Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Target sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,441 records · Page 80Linked to original sources
PubMed2006

The enteropathogenic Escherichia coli (EPEC) Map effector is imported into the mitochondrial matrix by the TOM/Hsp70 system and alters organelle morphology.

Enteropathogenic Escherichia coli (EPEC) is a human intestinal pathogen and a major cause of diarrhoea, particularly among infants in developing countries. EPEC target the Map and EspF multifunctional effector proteins to host mitochondria - organelles that play crucial roles in regulating cellular processes such as programmed cell death (apoptosis). While both molecules interfere with the organelles ability to maintain a membrane potential, EspF plays the predominant role and is responsible for triggering cell death. To learn more about the Map-mitochondria interaction, we studied Map localization to mitochondria with purified mitochondria (from mammalian and yeast cells) and within intact yeast. This revealed that (i) Map targeting is dependent on the predicted N-terminal mitochondrial targeting sequence, (ii) the N-terminal 44 residues are sufficient to target proteins to mitochondria and (iii) Map import involves the mitochondrial outer membrane translocase (Tom22 and Tom40), the mitochondrial membrane potential, and the matrix chaperone, mtHsp70. These results are consistent with Map import into the mitochondria matrix via the classical import mechanism. As all known, Map-associated phenotypes in mammalian cells are independent of mitochondrial targeting, this may indicate that import serves as a mechanism to remove Map from the cytoplasm thereby regulating cytoplasmic function. Intriguingly, Map, but not EspF, alters mitochondrial morphology with deletion analysis revealing important roles for residues 101-152. Changes in mitochondrial morphology have been linked to alterations in the ability of these organelles to regulate cellular processes providing a possible additional role for Map import into mitochondria.

Amino Acid Motifs↗
PubMed2025

Integrating RNA sequencing with deep learning-based metabolic toxicity prediction: A new perspective on screening prioritized liquid crystal monomers.

Nearly 99 % of liquid crystal monomers (LCMs) toxicological data remains gaps, especially to aquatic organisms. Herein, this study proposes a rapid and high-throughput screening method for identifying priority LCMs in natural water. Using six fluorinated LCMs (LCMsF) with significant enrichment characteristics in zebrafish as examples, RNA sequencing revealed that LCMsF-induced metabolic disturbances are predominant, including 28 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway abnormalities attributed to 498 differentially expressed genes. Notably, the intricate sequencing process resulted in the inability to rapid identify additional 857 LCMsF that may induce metabolic disturbances. To address this, LCMsT-MTP, a predictive deep learning model based on RNA sequencing, was developed. This model integrates a comprehensive representation of LCMsF structures and metabolic toxicity target sequences. LCMsT-MTP improves upon traditional methods that are limited to single targets and mechanisms by facilitating the simultaneous identification of 21 metabolic toxicities induced by LCMsF. In addition, the LCMsT-MTP model was further applied to non-fluorinated LCMs (LCMsNone F) that satisfy the applicability domains test. Accordingly, a metabolic toxicity priority list of LCMs was proposed, with ∼95 % of LCMs classified as high or medium risk. Priority list validation by molecular dynamics confirmed that the interactions of LCMsF/LCMsNone F and metabolic toxicity targets in representative KEGG pathways were distinct.

Animals↗
PubMed2006

DNA topology and geometry in Flp and Cre recombination.

