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Results for “RNA sequencing analysis”

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At least 1,459 records · Page 81Linked to original sources
PubMed2006

Differentially regulated micro-RNAs and actively translated messenger RNA transcripts by tumor suppressor p53 in colon cancer.

PURPOSE: The aim of this study was to investigate the role of p53 in regulating micro-RNA (miRNA) expression due to its function as a transcription factor. In addition, p53 may also affect other cellular mRNA gene expression at the translational level either via its mediated miRNAs or due to its RNA-binding function. EXPERIMENTAL DESIGN: The possible interaction between p53 and miRNAs in regulating gene expression was investigated using human colon cancer HCT-116 (wt-p53) and HCT-116 (null-p53) cell lines. The effect of p53 on the expression of miRNAs was investigated using miRNA expression array and real-time quantitative reverse transcription-PCR analysis. RESULTS: Our investigation indicated that the expression levels of a number of miRNAs were affected by wt-p53. Down-regulation of wt-p53 via small interfering RNA abolished the effect of wt-p53 in regulating miRNAs in HCT-116 (wt-p53) cells. Global sequence analysis revealed that over 46% of the 326 miRNA putative promoters contain potential p53-binding sites, suggesting that some of these miRNAs were potentially regulated directly by wt-p53. In addition, the expression levels of steady-state total mRNAs and actively translated mRNA transcripts were quantified by high-density microarray gene expression analysis. The results indicated that nearly 200 cellular mRNA transcripts were regulated at the posttranscriptional level, and sequence analysis revealed that some of these mRNAs may be potential targets of miRNAs, including translation initiation factor eIF-5A, eIF-4A, and protein phosphatase 1. CONCLUSION: To the best of our knowledge, this is the first report demonstrating that wt-p53 and miRNAs interact in influencing gene expression and providing insights of how p53 regulates genes at multiple levels via unique mechanisms.

Cell Line, Tumor↗
PubMed2004

RNAProfile: an algorithm for finding conserved secondary structure motifs in unaligned RNA sequences.

The recent interest sparked due to the discovery of a variety of functions for non-coding RNA molecules has highlighted the need for suitable tools for the analysis and the comparison of RNA sequences. Many trans-acting non-coding RNA genes and cis-acting RNA regulatory elements present motifs, conserved both in structure and sequence, that can be hardly detected by primary sequence analysis alone. We present an algorithm that takes as input a set of unaligned RNA sequences expected to share a common motif, and outputs the regions that are most conserved throughout the sequences, according to a similarity measure that takes into account both the sequence of the regions and the secondary structure they can form according to base-pairing and thermodynamic rules. Only a single parameter is needed as input, which denotes the number of distinct hairpins the motif has to contain. No further constraints on the size, number and position of the single elements comprising the motif are required. The algorithm can be split into two parts: first, it extracts from each input sequence a set of candidate regions whose predicted optimal secondary structure contains the number of hairpins given as input. Then, the regions selected are compared with each other to find the groups of most similar ones, formed by a region taken from each sequence. To avoid exhaustive enumeration of the search space and to reduce the execution time, a greedy heuristic is introduced for this task. We present different experiments, which show that the algorithm is capable of characterizing and discovering known regulatory motifs in mRNA like the iron responsive element (IRE) and selenocysteine insertion sequence (SECIS) stem-loop structures. We also show how it can be applied to corrupted datasets in which a motif does not appear in all the input sequences, as well as to the discovery of more complex motifs in the non-coding RNA.

3' Untranslated Regions↗
PubMed1997

Endogenous retroviral transcripts in myocytes from spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) is a well studied animal model of genetic hypertension and heart disease of unknown cause. With the use of differential display, a transcript was found in SHR myocardium that on sequence analysis was identified as an endogenous retrovirus (ERV). ERV gene expression was greater than an order of magnitude increased in adult SHR hearts relative to age-matched normotensive Wistar-Kyoto rats and was further increased in hearts from SHR with heart failure. In situ hybridization studies demonstrated that increased ERV gene expression was localized to myocardial cells. Increases in ERV transcripts in SHR suggest a possible link between inherited proviral elements and genetic hypertensive heart disease.

