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Malaza fastuosus is a lavishly patterned skipper butterfly from a genus that has three described species, all endemic to the mainland of Madagascar. To our knowledge, M. fastuosus has not been collected for nearly 50 years. To evaluate the power of our techniques to recover DNA, we used a single foreleg of an at least 140-year-old holotype specimen from the collection of the Natural History Museum London with no destruction of external morphology to extract DNA and assemble a complete mitogenome from next generation sequencing reads. The resulting 15 540 bp mitogenome contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and an A+T rich region, similarly to other Lepidoptera mitogenomes. Here we provide the first mitogenome also for Trapezitinae (Rachelia extrusus). Phylogenetic analysis of available skipper mitogenomes places Malaza outside of Trapezitinae and Barcinae + Hesperiinae, with a possible sister relationship to Heteropterinae. Of these, at least Heteropterinae, Trapezitinae, and almost all Hesperiinae have monocot-feeding caterpillars. Malaza appears to be an evolutionarily highly distinct ancient lineage, morphologically with several unusual hesperiid features. The monotypic subfamily Malazinae Lees & Grishin subfam. nov. (type genus Malaza) is proposed to reflect this morphological and molecular evidence. Key words: next-generation sequencing, phylogeny, Madagascar, ancient DNA, Paul Mabille. Resume : Le Malaza fastuosus est une espece de papillon aux motifs tres colores qui appartient a un genre qui compte trois especes decrites, toutes indigenes de Madagascar. Au meilleur de la connaissance des auteurs, aucun specimen du M. fastuosus n'a ete collecte depuis pres de 50 ans. Pour evaluer la puissance des techniques d'extraction d'ADN, les auteurs ont employe une seule patte avant sur un specimen holotype vieux d'au moins 140 ans, conserve au sein de la collection du Natural History Museum London et qui ne presentait aucun signe morphologique de dommage exterieur, pour extraire de l'ADN et assembler un mitogenome complet a l'aide de sequencage de seconde generation. Le mitogenome obtenu compte 15 540 pb et contient 13 genes codant pour des proteines, 22 genes d'ARN de transfert, deux genes codant pour des ARN ribosomiques et une region riche enA+T, tout comme les autres mitogenomes de lepidopteres. Les auteurs rapportent egalement le premier mitogenome au sein des Trapezitinae (Rachelia extrusus). Une analyse phylogenetique parmi les autres mitogenomes d'hesperides place le genre Malaza a l'exterieur des Trapezitinae et Barcinae + Hesperiinae, mais avec une possible relation proche avec les Heteropterinae. Parmi celles-ci, les Heteropterinae, les Trapezitinae et presque tous les Hesperiinae presentent des chenilles qui se nourrissent de monocotyledones. Le genre Malaza semble former un groupe distinct et ancien sur le plan evolutif, mais qui presente plusieurs caracteristiques morphologiques inhabituelles presentes chez les Hesperiidae. Une sous-famille monotypique, Malazinae Lees & Grishin (genre type Malaza) est propose pour refleter les evidences morphologiques et moleculaires. [Traduit par la Redaction] Mots-cles : sequencage de seconde generation, phylogenie, Madagascar, ADN ancient, Paul Mabille., Introduction Its name translated from Malagasy as 'famous', Malaza Mabille, 1904 is probably the most unusual skipper genus from Madagascar. Three Malagasy endemic species of Malaza have been described (Lees [...]

Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly inherited diseases--where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function allele--is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapy for Charcot-Marie-Tooth disease type 2D (CMT2D), caused by dominant mutations in glycyl-tRNA synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks of age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild- type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least 1 year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof of concept for gene therapy approaches for dominant neuromuscular diseases., Introduction Personalized medicine seeks to provide optimized treatments for individuals based on the molecular characteristics of their specific disease. For example, cancer treatments are customized to the genetics of the [...]

