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 937 records · Page 52Linked to original sources
PubMed2006

Suppression of the Sendai virus M protein through a novel short interfering RNA approach inhibits viral particle production but does not affect viral RNA synthesis.

Short RNA interference is more and more widely recognized as an effective method to specifically suppress viral functions in eukaryotic cells. Here, we used an experimental system that allows suppression of the Sendai virus (SeV) M protein by using a target sequence, derived from the green fluorescent protein gene, that was introduced in the 3' untranslated region of the M protein mRNA. Silencing of the M protein gene was eventually achieved by a small interfering RNA (siRNA) directed against this target sequence. This siRNA was constitutively expressed in a cell line constructed by transduction with an appropriate lentivirus vector. Suppression of the M protein was sufficient to diminish virus production by 50- to 100-fold. This level of suppression had no apparent effect on viral replication and transcription, supporting the lack of M involvement in SeV transcription or replication control.

Cell Line, Tumor↗
PubMed1995

Helix-stabilizing agent, CC-1065, enhances suppression of translation by an antisense oligodeoxynucleotide.

The antitumor antibiotic CC-1065 is known to bind at selected sequences in the minor groove of duplex nucleic acids and to hyperstabilize the duplexes against thermal melting. These properties suggested that CC-1065 may enhance translation inhibition by antisense oligonucleotides directed against a specific mRNA. A 585 bp mRNA transcript containing the equine infectious anemia virus (EIAV) S2 gene and a portion of the env gene was prepared. Also, a complementary 20 mer antisense oligodeoxynucleotide (5'-TGTTGGGTAATAGG-GGTTGA-3') was prepared against a target sequence in the mRNA located near the translational initiation sites of the overlapping S2 and env genes. The center of the target sequence had an expected CC-1065 recognition sequence (5'-UAUUA-3'). Translation in the presence of CC-1065 and antisense was markedly suppressed compared with that of antisense alone. Addition of a sense 20 mer strand, with or without CC-1065, had little or no effect on translation. CC-1065 and related compounds may be useful as ligands for enhancing the stability of sense-antisense duplexes and for promoting the inhibition of translation by antisense oligonucleotides.

Antibiotics, Antineoplastic↗
PubMed1997

Strategems in vitro for gene therapies directed to dominant mutations.

A major difficulty associated with the design of gene therapies for autosomal dominant diseases is the immense intragenic heterogeneity often encountered in such conditions. In order to overcome such difficulties we have designed, and evaluated in vitro, three strategies which avoid a requirement to target individual mutations for genetic suppression. In the first, normal and mutant alleles are suppressed by targeting sequences in transcribed but untranslated regions of transcript (UTRs), enabling introduction of a replacement gene with the correct coding sequencing but altered UTRs to prevent suppression. A second approach involves suppression in coding sequence and concurrent introduction of a replacement gene by exploiting the degeneracy of the genetic code. A third strategy utilises intragenic polymorphism to suppress the disease allele specifically, the advantage being that a proportion of patients with different disease mutations have the same polymorphism. These approaches provide a wider choice of target sequence than those directed to single disease mutations and are appropriate for many mutations within a given gene. General methods for suppression may be directed towards the primary defect or a secondary effect associated with the disease process, such as apoptosis. Three general methods targeting the primary defect which circumvent problems of allelic genetic heterogeneity are explored in vitro using hammerhead ribozymes designed to target transcripts from the rhodopsin, peripherin and collagen 1A1 and 1A2 genes, extensive genetic heterogeneity being a feature of associated disease pathologies.

Alleles↗
PubMed1998

[The experimental study on competitive PCR for quantitation of herpesviruses].

A single pair of oligonucleotide primer selected within a highly conserved region of the DNA polymerase gene in herpesviruses was synthesized. The competitive template DNA purified from cytomegalovirus (CMV) DNA was used to carry out competitive PCR amplification with herpes simplex virus type 1 (HSV1) DNA (target sequences). And anti-HSV1 effects of acyclovir (ACV) was investigated by the method. The results showed that the efficacy of PCR amplification was equal to each other(the ratio of the quantity of competitor template with DNA to the target sequence was 1.5:1). As the concentration of ACV was increased, the quantity of HSV1, DNA was decreased. It suggests that this method is practicable and some defects of mutant template can be overcomed.

