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Highlights • Synanthropic rodents and shrews shed atypical EPEC (aEPEC), but not EHEC strains. • aEPEC strains of diverse origin can be lysogenized with a stx -depleted bacteriophage yielding stable, yet prophage-inducible lysogens. • Sequence comparison of two highly related murine and human aEPEC strains and their respective lysogens showed sequence identities of >90%. • Whole genome analysis revealed prophage integration into the highly variable tRNA integration site argW in all three lysogens. • aEPEC are progenitors of typical EHEC. Abstract Bacteriophages play an important role in the evolution of bacterial pathogens. A phage-mediated transfer of stx -genes to atypical enteropathogenic E. coli (aEPEC) which are prevalent in different hosts, would convert them to enterohemorrhagic E. coli (EHEC). We decided to confirm this hypothesis experimentally to provide conclusive evidence that aEPEC isolated from different mammalian hosts are indeed progenitors of typical EHEC which gain the ability to produce Shiga-Toxin by lysogeny with stx -converting bacteriophages, utilizing the model phage Φ3538 Δ stx 2 ::cat. We applied a modified in vitro plaque-assay, using a high titer of a bacteriophage carrying a deletion in the stx 2 gene (Φ3538 Δ stx 2 ::cat) to increase the detection of lysogenic conversion events. Three wild-type aEPEC strains were chosen as acceptor strains: the murine aEPEC-strain IMT14505 (sequence type (ST)28, serotype Ont:H6), isolated from a striped field mouse (Apodemus agrarius) in the surrounding of a cattle shed, and the human aEPEC-strain 910#00 (ST28, Ont:H6). The close genomic relationship of both strains implies a high zoonotic potential. A third strain, the bovine aEPEC IMT19981, was of serotype O26:H11 and ST21 (STC29). All three aEPEC were successfully lysogenized with phage Φ3538 Δ stx 2 ::cat. Integration of the bacteriophage DNA into the aEPEC host genomes was confirmed by amplification of chloramphenicol transferase (cat) marker gene and by Southern-Blot hybridization. Analysis of the whole genome sequence of each of the three lysogens showed that the bacteriophage was integrated into the known tRNA integration site argW , which is highly variable among E. coli. In conclusion, the successful lysogenic conversion of aEPEC with a stx -phage in vitro underlines the important role of aEPEC as progenitors of EHEC. Given the high prevalence and the wide host range of aEPEC acceptors, their high risk of zoonotic transmission should be recognized in infection control measures. [ABSTRACT FROM AUTHOR]

Coccidioidomycosis is a systemic fungal infection for which a vaccine has been sought for over fifty years. The avirulent Coccidioides posadasii strain, Δcps1 , which is missing a 6 kb gene, showed significant protection in mice. These studies explore conditions of protection in mice and elucidate the immune response. Mice were vaccinated with different doses and viability states of Δcps1 spores, challenged with virulent C. posadasii , and sacrificed at various endpoints, dependent on experimental objectives. Tissues from vaccinated mice were harvested for in vitro elucidation of immune response. Vaccination with viable Δcps1 spores was required for protection from lethal challenge. Viable spore vaccination produced durable immunity, lasting at least 6 months, and prolonged survival (≥6 months). The C. posadasii vaccine strain also protected mice against C. immitis (survival ≥ 6 months). Cytokines from infected lungs of vaccinated mice in the first four days after Cp challenge showed significant increases of IFN-γ, as did stimulated CD4 + spleen cells from vaccinated mice. Transfer of CD4 + cells, but not CD8 + or B cells, reduced fungal burdens following challenge. IFN-γ from CD4 + cells in vaccinated mice indicates a Th1 response, which is critical for host control of coccidioidomycosis. [ABSTRACT FROM AUTHOR]

In the present study, we sequenced the entire mitochondrial genome (mitogenome) of Ectropis oblique using PCR amplification and sequencing methods The entire mitogenome is 15,356 bp long, including 13 protein coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and A + T rich element. The base composition and gene arrangement are identical to those of other lepidopterans. Of these 37 genes, twenty three are resided on heavy strand, while fourteen located on light strand. The newly sequenced mitogenome displayed a biased Adenine/thymine 84.94% usage versus guanine/cytosine. The AT skewness is 0.030 and the GC skewness is −0.180. Twelve out of 13 PCGs initiated with canonical start codon (ATN), while cox1 started with CGA. The A + T rich element of E. oblique is 363 bp long and contains of many features common to lepidopteran insects, including the ‘ATAGA’ motif, a 22 bp poly (T) stretch and a microsatellite-like (AT) 8 element upstream of trnM . Further, this mitogenome comprises 15 intergenic spacer and overlapping regions. Phylogenetic analyses showed that E. oblique belongs to Geometridae, and that the monophyly of Lepidoptera superfamilies is well supported. [ABSTRACT FROM AUTHOR]

