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At least 1,423 records · Page 79Linked to original sources
PubMed2001

Identification of slide coagulase positive, tube coagulase negative Staphylococcus aureus by 16S ribosomal RNA gene sequencing.

AIMS: To ascertain the clinical importance of a strain of slide coagulase positive but tube coagulase negative Staphylococcus species isolated from the blood culture of a 43 year old patient with refractory anaemia with excessive blasts in transformation who had neutropenic fever. METHODS: The isolate was investigated phenotypically by standard biochemical methods using conventional biochemical tests and two commercially available systems, the Vitek (GPI) and API (Staph) systems. Genotypically, the 16S ribosomal RNA (rRNA) gene of the bacteria 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. RESULTS: Conventional biochemical tests did not reveal a pattern resembling a known Staphylococcus species. The Vitek system (GPI) showed that it was 94% S. simulans and 3% S. haemolyticus, whereas the API system (Staph) showed that it was 86.8% S. aureus and 5.1% S. warneri. 16S rRNA gene sequencing showed that there was a 0 base difference between the isolate and S. aureus, 28 base difference between the isolate and S. lugdunensis, 39 base difference between the isolate and S. schleiferi, 21 base difference between the isolate and S. haemolyticus, 41 base difference between the isolate and S. simulans, and 23 base difference between the isolate and S. warneri, indicating that the isolate was a strain of S. aureus. Vancomycin was subsequently prescribed and blood cultures taken four days after the start of treatment were negative. CONCLUSIONS: 16S rRNA gene sequencing was useful in ascertaining the clinical importance of the strain of slide coagulase positive but tube coagulase negative Staphylococcus species isolated from blood culture and allowing appropriate management.

Adult↗
PubMed2002

Complexity and species variation of the kidney-type glutaminase gene.

Increased expression of rat kidney-type glutaminase (KGA) during metabolic acidosis results from selective mRNA stabilization. This process is mediated by an 8-base AU-sequence that functions as a pH-response element (pHRE). LLC-PK1-FBPase+ cells, a pH-responsive porcine kidney cell line, express four distinct GA mRNAs. RNase H mapping indicated that three of the GA mRNAs are generated by use of alternative polyadenylation sites and are homologs of the rat KGA mRNA, while the fourth contains a different COOH-terminal coding and 3'-untranslated sequence. PCR cloning and sequencing established that the latter GA mRNA is the homolog of the human GAC mRNA. A rat GAC cDNA was also cloned from a rat kidney library. The 3'-untranslated regions of the GAC mRNAs, but not the porcine or human KGA mRNAs, contain identifiable pHREs. The human KGA gene spans 82 kb and is composed of 19 exons. The unique sequence from the hGAC cDNA is contained in a single exon. Thus in humans, alternative splicing of the initial transcript could produce two GA mRNAs, only one of which may be increased during acidosis.

Alternative Splicing↗
PubMed2005

Can Zipf's law be adapted to normalize microarrays?

BACKGROUND: Normalization is the process of removing non-biological sources of variation between array experiments. Recent investigations of data in gene expression databases for varying organisms and tissues have shown that the majority of expressed genes exhibit a power-law distribution with an exponent close to -1 (i.e. obey Zipf's law). Based on the observation that our single channel and two channel microarray data sets also followed a power-law distribution, we were motivated to develop a normalization method based on this law, and examine how it compares with existing published techniques. A computationally simple and intuitively appealing technique based on this observation is presented. RESULTS: Using pairwise comparisons using MA plots (log ratio vs. log intensity), we compared this novel method to previously published normalization techniques, namely global normalization to the mean, the quantile method, and a variation on the loess normalization method designed specifically for boutique microarrays. Results indicated that, for single channel microarrays, the quantile method was superior with regard to eliminating intensity-dependent effects (banana curves), but Zipf's law normalization does minimize this effect by rotating the data distribution such that the maximal number of data points lie on the zero of the log ratio axis. For two channel boutique microarrays, the Zipf's law normalizations performed as well as, or better than existing techniques. CONCLUSION: Zipf's law normalization is a useful tool where the Quantile method cannot be applied, as is the case with microarrays containing functionally specific gene sets (boutique arrays).

