11 in the 5′ domain and moderately fast modifications of helix 44 bases A1433 and A1434 in the 3′ minor domain. At a later time, enhancements occur with bases A181and A190 in helix 9, bases A325 and A327 in helix 13, and base C264 at the distal end of helix 11 in the 5′ domain of 16S rRNA. The modifications that occur in the stem region of helix 11 are distant from the binding site of protein S20, as determined from the crystal structure. Simultaneous addition of protein S17 with S20 to the complex significantly alters the modifications caused by protein S20 in the stem region of helix 11 but does not alter the remaining modifications. Our results indicate that protein S20 is binding to at least two alternate 16S rRNA sites during the early assembly process.

Accuracy Assessment of Protein-Based Docking Programs against RNA Targets

Ribonucleic acid (RNA) molecules play central roles in a variety of biological processes and, hence, are attractive targets for therapeutic intervention. In recent years, molecular docking techniques have become one of the most popular and successful approaches in drug discovery; however, almost all docking programs are protein based. The adaptability of popular docking programs in RNA world has not been systematically evaluated. This paper describes the comprehensive evaluation of two widely used protein-based docking programs--GOLD and Glide--for their docking and virtual screening accuracies against RNA targets. Using multiple docking strategies, both GOLD 4.0 and Glide 5.0 successfully reproduced most binding modes of the 60 tested RNA complexes. Applying different docking/scoring combinations, significant enrichments from the simulated virtual and fragment screening experiments were achieved against tRNA decoding A site of 16S rRNA (rRNA A-site). Our study demonstrated that current protein-based docking programs can fulfill general docking tasks against RNA, and these programs are very helpful in RNA-based drug discovery and design.

Aptazyme-Mediated Regulation of 16S Ribosomal RNA

Developing artificial genetic switches in order to control gene expression via an external stimulus is an important aim in chemical and synthetic biology. Here, we expand the application range of RNA switches to the regulation of 16S rRNA function in Escherichia coli. For this purpose, we incorporated hammerhead ribozymes at several positions into orthogonalized 16S rRNA. We observed that ribosomal function is remarkably tolerant toward the incorporation of large additional RNA fragments at certain sites of the 16S rRNA. However, ribozyme-mediated cleavage results in severe reduction of 16S rRNA stability. We carried out an in vivo screen for the identification of sequences acting as ligand-responsive RNA switches, enabling thiamine-dependent switching of 16S rRNA function. In addition to expanding the regulatory toolbox, the presented artificial riboswitches should prove valuable to study aspects of rRNA folding and stability in bacteria.

A molecular dynamics study on binding recognition between several 4,5 and 4,6‐linked aminoglycosides with A‐site RNA

A molecular dynamics (MD) simulation has been performed for two sets of aminoglycoside antibiotics bound with an RNA duplex corresponding to the aminoacyl-tRNA decoding site of the 16S rRNA to characterize the energetics and dynamics of binding for several aminoglycosides. The binding free energy, essential dynamics and hydration analysis have been conducted to characterize the dynamics' properties associated with the binding recognition between each set of antibiotics and the RNA duplex. We have built several dynamic models with reasonable binding free energies showing good correlation with the experimental data. We have also conducted a hydration analysis on some long residency water molecules detected as W8 and W49 sites around the U1406 . U1495 pair and which are found to be important in binding recognition and in causing some apparent stretch variations of this pair during the dynamic studies. In addition, we also find that the hydration sites with long residence time identified between the ring III of two 4,6-linked antibiotics (tobramycin and kanamycin) and phosphate oxygen atoms of G1405/U1406 may be worthy of further exploration for rational drug design.

