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PubMed2003

Identification of differentially expressed genes in keratoconus epithelium analyzed on microarrays.

PURPOSE: The present study was conducted to investigate differential gene expression in individual samples derived from fresh-frozen human keratoconus and normal corneal epithelium, using gene microarrays. METHODS: Total RNA was extracted (11 keratoconus and 8 normal samples), labeled, and hybridized to microarrays (GeneChip; Affymetrix, Inc., Santa Clara, CA). GeneChip data were validated by verifying the expression profiles of 10 genes by real-time PCR and by recalculation using dChip software (Wong Laboratory, Department of Biostatistics, Harvard School of Public Health, Boston, MA). Furthermore, 3 of the 10 encoded proteins were stained by immunohistochemistry. RESULTS: In comparison with normal cornea, the expression of 471 of the 5600 genes on the microarrays was changed in the keratoconus samples. This number was reduced to 47 with increased expression and 9 with decreased expression when more stringent selection parameters were applied. These genes are believed to be involved in keratoconus. Two of the candidate genes, lysyl oxidase and tissue inhibitor of metalloproteinase 3, are known to be involved in other eye diseases. Expression profiles were reproduced with the software dChip (Wong Laboratory) and real-time PCR. Increases in keratin 6 and 13 were also detected at the protein level. CONCLUSIONS: Keratoconus epithelium appears to be characterized by massive changes of the cytoskeleton, reduced extracellular matrix remodeling, altered transmembrane signaling, and modified cell-to-cell and cell-to-matrix interactions. Validation of gene expression with dChip analysis and real-time PCR indicates GeneChip to be a valid technique for investigation of epithelium from single dissected corneal samples. Association between alterations at RNA and protein levels was observed for some of the tested candidates.

Adult↗
PubMed1993

Nucleotide sequence analysis of an Andean potato mottle virus middle component RNA cDNA clone: comparisons of the encoded proteins with those of other comoviruses.

Andean potato mottle virus (APMV) is a comovirus whose genomic structure consists of two plus-strand RNA molecules (M- and B-RNA). Here we report the nucleotide sequence analysis of an APMV M-RNA cDNA clone with 3,669 nucleotide (nt) residues, exclusive of the polyadenylate at the 3' end, covering approximately 99% of the APMV M-RNA. The first initiation codon in register translates from nt 194 to 3185 a polyprotein of 997 amino acid (aa) residues. A second initiation codon in register, beginning at nt position 416, translates a polyprotein of 923 aa. The cleavage sites used in the processing of polyprotein were identified in the long open reading frame by N-terminal microsequencing of the large coat protein (LCP) and the small coat protein (SCP). These dipeptide cleavage sites are Q/M for the LCP and Q/F for the SCP. In a comparison of the deduced APMV polyprotein aa sequence with those of four other comoviruses, the coding regions for the putative movement protein, LCP and SCP, were found similar in length in all five species. Multiple alignment of the M-RNA sequences for each of the three genes from the five comoviruses revealed different degrees of homology. APMV was always the least homologous of the five comoviruses, showing significant aa substitutions in positions where the other comoviruses have identical residue or conservative substitutions.

Amino Acid Sequence↗
PubMed2001

Microarrays for studying the host transcriptional response to microbial infection and for the identification of host drug targets.

It is essential to understand the molecular basis of a host's response to microbial infection so that disease and tissue damage can be prevented. Modulation of host RNA expression is a critical set of molecular changes that occur upon infection. Global analysis of gene expression should provide an understanding of host RNA transcriptional changes that occur upon host-pathogen interaction. This series of articles focuses on the use of microarrays for analyzing the transcriptional response of a host to microbial infection and for drug target identification.

Animals↗
PubMed2005

Cooperativity of paired oligonucleotide probes for microarray hybridization assays.

Synthetic DNA probes attached to microarrays usually range in length from 25 to 70 nucleotides. There is a compromise between short probes with lower sensitivity, which can be accurately synthesized in higher yields, and long probes with greater sensitivity but lower synthesis yields. Described here are microarrays printed with spots containing a mixture of two short probes, each designed to hybridize at noncontiguous sites in the same targeted sequence. We have shown that, for a printed microarray, mixed probe spots containing a pair of 30mers show significantly greater hybridization than spots containing a single 30mer and can approach the amount of hybridization to spots containing a 60mer or a 70mer. These spots with mixed oligonucleotide probes display cooperative hybridization signals greater than those that can be achieved by either probe alone. Both the higher synthesis yields of short probes and the greater sensitivity of long oligonucleotides can be utilized. This strategy provides new design options for microarray hybridization assays to detect RNA abundance, RNA splice variants, or sequence polymorphisms.

