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At least 1,621 records · Page 90Linked to original sources
PubMed2001

Characterization of BseMII, a new type IV restriction-modification system, which recognizes the pentanucleotide sequence 5'-CTCAG(N)(10/8)/.

We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5'-CTCAG sequence, and the nucleotide sequence of the genes encoding them. The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3'-protruding end. Magnesium ions and S:-adenosyl-L-methionine (AdoMet) are required for cleavage. S:-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5'-CTCAG-3'/5'-CTGAG-3'. Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5'-CTCAG strand of the target duplex. The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-L-methionine binding and catalysis. According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.

Adenosine↗
PubMed2005

Inhibition of human-type 1 3beta-hydroxysteroid deshydrogenase/Delta(5)-Delta(4)-isomerase expression using siRNA.

Specific inhibition of type 1 3beta-HSD is of particular interest since it will allow us to control the formation of androgens and estrogens in peripheral target tissues without affecting type 2 3beta-HSD, which is responsible for the biosynthesis of glucocorticoids and mineralocorticoids in the adrenals. The high homology between types 1 and 2 3beta-HSD is a major difficulty in the development of specific inhibitors through classical chemical synthesis. In this report, we describe the use of small interference RNA (siRNA) to specifically inhibit human type 1 3beta-HSD. We have constructed three DNA vector-based RNAi vectors that allow us to produce three RNA duplexes of 21 nucleotides targeting three different coding regions of human type 1 3beta-HSD mRNA. The resulting constructs were co-transfected into HEK-293 cells with a vector expressing type 1 3beta-HSD. The results indicate that while the two duplexes that target sequences in the 5'-region do not have a strong inhibitory effect, the duplex that targets the 3'-region efficiently inhibits 3beta-HSD activity. Up to 98% inhibition has been observed. To our knowledge, this is the first report showing successful inhibition of steroidogenic enzymes using siRNA technology.

3-Hydroxysteroid Dehydrogenases↗
PubMed1996

Distribution of programming in a rapid aimed sequential movement.

Studies indicate that rapid sequential movements are preprogrammed and that preprogramming increases with complexity, but more complex sequences that require on-line programming have been seldom been studied. The purpose of this investigation was to determine whether on-line programming occurs in a 7-target sequence in which there is a unique target constraint and if so, to determine how different task constraints affect the distribution of additional programming. Subjects contacted seven targets with a hand-held stylus as quickly as possible while maintaining a 90% hit rate. Initiation-band execution-timing patterns and movement kinematics were measured to determine when the additional programming took place. Results indicated that additional programming occurred before initiation and during movement to the first target when the constraint required more spatial accuracy (small target). A different type of unique target (a triple hit of one target) caused the additional programming to occur on-line one or two segments before its execution. Different positions of the unique target also affected timing patterns. Results were discussed in terms of: (1) capacity of processing; (2) control of movement variance; and (3) mean velocity as a programmed parameter in sequential aiming movements.

Adult↗
PubMed1993

Multiple and sensitive fluorescence in situ hybridization with rhodamine-, fluorescein-, and coumarin-labeled DNAs.

We have tested the use of several newly developed red, green, and blue fluorescent dUTPs in direct, multiple, and sensitive fluorescence in situ hybridization procedures. Among the ones tested, the tetramethylrhodamine-dUTP proved to give the best sensitivity; using conventional epifluorescence microscopy, cosmids could be visualized with a hybridization efficiency of 90%. Fluorescein-dUTP permitted visual cosmid detection with 50% efficiency, and, to the human eye, the green signals appeared less bright. With a blue fluorescent coumarin-labeled dUTP, only highly repetitive target sequences could be visualized directly. Imaging with a cooled CCD (charged coupled device) camera for prolonged integration times permitted localization of small targets using probes labeled with red and green fluorochromes. Visualization of a 1.8-kb single copy sequence with a rhodamine-labeled cDNA proved feasible. The strategy of identifying hybridization sites with multiple colors was successfully applied for multiple hybridizations to repetitive and unique targets, using the red and green labels directly and the biotin label indirectly with one layer of avidin-coumarin.

Cells, Cultured↗
PubMed1998

Insertion specificity and trans-activation of IS801.

