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At least 127 records · Page 7Linked to original sources
PubMed2004

Nucleosomal locations of dominant DNA sequence motifs for histone-DNA interactions and nucleosome positioning.

DNA sequence is an important determinant of the positioning, stability, and activity of nucleosomes, yet the molecular basis of these effects remains elusive. A "consensus DNA sequence" for nucleosome positioning has not been reported and, while certain DNA sequence preferences or motifs for nucleosome positioning have been discovered, how they function is not known. Here, we report that an unexpected observation concerning the reassembly of nucleosomes during salt gradient dialysis has allowed a breakthrough in our efforts to identify the nucleosomal locations of the DNA sequence motifs that dominate histone-DNA interactions and nucleosome positioning. We conclude that a previous selection experiment for high-affinity, nucleosome-forming DNA sequences exerted selective pressure chiefly on the central stretch of the nucleosomal DNA. This observation implies that algorithms for aligning the selected DNA sequences should seek to optimize the alignment over much less than the full 147 bp of nucleosomal DNA. A new alignment calculation implemented these ideas and successfully aligned 19 of the 41 sequences in a non-redundant database of selected high-affinity, nucleosome-positioning sequences. The resulting alignment reveals strong conservation of several stretches within a central 71 bp of the nucleosomal DNA. The alignment further reveals an inherent palindromic symmetry in the selected DNAs; it makes testable predictions of nucleosome positioning on the aligned sequences and for the creation of new positioning sequences, both of which are upheld experimentally; and it suggests new signals that may be important in translational nucleosome positioning.

Algorithms↗
PubMed2002

Radiation-induced DNA breaks in different human satellite DNA sequence areas, analyzed by DNA breakage detection-fluorescence in situ hybridization.

Human blood leukocytes were exposed to X rays to analyze the initial level of DNA breakage induced within different satellite DNA sequence areas and telomeres, using the DNA breakage detection-FISH procedure. The satellite DNA families analyzed comprised alphoid sequences, satellite 1, and 5-bp classical satellite DNA sequences from chromosome 1 (D1Z1 locus), from chromosome 9 (D9Z3 locus), and from the Y chromosome (DYZ1 locus). Since the control hybridization signal was quite different in each of the DNA targets, the relative increase in whole fluorescence intensity with respect to unirradiated controls was the parameter used for comparison. Irradiation of nucleoids obtained after protein removal demonstrated that the alkaline unwinding solution generates around half the amount of signal when breaks are present in the 5-bp classical DNA satellites as when the same numbers of breaks are present the genome overall, whereas the signal is slightly stronger when the breaks are within the alphoids or satellite 1 sequences. After correction for differences in sensitivity to the alkaline unwinding-renaturation, DNA housed in chromatin corresponding to 5-bp classical satellites proved to be more sensitive to breakage than the overall genome, whereas DNA in the chromatin corresponding to alphoids or satellite 1 showed a sensitivity similar to that of the whole genome. The minimum detectable dose was 0.1 Gy for the whole genome, 0.2 Gy for alphoids and satellite 1, and 0.4 Gy for the 5-bp classical satellites. Telomeric DNA sequences appeared to be maximally labeled in unirradiated cells. Thus telomeric ends behave like DNA breaks, constituting a source of background in alkaline unwinding assays.

Chromatin↗
PubMed2004

Interactive DNA sequence and structure design for DNA nanoapplications.

DNA sequence and structure design is very important for DNA nanoapplications. A computer-aided design tool is needed for exploring DNA sequence and structure of interests before experimental synthesis, which is a time- and labor-consuming process. In this paper, an interactive DNA sequence and structure design software tool called DNA shop is proposed and implemented. The visualization tool can generate DNA structures by specifying, selecting, and moving DNA sequences around and display corresponding structures. Using the tool, DNA sequence and structure can be visually inspected in three-dimensional space before experimental studies.

Algorithms↗
PubMed1988

Continuous, on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores.