Bacillus subtilis↗
PubMed2003

Protection of lungs from hyperoxic injury: gene expression analysis of cyclosporin A therapy.

We have previously shown that cyclosporin A (CsA), an inhibitor of protein phosphatase 2B (calcineurin), attenuates hyperoxia-induced reductions in murine lung compliance. CsA protected against hyperoxia-induced changes in neutrophil infiltration, capillary congestion, edema, and hyaline membrane formation. Gene expression studies were conducted to identify the gene expression patterns underlying the protective effects of CsA during hyperoxic lung injury. After 72 h of simultaneous treatment with >95% oxygen and CsA (50 mg x kg(-1) x day(-1)), RNA was isolated from murine lungs. RNA from treated and untreated lungs was reverse transcribed to cDNA, competitively hybridized, and used to probe 8,734 complimentary DNAs on the Incyte mouse GEM 1 array. Several known genes and expressed sequence tags (ESTs) showed increased (GenBank accession numbers: AA125385, AA241295, W87197, syntaxin, and cyclin G) or decreased [AA036517, AA267567, AA217009, W82577, uteroglobin, stromal cell-derived factor 1, and surfactant protein C (SP-C)] expression after hyperoxia. Hyperoxia-stimulated reductions in SP-C gene expression were confirmed through Northern blot analysis. The increase in gene expression of one expressed sequence tag (AA125385) with hyperoxia was reversed by CsA treatment. Sequence data demonstrated that this EST has high homology to murine cyclin B1. Western blot analysis did not demonstrate any changes in distal lung cyclin B1 expression after hyperoxia. Protein expression of cyclin B1 in the distal lung was observed in the endothelial cells, bronchiolar epithelial cells, and both the type I and type II alveolar epithelial cells. Further analysis of cyclin B1 may elucidate the protective actions of CsA in hyperoxic injury.

Animals↗
PubMed1987

Characterization of murine cDNAs encoding P-57, a neural-specific calmodulin-binding protein.

Polyclonal antibodies raised against bovine brain P-57, a neural-specific calmodulin-binding protein, were used to isolate murine cDNAs encoding P-57 from murine brain cDNA libraries in the expression vector lambda gt 11. Two of the overlapping clones contained an open reading frame encoding a polypeptide of 227 amino acid residues (predicted Mr, 23,635), a 163-nucleotide 5'-untranslated sequence, and a 403-nucleotide 3'-untranslated sequence. Hydrophobicity analysis of the predicted polypeptide indicated the lack of any considerable stretch of hydrophobic residues that may span the membrane. This is consistent with prior data suggesting that P-57 exists in a soluble, as well as a membrane-associated, form. The predicted amino acid composition of P-57 is rather unusual in that it is highly enriched in alanine, glutamic acid, and lysine residues, and relatively enriched with proline residues. This amino acid composition accounts for the very low helical content of the predicted polypeptide. A search of the GenBank and EMBL sequence data banks (GenBank Inc., release 44.0 (August, 1986); European Molecular Biology Library, release 8.0 (April, 1986] indicated that the P-57 nucleotide sequence shows no significant homology to any reported sequences. RNA blot analysis of brain, heart, liver, and testes RNA revealed that cDNAs detect P-57 transcripts of 1.5 kilobases in brain, but not in other tissues. Genome blot analysis was consistent with P-57 being encoded by a single or small number of genes. These data demonstrate that the accumulation of this novel calmodulin-binding polypeptide in neural tissue is controlled primarily at the level of RNA abundance.

Amino Acid Sequence↗
PubMed2005

An evaluation of linear RNA amplification in cDNA microarray gene expression analysis.

DNA microarray is becoming a common tool across a broad range of disciplines, particularly in the basic and clinical biomedical sciences. However, the limitation of this technology is the requirement for relatively large amount of RNA for labeling and hybridization. T7-based linear amplification technique can overcome this limitation and enables consumption of very low amount of samples. In this study, we utilized a low RNA input fluorescent linear amplification kit from Agilent to amplify 0.2mug total RNA and compared the gene expression profiles generated from amplified aRNA and total RNA. Our results demonstrated that nanograms total RNA can be amplified reproducibly with this protocol and can generate gene expression profiles comparable with unamplified total RNA. Furthermore, quantitative real-time PCR measurements of a set of 10 genes showed good correlation with amplified aRNA arrays.

