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PubMed2000

Probes for detection of specific DNA sequences at the single-molecule level.

A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.

Base Sequence↗
PubMed2002

Highly sensitive and specific detection of viable Escherichia coli in drinking water.

A highly sensitive and specific assay method was developed for the detection of viable Escherichia coli as an indicator organism in water, using nucleic acid sequence-based amplification (NASBA) and electrochemiluminescence (ECL) analysis. Viable E. coli were identified via a 200-nt-long target sequence from mRNA (clpB) coding for a heat shock protein. In the detection assay, a heat shock was applied to the cells prior to disruption to induce the synthesis of clpB mRNA and the mRNA was extracted, purified, and finally amplified using NASBA. The amplified mRNA was quantified with an ECL detection system after hybridization with specific DNA probes. Several disruption methods were investigated to maximize total RNA extracted from viable cells. Optimization was also carried out regarding the design of NASBA primer pairs and detection probes, as well as reaction and detection conditions. Finally, the assay was tested regarding sensitivity and specificity. Analysis of samples revealed that as few as 40 E. coli cells/mL can be detected, with no false positive signals resulting from other microorganisms or nonviable E. coli cells. Also, it was shown that a quantification of E. coli cells was possible with our assay method.

DNA Primers↗
PubMed2001

Local thermodynamic stability scores are well represented by a non-central student's t distribution.

Local folding in mRNAs is closely associated w ith biological functions. In this study, we reveal the whole distribution of local thermodynamic stability in the complete genome of the poliovirus P3/Leon/37 and the single-stranded RNA sequences that corresponds to the nucleotide sequence of the complete genome sequence (1 667 867 bp) of Helicobacter pylori (H. pylori) strain 26695. Local thermodynamic stability in the RNA sequences is measured by two standard z -scores, significance score and stability score. To estimate the distribution of thermodynamic stability, a model based on the non-central Student's t distribution has been developed. Significant patterns of extremes that are either much more stable or unstable than expected by chance are detected. Our results indicate that the highly stable and statistically more significant folding regions are predominantly in non-coding sequences in the two genome sequences. Moreover, the highly unstable folding regions, on the contrary, are predominantly in the protein coding sequences of H. pylori. The observed differences across the complete genomic sequences are statistically very significant by a chi2-test. These extreme patterns may be useful in searching for target sequences for long-chain antisense RNA and for locating potential RNA functional elements involved in the regulation of gene expression including translation, mRNA localization and metabolism.

Animals↗
PubMed1990

DNA binding by mutants of Tn21 resolvase with DNA recognition functions from Tn3 resolvase.

Substitution of amino acids within the section of Tn21 resolvase that corresponds to a helix-turn-helix structure, with the equivalent residues from Tn3 resolvase, yields proteins that retain the ability to mediate recombination between res sites from Tn21. These proteins had no recombinational activity on res sites from Tn3, even when the complete recognition helix had been exchanged. In this study, the binding of these mutants of Tn21 resolvase to DNA fragments containing res from either Tn21 or Tn3 was analysed by DNase I footprinting and by gel retardation. With DNA containing res from Tn21, the mutants bound to all three of the binding sites for resolvase (I, II, and III) but with a lower affinity than wild-type Tn21 resolvase. No complexes were detected between Tn3 resolvase and Tn21 DNA. With DNA containing res from Tn3, both the mutants and wild-type Tn21 resolvase bound to sites II and III, forming similar complexes to those with Tn3 resolvase: some of the mutants had higher affinities for these two sites on Tn3 DNA than on Tn21 DNA. In contrast, at site I in res from Tn3 (the location of the recombinational cross-over), the derivatives of Tn21 resolvase formed aberrant complexes whose structures differed radically from that with Tn3 resolvase. Alterations in the amino acid sequence of resolvase, within the helix-turn-helix region, therefore modulate the affinity of the protein for its target sequence in the DNA, but the specificity of resolvase for recombination at its cognate res sites is determined by the resultant organization of the DNA-protein complex.

Base Sequence↗
PubMed2003

Imprint of somatic hypermutation differs in human immunoglobulin heavy and lambda chain variable gene segments.

