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At least 235 records · Page 13Linked to original sources
PubMed1993

Bacteriophage T4 Alc protein: a transcription termination factor sensing local modification of DNA.

Bacteriophage T4 Alc protein participates in shutting off host transcription after infection of E. coli. It is demonstrated that Alc acts as a site-specific termination factor. The Alc sites occur frequently in E. coli DNA, resulting in early cessation of elongation in several tested transcription units. Alc-dependent termination requires unimpeded propagation of the elongating complex as it approaches the Alc site. Temporary halting of RNA polymerase within 10-15 bp before the Alc site prevents termination. Bacteriophage T4 transcription is protected from the action of Alc by overall substitution of cytosine with 5-hydroxymethyl cytosine in T4 DNA. In vitro methylation of CpG sequences in the vicinity of an Alc site abolishes the effect of Alc. Thus, Alc-dependent termination involves local sensing of the state of cytosine modification and a short-term "memory" of recent pausing.

Bacteriophage T4↗
PubMed2006

Radiation and new molecular agents part I: targeting ATM-ATR checkpoints, DNA repair, and the proteasome.

In response to DNA breaks, human cells delay their progression through the G1, S, and G2 phases of the cell cycle. This response requires the coordinated effort of the ATM-CHK2-p53 and ATR-CHK1 DNA damage-sensing pathways and DNA repair (eg, DNA-PK and RAD51 complexes). The turnover of many of these DNA damage-associated proteins is controlled by the 26S proteasome. In this article, we review molecular strategies that target each of these pathways using silencing RNA (siRNA), antisense, or small-molecule inhibition. Although these agents can radiosensitize tumor cells, little data are available regarding potential effects on normal tissues to determine the potential therapeutic ratio of these strategies after fractionated radiotherapy. Clinical trials using such agents will require novel correlative science endpoints to track DNA repair and cell-cycle arrest and will need careful assessment of normal tissue toxicity and stability.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2005

Mitochondrial transcription factor A in the maintenance of mitochondrial DNA: overview of its multiple roles.

Mitochondria have their own genome, which is essential for proper oxidative phosphorylation needed for a large part of ATP production in a cell. Although mitochondrial DNA-less (rho0) cells can survive under special conditions, the integrity of the mitochondrial genome is critical for survival of multicellular organisms. Mitochondrial transcription factor A (TFAM), originally cloned as transcription factor, is essential for the maintenance of mtDNA. Recently, it has become known that TFAM plays critical roles in multiple aspects to maintain the integrity of mitochondrial DNA: transcription, replication, nucleoid formation, damage sensing, and DNA repair. The effects of TFAM in these aspects are intimately related to each other and to function as a whole for the purpose of maintenance of mtDNA.

Animals↗
PubMed2005

A single complementary-sense transcript of a geminiviral DNA beta satellite is determinant of pathogenicity.

Small circular single-stranded DNA satellites, termed DNAbeta, have recently been found associated with some geminivirus infections. The DNA beta associated with Cotton leaf curl virus is responsible for symptom expression of a devastating disease in Pakistan. Mutagenesis of DNA beta revealed that the complementary-sense open reading frame (ORF) betaC1 is required for inducing disease symptoms in Nicotiana tabacum. An ORF present on the virion-sense strand betaV1 appeared to have no role in pathogenesis. Tobacco plants transformed with a betaC1 ORF under the control of the Cauliflower mosaic virus 35S promoter or with a dimeric DNA beta exhibited severe disease-like phenotypes, while plants transformed with a mutated version of betaC1 appeared normal. Northern blot analysis of RNA from the transgenic plants, using strand-specific probes, identified a single complementary-sense transcript. The transcript carries the full betaC1 ORF encoding a 118-amino acid product. It maps to the DNA beta at nucleotide position 186 to 563 and contains a polyadenylation signal 18 nt upstream of the stop codon. A TATA box is located 43 nt upstream of the start codon. Our results indicate that betaC1 protein is responsible for DNA beta-induced disease symptoms.

Amino Acid Sequence↗
PubMed2005

Fabrication of DNA microarrays onto polymer substrates using UV modification protocols with integration into microfluidic platforms for the sensing of low-abundant DNA point mutations.

