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PubMed1998

Use of subtractive hybridization to design habitat-based oligonucleotide probes for investigation of natural bacterial communities.

We describe a rapid oligonucleotide probe design strategy based on subtractive hybridization which yields probes for 16S rRNA or rRNA genes of individual members of microbial communities that are specific within the context of those communities. This strategy circumvents the need to sequence many similar or identical clones of dominant members of a community. Radioactively labeled subfragments of a cloned 16S rRNA gene sequence for which a probe is required (target) were hybridized with biotinylated total 16S ribosomal DNA (rDNA) amplified from the microbial community, and the hybrids formed were subsequently discarded. The remaining enriched fragments were used to screen a library consisting of cloned subfragments of the target sequence by colony hybridization in order to identify the variable regions of the 16S rRNA gene with the required specificity. The sequencing of random clones in one 16S rDNA library demonstrated that only those clones with 100% sequence identity with the probe fragment were detected by it. Moreover, sequencing of other, randomly selected, probe-positive clones revealed 100% sequence identity with the probe. Probes developed in this way tended to correspond to more variable regions of the 16S rRNA if the target sequences were similar to the sequences of other clones in the library and to less variable regions if the target sequences were phylogenetically isolated within the clone library. Although the absolute specificity of the latter probes, as assessed by comparison with available database sequences, was lower than the absolute specificity of the probes from the more variable regions, they were specific within the context of the environmental samples from which they were derived.

Bacteria↗

PubMed2006

Identification of potential pharmacological and toxicological targets differentiating structural analogs by a combination of transcriptional profiling and promoter analysis in LS-180 and Caco-2 adenocarcinoma cell lines.

Detecting and understanding the potential for off-target pharmacological effects is critical in the optimization of lead compounds in drug discovery programs. Compound-mediated activation of the pregnane X receptor (PXR; NR1I2), a key regulator for drug metabolism genes, is often monitored to avoid potential drug-drug interactions. Two structural analogs, MRL-1 and MRL-2, were determined to be equivalent PXR activators in trans-activation assays. To differentiate these two PXR activators, their transcriptional effects were examined in PXR-sufficient (LS180) and PXR-deficient (Caco-2) adenocarcinoma cell lines. Both compounds regulated drug-management genes (e.g. CYP3A4, CYP2B6, UGT1A1 and ABCB1) in LS180 cells, but not in PXR-deficient Caco-2 cells. The potency of MRL-1 and MRL-2 on PXR activation was again equivalent as revealed by a set of 113 genes that were regulated by four prototypical PXR agonists (rifampicin, ritonavir, troglitazone and dexamethasone) in the LS180 cells. The specificity of the PXR signature genes was supported by the enrichment of putative PXR binding sites uncovered by sequence-based promoter analyses. Interestingly, an additional off-target activity of MRL-2 was suggested where sterol response element binding protein binding sites were found enriched in a subset of PXR signature genes. These genes, involved in cholesterol and fatty acid synthesis, were significantly regulated by ritonavir, chlorpromazine and MRL-2, which were linked to the manifestation of phospholipidosis. The present study demonstrates the utility of our approach in the differentiation and selection of lead compounds for drug development.

Benzopyrans↗

PubMed1997

Nramp1 locus encodes a 65 kDa interferon-gamma-inducible protein in murine macrophages.

