Inverted Elements Research Articles

Broadband dual planar inverted F-antenna for wireless local area networks/worldwide interoperability for microwave access and lower-band ultra wideband wireless applications

An extension of the planar inverted F-antenna (PIFA) concept is proposed, combining two such antennas (one of them fed parasitically) to extend the usable bandwidth to cover the WiFi wireless local area network, worldwide interoperability for microwave access and lower-band ultra wideband spectra. The broad impedance bandwidth is achieved by combining a driven slotted PIFA, having bandwidth-enhanced rectangular strip feed, with a parasitic inverted F-shaped element, hence describable as a dual PIFA. The geometry parameters of these two radiators are selected to enhance the impedance bandwidth and hence to encompass the entire required set of operating frequency bands. The overall optimised dimensions of the antenna are 30 mm 15 mm 8 mm, thus making it compatible with installation on portable wireless electronic devices. The results show that the proposed antenna can achieve a gain between 2.0 and 4.0 dBi across the entire impedance bandwidth 2.4 6.2 GHz (i.e. 88.4 relative bandwidth) for a reflection coefficient of | S 11 | <; 10 dB. The radiation patterns of the antenna show low-gain broad-beam coverage, as is essential for a wireless electronic device. Computed and experimental results are compared and are shown to be in satisfactory agreement.

Insertion of miniature subterminal inverted repeat-like elements in diapause-regulated genes in the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Determining the genomic structure of diapause-associated transcripts (DAT) -2 and -3 led to the isolation of four novel miniature subterminal inverted repeat-like elements (MSITE): Mild-1, -2, -3 and -4. Mild-1a is inserted within the first intron of dia- pause protein-1. Mild-1a is 284 bp in length, has a 14 bp target site duplication and three sets of subterminal inverted repeats. The second element, Mild-2a, is inserted within the 3' terminus of Mild-1a. Mild-2a is 29 bp long with a 3 bp target site duplication and one set of subterminal inverted repeats. Using primers based on Mild-1, genomic clones were developed leading to the isolation of Mild-3a. Mild-3a shares 60% identity with Mild-1a, is 253 bp long, has a 9 bp target site duplication and has one set of subterminal inverted repeats. Mild-4a is inserted within the first intron of DAT-2 and is 227 bp in length with a 12 bp target site duplication. Mild-4a appears to be an intermediate form between a miniature inverted repeat transposable element (MITE) and a MSITE because the 5' inverted repeat is terminal (i.e., adjacent to the target site duplication) as in MITEs, but the 3' inverted repeat is separated (in this case, by 33 bp) from the 3' target site duplication as in MSITEs. The target site duplications of Mild-1, -3 and -4 families share a common conserved core of AATTT. All of the transposable elements are AT rich and are able to form hairpin structures. Within the promoter region of DAT-3 is a 163 bp sequence (Mild-1b) that shares 77% identity to the 3' terminus of Mild-1a. Mild-4a has iden- tity to 25 and 53 bp regions within the promoter of the juvenile hormone esterase B gene. Southern blot analysis revealed the pres- ence of Mild-1 and -3 elements in both Leptinotarsa decemlineata and Leptinotarsa juncta indicating that these elements are ancestral to the L. decemlineata, L. juncta separation.

Activity of the Rhodopseudomonas palustris p-Coumaroyl-Homoserine Lactone-Responsive Transcription Factor RpaR

The Rhodopseudomonas palustris transcriptional regulator RpaR responds to the RpaI-synthesized quorum-sensing signal p-coumaroyl-homoserine lactone (pC-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the rpaI promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by pC-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His(6)-RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the rpaI transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although pC-HSL-RpaR bound to rpaI promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the rpaI operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is pC-HSL-RpaR-activated antisense transcription of rpaR. These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon.

Characterization of AhMITE1 transposition and its association with the mutational and evolutionary origin of botanical types in peanut (Arachis spp.)

