Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA sensing”

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 73 records · Page 4Linked to original sources
PubMed2006

Initial mechanistic studies of antisense targeting in cells.

UNLABELLED: The continued development of antisense targeting will require a better understanding of the mechanism. METHODS: We performed initial studies of the mechanism of intracellular antisense targeting through measurements of in situ transcription, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), 32P-labeled uridine-5'-triphosphate (alpha-32P-UTP) incorporation, nuclear accumulations of 99mTc-labeled DNAs, and messenger RNA (mRNA) transcription rate. As reported earlier, an antisense DNA against the mdr1 mRNA coding for P-glycoprotein (Pgp) and its sense DNA control were used in KB-G2 (Pgp++) cells. RESULTS: Definitive evidence for antisense targeting was obtained by in situ transcription showing complementary DNA elongation in cells exposed to antisense DNA, acting therefore as an intracellular PCR primer of mdr1 mRNA, but not in cells exposed to sense DNA. Immunofluorescence staining showed higher accumulations of antisense versus sense DNAs in KB-G2 cells. Transnuclear migration was confirmed by higher accumulations in the nucleus compared with the cytoplasm in cells incubated with 99mTc-labeled antisense DNA. However, the observed specific accumulations of antisense DNAs of about 10(6) per cell over 10 h could not be explained by a feedback mechanism upregulating transcription in cells exposed to antisense DNA as no increase in mRNA levels was detected by both RT-PCR and 32P-UTP in these cells. To explore an alternative hypothesis, a novel approach using 99mTc-labeled antisense DNA as a probe of total mRNA from cells previously saturated with unlabeled antisense DNA was used to estimate the transcription rate. Compared with controls, mdr1 mRNA levels were found to be initially low after saturation and to recover at about 2,000 copies per minute per cell. If persistent, this transcription rate would provide 10(6) mRNAs in 10 h. CONCLUSION: The results of all studies are consistent with antisense as the mechanism of targeting. Though a feedback mechanism leading to upregulation of mRNA transcription is an unlikely explanation for the high specific accumulations, our results may be explained if antisense DNAs are targeting mdr1 mRNAs produced at high transcription rates. If the target is primarily pre-mRNA in the nucleus rather than mature mRNA in the cytoplasm, this would provide as well an explanation for the observed migration of 99mTc-labeled antisense DNA into the nucleus.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Photoregulation of RNA digestion by RNase H with azobenzene-tethered DNA.

RNA digestion by RNase H, which is responsible for the antisense effect, was efficiently photoregulated by use of the duplex of azobenzene-tethered sense DNA and native antisense DNA. In the dark, RNA digestion was suppressed because antisense DNA was strongly hybridized with azobenzene-tethered sense DNA, and accordingly RNA was isolated. On UV irradiation, antisense DNA was released from the azobenzene-tethered DNA due to the trans-to-cis isomerization and hybridized with RNA, which was digested by RNase H.

Azo Compounds↗
PubMed1985

The nucleotide sequence of cloned wheat dwarf virus DNA.

Restriction analysis and cloning of virus-specific double-stranded DNA isolated from plants infected with wheat dwarf virus (WDV) indicated that the virus genome, like that of maize streak virus (MSV), consists of a single DNA circle. The complete nucleotide sequence of cloned WDV DNA (2749 nucleotides) has been determined. Comparison of the potential coding regions in WDV DNA with those in the DNA of two strains of MSV suggests that these viruses encode at least two functional proteins, the coat protein read in the virion (+) DNA sense and a composite protein, formed from two open reading regions, in the complementary (-) DNA sense. Although WDV and MSV are serologically unrelated their coat proteins showed 35% direct amino acid sequence and their DNAs showed 46% nucleotide sequence homology. There was too little homology between the DNAs of WDV and those of two geminiviruses with bipartite genomes, cassava latent virus (CLV) and tomato golden mosaic virus (TGMV), to align the sequences. However comparison of the amino acid sequences of predicted proteins of WDV, MSV, TGMV and CLV revealed clear relationships between these viruses and suggested that the monopartite and the bipartite geminiviruses have a common ancestral origin. Four inverted repeat sequences which have the potential to form hairpin structures of deltaG >/= -14 kcal/mol were detected in WDV DNA. The sequence TAATATTAC present in the loop of one of these hairpins is conserved in similar putative structures in MSV DNA and in both DNA components of CLV and TGMV and may function as a recognition sequence for a protein involved in virus DNA replication.

