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.

SourceResearch connectionFrom yearThrough year

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

At least 1,351 records · Page 75Linked to original sources

PubMed1995

RAG-1 mutations that affect the target specificity of V(D)j recombination: a possible direct role of RAG-1 in site recognition.

The RAG-1 protein plays an essential role in V(D)j recombination, but its exact function has not yet been defined. Here we report that a particular mutation in RAG-1 affects recombination by altering the specificity of target sequence usage. Recombination mediated by wild-type RAG-1 is tolerant of a wide range of coding sequences adjacent to the recombination signal. With the mutant RAG-1, recombination is much more demanding; efficient recombination is only found when particular dinucleotides are adjacent to the signal sequence heptamer. The mutant is also more sensitive than wild-type RAG-1 to certain alterations within the signal sequence. We suggest that the RAG-1 protein may interact physically with the target DNA at the coding-signal sequence border.

Base Sequence↗

PubMed2004

Recent applications of RNAi in mammalian systems.

RNAi is a powerful cellular mechanism that involves targeted destruction of mRNAs. Although the phenomenon was first discovered in plants and lower eukaryotic organisms, it was later discovered as an important genetic regulatory mechanism in mammalian cells. RNAi is triggered by double stranded RNAs that are cleaved into short 21-23 base pair duplexes by an RNAse III type enzyme called Dicer. The short RNAs, termed small interfering RNAs (siRNAs), act as triggers for targeted RNA degradation. One of the two strands is selectively incorporated into a complex of proteins called the RNA induced silencing complex, or RISC. The incorporated small RNA guides the complex to the complementary target sequence, and this event is followed by endonucleolytic cleavage of the target and recycling of RISC. In mammalian cells, siRNAs do not activate interferon pathway genes, thereby making these powerful tools for sequence specific knockdown of RNAs. In this article we review the methods for programming mammalian cells with siRNAs, and overview a number of applications ranging from targeting oncogenes to inhibiting viral replication. The article also summarizes some important biological conclusions that can be drawn from selective downregulation of certain mRNA targets and addresses potential uses of RNAi as a new therapeutic modality.

Animals↗

PubMed1996

On signal sequence polymorphisms and diseases of distribution.

We report a previously unappreciated property of the signals that target organelle-specific proteins to their subcellular sites of action. Such targeting sequences are shown to be polymorphic. We discovered this polymorphism when we cloned the mitochondrial manganese-containing superoxide dismutase from cell lines of normal individuals and patients with genetic diseases of premature aging and compared their sequences to each other and to those previously reported. The polymorphism consists of a single nucleotide change in the region of the DNA that encodes the signal sequence such that either an alanine or valine is present. Subsequently, eight cell lines were analyzed and all three possible combinations of the two signal sequences were observed. Such signal sequence polymorphisms could result in diseases of distribution, where essential proteins are not properly targeted, thereby leading to absolute or relative deficiencies of critical enzymes within specific cellular compartments. Progeria and related syndromes may be diseases of distribution.

Base Sequence↗

PubMed2006

Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals.

A series of antisense oligonucleotides (ASOs) containing either 2'-O-methoxyethylribose (MOE) or locked nucleic acid (LNA) modifications were designed to investigate whether LNA antisense oligonucleotides (ASOs) have the potential to improve upon MOE based ASO therapeutics. Some, but not all, LNA containing oligonucleotides increased potency for reducing target mRNA in mouse liver up to 5-fold relative to the corresponding MOE containing ASOs. However, they also showed profound hepatotoxicity as measured by serum transaminases, organ weights and body weights. This toxicity was evident for multiple sequences targeting three different biological targets, as well as in mismatch control sequences having no known mRNA targets. Histopathological evaluation of tissues from LNA treated animals confirmed the hepatocellular involvement. Toxicity was observed as early as 4 days after a single administration. In contrast, the corresponding MOE ASOs showed no evidence for toxicity while maintaining the ability to reduce target mRNA. These studies suggest that while LNA ASOs have the potential to improve potency, they impose a significant risk of hepatotoxicity.