Age Factors↗
PubMedDate not supplied

Atypical Chryseobacterium meningosepticum and meningitis and sepsis in newborns and the immunocompromised, Taiwan.

From 1996 to 1999, 17 culture-documented systemic infections due to novel, atypical strains of Chryseobacterium meningosepticum occurred in two newborns and 15 immunocompromised patients in a medical center in Taiwan. All clinical isolates, which were initially misidentified as Aeromonas salmonicida by an automated bacterial identification system, were resistant to a number of antimicrobial agents. The isolates were characterized as atypical strains of C. meningosepticum by complete biochemical investigation, 16S rRNA gene sequence analysis, cellular fatty acid analysis, and random amplified polymorphic DNA fingerprinting (RAPD). This is the first report of a cluster of atypically variant strains of C. meningosepticum, which may be an emerging pathogen in newborns and the immunocompromised.

Adolescent↗
PubMed1995

Identification of two novel arginine binding DNAs.

RNA tertiary structure is known to play critical roles in RNA-protein recognition and RNA function. To examine how DNA tertiary structure might relate to RNA structure, we performed in vitro selection experiments to identify single-stranded DNAs that specifically bind arginine, and compared the results with analogous experiments performed with RNA. In the case of RNA, a motif related to the arginine binding site in human immunodeficiency virus TAR RNA was commonly found, whereas in the case of DNA, two novel motifs and no TAR-like structures were found. One DNA motif, found in approximately 40% of the cloned sequences, forms of hairpin structure with a highly conserved 10 nucleotide loop, whereas the second motif is especially rich in G residues. Chemical interference and mutagenesis experiments identified nucleotides in both motifs that form specific arginine binding sites, and dimethylsulfate footprinting experiments identified single guanine residues in both that are protected from methylation in the presence of arginine, suggesting possible sites of arginine contact or conformational changes in the DNAs. Circular dichroism experiments indicated that both DNAs undergo conformational changes upon arginine binding and that the arginine guanidinium group alone is responsible for binding. A model for the G-rich motif is proposed in which mixed guanine and adenine quartets may form a novel DNA structure. Arginine binding DNAs and RNAs should provide useful model systems for studying nucleic acid tertiary structure.

Arginine↗
PubMed2003

Saccharomyces cerevisiae Set1p is a methyltransferase specific for lysine 4 of histone H3 and is required for efficient gene expression.

Several homologues of the Drosophila Su(var)3-9 protein were recently reported to methylate lysine 9 of histone H3. Whereas this methylation signal served to recruit heterochromatin-associated proteins to transcriptionally silenced regions, histone H3 methylated at lysine 4 was associated with transcriptionally active areas of the genome. These findings suggested that the interplay between lysine 4 and 9 methylation is crucial in eukaryotic gene regulation. Here we provide evidence that Saccharomyces cerevisiae Set1p is a methyltransferase specific for lysine 4 of histone H3. In addition, we show that the absence of Set1p and lysine 4 methylation result in decreased transcription of approximately 80% of the genes in S. cerevisiae. Hierarchical clustering analysis of the set1(-) expression profile revealed a correspondence to that of a mad2(-) strain, suggesting that the transcriptional defect in the set1(-) strain may be due to changes in chromatin structure. These findings establish a central role for methylation of histone H3 lysine 4 in transcriptional regulation.

Amino Acid Sequence↗
PubMed2000

Differential gene expression in the activation and maturation of human monocytes.

Differential-display or RNA fingerprint was applied to identify genes differentially expressed in monocyte maturation induced by an immunomodulating peptide on human peripheral blood mononuclear cells. Two unknown sequences (06c22 and 06c71) and p21 protein (cyclin dependent kinase inhibitor) were repressed, and three genes activated: Cathepsin D, DRP2 (dihydropirimidinase related protein 2), and gp91phox (91-kDa subunit of citochrome b(558)). Phenotype of evolving monocytes was analyzed by flow cytometry and mRNA level of identified genes determined by reverse transcription-PCR. The expression pattern of identified genes seemed to correlate with different monocyte subsets, monocyte-derived cells, and expected functional changes. After peptide addition, immature monocytes were initially activated, increasing the expression of CD25, CD69, and HLA-DR markers. This was accompanied by repression of p21 and the two unknown sequences, along with the simultaneous activation of Cathepsin D and DRP2. Later, the differentiation marker CD16 rose, and gp91phox gene expression activated. Further maturation led certain monocytes to express marker CD23 and gp91phox expression to reach a maximum, while Cathepsin D and DRP2 dropped to preactivation levels. Results reflect part of the evolution of immature monocytes toward macrophages and monocyte-derived dendritic cell precursors.