Amino Acid Sequence↗
PubMed1989

A block of transcription elongation by RNA polymerase II at synthetic sites in vitro.

We have previously suggested that transcription elongation by RNA polymerase II can be blocked when the nascent RNA is folded into a stem-and-loop structure followed by polyuridines. As an approach to test this suggestion in vitro, several GC-rich deoxyoligonucleotides with dyad symmetries were chemically synthesized and inserted following the adenovirus 2 major late promoter. These constructs were transcribed in vitro using HeLa whole cell extract. The transcripts of the synthetic inserts can potentially form stem-and-loop structures with destabilization energy from 0 to -48 kcal followed by 3, 5, and 8 U residues. The results obtained show that transcription elongation is blocked by these synthetic inserts and that the extent of the elongation block is directly correlated to the stabilities of the potential stem-and-loop structure and the proceeding number of U residues. Three levels of elongation blocks were observed: a brief pause of the polymerase occurs when the RNA could be folded into a secondary structure or when there were 5-6 T residues on the sense DNA strand. An extended pause occurred when the number of T residues on the sense DNA strand was increased to 8. Transcription termination, with a partial release of the attenuated transcript occurred when a stable RNA secondary structure (delta G = -48 kcal) was followed by 8 U residues. The relevancy of these in vitro results to the in vivo mechanism of a transcription elongation block is discussed.

Base Sequence↗
PubMed2003

Role of Pax2 in apoptosis resistance and proinvasive phenotype of Kaposi's sarcoma cells.

In this study, we found that Kaposi's sarcoma cells but not human microvascular endothelial cells expressed PAX2, a gene coding for a transcription factor involved both in organogenesis and tumorigenesis. Moreover, Pax2 was frequently expressed, on spindle-shaped cells, in Kaposi's sarcoma lesions. We cloned PAX2 from Kaposi's sarcoma cells and obtained antisense and sense DNA. Transfection of Kaposi's sarcoma cells with antisense DNA, which suppressed Pax2 protein expression, reduced cell growth and survival and enhanced the sensitivity of Kaposi's sarcoma cells to apoptosis induced by serum deprivation or vincristine treatment. In addition, antisense transfection inhibited the cell motility, the invasion of Matrigel, and the spindle shape morphology, which are characteristics of Kaposi's sarcoma cells. Moreover, the alphavbeta3 integrin, known to be involved in tumor invasion, was down-regulated. To evaluate the possible role of Pax2 expression in the endothelial origin of Kaposi's sarcoma cells, human microvascular endothelial cells were transfected with sense DNA. Endothelial cells transfected with sense PAX2 acquired spindle shape morphology, showed enhanced motility and Matrigel invasion, and displayed an enhanced expression of alphavbeta3 integrin. In conclusion, the expression of Pax2 by Kaposi's sarcoma cells correlated with an enhanced resistance against apoptotic signals and with the proinvasive phenotype. Moreover, PAX2-transfected endothelial cells acquired a phenotype resembling that of Kaposi's lesional cells, suggesting a role of this embryonic gene in tumorigenesis.

Aged↗
PubMed1986

Estimation of gene expression in heterocysts of Anabaena variabilis by using DNA-RNA hybridization.