Acyclovir↗
PubMed1994

Fluorescence energy transfer between two triple helix-forming oligonucleotides bound to duplex DNA.

An 11-mer oligopyrimidine was covalently linked via its 5'-phosphate to an acridine derivative (acridine-11-mer), and a 13-mer was covalently linked via its 3'-phosphate to an ethidium derivative (13-mer-ethidium). Each of them formed a triple helix with a 31-bp DNA fragment containing two oligopurine-oligopyrimidine sequences, 11 and 13 bp in length, separated by a variable number of base pairs. When both oligonucleotides were bound to the 31-bp DNA fragment, fluorescence energy transfer (FET) from acridine to ethidium was observed, as revealed by a quenching of acridine fluorescence and a sensitized ethidium emission. FET was temperature-dependent and occurred only when both oligonucleotides were simultaneously bound to the DNA matrix. A single base-pair change in one of the target sequences strongly reduced the energy-transfer efficiency. This method was used to discriminate between a fully complementary and a mismatched target sequence.

Base Sequence↗
PubMed1996

Triplex-forming oligonucleotides trigger conformation changes of a target hairpin sequence.

We used a DNA duplex formed between the 5' end of a 69mer (69T) and an 11mer (OL7) as a substrate for BamHI. The former oligonucleotide folds into a hairpin structure, the stem of which contains a stretch of pyrimidines in one strand and consequently a stretch of purines in the other strand. The oligomer 69T was used as a target for complementary oligodeoxypyrimidines made of 10 nt (OL1), 16 nt (OL5) or 26 nt (OL2) which can engage the same 10 pyrimidine-purine-pyrimidine triplets with the 69T hairpin stem. Although the binding site of OL7 did not overlap that of OL1, OL2 or OL5, the BamHI activity on 69T-OL7 complexes was drastically modified in the presence of these triplex-forming oligomers: OL1 abolished the cleavage by BamHI whereas OL5 and OL2 strongly increased it. Using footprinting assays and point-mutated oligonucleotides we demonstrated that these variations were due to different conformations of the 69T-OL7 complex induced by the binding of oligomers OL1, OL2 or OL5. Therefore, oligonucleotides can act as structural switchers, offering one additional mode for modulating gene expression.

Animals↗
PubMed1995

Generation of entire human papillomavirus genomes by long PCR: frequency of errors produced during amplification.

Recently, several improvements of traditional PCR techniques have facilitated the amplification of significantly longer DNA target sequences. Here we report an improved method for amplification of entire human papillomavirus (HPV) genomes. Using rTth DNA polymerase, XL (Perkin-Elmer, Foster City CA), and the accompanying XL PCR buffer system, we have successfully amplified 8-kb genomes from approximately 10 copies of input reference strain HPV16 DNA. This long PCR (LPCR) method was subsequently used to amplify the entire HPV16 genome from clinical specimens. The fidelity with which the rTth DNA polymerase XL amplified target sequences under our chosen amplification conditions was estimated by partial sequencing of cloned LPCR products generated from cloned reference strain HPV16 genomes. A region spanning the HPV16 E6, E7, and part of the E1 open reading frames (ORFs) was sequenced in 29 clones. A total of 33 nucleotide substitutions were observed in the 23.5 kb sequenced. This corresponds to an error frequency of approximately one error per 700 bases. Finally, LPCR methods were used to amplify entire, novel HPV genomes from clinical specimens. LPCR primer pairs were designed for amplification of seven potentially novel HPV types. Amplicons of approximately 8 kb were generated from five of the seven HPV types targeted. One of the LPCR-generated novel genomes, CP141, was subsequently cloned and a partial sequence was determined.

Amino Acid Sequence↗
PubMed1995

Technical report. Optimizing probe selection in directed heteroduplex analysis using HDprobe 1.1.