Animals↗

PubMed1993

Cloning and characterization of a cDNA encoding the cytosolic copper/zinc-superoxide dismutase from sweet potato tuberous root.

A full-length cDNA clone encoding a putative copper/zinc-superoxide dismutase (SOD) of sweet potato, Ipomoea batatas (L.) Lam. cv Tainong 57, was isolated from a cDNA library constructed in lambda gt10 from tuber root mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 152 amino acid residues. The deduced amino acid sequence showed higher homology (78-86%) with the sequence of the cytosolic SOD than that of the chloroplast SOD from other plant species. The residues required for coordinating copper and zinc are conserved as they are among all reported Cu/Zn-SOD sequences. In addition, it lacks recognizable plastic or mitochondrial targeting sequences. These data suggest that the isolated sweet potato clone encodes a cytosolic Cu/Zn-SOD.

Amino Acid Sequence↗

PubMed1992

Molecular cloning of a cDNA encoding aspartate aminotransferase-P2 from lupin root nodules.

Two isoenzymic forms of aspartate aminotransferase are present in the plant fraction of developing lupin root nodules. One of these forms, aspartate aminotransferase-P2 (AAT-P2), increases dramatically with the onset of biological nitrogen fixation and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. A day 18 lupin nodule cDNA library in the lambda ZapII vector was immunoscreened with a monoclonal antibody specific for AAT-P2 and yielded two near-full-length 1700 bp clones. These clones were sequenced. Amino acid sequences from three peptides derived from immunopurified AAT-P2 were aligned, and showed 100% homology with the amino acid sequence deduced from the cDNA clones. The DNA sequence showed 50% homology with AAT sequences from a range of animal sources. Conversion of the clones to the phagemid form allowed their expression in Escherichia coli where both exhibited enzyme activity that could be immunoprecipitated with AAT-P2-specific monoclonal antibodies. Western blot analysis revealed protein moieties with molecular masses of 39, 43, 45 and 55 kDa. The 5' end of the clones coded for a hydrophobic leader sequence of about 50 amino acids indicative of a targeting sequence and consistent with the plastid localisation of nodule AAT-P2.

Amino Acid Sequence↗

PubMed2006

Synthesis and biological properties of sequence-specific DNA-alkylating pyrrole-imidazole polyamides.

In recent years, many diseases including cancer and hereditary and viral diseases have been understood at the DNA sequence level. Direct control of the expression level of a specific gene would provide a promising approach for knowledge-based therapy. N-Methylpyrrole (Py) and N-methylimidazole (Im) polyamides are a new type of small compound that precisely bind to the minor groove of the DNA duplex in a sequence-specific fashion and recruit alkylating agents to the target sequence. We designed and synthesized a series of sequence-specific alkylating Py-Im polyamide conjugates that selectively alkylate predetermined DNA sequences. We have demonstrated that sequence-specific alkylating agents possess gene-silencing activities and a promising potency against human cancer cell lines as well as against xenografts of human cancer cell lines. In this Account, we focus on recent progress in alkylating Py-Im polyamides with regard to sequence specificity and biological activities and the future direction of rational molecular design of genetic switches in the postgenome era is described.

Alkylation↗

PubMed1990

Identification of glycoinositol phospholipid linked and truncated forms of the scrapie prion protein.

Analysis of carboxy-terminal peptides derived from endoproteinase Lys-C digests of the scrapie isoform of the hamster prion protein revealed that the majority of the molecules are glycoinositol phospholipid linked through ethanolamine attached at serin-231. However, approximately 15% of PrPSc had a carboxy-terminal peptide that ends at glycine-228. It is intriguing that this glycine is part of the PrP sequence Gly-Arg-Arg, which is an established target sequence for the proteolysis and release of bioactive peptides from larger precursors. The mechanism of formation, as well as the role of the truncated carboxy terminus in the dissemination and neuropathology of scrapie, remains to be determined.

Amino Acid Sequence↗

PubMed1994

Alternate-strand DNA triple-helix formation using short acridine-linked oligonucleotides.