The transposable element IS801, isolated from plasmid pMMC7105 of Pseudomonas syringae pv. phaseolicola, transposes in Escherichia coli to plasmid targets, expressing a relatively relaxed target specificity. The target sequences are tetramers with homology with the left terminus (GAAC) of the transposing unit, the alternative targets being GAAC, GGAC, CAAG, and CGAC. In the areas flanking IS801 in 13 different locations, no similarities other than the target tetramer were observed. The transposase is physically and functionally separable from the transposing unit since transposition of constructs carrying marker genes occurs with the transposase expressed in trans. The IS801 transposase shows amino acid sequence homology to the transposases of the E. coli elements IS91 and IS1294. These transposases contain conserved amino acid motifs found in the replicases of certain plasmids that replicate as rolling circles.

Base Sequence↗
PubMed2023

FLASH-TB: an Application of Next-Generation CRISPR to Detect Drug Resistant Tuberculosis from Direct Sputum.

Offering patients with tuberculosis (TB) an optimal and timely treatment regimen depends on the rapid detection of Mycobacterium tuberculosis (Mtb) drug resistance from clinical samples. Finding Low Abundance Sequences by Hybridization (FLASH) is a technique that harnesses the efficiency, specificity, and flexibility of the Cas9 enzyme to enrich targeted sequences. Here, we used FLASH to amplify 52 candidate genes probably associated with resistance to first- and second-line drugs in the Mtb reference strain (H37Rv), then detect drug resistance mutations in cultured Mtb isolates, and in sputum samples. 92% of H37Rv reads mapped to Mtb targets, with 97.8% of target regions covered at a depth ≥ 10X. Among cultured isolates, FLASH-TB detected the same 17 drug resistance mutations as whole genome sequencing (WGS) did, but with much greater depth. Among the 16 sputum samples, FLASH-TB increased recovery of Mtb DNA compared with WGS (from 1.4% [IQR 0.5-7.5] to 33% [IQR 4.6-66.3]) and average depth reads of targets (from 6.3 [IQR 3.8-10.5] to 1991 [IQR 254.4-3623.7]). FLASH-TB identified Mtb complex in all 16 samples based on IS1081 and IS6110 copies. Drug resistance predictions for 15/16 (93.7%) clinical samples were highly concordant with phenotypic DST for isoniazid, rifampicin, amikacin, and kanamycin [15/15 (100%)], ethambutol [12/15 (80%)] and moxifloxacin [14/15 (93.3%)]. These results highlighted the potential of FLASH-TB for detecting Mtb drug resistance from sputum samples.

Humans↗
PubMed2001

Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference.

Group II intron mobility occurs by a target DNA-primed reverse transcription mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer to reverse transcribe the inserted intron RNA. The group II intron endonuclease, which mediates this process, is an RNP particle that contains the intron-encoded protein and the excised intron RNA and uses both cooperatively to recognize DNA target sequences. Here, we analyzed the interaction of the Lactococcus lactis Ll.LtrB group II intron endonuclease with its DNA target site by DNA footprinting and modification-interference approaches. In agreement with previous mutagenesis experiments showing a relatively large target site, DNase I protection extends from position -25 to +19 from the intron-insertion site on the top strand and from -28 to +16 on the bottom strand. Our results suggest that the protein first recognizes a small number of specific bases in the distal 5'-exon region of the DNA target site via major-groove interactions. These base interactions together with additional phosphodiester-backbone interactions along one face of the helix promote DNA unwinding, enabling the intron RNA to base-pair to DNA top-strand positions -12 to +3 for reverse splicing. Notably, DNA unwinding extends to at least position +6, somewhat beyond the region that base-pairs with the intron RNA, but is not dependent on interaction of the conserved endonuclease domain with the 3' exon. Bottom-strand cleavage occurs after reverse splicing and requires recognition of a small number of additional bases in the 3' exon, the most critical being T+5 in the now single-stranded downstream region of the target site. Our results provide the first detailed view of the interaction of a group II intron endonuclease with its DNA target site.

Adenine↗
PubMed1996

Binding of mitochondrial presequences to yeast cytosolic heat shock protein 70 depends on the amphiphilicity of the presequence.