Algorithms↗
PubMed2001

A novel insulinoma tumor suppressor gene locus on chromosome 22q with potential prognostic implications.

The molecular mechanisms contributing to the tumorigenesis of insulinomas are still poorly understood. As moderate to high rates of LOH have been found on chromosome 22q in gastrinomas, we performed a finer deletion mapping study of chromosome 22q with 8 microsatellite markers in 15 insulinomas (4 malignant and 11 benign). Fourteen of 15 (93%) insulinomas revealed LOH on chromosome 22q, whereas the shortest region of overlap implicated a deletion of approximately 700 kb at 22q12.1-q12.2 with an LOH rate of up to 57% (8 of 14). Although the expressed sequence tag marker A006E25 that is localized in the hSNF5/INI1 gene on 22q11.2 revealed LOH in 50% of informative cases (7 of 14), no alterations in this gene could be identified by single strand conformational polymorphism analysis, direct DNA sequencing, or RNA expression analysis. Remarkably, the four malignant tumors showed a common deleted region between markers D22S345 and D22S1144 compared with none of the 11 benign insulinomas. The observed high frequency of chromosome 22q12 deletions in insulinomas is suggestive for a region compatible with harboring a tumor suppressor gene. The hSNF5/INI1 gene is most likely not the candidate gene, because no alterations could be identified. The distinct pattern of allelic loss identified in this chromosomal region appears to be an attractive candidate marker for further evaluation with regard to the discrimination between benign and malignant insulinomas.

Adult↗
PubMed2004

Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits.

The 18S rRNA of the small eukaryotic ribosomal subunit contains several expansion segments. Electron microscopy data indicate that two of the largest expansion segments are juxtaposed in intact 40S subunits, and data from phylogenetic sequence comparisons indicate that these two expansion segments contain complementary sequences that could form a direct tertiary interaction on the ribosome. We have investigated the secondary structure of the two expansion segments in the region around the putative tertiary interaction. Ribosomes from yeast, wheat, and mouse-three organisms representing separate eukaryotic kingdoms-were isolated, and the structure of ES3 and part of the ES6 region were analyzed using the single-strand-specific chemical reagents CMCT and DMS and the double-strand-specific ribonuclease V1. The modification patterns were analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The investigated sequences were relatively exposed to chemical and enzymatic modification. This is in line with their indicated location on the surface at the solvent side of the subunit. The complementary ES3 and ES6 sequences were clearly inaccessible to single-strand modification, but available for cleavage by double-strand-specific RNase V1. The results are compatible with a direct helical interaction between bases in ES3 and ES6. Almost identical results were obtained with ribosomes from the three organisms investigated.

Animals↗
PubMed2006

Efficient prediction of siRNAs with siRNArules 1.0: an open-source JAVA approach to siRNA algorithms.

RNAi interference and siRNA have become useful tools for investigation of gene function. However, the discovery that not all siRNA are equally efficient made necessary screens or design algorithms to obtain high activity siRNA candidates. Several algorithms have been published, but they remain inefficient, obscure, or commercially restricted. This article describes an open-source JAVA program that is surprisingly efficient at predicting active siRNAs (Pearson correlation coefficient r = 0.52, n = 526 siRNAs). Furthermore, this version 1.0 sets the stage for further improvement of the free code by the open-source community (http://sourceforge.net/).

Algorithms↗
PubMed1997

The protein cofactor allows the sequence of an RNase P ribozyme to diversify by maintaining the catalytically active structure of the enzyme.

