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At least 721 records · Page 40Linked to original sources
PubMed2003

Down modulation of topoisomerase I affects DNA repair efficiency.

Topoisomerase I (topo I) relaxes supercoiled DNA through a breakage/rejoining reaction which involves a transient covalent bond between topo I and the 3' end of the cleaved DNA strand. Topo I activity is now shown to be involved in DNA damage/repair pathway in vivo. Down regulating topo I levels using anti-sense RNA approach inhibits repair of UV-induced DNA lesions, negatively affects clonogenic survival following UV-irradiation, and reduces the formation of repair patches at the cytological level. Finally, topo I is actively recruited onto genomic DNA following DNA damage by UV light without inducing ubiquitin-dependent degradation of topo I. Thus, topo I activity is important, possibly required, for pre- or post-DNA damage processing in nucleotide excision repair (NER).

Camptothecin↗
PubMed2005

Calcium receptor is functionally expressed in rat neonatal ventricular cardiomyocytes.

Both intra- and extracellular calcium play multiple roles in the physiology and pathophysiology of cardiomyocytes, especially in stimulus-contraction coupling. The intracellular calcium level is closely controlled through the concerted actions of calcium channels, exchangers, and pumps; however, the expression and function(s) of the so-called calcium-sensing receptor (CaR) in the heart remain less well characterized. The CaR is a seven-transmembrane receptor, which, in response to noncovalent binding of extracellular calcium, activates intracellular effectors, including G proteins and extracellular signal-regulated kinases (ERK1/2). We have shown that cultured neonatal cardiomyocytes express the CaR messenger RNA and the CaR protein. Furthermore, increasing concentrations of extracellular calcium and a type II CaR activator "calcimimetic" caused inositol phosphate (IP) accumulation, downregulated tritiated thymidine incorporation, and supported ERK1/2 phosphorylation, suggesting that the CaR protein is functionally active. Interestingly, the calcimimetic induced a more rapid ERK1/2 phosphorylation than calcium and left-shifted the IP concentration-response curve for extracellular calcium, supporting the hypothesis that CaR is functionally expressed in cardiac myocytes. This notion was underscored by studies using a virus containing a dominant-negative CaR construct, because this protein blunted the calcium-induced IP response. In conclusion, we have shown that the CaR is functionally expressed in neonatal ventricular cardiomyocytes and that the receptor activates second messenger pathways, including IP and ERK, and decreases DNA synthesis. A specific calcium-sensing receptor on cardiac myocytes could play a role in regulating cardiac development, function, and homeostasis.

Animals↗
PubMedDate not supplied

Evidence of antisense tumor targeting in mice.

Even though increased accumulations of radiolabeled antisense DNAs compared to control DNAs are becoming a routine observation in cultured tumor cells, trustworthy evidence of tumor targeting in vivo by an antisense mechanism remains elusive. The goal of this study was to obtain convincing evidence of antisense tumor targeting in nude mice by using two different tumors and both intratumoral (i.t.) and intravenous (i.v.) administration of radiolabeled antisense and control sense DNAs. Both the MDR++ cell line KB-G2 and its parent MDR+ cell line KB-31 were used in this study. The antisense (AS) DNA was directed against the AUG start codon of the MDR1 mRNA and, along with the sense (S) control DNA, was a uniform phosphorothioate administered naked. In previous cell culture studies from our laboratories, the accumulation of this AS DNA was strikingly high in KB-G2 cells and only average in KB-31 cells, a fact we attribute to the 1000-fold higher expression by RT-PCR of MDR1 mRNA in the former cell line. In this study, both DNAs were radiolabeled with (99m)Tc via MAG3 and administered i.t. or i.v. at 1 microg (100 microCi) per animal 24 h prior to sacrifice and dissection in mice bearing thigh tumors of about 1 g. Following i.t. administration, no statistically significant differences (Student's t test, p < 0.05, N = 4) between the AS and S DNA biodistributions in normal tissues were observed except in the KB-G2 mice in which muscle levels were lower for the S control. In contrast, tumor levels in the KB-G2 animals were significantly higher for the AS DNA vs S DNA (14.7 vs 8.5% ID/g) while this difference (8.6 vs 4.3% ID/g) was insignificant in the KB-31 animals. The whole body images obtained just prior to sacrifice clearly show improved targeting of AS DNA vs S DNA in the KB-G2 but not the KB-31 animals. Calculations based on these results show that about 60 000 AS DNAs accumulated specifically (i.e. AS DNA - S DNA) per KB-G2 tumor cell following i.t. administration. When administered i.v. rather than i.t., higher tumor levels in KB-G2 animals compared to KB-31 were not observed, most likely because of the lower dosage reaching the tumors. When the KB-G2 and KB-31 results are combined, no statistically significant differences between the AS and S DNA biodistributions in normal tissues were observed except in blood in which S DNA levels were higher and in spleen in which they were lower. In contrast, tumor levels were significantly higher for the AS DNA vs S DNA (0.100 vs 0.063% ID/g). Calculations based on these results show that about 400 AS DNAs accumulated specifically per tumor cell following i.v. administration. Therefore evidence for tumor targeting in vivo by an antisense mechanism has been obtained in that statistically higher tumor accumulations of the (99m)Tc-AS DNA were observed compared to the control (99m)Tc-S DNA both following i.t. and i.v. administrations. The successful localization of AS DNA in tumor demonstrates that in vivo AS targeting of tumor is feasible although improvements in tumor delivery and normal tissue clearance are needed for practical antisense imaging.