Nearly complete rRNA genes assembled from across the metazoan animals: Effects of more taxa, a structure-based alignment, and paired-sites evolutionary models on phylogeny reconstruction

This study (1) uses nearly complete rRNA-gene sequences from across Metazoa (197 taxa) to reconstruct animal phylogeny; (2) presents a highly annotated, manual alignment of these sequences with special reference to rRNA features including paired sites (http://purl.oclc.org/NET/rRNA/Metazoan_alignment) and (3) tests, after eliminating as few disruptive, rogue sequences as possible, if a likelihood framework can recover the main metazoan clades. We found that systematic elimination of ∼6% of the sequences, including the divergent or unstably placed sequences of cephalopods, arrowworm, symphylan and pauropod myriapods, and of myzostomid and nemertodermatid worms, led to a tree that supported Ecdysozoa, Lophotrochozoa, Protostomia, and Bilateria. Deuterostomia, however, was never recovered, because the rRNA of urochordates goes (nonsignificantly) near the base of the Bilateria. Counterintuitively, when we modeled the evolution of the paired sites, phylogenetic resolution was not increased over traditional tree-building models that assume all sites in rRNA evolve independently. The rRNA genes of non-bilaterians contain a higher % AT than do those of most bilaterians. The rRNA genes of Acoela and Myzostomida were found to be secondarily shortened, AT-enriched, and highly modified, throwing some doubt on the location of these worms at the base of Bilateria in the rRNA tree—especially myzostomids, which other evidence suggests are annelids instead. Other findings are marsupial-with-placental mammals, arrowworms in Ecdysozoa (well supported here but contradicted by morphology), and Placozoa as sister to Cnidaria. Finally, despite the difficulties, the rRNA-gene trees are in strong concordance with trees derived from multiple protein-coding genes in supporting the new animal phylogeny.

Interaction of Aminoglycosides with Human Mitochondrial 12S rRNA Carrying the Deafness-Associated Mutation

The mitochondrial 12S rRNA A1555G mutation is one of the important causes of aminoglycoside-induced and nonsyndromic hearing loss. Here we employed an RNA-directed chemical-modification approach to understanding the pathogenesis of aminoglycoside-induced hearing loss. The patterns of chemical modification of RNA oligonucleotides carrying the A1555G mutation by dimethyl sulfate (DMS) were distinct from those of the RNA oligonucleotides carrying wild-type sequence in the presence of aminoglycosides. In the RNA analogue carrying the A1555G mutation, reduced reactivity to DMS occurred in base G1555 as well as in bases C1556 and A1553 in the presence of paromomycin, neomycin, gentamicin, kanamycin, tobramycin, or streptomycin. In particular, base G1555 exhibited marked but similar levels of protection in the presence of 0.1 microM to 100 microM neomycin, gentamicin, or kanamycin. In contrast, the levels of protection in base G1555 appeared to be correlated with the concentration of paromycin, tobramycin, or streptomycin. Furthermore, increasing reactivities to DMS in the presence of these aminoglycosides were observed for bases A1492, C1493, C1494, and A1557 in the RNA analogue carrying the A1555G mutation. These data suggested that the A1555G mutation altered the binding properties of aminoglycosides at the A site of 12S rRNA and led to local conformational changes in 12S rRNA carrying the A1555G mutation. The interaction between aminoglycosides and 12S rRNA carrying the A1555G mutation provides new insight into the pathogenesis of aminoglycoside ototoxicity.

Selection of Peptides That Target the Aminoacyl-tRNA Site of Bacterial 16S Ribosomal RNA

For almost five decades, antibiotics have been used successfully to control infectious diseases caused by bacterial pathogens. More recently, however, two-thirds of bacterial pathogens exhibit resistance and are continually evolving new resistance mechanisms against almost every clinically used antibiotic. Novel efforts are required for the development of new drugs or drug leads to combat these infectious diseases. A number of antibiotics target the bacterial aminoacyl-tRNA site (A site) of 16S rRNA (rRNA). Mutations in the A-site region are known to cause antibiotic resistance. In this study, a bacterial (Escherichia coli) A-site rRNA model was chosen as a target to screen for peptide binders. Two heptapeptides, HPVHHYQ and LPLTPLP, were selected through M13 phage display. Both peptides display selective binding to the A-site 16S rRNA with on-bead fluorescence assays. Dissociation constants (Kd's) of the amidated peptide HPVHHYQ-NH2 to various A-site RNA constructs were determined by using enzymatic footprinting, electrospray ionization mass spectrometry (ESI-MS), and isothermal titration calorimetry (ITC) under a variety of buffer and solution conditions. HPVHHYQ-NH2 exhibits moderate affinity for the A-site RNA, with an average Kd value of 16 microM. In addition, enzymatic footprinting assays and competition ESI-MS with a known A-site binder (paromomycin) revealed that peptide binding occurs near the asymmetric bulge at positions U1495 and G1494 and leads to increased exposure of residues A1492 and A1493.

