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PubMed1992

PCR regimen for enhanced specificity and yield of targeted genomic DNA sequences: ras and p53.

By weighting the PCR reaction in favor of specificity for the target sequence in the beginning cycles and for continued efficient amplification of the sequence into later cycles, we were able to show an improvement in the specificity and quantity of amplified ras and p53 sequences. Increased purity and yield of specific products favorably enhanced post-PCR evaluation and interpretation of results using direct sequencing and single-stranded conformation polymorphism (SSCP) analysis when point mutations were present in DNA from tumor cell lines and tissues.

Base Sequence↗
PubMed2005

Site-selective RNA cleavage by DNA bearing a base pair-mimic nucleoside.

We have synthesized the deoxyadenosine derivative tethering a phenyl group (X), which mimics the Watson-Crick A/T base pair. The RNA/DNA hybrid duplexes containing X in the middle of the DNA sequence showed a similar thermal stability regardless of the ribonucleotide species (A, G, C, or U) opposite to X, probably because of the phenyl group stacking inside of the duplex accompanied by the opposite ribonucleotide base flipped in an extrahelical position. The RNA strand hybridized with the DNA strand bearing X was cleaved on the 3'-side of the ribonucleotide opposite to X in the presence of MgCl2, and the RNA sequence to be cleaved was not restricted. The site-specific RNA hydrolysis suggests that the DNA strand bearing X has the advantage of the site-selective base flipping in the target sequence and the development of a "universal deoxyribozyme" to exclusively cleave a target RNA sequence.

Adenosine↗
PubMed2006

Sequence comparison and protein structure prediction.

Sequence comparison is a major step in the prediction of protein structure from existing templates in the Protein Data Bank. The identification of potentially remote homologues to be used as templates for modeling target sequences of unknown structure and their accurate alignment remain challenges, despite many years of study. The most recent advances have been in combining as many sources of information as possible--including amino acid variation in the form of profiles or hidden Markov models for both the target and template families, known and predicted secondary structures of the template and target, respectively, the combination of structure alignment for distant homologues and sequence alignment for close homologues to build better profiles, and the anchoring of certain regions of the alignment based on existing biological data. Newer technologies have been applied to the problem, including the use of support vector machines to tackle the fold classification problem for a target sequence and the alignment of hidden Markov models. Finally, using the consensus of many fold recognition methods, whether based on profile-profile alignments, threading or other approaches, continues to be one of the most successful strategies for both recognition and alignment of remote homologues. Although there is still room for improvement in identification and alignment methods, additional progress may come from model building and refinement methods that can compensate for large structural changes between remotely related targets and templates, as well as for regions of misalignment.

Computational Biology↗
PubMedDate not supplied

Related function of mouse SOX3, SOX9, and SRY HMG domains assayed by male sex determination.

Sox genes encode proteins related to each other, and to the sex determining gene Sry, by the presence of a DNA binding motif known as the HMG domain. Although HMG domains can bind to related DNA sequences, Sox gene products may achieve target gene specificity by binding to preferred target sequences or by interacting with specific partner proteins. To assess their functional similarities, we replaced the HMG box of Sry with the HMG box of Sox3 or Sox9 and tested whether these constructs caused sex reversal in XX mice. Our results indicate that such chimeric transgenes can functionally replace Sry and elicit development of testis cords, male patterns of gene expression, and elaboration of male secondary sexual characteristics. This implies that chimeric SRY proteins with SOX HMG domains can bind to and regulate SRY target genes and that potential SRY partner factor interactions are not disrupted by HMG domain substitutions. genesis 28:111-124, 2000.

Amino Acid Sequence↗
PubMed2005

Enhancement of DNA immobilization and hybridization on gold electrode modified by nanogold aggregates.

Gold electrodes modified by nanogold aggregates (nanogold electrode) were obtained by the electrodeposition of gold nanoparticles onto planar gold electrode. The Electrochemical response of single-stranded DNA (ssDNA) probe immobilization and hybridization with target DNA was measured by cyclic voltammograms (CV) using methylene blue (MB) as an electroactive indicator. An improving method using long sequence target DNA, which greatly enhanced the response signal during hybridization, was studied. Nanogold electrodes could largely increase the immobilization amount of ssDNA probe. The hybridization amount of target DNA could be increased several times for the manifold nanogold electrodes. The detection limit of nanogold electrode for the complementary 16-mer oligonucleotide (target DNA1) and long sequence 55-mer oligonucleotide (target DNA2) could reach the concentration of 10(-9) mol/L and 10(-11) mol/L, respectively, which are far more sensitive than that of the planar electrode.

