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At least 1,261 records · Page 70Linked to original sources
PubMed2005

Establishment of a screening system for selection of siRNA target sites directed against hepatitis B virus surface gene.

To study the inhibitory effects of plasmid-derived small interfering RNA (siRNA) and synthetic siRNA on the expression of the hepatitis B virus surface (HBs) gene, three plasmid-derived siRNAs and one synthetic siRNA that complement the coding region of the HBs gene were prepared. The HBs expression plasmid pHBs-EGFP was also constructed. HeLa cells were co-transfected with pHBs-EGFP and the above siRNAs. The HBs mRNA quantities were measured by reverse-transcription PCR, and the level of HBs-EGFP fusion protein was quantified by fluorescent microscope. The concentrations of the hepatitis B virus surface antigen (HBsAg) derived from the culture supernatant of transfected HepG2.2.15 cells were measured by an enzyme-linked immunosorbent assay (ELISA) kit. The results showed that the three plasmid-derived siRNAs and the synthetic siRNA can effectively reduce the quantities of HBs mRNA and protein. The plasmid-derived siRNA psiRNA1 was found to be the most effective inhibitor of HBs expression. It can inhibit HBs-EGFP expression by 63.3% and suppress HBs mRNA by 75.6%. To further substantiate the above observations, psiRNA1 was transfected into HepG2.2.15 cells (an HBV secreting cell line). The transfections resulted in almost complete blockage of HBsAg production, whereas control vector-transfected cells secreted high levels of HBsAg 7 days post-transfection. In conclusion, our data suggests that RNA interference (RNAi) is an efficient approach for reducing the level of HBs transcripts and proteins and for suppressing HBsAg production.

Base Sequence↗
PubMed1993

Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms.

To facilitate genus and species level identification of a broad range of bacteria without the requirement of presumptive identification, we have developed a unified set of primers and polymerase chain reaction conditions to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. Spacer regions within these loci show a significant level of length and sequence polymorphism across both genus and species lines. A generic pair of priming sequences was selected for the amplification of these polymorphisms from highly conserved sequences in the 16S and 23S genes occurring adjacent to these polymorphic regions. This single set of primers and reaction conditions was used for the amplification of 16S-23S spacer regions for over 300 strains of bacteria belonging to eight genera and 28 species or serotypes, including Listeria, Staphylococcus, and Salmonella species and additional species related to these pathogenic organisms. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the species of bacteria within the test group. Unique elements in the amplification product patterns generally clustered at the species level, although some genus-specific characteristics were also observed. On the basis of the results obtained with our test group of 300 bacterial strains, amplification of the 16S-23S ribosomal spacer region is a suitable process for generating a data base for use in a polymerase chain reaction-based identification method, which can be comprehensively applied to the bacterial kingdom.

Base Sequence↗
PubMed1998

Hepatitis G virus infection in Amerindians and other Venezuelan high-risk groups.

Recently, a new virus related to flaviviruses, the hepatitis G virus (HGV), or GBV-C virus, was discovered as a putative blood-borne human pathogen. HGV RNA (NS5 region) was amplified by reverse transcription-nested PCR in the sera of 6 of 64 (9%) hemodialysis patients; 2 of 80 (2.5%) West Yukpa Amerindians, a population with a high rate of HBV infection but negative for HCV infection; and 1 patient with an acute episode of non-A, non-B, non-C hepatitis (NABCH). The patterns of single-strand conformation polymorphism of the amplified products were unique among different specimens and similar on follow-up for hemodialysis patients. All patients tested remained HGV RNA positive 1 and 2 years later, without major sequence variation, except for the NABCH patient, for whom a double infection and an apparent clearance of the original dominant variant was observed after 2 years. The sequences of the NS5 amplified products demonstrated 85 to 90% identity with other reported HGV sequences.

Base Sequence↗
PubMed2000

Mutations of the kissing-loop dimerization sequence influence the site specificity of murine leukemia virus recombination in vivo.