Hydrogen bonding and packing density are factors most strongly connected to limiting sites of high flexibility in the 16S rRNA in the 30S ribosome.

BackgroundConformational flexibility in structured RNA frequently is critical to function. The 30S ribosomal subunit exists in different conformations in different functional states due to changes in the central part of the 16S rRNA. We are interested in evaluating the factors that might be responsible for restricting flexibility to specific parts of the 16S rRNA using biochemical data obtained from the 30S subunit in solution. This problem was approached taking advantage of the observation that there must be a high degree of conformational flexibility at sites where UV photocrosslinking occurs and a lack of flexibility inhibits photoreactivity at many other sites that are otherwise suitable for reaction.ResultsWe used 30S x-ray structures to quantify the properties of the nucleotide pairs at UV- and UVA-s4U-induced photocrosslinking sites in 16S rRNA and compared these to the properties of many hundreds of additional sites that have suitable geometry but do not undergo photocrosslinking. Five factors that might affect RNA flexibility were investigated – RNA interactions with ribosomal proteins, interactions with Mg2+ ions, the presence of long-range A minor motif interactions, hydrogen bonding and the count of neighboring heavy atoms around the center of each nucleobase to estimate the neighbor packing density. The two factors that are very different in the unreactive inflexible pairs compared to the reactive ones are the average number of hydrogen bonds and the average value for the number of neighboring atoms. In both cases, these factors are greater for the unreactive nucleotide pairs at a statistically very significant level.ConclusionThe greater extent of hydrogen bonding and neighbor atom density in the unreactive nucleotide pairs is consistent with reduced flexibility at a majority of the unreactive sites. The reactive photocrosslinking sites are clustered in the 30S subunit and this indicates nonuniform patterns of hydrogen bonding and packing density in the 16S rRNA tertiary structure. Because this analysis addresses inter-nucleotide distances and geometry between nucleotides distant in the primary sequence, the results indicate regional and global flexibility of the rRNA.

Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA

Peptide nucleic acid (PNA) targeted to the functional domains of 23S rRNA can inhibit translation and cell growth. However, effective inhibition of translation and cell growth using 16S rRNA-targeted PNA has still not been achieved. Here, we report that PNA targeted to the functional site of 16S rRNA could inhibit both gene expression in vitro and bacterial growth in pure culture with sequence specificity. We used 10-mer PNAs conjugated with a cell-penetrating peptide, which targeted the mRNA binding site at the 3' end of 16S rRNA. Using 0.6 microM of the peptide-PNAs, cell-free ss-galactosidase production decreased by 50%, whereas peptide-PNAs with one or two mismatches to the target sequence showed much weaker inhibition effects. To determine the growth inhibition and bactericidal effects of the peptide-PNA conjugate, we performed OD measurement and viable cell counting. We observed dose- and sequence-dependent inhibition of cell growth and bactericidal effects. These growth inhibitory effects are observed both in the Gram-negative bacterium of Escherichia coli and the Gram-positive bacteria Bacillus subtilis and Corynebacterium efficiens, although inhibitory concentrations were different for each bacterial species. These results present possibilities for 16S rRNA sequence-based specific bacterial growth inhibition using a peptide-PNA conjugate.