AhMITE1 is an active miniature inverted repeat transposable element (MITE) in peanut (Arachis hypogaea L). Its transpositional activity from a particular (FST1-linked) site within the peanut genome was checked using AhMITE1-specifc PCR, which used a forward primer annealing to the 5′-flanking sequence and a reverse primer binding to AhMITE1. It was found that transposition activation was induced by stresses such as ethyl methane sulfonate (EMS), gamma irradiation, environmental conditions, and tissue culture. Excision and insertion of AhMITE1 at this particular site among the mutants led to gross morphological changes resembling alternate subspecies or botanical types. Analysis of South American landraces revealed the presence of AhMITE1 at the site among most of the spp. fastigiata types, whereas the element was predominantly missing from spp. hypogaea types, indicating its strong association. Four accessions of the primitive allotetraploid, A. monticola were devoid of AhMITE1 at the site, indicating only recent activation of the element, possibly because of the “genomic shock” resulting from hybridization followed by allopolyploidization.

A novel class of miniature inverted repeat transposable elements (MITEs) that contain hitchhiking (GTCY)n microsatellites

The movement of miniature inverted repeat transposable elements (MITEs) modifies genome structure and function. We describe the microsatellite-associated interspersed nuclear element 2 (MINE-2), that integrates at consensus WTTTT target sites, creates dinucleotide TT target site duplications (TSDs), and forms predicted MITE-like secondary structures; a 5' subterminal inverted repeat (SIR; AGGGTTCCGTAG) that is partially complementary to a 5' inverted repeat (IR; ACGAAGCCCT) and 3'-SIRs (TTACGGAACCCT). A (GTCY)(n) microsatellite is hitchhiking downstream of conserved 5'MINE-2 secondary structures, causing flanking sequence similarity amongst mobile microsatellite loci. Transfection of insect cell lines indicates that MITE-like secondary structures are sufficient to mediate genome integration, and provides insight into the transposition mechanism used by MINE-2s.

Analysis of SCAR marker nucleotide sequences in maize ( Zea mays L.) somaclones

SCAR (sequence characterized amplified region) markers allow the reliable identification of unique somaclonal variations. Six SCAR markers were developed previously and were thought to be exclusively characteristic of eight maize somaclones. However, we detected two of these markers in maize lines and a cultivar unrelated to the progenitor line of the somaclones. Therefore, we sequenced these markers and performed bioinformatic searches to understand the molecular events that may underlie the variability observed in the somaclones. All changes were found in noncoding sequences and were induced by different molecular events, such as the insertion of long terminal repeat (LTR) transposon(s), precise miniature inverted repeat transposable element (MITE) excision, microdeletion, recombination, and a change in the pool of mitochondrial DNA. For example, the SCAR marker QR is represented by the two variants QR-A and QR-2. The sequences of the two variants were similar, except for a 457-bp fragment found only in QR-A; this region was denoted as Q. Region Q was flanked by the direct 3-bp repeat 5′-TAA-3′ (target site duplication; TSD) and the inverted 14-bp repeat 5′-GGGCCTGTTTGGAA-3′ (terminal inverted repeats; TIRs). These features confer the Q region with similarity to the nonautonomic Tourist-like MITE. In two groups of independently produced somaclones, the same features (morphological, molecular) were variable, which confirms the theory of ‘hot spots’ occurring in the genome. The distribution of one of the SCAR markers was confirmed using Southern blot hybridization. The presence of the same molecular markers in the somaclones and in different non-somaclonal maize variants suggests that in some cases, the same mechanisms determine both in vitro and in vivo variability and that cell culture enhances the rate of heritable genomic changes that naturally occur in living organisms.