The murine Nramp1 (natural-resistance-associated macrophage protein) locus, formerly known as Ity/Lsh/Bcg, was isolated previously on the basis of chromosomal location, and as conferring natural resistance to infection against intracellular macrophage pathogens. The gene encodes a transporter molecule of unknown function. We have prepared polyclonal antisera against the C-terminal 35 amino acids of murine Nramp1. This serum is reactive towards a 65 kDa protein, expressed in murine macrophage cells from resistant or susceptible mice stimulated with interferon-gamma and lipopolysaccharide, but not in non-macrophage cells. Evidence indicates that Nramp1 is localized in a subcellular membrane rather than at the cell surface. This evidence includes: the identification of conserved endocytic targeting motifs following inspection of human and murine Nramp sequences; the enrichment of Nramp1, following magnetic selection of phagolysosomal vesicles from activated macrophages that were allowed to phagocytose magnetic, IgG-coated beads; confocal microscopy. These studies place Nramp1 on a membrane in close proximity to obligate intracellular pathogens. A link between Nramp1 and divalent-cation transport is suggested by sequence similarity with yeast SMF1. Evidence showing modulation of Nramp1 protein levels by iron chelation provides a direct link with Nramp1 function and divalent-cation metabolism.

Amino Acid Sequence↗

PubMed2002

Transgenic barley grain overexpressing thioredoxin shows evidence that the starchy endosperm communicates with the embryo and the aleurone.

Homozygous lines of barley overexpressing a wheat thioredoxin h transgene (up to 30-fold) were generated earlier by using a B(1)-hordein promoter with a signal peptide sequence for targeting to the protein body and found to be enriched in starch debranching enzyme (pullulanase). Here, we describe the effect of biochemically active, overexpressed thioredoxin h on germination and the onset of alpha-amylase activity. Relative to null segregant controls lacking the transgene, homozygotes overexpressing thioredoxin h effected (i) an acceleration in the rate of germination and appearance of alpha-amylase activity with a 1.6- to 2.8-fold increase in gibberellin A(1) (GA(1)) content; (ii) a similar acceleration in the appearance of the alpha-amylase activity in deembryonated transgenic grain incubated with gibberellic acid; (iii) a 35% increase in the ratio of relative reduction (abundance of SH) of the propanol soluble proteins (hordein I fraction); and (iv) an increase in extractable and soluble protein of 5-12% and 11-35%, respectively. Thioredoxin h, which was highly reduced in the dry grain, was degraded in both the null segregant and homozygote after imbibition. The increase in alpha-amylase activity and protein reduction status was accompanied by a shift in the distribution of protein from the insoluble to the soluble fraction. The results provide evidence that thioredoxin h of the starchy endosperm communicates with adjoining tissues, thereby regulating their activities, notably by accelerating germination of the embryo and the appearance of alpha-amylase released by the aleurone.

Desiccation↗

PubMed2025

De novo transcriptome assembly and gene expression analysis of Cnidium officinale under high-temperature conditions.

BACKGROUND: The medicinal plant Cnidium officinale (CO) is widespread in Northeast Asia and vulnerable to heat stress. The naturally occurring composition of pharmacological ingredients of CO results in overall physiological consequences; therefore, it is crucial to have a comprehensive understanding of metabolic response to ambient heat in terms of acclimation to estimate how much CO is exposed to threatening environmental conditions. RESULTS: Transcriptome analysis is critical for understanding the consequences of long-term physiological adaptation of CO to abiotic stress. However, transcriptome analysis on this species, particularly under prolonged stress conditions, has remained limited. We employed a temperature gradient tunnel (TGT) to subject CO to high-temperature exposure for four months, enabling us to observe the cumulative effects of heat and assess its acclimation mechanisms. In the absence of genome sequencing data, we performed de novo transcriptome assembly and compared DEGs from temperature treatment plots of a TGT and a growth chamber (GC). Since interpreting transcriptomic data can be complex, we employed a sequential analytical approach, including DEG clustering, GO enrichment, KEGG pathway mapping, miRNA-target gene analysis, and multiple rounds of RNA sequencing validation. DEGs were classified into two categories: genes exhibiting significant fold changes and genes showing significant count changes rather than fold changes. Then, we analyzed the functional roles of DEGs to determine which pathways respond to ambient and stressful high temperatures and validated the findings through cross-comparison with GC. Additionally, we conducted miRNA analysis to investigate post-transcriptional regulation under high temperatures. CO grown under higher ambient temperatures exhibited slight upregulation of pathways related to protein stability and turnover, ABA biosynthesis, and energy production, such as photosynthesis and oxidative phosphorylation. However, under extreme heat stress, most metabolic pathways were downregulated except for those involved in transcription, translation, oxidative phosphorylation and the biosynthesis of cutin, suberin, and wax. CONCLUSION: This study demonstrated that proper clustering of genes based on expression levels and fold changes in two different experimental conditions, along with pathway mapping, may provide a comprehensive understanding of CO's response to heat stress. These insights could contribute to future research on heat tolerance and crop improvement.