Somatic hypermutation (SHM) introduces mutations into immunoglobulin (Ig) variable gene segments, thus diversifying the B cell repertoire prior to positive selection of high affinity variants during maturation of T cell-dependent B cell responses. Somatic hypermutation of Ig heavy chain generates predominantly single base substitutions, favoring transition rather than transversion substitutions, and tends to direct mutations to specific 4-mer target sequences with G in second and C in third position. Here we have analyzed heavily mutated, nonproductively rearranged Ig lambda chain variable gene segments from human intestinal plasma cells, controlling for germline composition of the genes and local sequence variability. We have observed significant G.C strand bias in IgV(lambda), and differences in some di- and trinuleotide target preferences in IgV(lambda) compared to IgV(H). There is also a significant tendency to accumulate adjacent triplet mutations in IgV(lambda), which is not evident in IgV(H) in normal circumstances. These observations suggest that some aspect of the mechanism of somatic hypermutation operates differently in human immunoglobulin heavy and lambda light chain variable gene segments.

Aged↗
PubMed1997

Antisense oligonucleotides induce functional deletion of ligand gated ion channels in cultured neurons and brain explants.

The in situ application of the antisense technique for the study of ligand gated channels is discussed here. Using antisense oligodeoxynucleotides to downregulate a gene of interest means being confronted with a number of choices that will determine the success. These include choosing a target sequence, considering chemical modifications of the oligo as well as its length and estimation of the turnover of the target protein in order to set up the treatment schedule. In this paper a short overview of technical aspects of the antisense approach on primary cultured neurons and brain slice cultures is presented. In addition, the effects of antisense oligos on the expression of neuronal nicotinic acetylcholine receptors and GABA(A) receptors are discussed: Patch-clamp recordings of neurons treated with specific antisense oligos targeted at individual subunits showed a clear downregulation of the expression of native ligand gated channels. Moreover, in a number of experiments novel channel types with altered properties were observed following antisense treatment. Thus, non-targeted channel subunits that remain expressed after antisense deletion, may aggregate to form novel channel types that are normally not present. Alternatively, the translational arrest of a protein may be accompanied by compensatory changes in the synthesis and/or targeting of other channel subunits to the cell surface. The antisense technique enables identification of the functional contribution of individual channel subunits to endogenous channel activity in the central nervous system. As such it paves the way to the elucidation of in vivo channel-subunit composition and channel functions, of post- as well as pre-synaptic ligand gated channel receptors.

Brain↗
PubMed2005

High-frequency gene targeting in Arabidopsis plants expressing the yeast RAD54 gene.

Gene targeting, which is homologous recombination-mediated integration of an extra-chromosomal DNA segment into a chromosomal target sequence, enables the precise disruption or replacement of any gene. Despite its value as a molecular genetic tool, gene targeting remains an inefficient technology in most species. We report that expression of the yeast RAD54 gene, a member of the SWI2/SNF2 chromatin remodeling gene family, enhances gene targeting in Arabidopsis by one to two orders of magnitude, from 10(-4) to 10(-3) in WT plants to 10(-2) to 10(-1). We show that integration events, detected with an assay based on the use of a fluorescent seed marker, are precise and germinally transmitted. These findings suggest that chromatin remodeling is rate-limiting for gene targeting in plants and improves the prospects for using gene targeting for the precise modification of plant genomes.

Agrobacterium tumefaciens↗
PubMed1999

Isopentenyl diphosphate biosynthesis via a mevalonate-independent pathway: isopentenyl monophosphate kinase catalyzes the terminal enzymatic step.

In plants, the biosynthesis of isopentenyl diphosphate, the central precursor of all isoprenoids, proceeds via two separate pathways. The cytosolic compartment harbors the mevalonate pathway, whereas the newly discovered deoxyxylulose 5-phosphate pathway, which also operates in certain eubacteria, including Escherichia coli, is localized to plastids. Only the first two steps of the plastidial pathway, which involve the condensation of pyruvate and glyceraldehyde 3-phosphate to deoxyxylulose 5-phosphate followed by intramolecular rearrangement and reduction to 2-C-methylerythritol 4-phosphate, have been established. Here we report the cloning from peppermint (Mentha x piperita) and E. coli, and expression, of a kinase that catalyzes the phosphorylation of isopentenyl monophosphate as the last step of this biosynthetic sequence to isopentenyl diphosphate. The plant gene defines an ORF of 1,218 bp that, when the proposed plastidial targeting sequence is excluded, corresponds to approximately 308 aa with a mature size of approximately 33 kDa. The E. coli gene (ychB), which is located at 27.2 min of the chromosomal map, consists of 852 nt, encoding a deduced enzyme of 283 aa with a size of 31 kDa. These enzymes represent a conserved class of the GHMP family of kinases, which includes galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase, with homologues in plants and several eubacteria. Besides the preferred substrate isopentenyl monophosphate, the recombinant peppermint and E. coli kinases also phosphorylate isopentenol, and, much less efficiently, dimethylallyl alcohol, but dimethylallyl monophosphate does not serve as a substrate. Incubation of secretory cells isolated from peppermint glandular trichomes with isopentenyl monophosphate resulted in the rapid production of monoterpenes and sesquiterpenes, confirming that isopentenyl monophosphate is the physiologically relevant, terminal intermediate of the deoxyxylulose 5-phosphate pathway.