A Novel Mechanism Involving Coordinated Regulation of Nuclear Levels and Acetylation of NF-YA and Bcl6 Activates RGS4 Transcription

Neuronally enriched RGS4 plays a critical role attenuating G protein signaling in brain, although the mechanisms regulating RGS4 expression are unknown. Here we describe a novel mechanism for transcriptional activation of RGS4 in neuron-like PC6 cells, where RGS4 is markedly induced during confluence-induced growth arrest. Transcriptional activation of RGS4 in confluent PC6 cells was accompanied by impaired G(i/o)-dependent MAPK activation. In the human RGS4 gene promoter, we identified three phylogenetically conserved cis-elements: an inverted CCAAT box element (ICE), a cAMP response element, and a B-cell lymphoma 6 (Bcl6)-binding site. The ICE and the cAMP response element mediate activation, and the Bcl6 site mediates repression of RGS4 transcription. Activation of RGS4 transcription in confluent PC6 cells is accompanied by increases in NF-YA and C/EBPβ and decreases in Bcl6 levels in the nucleus. Increases in NF-YA and C/EBPβ lead to their increased binding to the RGS4 promoter in vivo, and dominant negative forms of these proteins repressed RGS4 promoter activity. Acetylation of NF-YA and Bcl6 were increased in postconfluent cells. Trichostatin A stimulation of RGS4 promoter activity, accompanied by increased binding of NF-YA and decreased binding of Bcl6 to the promoter, was abolished by mutation of the ICE and enhanced by mutation of the Bcl6 site. These findings demonstrate a dynamic and coordinated regulation of nuclear levels and acetylation status of trans-acting factors critical in determining the off/on state of the RGS4 promoter.

Small RNAs, DNA methylation and transposable elements in wheat

BackgroundMore than 80% of the wheat genome is composed of transposable elements (TEs). Since active TEs can move to different locations and potentially impose a significant mutational load, their expression is suppressed in the genome via small non-coding RNAs (sRNAs). sRNAs guide silencing of TEs at the transcriptional (mainly 24-nt sRNAs) and post-transcriptional (mainly 21-nt sRNAs) levels. In this study, we report the distribution of these two types of sRNAs among the different classes of wheat TEs, the regions targeted within the TEs, and their impact on the methylation patterns of the targeted regions.ResultsWe constructed an sRNA library from hexaploid wheat and developed a database that included our library and three other publicly available sRNA libraries from wheat. For five completely-sequenced wheat BAC contigs, most perfectly matching sRNAs represented TE sequences, suggesting that a large fraction of the wheat sRNAs originated from TEs. An analysis of all wheat TEs present in the Triticeae Repeat Sequence database showed that sRNA abundance was correlated with the estimated number of TEs within each class. Most of the sRNAs perfectly matching miniature inverted repeat transposable elements (MITEs) belonged to the 21-nt class and were mainly targeted to the terminal inverted repeats (TIRs). In contrast, most of the sRNAs matching class I and class II TEs belonged to the 24-nt class and were mainly targeted to the long terminal repeats (LTRs) in the class I TEs and to the terminal repeats in CACTA transposons. An analysis of the mutation frequency in potentially methylated sites revealed a three-fold increase in TE mutation frequency relative to intron and untranslated genic regions. This increase is consistent with wheat TEs being preferentially methylated, likely by sRNA targeting.ConclusionsOur study examines the wheat epigenome in relation to known TEs. sRNA-directed transcriptional and post-transcriptional silencing plays important roles in the short-term suppression of TEs in the wheat genome, whereas DNA methylation and increased mutation rates may provide a long-term mechanism to inactivate TEs.

Transcript expression and regulatory characteristics of a rice glycosyltransferase OsGT61-1 gene

Glycosyltransferases (GTs) catalyze the reaction of glycosylation of various cellular macromolecules. Rice genome contains 609 potential GT genes which have been grouped into 40 gene families according to CAZy (Carbohydrate Active Enzyme) database. Phylogenetic tree showed that the members of GT61 family are related across genera. Close relationship among various rice GT61 proteins was also reflected from MEME analysis. Full-length cDNA corresponding to MSU rice locus Os02g22380 and encoding for xylosyltransferase was obtained (referred to as OsGT61-1). OsGT61-1 transcript was noted to be ABA- and NaCl-responsive and its expression was more prominent in root than in shoot tissues. Experiments involving fusion of OsGT61-1 promoter (1410 bp upstream to ATG) with GUS reporter gene showed that GUS expression was in consonance with the transcript expression. 1410 bp OsGT61-1 promoter contains two miniature inverted repeat transposable element sequences namely Gaijin (144 bp) and Wanderer (211 bp). Using yeast as trans-host, we noted that these sequences negatively influence the downstream transcript expression.