In the filamentous cyanobacterium Anabaena variabilis, specialized cells called heterocysts occur in a regular pattern along the filament and are the sites of nitrogen fixation. We used two different types of DNA-excess RNA hybridization techniques to estimate the number of genes expressed in recently differentiated, mature heterocysts. In the first, RNA and DNA were incubated in a phosphate buffer at 60 degrees C, and the hybrids were separated from the unhybridized material by hydroxylapatite chromatography. In the second, the nucleic acids were incubated at 50 degrees C in a buffer containing 50% formamide, and the fraction of DNA in duplexes was assayed by S1 nuclease digestion. Both techniques revealed that approximately 65% of the A. variabilis genome was expressed in vegetative cells and 45% of the genome was expressed in heterocysts. Two experiments were conducted to estimate the number of heterocyst-specific mRNA transcripts. In one, hybridization of heterocyst RNA to a null DNA probe (DNA not transcribed in vegetative cells) revealed that heterocyst-specific transcripts were encoded by 25% of the DNA sense strand, representing approximately 1,000 genes (assuming each to be 1,500 nucleotides in length). The second approach, in which total cell DNA was hybridized to a mixture of heterocyst and vegetative cell RNA, indicated that 14.7% of the DNA sense strand, or about 600 genes, was transcribed exclusively in the heterocyst. The remaining 900 to 1,300 transcripts present in the heterocyst appeared to be constitutively produced in both vegetative cells and heterocysts. The heterocyst-specific transcripts were present in abundant copies in the cell, while transcripts that occurred in both cell types were present at much lower frequency.

Cyanobacteria↗
PubMed1997

Cell cycle regulation in irradiated and nonirradiated cells.

Exposure of cells to ionizing radiation induces damage in the DNA. The adverse consequences of such exposures depend on the amount of the DNA damage induced as determined by the absorbed dose, as well as by its form as determined by the linear energy transfer. In addition to physical determinants, biological factors critically affect radiation response. Cells have the ability to sense DNA damage, and to activate repair pathways that efficiently remove such damage and restore the integrity of the DNA. Highly sophisticated mechanisms further enable cells to actively stall growth and division after sensing DNA damage. Such inhibitory processes, termed checkpoints, may optimize repair and minimize the adverse consequences of DNA damage. We review the fundamental principles underlying checkpoints as they have emerged from active research in the last few years and discuss briefly their relevance to the practice of medical and radiation oncology.

Animals↗
PubMed1996

Sensing of DNA non-homology lowers the initiation of meiotic recombination in yeast.

BACKGROUND: Meiotic recombination between homologous chromosomes in the yeast Saccharomyces cerevisiae is initiated by the formation of DNA double-strand breaks (DSBs). The mechanism of DSB formation and the factors that determine their frequency and location have yet to be elucidated. Current studies of meiotic recombination are also concerned with the question of the functional relationship between DSB formation and the other meiotic processes of homology searching, pairing and synapsis of homologues. RESULTS: To test if DNA identity is required for high levels of DSBs and recombination, we have asked whether small DNA heterologies (140-547 bp) located within the well characterized ARG4 initiator of meiotic recombination, can affect DSB formation and gene conversion events in the ARG4 locus. The present physical and genetic analyses show that some heterologies reduced recombination frequencies without altering DSB formation, whereas others reduced both DSB and gene conversion frequencies. CONCLUSIONS: These results suggest that DNA heterologies overlapping a recombination initiator impair meiotic gene conversion at two levels. First, some heterologies affect the level of DSB formation, revealing the existence of an anti-initiation process sensing the presence of sequence non-homology between the homologous chromosomes. Second, heterologies can impair the successful processing of the recombination intermediates once DSBs are made. We present a model for interhomologue cross-talks involving chromosomal and DNA/DNA interactions.

Argininosuccinate Lyase↗
PubMedDate not supplied

Regulation of competence for genetic transformation in Streptococcus pneumoniae: a link between quorum sensing and DNA processing genes.

Competence for genetic transformation in pneumococcus depends on the coordinated functioning of a dispersed regulon responsible for production of proteins active in DNA binding, uptake, and recombination. This regulon is characterized by a shared noncanonical promoter consensus, TACGAATA, and is capable of 100-fold expression modulations. This review discusses recent evidence that its regulation depends on a novel sigma factor, itself controlled by an autostimulatory quorum sensing system that acts through an extracellular peptide signal.

Bacterial Proteins↗
PubMed1999

Transfection of anti-sense complementary DNA of human epidermal-growth-factor receptor attenuates the proliferation of human non-small-cell-lung-cancer cells.