Animals↗
PubMed2006

Distinct viral sequence elements are necessary for expression of Tomato golden mosaic virus complementary sense transcripts that direct AL2 and AL3 gene expression.

Transient expression studies using Nicotiana benthamiana protoplasts and plants have identified sequences important for transcription of complementary sense RNAs derived from Tomato golden mosaic virus (TGMV) DNA component A that direct expression of AL2 and AL3. Transcription of two complementary sense RNAs, initiating at nucleotides 1,935 (AL1935) and 1,629 (AL1629), is directed by unique sequences located upstream of each transcription initiation site. One element is located between 28 and 124 nucleotides (nt) upstream of the AL1935 transcription start site, which differs from a second element located 150 nt downstream, between 129 and 184 nt upstream of the AL1629 transcription start site. Transcription initiation at nucleotide 1,935 is lower than that at nucleotide 1,629 as determined by run-on transcription assays, and the resulting transcript is only capable of expressing AL3. The transcript initiating at nucleotide 1,629 is capable of directing expression of both AL2 and AL3, although expression of AL3 is up to fourfold greater than that for AL2. Nuclear factors purified from tobacco suspension cells bind to sequences upstream of both AL1935 and AL1629, correlating with the ability of these sequences to direct gene expression. Thus, in tobacco, regulatory sequences direct transcription of two unique TGMV messenger RNAs that differentially express AL2 and AL3.

Begomovirus↗
PubMed2000

Elastic stability of DNA configurations. I. General theory.

Results are presented in the theory of the elastic rod model for DNA, among which are criteria enabling one to determine whether a calculated equilibrium configuration of a DNA segment is stable in the sense that it gives a local minimum to the sum of the segment's elastic energy and the potential of forces acting on it. The derived stability criteria are applicable to plasmids and to linear segments subject to strong anchoring end conditions. Their utility is illustrated with an example from the theory of configurations of the extranucleosomal loop of a DNA miniplasmid in a mononucleosome, with emphasis placed on the influence that nicking and ligation on one hand, and changes in the ratio of elastic coefficients on the other, have on the stability of equilibrium configurations. In that example, the configurations studied are calculated using an extension of the method of explicit solutions to cases in which the elastic rod modeling a DNA segment is considered impenetrable, and hence excluded volume effects and forces arising from self-contact are taken into account.

Biophysical Phenomena↗
PubMed1999

A novel PCR-based approach for the detection of the Huntington disease associated trinucleotide repeat expansion.

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder associated with expansions of an unstable CAG trinucleotide repeat in exon 1 of the IT15 gene. In normal individuals, IT15 contains up to 35 CAG repeats, while in affected the repeat length is >36. Polymerase chain reaction (PCR) is used to estimate the number of CAG repeats but may be inefficient in long repeats because of the high C+G content of the HD locus. We present a novel PCR approach for the diagnosis of HD, which permits direct visualization of the amplified products on agarose gel, using ethidium bromide. It is based on the methylation-sensitive conversion of C residues to U by bisulfite treatment of single-stranded DNA and subsequent amplification of the sense strand with specific primers. The bisulfite treatment dramatically reduces the C + G content of the region; thus, the high Tm and stable secondary structures are no longer obstacles to PCR. In both normal and affected individuals, UAG repeats (5'- CAG-3', before bisulfite treatment) in the sense strand can easily be amplified and visualized on a gel by ethidium bromide staining. The method has considerable advantages compared with other described PCR-based diagnostic tests for HD.