Eleusine indica , also known as goosegrass, is a serious weed in at least 42 countries. In this paper we report the complete plastid genome sequence of goosegrass obtained by de novo assembly of paired-end and mate-paired reads generated by Illumina sequencing of total genomic DNA. The goosegrass plastome is a circular molecule of 135,151 bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 20,919 bases. The large (LSC) and the small (SSC) single-copy regions span 80,667 bases and 12,646 bases, respectively. The plastome of goosegrass has 38.19% GC content and includes 108 unique genes, of which 76 are protein-coding, 28 are transfer RNA, and 4 are ribosomal RNA. The goosegrass plastome sequence was compared to eight other species of Poaceae. Although generally conserved with respect to Poaceae, this genomic resource will be useful for evolutionary studies within this weed species and the genus Eleusine . [ABSTRACT FROM AUTHOR]

Elucidating the genomic features influencing prokaryotic growth rates has always been a study of interest. Previously, it was observed that overlapping genes (OGs) play a crucial role in the prokaryotic genome size reduction. This study is focused to explore whether OGs act as a potential correlate of prokaryotic growth rates. For this purpose, we compiled a dataset of 25 archaeal and 117 eubacterial genomes and analyzed the inter-correlation between the proportion of overlapping regions in these genomes with their growth rates. Here, we observed that the proportion of overlapping region holds a significant negative correlation with generation time in archaeal domain, whereas no correlation was observed in the eubacterial domain. However, after masking the effect of tRNA, rRNA multiplicity and environmental diversity, OGs show an independent effect over growth rates in the eubacterial domain as well as in the archaeal domain. Moreover, the influence of OGs on prokaryotic growth rates provides different delineations in archaeal and eubacterial domains. In archaea, both long overlap frequency (LOF) and short overlap frequency (SOF) influence the growth rates by increasing the degree of operonization. On the contrary, in the case of bacteria, neither SOF nor LOF plays any significant role in achieving faster growth rates. [ABSTRACT FROM AUTHOR]

Target identification is highly instructive in defining the biological roles of microRNAs. However, little is known about other small noncoding RNAs; for example, tRNA-derived RNA Fragments (tRFs). Some tRFs exhibit a gene-silencing mechanism distinctly different from that of typical microRNAs. We recently demonstrated that a respiratory syncytial virus (RSV)-induced tRF, called tRF5-GluCTC, promotes RSV replication. RSV is the single most important cause of lower respiratory tract infection in children. By using biochemical screening and bioinformatics analyses, we have identified apolipoprotein E receptor 2 (APOER2) as a target of tRF5-GluCTC. The 3′-portion of tRF5-GluCTC recognizes a target site in the 3′-untranslated region of APOER2 and suppresses its expression. We have also discovered that APOER2 is an anti-RSV protein whose suppression by tRF5-GluCTC promotes RSV replication. Our report represents the first identification of a natural target of a tRF and illustrates how a virus utilizes a host tRF to control a host gene to favor its replication. [ABSTRACT FROM AUTHOR]

Transcriptional regulation of ribosome and tRNA synthesis plays a central role in determining protein synthetic capacity and is tightly controlled in response to nutrient availability and cellular stress. In Saccharomyces cerevisiae, the regulation of ribosome and tRNA synthesis was recently shown to involve the Cdc-like kinase Kns1 and the GSK-3 kinase Mck1. In this study, we explored additional roles for these conserved kinases in processes connected to the target of rapamycin complex 1 (TORC1). We conducted a synthetic chemical-genetic screen in a kns1Δ mck1Δ strain and identified many novel rapamycin-hypersensitive genes. Gene ontology analysis showed enrichment for TORC1-regulated processes (vesicle-mediated transport, autophagy, and regulation of cell size) and identified new connections to protein complexes including the protein kinase CK2. CK2 is considered to be a constitutively active kinase and in budding yeast, the holoenzyme comprises two regulatory subunits, Ckb1 and Ckb2, and two catalytic subunits, Cka1 and Cka2. We show that Ckb1 is differentially phosphorylated in vivo and that Kns1 mediates this phosphorylation when nutrients are limiting and under all tested stress conditions. We determined that the phosphorylation of Ckb1 does not detectably affect the stability of the CK2 holoenzyme but correlates with the reduced occupancy of Ckb1 on tRNA genes after rapamycin treatment. Thus, the differential occupancy of tRNA genes by CK2 is likely to modulate its activation of RNA polymerase III transcription. Our data suggest that TORC1, via its effector kinase Kns1, may regulate the association of CK2 with some of its substrates by phosphorylating Ckb1. [ABSTRACT FROM AUTHOR]