The Flp recombinase of yeast and the Cre recombinase of bacteriophage P1 both belong to the lambda-integrase (Int) family of site-specific recombinases. These recombination systems recognize recombination-target sequences that consist of two 13bp inverted repeats flanking a 6 or 8bp spacer sequence. Recombination reactions involve particular geometric and topological relationships between DNA target sites at synapsis, which we investigate using nicked-circular DNA molecules. Examination of the tertiary structure of synaptic complexes formed on nicked plasmid DNAs by atomic-force microscopy, in conjunction with detailed topological analysis using the mathematics of tangles, shows that only a limited number of recombination-site topologies are consistent with the global structures of plasmids bearing directly and inversely repeated sites. The tangle solutions imply that there is significant distortion of the Holliday-junction intermediate relative to the planar structure of the four-way DNA junction present in the Flp and Cre co-crystal structures. Based on simulations of nucleoprotein structures that connect the two-dimensional tangle solutions with three-dimensional models of the complexes, we propose a recombination mechanism in which the synaptic intermediate is characterized by a non-planar, possibly near-tetrahedral, Holliday-junction intermediate. Only modest conformational changes within this structure are needed to form the symmetric, planar DNA junction, which may be characteristic of shorter-lived intermediates along the recombination pathway.

Base Sequence↗
PubMed1990

The segment-specific gene Krox-20 encodes a transcription factor with binding sites in the promoter region of the Hox-1.4 gene.

Krox-20 is a mouse zinc finger gene expressed in a segment-specific manner in the early central nervous system, which makes it a potential developmental control gene. In this report, we show that the Krox-20 protein binds in vitro to two specific DNA sites located upstream from the homeobox containing gene Hox-1.4. The nucleotide sequence recognized by Krox-20 is closely related to the Sp1 target sequence, which is consistent with the similarity existing between the zinc fingers of the two proteins. In co-transfection experiments in cultured cells, Krox-20 dramatically activates transcription from the herpes simplex virus thymidine kinase promoter when an oligomer of its binding site is present in cis close to the promoter. Analysis of mutated binding sites demonstrates that the level of activation by Krox-20 correlates with the affinity of the protein for the mutant sequence. These data indicate that Krox-20 constitutes a sequence-specific DNA-binding transcription factor. Parallel analysis of the expression of Krox-20 and Hox-1.4 in the neural tube by in situ hybridization revealed no overlap, arguing against direct interactions between these two genes. The possible involvement of Krox-20 in the regulation of the transcription of other homeobox genes is discussed in view of their respective patterns of expression.

Animals↗
PubMed2006

Assessing a novel approach for predicting local 3D protein structures from sequence.

We developed a novel approach for predicting local protein structure from sequence. It relies on the Hybrid Protein Model (HPM), an unsupervised clustering method we previously developed. This model learns three-dimensional protein fragments encoded into a structural alphabet of 16 protein blocks (PBs). Here, we focused on 11-residue fragments encoded as a series of seven PBs and used HPM to cluster them according to their local similarities. We thus built a library of 120 overlapping prototypes (mean fragments from each cluster), with good three-dimensional local approximation, i.e., a mean accuracy of 1.61 A Calpha root-mean-square distance. Our prediction method is intended to optimize the exploitation of the sequence-structure relations deduced from this library of long protein fragments. This was achieved by setting up a system of 120 experts, each defined by logistic regression to optimize the discrimination from sequence of a given prototype relative to the others. For a target sequence window, the experts computed probabilities of sequence-structure compatibility for the prototypes and ranked them, proposing the top scorers as structural candidates. Predictions were defined as successful when a prototype <2.5 A from the true local structure was found among those proposed. Our strategy yielded a prediction rate of 51.2% for an average of 4.2 candidates per sequence window. We also proposed a confidence index to estimate prediction quality. Our approach predicts from sequence alone and will thus provide valuable information for proteins without structural homologs. Candidates will also contribute to global structure prediction by fragment assembly.

Amino Acid Sequence↗
PubMed2001

Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure.