The proliferation of human non-small-cell-lung-cancer (NSCLC) cells is regulated by the epidermal-growth-factor-receptor (EGFR)-mediated autocrine loop that interacts with transforming growth factor-alpha (TGF-alpha) of autocrine or paracrine origin. We have shown that EGFR expression is elevated in the brain metastatic variant of human NSCLC cells H226Br, which thereby acquire their increased sensitivity toward exogenous TGF-alpha. To determine detailed cell-phenotype changes as a result of EGFR down-regulation, H226Br cells were transfected with a human EGFR-cDNA construct encompassing an N-terminal fragment (1.8 kb) in anti-sense orientation downstream of the cytomegalovirus (CMV) promoter. The EGFR transcript expressed in the 3'-5' direction is expected to neutralize EGFR mRNA and to reduce protein expression correspondingly. The established cell lines resistant to G418 were shown integrated with the transfected construct and their proliferation rates reduced as compared with the parental cells and with those transfected with vector alone. Down-regulated EGFR expression in cells with the anti-sense construct can be confirmed by Scatchard analysis and suppressed EGFR kinase activity. The restrained-growth phenotype is also demonstrated in the prolonged G2-M phase during the cell cycle, and correlated with impairment of cell proliferation. This finding suggests that EGFR over-expression is critical in maintaining the malignant phenotype of NSCLC cells, thereby providing a valuable biomarker and potential target prevention for lung-cancer-cell proliferation.

Carcinoma, Non-Small-Cell Lung↗
PubMed2006

Pausing during reverse transcription increases the rate of retroviral recombination.

Retroviruses package two copies of genomic RNA into viral particles. During the minus-sense DNA synthesis step of reverse transcription, the nascent DNA can transfer multiple times between the two copies of the genome, resulting in recombination. The mechanism for this process is similar to the process of obligate strand transfers mediated by the repeat and primer binding site sequences. The location at which the DNA 3' terminus completely transfers to the second RNA strand defines the point of crossover. Previous work in vitro demonstrated that reverse transcriptase pausing has a significant impact on the location of the crossover, with a proportion of complete transfer events occurring very close to pause sites. The role of pausing in vivo, however, is not clearly understood. By employing a murine leukemia virus-based single-cycle infection assay, strong pausing was shown to increase the probability of recombination, as reflected in the reconstitution of green fluorescent protein expression. The infection assay results were directly correlated with the presence of strong pause sites in reverse transcriptase primer extension assays in vitro. Conversely, when pausing was diminished in vitro, without changing the sequence of the RNA template involved in recombination, there was a significant reduction in recombination in vivo. Together, these data demonstrate that reverse transcriptase pausing, as observed in vitro, directly correlates with recombination during minus-sense DNA synthesis in vivo.

DNA, Viral↗
PubMed2005

Specific molecule localization in microchannel laminar flow and its application for non-immobilized-probe analysis.

Microfluidic systems enable superior control of fluidics. We have developed a novel size-separation method utilizing secondary flow within a microchannel. Using confocal fluorescence microscopy and computer simulation, we confirmed that separation occurred as a result of specific molecular localization in the curving part of the microchannel. Maximum separation efficiency was achieved by optimizing microchannel design and flow rate for individual separation targets. In addition, more effective separation was achieved by use of plural microchannel curves. This method was used for sequence-selective DNA sensing. Double-stranded DNA formed by hybridization between target DNA and a complementary probe had different elution profiles from those of the single-stranded non-complementary sequence. Moreover, the response depends on the length of the DNA molecules. This method does not require immobilization of either probe or target DNA, because all reactions occurred in the solution phase. Such features may reduce experimental error and the difference between data from different operators.

Biosensing Techniques↗
PubMed2004

Luminescent pH sensing and DNA binding properties of a novel ruthenium(II) complex.

A new Ru(II) complex, [Ru(bpy)(2)(dhipH3)](ClO4)(2) (in which bpy=2,2'-bipyridine, dhipH(3)=3,4-dihydroxy-imidado[4,5-f][1,10]-phenanthroline), was synthesized and characterized, and the pH effect on the emission spectra of the complex was studied. The interaction of the complex with calf thymus DNA was investigated by UV-visible and emission spectroscopy, and viscosity measurements. The results suggest that the complex acted as a sensitive luminescent pH sensor and a strong ct-DNA intercalator with an intrinsic binding constant of (4.0+/-0.7) x 10(5) M(-1) in buffered 50 mM NaCl.