Adsorption↗
PubMed2005

Use of a suspension array for rapid identification of the varieties and genotypes of the Cryptococcus neoformans species complex.

Cryptococcus neoformans is an encapsulated fungal pathogen known to cause severe disease in immunocompromised patients. The disease, cryptococcosis, is mostly acquired by inhalation and can result in a chronic meningoencephalitis, which can be fatal. Here, we describe a molecular method to identify the varieties and genotypic groups within the C. neoformans species complex from culture-based assays. The method employs a novel flow cytometer with a dual laser system that allows the simultaneous detection of different target sequences in a multiplex and high-throughput format. The assay uses a liquid suspension hybridization format with specific oligonucleotide probes that are covalently bound to the surface of fluorescent color-coded microspheres. Biotinylated target amplicons, which hybridized to their complementary probe sequences, are quantified by the addition of the conjugate, streptavidin R-phycoerythrin. In this study we developed and validated eight probes derived from sequence analysis of the intergenic spacer region of the rRNA gene region. The assay proved to be specific and sensitive, allowed discrimination of a 1-bp mismatch with no apparent cross-reactivity, and detected 10(1) to 10(3) genome copies. The described protocol, which can be used directly with yeast cells or isolated DNA, can be undertaken in less than 1 h following PCR amplification and permits identification of species in a multiplex format. In addition to a multiplex capability, the assay allows the simultaneous detection of target sequences in a single reaction. The accuracy, speed, flexibility, and sensitivity of this technology are a few of the advantages that will make this assay useful for the diagnosis of human cryptococcal infections and other pathogenic diseases.

Cryptococcus neoformans↗
PubMed2002

A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast.

Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of approximately 0.5%; the positive clones are identified by PCR or colony hybridization. This paper describes a novel TAR cloning procedure that selects positive clones by positive and negative genetic selection. This system utilizes a TAR vector with two targeting hooks, HIS3 as a positive selectable marker, URA3 as a negative selectable marker and a gene-specific sequence called a loop sequence. The loop sequence lies distal to a targeting hook sequence in the chromosomal target, but proximal to the targeting hook and URA3 in the TAR vector. When this vector recombines with chromosomal DNA at the gene-specific targeting hook, the recombinant YAC product carries two copies of the loop sequence, therefore, the URA3 negative selectable marker becomes mitotically unstable and is lost at high frequency by direct repeat recombination involving the loop sequence. Positive clones are identified by selecting against URA3. This method produces positive YAC recombinants at a frequency of approximately 40%. This novel TAR cloning method provides a powerful tool for structural and functional analysis of complex genomes.

Animals↗
PubMed2000

Host sequences flanking the human T-cell leukemia virus type 1 provirus in vivo.

Human pathogenic retroviruses do not have common loci of integration. However, many factors, such as chromatin structure, transcriptional activity, DNA-protein interaction, CpG methylation, and nucleotide composition of the target sequence, may influence integration site selection. These features have been investigated by in vitro integration reactions or by infection of cell lines with recombinant retroviruses. Less is known about target choice for integration in vivo. The present study was conducted in order to assess the characteristics of cellular sequences targeted for human T-cell leukemia virus type 1 (HTLV-1) integration in vivo. Sequencing integration sites from >/=200 proviruses (19 kb of sequence) isolated from 29 infected individuals revealed that HTLV-1 integration is not random at the level of the nucleotide sequence. The virus was found to integrate in A/T-rich regions with a weak consensus sequence at positions within and without of the hexameric repeat generated during integration. These features were not associated with a preference for integration near active regions or repeat elements of the host chromosomes. Most or all of the regions of the genome appear to be accessible to HTLV-1 integration. As with integration in vitro, integration specificity in vivo seems to be determined by local features rather than by the accessibility of specific regions.

AT Rich Sequence↗
PubMed1998

Deposition of storage proteins.