The interactions between a yeast cytosolic hsp70, Ssa1p, and various synthetic peptides, including mitochondrial presequences, have been studied. The interactions were monitored both indirectly, by measuring the effects of the presequences on the ATPase activity and oligomeric state of the enzyme, and directly, by measuring the increased steady-state fluorescence polarization of fluorescent derivatives of the presequences as they bind to Ssa1p. The presequences are all able to convert Ssa1p from an oligomeric to a monomeric form in a concentration-dependent manner. The presequences are also able to stimulate the ATPase activity of the enzyme at similar concentrations. Quantification of the binding by fluorescence polarization showed that the affinity for Ssa1p is directly related to the physical properties of the presequences. The most amphiphilic presequences, as measured by retention times on reversed-phase high pressure liquid chromatography or surface activity in lipid monolayers, had the highest affinity for Ssa1p. The least amphiphilic presequences, which had previously been shown to be ineffective as mitochondrial targeting sequences, had relatively low affinity for Ssa1p. The results show that Ssa1p interacts with a broad range of amino acid sequences and that the strength of these interactions is related to the physical properties of the sequence. That the physical properties recognized by Ssa1p are identical to those necessary for the targeting function of mitochondrial presequences suggests that Ssa1p may interact with mitochondrial precursor proteins in the cytosol. The interactions may serve a variety of purposes: the maintenance of precursors in translocation-competent forms, the prevention of improper association of precursors with non-mitochondrial membranes, and the delivery of precursors to the mitochondrial surface.

Adenosine Triphosphatases↗
PubMed2002

Allele-specific genotype detection of factor V Leiden mutation from polymerase chain reaction amplicons based on label-free electrochemical genosensor.

An electrochemical genosensor for the genotype detection of allele-specific factor V Leiden mutation from PCR amplicons using the intrinsic guanine signal is described. The biosensor relies on the immobilization of the 21-mer inosine-substituted oligonucleotide capture probes related to the wild-type or mutant-type amplicons, and these probes are hybridized with their complementary DNA sequences at a carbon paste electrode (CPE). The extent of hybridization between the probe and target sequences was determined by using the oxidation signal of guanine in connection with differential pulse voltammetry (DPV). The guanine signal was monitored as a result of the specific hybridization between the probe and amplicon at the CPE surface. No label-binding step was necessary, and the appearance of the guanine signal shortened the assay time and simplified the detection of the factor V Leiden mutation from polymerase chain reaction (PCR)-amplified amplicons. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the guanine signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized to detect down to 51.14 fmol/mL target DNA. With the help of the appearance of the guanine signal, the yes/no system is established for the electrochemical detection of allele-specific mutation on factor V for the first time. Features of this protocol are discussed and optimized.

Alleles↗
PubMed1996

Internal targeting signal of the BCS1 protein: a novel mechanism of import into mitochondria.

The BCS1 protein is anchored in the mitochondrial inner membrane via a single transmembrane domain and has an N(out)-C(in) topology. Unlike the majority of nuclear encoded mitochondrial preproteins, the BCS1 protein does not contain an N-terminal targeting sequence. A positively charged segment of amino acids which is located immediately C-terminal to the transmembrane domain acts as an internal targeting signal. In order to function, we postulate that this sequence co-operates with the transmembrane domain to form a tight hairpin loop structure. This loop is translocated across the inner membrane via the MIM/mt-Hsp70 machinery in a membrane potential-dependent manner. This novel mechanism of import and sorting of the BCS1 protein is proposed to represent a more general mechanism used by a number of inner membrane proteins.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2006

Determination of genomic copy number with quantitative microsphere hybridization.