Amino Acid Sequence↗
PubMed1995

Silencer elements modulate the expression of the gene for the neuron-glia cell adhesion molecule, Ng-CAM.

The combined factors that regulate the expression of cell adhesion molecules (CAMs) during development of the nervous system are largely unknown. To identify such factors for Ng-CAM, the neuron-glia CAM, constructs containing portions of the 5' end of the Ng-CAM gene were examined for activity after transfection into N2A neuroblastoma and NIH3T3 cells. Positive regulatory elements active in both cell types included an Ng-CAM proximal promoter with SP1 and cAMP response element motifs extending 447 base pairs upstream of a single RNA start site and a region within the first exon corresponding to 5'-untranslated sequences. Negative regulatory elements included five neuron-restrictive silencer elements (NRSEs) and a binding site for Pax gene products in a 305-base pair segment of the first intron. Constructs containing the promoter together with the entire first intron were active in N2A cells but were silenced in NIH3T3 cells. This silencer activity was mapped to the NRSEs. In contrast, the Pax motif inhibited activity of Ng-CAM constructs in both cell types. The DNA elements defined in these transfection experiments were examined for their ability to bind nuclear factors. The region within the first exon formed a DNA-protein complex after exposure to nuclear extracts prepared from both NIH3T3 and N2A cells. The NRSE region formed a more prominent complex with proteins prepared from NIH3T3 cells than it did with extracts from N2A cells. A member of the Pax protein family, Pax-3 bound to the Pax motif. Mutations introduced within the Pax motif in its ATTA sequence eliminated this binding whereas mutations in its GTTCC sequence did not, suggesting that paired homeodomain interactions are important for the recognition of Pax-3 by this DNA target sequence. The combined data suggest that negative regulation by NRSEs and Pax proteins may play a key role in the place-dependent expression patterns of Ng-CAM during development.

3T3 Cells↗
PubMed1998

FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes.

The fate of fluorescently labeled pre-nsL-TP (Cy3-pre-nsL-TP) microinjected into BALB/c 3T3 fibroblasts was investigated by confocal laser scanning microscopy. The protein exhibited a distinct punctate fluorescence pattern and colocalized to a high degree with the immunofluorescence pattern for the peroxisomal enzyme acyl-CoA oxidase. Proteolytic removal of the C-terminal leucine of the putative peroxisomal targeting sequence (AKL) resulted in a diffuse cytosolic fluorescence. These results indicate that microinjected Cy3-pre-nsL-TP is targeted to peroxisomes. The association of nsL-TP with peroxisomal enzymes was investigated in cells by measuring fluorescence resonance energy transfer (FRET) between the microinjected Cy3-pre-nsL-TP and Cy5-labeled antibodies against the peroxisomal enzymes acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, bifunctional enzyme, PMP70 and catalase. The technique of photobleaching digital imaging microscopy (pbDIM), used to quantitate the FRET efficiency on a pixel-by-pixel basis, revealed a specific association of nsL-TP with acyl-CoA oxidase, 3-ketoacyl-CoA thiolase and bifunctional enzyme in the peroxisomes. These observations were corroborated by subjecting a peroxisomal matrix protein fraction to affinity chromatography on Sepharose-immobilized pre-nsL-TP. Acyl-CoA oxidase was retained. These studies provide strong evidence for a role of nsL-TP in the regulation of peroxisomal fatty acid beta-oxidation, e.g. by facilitating the presentation of substrates and/or stabilization of the enzymes.

3T3 Cells↗
PubMed2002

A directional nucleation-zipping mechanism for triple helix formation.