Conflict and resolution between phylogenies inferred from molecular and phenotypic data sets for hagfish, lampreys, and gnathostomes

One of the most problematic issues in vertebrate phylogenetics is the disagreement between phenotypic and molecular inferences regarding the relationships among hagfishes, lampreys, and gnathostomes. Phenotypic characters support monophyly of lampreys and gnathostomes, whereas nearly all published analyses of molecular data sets support monophyly of hagfishes and lampreys. In this study I present results of phylogenetic analyses of combined phenotypic and molecular data sets that focus on relationships among hagfishes, lampreys, and gnathostomes. Maximum parsimony analyses of 115 phenotypic characters combined with 4,638 rRNA sites and more than 10,000 amino acids each result in monophyly of lampreys and gnathostomes, demonstrating that the addition of relatively few phenotypic characters can alter phylogenetic inferences from large molecular data sets. On the other hand, Bayesian analyses of the combined data sets support monophyly of hagfish and lampreys, indicating that model-based analyses may be prone to data "swamping," where the phylogenetic signal of the larger molecular data sets overwhelm the signal present in the much smaller phenotypic data set. Nodes that relate hagfish and lampreys were recovered at a low frequency in parametric bootstrapping analyses, indicating that the timing of diversification among hagfishes, lampreys, and gnathostomes has created a difficult phylogenetic problem for molecular data. The fact that addition of relatively few phenotypic characters can alter phylogenetic inferences of cyclostome monophyly obtained from molecular data sets, and the inability of simulated data sets to recover key nodes in the craniate phylogeny provide reasons to view the strong support for cyclostome monophyly inferred from molecular data sets with a measured degree of skepticism.

The amino terminal domain from Mrt4 protein can functionally replace the RNA binding domain of the ribosomal P0 protein.

In Saccharomyces cerevisiae, the Mrt4 protein is a component of the ribosome assembly machinery that shares notable sequence homology to the P0 ribosomal stalk protein. Here, we show that these proteins can not bind simultaneously to ribosomes and moreover, a chimera containing the first 137 amino acids of Mrt4 and the last 190 amino acids from P0 can partially complement the absence of the ribosomal protein in a conditional P0 null mutant. This chimera is associated with ribosomes isolated from this strain when grown under restrictive conditions, although its binding is weaker than that of P0. These ribosomes contain less P1 and P2 proteins, the other ribosomal stalk components. Similarly, the interaction of the L12 protein, a stalk base component, is affected by the presence of the chimera. These results indicate that Mrt4 and P0 bind to the same site in the 25S rRNA. Indeed, molecular dynamics simulations using modelled Mrt4 and P0 complexes provide further evidence that both proteins bind similarly to rRNA, although their interaction with L12 displays notable differences. Together, these data support the participation of the Mrt4 protein in the assembly of the P0 protein into the ribosome and probably, that also of the L12 protein.

Analysis of C/D box snoRNA genes in vertebrates: The number of copies decreases in placental mammals

C/D box small nucleolar RNAs (snoRNAs) guide site-specific 2′-O-methylation of RNAs. Nearly all C/D box snoRNAs with known targets are involved in rRNA modification. In vertebrates, snoRNAs are encoded in introns of various genes and their processing is coupled with splicing of host gene pre-mRNA. Here, the genes encoding C/D box snoRNAs that guide 2′-O-methylation of rRNA were identified and analyzed in vertebrate genomes. The number of copies of most C/D box snoRNA genes proved to be lower in placental mammals compared to other vertebrates. This can be due to smaller oocytes and accordingly lower number of ribosomes in them in eutherians. The targets of snoRNAs encoded by single-copy and multiple-copy genes proved to have different distribution in rRNAs. The causes of this difference are discussed. In some cases, the transcripts of homologous C/D box RNA genes were shown to guide the modification of neighboring nucleotides in rRNA. C/D box snoRNA pseudogenes were found in all vertebrate classes. Three novel C/D box snoRNAs were found in Xenopus tropicalis that may guide 2′-O-methylation of Xenopus-specific rRNA sites. A list of 922 annotated C/D box snoRNA genes is presented.

