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At least 469 records · Page 26Linked to original sources
PubMed1990

Detection of heteroduplex DNA molecules among the products of Saccharomyces cerevisiae meiosis.

We have used denaturant-gel electrophoresis to provide a physical demonstration of heteroduplex DNA in the products of yeast meiosis. We examined heteroduplex formation at arg4-nsp, a G.C----C.G transversion that displays a moderately high level of postmeiotic segregation. Of the two possible arg4-nsp/ARG4 mismatches (G.G and C.C), only C.C was detected in spores from mismatch repair-competent (Pms1+) diploids. In contrast, C.C and G.G were present at nearly equal levels in spores from Pms1- diploids. These results confirm previous suggestions that postmeiotic segregation spores contain heteroduplex DNA at the site of the marker in question, that C.C is repaired less frequently than is G.G, and that the PMS1 gene product plays a role in mismatch correction. Combined with the observation that Pms1+ ARG4/arg4-nsp diploids produce 3 times more 3+:5m (wildtype:mutant) tetrads (+, +, +/m, m) than 5+:3m tetrads (+, +/m, m, m), these results indicate that, during meiosis, formation of heteroduplex DNA at ARG4 involves preferential transfer of the sense (nontranscribed) strand of the DNA duplex.

Base Sequence↗
PubMed2006

HP1 proteins are essential for a dynamic nuclear response that rescues the function of perturbed heterochromatin in primary human cells.

Cellular information is encoded genetically in the DNA nucleotide sequence and epigenetically by the "histone code," DNA methylation, and higher-order packaging of DNA into chromatin. Cells possess intricate mechanisms to sense and repair damage to DNA and the genetic code. However, nothing is known of the mechanisms, if any, that repair and/or compensate for damage to epigenetically encoded information, predicted to result from perturbation of DNA and histone modifications or other changes in chromatin structure. Here we show that primary human cells respond to a variety of small molecules that perturb DNA and histone modifications by recruiting HP1 proteins to sites of altered pericentromeric heterochromatin. This response is essential to maintain the HP1-binding kinetochore protein hMis12 at kinetochores and to suppress catastrophic mitotic defects. Recruitment of HP1 proteins to pericentromeres depends on histone H3.3 variant deposition, mediated by the HIRA histone chaperone. These data indicate that defects in pericentromeric epigenetic heterochromatin modifications initiate a dynamic HP1-dependent response that rescues pericentromeric heterochromatin function and is essential for viable progression through mitosis.

Cell Cycle Proteins↗
PubMed1988

Quantification of alpha-amylase mRNA in Bacillus subtilis by nucleic acid sandwich hybridization.

A novel hybridization test for quantification of mRNA from bacterial cells lysed directly in the culture medium was developed and optimized. The method uses two adjacent probes from a DNA fragment of interest in a sandwich hybridization. An unlabeled probe is immobilized on a solid support to capture homologous nucleic acids in the test solution. Hybrid detection is performed with the other, labeled, probe, which can bind to the filter only as a sample-mediated hybrid. We used the Bacillus amyloliquefaciens alpha-amylase gene and its mRNA as models for characterizing gene expression in Bacillus subtilis. To confirm the specificity of mRNA hybridization, nondenatured nucleic acid samples were allowed to react simultaneously with two filters: the mRNA-specific filter containing anti-sense single-stranded DNA and the background filter containing single-stranded DNA of the mRNA sense, respectively. We also developed a rapid enzymatic lysis procedure for B. subtilis, allowing complete degradation of late stationary phase cells grown in rich culture medium, while retaining mRNA molecules of high integrity. The applicability of this rapid lysis procedure and the hybridization method for mRNA analyses in long-term fermentations was demonstrated.

Bacillus subtilis↗
PubMed1998

p53-independent DNA repair and cell cycle arrest in embryonic stem cells.