Small noncoding RNAs (sncRNAs) mediate a variety of cellular functions in animals and plants. Deep sequencing has made it possible to obtain highly detailed information on the types and abundance of sncRNAs in biological specimens, leading to the discovery that sncRNAs circulate in the blood of humans and mammals. The most abundant types of circulating sncRNAs are microRNAs (miRNAs), 5′ transfer RNA (tRNA) halves, and YRNA fragments, with minute amounts of other types that may nevertheless be significant. Of the more abundant circulating sncRNAs only miRNAs have well described functions, but characteristics of the others suggest specific processing and secretion as complexes that protect the RNA from degradation. The properties of circulating sncRNAs are consistent with their serving as signaling molecules, and investigations of circulating miRNAs support the view that they can enter cells and regulate cellular functions. The serum levels of specific sncRNAs change markedly with age, and these changes can be mitigated by calorie restriction (CR), indicating that levels are under physiologic control. The ability of circulating sncRNAs to transmit functions between cells and to regulate a broad spectrum of cellular functions, and the changes in their levels with age, implicate them in the manifestations of aging. Our understanding of the functions of circulating sncRNA, particularly in relation to aging, is currently at a very early stage; results to date suggest that more extensive investigation will yield important insights into mechanisms of aging. [ABSTRACT FROM AUTHOR]

In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459-469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae is a causative agent of nosocomial infections such as pneumonia, meningitis, and septicemia. Penicillin resistance in S. pneumoniae depends in part upon MurM, an aminoacyl-tRNA ligase that attaches L-serine or L-alanine to the stem peptide lysine of Lipid II in cell wall peptidoglycan. To investigate the exact substrates the translation machinery provides MurM, quality control by alanyl-tRNA synthetase (AlaRS) was investigated. AlaRS mischarged serine and glycine to tRNAAla, as observed in other bacteria, and also transferred ala-nine, serine, and glycine to tRNAPhe. S. pneumoniae tRNAPhe has an unusual U4:C69 mismatch in its acceptor stem that prevents editing by phenylalanyl-tRNA synthetase (PheRS), leading to the accumulation of misaminoacylated tRNAs that could serve as substrates for translation or for MurM. Although the peptidoglycan layer of S. pneumoniae tolerates a combination of both branched and linear muropeptides, deletion of MurM results in a reversion to penicillin sensitivity in strains that were previously resistant. However, because MurM is not required for cell viability, the reason for its functional conservation across all strains of S. pneumoniae has remained elusive. We now show that MurM can directly function in translation quality control by acting as a broad specificity lipid-independent trans editing factor that deacylates tRNA. This activity of MurM does not require the presence of its second substrate, Lipid II, and can functionally substitute for the activity of widely conserved editing domain homologues of AlaRS, termed AlaXPs proteins, which are themselves absent from S. pneumoniae. [ABSTRACT FROM AUTHOR]

Recognition strategies for tRNA aminoacylation are ancient and highly conserved, having been selected very early in the evolution of the genetic code. In most cases, the trinucleotide anticodons of tRNA are important identity determinants for aminoacylation by cognate aminoacyl-tRNA synthetases. However, a degree of ambiguity exists in the recognition of certain tRNAIle isoacceptors that are initially transcribed with the methioninespecifying CAU anticodon. In most organisms, the C34 wobble position in these tRNAIle precursors is rapidly modified to lysidine to prevent recognition by methionyl-tRNA synthetase (MRS) and production of a chimeric Met-tRNAIle that would compromise translational fidelity. In certain bacteria, however, lysidine modification is not required for MRS rejection, indicating that this recognition strategy is not universally conserved and may be relatively recent. To explore the actual distribution of lysidine-dependent tRNAIle rejection by MRS, we have investigated the ability of bacterial MRSs from different clades to differentiate cognate tRNACAU Met from near-cognate tRNACAU Ile. Discrimination abilities vary greatly and appear unrelated to phylogenetic or structural features of the enzymes or sequence determinants of the tRNA. Our data indicate that tRNAIle identity elements were established late and independently in different bacterial groups. We propose that the observed variation in MRS discrimination ability reflects differences in the evolution of genetic code machineries of emerging bacterial clades. [ABSTRACT FROM AUTHOR]