Of the sequence comparison methods, profile-based methods perform with greater selectively than those that use pairwise comparisons. Of the profile methods, hidden Markov models (HMMs) are apparently the best. The first part of this paper describes calculations that (i) improve the performance of HMMs and (ii) determine a good procedure for creating HMMs for sequences of proteins of known structure. For a family of related proteins, more homologues are detected using multiple models built from diverse single seed sequences than from one model built from a good alignment of those sequences. A new procedure is described for detecting and correcting those errors that arise at the model-building stage of the procedure. These two improvements greatly increase selectivity and coverage. The second part of the paper describes the construction of a library of HMMs, called SUPERFAMILY, that represent essentially all proteins of known structure. The sequences of the domains in proteins of known structure, that have identities less than 95 %, are used as seeds to build the models. Using the current data, this gives a library with 4894 models. The third part of the paper describes the use of the SUPERFAMILY model library to annotate the sequences of over 50 genomes. The models match twice as many target sequences as are matched by pairwise sequence comparison methods. For each genome, close to half of the sequences are matched in all or in part and, overall, the matches cover 35 % of eukaryotic genomes and 45 % of bacterial genomes. On average roughly 15% of genome sequences are labelled as being hypothetical yet homologous to proteins of known structure. The annotations derived from these matches are available from a public web server at: http://stash.mrc-lmb.cam.ac.uk/SUPERFAMILY. This server also enables users to match their own sequences against the SUPERFAMILY model library.

Amino Acid Sequence↗
PubMed1995

Isolation, expression, and evolution of the gene encoding mitochondrial elongation factor Tu in Arabidopsis thaliana.

We have characterized a second nuclear gene (tufM) in Arabidopsis thaliana that encodes a eubacterial-like protein synthesis elongation factor Tu (EF-Tu). This gene does not closely resemble the previously described Arabidopsis nuclear tufA gene, which encodes the plastid EF-Tu, and does not contain sequence elements found in all cyanobacterial and plastid tufA genes. However, the predicted amino acid sequence includes an N-terminal extension which resembles an organellar targeting sequence and shares three unique sequence elements with mitochondrial EF-Tu's, from Saccharomyces cerevisiae and Homo sapiens, suggesting that this gene encodes the Arabidopsis mitochondrial EF-Tu. Consistent with this interpretation, the gene is expressed at a higher level in flowers than in leaves. Phylogenetic analysis confirms the mitochondrial character of the sequence and indicates that the human, yeast, and Arabidopsis tufM genes have undergone considerably more sequence divergence than their cytoplasmic counterparts, perhaps reflecting a cross-compartmental acceleration of gene evolution for components of the mitochondrial translation apparatus. As previously observed for tufA, the tufM gene is present in one copy in Arabidopsis but in several copies in other species of crucifers.

Amino Acid Sequence↗
PubMed1992

RNA editing in trypanosomes. The us(e) of guide RNAs.

Guide RNAs are encoded in maxicircle and minicircle DNA of trypanosome mitochondria. They play a pivotal role in RNA editing, a process during which the nucleotide sequence of mitochondrial RNAs is altered by U-insertion and deletion. Guide RNAs vary in length from 35 to 78 nucleotides, which correlates with the variation in length of the three functionally important regions of which they are composed: (i) a 4-14 nucleotide 'anchor' sequence embedded in the 5' region, which is complementary to a target sequence on the pre-edited RNA downstream of an editing domain, (ii) a middle part containing the editing information, which ranges from guiding the insertion of just one U into one site to that of the insertion of 32 Us into 10 sites, and (iii) a 5-24 nucleotide 3' terminal oligo [U] extension. Moreover, a variable uridylation site creates gRNAs containing a varying segment of editing information for the same domain. Comparison of different guide RNAs demonstrates that, besides the U-tail, they have no obvious common primary and secondary sequence motifs, each particular sequence being unique. The occurrence in vivo and the synthesis in vitro of chimeric molecules, in which a guide RNA is covalently linked through its 3' U-tail to an editing site of a pre-edited RNA, suggests that RNA editing occurs by consecutive transesterification reactions and is evidence that the guide RNAs not only provide the genetic information, but also the Us themselves.

Animals↗
PubMed2005

Identification of two distinct intracellular localization signals in STT3-B.