Base Sequence↗
PubMed2002

Generation of RNA aptamers to the G-protein-coupled receptor for neurotensin, NTS-1.

G-protein-coupled receptors (GPCRs) are integral membrane proteins involved in signal transduction and constitute major drug targets for disease therapy. Aptamers, which are globular RNA or DNA molecules evolved to specifically bind a target, could represent a valuable tool with which to probe the role of such receptors in normal tissue and disease pathology and for cocrystallization with receptors for structure determination by X-ray crystallography. Using the bacterially expressed rat neurotensin receptor NTS-1 as an example, we describe a strategy for the generation of GPCR-specific RNA aptamers. Seven rounds of a "subtractive," paramagnetic bead-based selection protocol were used to enrich for neurotensin receptor-specific aptamers, while circumventing the evolution of aptamers reactive to minor protein contaminants. Representatives of each aptamer family were analyzed in Escherichia coli membrane nitrocellulose filter binding assays. Eight aptamers demonstrated specificity for the neurotensin receptor. One aptamer, P19, was characterized in detail and shown to bind to both the rat receptor and the human receptor with nanomolar affinity. P19 was also shown to interact with rat neurotensin receptor expressed in CHO cells, in both membrane preparations and intact cells. P19 represents the first example of a GPCR-specific RNA aptamer.

Animals↗
PubMedDate not supplied

Congenital transcobalamin II deficiency due to errors in RNA editing.

Transcobalamin II (TCII) is a plasma protein essential for the transport and cellular uptake of vitamin B12 (B12; cobalamin, Cbl). Congenital deficiency of functional TCII is an autosomal recessive genetic disorder that results in clinical B12 deficiency usually within several months following birth. In this report, we describe the molecular basis for TCII deficiency in two patients who developed a megaloblastic anemia in early infancy. The serum of both patients contained immunoreactive TCII that did not bind [57Co]Cbl. The fibroblasts from each patient secreted a similarly nonfunctional TCII, yet full-length TCII transcripts were identified by Northern blot. Overlapping cDNA fragments were generated by reverse transcription-polymerase chain reaction and several mutations were identified in the coding region of the cDNA, one of which was common to both patients. However, amplification of the corresponding regions of the gene from genomic DNA failed to identify these mutations. These findings were confirmed by replicate analyses and support the proposal that a variance in RNA editing is the likely mechanism for the mutations that resulted in the expression of a nonfunctional TCII protein in these patients.

Anemia, Megaloblastic↗
PubMed2001

A new protein expressed in bone marrow cells and osteoblasts with implication in osteoblast recruitment.

To study osteoblast recruitment from bone marrow cells, a rat femur cDNA library was screened by in situ hybridization for novel mRNA sequences that are frequently expressed in both marrow cells and osteoblasts. One isolated clone, called RP59, is described here. Northern blots indicated two bands of 2.6 and 2.8 kb in femur and spleen, tissues containing high amounts of immature mesenchymal cells, and no or little expression in other tissues. The cDNA sequence revealed a reading frame for a repetitive protein composed of arrays of 14-mers and phased phosphorylation sites. Antisera versus RP59 detected a single band of 90 kDa by Western blotting of femur extract. Immunohistochemistry indicated strong RP59 presence in the cytoplasm of bone marrow cells and weaker presence in nuclei of osteoblasts. Intermediate stages were found between strongly labeled, round, free bone marrow cells and weaker labeled, fibroblast-like young osteoblasts associated with bone matrix. These data indicated that marrow cells with high RP59 content were recruited into growing bone tissue. RP59 may help to study the transition of bone marrow cell to osteoblast in more detail.

Amino Acid Sequence↗
PubMed1996

Typing of Mycoplasma pneumoniae by nucleic acid sequence-based amplification, NASBA.