Plants store amino acids for longer periods in the form of specific storage proteins. These are deposited in seeds, in root and shoot tubers, in the wood and bark parenchyma of trees and in other vegetative organs. Storage proteins are protected against uncontrolled premature degradation by several mechanisms. The major one is to deposit the storage proteins into specialized membrane-bounded storage organelles, called protein bodies (PB). In the endosperm cells of maize and rice prolamins are sequestered into PBs which are derived from the endoplasmic reticulum (ER). Globulins, the typical storage proteins of dicotyledonous plants, and prolamins of some cereals are transported from the ER through the Golgi apparatus and then into protein storage vacuoles (PSV) which later become transformed into PBs. Sorting and targeting of storage proteins begins during their biosynthesis on membrane-bound polysomes where an N-terminal signal peptide mediates their segregation into the lumen of the ER. After cleavage of the signal peptide, the polypeptides are glycosylated and folded with the aid of chaperones. While still in the ER, disulfide bridges are formed which stabilize the structure and several polypeptides are joined to form an oligomer which has the proper conformation to be either deposited in ER-derived PB or to be further transferred to the PSV. At the trans-Golgi cisternae transport vesicles are sequestered which carry the storage proteins to the PSV. Several storage proteins are also processed after arriving in the PSVs in order to generate a conformation that is capable of final deposition. Some storage protein precursors have short N- or C-terminal targeting sequences which are detached after arrival in the PSV. Others have been shown to have internal sequence regions which could act as targeting information. In some cases positive targeting information is known to mediate sorting into the PSV whereas in other cases aggregation and membrane association seem to be major sorting mechanisms.

Amino Acid Sequence↗
PubMed1991

'Pseudo' domains in phage-encoded DNA methyltransferases.

5-Cytosine-DNA-methyltransferases, which are found in many organisms ranging from bacteriophages to mammals, transfer a methyl group from S-adenosylmethionine to the carbon-5 of a cytosine residue in specific DNA target sequences. Some phage-encoded methyltransferases methylate more than one sequence: these enzymes contain several independent target-recognizing domains each responsible for recognizing a different site. The amino-acid sequences of these multispecific methyltransferases reveal that some enzymes in addition carry domains that do not contribute to the enzymes' methylation potential, but strongly resemble previously identified target-recognizing domains. Here we show that introducing defined amino-acid alterations into these inactive domains endows these enzymes with additional methylation specificities. Gel retardation analysis demonstrates that these novel methylation specificities correlate with the acquisition of additional DNA-binding potential of the proteins.

Amino Acid Sequence↗
PubMed2025

Targeted Next-Generation Sequencing in Rare Diseases.

Targeted next-generation sequencing (NGS) in rare disease focuses on genetic analysis of specific regions in genome that are linked to a rare disease. In addition to library preparation, sequencing, and data analysis, targeted NGS includes an additional step of target enrichment of selected genes and regions. It allows for more sensitive and profound sequencing, as it is a fast and cost-effective approach with less data burden and is therefore often a method of choice for identifying rare variants in known genes, especially in diagnostics of rare diseases. Several in silico tools address the pathogenicity predictions of rare variants of unknown significance (VUS) and can therefore facilitate clinical interpretation.

Rare Diseases↗
PubMed2003

Profiling substrate phosphorylation at the phosphopeptide level.

The identification of substrates is a key aspect in the study of the biological function of protein kinases. The procedure here described is aimed at profiling substrate phosphorylation at the phosphopeptide level by sequentially involving (i). the assessment of the in vitro activity of individual protein kinases on a complex mix of immobilized proteins, (ii). the fractionation of the phosphopeptides being released upon proteolysis of substrates, and (iii). the final identification of the targeted sequences. In particular, the protein sample is spotted onto nitrocellulose membrane and then subjected to a solid-phase kinase assay in the presence of [32P]ATP, prior to solid-phase proteolytic digestion and two-dimensional phosphopeptide mapping. Radiolabeled phosphopeptides are subsequently isolated and sequenced to identify the substrates being targeted by the examined protein kinase. Using the gamma-isotype of p21-activated protein kinase (gamma-PAK) and its known in vitro substrates, I verified that both the specificity of substrate phosphorylation and its efficiency are similar upon solid- and liquid-phase conditions. To demonstrate the feasibility of the overall experimental system, I then employed a fairly crude cell extract as a source of candidate substrates and successfully identified the sequence of a putative substrate of gamma-PAK.