The STT3 subunit of the oligosaccharyltransferase complex plays a critical role in the N-glycosylation process. From Arabidopsis thaliana to Homo sapiens, two functional STT3 isoforms have been identified, STT3-A and STT3-B. We report that the last transmembrane (TM) segment of STT3-B corresponds to a topogenic determinant that is sufficient for proper integration and orientation of STT3-B C-terminal domain. Notably, the last TM segment of STT3-A and -B isoforms present major differences in amino acid sequence and predicted 3D structure. We also identified a bipartite nuclear targeting sequence in the C-terminal tail of STT3-B that is absent in STT3-A. The latter sequence is sufficient to induce nucleolar localization of a reporter protein. Our results show that STT3-A and -B display two structural differences that may have a drastic influence on their function and might account for the remarkable evolutionary conservation of the two STT3 paralogs.

Amino Acid Sequence↗
PubMed2005

Nanoparticle-based DNA biosensor for visual detection of genetically modified organisms.

Although screening of raw ingredients and food products for genetically modified organisms (GMO) may be accomplished by detecting either the exogenous DNA or the novel protein, DNA is the preferred analyte because of its superior stability during food processing. The development of DNA biosensors is of increasing importance due to the growing demand for rapid and reliable methods for GMO detection. We report the first DNA biosensor in a dry-reagent dipstick configuration for visual detection and confirmation of GMO-related sequences by hybridization within minutes. The sensor is disposable and does not require special instrumentation. It detects the 35S promoter and nopaline synthase (NOS) terminator sequences that are present in the majority of transgenic plants. The target sequences are amplified by the polymerase chain reaction (PCR) and hybridized (7min) with probes bearing oligo(dA) tail. The biotinylated product is applied to the sensor followed by immersion in the appropriate buffer. Migration of the buffer rehydrates gold nanoparticles conjugated to oligo(dT), which hybridize with the oligo(dA) tails. The hybrids are captured by immobilized streptavidin at the test zone of the sensor giving a characteristic red line due to the accumulation of the nanoparticles. The excess of nanoparticle conjugates are captured at the control zone by immobilized oligo(dA) strands. Amplified 35S or NOS DNA is detectable at 0.16nM. Soybean powder certified reference material with 0.1% GMO content is clearly detectable after 35 and 40 amplification cycles for 35S and NOS sequence, respectively. The sensor was also applied to real samples from various sources.

Biosensing Techniques↗
PubMed2000

Identification of an optimal Ncx binding sequence required for transcriptional activation.

The Ncx gene encodes a homeobox containing transcription factor that belongs to the Hox11 gene family. We determined specific Ncx protein binding consensus DNA sequences. Optimal Ncx binding sequences were 5'-CGGTAATTGG-3' (TAAT core) and 5'-CGGTAAGTGG-3' (TAAG core), which coincided with the Hox11 binding sequence. Both Ncx and Hox11 could bind to the TAAT and the TAAG core oligonucleotide in vitro. However, they could efficiently transactivate the reporter plasmid linked to the TAAT core sequence but not to the TAAG core sequence. Thus, Ncx and Hox11 act as transcriptional activators via their target sequence, 5'-CGGTAATTGG-3'.

Binding Sites↗
PubMed2003

The PmSOD1 gene of the protistan parasite Perkinsus marinus complements the sod2Delta mutant of Saccharomyces cerevisiae, and directs an iron superoxide dismutase to mitochondria.