Nucleic acid sequence-based amplification, NASBA, is an isothermal amplification technique for nucleic acids and was used for typing a collection of 24 Mycoplasma pneumoniae strains. A set of primers was chosen from the 16S rRNA sequence alignment of Mycoplasma species. The nucleotide sequences of the (-)RNA amplicons were determined for M. pneumoniae strains M15/83 (type 1) and FH (type 2), and revealed a one-point difference at the 16S rRNA level between the two types. Based on this result, two type-specific probes were constructed. The probes were hybridized in solution with the amplified nucleic acids of 24 M. pneumoniae strains in an enzyme-linked gel assay (ELGA). The results obtained by NASBA-based typing are in agreement with the classification of the 24 M. pneumoniae strains into two types by other typing methods, confirming the reliability of this technique.

Bacterial Typing Techniques↗
PubMed2001

Enhanced accumulation of coronavirus defective interfering RNA from expressed negative-strand transcripts by coexpressed positive-strand RNA transcripts.

Expression of negative-strand murine coronavirus mouse hepatitis virus (MHV) defective interfering (DI) RNA transcripts in MHV-infected cells results in the accumulation of positive-strand DI RNAs (M. Joo et al., 1996, J. Virol. 70, 5769-5776). However, the expressed negative-strand DI RNA transcripts are poor templates for positive-strand DI RNA synthesis. The present study demonstrated that DI RNA accumulation from the expressed negative-strand DI RNA transcripts in MHV-infected cells was enhanced by the coexpression of complementary RNA transcripts that correspond to the 5' region of positive-strand DI RNA. The positive-strand RNA transcripts corresponding to the 5' end-most 0.7-2.0 kb DI RNA had a similar enhancement effect. The coexpressed positive-strand RNA transcripts lacking the leader sequence or those containing only the leader sequence failed to demonstrate this enhancement effect, demonstrating that the presence of the leader sequence in the coexpressed positive-strand RNA transcripts was necessary, but not sufficient, for the enhancement of DI RNA accumulation from the coexpressed negative-strand DI RNA transcripts. Negative-strand DI RNA transcripts that were coexpressed with the partial-length positive-strand RNA transcripts were no more stable than those expressed alone, suggesting that a higher stability of the expressed negative-strand RNA transcripts was an unlikely reason for the higher DI RNA accumulation in cells coexpressing two complementary DI RNA transcripts. Sequence analyses unexpectedly demonstrated that the leader sequence of the majority of accumulated DI RNAs switched to helper virus derived leader sequence, suggesting that enhancement of DI RNA accumulation was mediated by the efficient utilization of helper virus derived leader sequence for DI RNA synthesis. Furthermore, our data suggested that this leader switching, a type of homologous RNA-RNA recombination, occurred during positive-strand DI RNA synthesis and that MHV positive-strand RNA synthesis mechanism may have a preference toward recognizing double-stranded RNA structures over single-stranded negative-strand RNA to produce positive-strand DI RNAs.

5' Untranslated Regions↗
PubMed2003

Detection of RNA viruses in sudden infant death (SID).

Continuing previous work, reverse transcriptase polymerase chain reaction approaches for the detection of RNA viruses (influenza A+B, parainfluenza virus 3, respiratory syncytial virus) were developed. A total of 118 infant deaths, including 13 cases of non-natural death (nND), 78 cases of natural death (ND) without morphological signs of interstitial pneumonia (IP), 27 cases showing IP (ND+IP), were investigated using frozen lung tissue ( N=100) and paraffin-embedded material ( N=18). In five of the autopsy cases (ND+IP) the influenza B virus genome could be detected and the other types of viruses were completely negative. Together with previous results (detection of adenoviruses and cytomegaloviruses) in the same groups, the frequency of virus detection in the cases with IP was 48% compared with 14% in the ND without IP and 7% in the nND. Significant differences in the frequency of virus detection were also obtained when the cases were divided in SIDS and non-SIDS. The results obtained indicate an association between IP and some viruses and support the hypothesis that respiratory virus infections could act as trigger in sudden infant death.

Autopsy↗
PubMed2002

Occurrence and localization of uroguanylin in the aging human prostate.