To study the effect proteins have on the catalysis and evolution of RNA enzymes, we simulated evolution of RNase P catalytic M1 RNA in vitro, in the presence and absence of its C5 protein cofactor. In the presence of C5, functional M1 sequence variants (not catalytically active in the absence of C5) were selected in addition to those identical to M1. C5 maintains the catalytically active structure of the variants and allows for an enhanced spectrum of M1 molecules to function in the context of a ribonucleoprotein (RNP) complex. The generation of an RNP enzyme, requiring both RNA and protein components, from a catalytically active RNA molecule has implications for how modern RNP complexes evolved from ancestral RNAs.

Bacterial Proteins↗
PubMed1997

Mammalian prothymosin alpha links to tRNA in Escherichia coli cells.

Prothymosin alpha, a small and highly acidic nuclear protein related to cell proliferation, is known to be covalently attached to a small unidentified cytoplasmic RNA in mammalian cells. Here we demonstrate that recombinant rat prothymosin a links covalently to an RNA when overproduced in Escherichia coli cells. The RNA species of this complex is represented by a wide range of bacterial tRNAs. tRNA(Lys), tRNA(3Ser), tRNA(2Ile), and tRNA(mMet) were identified by sequencing. Prothymosin alpha appears to be linked to the 5' terminus of tRNA. tRNA attachment site lies close to the carboxy-terminus of prothymosin alpha. Furthermore, the carboxy-terminal peptide of prothymosin alpha is also competent for tRNA binding. The site of tRNA attachment coincides with the nuclear localization signal of prothymosin alpha, and tRNA binding might be expected to affect subcellular localization of this protein.

Amino Acid Sequence↗
PubMed1997

Ribosomal binding of modified tRNA anticodons related to thermal stability.

The physicochemical contributions of modified nucleosides to tRNA functions are not well understood. In order to determine the contributions of specific modifications to tRNA stability as well as to ribosomal binding, ten variously modified yeast tRNA(Phe) anticodon stems and loops (tRNA(Phe)AC) were synthesized. Thermal denaturation studies on these synthetic 17mers show dramatic stabilization (or destabilization) by the presence of the various naturally occurring nucleoside modifications. Adapting a novel molecular biology approach (initially pioneered by Moazed and Noller), the interactions of these variously modified anticodons with the E. coli 16S rRNA "P-site" residues are being quantitated. The binding (affinity) constant (kD) of the tRNA(Phe)AC to the 8 of the ten 16S rRNA nucleosides that interact with tRNA and synthetic anticodons are being examined. We postulate that the "stabilizing" modifications (m1G37, psi 39, and m5C40) in the presence of an "open loop" will dramatically increase the binding affinity of the tRNA(Phe)AC to the 30S E. coli ribosomal subunit when compared to unmodified tRNA(Phe)AC. On the other hand, "destabilizing" modifications are expected to reduce the binding affinity of the tRNA(Phe)AC to the E. coli 30S ribosomal subunit. The results from these experiments have demonstrated the importance of nucleoside modifications to tRNA stability and ribosomal binding affinity, and will relate the structural contributions of nucleoside modifications to tRNA function.

Anticodon↗
PubMed2025

Functional phenotyping of genomic variants using joint multiomic single-cell DNA-RNA sequencing.

Genetic variants (both coding and noncoding) can impact gene function and expression, driving disease mechanisms such as cancer progression. The systematic study of endogenous genetic variants is hindered by inefficient precision editing tools, combined with technical limitations in confidently linking genotypes to gene expression at single-cell resolution. We developed single-cell DNA-RNA sequencing (SDR-seq) to simultaneously profile up to 480 genomic DNA loci and genes in thousands of single cells, enabling accurate determination of coding and noncoding variant zygosity alongside associated gene expression changes. Using SDR-seq, we associate coding and noncoding variants with distinct gene expression in human induced pluripotent stem cells. Furthermore, we demonstrate that in primary B cell lymphoma samples, cells with a higher mutational burden exhibit elevated B cell receptor signaling and tumorigenic gene expression. SDR-seq provides a powerful platform to dissect regulatory mechanisms encoded by genetic variants, advancing our understanding of gene expression regulation and its implications for disease.