The facultative intracellular oyster parasite, Perkinsus marinus, taxonomically related to both dinoflagellates and apicomplexan parasites, possesses at least two distinct genes (PmSOD1 and PmSOD2) predicted to encode iron-containing superoxide dismutases (Fe-SOD). The present study demonstrates that PmSOD1 complements a Saccharomyces cerevisiae mutant lacking the mitochondrial manganese-containing SOD (Mn-SOD), whereas PmSOD2 complements an Escherichia coli mutant lacking genes for cytosolic SOD activities. Mitochondria isolated from complemented yeast contain an SOD activity susceptible to inhibition by hydrogen peroxide, but resistant to cyanide, both characteristics of Fe-SODs. In cultured P. marinus trophozoites, indirect immunofluorescence using anti-PmSOD1 antibodies shows colocalization of PmSOD1 product with the mitochondrial marker MitoTracker Red. Further analysis of the leader sequence of the predicted PmSOD1 product revealed similarities to a mitochondrial targeting domain, an unusual observation for Fe-SODs, which are typically localized in the cytoplasm. These results suggest that PmSOD1 encodes a mitochondrial Fe-SOD, which may contribute to P. marinus resistance to exogenous oxidative damage in host phagocytes. The present study constitutes the first report of an endogenous Fe-SOD that is directed to the mitochondria, and suggests that mitochondria targeting sequences have been conserved among diverse branches of the eukaryotes, including the early protista. It also illustrates the potential of complementation-based approaches for further gene discovery and characterization in P. marinus.

Amino Acid Sequence↗
PubMed1999

Identification and characterization of the putative retinoblastoma control element of the rat insulin-like growth factor binding protein-2 gene.

The authors previously identified a silencer of the rat IGFBP-2 gene. Sequence examination of the silencer has revealed that it contains the target sequence for the pRb (retinoblastoma) tumour suppressor gene, referred to as the retinoblastoma control element (RCE) which is frequently found in the regulatory element of cellular oncogenes and growth factors. The presence of RCE suggests that the IGFBP-2 gene may be regulated by the pRb tumour suppressor gene. An in vitro gel retardation assay has shown that the putative RCEs from the IGFBP-2 gene are complexed with multiple nuclear factors from the rat liver BRL-3A cells. These DNA-protein complexes were not detected with the nuclear extracts from the cells that were growth arrested at the G1/S border of the cell cycle by high cell density. Using specific antibodies, Sp1 was shown to be one of the components for the multiple DNA-protein complex while pRb does not appear to be directly involved in the formation of the complex.

Animals↗
PubMed2000

Genomic cloning and characterization of mitochondrial elongation factor Tu (EF-Tu) gene (tufM) from maize (Zea mays L.).

We have cloned and characterized a mitochondrial elongation factor Tu (EF-Tu) gene (tufM) in maize (Zea mays L.). This maize tufM gene encoded a polypeptide of 452 amino acid residues, consisting of a putative transit peptide of 55 residues and a mature EF-Tu of 397 residues. The coding region was composed of 12 exons and 11 introns that ranged from 76 to 1673bp in length. The deduced amino acid sequence showed 85.9% and 61.2% identity with Arabidopsis mitochondrial EF-Tu and Arabidopsis chloroplast EF-Tu sequence respectively. The transcription initiation site was determined to be 165bp upstream of the AUG initiation codon by primer extension analysis. Southern blot analysis revealed that the cloned EF-Tu gene was encoded by the members of small gene family in maize. Although this gene does not resemble the Arabidopsis nuclear tufA gene, which encodes the plastid EF-Tu, and does not contain sequence elements found in all cyanobacterial and plastid tufA genes, the predicted amino acid sequence includes an N-terminal extension that resembles a mitochondrial targeting sequence, and shares three unique sequence elements with mitochondrial EF-Tu's from Arabidopsis thaliana, Saccharomyces cerevisiae, and Homo sapiens. Therefore, we concluded that this gene encodes the maize mitochondrial EF-Tu.

Amino Acid Sequence↗
PubMed1992

Variations in alphoid DNA sequences escape detection of aneuploidy at interphase by FISH technique.

The advent of a new staining technique, termed fluorescence in situ hybridization (FISH), allows the rapid identification of the genomic constitution of an individual with aneuploidy even in interphase nuclei through the use of a series of chromosome-specific DNA probes, an approach termed "interphase cytogenetics." However, alphoid DNA sequences of every centromere are polymorphic (heteromorphic), and the number of targeted sequences may be below the detection level of a specific DNA probe, thus escaping detection and resulting in the imprecise identification of the chromosomal constitution at interphase. The limitations associated with the FISH technique have dire consequences which are emphasized here with an example in which the presence of an additional chromosome 21 in two siblings born consecutively with trisomy 21 (Down syndrome) was not detected by "interphase cytogenetics." The copy number of alphoid DNA sequences of one of the paternal chromosomes 21 was low and resulted in discordance between domain numbers at interphase and actual chromosome numbers at metaphase in both children. This is an isolated incident that could have led to a misdiagnosis if FISH were the only test employed. Although the advantages of this technology are undeniably enormous, the present finding has made it apparent that precise standards and reliability of the procedure must be established prior to its routine application.