We describe the microfabrication and operational characteristics of a simple flow-through biochip sensor capable of detecting low abundant point mutations in K-ras oncogenes from genomic DNA, which carry high diagnostic value for colorectal cancers. The biochip consisted of an allele-specific ligase detection reaction (LDR) coupled to a universal array for interrogating multiple mutations simultaneously from a clinical sample. The integrated sensing platform was micro-manufactured from two different polymers, polycarbonate, PC, which was used for the LDRs, and poly(methyl methacrylate), PMMA, which was used to build the microarray. Passive elements were hot embossed into the PC and PMMA microchips and then, the chips assembled into a three-dimensional architecture with the interconnect fabricated from an elastomer, poly(dimethylsiloxane), PDMS, to produce a leak-free connection between the biochips. The array in PMMA was produced using a photomodification process, which involved three steps; (1) UV (254 nm) exposure of the polymer surface; (2) EDC coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond) and; (3) washing of the surface. The LDR/hybridization flow-through biochip performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time=19.1 min) and could screen multiple mutations simultaneously for high throughput applications at a level of one mutant sequence in 100 wild-type sequences.

Biosensing Techniques↗
PubMed2004

Concerted effect of transforming growth factor-beta, cyclin inhibitor p21, and c-myc on smooth muscle cell proliferation.

Increased aortic smooth muscle cell (SMC) proliferation is a key event in the pathogenesis of atherosclerosis. Transforming growth factor-beta (TGF-beta) is one of the potent inhibitors of SMC proliferation. The purpose of this study was 1) to explore the effect of TGF-beta inhibition on proliferation of SMC and expression of growth regulatory molecules like p21 and c-myc and 2) to determine whether restoration of cell cycle regulatory molecules normalizes the altered proliferation. To test the role of TGF-beta in SMC proliferation, using antisense plasmid DNA, we inhibited TGF-beta gene from aortic SMC, which resulted in a significant increase (P < 0.03) in proliferation (studied by quantifying new DNA synthesis with [(3)H]thymidine uptake assay). In TGF-beta-altered SMC (TASMC), the mRNA expression (studied by RT-PCR) of c-myc was increased whereas that of the cyclin inhibitor p21 was completely inhibited. Using p21 sense plasmid DNA, we transfected p21 gene in TASMC, which restored p21 mRNA and protein expression and decreased proliferation (P < 0.002) in TASMC. Similar treatment with c-myc antisense oligonucleotides significantly (P < 0.001) decreased the proliferation of TASMC. TASMC also exhibited alteration in morphological changes in SMC but returned to normal with treatment of p21 and TGF-beta sense plasmid DNA. Two-dimensional gel electrophoresis analysis of SMC and TASMC demonstrated differential expression of proteins relevant to cellular proliferation and atherosclerosis. This study uniquely analyzes the effect of TGF-beta at the molecular level on proliferation of SMC and on cell cycle regulatory molecules, implicating their potential role in the pathogenesis of atherosclerosis.

Aorta↗
PubMed1993

DNA-DNA subtractive cDNA cloning using oligo(dT)30-Latex and PCR: identification of cellular genes which are overexpressed in senescent human diploid fibroblasts.

We previously reported an efficient method for subtractive cDNA cloning using oligo(dT)30-Latex and polymerase chain reaction (PCR) (E. Hara et al., Nucleic Acids Res. 19, 7097-7104, 1991). The subtraction was performed by hybridization between mRNA of cell type B and the cDNA made from mRNA of cell type A using an oligo(dT)30 primer covalently linked to Latex particles in an Eppendorf tube. The mRNA common to both types of cells could be removed by a brief centrifugation. In the present paper, the method was improved by using the sense strand DNA instead of mRNA for hybridization to cDNA covalently linked to the particles to minimize mRNA degradation and by optimizing the hybridization condition. The sense strand DNA was made from cDNA-oligo(dT)30-Latex by asymmetric PCR. Using the improved method, a subtractive cDNA library with longer cDNA inserts was successfully constructed with higher probability than the original method.

Animals↗
PubMed1988

Mono- through hexanucleotide composition of the sense strand of yeast DNA: a Markov chain analysis.