Base Sequence↗
PubMed1999

A kinetic proofreading mechanism for disentanglement of DNA by topoisomerases.

Cells must remove all entanglements between their replicated chromosomal DNAs to segregate them during cell division. Entanglement removal is done by ATP-driven enzymes that pass DNA strands through one another, called type II topoisomerases. In vitro, some type II topoisomerases can reduce entanglements much more than expected, given the assumption that they pass DNA segments through one another in a random way. These type II topoisomerases (of less than 10 nm in diameter) thus use ATP hydrolysis to sense and remove entanglements spread along flexible DNA strands of up to 3,000 nm long. Here we propose a mechanism for this, based on the higher rate of collisions along entangled DNA strands, relative to collision rates on disentangled DNA strands. We show theoretically that if a type II topoisomerase requires an initial 'activating' collision before a second strand-passing collision, the probability of entanglement may be reduced to experimentally observed levels. This proposed two-collision reaction is similar to 'kinetic proofreading' models of molecular recognition.

DNA↗
PubMed1986

Inhibition of retroviral replication by anti-sense RNA.

We tested the effect of anti-sense RNA on the replication of avian retroviruses in cultured cells. The replication of a recombinant retrovirus carrying a neomycin resistance gene (neor) in the anti-sense orientation was blocked when the cells expressed high steady-state levels of RNA molecules with neor in sequence in the sense was blocked when the cells expressed high steady-state levels of RNA molecules with neor sequences in the sense orientation, i.e., complementary to the viral sequence. Viral DNA bearing neor sequences was not detected specifically in host cells where this anti-sense RNA inhibition of viral replication occurred. These observations suggest that anti-sense RNA inhibition may be a useful strategy for the inhibition of retroviral infections.

Animals↗
PubMed1987

Analysis of Aleutian disease of mink parvovirus infection using strand-specific hybridization probes.

A 7- to 95-map unit segment of DNA from Aleutian disease of mink parvovirus (ADV) was subcloned into a bacteriophage SP6 based transcription vector and used to produce radiolabeled viral RNA transcripts corresponding to either 'plus' or 'minus' sense. The radiolabeled transcripts were reacted against Southern blots of whole cell DNA from ADV infected cell cultures as hybridization probes. The 'plus' sense RNA probe hybridized both to duplex replicative forms (RFs) as well as to single-stranded virion DNA (SS DNA), which is 'minus' in sense. In contrast, the 'minus' sense RNA probe reacted preferentially with the duplex RFs. When these probes were tested against DNA extracted from mink infected with the virulent ADV-Utah I strain, RFs were detected at 10 days after infection in mesenteric lymph node, liver, spleen and gut, but only in gut and mesenteric lymph node at 43 days. SS DNA was noted in these tissues at 10, 43 and 60 days, and was more abundant than RFs. Only SS DNA at very low levels was observed in bone marrow cells. Serum contained large amounts of SS DNA (probably in virions) at 10 days, less at 43 days, and no detectable DNA at 60 days. These findings suggest that ADV replication may have occurred in the gut as well as lymphoreticular tissues, and that bone marrow was not a major site of ADV replication.

Aleutian Mink Disease↗
PubMed1996

Structure and function of Escherichia coli met repressor: similarities and contrasts with trp repressor.

Transcription of genes encoding enzymes for the biosynthesis of methionine and trytophan in Escherichia coli is regulated by the ligand-activated met and trp repressors. X-ray crystallographic studies show how these two small proteins, although similar in size and function, have totally different three-dimensional structures and specifically recognize their respective DNA operator sequences in different ways. A common feature is that both repressors bind as cooperative arrays to tandem repeats of 8 base-pair 'Met' or 'Trp boxes' respectively, and the consensus sequences share the rare tetranucleotide CTAG. A series of structural and functional studies have shown how the two repressors discriminate between their operators, using a combination of direct contacts between side chains and bases, and indirect sensing of conformational properties of the DNA.