A detailed kinetic study of triple helix formation was performed by surface plasmon resonance. Three systems were investigated involving 15mer pyrimidine oligonucleotides as third strands. Rate constants and activation energies were validated by comparison with thermodynamic values calculated from UV-melting analysis. Replacement of a T.A base pair by a C.G pair at either the 5' or the 3' end of the target sequence allowed us to assess mismatch effects and to delineate the mechanism of triple helix formation. Our data show that the association rate constant is governed by the sequence of base triplets on the 5' side of the triplex (referred to as the 5' side of the target oligopurine strand) and provides evidence that the reaction pathway for triple helix formation in the pyrimidine motif proceeds from the 5' end to the 3' end of the triplex according to the nucleation-zipping model. It seems that this is a general feature for all triple helices formation, probably due to the right-handedness of the DNA double helix that provides a stronger base stacking at the 5' than at the 3' duplex-triplex junction. Understanding the mechanism of triple helix formation is not only of fundamental interest, but may also help in designing better triple helix-forming oligonucleotides for gene targeting and control of gene expression.

DNA↗
PubMed1991

Effect of RNA secondary structure on polyadenylation site selection.

Functional polyadenylation [poly(A)] sites consist of two sequence elements, the AAUAAA and G/U box signals, that closely flank the site of mRNA 3'-end formation. In agreement with previous results, random sequence insertions between the AAUAAA and G/U box signals were observed to inhibit poly(A) site function. However, sequence insertions of similar size that were predicted to form RNA stem-loop structures were found to have little effect on the efficiency of polyadenylation and instead induced a 3' shift in the site of polyadenylation that was equal to the length of the inserted stem-loop. The in vivo utilization of a poly(A) site bearing an internal RNA stem-loop structure was inhibited by mutations that destabilized the predicted stem but was restored by compensatory mutations. These results strongly support the hypothesis that the appropriate spacing of the AAUAAA and G/U box signals is critical for poly(A) site function. Sequence insertions that are able to form RNA secondary structures that maintain the correct spacing of these two RNA target sequences are well tolerated, whereas sequence insertions that disturb this spacing inhibit poly(A) site recognition. It is proposed that the effect of sequence insertions on poly(A) site function may be sufficiently predictable to allow the development of an assay for in vivo RNA secondary structure that uses poly(A) site selection as a readout.

Animals↗
PubMed2002

A targeted inhibition of DNA-dependent protein kinase sensitizes breast cancer cells following ionizing radiation.

A major mechanism by which cancer cells become resistant to ionizing radiation (IR) and chemotherapy drugs is by enhanced DNA repair of the lesions; therefore, through inhibition of DNA repair pathways that tumor cells rely on to escape chemotherapy, we expect to increase the killing of cancer cells and reduce drug resistance. DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase essential for DNA repair as well as sensing and transmitting a damage signal to downstream targets leading to cell cycle arrest. We used a peptide cotherapy strategy to see whether a targeted inhibition of DNA-PK activity sensitizes breast cancer cells in response to IR or chemotherapy drug. A synthesized peptide representing the C terminus of Ku80 (HNI-38) selectively targeted and disrupted interaction between Ku complex and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) as well as the DNA binding activity of Ku that led to the inhibition of DNA-PK activity and reduction in double-stranded DNA break (dsb) repair activity. Furthermore, a peptide-based inhibitor with target sequence effectively inhibited the growth of breast cancer cells only in the presence of DNA damage, suggesting that the target peptide sensitizes cancer cells through blocking dsb DNA repair activity. Together, this study not only validates the involvement of the C terminus of Ku80 in Ku's DNA termini binding and interaction with DNA-PKcs, but also a supports physiological role for DNA-PK in IR or chemotherapy drug resistance of cancer cells.

Antineoplastic Agents↗
PubMed1995

The hsp70 gene family of Neurospora crassa: cloning, sequence analysis, expression, and genetic mapping of the major stress-inducible member.

The gene encoding the major heat shock-inducible member of the HSP70 family of Neurospora crassa was cloned and characterized. The 5' nontranscribed region shows the presence of consensus sequence motifs resembling the classical heat shock elements found in many heat shock-responsive eukaryotic promoters, as well as metal-responsive-element sequences. The coding region of the gene contains four introns with boundaries and internal consensus motifs typical of genes of filamentous fungi. None of the other stress-inducible hsp70 genes of fungal origin have, so far, been reported to contain introns. The sequence adjoining the transcriptional initiation zone shows the presence of prominent CT-rich stretches, characteristic of highly expressed fungal genes. The deduced amino acid sequence corresponds to a 646-residue polypeptide, with a calculated molecular mass of 70,561 Da and an average pI of 6.01, exhibiting strong sequence homology with many other eukaryotic HSP70s, with typical HSP70 family signatures 1 and 2 and a bipartite nuclear targeting sequence. Experiments with primer extension revealed the presence of one minor and two major transcriptional start sites. This gene, designated hsps-1, was mapped to a locus on the left arm of linkage group II, in close proximity to the AR-30 translocation breakpoint.