Evaluation Studies as Topic↗
PubMed2005

A heuristic approach for detecting RNA H-type pseudoknots.

MOTIVATION: RNA H-type pseudoknots are ubiquitous pseudoknots that are found in almost all classes of RNA and thought to play very important roles in a variety of biological processes. Detection of these RNA H-type pseudoknots can improve our understanding of RNA structures and their associated functions. However, the currently existing programs for detecting such RNA H-type pseudoknots are still time consuming and sometimes even ineffective. Therefore, efficient and effective tools for detecting the RNA H-type pseudoknots are needed. RESULTS: In this paper, we have adopted a heuristic approach to develop a novel tool, called HPknotter, for efficiently and accurately detecting H-type pseudoknots in an RNA sequence. In addition, we have demonstrated the applicability and effectiveness of HPknotter by testing on some sequences with known H-type pseudoknots. Our approach can be easily extended and applied to other classes of more general pseudoknots. AVAILABILITY: The web server of our HPknotter is available for online analysis at http://bioalgorithm.life.nctu.edu.tw/HPKNOTTER/ CONTACT: cllu@mail.nctu.edu.tw, chiu@cc.nctu.edu.tw

Algorithms↗
PubMed1997

Influenza virus M1 protein binds to RNA through its nuclear localization signal.

The RNA-binding activity of influenza A virus M1 protein was studied by cross-linking the protein to viral RNA followed by sequence analysis of the oligoribonucleotide bound to the protein as well as sequence analysis of the M1 peptide bound to the RNA. M1 was found to bind to RNA without any RNA sequence specificity, as verified in a series of filter-binding experiments using a large variety of nucleic acids including DNA. The peptide sequence that bound to the RNA was the RKLKR nuclear localization signal of M1. Site-specific mutagenesis of recombinant M1 showed that most of the basic residues in that region had to be mutated in order to inhibit RNA-binding. We also constructed an M1 mutant that no longer bound to RNA but which was still able to inhibit the in vitro transcription activity of isolated viral ribonucleoprotein, albeit to a lower extent. Mutation of the zinc-binding sequence had no effect on RNA-binding or transcription-inhibition activity.

Amino Acid Sequence↗
PubMed2004

Gene expression analysis in platelets from a single donor: evaluation of a PCR-based amplification technique.

BACKGROUND: Genetic analysis of platelet mRNA may facilitate the diagnosis of disorders affecting the megakaryocytic-platelet lineage. Its use, however, is limited by the exceptionally small yield of platelet mRNA and the risk of leukocyte contamination during platelet preparation. METHODS: We depleted platelet suspensions of leukocytes by filtration and used a PCR-based RNA amplification step [switching mechanism at the 5' end of RNA templates (SMART)]. We tested the reliability and precision of the RNA amplification procedure by use of real-time PCR to measure quantities of specific transcripts: von Willebrand factor (vWF), A-subunit of coagulation factor XIII (F13A), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Microarray analysis was performed on platelet RNA with and without amplification. RESULTS: Microgram quantities of platelet-specific cDNAs were produced from as little as 50 ng of total platelet RNA or 40 mL of whole blood. At cycle numbers <16, amplification of all transcripts tested was exponential with slightly more efficient amplification of low-abundance transcripts. Expression profiling of 9850 genes gave identical results for 9815 genes (1576 positive/8239 negative). Eight transcripts failed to be amplified by the SMART procedure. Expression of vWF, F13A, and GAPDH transcripts showed only minor day-to-day variations in three healthy individuals. CONCLUSION: The proposed protocol makes extremely small amounts of platelet RNA available for gene expression analysis in single patients.

Blood Platelets↗
PubMed2004

A rapid filtration apparatus for harvesting cells under controlled conditions for use in genome-wide temporal profiling studies.

Gene expression can respond rapidly to changes in environmental conditions. To effectively monitor these responses, we built a filtration apparatus that allows for the rapid harvesting and processing of moderate volumes of yeast cells under controlled atmospheric conditions (e.g., anaerobic conditions). Harvesting by filtration offers several advantages over that by centrifugation, especially when rapid, repeated sampling of dilute cultures is required. A number of different filter membranes, including cellulose acetate, mixed esters of cellulose, regenerated cellulose, polycarbonate, and polyvinylidene fluoride, were assayed for harvest efficiency and the quality of RNA obtained by hot-phenol extraction from cells directly adhering to the membranes. To determine the suitability of the RNA for microarray analyses, we quantified both cDNA yield from reverse transcription and the indirect coupling of Cyan dyes. In general, filtration times, cell yields, and RNA quality were similar among the filters examined, although some media components (e.g., antifoam) can cause fouling of smaller-pore-sized filters. Thus, choice of a membrane will depend on the particular medium, ease of filter handling, or on other experimental considerations. We routinely use this filtration apparatus with Osmonics 1.2 microm cellulose acetate filters for isolating RNA for genome-wide temporal profiling analyses.