The role of p53 in DNA repair and cell cycle checkpoint after ultraviolet irradiation was investigated in an embryonic stem cell line homozygous for a targeted deletion of p53. Results indicate that loss of p53 does not alter the capacity of ES cells to respond to DNA damage. Wild-type and p53-deficient cells showed similar cessation of DNA synthesis after UV damage and similar ultimate capacity to repair a transiently transfected reporter plasmid. Interestingly, in the absence of DNA damaging treatment, the transit of p53-deficient cells through S phase was slower than wild-type cells. We suggest that this may result from the absence of a p53-dependent response to endogenous DNA damage: without p53 sensing endogenous damage leading to immediate repair, such damage may persist and thus delay DNA synthesis.

Animals↗
PubMed2003

Analysis of the DNA joining repertoire of Chlorella virus DNA ligase and a new crystal structure of the ligase-adenylate intermediate.

Chlorella virus DNA ligase is the smallest eukaryotic ATP-dependent DNA ligase known; it suffices for yeast cell growth in lieu of the essential yeast DNA ligase Cdc9. The Chlorella virus ligase-adenylate intermediate has an intrinsic nick sensing function and its DNA footprint extends 8-9 nt on the 3'-hydroxyl (3'-OH) side of the nick and 11-12 nt on the 5'-phosphate (5'-PO4) side. Here we establish the minimal length requirements for ligatable 3'-OH and 5'-PO4 strands at the nick (6 nt) and describe a new crystal structure of the ligase-adenylate in a state construed to reflect the configuration of the active site prior to nick recognition. Comparison with a previous structure of the ligase-adenylate bound to sulfate (a mimetic of the nick 5'-PO4) suggests how the positions and contacts of the active site components and the bound adenylate are remodeled by DNA binding. We find that the minimal Chlorella virus ligase is capable of catalyzing non-homologous end-joining reactions in vivo in yeast, a process normally executed by the structurally more complex cellular Lig4 enzyme. Our results suggest a model of ligase evolution in which: (i) a small 'pluripotent' ligase is the progenitor of the much larger ligases found presently in eukaryotic cells and (ii) gene duplications, variations within the core ligase structure and the fusion of new domains to the core structure (affording new protein-protein interactions) led to the compartmentalization of eukaryotic ligase function, i.e. by enhancing some components of the functional repertoire of the ancestral ligase while disabling others.

Adenosine Monophosphate↗
PubMed1999

Recognition of target DNA and transcription activation by the CO-sensing transcriptional activator CooA.

CooA from Rhodospirillum rubrum is a heme-based CO-sensing transcriptional activator, in which CO acts as a physiological effector. In this study, we examined the mechanism of site-specific recognition and transcriptional activation by CooA by elucidating the transcriptional activator activity of the mutant CooA proteins and the chimeric proteins derived from CRP and CooA and the promoter activity of the mutant promoters. Site-directed mutagenesis has revealed that Arg(177), Gln(178), and Ser(181) on the recognition helix of the helix-turn-helix motif in CooA are responsible for the site-specific recognition. The side chains of these amino acid residues at positions 177, 178, and 181 are believed to be hydrogen bonding to the G:A, T:A, and C:G pairs at positions 2/15, 3/14, and 4/13 in the CooA-dependent promoters to recognize the DNA site for CooA. The properties of the CRP/CooA chimeric proteins constructed in this work suggest that CooA activates transcription by a similar mechanism to that of CRP at Class II CRP-dependent promoters.

Amino Acid Sequence↗
PubMed1997

Different DNA contact schemes are used by two winged helix proteins to recognize a DNA binding sequence.