Background Lentibacillus species are gram variable aerobic bacteria that live primarily in halophilic environments. Previous reports have shown that bacteria belonging to this species are primarily isolated from salty environments or food. We isolated a bacterial strain CBA3610, identified as a novel species of the genus Lentibacillus, from a human fecal sample. In this report, the whole genome sequence of Lentibacillus sp. CBA3610 is presented, and genomic analyses are performed. Results Complete genome sequence of strain CBA3610 was obtained through PacBio RSII and Illumina HiSeq platforms. The size of genome is 4,035,571 bp and genes estimated to be 4714 coding DNA sequences and 64 tRNA and 17 rRNA were identified. The phylogenetic analysis confirmed that it belongs to the genus Lentibacillus. In addition, there were genes related to antibiotic resistance and virulence, and genes predicted as CRISPR and prophage were also identified. Genes related to osmotic stress were found according to the characteristics of halophilic bacterium. Genomic differences from other Lentibacillus species were also confirmed through comparative genomic analysis. Conclusions Strain CBA3610 is predicted to be a novel candidate species of Lentibacillus through phylogenetic analysis and comparative genomic analysis with other species in the same genus. This strain has antibiotic resistance gene and pathogenic genes. In future, the information derived from the results of several genomic analyses of this strain is thought to be helpful in identifying the relationship between halophilic bacteria and human gut microbiota. Keywords: Lentibacillus sp. CBA3610, Complete genome sequence, Gut microbiota, Halophile, Author(s): Seung Woo Ahn[sup.1], Se Hee Lee[sup.1], Hong-Seok Son[sup.2], Seong Woon Roh[sup.1] and Yoon-E Choi[sup.3] Background Lentibacillus is a gram-variable, aerobic or facultatively anaerobic, and halophilic bacterial genus of the [...]

Background Enterobacter cloacae complex (ECC) bacteria, such as E. cloacae, E. sichuanensis, E. kobei, and E. roggenkampii, have been emerging as nosocomial pathogens. Many strains isolated from medical clinics were found to be resistant to antibiotics, and in the worst cases, acquired multidrug resistance. We present the whole genome sequence of SGAir0282, isolated from the outdoor air in Singapore, and its relevance to other ECC bacteria by in silico genomic analysis. Results Complete genome assembly of E. sichuanensis strain SGAir0282 was generated using PacBio RSII and Illumina MiSeq platforms, and the datasets were used for de novo assembly using Hierarchical Genome Assembly Process (HGAP) and error corrected with Pilon. The genome assembly consisted of a single contig of 4.71 Mb and with a G+C content of 55.5%. No plasmid was detected in the assembly. The genome contained 4371 coding genes, 83 tRNA and 25 rRNA genes, as predicted by NCBI's Prokaryotic Genome Annotation Pipeline (PGAP). Among the genes, the antibiotic resistance related genes were included: Streptothricin acetdyltransferase (SatA), fosfomycin resistance protein (FosA) and metal-dependent hydrolases of the beta-lactamase superfamily I (BLI). Conclusion Based on whole genome alignment and phylogenetic analysis, the strain SGAir0282 was identified to be Enterobacter sichuanensis. The strain possesses gene clusters for virulence, disease and defence, that can also be found in other multidrug resistant ECC type strains. Keywords: Airborne bacteria, Enterobacter cloacae complex, Whole genome sequencing, Multidrug resistant, Author(s): Akira Uchida[sup.1], Hie Lim Kim[sup.1,2], Rikky W. Purbojati[sup.1], Vineeth Kodengil Vettath[sup.1], Anjali B. Gupta[sup.1], James N. I. Houghton[sup.1], Caroline Chénard[sup.1], Anthony Wong[sup.1], Megan E. Clare[sup.1], Kavita K. Kushwaha[sup.1], Deepa [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tins.2009.11.001 Byline: William W. Motley (1)(2), Kevin Talbot (2)(3), Kenneth H. Fischbeck (1) Abstract: Charcot-Marie-Tooth disease type 2D, a hereditary axonal neuropathy, is caused by mutations in glycyl-tRNA synthetase (GARS). The mutations are distributed throughout the protein in multiple functional domains. In biochemical and cell culture experiments, some mutant forms of GARS have been indistinguishable from wild-type protein, suggesting that these in vitro tests might not adequately assess the aberrant activity responsible for axonal degeneration. Recently, mouse and fly models have offered new insights into the disease mechanism. There are still gaps in our understanding of how mutations in a ubiquitously expressed component of the translation machinery result in axonal neuropathy. Here, we review recent reports, weigh the evidence for and against possible mechanisms and suggest areas of focus for future work. Author Affiliation: (1) Neurogenetics Branch, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), 35 Convent Drive, Bethesda, MD 20892-3705, USA (2) MRC Functional Genomics Unit, University of Oxford, Oxford, UK (3) Department of Clinical Neurology, University of Oxford, John Radcliffe Hospital, Oxford, UK