Amino Acid Sequence↗
PubMed2002

Molecular characterisation of bacteria in a wetland used to remove ammoniacal-N from landfill leachate.

Bacteria associated with leachate from a constructed, surface-flow wetland effective at removing ammonium-N, were characterised using molecular methods which bypass the need for cultivation. DNA was extracted from the sample and bacterial 16S rDNA sequences amplified and cloned. Ninety-six individual clones were re-amplified and analysed by restriction digestion and targeted sequencing. A collector's curve was used to estimate the total number of bacterial groups (operational taxonomic units, OTUs) in the sample at 47. Thirty-six clones representing 28 OTUs were sequenced and characterised by aligning them against published sequences. Of these, 10 were confidently identified, whilst for the remaining 18, the closest match was obtained. The sequences in 64% of the OTUs were similar to those of the Proteobacteria but no classical nitrifying bacteria were identified. However, alkaliphilic Methylomicrobium sp. that oxidise ammonium and bacteria from the genus Alcaligenes, at least one species of which is capable of heterotrophic nitrification, were detected.

DNA, Bacterial↗
PubMed2002

Species identification of mustelids by comparing partial sequences on mitochondrial DNA from fecal samples.

Nucleotide sequences of cytochrome b/tRNA/D-loop region on mitochondrial DNA of mustelids feces were compared to identify species. PCR amplification of target sequence for 47 (24.9%) feces and species identification of five feces (2.6%) out of 189 feces, collected at several study sites in Hokkaido, were successful. Species of three feces were Martes zibellina and those of other two feces were Martes melampus and Mustela itatsi. The low success rate of identification appeared to be due to failure of PCR amplification by inhibitors in feces. It was suggested that the method used in this study was useful for not only identify mustelids species, but also analyzing their genetic relationships.

Animals↗
PubMed2000

A leaf-peroxisomal protein, hydroxypyruvate reductase, is produced by light-regulated alternative splicing.

Hydroxypyruvate reductase (HPR) is localized in leaf peroxisomes in plants, and it plays an important role in the glycolate pathway of photorespiration. In this laboratory, two highly homologous cDNAs for pumpkin HPR (HPR1 and HPR2) have been obtained, and appear to be produced from the same primary transcript by alternative splicing. Analyses at the mRNA level showed that the amounts of the two HPR mRNAs is changed in response to light, suggesting that light changes the splicing pattern of HPR pre-mRNA from almost equal amounts of two HPR mRNAs to greater production of HPR2 mRNA. From the sequences of the two HPR cDNAs, the HPR1 protein, but not the HPR2 protein, was found to have a targeting sequence into peroxisomes at the carboxy terminus. Analyses of transgenic Arabidopsis thaliana expressing fusion proteins with green fluorescent protein confirmed the different subcellular localizations of the two HPR proteins. These findings indicate the presence of light-regulated alternative splicing of HPR pre-mRNA, which controls the subcellular localizations of two HPR proteins in pumpkin cells.

Alcohol Oxidoreductases↗
PubMed1997

Sequence-specific control of gene expression by antigene and clamp oligonucleotides.

Control of gene expression at the transcriptional level can be achieved with triplex-forming oligonucleotides provided that the target sequence is accessible within the chromatin structure of cell nuclei. Using oligonucleotide-psoralen conjugates as probes we have shown that the promoter region of the gene encoding the alpha subunit of the interleukin 2 receptor and the polypurine tract of integrated HIV provirus can form sequence-specific, triple-helical complexes in cell cultures. Oligonucleotide-intercalator conjugates can inhibit transcription initiation by competing with transcription factor binding. Oligonucleotide analogues containing N3'-->P5' phosporamidate linkages form stable triple helices that are able to arrest transcription at the elongation step. A triple helix can also be formed on a single-stranded target by clamp oligonucleotides. A clamp targeted to the polypurine tract of HIV RNA is able to block reverse transcription of the viral RNA.

Animals↗

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