The hepatocyte nuclear factor 3 (HNF-3)/fork head (fkh) family contains a large number of transcription factors which recognize divergent DNA sequences via a winged-helix binding motif. In this report we present studies on the DNA binding properties of winged-helix HNF-3/fkh homologues 1 (HFH-1) and 2 (HFH-2) which recognize a shared DNA binding site with different affinities. To explore how HFH-1 and HFH-2 proteins recognize this DNA binding sequence, the binding affinities of these two HFH proteins toward a series of DNA sites containing a single strand trimer loop insertion at different positions were studied. This insertion induces a bend of approximately 80 degrees in the DNA binding site (prebending). HFH-1 and HFH-2 were shown to recognize DNA sites prebent at many nucleotide positions on both strands of the DNA sequence. Both HFH-1 and HFH-2 were more sensitive to mismatch insertions on the sense strand of the DNA binding site, especially within the AAAATAAC sequence. Our data suggest that the recognition helix (helix 3) recognizes the AAAATAAC sequence and that the helix 3/DNA interaction results in bending of the DNA which narrows the major groove in the AAAATAAC sequence. Furthermore, the binding affinities of HFH-1 and HFH-2 toward DNA binding sites with base-pair reversion in the AAAATAAC sequence was also investigated. Different patterns of response from HFH-1 and HFH-2 to both prebent and base-pair reverted binding sites was observed. Our results demonstrate that even two highly conserved members of the winged-helix family may contact the same DNA sequence differently.

Binding Sites↗
PubMed1990

Phosphate-methylated DNA aimed at HIV-1 RNA loops and integrated DNA inhibits viral infectivity.

Phosphate-methylated DNA hybridizes strongly and specifically to natural DNA and RNA. Hybridization to single-stranded and double-stranded DNA leads to site-selective blocking of replication and transcription. Phosphate-methylated DNA was used to interrupt the life cycle of the human immunodeficiency virus type-1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). Both antisense and sense phosphate-methylated DNA 20-nucleotide oligomers, targeted at the transactivator responsive region and the primer binding site, caused complete inhibition of viral infectivity at a low concentration. Hybridization of phosphate-methylated DNA with folded and unfolded RNA was studied by ultraviolet and proton nuclear magnetic resonance spectroscopy. The combined results of hybridization studies and biological experiments suggest that the design of effective antisense phosphate-methylated DNA should focus on hairpin loop structures in the viral RNA. For sense systems, the 5' end of the integrated viral genome is considered to be the important target site.

Anticodon↗
PubMed2001

Calcium-regulated DNA binding and oligomerization of the neuronal calcium-sensing protein, calsenilin/DREAM/KChIP3.

Calsenilin/DREAM/KChIP3, a member of the recoverin branch of the EF-hand superfamily, interacts with presenilins, serves as a calcium-regulated transcriptional repressor, and interacts with A-type potassium channels. Here we report physicochemical characterization of calcium binding, oligomerization, and DNA binding of human calsenilin/DREAM/KChIP3. Equilibrium Ca(2+) binding measurements indicate that the protein binds 3 Ca(2+) with a dissociation constant of 14 microM and a Hill coefficient of 0.7. Dynamic light scattering and size exclusion chromatography show that the Ca(2+)-bound protein exists as a dimer at protein concentrations lower than 150 microM and forms a tetramer at concentrations above 200 microM. The Ca(2+)-free protein is a tetramer in the concentration range 20-450 microM. Isothermal titration calorimetry and dynamic light scattering indicate that the Ca(2+)-free protein tetramer binds endothermically (DeltaH = +25 kcal/mol) to four molecules of DNA derived from the downstream regulatory element (DRE) of either the prodynorphin or c-fos genes. One DRE molecule binds tightly to the protein with a dissociation constant (K(d)) of 75 nM, and the other three bind more weakly (K(d) = 640 nM). No significant DNA binding was observed for the Ca(2+)-bound protein. The N-terminal protein fragment (residues 1-70) binds nonspecifically to DRE in a Ca(2+)-independent manner, whereas a C-terminal fragment containing the four EF-hands (residues 65-256) binds DRE (K(d) = 200 nM) in a Ca(2+)-regulated and sequence-specific fashion. The C-terminal fragment is a tetramer in the Ca(2+)-free state and dissociates into dimers at saturating Ca(2+) levels.

Amino Acid Sequence↗
PubMed2006

Functional characterization of Drosophila Translin and Trax.