Peptide Mapping↗
PubMed2006

Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes.

Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation.

Animals↗
PubMed2003

Cloning and identification of a novel human RNPC3 gene that encodes a protein with two RRM domains and is expressed in the cell nucleus.

The RNA recognition motifs (RRM) domain is one of the most common eukaryotic protein folds. Proteins containing RRM domains function in important steps of posttranscriptional regulation of gene expression and are involved in processing and transport of mRNA precursors. Here we describe the cloning and characterization of a novel human RNPC3 gene containing two RNA recognition motifs. The 1870 bp cDNA encodes a protein with 517 amino acids. It also contains two bipartite nuclear targeting sequences, which is important for nuclear targeting for proteins, especially those functioning in the cell nucleus. The GFP location of the RNPC3 gene product shows that this protein is located in the cell nucleus. RT-PCR reveals that it is abundantly expressed in kidney and pancreas.

Amino Acid Motifs↗
PubMed2005

Microarray-based detection and typing of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) is the most economically important veterinary pathogen because of its highly infectious nature and the devastating effects the virus has on the livestock industry. Rapid diagnostic methods are needed for detection and typing of FMDV serotypes and differentiation from other viruses causing vesicular diseases. We developed a microarray-based test that uses a FMD DNA chip containing 155 oligonucleotide probes, 35-45 base pair (bp) long, virus-common and serotype-specific, designed from the VP3-VP1-2A region of the genome. A set of two forward primers and one reverse primer were also designed to allow amplification of approximately 1100 bp of target sequences from this region. The amplified target was labelled with Alexa-Fluor 546 dye and applied to the FMD DNA chip. A total of 23 different FMDV strains representing all seven serotypes were detected and typed by the FMD DNA chip. Microarray technology offers a unique capability to identify multiple pathogens in a single chip.

Animals↗
PubMed2002

Real-time fluorescence PCR assays for detection and characterization of Shiga toxin, intimin, and enterohemolysin genes from Shiga toxin-producing Escherichia coli.

PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx(1) and stx(2)) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx(1), eae, and E-hly genes and 96 and 100%, respectively, for the stx(2) gene. No stx(2) genes were detected from 10 stx(2f)-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains.

Adhesins, Bacterial↗
PubMed1988

Random mutagenesis using degenerate oligodeoxyribonucleotides.

An efficient method for random mutagenesis was applied to a 75-bp target sequence. The mutational changes in the target region are introduced by the technique of oligodeoxyribonucleotide(oligo)-directed, site-specific mutagenesis using mixtures of degenerate oligos. These are designed in such a way that they carry with a high probability randomly distributed substitutions, which are introduced into the oligos by utilizing appropriate concentrations of all four nucleotide precursors during each chain elongation step. These mixtures of degenerate oligos were hybridized to the appropriate M13-hybrid ss-template and then extended in vitro using PolIk. In order to avoid any bias artificially created by the Escherichia coli mismatch repair system, homoduplex molecules were synthesized in vitro according to the method of Taylor et al. [Nucleic Acids Res. 13 (1985) 8765-8785]. After transformation of the appropriate E. coli host, M13 plaques were randomly analysed by DNA sequencing. Using appropriate preparations of template DNA and oligos we attained mutagenesis efficiencies in the range of 20-50%. The analysis of 85 different mutants revealed that the distribution of the mutations is random and that all expected substitutions occur with about the same probability.

Base Sequence↗
PubMed1997

A 38 bp repeat sequence within the pea seed storage protein promoter of legA is a binding site for a nuclear DNA-binding protein.

This paper describes a target sequence for a pea seed nuclear DNA-binding protein (of Pisum sativum) present at the cell expansion phase. Electromobility shift assays as well as in situ copper-phenanthroline footprinting have shown that this protein binds to an imperfect repeat sequence of the legA promoter located -404 tot -367 relative to the transcriptional start site, a region designated USR1 (-549 tot -316). Competition assays showed that the same or a related protein binds to USR2 located at -833 to -582. Another protein that does not recognize the repeat sequence as a target site binds adjacent but upstream to the repeat sequence in USR1. It is suggested that these protein-DNA interactions are initial events in the assembly of a transcriptional activation complex.

Base Composition↗