We have used DNAse I footprinting to examine the formation of intermolecular DNA triple helices at sequences containing adjacent blocks of purines and pyrimidines. The target sites G6T6.A6C6 and T6G6.C6A6 were cloned into longer DNA fragments and used as substrates for DNAse I footprinting, which examined the binding of the acridine (Acr)-linked oligonucleotides Acr-T5G5 and Acr-G5T5 respectively. These third strands were designed to incorporate both G.GC triplets, with antiparallel Gn strands held together by reverse Hoogsteen base pairs, and T.AT triplets, with the two T-containing strands arranged antiparallel to each other. We find that Acr-T5G5 binds to the target sequence G6T6.-A6C6, in the presence of magnesium at pH 7.0, generating clear DNAse I footprints. In this structure the central guanine is not recognized by the third strand and is accessible to modification by dimethyl sulphate. Under these conditions no footprint was observed with Acr-G5T5 and T6G6.C6A6, though this triplex was evident in the presence of manganese chloride. Manganese also facilitated the binding of Acr-T5G5 to a second site in the fragment containing the sequence T6G6.C6A6. This represents interaction with the sequence G4ATCT6, located at the boundary between the synthetic insert and the remainder of the fragment, and suggests that this bivalent metal ion may stabilize triplexes that contain one or two mismatches. Manganese did not affect the interaction of either oligonucleotide with G6T6.A6C6.

Acridines↗

PubMed2003

Linear plasmid SLP2 of Streptomyces lividans is a composite replicon.

SLP2 is a 50 kb linear plasmid in Streptomyces lividans that contains short (44 bp) terminal inverted repeats and covalently bound terminal proteins. The nucleotide sequence of SLP2 was determined. The rightmost 15.4 kb sequence is identical to that of the host chromosome, including the Tn4811 sequence at the border, which is interrupted by an insertion sequence (IS) element in SLP2. Examination of the flanking target sequences of Tn4811 suggests a previous recombinational event there. The 43 putative protein coding sequences contained many involved in replication (including two terminal protein homologues), partitioning, conjugal transfer and intramycelial spread. The terminally located helicase-like gene ttrA was necessary for conjugal transfer. The two telomeres diverge significantly in primary sequence, while preserving similar secondary structures. Mini-linear plasmids containing these telomeres replicated in S. lividans using the chromosomally encoded terminal protein. In addition, two pseudotelomere sequences are present near the left telomere. The G+C content and GC or AT skew profiles exhibit complex distributions. These, plus the inferred recombination at the right arm, indicate that SLP2 has evolved through rounds of exchanges involving at least three replicons.

Bacterial Proteins↗

PubMed1994

Wheat acetyl-coenzyme A carboxylase: cDNA and protein structure.

cDNA fragments encoding part of wheat (Triticum aestivum) acetyl-CoA carboxylase (ACC; EC 6.4.1.2) were cloned by PCR using primers based on the alignment of several biotin-dependent carboxylases. A set of overlapping clones encoding the entire wheat ACC was then isolated by using these fragments as probes. The cDNA sequence contains a 2257-amino acid reading frame encoding a 251-kDa polypeptide. The amino acid sequence of the most highly conserved domain, corresponding to the biotin carboxylases of prokaryotes, is 52-55% identical to ACC of yeast, rat, and diatom. Identity with the available C-terminal amino acid sequence of maize ACC is 66%. The biotin attachment site has the typical eukaryotic EVMKM sequence. The cDNA does not encode an obvious chloroplast targeting sequence. Various cDNA fragments hybridize in Northern blots to a 7.9-kb mRNA. Southern analysis with cDNA probes revealed multiple hybridizing fragments in hexaploid wheat DNA. Some of the wheat cDNA probes also hybridize with ACC-specific DNA from other plants, indicating significant conservation among plant ACCs.

Acetyl-CoA Carboxylase↗

PubMed1999

Padlock oligonucleotides for duplex DNA based on sequence-specific triple helix formation.

