Classification of Escherichia coli K-12 ribosome binding sites. An error-control coding model.
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Binding Sites↗
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Phylogeny of spore-forming lactic acid bacteria based on 16S rRNA gene sequences.
The phylogeny of spore-forming lactic acid bacteria was investigated on the basis of 16S rRNA gene sequences. Sixteen strains were separated into three lines of descent; one consisted of 14 strains assigned to Sporolactobacillus spp. and Bacillus spp., and the other two each consisted of "Sporolactobacillus dextrus" and Bacillus coagulans. Strains of all the first lineage but one composed a cluster of similarity values of 97.2% and higher, and were represented by the type strain of S. inulinus. The cluster was further separated into five subclusters, four catalase negative and one positive. The definition of the genus Sporolactobacillus should be amended to accommodate catalase positive strains. Spore-forming lactic acid bacteria originated at different phylogenetic positions, and would have evolved convergently in the area of Bacillus.
Bacillus↗
Determination of 23S rRNA sequences from members of the genus Streptococcus and characterization of genetically distinct organisms previously identified as members of the Streptococcus anginosus group.
The 23S rRNA gene sequences of eight type strains of the genus Streptococcus were determined. Three species-specific probes for the three members of the Streptococcus anginosus group and one group-specific probe for these three species were designed. The PCR-amplified 23S rRNA gene sequences of 28 clinical strains were examined by hybridization with the four oligonucleotide probes. They were suspected to be members of the S. anginosus group by biochemical tests; however, only 21 strains (75%) hybridized to one of the three species probes. Seven biochemically atypical strains did not hybridize to these species-specific probes. Of the seven strains, three hybridized to the group-specific probe. The 23S ribosomal RNA sequences of these three strains differed by three bases within the 20-base probe area of S. anginosus. The phylogenetic tree of the 16S rRNA of these three strains located them within the branch of S. anginosus. The remaining four strains, which did not hybridize to group- or species-specific probes, had a raffinose-positive and VP-negative phenotype. The probe hybridization results showed that these four strains did not belong to the S. anginosus group and were closely related to S. parasanguis according to partial sequences of their 16S rRNA genes.
Base Sequence↗
Elucidation of the small RNA component of the transcriptome.
Small RNAs play important regulatory roles in most eukaryotes, but only a small proportion of these molecules have been identified. We sequenced more than two million small RNAs from seedlings and the inflorescence of the model plant Arabidopsis thaliana. Known and new microRNAs (miRNAs) were among the most abundant of the nonredundant set of more than 75,000 sequences, whereas more than half represented lower abundance small interfering RNAs (siRNAs) that match repetitive sequences, intergenic regions, and genes. Individual or clusters of highly regulated small RNAs were readily observed. Targets of antisense RNA or miRNA did not appear to be preferentially associated with siRNAs. Many genomic regions previously considered featureless were found to be sites of numerous small RNAs.
Arabidopsis↗
Comparison of gene expression profiles of Candida albicans azole-resistant clinical isolates and laboratory strains exposed to drugs inducing multidrug transporters.
Azole resistance in Candida albicans can be due to upregulation of multidrug transporters belonging to ABC (ATP-binding cassette) transporters (CDR1 and CDR2) or major facilitators (CaMDR1). Upregulation of these genes can also be achieved by exposure to fluphenazine, resulting in specific upregulation of CDR1 and CDR2 and by exposure to benomyl, resulting in specific CaMDR1 upregulation. In this study, these two different states of gene upregulation were used to determine coregulated genes that often share similar functions or similar regulatory regions. The transcript profiles of a laboratory strain exposed to these drugs were therefore determined and compared with those of two matched pairs of azole-susceptible and -resistant strains expressing CDR1 and CDR2 (CDR strains) or CaMDR1 (MDR isolates). The results obtained revealed that, among 42 commonly regulated genes (8.6% of all regulated genes) between fluphenazine-exposed cells and CDR isolates, the most upregulated were CDR1 and CDR2 as expected, but also IFU5, RTA3 (which encodes putative membrane proteins), HSP12 (which encodes heat shock protein), and IPF4065 (which is potentially involved in stress response). Interestingly, all but HSP12 and IPF4065 contain a putative cis-acting drug responsive element in their promoters. Among the 57 genes (11.5% of all regulated genes) commonly regulated between benomyl-exposed cells and MDR isolates, the most upregulated were CaMDR1 as expected but also genes with oxido-reductive functions such as IFD genes, IPF5987, GRP2 (all belonging to the aldo-keto reductase family), IPF7817 [NAD(P)H oxido-reductase], and IPF17186. Taken together, these results show that in vitro drug-induced gene expression only partially mimics expression profiles observed in azole-resistant clinical strains. Upregulated genes in both drug-exposed conditions and clinical strains are drug resistance genes but also genes that could be activated under cell damage conditions.