Humans↗
PubMed2005

Suppression of Epstein-Barr nuclear antigen 1 (EBNA1) by RNA interference inhibits proliferation of EBV-positive Burkitt's lymphoma cells.

PURPOSE: Epstein-Barr virus (EBV) is associated with the development of several lymphoid and epithelial malignancies, including Burkitt's lymphoma. The EBV latent protein, EBV Nuclear Antigen 1 (EBNA1), is detectable in almost all types of EBV-associated tumors and is essential for replication and maintenance of the latent episome of EBV. We here examined whether the RNA interference (RNAi) technique could be employed to suppress expression of EBNA1 in EBV-positive Burkitt's lymphoma cells. METHODS: A Raji cell line expressing small hairpin RNAs (shRNAs) against EBNA1 was established and EBNA1 mRNA level was determined by real-time RT-PCR analysis. We investigated the effects of EBNA1 silence on lymphoma cell growth and cell cycle progression. RESULTS: Transfection of an EBNA1 RNAi plasmid resulted in substantial loss of EBNA1 mRNA and significantly inhibited proliferation of Raji cells relative to the control plasmid case. Suppression of EBNA1 was also associated with downregulation of EBV oncogene EBNA2, a decreased PCNA labeling index and increased G0/G1 fraction in cell cycle analysis. CONCLUSIONS: These findings point to potential therapeutic applications for vector-mediated siRNA delivery to control EBV-associated malignant disorders.

Apoptosis↗
PubMed1997

Proteome research: complementarity and limitations with respect to the RNA and DNA worlds.

A methodological overview of proteome analysis is provided along with details of efforts to achieve high-throughput screening (HTS) of protein samples derived from two-dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequence information, mass spectrometry has a key role to play in achieving HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700-1000 protein (genes) per day to batteries of mass spectrometers or liquid chromatography (LC)-based analyses are well advanced, as are efforts to produce high density gridded arrays containing > 1000 proteins on a single matrix assisted laser desorption ionisation/time-of-flight (MALDI-TOF) sample stage. High sensitivity HTS of proteins is proposed by employing principally mass spectrometry in an hierarchical manner: (i) MALDI-TOF-mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analysis of peptides with respect to predicted fragmentation patterns or by sequence tagging; and (iii) ESI/MS/MS for peptide sequencing. Genomic sequences when complemented with information derived from hybridisation assays and proteome analysis may herald in a new era of holistic cellular biology. The current preoccupation with the absolute quantity of gene-product (RNA and/or protein) should move backstage with respect to more molecularly relevant parameters, such as: molecular half-life; synthesis rate; functional competence (presence or absence of mutations); reaction kinetics; the influence of individual gene-products on biochemical flux; the influence of the environment, cell-cycle, stress and disease on gene-products; and the collective roles of multigenic and epigenetic phenomena governing cellular processes. Proteome analysis is demonstrated as being capable of proceeding independently of DNA sequence information and aiding in genomic annotation. Its ability to confirm the existence of gene-products predicted from DNA sequence is a major contribution to genomic science. The workings of software engines necessary to achieve large-scale proteome analysis are outlined, along with trends towards miniaturisation, analyte concentration and protein detection independent of staining technologies. A challenge for proteome analysis into the future will be to reduce its dependence on two-dimensional (2-D) gel electrophoresis as the preferred method of separating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing differential display, high density expression arrays, expressed sequence tags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular biology.

Animals↗
PubMed2005

Skewed T-cell receptor BV14 and BV16 expression and shared CDR3 sequence and common sequence motifs in synovial T cells of rheumatoid arthritis.