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ajhg.2009.04.013 Byline: Luisa Pereira (1)(2), Fernando Freitas (1), Veronica Fernandes (1), Joana B. Pereira (1), Marta D. Costa (1), Stephanie Costa (1), Valdemar Maximo (1)(2), Vincent Macaulay (3), Ricardo Rocha (4), David C. Samuels (5) Abstract: We analyzed the current status (as of the end of August 2008) of human mitochondrial genomes deposited in GenBank, amounting to 5140 complete or coding-region sequences, in order to present an overall picture of the diversity present in the mitochondrial DNA of the global human population. To perform this task, we developed mtDNA-GeneSyn, a computer tool that identifies and exhaustedly classifies the diversity present in large genetic data sets. The diversity observed in the 5140 human mitochondrial genomes was compared with all possible transitions and transversions from the standard human mitochondrial reference genome. This comparison showed that tRNA and rRNA secondary structures have a large effect in limiting the diversity of the human mitochondrial sequences, whereas for the protein-coding genes there is a bias toward less variation at the second codon positions. The analysis of the observed amino acid variations showed a tolerance of variations that convert between the amino acids V, I, A, M, and T. This defines a group of amino acids with similar chemical properties that can interconvert by a single transition. Author Affiliation: (1) Instituto de Patologia e Imunologia Molecular Universidade do Porto (IPATIMUP), Porto 4200-465, Portugal (2) Faculdade de Medicina da Universidade do Porto, Porto 4200-465, Portugal (3) Department of Statistics, University of Glasgow, Glasgow G12 8QQ, UK (4) Departamento de Ciencias de Computadores, Faculdade de Ciencias da Universidade do Porto, Porto 4169-007, Portugal (5) Center for Human Genetics Research, Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA Article History: Received 5 February 2009; Revised 10 April 2009; Accepted 20 April 2009 Article Note: (miscellaneous) Published online: May 7, 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.11.016 Byline: Florian Brandt (1)(2), Stephanie A. Etchells (2), Julio O. Ortiz (1), Adrian H. Elcock (4), F. Ulrich Hartl (2)(3), Wolfgang Baumeister (1)(3) Keywords: PROTEINS; RNA Abstract: Recent advances have led to insights into the structure of the bacterial ribosome, but little is known about the 3D organization of ribosomes in the context of translating polysomes. We employed cryoelectron tomography and a template-matching approach to map 70S ribosomes in vitrified bacterial translation extracts and in lysates of active E. coli spheroplasts. In these preparations, polysomal arrangements were observed in which neighboring ribosomes are densely packed and exhibit preferred orientations. Analysis of characteristic examples of polysomes reveals a staggered or pseudohelical organization of ribosomes along the mRNA trace, with the transcript being sequestered on the inside, the tRNA entrance sites being accessible, and the polypeptide exit sites facing the cytosol. Modeling of elongating nascent polypeptide chains suggests that this arrangement maximizes the distance between nascent chains on adjacent ribosomes, thereby reducing the probability of intermolecular interactions that would give rise to aggregation and limit productive folding. Author Affiliation: (1) Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, Martinsried 82152, Germany (2) Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, Martinsried 82152, Germany (3) Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universitat Munchen, Butenandtstr. 5-13, Munchen 81377, Germany (4) Department of Biochemistry, University of Iowa, 4-530 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242, USA Article History: Received 24 July 2008; Revised 12 October 2008; Accepted 13 November 2008 Article Note: (miscellaneous) Published: January 22, 2009

CCA-adding enzymes are specialized polymerases that add a specific sequence (C-C-A) to tRNA 3' ends without requiring a nucleic acid template. In some organisms, CCA synthesis is accomplished by the collaboration of evolutionary closely related enzymes with partial activities (CC and A addition). These enzymes carry all known motifs of the catalytic core found in CCA-adding enzymes. Therefore, it is a mystery why these polymerases are restricted in their activity and do not synthesize a complete CCA terminus. Here, a region located outside of the conserved motifs was identified that is missing in CC-adding enzymes. When recombinantly introduced from a CCA-adding enzyme, the region restores full CCA-adding activity in the resulting chimera. Correspondingly, deleting the region in a CCA-adding enzyme abolishes the A-incorporating activity, also leading to CC addition. The presence of the deletion was used to predict the CC-adding activity of putative bacterial tRNA nucleotidyltransferases. Indeed, two such enzymes were experimentally identified as CC-adding enzymes, indicating that the existence of the deletion is a hallmark for this activity. Furthermore, phylogenetic analysis of identified and putative CCadding enzymes indicates that this type of tRNA nucleotidyltransferases emerged several times during evolution. Obviously, these enzymes descend from CCA-adding enzymes, where the occurrence of the deletion led to the restricted activity of CC addition. A-adding enzymes, however, seem to represent a monophyletic group that might also be ancestral to CCA-adding enzymes. Yet, experimental data indicate that it is possible that A-adding activities also evolved from CCA-adding enzymes by the occurrence of individual point mutations. tRNA maturation | CCA-adding enzyme | flexible loop