ATP Binding Cassette Transporter, Subfamily B↗
Fatal case of Listeria innocua bacteremia.
Listeria innocua is widespread in the environment and in food. This species has to date never been described in association with human disease. We report a case of fatal bacteremia caused by L. innocua in a 62-year-old patient.
Bacteremia↗
Rotavirus protein NSP3 (NS34) is bound to the 3' end consensus sequence of viral mRNAs in infected cells.
Interaction between viral proteins and RNAs has been studied in rotavirus-infected cells. The use of UV cross-linking followed by immunoprecipitation and labeling with T4 polynucleotide kinase allowed us to detect interactions between RNA and nonstructural viral proteins. The RNAs linked to the nonstructural protein NSP3 have been identified as rotavirus mRNAs, and the sequences of the RNase T1-protected fragments have been established. These sequences correspond to the 3' end sequence common to all rotavirus group A genes. We also show that the last 3' nucleotide is cross-linked to the protein and that monomeric and multimeric forms of NSP3 are bound to rotavirus mRNA. The role of NSP3 in rotavirus replication is discussed in the light of our results and by comparison with other RNA-binding proteins of members of the Reoviridae family.
Base Sequence↗
Sequence and structural elements at the 3' terminus of bovine viral diarrhea virus genomic RNA: functional role during RNA replication.
Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus in the family Flaviviridae, has a positive-stranded RNA genome consisting of a single open reading frame and untranslated regions (UTRs) at the 5' and 3' ends. Computer modeling suggested the 3' UTR comprised single-stranded regions as well as stem-loop structures-features that were suspected of being essentially implicated in the viral RNA replication pathway. Employing a subgenomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA replicon (S.-E. Behrens, C. W. Grassmann, H. J. Thiel, G. Meyers, and N. Tautz, J. Virol. 72:2364-2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3' UTR by experimental means and to investigate the significance of defined RNA motifs for the RNA replication pathway. Enzymatic and chemical structure probing revealed mainly the conserved terminal part (termed 3'C) of the DI9c 3' UTR containing distinctive RNA motifs, i.e., a stable stem-loop, SL I, near the RNA 3' terminus and a considerably less stable stem-loop, SL II, that forms the 5' portion of 3'C. SL I and SL II are separated by a long single-stranded intervening sequence, denoted SS. The 3'-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e.g., with upstream DI9c RNA sequences, were not detected under the experimental conditions used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS motifs do indeed play essential roles during RNA replication. Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as substitution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate. Conversely, SL I stems comprising compensatory base exchanges turned out to support replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c. In summary, these results indicate that RNA structure as well as sequence elements harbored within the 3'C region of the BVDV 3' UTR create a common cis-acting element of the replication process. The data further point at possible interaction sites of host and/or viral proteins and thus provide valuable information for future experiments intended to identify and characterize these factors.
3' Untranslated Regions↗
Complete genome sequence and analyses of the subgenomic RNAs of sweet potato chlorotic stunt virus reveal several new features for the genus Crinivirus.
The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3' terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5' ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae.
Amino Acid Sequence↗
Xenopus U3 snoRNA GAC-Box A' and Box A sequences play distinct functional roles in rRNA processing.
Mutations in the 5' portion of Xenopus U3 snoRNA were tested for function in oocytes. The results revealed a new cleavage site (A0) in the 3' region of vertebrate external transcribed spacer sequences. In addition, U3 mutagenesis uncoupled cleavage at sites 1 and 2, flanking the 5' and 3' ends of 18S rRNA, and generated novel intermediates: 19S and 18.5S pre-rRNAs. Furthermore, specific nucleotides in Xenopus U3 snoRNA that are required for cleavages in pre-rRNA were identified: box A is essential for site A0 cleavage, the GAC-box A' region is necessary for site 1 cleavage, and the 3' end of box A' and flanking nucleotides are required for site 2 cleavage. Differences between metazoan and yeast U3 snoRNA-mediated rRNA processing are enumerated. The data support a model where metazoan U3 snoRNA acts as a bridge to draw together the 5' and 3' ends of the 18S rRNA coding region within pre-rRNA to coordinate their cleavage.