Here we compare several methods for predicting oligonucleotide frequencies in 392 kb of yeast DNA. As in previous work on E. coli, a relatively simple equation based on tetranucleotide frequencies can be used in predicting the frequencies of longer oligonucleotides. For example, the mean of observed/expected abundances of 4,096 hexamers was 1.00 with a sample standard deviation of .18. This simple predictor arises by considering each base on the sense strand of yeast to depend only on the three bases 5' to it (a 3rd order Markov chain) and is more accurate in estimating oligonucleotide frequencies than other statistical methods examined. This equation is useful in predicting restriction enzyme fragment sizes, selecting restriction enzymes that cut preferentially in coding vs noncoding regions, and in constructing detailed physical maps of whole genomes. When ranked highest to lowest abundance, the observed frequencies of oligomers of a given length (up to 6 bases) are closely tracked by the predicted abundances of a 3rd or 4th order Markov chain. These ordered abundance curves have a power curve shape with a broad linear range with a sharp break at the top end of the curve. There is also a strong disparity between the most and least abundant oligomer with for example a 79-fold variation between the most and least abundant hexamer. The curves reveal a strong dependence of oligomer frequencies on base composition. Unlike E. Coli, there is no sharp downturn at the low end of the curves and hence, no class of oligomers rare relative to other oligomers of the same length.

Base Composition↗
PubMedDate not supplied

Psoralen-containing vinyl monomer for conjugation of double-helical DNA with vinyl polymers.

We have synthesized a vinyl monomer having a psoralen moiety, which can form a photoadduct with double-helical DNA. The monomer 1 was proved to have an ability to crosslink DNA double strands through a photochemical reaction when irradiated by UV light. The resulting DNA having vinyl groups was copolymerized with a comonomer such as acrylamide and N-isopropylacrylamide to give rise to a DNA-vinyl polymer conjugate. A conjugation based on covalent bondings was verified by using gel electrophoresis; the conjugation efficiency was found to be dependent upon concentration of the monomer which had been used in the antecedent photochemical reaction. This monomer will be a useful tool when anchoring double-helical DNA on polymeric materials for separating and sensing DNA-binding substances.

Acrylic Resins↗
PubMed1996

DNA recognition and structural specificities.

How a short DNA sequence interacts in a sequence specific manner with appropriate protein is understood only in certain systems for which high resolution crystal structures of the protein-DNA complexes are available. The base sequence of DNA is sensed directly (read-out) by the protein through the major or minor groove, while DNA shape also is sensed through multiple interactions with the sugar phosphate backbone. Several repressors, activators and restriction endonucleases complexed with their cognate DNA oligomers are now known and reviewed here. If the binding site on DNA has two fold symmetry, the protein interacts as dimer and uses a variety of structural motifs for specific interaction. The level of specificity of interaction is enhanced by flexibility and/or distortion in either the DNA or protein tertiary structure.

Animals↗
PubMed2006

Sensing metal ions with DNA building blocks: fluorescent pyridobenzimidazole nucleosides.

We describe novel fluorescent N-deoxyribosides (1 and 2) having 2-pyrido-2-benzimidazole and 2-quino-2-benzimidazole as aglycones. The compounds were prepared from the previously unknown heterocyclic precursors and Hoffer's chlorosugar, yielding alpha anomers as the chief products. X-ray crystal structures confirmed the geometry and showed that the pyridine and benzimidazole ring systems deviated from coplanarity in the solid state by 154 degrees and 140 degrees , respectively. In methanol compounds 1 and 2 had absorption maxima at 360 and 370 nm, respectively, and emission maxima at 494 and 539 nm. Experiments revealed varied fluorescence responses of the nucleosides to a panel of 17 monovalent, divalent, and trivalent metal ions in methanol. One or both of the nucleosides showed significant changes with 10 of the metal ions. The most pronounced spectral changes for ligand-nucleoside 1 included red shifts in fluorescence (Au(+), Au(3+)), strong quenching (Cu(2+), Ni(2+), Pt(2+)), and substantial enhancements in emission intensity coupled with red shifts (Ag(+), Cd(2+), Zn(2+)). The greatest spectral changes for ligand-nucleoside 2 included a red shift in fluorescence (Ag(+)), a blue shift (Cd(2+)), strong quenching (Pd(2+), Pt(2+)), and substantial enhancements in emission intensity coupled with a blue shift (Zn(2+)). The compounds could be readily incorporated into oligodeoxynucleotides, where an initial study revealed that they retained sensitivity to metal ions in aqueous solution and demonstrated possible cooperative sensing behavior with several ions. The two free nucleosides alone can act as differential sensors for multiple metal ions, and they are potentially useful monomers for contributing metal ion sensing capability to DNAs.

Benzimidazoles↗
PubMed2003

Peptides derived from the two regulatory domains of p53 are recognized by two p53-activating antibodies.