Gene Expression Profiling↗

PubMed2007

Nitrifying bacterial communities in an aquaculture wastewater treatment system using fluorescence in situ hybridization (FISH), 16S rRNA gene cloning, and phylogenetic analysis.

Aquaculture, especially shrimp farming, has played a major role in the growth of Thailand's economy in recent years, as well as in many South East Asian countries. However, the nutrient discharges from these activities have caused adverse impacts on the quality of the receiving waterways. In particular nitrogenous compounds, which may accumulate in aquaculture ponds, can be toxic to aquatic animals and cause environmental problems such as eutrophication. The mineralization process is well known, but certain aspects of the microbial ecology of nitrifiers, the microorganisms that convert ammonia to nitrate, are poorly understood. A previously reported enrichment of nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) from a shrimp farm inoculated in a sequencing batch reactor (SBR) was studied by molecular methods. The initial identification and partial quantification of the nitrifying bacteria (AOB and NOB) were carried out by fluorescence in situ hybridization (FISH) using previously published 16S rRNA-targeting oligonucleotide probes. The two dominant bacterial groups detected by FISH were from the Cytophaga-Flavobacterium-Bacteroides and Proteobacteria (beta subdivision) phyla. Published FISH probes for Nitrobacter and Nitrospira did not hybridize to any of the bacterial cells. Therefore it is likely that new communities of NOBs, differing from previously reported ones, exist in the enrichments. Molecular genetic techniques (cloning, sequencing, and phylogenetic analysis) targeting the 16S rRNA genes from the nitrifying enrichments were performed to identify putative AOBs and NOBs.

Animals↗

PubMed2001

Optimized RNA targets of two closely related triple KH domain proteins, heterogeneous nuclear ribonucleoprotein K and alphaCP-2KL, suggest Distinct modes of RNA recognition.

The KH domain mediates RNA binding in a wide range of proteins. Here we investigate the RNA-binding properties of two abundant RNA-binding proteins, alphaCP-2KL and heterogeneous nuclear ribonucleoprotein (hnRNP) K. These proteins constitute the major poly(C) binding activity in mammalian cells, are closely related on the basis of the structures and positioning of their respective triplicated KH domains, and have been implicated in a variety of post-transcriptional controls. By using SELEX, we have obtained sets of high affinity RNA targets for both proteins. The primary and secondary structures necessary for optimal protein binding were inferred in each case from SELEX RNA sequence comparisons and confirmed by mutagenesis and structural mapping. The target sites for alphaCP-2KL and hnRNP K were both enriched for cytosine bases and were presented in a single-stranded conformation. In contrast to these shared characteristics, the optimal target sequence for hnRNP K is composed of a single short "C-patch" compatible with recognition by a single KH domain whereas that for alphaCP-2KL encompassed three such C-patches suggesting more extensive interactions. The binding specificities of the respective SELEX RNAs were confirmed by testing their interactions with native proteins in cell extracts, and the importance of the secondary structure in establishing an optimized alphaCP-2KL-binding site was supported by comparison of SELEX target structure with that of the native human alpha-globin 3'-untranslated region. These data indicate that modes of macromolecular interactions of arrayed KH domains can differ even among closely related KH proteins and that binding affinities are substantially dependent on the presentation of the target site within the RNA secondary structure.