Sites of 18S rRNA contacting mRNA 3′ and 5′ of the P site codon in human ribosome: A cross-linking study with mRNAs carrying 4-thiouridines at specific positions

Long synthetic mRNAs were used to study the positioning of the E site codon, the 2nd and 3rd nucleotides of the A site bound codon and a nucleotide 3′ of this codon with respect to the 18S rRNA in the human 80S ribosome. The mRNAs contained a GAC triplet coding for Asp and a single 4-thiouridine residue (s 4U) upstream or downstream of the GAC codon. In the presence of tRNA Asp, the GAC codon of the mRNAs was targeted to the ribosomal P site thus placing s 4U in one of the following positions − 3, − 2, − 1, + 5, + 6 or + 7 with respect to the first nucleotide of the P site bound codon. It was found that mRNAs that bore s 4U in positions + 5 to + 7 cross-linked to the 18S rRNA nucleotides C1696, C1698 and 1820–1825, the distribution of cross-links among these targets depending on the position of s 4U. Cross-links of mRNAs containing s 4U in positions − 3 to − 1 were found in the region 1699–1704 of the 18S rRNA. In the absence of tRNA, all mRNAs cross-linked only to C1696 and C1698. Absence of the cross-linked nucleotides C1696 and C1698 in the case of mRNAs containing s 4U in positions − 3 to − 1 confirmed that tRNA Asp actually phased the mRNA on the ribosome.

Antisense Masking Reveals Contributions of mRNA-rRNA Base Pairing to Translation of Gtx and FGF2 mRNAs

We previously showed that a 9-nucleotide sequence from the 5' leader of the Gtx homeodomain mRNA facilitates translation initiation by base pairing to 18S rRNA. These earlier studies tested the Gtx element in isolation; we now assess the physiological relevance of this element in the context of two natural mRNAs that contain this sequence in their 5' leaders, Gtx itself and FGF2 (fibroblast growth factor 2). 2'-O-Methyl-modified RNA oligonucleotides were employed to block mRNA-rRNA base pairing by targeting either the Gtx-binding site in 18S rRNA or Gtx elements in recombinant mRNAs containing the Gtx or FGF2 5' leaders linked to a reporter cistron. Studies in cell-free lysates and transfected COS-7 cells showed that translation of mRNAs containing the Gtx or FGF2 5' leaders was decreased by > 50% when oligonucleotides targeting either the rRNA or mRNA were used. Specificity was demonstrated by showing that translation of the recombinant mRNAs was unaffected by control oligonucleotides. In addition, the specific oligonucleotides did not affect the translation of recombinant mRNAs in which the Gtx elements were mutated. Experiments performed using constructs containing Gtx and FGF2 5' leader and coding sequences ruled out possible effects of the reporter cistron. Furthermore, two-dimensional gel electrophoresis revealed that the oligonucleotides used in this study had little overall effect on the proteomes of cells transfected with these oligonucleotides. This study demonstrates that mRNA-rRNA base pairing affects the expression of two cellular mRNAs and describes a new approach for investigating putative mRNA-rRNA base pairing interactions in mammalian cells.

Mis-targeted methylation in rRNA can severely impair ribosome synthesis and activity

Eukaryotic rRNAs contain scores of two major types of nucleotide modifications, 2’-O-methylation (Nm) and pseudouridylation (Ψ). Both types are known to alter the stability and dynamics of RNA folding. In Eukaryotes, these modifications are created by small nucleolar RNPs (snoRNPs) with site-specificity provided by the snoRNA component. Little is yet known about the influence of such modifications on ribosome synthesis or activity, although in a few cases depletions of natural modifications have impaired ribosome function. Our previous work showed that targeting Nm modifications to non-natural sites in yeast rRNA can severely impair cell growth, however, the underlying basis of the interference effects were not described. Here, we show that targeting Nm formation to several individual sensitive sites in the peptidyl transferase center (PTC) strongly impairs ribosome accumulation and activity. Methylation was detected for all sites targeted, suggesting that the non-natural modification is the basis of the interference effects. For certain sensitive sites, the translation rate was reduced by 70-100%, due to: 1) a marked decrease (28-50%) in ribosomal subunits caused by slower pre-rRNA processing and mainly faster rRNA turn over and, 2) impaired activity of the surviving ribosomes. This last finding infers that the mis-targeted methylations compromise PTC function. The discovery that a new methylation can trigger robust rRNA degradation indicates that modification effects are monitored for quality control. These findings infer that nucleotide modifications can serve as evolutionary constraints and that snoRNPs mutations expected to occur in nature can cause human disease.