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2006.08.005 Byline: Alan G. Hinnebusch Abstract: Translation initiation in eukaryotes depends on many eukaryotic initiation factors (eIFs) that stimulate both recruitment of the initiator tRNA, Met-tRNA.sub.i.sup.Met, and mRNA to the 40S ribosomal subunit and subsequent scanning of the mRNA for the AUG start codon. The largest of these initiation factors, the eIF3 complex, organizes a web of interactions among several eIFs that assemble on the 40S subunit and participate in the different reactions involved in translation. Structural analysis suggests that eIF3 performs this scaffolding function by binding to the 40S subunit on its solvent-exposed surface rather than on its interface with the 60S subunit, where the decoding sites exist. This location of eIF3 seems ideally suited for its other proposed regulatory functions, including reinitiating translation on polycistronic mRNAs and acting as a receptor for protein kinases that control protein synthesis. Author Affiliation: Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.08.032 Byline: Andrei Korostelev (1), Sergei Trakhanov (1), Martin Laurberg (1), Harry F. Noller (1) Abstract: Our understanding of the mechanism of protein synthesis has undergone rapid progress in recent years as a result of low-resolution X-ray and cryo-EM structures of ribosome functional complexes and high-resolution structures of ribosomal subunits and vacant ribosomes. Here, we present the crystal structure of the Thermus thermophilus 70S ribosome containing a model mRNA and two tRNAs at 3.7 A resolution. Many structural details of the interactions between the ribosome, tRNA, and mRNA in the P and E sites and the ways in which tRNA structure is distorted by its interactions with the ribosome are seen. Differences between the conformations of vacant and tRNA-bound 70S ribosomes suggest an induced fit of the ribosome structure in response to tRNA binding, including significant changes in the peptidyl-transferase catalytic site. Author Affiliation: (1) Center for Molecular Biology of RNA and Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA Article History: Received 13 July 2006; Revised 10 August 2006; Accepted 30 August 2006 Article Note: (miscellaneous) Published online: September 7, 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.04.028 Byline: Ken-ichi Noma (1), Hugh P. Cam (1), Richard J. Maraia (2), Shiv I.S. Grewal (1) Abstract: Eukaryotic genome complexity necessitates boundary and insulator elements to partition genomic content into distinct domains. We show that inverted repeat (IR) boundary elements flanking the fission yeast mating-type heterochromatin domain contain B-box sequences, which prevent heterochromatin from spreading into neighboring euchromatic regions by recruiting transcription factor TFIIIC complex without RNA polymerase III (Pol III). Genome-wide analysis reveals TFIIIC with Pol III at all tRNA genes, many of which cluster at pericentromeric heterochromatin domain boundaries. However, a single tRNA.sup.phe gene with modest TFIIIC enrichment is insufficient to serve as boundary and requires RNAi-associated element to restrain heterochromatin spreading. Remarkably, we found TFIIIC localization without Pol III at many sites located between divergent promoters. These sites appear to act as chromosome-organizing clamps by tethering distant loci to the nuclear periphery, at which TFIIIC is concentrated into several distinct bodies. Our analyses uncover a general genome organization mechanism involving conserved TFIIIC complex. Author Affiliation: (1) Laboratory of Molecular Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA (2) Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA Article History: Received 28 November 2005; Revised 27 February 2006; Accepted 3 April 2006 Article Note: (miscellaneous) Published: June 1, 2006

Results show that the ribosome recycling factor binds to the ribosome in a different orientation than the conventional type exhibited by tRNA, indicating that its mechanism of action is not a mimicry of tRNA.