Bacterial Proteins↗
PubMed2003

Mobility of the Sinorhizobium meliloti group II intron RmInt1 occurs by reverse splicing into DNA, but requires an unknown reverse transcriptase priming mechanism.

The mobile group II introns characterized to date encode ribonucleoprotein complexes that promote mobility by a major retrohoming mechanism in which the intron RNA reverse splices directly into the sense strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase/maturase cleaves the antisense strand and uses it as primer for reverse transcription of the inserted intron RNA. Here, we show that the Sinorhizobium meliloti group II intron RmInt1, which encodes a protein lacking a DNA endonuclease domain, similarly uses both the intron RNA and an intron-encoded protein with reverse transcriptase and maturase activities for mobility. However, while RmInt1 reverse splices into both single-stranded and double-stranded DNA target sites, it is unable to carry out site-specific antisense-strand cleavage due to the lack of a DNA endonuclease domain. Our results suggest that RmInt1 mobility involves reverse splicing into double-stranded or single-stranded DNA target sites, but due to the lack of DNA endonuclease function, it requires an alternate means of procuring a primer for target DNA-primed reverse transcription.

Base Sequence↗
PubMed2006

Pathways that suppress programmed DNA breaks from progressing to chromosomal breaks and translocations.

Guarding the genome against internal and external assaults requires the coordinated interaction of multiple cellular networks to sense, respond to, and repair breaks in chromosomal DNA. Both external factors such as ionizing radiation or internal events like oxidative damage can cause DNA double stranded breaks (DSBs). DSBs are also part of the normal lymphocyte developmental program where they are an integral element of the mechanisms that generate a diverse immune repertoire in the context of V(D)J and immunoglobulin heavy chain (IgH) class switch recombination (CSR). DSBs initiate a cascade of cellular events that direct cells to pause and properly repair potentially lethal chromosomal breaks. Errors in the repair of both general and lymphocyte-specific DSBs can lead to oncogenic chromosomal translocations . Here, we review recent advances in understanding factors and protein complexes involved in the response to DNA DSBs with a focus on the B lymphocyte specific process of CSR.

Animals↗
PubMed2005

Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.

Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25-50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.

DNA, Intergenic↗
PubMed1991

Transcription of cRNA for in situ hybridization from polymerase chain reaction-amplified DNA.

The synthesis of cRNA probes for in situ hybridization is usually accomplished with transcription systems using cloning vectors that contain bacteriophage RNA polymerase promoters. In this report we describe an alternative technique for generating cRNA probes that obviates the need for cDNA cloning by using the polymerase chain reaction. The segment of DNA corresponding to the desired RNA sequence was amplified from genomic DNA by the polymerase chain reaction. The bacteriophage SP6 and T7 RNA polymerase promoters were incorporated into the amplified DNA by including the promoter sequences in the 5' termini of the oligonucleotides used to prime the polymerase chain reaction. The promoters permitted the subsequent transcription of sense and antisense cRNA from the amplified DNA. The antisense cRNA was radiolabeled to high specific activity for use as a probe in Northern and mRNA in situ hybridization analyses. This technique offers the advantages of speed and simplicity over conventional transcription systems, which require cDNA cloning. Bypassing the need for cloning in the synthesis of cRNA probes may facilitate the use of these probes in research and diagnostic applications of mRNA in situ hybridization.

Animals↗
PubMed2000

Partial cloning and sequencing of chick fibrillin-1 cDNA.

The recent identification of numerous matrix genes and gene products has allowed a detailed examination of their roles in development. Two of these extracellular matrix proteins, fibrillin-1 and fibrillin-2, are components of the elastin-associated microfibrils. Given what is known about the distribution of the fibrillins in normal tissues and the abnormalities that result when mutations occur, a basic hypothesis has emerged: fibrillin-1 is primarily responsible for load bearing and providing structural integrity, whereas fibrillin-2 may be a director of elastogenesis. Nevertheless, examination of phenotypes in disorders caused by mutations in fibrillin-1 or fibrillin-2 suggests some common functions. To better understand these similar and diverse roles, it would be helpful to examine these proteins during chick development. To accomplish this goal, it is first necessary to characterize the chick homologs of the known fibrillins. In this study, the partial chick FBN1 cDNA was identified by polymerase chain reaction-aided cloning as a first step toward elucidating these goals. Sequence analysis indicated that there is striking conservation between chick and mammalian fibrillin-1 at the DNA and protein levels. Antisense and sense riboprobes were synthesized and used in in situ hybridization in stage 14 chick embryos and high levels of FBN1 transcripts were observed in the heart.