Transposition of the Tourist-MITE mPing in yeast: an assay that retains key features of catalysis by the class 2 PIF/Harbinger superfamily

BackgroundPIF/Harbinger is the most recently discovered DNA transposon superfamily and is now known to populate genomes from fungi to plants to animals. Mobilization of superfamily members requires two separate element-encoded proteins (ORF1 and TPase). Members of this superfamily also mobilize Tourist-like miniature inverted repeat transposable elements (MITEs), which are the most abundant transposable elements associated with the genes of plants, especially the cereal grasses. The phylogenetic analysis of many plant genomes indicates that MITEs can amplify rapidly from one or a few elements to hundreds or thousands.The most active DNA transposon identified to date in plants or animals is mPing, a rice Tourist-like MITE that is a deletion derivative of the autonomous Ping element. Ping and the closely related Pong are the only known naturally active PIF/Harbinger elements. Some rice strains accumulate ~40 new mPing insertions per plant per generation. In this study we report the development of a yeast transposition assay as a first step in deciphering the mechanism underlying the amplification of Tourist-MITEs.ResultsThe ORF1 and TPase proteins encoded by Ping and Pong have been shown to mobilize mPing in rice and in transgenic Arabidopsis. Initial tests of the native proteins in a yeast assay resulted in very low transposition. Significantly higher activities were obtained by mutation of a putative nuclear export signal (NES) in the TPase that increased the amount of TPase in the nucleus. When introduced into Arabidopsis, the NES mutant protein also catalyzed higher frequencies of mPing excision from the gfp reporter gene. Our yeast assay retains key features of excision and insertion of mPing including precise excision, extended insertion sequence preference, and a requirement for two proteins that can come from either Ping or Pong or both elements.ConclusionsThe yeast transposition assay provides a robust platform for analysis of the mechanism underlying transposition catalyzed by the two proteins of PIF/Harbinger elements. It recapitulates all of the features of excision and reinsertion of mPing as seen in plant systems. Furthermore, a mutation of a putative NES in the TPase increased transposition both in yeast and plants.

Analysis of the dbpBA Upstream Regulatory Region Controlled by RpoS in Borrelia burgdorferi

Decorin-binding proteins B and A (DbpB and DbpA) are thought to play important roles in Borrelia burgdorferi pathogenesis by serving as adhesins for the extracellular matrix. It has been established that the expression of DbpBA is governed by the Rrp2-RpoN-RpoS regulatory pathway. However, the precise mechanism underlying the control of DbpBA expression has been unclear. In particular, it has been unknown whether RpoS influences DbpBA expression directly or indirectly (through an additional regulatory molecule[s]). Here, employing a wild-type B. burgdorferi strain and a dbpBA-deficient mutant, we analyzed the 5' genetic elements of the dbpBA operon using deletion analysis, coupled with luciferase reporter assays, quantitative reverse transcription PCR, and immunoblot analyses. A minimal promoter, encompassed within 70 bp upstream of the ATG start codon of dbpBA, was identified and found to be necessary and sufficient to initiate dbpBA transcription. The minimal dbpBA promoter was responsive to environmental stimuli such as temperature, pH, and whole blood. Two in silico-identified inverted repeat elements were not involved in the response of dbpBA expression to in vitro stimulation by environmental factors. The expression of dbpBA from the minimal promoter was abolished when rpoS was inactivated. In addition, the targeted mutagenesis of a C at position -14 within the extended -10 region of dbpBA, which has been postulated to be strategic for Esigma(S) binding in Escherichia coli, abolished dbpBA expression in B. burgdorferi. These combined data suggest that the Rrp2-RpoN-RpoS pathway controls dbpBA expression by the direct binding of RpoS to an RpoS-dependent promoter. However, given that there remains a distinct difference between the expression of DbpBA and other genes under the direct control of RpoS (e.g., OspC), our findings do not preclude the existence of another layer of gene regulation that may contribute to the modulation of DbpBA expression via an as-yet unknown mechanism.