Directed heteroduplex analysis (DHDA) has proven to be a powerful technique for rapid geotyping in human populations. This strategy should also have widespread utility in differentiating closely related organisms of medical and public health importance through identification of DNA sequence polymorphisms. Identifying an optimal probe sequence for use in DHDA has required empirical testing of both the positive and negative strands of a number of potential probes. To identify optimal probes more efficiently, a computer program has been developed that predicts the number of potential stable and unstable mismatches between a probe and its target sequences in DHDA. This information can then be used to predict--from among a group of potential probes--which one will be the most successful in differentiating closely related homologues of a targeted gene sequence. This approach was tested on a number of probe and target sequences derived from the mitochondrial NADH dehydrogenase subunit 4 gene of the West African black fly, Simulium damnosum sensu lato. The number of unstable mismatches predicted to occur in a given heteroduplex by the computer program was found to be important in differentiating closely related species. Therefore, this strategy is useful in identifying informative probes in the development of new DHDA-based assays.

Animals↗
PubMed1992

The polymerase chain reaction: an epidemiological tool to differentiate between two clusters of pathogenic Yersinia enterocolitica strains.

A primer set designed to amplify the enterotoxin (yst) gene of pathogenic Yersinia enterocolitica strains generated two different electrophoretic profiles of the target sequence when a collection of strains of worldwide origin was screened. Serovars O:1,3; O:2a,3; O:3; O:5,27 and O:9, known as European strains, produced a 200-bp fragment that matched the size of the target sequence. However, serovars O:4,32; O:8; O:13a,13b; O:20 and O:21, known as American strains, generated two fragments of 1.4 and 1.6 kb. The amplified products of one American strain were sequenced and the presence of the yst gene was confirmed in both fragments. Thus, the potential of the polymerase chain reaction to be used as an epidemiological tool in differentiation between the two clusters of pathogenic strains of Y. enterocolitica could be demonstrated.

Base Sequence↗
PubMed2000

Molecular adaptation of alanine:glyoxylate aminotransferase targeting in primates.

The intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is targeted to different organelles (mitochondria and/or peroxisomes) in different species. Possibly under the influence of dietary selection pressure, the subcellular distribution of AGT has changed on at least eight occasions during the evolution of mammals. AGT targeting is dependent on the variable use of two alternative transcription and translation initiation sites which determine whether or not the region encoding the N-terminal mitochondrial targeting sequence is contained within the open reading frame. In the present study, we sequenced the 5' region of the AGT gene, including both ancestral translation start sites, for 11 anthropoid primates and compared the results with data already available for two others. We show that while the more 3' of the two translation start sites is maintained in all species, the more 5' site has been lost in six species (five of seven catarrhines and one of six platyrrhines). In addition, the remaining two catarrhines, which have maintained the 5' translation start site, are predicted to have lost mitochondrial targeting by a different mechanism, possibly loss of the more 5' transcription start site. Analysis of the relative frequencies of nonsynonymous and synonymous mutations in the region encoding the extant or ancestral mitochondrial targeting sequences led us to suggest that there has been recent strong positive selection pressure to lose, or decrease the efficiency of, mitochondrial AGT targeting in several anthropoid lineages, and that the loss of mitochondrial targeting in this group of mammals is likely to have occurred on at least four, and possibly five, separate occasions.

5' Untranslated Regions↗
PubMed2007

A large genomic deletion in the PDHX gene caused by the retrotranspositional insertion of a full-length LINE-1 element.

The long interspersed element-1 (LINE-1 or L1) retrotransposition has altered the human genome in many ways. In particular, recent in vitro studies have demonstrated that the retrotranspositional insertion of L1 elements has resulted in significant genomic deletions. Here we provide evidence for its operation in the human genome by identifying a approximately 46-kb pathological genomic deletion in the PDHX gene directly linked to the insertion of a full-length L1 element, in a patient with pyruvate dehydrogenase complex (PDHc) deficiency. Both the deduced bottom and top strand cleavage sites in the PDHX gene coincide with the consensus L1 endonuclease (EN) target sequence 5'-TTTT/A-3', while the full-length L1 element is followed by a 67-bp poly(A) tail. Interestingly, two hairpin structures, potentially formed by the inverted repeats present immediately 5' to the top strand nick site and 3' to the bottom strand nick site, may have facilitated the accessibility of L1 EN to the target sequences and also brought the two otherwise distantly located sequences into close proximity. Since the L1 element inserted in the PDHX gene is full-length, we favor the model of the template jumping as opposed to that of the microhomology-mediated end-joining for linking the 5' end of the nascent L1 copy to its genomic target. Our finding not only serves as an important complement to the in vitro approaches to studying L1 retrotransposition, but also reveals a novel mechanism causing human genetic disease.