The vertebrate RNA and ssDNA-binding protein Translin has been suggested to function in a variety of cellular processes, including DNA damage response, RNA transport, and translational control. The Translin-associated factor X (Trax) interacts with Translin, and Trax protein stability depends on the presence of Translin. To determine the function of the Drosophila Translin and Trax, we generated a translin null mutant and isolated a trax nonsense mutation. translin and trax single and double mutants are viable, fertile, and phenotypically normal. Meiotic recombination rates and chromosome segregation are also not affected in translin and trax mutants. In addition, we found no evidence for an increased sensitivity for DNA double-strand damage in embryos and developing larvae. Together with the lack of evidence for their involvement in DNA double-strand break checkpoints, this argues against a critical role for Translin and Trax in sensing or repairing such DNA damage. However, Drosophila translin is essential for stabilizing the Translin interaction partner Trax, a function that is surprisingly conserved throughout evolution. Conversely, trax is not essential for Translin stability as trax mutants exhibit normal levels of Translin protein.

Amino Acid Sequence↗
PubMed2000

High-efficiency silencing of a beta-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation.

Two transgenic callus lines of rice, stably expressing a beta-glucuronidase (GUS) gene, were supertransformed with a set of constructs designed to silence the resident GUS gene. An inverted-repeat (i/r) GUS construct, designed to produce mRNA with self-complementarity, was much more effective than simple sense and antisense constructs at inducing silencing. Supertransforming rice calluses with a direct-repeat (d/r) construct, although not as effective as those with the i/r construct, was also substantially more effective in silencing the resident GUS gene than the simple sense and antisense constructs. DNA hybridisation analyses revealed that every callus line supertransformed with either simple sense or antisense constructs, and subsequently showing GUS silencing, had the silence-inducing transgenes integrated into the plant genome in inverted-repeat configurations. The silenced lines containing i/r and d/r constructs did not necessarily have inverted-repeat T-DNA insertions. There was significant methylation of the GUS sequences in most of the silenced lines but not in the unsilenced lines. However, demethylation treatment of silenced lines with 5-azacytidine did not reverse the post-transcriptional gene silencing (PTGS) of GUS. Whereas the levels of RNA specific to the resident GUS gene were uniformly low in the silenced lines, RNA specific to the inducer transgenes accumulated to a substantial level, and the majority of the i/r RNA was unpolyadenylated. Altogether, these results suggest that both sense- and antisense-mediated gene suppression share a similar molecular basis, that unpolyadenylated RNA plays an important role in PTGS, and that methylation is not essential for PTGS.

Azacitidine↗
PubMed1998

Antisense homology box-derived peptides represent a new class of endothelin receptor inhibitors.

Several peptides encoded by the sense and corresponding antisense DNA have been found to recognize and bind to each other. We developed software to search for sense-antisense regions within proteins taking into account the degeneracy of the genetic code, i.e., one amino acid can have several "antisense" counterparts. Using this approach, we searched endothelin receptor type A for intramolecular regions related in sense-antisense fashion. After locating these regions (termed "antisense homology boxes"), several corresponding peptides were synthesized. The four new ET(A) receptor fragment peptides ETR-P1 ("CALSVDRYRAVASW"), ETR-P3 ("QGIGPLITAIEI"), ETR-P4 ("IADNAERYSANLSSHV") and ETR-P6 ("LNRRNGSLRIALSEHLKNRREVA") reported here can inhibit ET-1 activity.

3T3 Cells↗
PubMed2025

Innate immune sensing and signaling: Co-opted for genome surveillance? Implications for tumorigenesis.