A method for sequencing DNA by using a difluoresceinated primer and laser excitation is described. Dideoxy protocols have been determined that provide sequences for 600 bases starting with base 1 with less than 1% error in a single load. Electrophoresis is at 20 W and the bands are detected 24 cm from the bottom of the loading well with a scanning fluorescence detector. Bands are imaged on a TV screen in two dimensions. The sequences can be read from the TV screen manually or semiautomatically by using a simple software program. The system allows more bases to be read with a lower error rate than any other reported automated sequencing method.

Animals↗
PubMed1984

Deletion of DNA sequence in a nononcogenic variant of Herpesvirus saimiri.

The 110-kilobase-pair stretch of unique sequence DNA of Herpesvirus saimiri is flanked by highly repetitive DNA. Detailed restriction endonuclease mapping has localized the left junction of repetitive and unique DNA to a 100-base-pair region. H. saimiri 11att, a replication competent nononcogenic variant of strain 11, has a deletion of 2.3 kilobase pairs of sequence information that spans this left junction of repetitive and unique DNA.

Animals↗
PubMed2003

A comparison of intraspecific patterns of DNA sequence variation in mitochondrial DNA, alpha-enolase, and MHC class II B loci in auklets (Charadriiformes: Alcidae).

Patterns of DNA sequence variation can be used to learn about mechanisms of organismal evolution, but only if mechanisms of sequence evolution are well understood. Although theories of molecular evolution are well developed, few empirical studies have addressed patterns and mechanisms of sequence evolution in nuclear genes within species. In the present study, we compared DNA sequences among three loci with different evolutionary constraints to determine the influences of effective population size, balancing selection, and linkage on intraspecific patterns of sequence variation. Specifically, we assessed the degree and nature of polymorphism in a 307-base pair (bp) fragment of the mitochondrial cytochrome b gene, intron VIII of the gene for alpha-enolase (a presumably neutral nuclear gene), and an approximately 600-bp fragment of an MHC class II B gene, including 155 bp of the hypervariable peptide binding region (a nuclear locus thought to be under balancing selection) for least and crested auklets (Aethia pusilla and A. cristatella; Charadriiformes: Alcidae). Transspecies polymorphism was found in both alpha-enolase and the MHC but not cytochrome b and, given estimates of effective population size, probably represents retained ancestral variation. Biases in nucleotide composition suggested that mutational bias, tRNA availability, and the secondary structure of mRNA and/or DNA may influence base usage. Several lines of evidence indicated that balancing selection may be acting on the MHC II B exon 2. However, no evidence of balancing selection was observed in the intron and exon sequences immediately downstream of MHC II B exon 2.

Animals↗
PubMed1980

A short primer for sequencing DNA cloned in the single-stranded phage vector M13mp2.

In this paper we describe the synthesis and cloning of a short segment of DNA complementary to the region immediately adjacent to the EcoRI insertion site in the single-stranded bacteriophage vector M13mp2. This segment is useful as a "universal" primer for DNA sequencing by the dideoxynucleotide chain termination method; the template can be any DNA species cloned in M13mp2 or its derivatives. The primer has been cloned into the tetracycline resistance gene of plasmid pBR322 as one strand of a 26 bp EcoRI/BamHI fragment. This fragment may be readily prepared from an EcoRI + BamHI restriction digest of the parent plasmid (designated pSP14) by a simple size fractionation.

Base Sequence↗
PubMed1985

A simple and rapid method for sequencing DNA.

A simplified technique for DNA sequence analysis has been developed, based on modification of a previous method [(1980) Methods Enzymol. 65, 499-560]. It employs an adsorptive immobilization of terminally labelled DNA on DEAE paper followed by G, A+G, C+T and C specific modification and cleavage reactions. This solid-phase technique is faster and more convenient than the original method. The efficiency is comparable. The total processing time taken to produce cleaved fragments loaded on a gel is less than 2 h.

Autoradiography↗
PubMed2006

Pattern locator: a new tool for finding local sequence patterns in genomic DNA sequences.