Adult↗
PubMed1995

COOH-terminal sequence motifs target the T cell protein tyrosine phosphatase to the ER and nucleus.

The noncatalytic domain of the human T cell protein tyrosine phosphatase (TCPTP) is alternatively spliced to generate a 45-kD form, p45TC, and a 48-kD form, p48TC (Champion-Arnaud et al., 1991; Mosinger et al., 1992). This manuscript concerns structural motifs in the noncatalytic segment of the enzyme responsible for targeting the two forms to different subcellular compartments. Endogenous and transiently expressed p48TC associates with the ER, as determined by sucrose gradient fractionation and indirect immunofluorescence, respectively. By contrast, p45TC localizes in the nucleus even though upon cell lysis it is not retained and fractionates with markers for soluble enzymes. Using fusion proteins consisting of beta-galactosidase and COOH-terminal fragments of p48TC, two motifs necessary for ER retention within a 70-residue targeting segment have been identified. These include the terminal 19 hydrophobic residues which comprise a potential membrane-spanning segment and residues 346-358 which encompass a cluster of basic amino acids that may represent another type of ER retention motif. The sequence RKRKR, which immediately precedes the splice junction, functions as a nuclear localization signal for p45TC.

Alternative Splicing↗
PubMed1998

Exchange of viral promoter/enhancer elements with heterologous regulatory sequences generates targeted hybrid long terminal repeat vectors for gene therapy of melanoma.

To generate transcriptionally targeted vectors, tissue-specific elements of the human tyrosinase promoter were exchanged with corresponding viral elements in the Moloney murine leukemia virus long terminal repeat (LTR). From these experiments, a vesicular stomatitis virus type G pseudotyped, hybrid LTR vector that contained three tyrosinase enhancer elements and gave high-level, tightly tissue-specific expression at high titers (3 x 10(7) CFU/ml) was constructed.

Animals↗
PubMed1992

Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA.

Carrier cattle infected with Babesia bovis are difficult to detect because of the low numbers of parasites that occur in peripheral blood. However, diagnosis of low-level infections with the parasite is important for evaluating the efficacies of vaccines and in transmission and epidemiological studies. We used the polymerase chain reaction (PCR) to amplify a portion of the apocytochrome b gene from the parasite and tested the ability of this method to detect carrier cattle. The target sequence is associated with a 7.4-kb DNA element in undigested B. bovis genomic DNA (as shown previously), and the amplified product was detected by Southern and dot blot hybridization. The assay was specific for B. bovis, since no amplification was detected with Babesia bigemina, Trypanosoma brucei, Anaplasma marginale, or leukocyte DNA. The target sequence was amplified in DNA from B. bovis Mexico, Texas, and Australia S and L strains, demonstrating the applicability of the method to strains from different geographic regions. The sensitivity of the method ranged from 1 to 10 infected erythrocytes extracted from 0.5 ml of blood. This sensitivity was about 1,000 times greater than that from the use of unamplified parasite DNA. By the PCR method, six B. bovis carrier cattle were detected 86% of the time (range, 66 to 100%) when they were tested 11 times, while with microscopic examination of thick blood smears, the same carrier cattle were detected only 36% of the time (range, 17 to 66%). The method provides a useful diagnostic tool for detecting B. bovis carrier cattle, and the sensitivity is significantly improved over that of current methods. The results also suggest that characteristics of the apocytchrome b gene may make this a valuable target DNA for PCR-based detection of other hemoparasites.

Animals↗
PubMed1990

Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes.

Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif+-Frankia strains only, were used to detect Frankia specific target sequences in RNA isolations from soil. A method is described for direct isolation of RNA from a loamy soil and a peat. Yields of about 10 ng RNA/g wet soil are obtained without detectable contamination with humic acids. Isolation of RNA after initial extraction of bacteria from soil resulted in significantly lower RNA yields, compared to the direct isolation procedure. Hybridization with both probes against rRNA isolations from Frankia-containing soil could detect target sequences within RNA isolations from 1 g wet soil with an estimated detection limit of 10(4) cells.

Actinomycetales↗
PubMed2005

Further characterization of human DNA polymerase delta interacting protein 38.