An oligonucleotide was circularized around double-stranded DNA thanks to triple helix formation. Short oligonucleotides are known to be able to form DNA triple helices by binding into the DNA major groove at an oligopurine.oligopyrimidine sequence. After sequence-specific recognition of a double-stranded DNA target through triple helix formation, the ends of the triplex-forming oligonucleotide were joined through the action of T4 DNA ligase, thus creating a circular DNA molecule catenated to the plasmid containing the target sequence. The labeling of the double-stranded DNA sequence has been carried out without any chemical or enzymatic modification of this sequence. These "padlock" oligonucleotides provide a tool to attach a noncovalent tag in an irreversible way to supercoiled plasmid or other double-stranded DNAs. Such a complex may find applications in the development of new techniques for duplex DNA detection or plasmid delivery methods for gene therapy.

Animals↗

PubMed1989

Xanthine dehydrogenase is transported to the Drosophila eye.

The rosy (ry) locus in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase. Mutants that have no enzyme activity are characterized by a brownish eye color phenotype reflecting a deficiency in the red eye pigment. This report demonstrates that enzyme which is synthesized in some tissue other than the eye is transported and sequestered at the eye. Previous studies find that no leader sequence is associated with this molecule but a peroxisomal targeting sequence has been noted, and the enzyme has been localized to peroxisomes. This represents a rare example of an enzyme involved in intermediary metabolism being transported from one tissue to another and may also be the first example of a peroxisomal protein being secreted from a cell.

Animals↗

PubMed2000

Purification and characterization of chloroplast dehydroascorbate reductase from spinach leaves.

Green leaves of plants require the high-level activity that can regenerate ascorbate during photosynthesis. One of such enzyme is dehydroascorbate reductase (DHAR), but the molecular and enzymological properties of the enzyme remain to be fully characterized. In this study, we showed that two major DHAR existed in spinach leaves. The two DHARs occupied at least over 90% of total DHAR activity. The amount of the two DHARs was almost the same. We purified both DHARs from spinach leaves. One form of DHAR originated in chloroplasts; the other occurred in the subcellular compartment other than chloroplasts. The chloroplast DHAR had Km values of 70 microM and 1.1 mM for dehydroascorbate and reduced glutathione, respectively. The specific activity of the purified enzyme corresponded to 360 micromol of ascorbate formed per milligram of protein per minute. These properties were quite different from those of trypsin inhibitor, which has been reported to be the plastid DHAR. The other DHAR had the very similar properties to those of chloroplast DHAR. Chloroplast and the other DHARs functioned as a monomer with molecular masses of 26 kDa and 25 kDa, respectively. cDNA for the chloroplast DHAR was cloned with the determined amino-terminal amino acid sequence. The primary sequence predicted from the cDNA included the plastid-targeting sequence. Finally, the significance of chloroplast DHAR in the regeneration of ascorbate is discussed.

Amino Acid Sequence↗

PubMed1990

The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator.

The mouse gene Krox-24 is transiently activated during cell cycle reentry. It encodes a protein with three zinc fingers similar to those of the transcription factor Sp1. Here we present a biochemical characterization of the gene products. Krox-24 mRNA is translated into two proteins of 82 and 88 kilodaltons, designated p82Krox-24 and p88Krox-24, respectively. p82Krox-24 is initiated at the first AUG codon of the open reading frame, whereas synthesis of p88Krox-24 starts at a non-AUG codon located upstream. Both proteins were synthesized in HeLa cells infected with recombinant vaccinia viruses expressing Krox-24 cDNAs. Under these conditions, they were found phosphorylated on serine residues and glycosylated. The availability of the proteins made possible the determination of the DNA recognition sequence. In vitro, Krox-24 bound specifically to the sequence 5'-GCG(C/G)GGGCG-3'. This sequence is similar but not identical to the Sp1 target sequence. Insertion of an oligomer for the binding site in cis, close to the herpes simplex virus thymidine kinase promoter, rendered this promoter responsive to Krox-24. Krox-24 is therefore a sequence-specific transcriptional activator. Krox-24-binding sites were found upstream of several serum-inducible genes, raising the possibility that Krox-24 is involved in the regulation of these genes.