Abstract: To investigate the features of the control region (CR) and the gene rearrangement in the mitochondrial (mt) genome of Thysanoptera insects, we sequenced the whole mt genome of the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae). The mt genome is a circular molecule with 14,889 nucleotides and an A+T content of 76.6%, and it has triplicate putative CRs. We propose that tandem duplication and deletion account for the evolution of the CR and the gene translocations. Intramitochondrial recombination is a plausible model for the gene inversions. We discuss the excessive duplicate CR sequences and the transcription of the rRNA genes, which are distant from one another and from the CR. Finally, we address the significance of the complicated mt genomes in Thysanoptera for the evolution of the CR and the gene arrangement of the mt genome. [Copyright &y& Elsevier]

Abstract: Aspergillus is a genus of mold fungi that includes more than 200 described species. Many members of the group are relevant pathogens and other species are economically important. Only one species has been analyzed for codon usage, and this was performed with a small number of genes. In this paper, we report the codon usage patterns of eight completely sequenced genomes which belong to this genus. The results suggest that selection for translational efficiency and accuracy are the major factors shaping codon usage in all of the species studied so far, and therefore they were active in the last common ancestor of the group. Composition and molecular distances analyses show that highly expressed genes evolve slower at synonymous sites. We identified a conserved core of translationally optimal codons and study the tRNA gene pool in each genome. We found that the great majority of preferred triplets match the respective cognate tRNA with more copies in the respective genome. We discuss the possible scenarios that can explain the observed differences among the species analyzed. Finally we highlight the biotechnological application of this research regarding heterologous protein expression. [Copyright &y& Elsevier]

Abstract: Eukaryotic cells are characterized by their content of intracellular membrane-bound organelles, including mitochondria as well as nuclei. These two DNA-containing compartments employ two distinct strategies for storage and readout of genetic information. The diploid nuclei of human cells contain about 6billion base pairs encoding about 25,000 protein-encoding genes, averaging 120kB/gene, packaged in chromatin arranged as a regular nucleosomal array. In contrast, human cells contain hundreds to thousands of copies of a ca.16kB mtDNA genome tightly packed with 13 protein-coding genes along with rRNA and tRNA genes required for their expression. The mtDNAs are dispersed throughout the mitochondrial network as histone-free nucleoids containing single copies or small clusters of genomes. This review will summarize recent advances in understanding the microscopic structure and molecular composition of mtDNA nucleoids in higher eukaryotes. This article is part of a Special Issue entitled: Mitochondrial Gene Expression. [Copyright &y& Elsevier]

Clin Microbiol Infect 2012; 18: E235-E237 Abstract The results of Entamoeba histolytica infections range from asymptomatic colonization to variable disease outcomes. However, markers that may predict infection outcomes are not known. Here, we investigated sequence types of a non-coding tRNA-linked locus R-R to identify surrogate markers that may show association with infection outcomes. Among 112 clinical samples-21 asymptomatic, 20 diarrhoea/dysentery and 71 liver abscesses-we identified 11 sequence types. Sequence type 5RR was mostly associated with asymptomatic samples, and sequence type 10RR was predominantly associated with the symptomatic (diarrhoea/dysentery and liver abscess) samples. This is the first report that identifies markers that may predict disease outcomes in E. histolytica infection. [ABSTRACT FROM AUTHOR]

The article provides an overview of several topics related to gene and cell therapy. Topics discussed include the association of gene regulation with tRNA-derived RNA fragments (tRFs), the focus on mitochondrial DNA with transcription activator like effector nucleases, and the attribution of broadened T-cell responses to tumor's viral infection.

The article discusses a study by Charles W. Carter and colleagues at the University of North Carolina at Chapel Hill which argued that peptides and RNA cooperated to develop the genetic code. The study explored the RNA world hypothesis which proposes that RNA molecules evolved before proteins. The researchers also demonstrated that Urzymes, the molecules derived from aminoacyl-tRNA synthetases, accelerate tRNA aminoacylation several times compared to uncatalyzed peptide synthesis rate.