Polymerase delta interacting protein 38 (PDIP38) was identified as a human DNA polymerase (pol) delta interacting protein through a direct interaction with p50, the small subunit of human pol delta. PDIP38 was also found to interact with proliferating cell nuclear antigen, which suggested that it might play a role in vivo in the processes of DNA replication and DNA repair in the nucleus. In order to characterize further this novel protein, we have examined its subcellular localization by the use of immunochemical and cellular fractionation techniques. These studies show that PDIP38 is a novel mitochondrial protein and is localized mainly to the mitochondria. PDIP38 was shown to possess a functional mitochondrial targeting sequence that is located within the first 35 N-terminal amino acid residues. The mature PDIP38 protein is about 50 amino acid residues smaller than the full-length precursor PDIP38 protein, consistent with it being processed by cleavage of the mitochondrial targeting sequence during entry into the mitochondria. His-tagged mature PDIP38 inhibited pol delta activity in vitro and interacted with human papillomavirus 16 E7 oncoprotein, suggesting that PDIP38 might play a role in the pol delta-mediated viral DNA replication. Although the localization of PDIP38 to the mitochondria suggests that it serves functions within the mitochondria, we cannot eliminate the possibility that it may be involved in pol delta-mediated DNA replication or DNA repair under certain conditions such as viral infection.

Cell Line↗
PubMed1995

PCR in situ: aspects which reduce amplification and generate false-positive results.

PCR in situ promises the ability to amplify and detect very low levels of target nucleic acid in tissues. Despite considerable effort, the technique is still technically difficult and has not yet proved to be reliable or reproducible. We have now identified a number of factors which can contribute to the poor amplification of the target DNA and to the generation of false-positive signals. These factors include the effects of fixation, reagent abstraction, DNA degradation, DNA end-labelling and product diffusion. We present evidence to show that formaldehyde fixation cross-links histones to DNA and thus restricts the subsequent amplification of target sequences by PCR. End-labelling of DNA occurs when direct incorporation is used to detect amplified products and this gives rise to false-positive signals. Amplified products can also diffuse out of cells and into neighbouring cells which do not contain target sequences. They can undergo re-amplification within these cells giving rise to false-positive signals. We believe considerable caution should be exercised in the interpretation of results generated using PCR in situ.

Autoradiography↗
PubMed2000

Abietadiene synthase from grand fir (Abies grandis): characterization and mechanism of action of the "pseudomature" recombinant enzyme.

The oleoresin secreted by grand fir (Abies grandis) is composed of resin acids derived largely from the abietane family of diterpene olefins as precursors which undergo subsequent oxidation of the C18-methyl group to a carboxyl function, for example, in the conversion of abieta-7,13-diene to abietic acid. A cDNA encoding abietadiene synthase has been isolated from grand fir and the heterologously expressed bifunctional enzyme shown to catalyze both the protonation-initiated cyclization of geranylgeranyl diphosphate to the intermediate (+)-copalyl diphosphate and the ionization-dependent cyclization of (+)-copalyl diphosphate, via a pimarenyl intermediate, to the olefin end products. Abietadiene synthase is translated as a preprotein bearing an N-terminal plastidial targeting sequence, and this form of the recombinant protein expressed in Escherichia coli proved to be unsuitable for detailed structure-function studies. Since the transit peptide-mature protein cleavage site could not be determined directly, a truncation series was constructed to delete the targeting sequence and prepare a "pseudomature" form of the enzyme that resembled the native abietadiene synthase in kinetic properties. Both the native synthase and the pseudomature synthase having 84 residues deleted from the preprotein converted geranylgeranyl diphosphate and the intermediate (+)-copalyl diphosphate to a nearly equal mixture of abietadiene, levopimaradiene, and neoabietadiene, as well as to three minor products, indicating that this single enzyme accounts for production of all of the resin acid precursors of grand fir. Kinetic evaluation of abietadiene synthase with geranylgeranyl diphosphate and (+)-copalyl diphosphate provided evidence for two functionally distinct active sites, the first for the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate and the second for the cyclization of (+)-copalyl diphosphate to diterpene end products, and demonstrated that the rate-limiting step of the coupled reaction sequence resides in the second cyclization process. The structural implications of these findings are discussed in the context of primary sequence elements considered to be responsible for binding the substrate and intermediate and for initiating the respective cyclization steps.

Isomerases↗