Cell Culture Techniques↗
PubMed2006

Detection and structural analysis of R-loops.

R-loops are structures where an RNA strand is base paired with one DNA strand of a DNA duplex, leaving the displaced DNA strand single-stranded. Stable R-loops exist in vivo at prokaryotic origins of replication, the mitochondrial origin of replication, and mammalian immunoglobulin (Ig) class switch regions in activated B lymphocytes. All of these R-loops arise upon generation of a G-rich RNA strand by an RNA polymerase upon transcription of a C-rich DNA template strand. These R-loops are of significant length. For example, the R-loop at the col E1 origin of replication appears to be about 140 bp. Our own lab has focused on class switch regions, where the R-loops can extend well over a kilobase in length. Here, methods are described for detection and analysis of R-loops in vitro and in vivo.

Animals↗
PubMed2006

Suspension array analysis of 16S rRNA from Fe- and SO(4)2- reducing bacteria in uranium-contaminated sediments undergoing bioremediation.

A 16S rRNA-targeted tunable bead array was developed and used in a retrospective analysis of metal- and sulfate-reducing bacteria in contaminated subsurface sediments undergoing in situ U(VI) bioremediation. Total RNA was extracted from subsurface sediments and interrogated directly, without a PCR step. Bead array validation studies with total RNA derived from 24 isolates indicated that the behavior and response of the 16S rRNA-targeted oligonucleotide probes could not be predicted based on the primary nucleic acid sequence. Likewise, signal intensity (absolute or normalized) could not be used to assess the abundance of one organism (or rRNA) relative to the abundance of another organism (or rRNA). Nevertheless, the microbial community structure and dynamics through time and space and as measured by the rRNA-targeted bead array were consistent with previous data acquired at the site, where indigenous sulfate- and iron-reducing bacteria and near neighbors of Desulfotomaculum were the organisms that were most responsive to a change in injected acetate concentrations. Bead array data were best interpreted by analyzing the relative changes in the probe responses for spatially and temporally related samples and by considering only the response of one probe to itself in relation to a background (reference) environmental sample. By limiting the interpretation of the data in this manner and placing it in the context of supporting geochemical and microbiological analyses, we concluded that ecologically relevant and meaningful information can be derived from direct microarray analysis of rRNA in uncharacterized environmental samples, even with the current analytical uncertainty surrounding the behavior of individual probes on tunable bead arrays.

Bacteria↗
PubMed2003

Validation of cDNA microarray gene expression data obtained from linearly amplified RNA.

BACKGROUND: DNA microarray technology has permitted the analysis of global gene expression profiles for several diseases, including cancer. However, standard hybridisation and detection protocols require micrograms of mRNA for microarray analysis, limiting broader application of this technology to small excisional biopsies, needle biopsies, and/or microdissected tissue samples. Therefore, linear amplification protocols to increase the amount of RNA have been developed. The correlation between the results of microarray experiments derived from non-amplified RNA and amplified samples needs to be evaluated in detail. METHODS: Total RNA was amplified and replicate hybridisation experiments were performed with linearly amplified (aRNA) and non-amplified mRNA from tonsillar B cells and the SUDHL-6 cell line using cDNA microarrays containing approximately 4500 genes. The results of microarray differential expression using either source of RNA (mRNA or aRNA) were also compared with those found using real time quantitative reverse transcription polymerase chain reaction (QRT-PCR). RESULTS: Microarray experiments using aRNA generated reproducible data displaying only small differences to data obtained from non-amplified mRNA. The quality of the starting total RNA template and the concentration of the promoter primer used to synthesise cDNA were crucial components of the linear amplification reaction. Approximately 80% of selected upregulated and downregulated genes identified by microarray analysis using linearly amplified RNA were confirmed by QRT-PCR using non-amplified mRNA as the starting template. CONCLUSIONS: Linear RNA amplification methods can be used to generate high fidelity microarray expression data of comparable quality to data generated by microarray methods that use non-amplified mRNA samples.

Gene Amplification↗
PubMed1993

Serum amyloid-P component of the Armenian hamster: gene structure and comparison with structure and expression of the SAP gene from Syrian hamster.