Uroguanylin, a peptide hormone highly expressed in the gastrointestinal tract, is implicated in the regulation of epithelial salt and water transport processes. Since little is known about a possible role of uroguanylin in the reproductive system, we investigated for the first time the occurrence of this peptide in the human prostate using specimens of benign prostatic hyperplasia. Northern blot analyses detected a single uroguanylin transcript of approximately 600 bp in prostate RNA. The uroguanylin expression was further investigated by reverse transcriptase polymerase chain reaction of prostate RNA with uroguanylin-specific primers. Sequencing of the fragments obtained indicated the presence of a uroguanylin molecule with a sequence identical to its intestinal counterpart. Furthermore, in situ hybridization and immunohistochemistry revealed that uroguanylin mRNA and peptide are confined to epithelial cells of the prostate glands. Comparison with the distribution pattern of immunoreactivity for prostate-specific antigen (PSA) showed a high degree of colocalization of uroguanylin- and PSA-immunoreactive cells. In addition, by western blotting techniques we detected the presence of high molecular weight uroguanylin-immunoreactive material in prostatic fluid. In conclusion, our study indicates that the human prostate glands synthesize and secrete (pro-)uroguanylin. We hypothesize that this hormone may play a novel role in the male reproductive tract.

Aged↗
PubMed2006

Decreased expression of insulin-like growth factor binding protein 7 in human colorectal carcinoma is related to DNA methylation.

PURPOSE: Insulin-like growth factor binding protein 7 (IGFBP-7) is considered a tumor suppressor in various cancers, but its role in colorectal cancer (CRC) is still uncertain. The aims of this study were to analyze the IGFBP-7 expression, and explore the mechanism responsible for the inactivation of IGFBP-7 in CRC. METHODS: mRNA expression was studied by RT-PCR and Northern blot analysis of cultured cells. Methylation status was analyzed by treatment with 5-aza-2'-deoxycytidine followed by sequencing of PCR products of sodium bisulfite-treated genomic DNA. IGFBP-7 protein expression was evaluated by immunohistochemistry (IHC) on tissue microarrays. RESULTS: mRNA expression was lost in six out of eight CRC cell lines as compared to normal colon cells. DNA methylation was found in the region of exon 1 and intron 1 of IGFBP-7. In tumor tissue, 107 out of 279 samples showed a negative expression of IGFBP-7 by IHC, which was significantly associated with poor prognosis. The analysis of 37 paired cancerous and normal mucosa samples confirmed the downregulation in the tumors, but revealed variable basal expression levels of IGFBP-7 in normal mucosal samples. CONCLUSIONS: DNA methylation is a mechanism responsible for IGFBP-7 gene silencing providing a target for therapeutic intervention of this tumor suppressor gene.

Cell Line, Tumor↗
PubMed2006

Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection.

The gut microbiotas of zebrafish and mice share six bacterial divisions, although the specific bacteria within these divisions differ. To test how factors specific to host gut habitat shape microbial community structure, we performed reciprocal transplantations of these microbiotas into germ-free zebrafish and mouse recipients. The results reveal that communities are assembled in predictable ways. The transplanted community resembles its community of origin in terms of the lineages present, but the relative abundance of the lineages changes to resemble the normal gut microbial community composition of the recipient host. Thus, differences in community structure between zebrafish and mice arise in part from distinct selective pressures imposed within the gut habitat of each host. Nonetheless, vertebrate responses to microbial colonization of the gut are ancient: Functional genomic studies disclosed shared host responses to their compositionally distinct microbial communities and distinct microbial species that elicit conserved responses.

Animals↗
PubMed2006

Rapid sequencing of the non-coding regions of influenza A virus.

The non-coding regions (NCRs) of influenza A virus gene segments play a crucial role in the viral replication cycle. Although the NCRs are considered to be conserved, some variation does exist, that affects viral replication. Therefore, a rapid method to sequence the 5' and 3' NCRs was designed. This method is based on ligation of viral RNA, RT reactions and subsequent PCR with primersets consisting of a gene segment specific primer and a primer designed across the junction of the 5' and 3' ends. These PCR fragments can be sequenced directly without the need for cloning PCR fragments first. This method was used to sequence the NCRs of A/Bilthoven/16190/68 (H3N2) and A/Turkey/Turkey/1/05 (H5N1).

Influenza A Virus, H3N2 Subtype↗