Conjugative Transferability of the A/C Plasmids fromSalmonella entericaIsolates That Possess or LackblaCMYin the A/C Plasmid Backbone

The objective of this study was to understand the conjugative transmissibility of resistance plasmids present in 205 Salmonella enterica isolates from bovine sources. Polymerase chain reaction (PCR)-based replicon typing was used to type plasmid replicons. Conjugation experiments were preformed in triplicate at 30 degrees C and 37 degrees C on solid medium. PCR mapping of the A/C transfer gene operon was done on 17 Salmonella Newport isolates that were only positive for A/C. Eighty-six percent (n = 177) of the Salmonella isolates were multidrug resistant (MDR) with resistance to 3-12 antimicrobial agents. Of these, 82% (n = 146) were resistant to extended-spectrum cephalosporins and possessed a bla(CMY) gene. A/C was the predominant replicon detected, present in 90% (n = 160) of the MDR isolates. Twenty-three percent (n = 37) of the A/C-positive strains were positive for a second replicon. Replicons coresident with A/C included I1, N, B/O, HI1, and HI2. Only 31% (n = 54) of the MDR isolates produced transconjugants, and most of these donors carried multiple replicons. A/C cotransferred with B/O, N, and I1 at both 30 degrees C and 37 degrees C and with HI2 at 30 degrees C. Seven Salmonella Newport isolates that produced transconjugants possessed only the single A/C replicon and lacked bla(CMY). PCR mapping of the A/C transfer gene operon in ten Salmonella Newport isolates that carried bla(CMY) revealed a bla(CMY) inverted repeat element integrated between the traA and traC genes. These results suggest that A/C may have been a conjugative plasmid before the integration of bla(CMY) into the transfer gene operon. Additionally, transfer deficient A/C replicons may be mobilized in the presence of certain compatible conjugative plasmids.

Characterization of WAP2 gene in Aegilops tauschii and comparison with homoeologous loci in wheat

Abstract The Q/q gene, also known as WAP2, is an important gene for wheat domestication and is a member of the AP2 (APETALA2) class of transcription factors. In the present study, we first isolated the WtAP2 allele (where the superscript “t” refers to the speciese source, in this case “tauschii”) on chromosome 5D from Aegilops tauschii Coss., the D‐genome donor species of common wheat. We found that WtAP2 and the AP2 gene from Arabidopsis share a central core of the AP2 polypeptide, a highly basic 10‐amino acid domain, and an AASSGF box, although there are many differences in the 37‐amino acid serine‐rich acidic domain and the remaining regions. In addition, WtAP2 was highly homologous to the homoeologous loci on 5A and 5B of wheat at both the nucleotide and amino acid level. However, there were some variations that are probably related to gene function. In the first AP2 domain, the amino acids VYL on the 5D and 5A loci were replaced with LLR on 5B. In the 37‐amino acid serine‐rich acidic domain, WtAP2 on 5D had an extra amino acid insertion. There was also a variation at the 329 amino acid position, which is thought to be related to the appearance of free‐threshing wheat. At this position, the amino acid is isoleucine on 5A for the Q allele and valine for the q allele, whereas the amino acid is leucine on 5D and 5B. Furthermore, a Stowaway miniature terminal inverted repeat element (MITE) insertion was present in the ninth intron of WAP2 on 5B of all common wheats and partial tetraploid Triticum turgidum wheats. These results provide new clues for studies into the evolutionary biology of WAP2 and the origin of common wheat.