T-lymphocytes play an important role in rheumatoid arthritis (RA). In this study, we evaluated the hypothesis that common T-cell receptor (TCR) structural features may exist among infiltrating T cells of different RA patients, if the TCR repertoire is shaped by interaction with common self or microbial antigens in the context of susceptible HLA genes in RA. Synovial lesion tissue (ST), synovial fluid (SF) and blood specimens from RA patients and controls were analyzed for TCR V gene repertoire by real-time PCR. There was highly skewed BV14 and BV16 usage in synovial T cells of RA as opposed to those of controls, which was accompanied with a trend for correlation between skewed BV16 and DRB1(*)0405. Immunoscope analysis of the V-D-J region of ST-derived T cells demonstrated oligoclonal and polyclonal expansion of BV14(+) and BV16(+) T cells. Detailed characterization using specific BV and BJ primers further revealed common clonotypes combining the same BV14/BV16, BJ and CDR3 length. DNA cloning and sequence analysis of the clonotypes confirmed identical CDR3 sequences and common CDR3 sequence motifs among different RA patients. The findings are important in the understanding of BV gene skewing and CDR3 structural characteristics among synovial infiltrating T cells of RA.

Arthritis, Rheumatoid↗
PubMedDate not supplied

[Real time PCR methodology for quantification of nucleic acids].

The polymerase chain reaction (PCR) has become an essential tool for molecular biologists and its introduction into nucleic acids detection systems has revolutionized the quantitative analysis of DNA and RNA. The technique has rapidly evolved over the last few years and the growing interest in quantitative applications of the PCR has favoured the development of real-time quantitative PCR. In this paper, we review, after presentation of the theorical aspects of PCR, the basic principles of real-time PCR with the introduction of the concept of threshold cycle. More precisely, we describe the novel assay formats that greatly simplify the protocols used for the detection of specific nucleic acids. We focus on the actual four technologies that enable sequence detection in a closed tube and that are SYBR Green I, TaqMan probes, Hybridization probes and Molecular Beacon probes. We then discuss the different quantification strategies in real time PCR and compare the competiting instruments on the market. The most important real-time PCR applications in clinical biology are also described.

Clinical Protocols↗
PubMed2005

Transcriptome response following administration of a live bacterial vaccine in Atlantic salmon (Salmo salar).

Antibacterial responses have been studied in Atlantic salmon following an acute intra peritoneal injection of a genetically attenuated (aroA(-)) strain of Aeromonas salmonicida known to elicit protective immunity. Three tissues were studied for transcriptional changes, the liver, head kidney and the gill. RNA was collected from fish 6, 12, 24 and 48 h following infection or at the same time points from fish injected with PBS as non-infected control. PCR-select cDNA subtraction libraries were constructed from pooled 24 and 48 h post infection RNA to identify up-regulated mRNAs. One thousand four hundred and eighty six cDNA clones were sequenced from enriched cDNA libraries, of which 71% had significant homologies to known functional proteins. Many of these clones have previously been un-characterised in Atlantic salmon. A salmonid cDNA microarray was used to further analyse the gene expression profile as the library construction in itself does not answer the dynamics of the response. The greatest increase in expression identified in the array analysis was a liver antibacterial peptide, hepcidin that was increased 11-fold following the challenge. A panel of clones were chosen for semiquantitative reverse transcriptase PCR from all time points sampled. These results indicated there were both temporal differences and tissue differences in the transcriptional response to bacterial exposure, potentially of relevance to the establishment of protection.

Aeromonas salmonicida↗
PubMed2006

Non-invasive genetic diagnosis of male infertility using spermatozoal RNA: KLHL10 mutations in oligozoospermic patients impair homodimerization.