The C-terminus of the transcription factor p53 seems to play an important role by controlling the specific DNA-binding activity, which is directly associated with sensing damaged DNA. Another region located in the N-terminus of the protein has also been shown to regulate the DNA-binding activity of the protein. This activity can be promoted by peptides derived from these two negative regulatory regions or by binding of antibodies directed against the C-terminus of the p53 protein. Using both phage display peptide and multiple peptide synthesis technologies, we demonstrated that mAbs HR231 and Pab421, two p53-activating antibodies, recognize peptides derived from the C-terminus of p53, as previously described, but also peptides from the N-terminus of the protein, suggesting that these peptides are part of a conformational epitope. Furthermore, the sequences of these peptides are located in the two negative regulatory regions identified on the p53 protein, which is consistent with the biological activity of mAbs HR231 and Pab421.

Amino Acid Sequence↗
PubMed2000

Sensing and responding to DNA damage.

DNA damage or stalled DNA replication can activate specific signal transduction pathways, termed checkpoints. Checkpoint activation can result in increased repair, induction of a transcriptional programme and inhibition of cell-cycle progression. Recent results have suggested possible mechanisms for the detection of specific DNA structures, provided further information on the organisation of the signal transduction cascade and demonstrated involvement of the checkpoint pathway in DNA repair.

Animals↗
PubMed2006

Epigenetic information in chromatin: the code of entry for DNA repair.

Epigenetic changes are important etiological factors of human cancer. Epigenetic information in chromatin (known as 'histone code') is a fascinating feature used by cells to extend and modulate the genetic (DNA) code. The histone code is thus proposed to be 'read' by cells to regulate accessibility to, and functions of, chromatin DNA. While the role of the epigenetic code involving chromatin modifying/remodeling complexes in transcriptional regulation is well established, it is only recently that these mechanisms have been implicated in DNA damage detection and DNA repair. However, how the components of the DNA damage sensing and repair machinery gain access to broken DNA in compacted chromatin remains a mystery. Recent studies provide important insights into DNA damage- and repair-specific modifications to histones and shed light on how the epigenetic code controls DNA repair.

Animals↗
PubMed1997

The DNA binding regulatory domain of p53: see the C.

The p53 tumor suppressor gene is a critical regulator of normal development involved in cell cycle control pathways, such as growth arrest, differentiation and apoptosis. The DNA binding activity of p53 is central to its function. In addition to the specific DNA binding activity that is confined to the "core" domain of the molecule, the C-terminus seems to play an important role in both controlling the specific as well as exhibiting a non-specific DNA binding activity, which is directly associated with sensing damaged DNA. The C-terminal DNA binding activity appears to be regulated by phosphorylation, glycosylation, splicing and binding of several factors. The C-terminus seems to recognize single and double stranded DNA breaks that occur during DNA replication and recombination, as well as following external DNA stress signals. Unless the cell manages to correct the DNA damage it has the tempting option to progress towards apoptosis. Imagine the C-terminus as a traffic light ensuring the safe "on going" through the cell cycle; in case damaged DNA could not be corrected, p53 dependent apoptosis or terminal differentiation "signs" are turned on!

Animals↗
PubMed1997

Two mRNAs are transcribed from banana bunchy top virus DNA-1.

We have mapped the mRNA transcripts of banana bunchy top virus (BBTV) DNA-1. Northern hybridization and 3' RACE analysis identified two poly-adenylated RNAs associated with BBTV DNA-1. Previously, one major ORF in the virion sense of DNA-1 had been identified, which encoded a putative replication protein (Rep). An mRNA was identified in BBTV infected bananas that was clearly transcribed from this Rep ORF. Further, a second transcript was identified which mapped to an ORF completely within the Rep ORF. This encoded a putative 5 kDa protein of unknown function. Both these transcripts were also identified in a tobacco plant that had been transformed with Agrobacterium tumefaciens harbouring a binary construct containing the Rep ORF from BBTV DNA-1. This Rep ORF was inserted 3' of a cauliflower mosaic virus 35S promoter and 5' of a vegetable storage protein terminator. The transcripts mapped from these tobacco plants were identical at the 3' end to the transcripts from BBTV infected banana plants. The site of polyadenylation for the Rep ORF was at base 963 immediately 3' of the translational stop codon confirming that the polyadenylation signals for this transcript were all within the ORF. However, the internal ORF had a large untranslated region of 272 bases with its site of polyadenylation at nucleotide 803 and a polyadenylation signal 3' of the translational stop codon. A possible upstream termination signal (A/TTGTAA) was identified and was conserved within BBTV DNA-1 sequences from different international isolates.

Agrobacterium tumefaciens↗