Amino Acid Sequence↗
PubMed2003

The protein complex composed of nickel-binding SrnQ and DNA binding motif-bearing SrnR of Streptomyces griseus represses sodF transcription in the presence of nickel.

Nickel-responsive transcriptional repression of sodF, which codes for iron- and zinc-containing superoxide dismutase of Streptomyces griseus, was mediated through an operator (-2 to +15) spanning over the 5' end (+1) of the transcript. Two open reading frames, SrnR (12,343 Da) and SrnQ (12,486 Da), with overlapping stop-start codons were identified downstream from sodF and found responsible for the repression of sodF. The deduced amino acid sequence of SrnR revealed a DNA binding motif and showed homology to the transcriptional regulators of ArsR family, whereas SrnQ did not show any similarity to any known proteins. When srnRQ DNA was maintained in trans in S. griseus on a multicopy plasmid, sodF transcription was highly repressed by nickel, but neither srnR nor srnQ alone showed the effect. Consistently, the sodF transcription of srnR-interrupted mutant was no longer repressed by nickel, which was complemented only with srnRQ DNA. Nickel-dependent binding of SrnR and SrnQ to the sodF operator DNA was observed only when the two proteins were provided together. The maximum protein-DNA interaction was shown when SrnR and SrnQ were present in one-to-one stoichiometric ratio. The two proteins appear to constitute an octamer composed of four subunits of each protein. SrnR directly interacted with SrnQ, and the protein interaction did not require nickel. The conformation of SrnQ was changed upon nickel binding, which was in the ratio of one Ni(2+) ion per protein molecule. A model is proposed in which SrnQ of the protein complex senses nickel and subsequently enhances the DNA binding activity of SrnR through the protein-protein interaction.

Amino Acid Motifs↗
PubMed2006

pH-sensing nano-crystals of carbonate apatite: effects on intracellular delivery and release of DNA for efficient expression into mammalian cells.

Two unique and fascinating properties of carbonate apatite which are well-known in hard tissue engineering, have been unveiled, for the first time, for the development of the simplest, but most efficient non-viral gene delivery device - ability of preventing the growth of crystals needed for high frequency DNA transfer across a plasma membrane and a fast dissolution rate for effective release of DNA during endosomal acidification, leading to a remarkably high transgene expression (5 to 100-fold) in mammalian cells compared to the widely used transfecting agents. Moreover, by modulating the crystal dissolution rate of carbonate apatite through incorporation of fluoride or strontium into it, transfection activity could be dramatically controlled, thus shedding light on a new barrier in the non-viral route, which was overlooked so far. Thus we have developed an innovative technology with significant insights, that would come as a promising tool for both basic research laboratories and clinical settings.

Animals↗
PubMed2006

Inhibition of Aurora A in response to DNA damage.

Mitotic kinases are the ultimate target of pathways sensing genotoxic damage and impinging on the cell cycle machinery. Here, we provide evidence that Aurora A (AurA) was inhibited upon generation of double-strand breaks in DNA. We demonstrate that AurA was not downstream of CDK1 and that inhibition of AurA and CDK1 by DNA damage occurred independently. Using a cell line functionally deficient in Chk2, a selective Chk1 inhibitor and siRNA to Chk1, we show that DNA-damage signals were delivered to AurA through a Chk1-dependent pathway. With regard to the molecular mechanism of AurA inhibition, we found that the point mutation Ser(342)>Ala rendered AurA resistant to inhibition by DNA damage. By means of two distinct approaches we examined the impact of reconstitution of AurA activity in DNA-damaged cells: (i) transient expression of wild-type and Ser(342)>Ala mutant, but not kinase-dead, AurA led to bypass of the DNA damage block; (ii) direct transduction of highly active wt-AurA into G2 arrested cells precisely after induction of DNA damage resulted in mitotic entry. We show that the mechanism through which AurA allowed entry into mitosis was reactivation of CDK1, thus indicating that AurA plays a key role upstream of CDK1. A model depicting the possible role of AurA at the onset of mitosis and upon DNA damage is presented.

Aurora Kinases↗