The genetic information of retroviruses is retained within a dimeric RNA genome held together by intermolecular RNA-RNA interactions near the 5' ends. Coencapsidation of retrovirus-derived RNA molecules allows frequent template switching of the virus-encoded reverse transcriptase during DNA synthesis in newly infected cells. We have previously shown that template shifts within the 5' leader of murine leukemia viruses occur preferentially within the kissing stem-loop motif, a cis element crucial for in vitro RNA dimer formation. By use of a forced recombination approach based on single-cycle transfer of Akv murine leukemia virus-based vectors harboring defective primer binding site sequences, we now report that modifications of the kissing-loop structure, ranging from a deletion of the entire sequence to introduction of a single point mutation in the loop motif, significantly disturb site specificity of recombination within the highly structured 5' leader region. In addition, we find that an intact kissing-loop sequence favors optimal RNA encapsidation and vector transduction. Our data are consistent with the kissing-loop dimerization model and suggest that a direct intermolecular RNA-RNA interaction, here mediated by palindromic loop sequences within the mature genomic RNA dimer, facilitates hotspot template switching during retroviral cDNA synthesis in vivo.

3T3 Cells↗
PubMed2006

Predicting antisense RNAs in the genomes of Escherichia coli and Salmonella typhimurium using promoter-search algorithm PlatProm.

A pattern recognition software PlatProm, which takes into consideration both sequence-specific and structure-specific features in the genetic environment of the promoter sites and identifies transcription start points with a very high accuracy was used to reveal potentially transcribed regions in the genomes of two bacterial species. Along with the expected promoters located upstream from coding sequences PlatProm identified several hundred of very similar signals in other intergenic regions and within coding sequences. Homologous genes of Escherichia coli and Salmonella typhimurium, containing potential promoters on the template strand are suggested as putative targets for regulations by antisense RNA-products (aRNAs).

Algorithms↗
PubMed2006

Microbial identification by mass cataloging.

BACKGROUND: The public availability of over 180,000 bacterial 16S ribosomal RNA (rRNA) sequences has facilitated microbial identification and classification using hybridization and other molecular approaches. In their usual format, such assays are based on the presence of unique subsequences in the target RNA and require a prior knowledge of what organisms are likely to be in a sample. They are thus limited in generality when analyzing an unknown sample.Herein, we demonstrate the utility of catalogs of masses to characterize the bacterial 16S rRNA(s) in any sample. Sample nucleic acids are digested with a nuclease of known specificity and the products characterized using mass spectrometry. The resulting catalogs of masses can subsequently be compared to the masses known to occur in previously-sequenced 16S rRNAs allowing organism identification. Alternatively, if the organism is not in the existing database, it will still be possible to determine its genetic affinity relative to the known organisms. RESULTS: Ribonuclease T1 and ribonuclease A digestion patterns were calculated for 1,921 complete 16S rRNAs. Oligoribonucleotides generated by RNase T1 of length 9 and longer produce sufficient diversity of masses to be informative. In addition, individual fragments or combinations thereof can be used to recognize the presence of specific organisms in a complex sample. In this regard, 140 strains out of 1,921 organisms (7.3%) could be identified by the presence of a unique RNase T1-generated oligoribonucleotide mass. Combinations of just two and three oligoribonucleotide masses allowed 54% and 72% of the specific strains to be identified, respectively. An initial algorithm for recovering likely organisms present in complex samples is also described. CONCLUSION: The use of catalogs of compositions (masses) of characteristic oligoribonucleotides for microbial identification appears extremely promising. RNase T1 is more useful than ribonuclease A in generating characteristic masses, though RNase A produces oligomers which are more readily distinguished due to the large mass difference between A and G. Identification of multiple species in mixtures is also feasible. Practical applicability of the method depends on high performance mass spectrometric determination, and/or use of methods that increase the one dalton (Da) mass difference between uracil and cytosine.

Algorithms↗
PubMed2006

GENCODE: producing a reference annotation for ENCODE.

BACKGROUND: The GENCODE consortium was formed to identify and map all protein-coding genes within the ENCODE regions. This was achieved by a combination of initial manual annotation by the HAVANA team, experimental validation by the GENCODE consortium and a refinement of the annotation based on these experimental results. RESULTS: The GENCODE gene features are divided into eight different categories of which only the first two (known and novel coding sequence) are confidently predicted to be protein-coding genes. 5' rapid amplification of cDNA ends (RACE) and RT-PCR were used to experimentally verify the initial annotation. Of the 420 coding loci tested, 229 RACE products have been sequenced. They supported 5' extensions of 30 loci and new splice variants in 50 loci. In addition, 46 loci without evidence for a coding sequence were validated, consisting of 31 novel and 15 putative transcripts. We assessed the comprehensiveness of the GENCODE annotation by attempting to validate all the predicted exon boundaries outside the GENCODE annotation. Out of 1,215 tested in a subset of the ENCODE regions, 14 novel exon pairs were validated, only two of them in intergenic regions. CONCLUSION: In total, 487 loci, of which 434 are coding, have been annotated as part of the GENCODE reference set available from the UCSC browser. Comparison of GENCODE annotation with RefSeq and ENSEMBL show only 40% of GENCODE exons are contained within the two sets, which is a reflection of the high number of alternative splice forms with unique exons annotated. Over 50% of coding loci have been experimentally verified by 5' RACE for EGASP and the GENCODE collaboration is continuing to refine its annotation of 1% human genome with the aid of experimental validation.