Infertility affects an estimated 7% of men worldwide, nearly a quarter of whom are diagnosed as idiopathic. The genetic etiologies of idiopathic male infertility are unknown, partly due to lack of simple diagnostic techniques. Moreover, the transmission risk of such genetic defects to offspring born from assisted reproductive techniques is increasingly becoming a concern for physicians and infertile couples. We explored the feasibility of obtaining full-length mRNAs from transcriptionally inert human spermatozoa in semen as a non-invasive diagnostic tool for identifying germline mutations in candidate infertility-associated genes. The efficacy of reverse-transcription PCR on spermatozoal RNA from infertile patients with wide-ranging sperm concentrations varied between 91 and 99% for multiple haploid germ cell-expressed genes. Using this methodology, we identified seven oligozoospermic patients with missense and splicing mutations in the germ cell-specific gene, KLHL10. Three of 270 (1.1%) severely oligozoospermic patients (<10(6) sperm/ml) harbor KLHL10 alterations that were absent in 394 controls and exhibited significant association (P=0.02). Two KLHL10 missense mutations (A313T and Q216P) resulted in impaired homodimerization with the wild-type protein in yeast interaction assays, suggesting a functional deficiency. This study demonstrates the utility of this approach for analysis of haploid germ cell-expressed genes regulating post-meiotic events including sperm maturation, motility and fertilization. The development of non-invasive techniques to analyze genetic defects of human spermatogenesis, previously possible only with invasive testis biopsies, provides important diagnostic and therapeutic implications for reproductive medicine.

Amino Acid Sequence↗
PubMed1989

Sites of initiation and pausing in the Escherichia coli rnpB (M1 RNA) transcript.

DNA sequences affecting the transcription of the Escherichia coli rnpB transcript encoding the catalytic M1 RNA subunit of RNase P have been analyzed. Previous work (Motamedi, H., Lee, Y., and Schmidt, F.J.) (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3959-3963) identified S1 nuclease protection products corresponding to transcripts originating upstream of the M1 RNA gene. Sequence analysis of the upstream region of rnpB identified three regions homologous to the E. coli consensus promoter sequence. In the present work, analysis of in vitro transcription products by S1 nuclease mapping indicated that all three promoter homologies were capable of directing transcription. The nearest promoter, P-1, was approximately 100 times more active than either of the upstream homologies P-2 and vivo experiments, wherein the three promoter homologies preceding rnpB were cloned into the galactokinase (GalK) expression vector pKO100. The promoter homology nearest to the M1 RNA gene directed the synthesis of GalK above background. The upstream promoter homologies did not direct the synthesis of GalK at a level greater than 1% of transcription from P-1. Deletion of the upstream homologies did not affect transcription from P-1. It was concluded that P-1 is responsible for essentially all M1 RNA transcription in vivo. Single-round transcription experiments in vitro detected strong NusA-independent transcriptional pausing at nucleotides +118 and +121 of the rnpB transcript, with a half-life of 27 s when concentrations of NTPs were near the average Km for elongation. Pausing at these points was eliminated by substitution of ITP for GTP in the transcription mixture. This suggests that pausing is dependent on transcript secondary structure. The position of pausing corresponds to that of a dual stem and loop structure of M1 RNA which has recently been proposed on the basis of phylogenetic sequence analysis.

Base Sequence↗
PubMed2005

Characterization of an exchangeable gene trap using pU-17 carrying a stop codon-beta geo cassette.

We have developed a new exchangeable gene trap vector, pU-17, carrying the intron-lox71-splicing acceptor (SA)-beta geo-loxP-pA-lox2272-pSP73-lox511. The SA contains three stop codons in-frame with the ATG of beta galactosidase/neomycin-resistance fusion gene (beta geo) that can function in promoter trapping. We found that the trap vector was highly selective for integrations in the introns adjacent to the exon containing the start codon. Furthermore, by using the Cre-mutant lox system, we successfully replaced the beta geo gene with the enhanced green fluorescent protein (EGFP) gene, established mouse lines with the replaced clones, removed the selection marker gene by mating with Flp-deleter mice, and confirmed that the replaced EGFP gene was expressed in the same pattern as the beta geo gene. Thus, using this pU-17 trap vector, we can initially carry out random mutagenesis, and then convert it to a gain-of-function mutation by replacing the beta geo gene with any gene of interest to be expressed under the control of the trapped promoter through Cre-mediated recombination.

Animals↗