We developed a novel quantitative microsphere suspension hybridization (QMH) assay for determination of genomic copy number by flow cytometry. Single copy (sc) products ranging in length from 62 to 2,304 nucleotides [Rogan et al., 2001; Knoll and Rogan, 2004] from ABL1 (chromosome 9q34), TEKT3 (17p12), PMP22 (17p12), and HOXB1 (17q21) were conjugated to spectrally distinct polystyrene microspheres. These conjugated probes were used in multiplex hybridization to detect homologous target sequences in biotinylated genomic DNA extracted from fixed cell pellets obtained for cytogenetic studies. Hybridized targets were bound to phycoerythrin-labeled streptavidin; then the spectral emissions of both target and conjugated microsphere were codetected by flow cytometry. Prior amplification of locus-specific target DNA was not required because sc probes provide adequate specificity and sensitivity for accurate copy number determination. Copy number differences were distinguishable by comparing the mean fluorescence intensities (MFI) of test probes with a biallelic reference probe in genomic DNA of patient samples and abnormal cell lines. Concerted 5' ABL1 deletions in patient samples with a chromosome 9;22 translocation and chronic myelogenous leukemia were confirmed by comparison of the mean fluorescence intensities of ABL1 test probes with a HOXB1 reference probe. The relative intensities of the ABL1 probes were reduced to 0.59+/-0.02 fold in three different deletion patients and increased 1.42+/-0.01 fold in three trisomic 9 cell lines. TEKT3 and PMP22 probes detected proportionate copy number increases in five patients with Charcot-Marie-Tooth Type 1a disease and chromosome 17p12 duplications. Thus, the assay is capable of distinguishing one allele and three alleles from a biallelic reference sequence, regardless of chromosomal context.

Cell Line↗
PubMed1996

Tandem repeating of the expression cartridge--a novel strategy to enhance the expression efficiency of cloned gene in Escherichia coli.

A novel strategy to enhance the expression efficiency of cloned target gene in Escherichia coli was developed. The whole expression cartridge, consisting of promoter, SD sequence, target gene and transcription terminator, was tandem repeatedly engineered into a expression plasmid. Consequently, the copy number of specific gene was increased substantially, leading to the improvement of expression efficiency. Using this approach, a recombinant plasmid, designed as pLYD, was constructed and transformated into the Escherichia coli strain DH5 alpha. Upon induction, the desired protein was synthesized in a considerable level and accumulated up to 63% of the total cell proteins. The present study revealed that tandem repeating of expression cartridge provided a convenient means to improve expression level efficiently.

DNA, Recombinant↗
PubMed2004

The status of structural genomics defined through the analysis of current targets and structures.

Structural genomics--large-scale macromolecular 3-dimenional structure determination--is unique in that major participants report scientific progress on a weekly basis. The target database (TargetDB) maintained by the Protein Data Bank (http://targetdb.pdb.org) reports this progress through the status of each protein sequence (target) under consideration by the major structural genomics centers worldwide. Hence, TargetDB provides a unique opportunity to analyze the potential impact that this major initiative provides to scientists interested in the sequence-structure-function-disease paradigm. Here we report such an analysis with a focus on: (i) temporal characteristics--how is the project doing and what can we expect in the future? (ii) target characteristics--what are the predicted functions of the proteins targeted by structural genomics and how biased is the target set when compared to the PDB and to predictions across complete genomes? (iii) structures solved--what are the characteristics of structures solved thus far and what do they contribute? The analysis required a more extensive database of structure predictions using different methods integrated with data from other sources. This database, associated tools and related data sources are available from http://spam.sdsc.edu.

Computational Biology↗
PubMed1987

Construction and use of chimeric SPR/phi 3T DNA methyltransferases in the definition of sequence recognizing enzyme regions.

Multispecific DNA methyltransferases (Mtases) of temperate Bacillus subtilis phages SPR and phi 3T methylate the internal cytosine of the sequence GGCC. They differ in their capacity to methylate additional sequences. These are CCGG and CC(A/T)GG in SPR and GCNGC in phi 3T. Introducing unique restriction sites at equivalent locations within the two genes facilitated the construction of chimeric genes. These expressed Mtase activity at a level comparable to that of the parental genes. The methylation specificity of chimeric enzymes was correlated with the location of chimeric fusions. This analysis, which also included the use of mutant genes, showed that domains involved in the recognition of target sequences unique to each enzyme [CCGG, CC(A/T)GG or GCNGC] are represented by the central non-conserved parts of the proteins, whilst recognition of the sequence (GGCC), which is a target for both enzymes, is determined by an adjacent conserved region.

Bacillus subtilis↗
PubMed1990

In vitro transcriptional activation, dimerization, and DNA-binding specificity of the Epstein-Barr virus Zta protein.