Chromosome Mapping↗
PubMed2003

Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1.

In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3'untranslated region (3' UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. Here, we show that a 34 nucleotide region of the glp-1 3' UTR contains two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to promote translation in the embryo. Furthermore, we show that the STAR/KH domain protein GLD-1 binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other regulatory factors is important to localize this Notch receptor, and contributes to the spatial organization of Notch signaling.

Animals↗
PubMed2003

Antagonistic signals within the COX2 mRNA coding sequence control its translation in Saccharomyces cerevisiae mitochondria.

Translation of the mitochondrially coded COX2 mRNA within the organelle in yeast produces the precursor of Cox2p (pre-Cox2p), which is processed and assembled into cytochrome c oxidase. The mRNA sequence of the first 14 COX2 codons, specifying the pre-Cox2p leader peptide, was previously shown to contain a positively acting element required for translation of a mitochondrial reporter gene, ARG8(m), fused to the 91st codon of COX2. Here we show that three relatively short sequences within the COX2 mRNA coding sequence, or structures they form in vivo, inhibit translation of the reporter in the absence of the positive element. One negative element was localized within codons 15 to 25 and shown to function at the level of the mRNA sequence, whereas two others are within predicted stem-loop structures formed by codons 22-44 and by codons 46-74. All three of these inhibitory elements are antagonized in a sequence-specific manner by reintroduction of the upstream positive-acting sequence. These interactions appear to be independent of 5'- and 3'-untranslated leader sequences, as they are also observed when the same reporter constructs are expressed from the COX3 locus. Overexpression of MRS2, which encodes a mitochondrial magnesium carrier, partially suppresses translational inhibition by each isolated negatively acting element, but does not suppress them in combination. We hypothesize that interplay among these signals during translation in vivo may ensure proper timing of pre-Cox2p synthesis and assembly into cytochrome c oxidase.

Amino Acid Sequence↗
PubMed2002

Genetic relatedness among Japanese, American and European isolates of viral hemorrhagic septicemia virus (VHSV) based on partial G and P genes.

Molecular virological analyses of 8 Japanese VHSV (viral hemorrhagic septicemia virus) isolates from wild and farmed Japanese flounder Paralichthys olivaceus were performed to investigate their genetic relatedness to American and European isolates of VHSV. Phylogenetic analyses based on the partial nucleotide sequences of G and P genes revealed that there are 2 genogroups of VHSV in Japan. The first one represented by the Obama25 isolate is closely related to the American isolates (Genogroup I) while the other, the KRRV9601 isolate, is closely related to the traditional European isolates (Genogroup III). The 2 types of Japanese VHSV showed differences in the relative mobility of the G protein and intensity of the antibody reaction on the P and M proteins. The Obama25 type of VHSV is widely distributed as a native virus in the coastal areas of western Japan and has been responsible for the occurrence of VHSV infection in farmed Japanese flounder while the KRRV9601 isolate is considered to have been introduced from a foreign country.

Animals↗
PubMed1977

Purification and characterization of polynucleotide phosphorylase from Escherichia coli. Probe for the analysis of 3' sequences of RNA.

A simple procedure for purifying polynucleotide phosphorylase from Escherichia coli cells by means of affinity chromatography on an RNA-Sepharose column is described. The purified enzyme preparation has a specific activity 3500-fold that of the crude extract and is essentially homogeneous, as determined by ultracentrifugation, polyacrylamide gel electrophoresis under denaturing conditions, isoelectric focusing and serological assays. It is virtually free of nuclease contamination, a property which permits its use in the synchronous phosphorolysis of RNA chains. The enzyme molecule is composed of three identical subunits of Mr = 84,000. Each subunit contains three cysteine residues, one of which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) whereas the two other groups are only exposed on denaturation of the protein. All three enzyme subunits participate in the processive phosphorolysis of the poly(A) tail of each globin mRNA chain. An advantageous method was developed for synchronous phosphorolysis of RNA molecules using a molar excess of polynucleotide phosphorylase immobilized onto Sepharose.