The methanogenic archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus contain a dual-specificity prolyl-tRNA synthetase (ProCysRS) that accurately forms both prolyl-tRNA (Pro-tRNA) and cysteinyl-tRNA (Cys-tRNA) suitable for in vivo translation. This intriguing enzyme may even perform its dual role in organisms that possess a canonical single-specificity cysteinyl-tRNA synthetase (CysRS), raising the question as to whether this latter aminoacyl-tRNA synthetase is indeed required for cell viability. To test the postulate that all synthetase genes are essential, we disrupted the cysS gene (encoding CysRS) of Methanococcus maripaludis. The knockout strain was viable under normal growth conditions. Biochemical analysis showed that the pure M. maripaludis ProCysRS was capable of forming Cys-tRNA, implying that the dual-specificity enzyme compensates in vivo for the loss of CysRS. The canonical CysRS has a higher affinity for cysteine than ProCysRS, a reason why M. maripaludis may have acquired cysS by a late lateral gene transfer. These data challenge the notion that all twenty aminoacyl-tRNA synthetases are essential for the viability of a cell.

A general approach to site-specific insertion of amino acid analogues into proteins in vivo would be the import into cells of a suppressor tRNA aminoacylated with the analogue of choice. The analogue would be inserted at any site in the protein specified by a stop codon in the mRNA. The only requirement is that the suppressor tRNA must not be a substrate for any of the cellular aminoacyl-tRNA synthetases. Here, we describe conditions for the import of amber and ochre suppressor tRNAs derived from Escherichia coli initiator tRNA into mammalian COS1 cells, and we present evidence for their activity in the specific suppression of amber (UAG) and ochre (UAA) codons, respectively. We show that an aminoacylated amber suppressor tRNA (supF) derived from the E. coli tyrosine tRNA can be imported into COS1 cells and acts as a suppressor of amber codons, whereas the same suppressor tRNA imported without prior aminoacylation does not, suggesting that the supF tRNA is not a substrate for any mammalian aminoacyl-tRNA synthetase. These results open the possibility of using the supF tRNA aminoacylated with an amino acid analogue as a general approach for the site-specific insertion of amino acid analogues into proteins in mammalian cells. We discuss the possibility further of importing a mixture of amber and ochre suppressor tRNAs for the insertion of two different amino acid analogues into a protein and the potential use of suppressor tRNA import for treatment of some of the human genetic diseases caused by nonsense mutations.

Research has been conducted on the structure of the specificity domain of the Bacillus subtilis RNase P RNA. The experiments have suggested that this structure is composed of a core, a functional unit of this domain, and a buttress module which provides the structural stability to the core module.

Results indicate that structure of the human tRNA(sup)Lys3UUU anticodon stem coupled with the modified amino acid t(sup)6A(sub)37 facilitates ribosome binding. Data indicate that the modified nucleoside influences anticodon loop conformation, which is mediated by t(sup)6A.

Results point out that it is critical for human tRNA(sup)LysUUU species to undergo anticodon domain modification for its effective binding to AAA-programmed ribosomes and wobbling to AAG codons.

The mitochondrial DNA (mtDNA) control region and flanking tRNAs were sequenced from 76 mice collected at 60 localities extending from Egypt through Turkey, Yemen, Iran, Afghanistan, Pakistan, and Nepal to eastern Asia. Segments of the Y chromosome and of a processed p53 pseudogene ([Psi]p53) were amplified from many of these mice and from others collected elsewhere in Eurasia and North Africa. The 251 mtDNA types, including 54 new ones reported here, now identified from commensal house mice (Mus musculus group) by sequencing this segment can be organized into four major lineages - domesticus, musculus, castaneus, and a new lineage found in Yemen. Evolutionary tree analysis suggested the domesticus mtDNAs as the sister group to the other three commensal mtDNA lineages and the Yemeni mtDNAs as the next oldest lineage. Using this tree and the phylogeographic approach, we derived a new model for the origin and radiation of commensal house mice whose main features are an origin in west-central Asia (within the present-day range of M. domesticus) and the sequential spreading of mice first to the southern Arabian Peninsula, thence eastward and northward into south-central Asia, and later from south-central Asia to north-central Asia (and thence into most of northern Eurasia) and to southeastern Asia. Y chromosomes with and without an 18-bp deletion in the Zfy-2 gene were detected among mice from Iran and Afghanistan, while only undeleted Ys were found in Turkey, Yemen, Pakistan, and Nepal. Polymorphism for the presence of a [Psi]p53 was observed in Georgia, Iran, Turkmenistan, Afghanistan, and Pakistan. Sequencing of a 128-bp [Psi]p53 segment from 79 commensal mice revealed 12 variable sites and implicated [greater than or equal to]14 alleles. The allele that appeared to be phylogenetically ancestral was widespread, and the greatest diversity was observed in Turkey, Afghanistan, Pakistan, and Nepal. Two mice provided evidence for a second [Psi]p53 locus in some commensal populations.