Serum amyloid P (SAP), a phylogenetically conserved pentraxin, is an integral component of all amyloid deposits. Regulation of expression of SAP gene expression is quite different in two related hamster species. In Syrian hamsters, the resting serum levels of SAP are determined by gender, and the direction of alteration following inflammation is divergent. In Armenian hamsters, SAP is not a prominent acute-phase reactant and there is no gender dimorphism of expression. The structure and expression of the SAP gene of the Armenian hamsters was investigated by isolation of genomic clones, nucleotide sequence analysis, and RNA studies. The gene structure of Armenian hamster SAP is similar to the genes of all other pentraxins studied. While the upstream regions of the SAP genes of Syrian and Armenian hamsters are quite similar, important differences in potential enhancer sites have been recognized by comparing the corresponding sequences of SAP genes from both species. Little alteration in hepatic levels of transcripts encoding SAP or CRP, the other pentraxin, were noted following administration of lipopolysaccharide to Armenian hamsters. This relative lack of response occurred despite a marked acute phase reaction documented for serum amyloid A mRNA levels.

Amino Acid Sequence↗
PubMed1998

Generation of infectious pancreatic necrosis virus from cloned cDNA.

We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of the Birnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3'-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3' end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.

Animals↗
PubMed2004

Microarray analysis of differentiation-specific gene expression during 3T3-L1 adipogenesis.

During cellular differentiation and development, it is recognized that many complex molecular mechanisms as well as precise patterns of differentially expressed genes occur in directing precursor cells toward a given lineage. Using microarray-based technology, we examined gene expression across the course of 3T3-L1 adipocyte differentiation. Total cellular RNA was isolated at times 0, 2, 8, 16, 24, 48, and 96 h following treatment with either standard hormonal inducers of differentiation; insulin, dexamethasone, isobutylmethylxanthine (IDX), or IDX plus trichostatin A (TsA), a histone deacetylase inhibitor and potent adipogenic inhibitor. cRNA was synthesized from cellular RNA and hybridized to high density Affymetrix MG_U74Av2 microarray gene chips containing 12,488 cDNA/Expressed Sequence Tags (ESTs) probe sets. From the IDX-only treated cells, all probe sets that were either unchanged or differentially expressed less than 2-fold throughout differentiation with respect to time 0 preadipocytes were excluded from further analyses. This selection resulted in a net of 1686 transcripts, 859 were increased in expression, and 827 were decreased in expression at least 2-fold across differentiation. To focus in on genes that were more specific to differentiation, the same analysis was performed on IDX plus TsA-treated non-differentiating cells and all probe sets from the IDX-only group that exhibited similar expression profiles in the non-differentiating TsA-treated group were excluded leaving a total of 1016 transcripts that were regulated only under differentiating conditions. Six hundred and thirty-six of these transcripts were elevated at least 2-fold and 380 exhibited a decrease in expression relative to time 0 preadipocytes. This group of genes was further analyzed using hierarchical clustering and self-organizing maps and resulted in the identification of numerous genes not previously known to be regulated during adipocyte differentiation. Many of these genes may well represent novel adipogenic mediators and markers of adipogenesis.

1-Methyl-3-isobutylxanthine↗
PubMed2004

RAG: RNA-As-Graphs database--concepts, analysis, and features.

MOTIVATION: Understanding RNA's structural diversity is vital for identifying novel RNA structures and pursuing RNA genomics initiatives. By classifying RNA secondary motifs based on correlations between conserved RNA secondary structures and functional properties, we offer an avenue for predicting novel motifs. Although several RNA databases exist, no comprehensive schemes are available for cataloguing the range and diversity of RNA's structural repertoire. RESULTS: Our RNA-As-Graphs (RAG) database describes and ranks all mathematically possible (including existing and candidate) RNA secondary motifs on the basis of graphical enumeration techniques. We represent RNA secondary structures as two-dimensional graphs (networks), specifying the connectivity between RNA secondary structural elements, such as loops, bulges, stems and junctions. We archive RNA tree motifs as 'tree graphs' and other RNAs, including pseudoknots, as general 'dual graphs'. All RNA motifs are catalogued by graph vertex number (a measure of sequence length) and ranked by topological complexity. The RAG inventory immediately suggests candidates for novel RNA motifs, either naturally occurring or synthetic, and thereby might stimulate the prediction and design of novel RNA motifs. AVAILABILITY: The database is accessible on the web at http://monod.biomath.nyu.edu/rna

Algorithms↗