Animals↗

PubMed2003

The fragile X mental retardation protein binds and regulates a novel class of mRNAs containing U rich target sequences.

Fragile X syndrome is a common form of inherited mental retardation caused by the absence of the fragile X mental retardation protein (FMRP). It has been hypothesized that FMRP is involved in the processing and/or translation of mRNAs. Human and mouse target-mRNAs, containing purine quartets, have previously been identified. By using cDNA-SELEX (systematic evolution of ligands by exponential enrichment), we identified another class of human target-mRNAs which contain U rich sequences. This technique, in contrast to oligonucleotide-based SELEX, allows the identification of FMRP targets directly from mRNA pools. Many of the proteins encoded by the identified FMRP targets have been implicated in neuroplasticity. Steady state levels of target-mRNAs were unchanged in the brain of fragile X mice. However, levels of two target-encoded proteins, an L-type calcium channel subunit and MAP1B, were downregulated in specific brain regions suggesting a defect in the expression of target-encoded proteins in fragile X syndrome.

Adenosine Triphosphatases↗

PubMed1993

In vitro evolution of functional nucleic acids: high-affinity RNA ligands of HIV-1 proteins.

SELEX (Systematic Evolution of Ligands by EXponential enrichment) is a protocol for isolating, from a pool of variant nucleic acid sequences, high-affinity ligands to a target protein [Tuerk and Gold, Science 249 (1990) 505-510]. This procedure involves cycles of affinity selection by a target molecule from a heterogeneous population of nucleic acids, replication of the bound species (the ligands), and in vitro transcription to generate an enriched pool of RNA. We have used the SELEX procedure to obtain high-affinity RNA ligands against the reverse transcriptase and the Rev and Tat proteins of human immunodeficiency virus 1 (HIV-1). Through sequence comparisons within the collection of ligands isolated for each of these target proteins, we derive consensus descriptions of what secondary structure and primary sequences are required for binding. These descriptions serve as the starting point for the ultimate development of compounds intended to alter the course of HIV-1 infection.

Base Sequence↗

PubMed1999

Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome.

The genomes of higher plants and animals are highly differentiated, and are composed of a relatively small number of genes and a large fraction of repetitive DNA. The bulk of this repetitive DNA constitutes transposable, and especially retrotransposable, elements. It has been hypothesized that most of these elements are heavily methylated relative to genes, but the evidence for this is controversial. We show here that repeat sequences in maize are largely excluded from genomic shotgun libraries by the selection of an appropriate host strain because of their sensitivity to bacterial restriction-modification systems. In contrast, unmethylated genic regions are preserved in these genetically filtered libraries if the insert size is less than the average size of genes. The representation of unique maize sequences not found in plant reference genomes is also greatly enriched. This demonstrates that repeats, and not genes, are the primary targets of methylation in maize. The use of restrictive libraries in genome shotgun sequencing in plant genomes should allow significant representation of genes, reducing the number of reactions required.

Cloning, Molecular↗

PubMed2003

Constructing gene-enriched plant genomic libraries using methylation filtration technology.

Full genome sequencing in higher plants is a very difficult task, because their genomes are often very large and repetitive. For this reason, gene targeted partial genomic sequencing becomes a realistic option. The method reported here is a simple approach to generate gene-enriched plant genomic libraries called methylation filtration. This technique takes advantage of the fact that repetitive DNA is heavily methylated and genes are hypomethylated. Then, by simply using an Escherichia coli host strain harboring a wild-type modified cytosine restriction (McrBC) system, which cuts DNA containing methylcytosine, repetitive DNA is eliminated from these genomic libraries, while low copy DNA (i.e., genes) is recovered. To prevent cloning significant proportions of organelle DNA, a crude nuclear preparation must be performed prior to purifying genomic DNA. Adaptor-mediated cloning and DNA size fractionation are necessary for optimal results.

DNA, Plant↗

PubMed2003

Genome-wide identification of in vivo Drosophila Engrailed-binding DNA fragments and related target genes.