Characterization of Renibacterium salmoninarum with reduced susceptibility to macrolide antibiotics by a standardized antibiotic susceptibility test

Three cohorts of juvenile and subadult Chinook salmon Oncorhynchus tshawytscha received multiple treatments with macrolide antibiotics for bacterial kidney disease (BKD) during rearing in a captive broodstock program. A total of 77 mortalities among the cohorts were screened for Renibacterium salmoninarum, the etiologic agent of BKD, by agar culture from kidney, and isolates from 7 fish were suitable for growth testing in the presence of macrolide antibiotics. The minimum inhibitory concentration (MIC) of erythromycin and azithromycin was determined by a modification of the standardized broth assay using defined medium. The American Type Culture Collection (ATCC) type strain 33209 exhibited a MIC of 0.008 microg m(-1) to either erythromycin or azithromycin. Isolates from 3 fish displayed MICs identical to the MICs for the ATCC type strain 33209. In contrast, isolates from 4 fish exhibited higher MICs, ranging between 0.125 and 0.250 microg ml(-1) for erythromycin and between 0.016 and 0.031 microg ml(-1) for azithromycin. Sequence analysis of the mutational hotspots for macrolide resistance in the 23S rDNA gene and the open reading frames of ribosomal proteins L4 and L22 found identical sequences among all isolates, indicating that the phenotype was not due to mutations associated with the drug-binding site of 23S rRNA. These results are the first report of R. salmoninarum with reduced susceptibility to macrolide antibiotics isolated from fish receiving multiple antibiotic treatments.

Ribosomal RNAs are tolerant toward genetic insertions: evolutionary origin of the expansion segments

Ribosomal RNAs (rRNAs), assisted by ribosomal proteins, form the basic structure of the ribosome, and play critical roles in protein synthesis. Compared to prokaryotic ribosomes, eukaryotic ribosomes contain elongated rRNAs with several expansion segments and larger numbers of ribosomal proteins. To investigate architectural evolution and functional capability of rRNAs, we employed a Tn5 transposon system to develop a systematic genetic insertion of an RNA segment 31 nt in length into Escherichia coli rRNAs. From the plasmid library harboring a single rRNA operon containing random insertions, we isolated surviving clones bearing rRNAs with functional insertions that enabled rescue of the E. coli strain (Δ7rrn) in which all chromosomal rRNA operons were depleted. We identified 51 sites with functional insertions, 16 sites in 16S rRNA and 35 sites in 23S rRNA, revealing the architecture of E. coli rRNAs to be substantially flexible. Most of the insertion sites show clear tendency to coincide with the regions of the expansion segments found in eukaryotic rRNAs, implying that eukaryotic rRNAs evolved from prokaryotic rRNAs suffering genetic insertions and selections.

Nomenclature of Plasmid-Mediated 16S rRNA Methylases Responsible for Panaminoglycoside Resistance