Chromatography, Affinity↗
PubMed1994

From RNA to sequenced clones within three days: a complete protocol.

Detection of specific mRNA transcripts by the reverse transcription/polymerase chain reaction (RT/PCR) technique has become increasingly important. The technique is fast and has a very high resolution. Cloning of these PCR fragments into vectors is sometimes necessary for identification of alternative splicing products, for bacterial expression or for generation of a DNA probe. Here we present a complete protocol for RT/PCR, cloning and sequencing of PCR, cloning and sequencing of PCR products beginning with the total RNA and ending with the DNA sequence within three days. To illustrate the procedure as an example, a fragment of the human glyceraldehyde-3-phosphate dehydrogenase mRNA was amplified from total RNA, cloned and partially sequenced. The protocol has been optimized for small scale to facilitate handling and to reduce costs.

Base Sequence↗
PubMed1995

Posttranscriptional regulation of insulin-like growth factor II mRNA.

The insulin-like growth factor II (IGF-II) gene generates multiple mature transcripts with different 5' untranslated regions (5'UTR) but identical coding regions and 3'UTRs. We have analysed the translational regulation and decay of the transcripts. Human IGF-II mRNAs provide a provocative example of translational discrimination in which only the minor 4.8 kb mRNA is actively engaged in protein synthesis while the major 6.0 kb mRNA is present in a 100S RNP particle. The 6.0 kb mRNA exhibits a structured 5'UTR of 1170 nucleotides that function as a cis-acting translational attenuator. IGF-II transcripts are processed by endonucleolytic cleavage 1209 and 2183 nucleotides downstream from the translation termination codons in the rat and human, respectively. The cleavage site is situated in a highly conserved and structured domain that exhibits two large hairpins and an intramolecular guanosine quadruplex. The structural elements may provide binding sites for trans-acting factors and ensure that the cleavage site is not sequestered in stable RNA structures. Since expression of IGF-II is initiated from minimal promoters and finished by constitutive secretion from the cell, regulatory events governing IGF-II production are likely to be implemented between the transcriptional and the posttranslational level. The combined effect of translational discrimination and endonucleolysis may therefore play an important role in the regulation of IGF-II expression.

Animals↗
PubMed1998

Identification of an alternative open reading frame ("hidden gene"?) stringently required for infectivity of poliovirus cDNA clones.

Translation of the uncapped poliovirus RNA starts at the AUG triplet spanning positions 741-743, and proceeds uninterrupted for almost the entire length of the genome. Such a cap-independent mechanism of internal initiation of translation determines that a long, extra-cistronic region extends between the 5'-end and the main open reading frame (ORF). We have identified 10 short ORFs initiated by the alternative translation initiation codons ACG, AUA, and GUG in the 5'-terminal extra-cistronic region (5'-ECR) of poliovirus RNA. Mutations introduced in all but one of these mini-cistrons had no effect on the infectivity of full-length cDNA clones, except when they modified a "hidden frame" spanning between nucleotides 157-192 (starting triplet: ACG). The mini cistron 157-192 is conserved in position, length and sequence in the genome of all types and strains of poliovirus. Adaptation to rat (Lansing) or mouse (variant of Sabin 2) is accompanied by a consistent pattern of changes in the primary sequence of this "hidden frame". The substitutions that abrogated the infectivity of cDNA clones were not expected to modify the predicted secondary structure of the 5'-ECR, and they did not alter the ability of the IRES to direct internal initiation of translation in bi-cistronic mRNAs. The infectivity of the mutated poliovirus cDNAs could be complemented in trans by co-transfecting the target COS-1 cells with an expression vector containing just the 5'-ECR of poliovirus type 2 (Lansing strain). The infectivity of poliovirus cDNA could be restored by co-transfecting short RNA transcripts of the wt 5'-ECR (Lansing), suggesting that the complementation in trans indeed requires the expression of the helper cDNA clone.

Animals↗