Chromatin immunoprecipitation after UV crosslinking of DNA/protein interactions was used to construct a library enriched in genomic sequences that bind to the Engrailed transcription factor in Drosophila embryos. Sequencing of the clones led to the identification of 203 Engrailed-binding fragments localized in intergenic or intronic regions. Genes lying near these fragments, which are considered as potential Engrailed target genes, are involved in different developmental pathways, such as anteroposterior patterning, muscle development, tracheal pathfinding or axon guidance. We validated this approach by in vitro and in vivo tests performed on a subset of Engrailed potential targets involved in these various pathways. Finally, we present strong evidence showing that an immunoprecipitated genomic DNA fragment corresponds to a promoter region involved in the direct regulation of frizzled2 expression by engrailed in vivo.

Animals↗

PubMed2003

Targeted cytosine methylation for in vivo detection of protein-DNA interactions.

We report a technique, named targeted gene methylation (TAGM), for identifying in vivo protein-binding sites in chromatin. M.CviPI, a cytosine-5 DNA methyltransferase recognizing GC sites, is fused to a DNA-binding factor enabling simultaneous detection of targeted methylation, factor footprints, and chromatin structural changes by bisulfite genomic sequencing. Using TAGM with the yeast transactivator Pho4, methylation enrichments of up to 34- fold occur proximal to native Pho4-binding sites. Additionally, significant selective targeting of methylation is observed several hundred nucleotides away, suggesting the detection of long-range interactions due to higher-order chromatin structure. In contrast, at an extragenic locus lacking Pho4-binding sites, methylation levels are at the detection limit at early times after Pho4 transactivation. Notably, substantial amounts of methylation are targeted by Pho4-M.CviPI under repressive conditions when most of the transactivator is excluded from the nucleus. Thus, TAGM enables rapid detection of DNA-protein interactions even at low occupancies and has potential for identifying factor targets at the genome-wide level. Extension of TAGM from yeast to vertebrates, which use methylation to initiate and propagate repressed chromatin, could also provide a valuable strategy for heritable inactivation of gene expression.

Binding Sites↗

PubMed1996

PEST sequences and regulation by proteolysis.

In 1986, we proposed that polypeptide sequences enriched in proline (P), glutamic acid (E), serine (S) and threonine (T) target proteins for rapid destruction. For much of the past decade there were only sporadic experimental tests of the hypothesis. This situation changed markedly during the past two years with a number of papers providing strong evidence that PEST regions do, in fact, serve as proteolytic signals. Here, we briefly review the properties of PEST regions and some interesting examples of the conditional nature of such signals. Most of the article, however, focuses on recent experimental support for the hypothesis and on mechanisms responsible for the rapid degradation of proteins that contain PEST regions.

Amino Acid Sequence↗

PubMed2026

Zhiling Jiangya decoction treats hypertension in rats: An integrative study of network pharmacology, immune infiltration, molecular simulation, and 16S rDNA sequencing.