Transpositional activation of mPing in an asymmetric nuclear somatic cell hybrid of rice and Zizania latifolia was accompanied by massive element loss

We have reported previously that the most active miniature inverted terminal repeat transposable element (MITE) of rice, mPing, was transpositionally mobilized in several rice recombinant inbred lines (RILs) derived from an introgressive hybridization between rice and wild rice (Zizania latifolia Griseb.). To further study the phenomenon of hybridization-induced mPing activity, we undertook the present study to investigate the element's behavior in a highly asymmetric somatic nuclear hybrid (SH6) of rice and Z. latifolia, which is similar in genomic composition to that of the RILs, though probably contains more introgressed alien chromatins from the donor species than the RILs. We found that mPing, together with its transposase-donor, Pong, underwent rampant transpositional activation in the somatic hybrid (SH6). Because possible effects of protoplast isolation and cell culture can be ruled out, we attribute the transpositional activation of mPing and Pong in SH6 to the process of asymmetric somatic hybridization, namely, one-step introgression of multiple chromatin segments of the donor species Z. latifolia into the recipient rice genome. A salient feature of mPing transposition in the somatic hybrid is that the element's activation was accompanied by massive loss of its original copies, i.e., abortive transpositions, which was not observed in previously reported cases of mPing activity. These data not only corroborated our earlier finding that wide hybridization and introgression may trigger transpositional activation of otherwise quiescent transposable elements, but also suggest that transpositional mobilization of a MITE like mPing can be accompanied by dramatic reduction of its original copy numbers under certain conditions, thus provide novel insights into the dynamics of MITEs in the course of genome evolution.

Novel Means for Spreading Resistance Traits among Gram-Negative Pathogens

A newly recognized mobile element named “integron mobilization unit” (IMU) is a novel means for spreading antibiotic resistance genes, thus adding to the roster of molecular mechanisms for disseminating resistance traits to a broad variety of gram-negative bacterial pathogens, according to Johann D. Pitout of the University of Calgary in Calgary, Alberta, Canada, Patrice Nordmann of INSERM in Paris, France, and the nearby Hopital de Bicetre, and their collaborators. Although not a conventional transposon, the IMU behaves much like a miniature inverted transposable element (MITE), marking the first time that such an element was found to mobilize clinically relevant resistance genes, the researchers point out. This instance also appears to be the first report of the GES-5 class A carbapenemase being detected in North America. Details appear in the June 2009 Antimicrobial Agents and Chemotherapy (53:2492–2498).

Integron Mobilization Unit as a Source of Mobility of Antibiotic Resistance Genes

Antibiotic resistance genes are spread mostly through plasmids, integrons (as a form of gene cassettes), and transposons in gram-negative bacteria. We describe here a novel genetic structure, named the integron mobilization unit (IMU), that has characteristics similar to those of miniature inverted transposable elements (MITEs). Two IMUs (288 bp each) were identified from a carbapenem-resistant Enterobacter cloacae isolate that formed a composite structure encompassing a defective class 1 integron containing the carbapenem resistance gene bla(GES-5). This beta-lactamase gene was located on a 7-kb IncQ-type plasmid named pCHE-A, which was sequenced completely. The plasmid pCHE-A was not self conjugative but was mobilizable, and it was successfully transferred from E. cloacae to Pseudomonas aeruginosa. The in silico analysis of the extremities of the IMU elements identified similarities with those of insertion sequence ISSod9 from Shewanella oneidensis MR-1. The mobilization of the IMU composite structure was accomplished by using the transposase activity of ISSod9 that was provided in trans. This is the first identification of MITE-type structures as a source of gene mobilization, implicating here a clinically relevant antibiotic resistance gene.

Repetitive genome elements in a European corn borer, Ostrinia nubilalis, bacterial artificial chromosome library were indicated by bacterial artificial chromosome end sequencing and development of sequence tag site markers: implications for lepidopteran genomic research