DNA↗
PubMedDate not supplied

DNA-responsive hydrogels that can shrink or swell.

Molecule-responsive hydrogels are reputed to be smart materials because of their unique properties. We recently reported that hydrogels containing directly grafted single-stranded (ss) DNA or ssDNA-polyacrylamide conjugate in a semi-interpenetrating network (semi-IPN) manner that "only shrunk" by the addition of ssDNA samples. To date, however, no DNA-responsive hydrogels have been reported capable of "swelling" in response to specific DNAs. Smart materials capable of both shrinking and swelling in response to specific DNAs would be very useful in biochemical and biomedical applications. Here, we show a novel "shrinking or swelling" DNA-responsive mechanism. Novel hybrid hydrogels containing rationally designed ssDNA as the cross-linker were capable of shrinking or swelling in response to ssDNA samples and recognizing a single base difference in the samples. On the basis of the results presented in this paper, it is envisioned that these novel hybrid hydrogels could function and have potential in applications such as DNA-sensing devices and DNA-triggered actuators.

Acrylic Resins↗
PubMed2005

Nanomechanical sensing of DNA sequences using piezoresistive cantilevers.

A microfabricated cantilever with an internal piezoresistive component has been sensitized with thiol tethered ss-DNA strands and utilized for an in situ, label-free, highly specific, and rapid DNA detection assay. The generation of a differential surface stress onto the functionalized cantilever surface upon target recognition has allowed nanomechanical identification of 12-nucleotide complementary DNA probes with single base mismatch discrimination (sensitivity of 0.2 microM). Interestingly, utilization of an overhang extension distal to the surface enhanced the sensitivity to the 0.01 microM level. The cantilever was functionalized by inkjet printing technology. Replacing the capture probe with locked nucleic acid (LNA) resulted in a faster target probe capture kinetics compared to DNA-DNA hybridization. The capabilities of the piezoresistive cantilever indicate future ergonomic convenience via miniaturization alternative to the conventional laser-based detection method for portable on-site applications.

Base Sequence↗
PubMed1979

Split genes and RNA splicing.

A number of genes in higher organisms and in their viruses appear to be split. That is, they have "nonsense" stretches of DNA interspersed within the sense DNA. The cell produces a full RNA transcript of this DNA, nonsense and all, and then appears to splice out the nonsense sequences before sending the RNA to the cytoplasm. In this article what is known about these intervening sequences and about the processing of the RNA is outlined. Also discussed is their possible use and how they might have arisen in evolution.

Animals↗
PubMed2004

The functions of budding yeast Sae2 in the DNA damage response require Mec1- and Tel1-dependent phosphorylation.

DNA damage checkpoint pathways sense DNA lesions and transduce the signals into appropriate biological responses, including cell cycle arrest, induction of transcriptional programs, and modification or activation of repair factors. Here we show that the Saccharomyces cerevisiae Sae2 protein, known to be involved in processing meiotic and mitotic double-strand breaks, is required for proper recovery from checkpoint-mediated cell cycle arrest after DNA damage and is phosphorylated periodically during the unperturbed cell cycle and in response to DNA damage. Both cell cycle- and DNA damage-dependent Sae2 phosphorylation requires the main checkpoint kinase, Mec1, and the upstream components of its pathway, Ddc1, Rad17, Rad24, and Mec3. Another pathway, involving Tel1 and the MRX complex, is also required for full DNA damage-induced Sae2 phosphorylation, that is instead independent of the downstream checkpoint transducers Rad53 and Chk1, as well as of their mediators Rad9 and Mrc1. Mutations altering all the favored ATM/ATR phosphorylation sites of Sae2 not only abolish its in vivo phosphorylation after DNA damage but also cause hypersensitivity to methyl methanesulfonate treatment, synthetic lethality with RAD27 deletion, and decreased rates of mitotic recombination between inverted Alu repeats, suggesting that checkpoint-mediated phosphorylation of Sae2 is important to support its repair and recombination functions.

Alleles↗