Amino Acid Sequence↗

PubMed1993

XrpFI, an amphibian transcription factor composed of multiple polypeptides immunologically related to the GA-binding protein alpha and beta subunits, is differentially expressed during Xenopus laevis development.

XrpFI, first identified in the extract of Xenopus laevis oocyte nuclei, binds to a proximal sequence of the L14 ribosomal protein gene promoter. Its target sequence, 5'-TAACCGGAAGTTTGT-3', is required to fully activate the promoter, and the two G's of the central motif are essential for factor binding and transcriptional activation; our data also suggest that XrpFI may play a role in cap site positioning. The binding site of XrpFI is homologous to the sequence recognized by the family of ets genes. Antibodies specific for Ets-1 and Ets-2 proteins did not react with XrpFI, but those raised against the rat alpha and beta GA-binding proteins both supershifted the retarded bands formed by XrpFI. The Xenopus polypeptides related to GA-binding protein alpha interact with DNA both as monomers and as heterodimers associated with beta-related proteins. Oocyte nuclei contain multiple forms of alpha- and beta-related proteins: the alpha-like proteins remain throughout development, while the pattern of the beta species changes in the embryonic stages examined. beta-like proteins are undetectable in the cleavage period up to the neurula stage, but at later stages, when ribosomal protein genes are actively transcribed, two beta-related polypeptides reappear.

Animals↗

PubMed2004

An analysis of mismatched duplex DNA unzipping through a bacterial nanopore.

A 50-base Guide strand was synthesized that consisted of a central 10-base probe sequence flanked by two tracts of 20 adenine residues. Target sequences of 10 bases containing up to three mismatches were prepared and hybridized to the Guide strand in 1 M KCl. The transport of these constructs through single alpha-hemolysin pores was analysed by measuring the current blockade as a function of time. Complementary dsDNA takes significantly longer (840 +/- 60 micro s) to pass through the pore than a sequence of the same length containing a single (590 +/- 45 micro s) and a double (270 +/- 50 micro s) mismatch. Constructs involving three mismatches were indistinguishable from Guide ssDNA transport (120 +/- 30 micro s). The results suggest that dsDNA must unzip as it is transported through the nanopore. Duplexes containing mismatches unzip more quickly and can be distinguished from those with perfect complementarity.

Adenine↗

PubMed1999

Protalign: a 3-dimensional protein alignment assessment tool.

Protein fold recognition (sometimes called threading) is the prediction of a protein's 3-dimensional shape based on its similarity to a protein of known structure. Fold predictions are low resolution; that is, no effort is made to rotate the protein's component amino acid side chains into their correct spatial orientations. The goal is simply to recognize the protein family member that most closely resembles the target sequence of unknown structure and to create a sensible alignment of the target to the known structure (i.e., a structure-sequence alignment). To facilitate this type of structure prediction, we have designed a low resolution molecular graphics tool. ProtAlign introduces the ability to interact with and edit alignments directly in the 3-dimensional structure as well as in the usual 2-dimensional layout. It also contains several functions and features to help the user assess areas within the alignment. ProtAlign implements an open pipe architecture to allow other programs to access its molecular graphics capabilities. In addition, it is capable of "driving" other programs. Because amino acid side chain orientation is not relevant in fold recognition, we represent amino acid residues as abstract shapes or glyphs much like Lego (tm) blocks and we borrow techniques from comparative flow visualization using streamlines to provide clean depictions of the entire protein model. By creating a low resolution representation of protein structure, we are able to at least double the amount of information on the screen. At the same time, we create a view that is not as busy as the corresponding representations using traditional high resolution visualization methods which show detailed atomic structure. This eliminates distracting and possibly misleading visual clutter resulting from the mapping of protein alignment information onto a high resolution display of the known structure. This molecular graphics program is implemented in Open GL to facilitate porting to other platforms.

Amino Acid Sequence↗