DNA Probes↗
PubMed2006

Detection of terminal mismatches on DNA duplexes with fluorescent oligonucleotides.

This paper describes the design of terminal-mismatch discriminating fluorescent oligonucleotides (TMDFOs). The method is based on the use of sets of oligo-2'-deoxyribonucleotide probes linked via their 5'-ends, and varying-sized flexible polymethylene chains, to thiazole orange, with the linker being attached to the benzothiazole moiety. The sequence of each set of labelled probes was identical and complementary to the sequence to be analyzed on the single-stranded nucleic acid target except at the interrogation position, located at the 5'-end of the probes in a position adjacent to the attachment site of the label, where each of the four nucleic bases were incorporated. This work allowed the selection of probes showing, upon their hybridization with the target sequence, good discrimination between the matched and the mismatched duplexes under non-stringent conditions, with the mismatched duplexes being more fluorescent than the perfectly matched ones.

Base Pair Mismatch↗
PubMed2000

Mechanisms of mitochondrial protein import.

Mitochondria import most of their proteins from the cytosol. Precursor forms of most matrix proteins as well as some IM and IMS proteins are synthesized on cytoplasmic ribosomes with N-terminal cleavable signal sequences. Many other mitochondrial proteins including IM carrier proteins contain internal targeting sequences. Three multisubunit translocases, one in the OM and two in the IM, participate in the import process. These translocases co-operate with cytosolic chaperones, chaperone-like soluble proteins in the IMS as well as chaperones in the matrix. Insertion of carrier proteins into the IM only requires a membrane potential. On the other hand, translocation of preproteins across the IM into the matrix requires (i) a membrane potential, (ii) GTP hydrolysis, which occurs at the outer side of the IM, and (iii) ATP-dependent interactions occurring at the matrix side. Following import, the cleavable signal sequence of most preproteins is removed in one step by the MPP. In some cases, removal of the signal sequence is achieved in two steps; first by MPP and second by either mitochondrial intermediate peptidase or by IM peptidases. Imported proteins must be folded properly to perform their functions.

Animals↗
PubMed1995

Nuclear localization signals of human and Thermoplasma proteasomal alpha subunits are functional in vitro.

Proteasomes are located both in the nuclei and in the cytoplasm of eukaryotic cells. Active transport of these complexes through the nuclear pores has been proposed to be mediated by nuclear localization signals (NLS), which have been found in several of the alpha-type proteasomal subunits. We have tested three different putative NLS sequences from human alpha-type proteasomal subunits (Hsc iota, Hsc9, and Hsc3), as well as a putative NLS-type sequence from the archaeon Thermoplasma acidophilum, for their ability to direct non-nuclear proteins to the nucleus. Synthetic peptides containing these putative NLS sequences were generated and conjugated to large fluorescent reporter molecules: allophycocyanin or fluorescein-labeled bovine serum albumin. The conjugates were introduced into digitonin-permeabilized HeLa and 3T3 cells in the presence of cell lysate and ATP, and nuclear import was monitored by fluorescence microscopy. All three putative NLS sequences from human proteasomal subunits were able to direct the reporter molecules to the nucleus in both cell types, although differences in efficiency were observed. Substitution of threonine for the first lysine residue of the eukaryotic NLS motifs inhibited nuclear import completely. Interestingly, the putative NLS sequence found in T. acidophilum was also functional as a nuclear targeting sequence.

3T3 Cells↗