Production of 16S rRNA methylase has recently drawn attention as a novel aminoglycoside resistance mechanism in pathogenic gram-negative bacteria (1). It confers very-high-level resistance to all aminoglycosides that are currently available for parenteral formulation. Six distinct genes, rmtA, rmtB, rmtC, rmtD, armA, and npmA, encoding their respective enzymes have been identified in clinical and veterinary strains from various geographic areas, including East Asia, Europe, and the Americas, since 2003 (1, 10). NpmA is the only enzyme among them that methylates residue A1408, whereas the others methylate residue G1405, both within the aminoacyl site (A site) of the 16S rRNA (7, 10). All six genes are confirmed to be or are likely to locate on plasmids (3, 4, 10, 11, 12, 14). Recent findings also indicate that some of these genes are capable of crossing the barrier between glucose-fermenting and nonfermenting species. For instance, armA has been identified in both members of the family Enterobacteriaceae and in Acinetobacter baumannii (5, 13), and rmtD has been identified in Klebsiella pneumoniae and Pseudomonas aeruginosa (unpublished data). We will likely see an increasing number of reports about this resistance mechanism, including identification of genes encoding new 16S rRNA methylases. Historically, the nomenclature of genes and enzymes for many resistance mechanisms has become complicated and nonsystematic (6). An extreme example is that of aminoglycoside acetyltransferases, where new gene names are arbitrarily assigned from one of the two coexisting nomenclature systems (9). The situation is somewhat better with β-lactamases and macrolide resistance genes, due to a registry and guidelines, respectively (http://www.lahey.org/Studies/) (8). To prevent confusion over the nomenclature of 16S rRNA methylases, we would like to propose practical rules for the nomenclature of these enzymes, which shall apply to any relevant enzymes to be identified in the future. Currently, the highest and lowest identities of amino acid sequences among the G1405 16S rRNA methylases are 81.7% between RmtA and RmtB and 25.8% between ArmA and RmtD, respectively (2, 3). On the other hand, identities lower than 10% are observed between the G1405 16S rRNA methylases and the NpmA that methylates A1408 (10) (Table ​(Table).). Thus, we propose the following rules. A gene that has an amino acid identity greater than 95% with the closest known 16S rRNA methylase gene will be assigned a variant number starting from two in the order of the dates on which the sequences are deposited in the GenBank/EBML/DDBJ, e.g., rmtA2 and then rmtA3, analogous to the nomenclature of the qnr genes. A gene that has between 50 and 95% amino acid identity with the closest known 16S rRNA methylase gene will be assigned a new alphabet letter according to the closest existing gene name, e.g., rmtE, rmtF, armB, or armC, provided that the gene is shown to confer a consistent aminoglycoside resistance profile. A gene that has either an amino acid identity of less than 50% with the closest known 16S rRNA methylase gene or that is proven to methylate a new residue of 16S rRNA may be assigned a brand new gene name, like npmA, contingent upon demonstration of 16S rRNA methylation activity of the gene product and attributable resistant phenotype. Data regarding 16S rRNA methylase genes in pathogenic bacteria will be accumulated and provided at the following website http://www.nih.go.jp/niid/16s_database/index.html. TABLE 1. Identity of amino acid residues among the sequences of plasmid-mediated 16S rRNA methylases

New polymorphic mtDNA restriction site in the 12S rRNA gene detected in Tunisian patients with non-syndromic hearing loss

The 12S rRNA gene was shown to be a hot spot for aminoglycoside-induced and non-syndromic hearing loss since several deafness-associated mtDNA mutations were identified in this gene. Among them, we distinguished the A1555G, the C1494T and the T1095C mutations and C-insertion or deletion at position 961. One hundred Tunisian patients with non-syndromic hearing loss and 100 hearing individuals were analysed in this study. A PCR-RFLP analysis with HaeIII restriction enzyme showed the presence of the A1555G mutation in the 12S rRNA gene in only one out of the 100 patients. In addition, PCR-RFLP and radioactive PCR revealed the presence of a new HaeIII polymorphic restriction site in the same gene of 12S rRNA site in 4 patients with non-syndromic hearing loss. UVIDOC-008-XD analyses showed the presence of this new polymorphic restriction site with a variable heteroplasmic rates at position +1517 of the human mitochondrial genome. On the other hand, direct sequencing of the entire mitochondrial 12S rRNA gene in the 100 patients and in 100 hearing individuals revealed the presence of the A750G and A1438G polymorphisms and the absence of the C1494T, T1095C and 961insC mutations in all the tested individuals. Sequencing of the whole mitochondrial genome in the 4 patients showing the new HaeIII polymorphic restriction site revealed only the presence of the A8860G transition in the MT-ATP6 gene and the A4769G polymorphism in the ND2 gene.