Animals↗
Endocarditis caused by Haemophilus parainfluenzae identified by 16S ribosomal RNA sequencing.
Haemophilus parainfluenzae, a human commensal, is an infrequent cause of serious disease. A case of endocarditis caused by this organism in a five year old boy with complex congenital heart disease is reported. The course of this disease was very aggressive, leading to heart failure, disseminated intravascular coagulation and multiorgan failure in spite of appropriate antibiotics and surgical intervention. The difficulties in the detection and identification of H parainfluenzae using conventional culture based technology, and the potential role of molecular techniques, are highlighted.
Anti-Bacterial Agents↗
Identification by 16S ribosomal RNA gene sequencing of Arcobacter butzleri bacteraemia in a patient with acute gangrenous appendicitis.
AIMS: To identify a strain of Gram negative facultative anaerobic curved bacillus, concomitantly isolated with Escherichia coli and Streptococcus milleri, from the blood culture of a 69 year old woman with acute gangrenous appendicitis. The literature on arcobacter bacteraemia and arcobacter infections associated with appendicitis was reviewed. METHODS: The isolate was phenotypically investigated by standard biochemical methods using conventional biochemical tests. Genotypically, the 16S ribosomal RNA (rRNA) gene of the bacterium was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank by multiple sequence alignment. Literature review was performed by MEDLINE search (1966-2000). RESULTS: The bacterium grew on blood agar, chocolate agar, and MacConkey agar to sizes of 1 mm in diameter after 24 hours of incubation at 37 degrees C in 5% CO2. It grew at 15 degrees C, 25 degrees C, and 37 degrees C; it also grew in a microaerophilic environment, and was cytochrome oxidase positive and motile, typically a member of the genus arcobacter. Furthermore, phenotypic testing showed that the biochemical profile of the isolate did not fit into the pattern of any of the known arcobacter species. 16S rRNA gene sequencing showed one to two base differences between the isolate and A butzleri, but 35 to 39 base differences between the isolate and A cryaerophilus, indicating that the isolate was a strain of A butzleri. Only three cases of arcobacter bacteraemia with detailed clinical characteristics were found in the English literature. The sources of the arcobacter species in the three cases were largely unknown, although the gastrointestinal tract is probably the portal of entry of the A butzleri isolated from the present case because the two concomitant isolates (E coli and S milleri) in the blood culture were common flora of the gastrointestinal tract. In addition, A butzleri has previously been isolated from the abdominal contents or peritoneal fluid of three patients with acute appendicitis. CONCLUSIONS: 16S rRNA gene sequencing was useful in the identification of the strain of A butzleri isolated from the blood culture of a patient with acute gangrenous appendicitis. Arcobacter bacteraemia is rare. Further studies using selective medium for the delineation of the association between A butzleri and acute appendicitis are warranted.
Acute Disease↗
Properties of the genus Tatlockia. Differentiation of Tatlockia (Legionella) maceachernii and micdadei from each other and from other legionellae.
The biochemical reactions, carbohydrate content, and 16S-rRNA sequences of Tatlockia (Legionella) maceachernii and Tatlockia micdadei strains were studied. Except for catalase activity, Tatlockia strains were relatively inert in the biochemical tests commonly used in clinical laboratories. Phenotypically, the two Tatlockia species could be distinguished from other legionellae by the presence of yersiniose A, by their inability to hydrolyze hippurate or starch, by the absence of colony or media fluorescence, and by the absence of distinct browning of tyrosine-containing medium. These two species differed from one another by the production of acetoin by T. micdadei but not by T. maceachernii. Gelatinase activity, which had been reported in T. maceachernii, was observed in only one of the four strains studied. The 16S-rRNA sequences and carbohydrate profiles of T. maceachernii and T. micdadei were essentially identical. In preparing the RNA for study, it was noted that the 23S rRNA was fragmented in all T. maceachernii strains tested, while the 23S rRNA of T. micdadei strains was intact. Among the legionellae studied, T. maceachernii was most closely related to T. micdadei.
Bacterial Typing Techniques↗
Translation regulation of intron-containing genes in chloroplasts.