The European corn borer, Ostrinia nubilalis, is a serious pest of food, fiber, and biofuel crops in Europe, North America, and Asia and a model system for insect olfaction and speciation. A bacterial artificial chromosome library constructed for O. nubilalis contains 36 864 clones with an estimated average insert size of >or=120 kb and genome coverage of 8.8-fold. Screening OnB1 clones comprising approximately 2.76 genome equivalents determined the physical position of 24 sequence tag site markers, including markers linked to ecologically important and Bacillus thuringiensis toxin resistance traits. OnB1 bacterial artificial chromosome end sequence reads (GenBank dbGSS accessions ET217010 to ET217273) showed homology to annotated genes or expressed sequence tags and identified repetitive genome elements, O. nubilalis miniature subterminal inverted repeat transposable elements (OnMITE01 and OnMITE02), and ezi-like long interspersed nuclear elements. Mobility of OnMITE01 was demonstrated by the presence or absence in O. nubilalis of introns at two different loci. A (GTCT)n tetranucleotide repeat at the 5' ends of OnMITE01 and OnMITE02 are evidence for transposon-mediated movement of lepidopteran microsatellite loci. The number of repetitive elements in lepidopteran genomes will affect genome assembly and marker development. Single-locus sequence tag site markers described here have downstream application for integration within linkage maps and comparative genomic studies.

Complete Genome Sequence of the Anaerobic, Protein-Degrading Hyperthermophilic Crenarchaeon Desulfurococcus kamchatkensis

Desulfurococcus kamchatkensis is an anaerobic organotrophic hyperthermophilic crenarchaeon isolated from a terrestrial hot spring. Its genome consists of a single circular chromosome of 1,365,223 bp with no extrachromosomal elements. A total of 1,474 protein-encoding genes were annotated, among which 205 are exclusive for D. kamchatkensis. The search for a replication origin site revealed a single region coinciding with a global extreme of the nucleotide composition disparity curve and containing a set of crenarchaeon-type origin recognition boxes. Unlike in most archaea, two genes encoding homologs of the eukaryotic initiator proteins Orc1 and Cdc6 are located distantly from this site. A number of mobile elements are present in the genome, including seven transposons representing IS607 and IS200/IS605 families and multiple copies of miniature inverted repeat transposable elements. Two large clusters of regularly interspaced repeats are present; none of the spacer sequences matches known archaeal extrachromosomal elements, except one spacer matches the sequence of a resident gene of D. kamchatkensis. Many of the predicted metabolic enzymes are associated with the fermentation of peptides and sugars, including more than 30 peptidases with diverse specificities, a number of polysaccharide degradation enzymes, and many transporters. Consistently, the genome encodes both enzymes of the modified Embden-Meyerhof pathway of glucose oxidation and a set of enzymes needed for gluconeogenesis. The genome structure and content reflect the organism's nutritionally diverse, competitive natural environment, which is periodically invaded by viruses and other mobile elements.

Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs

Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by a complex of TRBP and Dicer. dgcr8Delta/Delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1Delta/Delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild-type, dgcr8Delta/Delta, and dicer1Delta/Delta mESCs. Several of the resulting DGCR8-independent, Dicer-dependent RNAs were noncanonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of shRNAs. Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small RNA-generating pathways and show that mammalian siRNAs exist in cell types other than oocytes.

Transactivation of the Hepatitis B Virus Core Promoter by the Nuclear Receptor FXRα

Hepatitis B virus (HBV) core promoter activity is positively and negatively regulated by nuclear receptors, a superfamily of ligand-activated transcription factors, via cis-acting sequences located in the viral genome. In this study, we investigated the role of farnesoid X receptor alpha (FXRalpha) in modulating transcription from the HBV core promoter. FXRalpha is a liver-enriched nuclear receptor activated by bile acids recognizing hormone response elements by forming heterodimers with retinoid X receptor alpha (RXRalpha). Electrophoretic mobility shift assays demonstrated that FXRalpha-RXRalpha heterodimers can bind two motifs on the HBV enhancer II and core promoter regions, presenting high homology to the consensus (AGGTCA) inverted repeat FXRalpha response elements. In transient transfection of the human hepatoma cell line Huh-7, bile acids enhanced the activity of a luciferase reporter containing the HBV enhancer II and core promoter sequences through FXRalpha. Moreover, using a greater-than-genome-length HBV construct, we showed that FXRalpha also increased synthesis of the viral pregenomic RNA and DNA replication intermediates. The data strongly suggest that FXRalpha is another member of the nuclear receptor superfamily implicated in the regulation of HBV core promoter activity and that bile acids could play an important role in the natural history of HBV infection.