Innate immune signaling is traditionally associated with the response to pathogenic infection. However, emerging evidence suggests that nuclear innate immune sensors and their downstream pathways may also serve as a critical mechanism for genome surveillance. This review explores a model in which DNA sensors such as mouse IFI204 and IFI205 (IFI16 in humans) localize to replication forks, where they detect endogenous aberrant DNA structures and initiate an interferon-stimulated gene (ISG) transcriptional program. A key output of this transcriptional program is ISG15, which we find conjugated to fork-associated proteins and facilitates recruitment of the replication fork protection complex, thereby stabilizing replication forks under physiological conditions. We discuss how nuclear innate immune sensors mediate replication stress sensing and examine the broad consequences of downstream ISG transcription across diverse contexts-including its impact on genome stability and its dual roles in modulating tumor cell behavior and the tumor microenvironment. These findings suggest that the innate immune system, through its nuclear DNA sensing arm, may be evolutionarily co-opted for genome surveillance and may influence tumor initiation and therapy resistance. Understanding how innate immune signaling intersects with replication stress could offer mechanistic insights into tumor development and reveal novel therapeutic targets.

Humans↗
PubMed1988

Localization of vasopressin mRNA in the rat brain using in situ hybridization with short cRNA probes made from synthetic DNA templates.

Short antisense and sense RNA sequences were transcribed from annealed double stranded oligodeoxynucleotide templates containing the T7 RNA polymerase promotor sequence and its complementary sequence, together with 5' overhanging sequence corresponding to either the sense or antisense sequences of part of the rat vasopressin gene to produce after transcription 35S-labelled sense or antisense RNA for use in situ hybridization histochemistry. Labelled antisense RNA coding for a vasopressin sequence visualized sites of vasopressin mRNA expression (as did the 35S-labelled antisense oligodeoxynucleotide sequence), whereas sense RNA sequences revealed no specific sites of hybridization. This method represents an accessible, convenient and general method for the generation of cRNA probes suitable for use in situ hybridization.

Animals↗
PubMed2000

Hybridization assay at a disposable electrochemical biosensor for the attomole detection of amplified human cytomegalovirus DNA.

A disposable electrochemical biosensor for the detection of DNA sequences related to the human cytomegalovirus (HCMV) is described. The sensor relies on the adsorption of an amplified human cytomegalovirus DNA strand onto the sensing surface of a screen-printed carbon electrode, and to its hybridization to a complementary single-stranded biotinylated DNA probe. The extent of hybrids formed was determined with streptavidin conjugated to horseradish peroxidase. The peroxidase label was indirectly quantified by measuring the amount of the chromophore and electroactive product 2,2'-diaminoazobenzene generated from the o-phenylenediamine substrate. The intensity of differential pulse voltammetric peak currents resulting from the reduction of the enzyme-generated product was related to the number of target HCMV-amplified DNA molecules present in the sample, and the results were compared to those obtained with standard methods, i.e., agarose gel electrophoresis quantification and colorimetric hybridization assay in a microtiter plate. A detection limit of 0.6 amol/ml of HCMV-amplified DNA fragment was obtained with the electrochemical DNA biosensor. The electrochemical method was 23,000-fold more sensitive than the gel electrophoresis technique and 83-fold more sensitive than the colorimetric hybridization assay in a microtiter plate.

Biosensing Techniques↗
PubMed2003

Characterization of a series of far-red-absorbing thiobarbituric acid oxonol derivatives as fluorescent probes for biological applications.

Chromophores that absorb in the far-red region of the spectrum are increasingly being utilized for applications in the biosciences. We have synthesized and evaluated a novel series of fluorescent oxonols based on thiobarbituric acids containing aryl and heteroaryl substituents. The novel chromophores possess narrow absorption spectra ( approximately 40-nm bandwidths), reasonable Stokes shifts ( approximately 25 nm), and quantum yields of up to 0.67 in organic solvents and 0.3 in aqueous solvents, with absorption wavelength maxima at 620-640 nm. The spectral properties of the compounds are sensitive to base and exhibit a loss of far-red absorbance that is concentration and time dependent. Derivatives have been synthesized that can be used for the labeling of macromolecules such as proteins and DNA. The probes show environment sensitivity and the oligonucleotide conjugates sense the formation of duplex DNA. These novel far-red fluorophores have potential applications in diagnostic and research applications.