OBJECTIVE: This study integrated network pharmacology, immune infiltration analysis, molecular docking, molecular dynamics simulation, ADMET prediction, 16S rDNA sequencing, and rat experiments to elucidate the potential mechanisms underlying the antihypertensive effects of Zhiling Jiangya Decoction (ZLJYD). METHODS: Active compounds and their potential targets were screened from the PubChem, TCMSP, NovoPro, and SwissTargetPrediction databases. Hypertension-related targets were retrieved from the OMIM and GeneCards databases, and overlapping targets were identified. The STRING database and Cytoscape 3.10.1 software were used to construct a protein-protein interaction network and a herb-component-target-disease network. Gene Ontology functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed to identify the key biological processes and signaling pathways involved. Using the CIBERSORT algorithm combined with correlation analysis, we investigated the association between key targets and immune cell infiltration. Molecular docking, molecular dynamics simulations, and ADMET predictions were performed to assess the binding stability and pharmacokinetic properties of the main compounds with their corresponding targets. Finally, the antihypertensive efficacy of ZLJYD was validated using a spontaneously hypertensive rat model, and alterations in gut microbiota were analyzed using 16S rDNA sequencing. RESULTS: A total of 123 active compounds and 267 hypertension-related targets of ZLJYD were identified. Enrichment analysis revealed that these targets were primarily associated with the PI3K-Akt signaling pathway and lipid and atherosclerosis pathways. Immune infiltration analysis suggested that the therapeutic effects of ZLJYD may involve the regulation of follicular helper T cells, naïve B cells, and naïve CD4⁺ T cells. Molecular docking and dynamics simulations supported the stable binding of key compounds to their target proteins, while ADMET predictions indicated favorable pharmacokinetic properties and safety profiles. Rat experiments demonstrated that ZLJYD significantly reduced blood pressure in spontaneously hypertensive rats, partially alleviated gut microbiota dysbiosis, and altered microbial community structure and phylogenetic diversity. CONCLUSION: This study systematically elucidates the potential mechanisms underlying the antihypertensive effects of ZLJYD through multiple components, targets, and pathways, particularly immune regulation and gut microbiota remodeling. These findings provide mechanistic insights into its potential therapeutic application.

16S rDNA sequencing↗

PubMed2006

CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding.

CUG-BP1 [CUG-binding protein 1 also called CELF (CUG-BP1 and ETR3 like factors) 1] is a human RNA-binding protein that has been implicated in the control of splicing and mRNA translation. The Xenopus homologue [EDEN-BP (embryo deadenylation element-binding protein)] is required for rapid deadenylation of certain maternal mRNAs just after fertilization. A variety of sequence elements have been described as target sites for these two proteins but their binding specificity is still controversial. Using a SELEX (systematic evolution of ligand by exponential enrichment) procedure and recombinant CUG-BP1 we selected two families of aptamers. Surface plasmon resonance and electrophoretic mobility-shift assays showed that these two families differed in their ability to bind CUG-BP1. Furthermore, the selected high-affinity aptamers form two complexes with CUG-BP1 in electrophoretic mobility assays whereas those that bind with low affinity only form one complex. The validity of the distinction between the two families of aptamers was confirmed by a functional in vivo deadenylation assay. Only those aptamers that bound CUG-BP1 with high affinity conferred deadenylation on a reporter mRNA. These high-affinity RNAs are characterized by a richness in UGU motifs. Using these binding site characteristics we identified the Xenopus maternal mRNA encoding the MAPK (mitogen-activated protein kinase) phosphatase (XCl100alpha) as a substrate for EDEN-BP. In conclusion, high-affinity CUG-BP1 binding sites are sequence elements at least 30 nucleotides in length that are enriched in combinations of U and G nucleotides and contain at least 4 UGU trinucleotide motifs. Such sequence elements are functionally competent to target an RNA for deadenylation in vivo.

3' Untranslated Regions↗

PubMed2005

Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments.

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.

Base Sequence↗

PubMed1993

Genomic subtractive hybridization to isolate species-specific DNA sequences in insects.

Selective enrichment has been used in a number of instances for the isolation of species-specific sequences in prokaryotes. This paper reports the successful application of the technique to insects. Genomic probes were derived to the target species D. funebris and D. simulans. The method involves the biotinylation of non-target 'driver' DNA prepared from the closely related species D. melanogaster and its hybridization to homologous sequences in the target DNA. Hybrid molecules were removed from the reaction by incubation with streptavidin followed by phenol extraction, leaving a preparation enriched for target fragments. All DNA fragments isolated in the D. funebris experiments proved to be specific to that species. Five out of twenty-four fragments screened in the D. simulans experiments were specific when screened with homologous DNA and genomic DNA from its sibling species, D. melanogaster.

Animals↗