The Epstein-Barr virus BZLF1 immediate-early gene encodes a transcriptional activator protein, Zta, which acts as a key regulatory switch in the transition between the latent and lytic viral life cycle. In this work, full-length Zta was expressed at high levels in Escherichia coli and purified to homogeneity by DNA affinity chromatography. The bacterial protein bound to specific target sequences (Zta response elements) and activated transcription in vitro from an Epstein-Barr virus early target promoter (BHLF1). Zta bound to DNA as a dimer. The formation of a heterodimer with a Zta deletion mutant was detected by gel electrophoresis mobility shift assays. Footprinting analysis on the BHLF1, BZLF1, and simian virus 40 control regions revealed multiple binding sites with no simple consensus sequence. Zta bound upstream from its own promoter at low concentrations, while at high concentrations it bound at the transcription start site, suggesting that it may activate and then autoregulate its own expression. These results demonstrate that Zta is a sequence-specific DNA-binding transcription factor.

Amino Acid Sequence↗
PubMed2001

Design and optimization of camptothecin conjugates of triple helix-forming oligonucleotides for sequence-specific DNA cleavage by topoisomerase I.

To achieve a sequence-specific DNA cleavage by topoisomerase I, derivatives of the antitumor drug camptothecin have been covalently linked to triple helix-forming oligonucleotides that bind in a sequence-specific manner to the major groove of double-helical DNA. Triplex formation at the target sequence positions the drug selectively at the triplex site, thereby stimulating topoisomerase I-mediated DNA cleavage at this site. In a continuous effort to optimize this strategy, a broad set of conjugates consisting of (i) 16-20-base-long oligonucleotides, (ii) alkyl linkers of variable length, and (iii) camptothecin derivatives substituted on the A or B quinoline ring were designed and synthesized. Analysis of the cleavage sites at nucleotide resolution reveals that the specificity and efficacy of cleavage depends markedly on the length of both the triple-helical structure and the linker between the oligonucleotide and the poison. The optimized hybrid molecules induced strong and highly specific cleavage at a site adjacent to the triplex. Furthermore, the drug-stabilized DNA-topoisomerase I cleavage complexes were shown to be more resistant to salt-induced reversal than the complexes induced by camptothecin alone. Such rationally designed camptothecin conjugates could provide useful antitumor drugs directed selectively against genes bearing the targeted triplex binding site. In addition, they represent a powerful tool to probe the molecular interactions in the DNA-topoisomerase I complex.

Base Sequence↗
PubMed1999

N-terminal and C-terminal plasma membrane anchoring modulate differently agonist-induced activation of cytosolic phospholipase A2.

The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA2 undergoes calcium-dependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA2 activation, we constructed chimeras of cPLA2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA2) or the C-terminal targeting signal of K-Ras4B (cPLA2-Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the alpha2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [3H]arachidonic acid, indicating that constitutive association of cPLA2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA2 increased [3H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA2-Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA2. The lack of stimulation of cPLA2-Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.

Amino Acid Sequence↗
PubMed1994

The interaction of DNA with the DNA-binding domain encoded by the Drosophila gene fork head.

The Drosophila gene fork head (fkh) encodes a nuclear protein which shares sequence similarity with the rat hepatocyte-enriched transcription factor family HNF3 alpha-gamma. The sequence similarity is restricted to the region that has been defined as the DNA-binding domain of these proteins, termed the fork head domain. In this study, we investigate the structural properties of the fork head domain of the prototype of this protein family encoded by fkh and its interaction with DNA. The core sequence required for DNA binding of the fork head domain consists of 114 amino acids and represents a stable and highly compact monomer of globular structure with an alpha-helix content of 37%. The fork head domain binds specifically to the DNA target sequence of HNF3 alpha-gamma and to an enhancer element that is derived from a regulatory sequence of an in vivo Drosophila target gene. The specific interaction between the DNA-binding domain of the fkh-encoded protein and its target DNA is mediated by two contact regions which are separated from each other by one turn of the DNA. Our data are consistent with a structural model which derived rom X-ray diffraction analysis of the DNA-binding domain of HNF3 gamma. Differences concerning the DNA contact sites between the DNA-binding domain of the fkh-encoded protein and the HNF3 protein family are discussed.

Animals↗