Amino Acid Sequence↗
PubMed1990

A comparison of DNA adduct formation in white blood cells and internal organs of mice exposed to benzo[a]pyrene, dibenzo[c,g]carbazole, safrole and cigarette smoke condensate.

Measurement of tissue/cell DNA adducts represents a suitable monitor of carcinogen exposure because the majority of chemical mutagens/carcinogens react with DNA, forming covalent adducts, a key event in the initiation of chemical carcinogenesis. Investigations of DNA-adduct formation in vivo in white blood cells (WBC) versus target tissues, i.e. internal organs for most carcinogens, is expected to yield useful information about the suitability of WBC for biomonitoring and risk assessment. For this purpose, female ICR mice were given 0.4 mmole/kg benzo[a]pyrene (BP), 0.045 mmole/kg dibenzo[c,g]carbazole (DBC) or 2.47 mmole/kg safrole by oral gavage or 4 daily doses (equivalent to 3 cigarettes) of cigarette-smoke condensate (CSC) by topical application. At 24 h after dosing, DNA adducts were detected by a nuclease P1-enhanced 32P-postlabeling assay [M.V. Reddy and K. Randerath, Carcinogenesis, 7 (1986) 1543] in WBC and internal tissues treated with individual carcinogens, while CSC treatment elicited aromatic adducts in most tissues but not in WBC. Adduct patterns of WBC DNA were qualitatively similar to those of internal organs, but adduct amounts varied. BP, a systemic carcinogen, bound nearly as much to WBC DNA as to target-tissue DNA samples; whereas the liver carcinogens, DBC and safrole, bound to WBC DNA considerably less (22- and 51-fold, respectively) compared with liver DNA. The number of adducts in 10(7) nucleotides of WBC, liver, lung, kidney and spleen DNA, respectively, were: 2, 5, 3, 2 and 3 with BP; 6, 131, 6, 14 and 4 with DBC; 5, 238, 3, 5 and 0.6 with safrole. For CSC, these values were 0, 1 and 0.02 in WBC, lung and spleen, respectively. Our results show that carcinogen binding to WBC DNA does not reflect binding to target-tissue DNA in a quantitative sense for the carcinogens studied except for BP, and that WBC are not suitable surrogates for monitoring CSC exposure by DNA-adduct measurement after topical application. The CSC data in mice was consistent with the previous findings in humans that smokers' tissues but not WBC show smoking-related bulky/aromatic DNA adducts, as measured by 32P-postlabeling.

Animals↗
PubMed2000

Synthesis of virus-specific high-mobility DNA after temperature upshift of SC-1 cells chronically infected with moloney murine leukemia virus mutant ts1.

Premature termination products of reverse transcription that consist physically of viral minus-sense single-stranded DNA that is shorter than one long terminal repeat and partial DNA duplexes are dramatically increased in the central nervous system (CNS) of FVB/N mice that are infected by ts1, a temperature-sensitive mutant of Moloney murine leukemia virus. Due to their migration in agarose gels, these incomplete physical forms of DNA have been designated high-mobility (HM) DNA. In non-CNS tissues, the level of HM DNA is either low or not detectable. In order to determine the conditions that are necessary for the synthesis of HM DNA in vivo, we have characterized the physical forms of HM DNA that were synthesized in vitro in chronically infected SC-1 cells after temperature upshift. At the permissive temperature of 34 degrees C, the chronically infected SC-1 cells did not synthesize HM DNA. After temperature upshift of the cultured cells from 34 to 37 degrees C, the chronically infected SC-1 cells developed extremely high levels of HM DNA. Following temperature downshift of the cultured cells from 37 to 34 degrees C, a decrease in the level of HM DNA and an increase in the level of unintegrated linear proviral DNA occurred simultaneously. These results suggested that the accumulation of HM DNA both in vitro and in vivo may be the result of superinfection.

Animals↗