The paper provides a short description of the originally developed algorithm for searching of the conservative protein-RNA binding sites. The algorithm is applied to analyze chloroplast genes. The candidate protein-RNA binding sites were detected upstream of atpF, petB, clpP, psaA, psbA, and psbB genes in many chloroplasts of algae and plants. We suggest that some of these sites are involved in suppressing translation until splicing is completed.
Algorithms↗
Erythroid-induced commitment of K562 cells results in clusters of differentially expressed genes enriched for specific transcription regulatory elements.
Understanding regulation of fetal and embryonic hemoglobin expression is critical, since their expression decreases clinical severity in sickle cell disease and beta-thalassemia. K562 cells, a human erythroleukemia cell line, can differentiate along erythroid or megakaryocytic lineages and serve as a model for regulation of fetal/embryonic globin expression. We used microarray expression profiling to characterize transcriptomes from K562 cells treated for various times with hemin, an inducer of erythroid commitment. Approximately 5,000 genes were expressed irrespective of treatment. Comparative expression analysis (CEA) identified 899 genes as differentially expressed; analysis by the self-organizing map (SOM) algorithm clustered 425 genes into 8 distinct expression patterns, 322 of which were shared by both analyses. Differential expression of a subset of genes was validated by real-time RT-PCR. Analysis of 5'-flanking regions from differentially expressed genes by PAINT v3.0 software showed enrichment in specific transcription regulatory elements (TREs), some localizing to different expression clusters. This finding suggests coordinate regulation of cluster members by specific TREs. Finally, our findings provide new insights into rate-limiting steps in the appearance of heme-containing hemoglobin tetramers in these cells.
5' Flanking Region↗
Transient loss of T-cell reactivity in a patient with hepatitis C virus reinfection.
BACKGROUND/AIMS: Self-limited acute hepatitis C virus (HCV) infection with spontaneous recovery is only rarely observed in clinical practice. All existing studies correlate strong cellular immune responses to the recovery from HCV infection. CASE REPORT: Here, we present a 49-year-old haemophiliac, who had successfully recovered from an acute hepatitis, which was classified retrospectively as HCV infection based on his antibody profiles. This patient was reinfected with HCV 18 years later from an exogenous source, and successfully recovered from this reinfection within 2 months. After his first hepatitis the patient displayed strong cellular responses against recombinant HCV proteins. During reinfection, T-cell proliferation was markedly reduced, while HCV antibody titres increased. However, E2 antibodies were consistently not detectable. T-cell proliferation returned to the pre-reinfection level only several months after loss of viraemia. DISCUSSION: Our observations resulted in the unexpected finding that our patient cleared HCV reinfection despite an apparent loss of his pre-existing T-cell reactivity in the peripheral blood.
Hemophilia A↗
Integrated siRNA design based on surveying of features associated with high RNAi effectiveness.
BACKGROUND: Short interfering RNAs have allowed the development of clean and easily regulated methods for disruption of gene expression. However, while these methods continue to grow in popularity, designing effective siRNA experiments can be challenging. The various existing siRNA design guidelines suffer from two problems: they differ considerably from each other, and they produce high levels of false-positive predictions when tested on data of independent origins. RESULTS: Using a distinctly large set of siRNA efficacy data assembled from a vast diversity of origins (the siRecords data, containing records of 3,277 siRNA experiments targeting 1,518 genes, derived from 1,417 independent studies), we conducted extensive analyses of all known features that have been implicated in increasing RNAi effectiveness. A number of features having positive impacts on siRNA efficacy were identified. By performing quantitative analyses on cooperative effects among these features, then applying a disjunctive rule merging (DRM) algorithm, we developed a bundle of siRNA design rule sets with the false positive problem well curbed. A comparison with 15 online siRNA design tools indicated that some of the rule sets we developed surpassed all of these design tools commonly used in siRNA design practice in positive predictive values (PPVs). CONCLUSION: The availability of the large and diverse siRNA dataset from siRecords and the approach we describe in this report have allowed the development of highly effective and generally applicable siRNA design rule sets. Together with ever improving RNAi lab techniques, these design rule sets are expected to make siRNAs a more useful tool for molecular genetics, functional genomics, and drug discovery studies.
Algorithms↗
Design and cloning strategies for constructing shRNA expression vectors.
BACKGROUND: Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). RESULTS: We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20-40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression. CONCLUSION: In this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process.
Base Sequence↗