UNLABELLED: We present a new tool for finding local sequence patterns in long DNA sequences. The program, Pattern Locator, uses an intuitive syntax for pattern description, and provides more flexibility than existing programs by allowing combinations of specific nucleotide sequences, direct and inverted repeats, variable length tandem repeats of subpatterns, and a specified number of errors in any part of the pattern. AVAILABILITY: The program is available for download and as a web service accessible through a CGI interface at http://www.cmbl.uga.edu/software.html. The source code is written in C and distributed under the GNU General Public License.

Algorithms↗
PubMed1997

Rescuing corrupted gel files from Model 377 and 373 DNA Sequencers.

Automated DNA sequencing requires the intensive use of computers to handle the large amount of data taken. When a computer failure occurs and the data are no longer accessible, all the expense and effort that went into the sequencing experiment is lost. By using the data storage architecture of Macintosh computers to our advantage, we may prevent this loss in the case of automatic sequencers from PE Applied Biosystems. The software required to allow the experimenter to do this has been written and is available free of charge.

Automation↗
PubMed2002

DNA sequence variants in epithelium-specific ETS-2 and ETS-3 are not associated with asthma.

Epithelium-specific ETS-2 and ETS-3 are transcription factors that have been proposed as asthma candidate genes. To investigate the association of sequence variants in these genes with asthma, we conducted a case-control association analysis in a sample of 311 white subjects with asthma and 177 white subjects without asthma. Common polymorphisms in these genes were detected by sequencing DNA from 32 cell lines obtained from Coriel (Camden, NJ). Seven noncoding or synonymous single-nucleotide polymorphisms were detected: three in epithelium-specific ETS-2 and four in epithelium-specific ETS-3. Subjects were genotyped at all loci by mass spectroscopy. To ensure the suitability of our control subjects, we also genotyped subjects at 49 unlinked polymorphisms evenly distributed throughout the autosomes and found no evidence of population stratification. Logistic regression adjusted for age and sex suggested a weak association of one epithelium-specific ETS-2 polymorphism with asthma diagnosis (odds ratio = 1.89, 95% confidence interval = 1.13-3.18, p = 0.02). Total serum immunoglobulin E and FEV1 predicted levels were not associated with any of the polymorphisms. Extended haplotyping indicated linkage disequilibrium in these genes; however, no association or epistatic interaction was found. This study suggests that epithelium-specific ETS-2 and ETS-3 genes are unlikely to contain polymorphic loci that have a major impact on asthma susceptibility in our population.

Adolescent↗
PubMed1994

Error-prone replication of repeated DNA sequences by T7 DNA polymerase in the absence of its processivity subunit.

We have examined the effect of thioredoxin, an accessory protein that confers high processivity to bacteriophage T7 DNA polymerase, on the fidelity of DNA synthesis. In the presence of thioredoxin, exonuclease-proficient T7 DNA polymerase is highly accurate. In fidelity assays that score errors that revert M13mp2 lacZ alpha-complementation mutants, error rates are < or = 2.2 x 10(-6) for base substitution and < or = 3.7 x 10(-7) and < or = 4.5 x 10(-7) for frameshifts that revert mutations in the +1 and -1 reading frames, respectively. Rates are more than 10-fold higher during synthesis by polymerase.thioredoxin complex lacking 3'-->5' exonuclease activity, demonstrating that frameshift as well as substitution errors are subject to proofreading. The contribution of thioredoxin to accuracy has been examined by comparing the fidelity of the exonuclease-deficient polymerase in the presence or absence of the accessory protein. Thioredoxin either enhances or reduces fidelity, depending on the type of error considered. In the absence of thioredoxin, T7 DNA polymerase is 3-fold more accurate for base substitutions and > or = 27-fold and 9-fold more accurate, respectively, for 1- and 2-nt deletion errors at nonreiterated nucleotide sequences. Higher fidelity for all three errors may reflect the inability of the polymerase to continue synthesis from the premutational intermediates in the absence of the accessory protein. In marked contrast, the rate for frameshift errors wherein one or more nucleotides has been added to a repeated DNA sequence increases 46-fold when thioredoxin is absent from the polymerization reaction. The error rate increases as the length of the repeated sequence increases, consistent with a model where strand slippage creates misaligned template-primers. Thus, replicative expansion of repetitive sequences occurs in the absence of a replication accessory protein.

Base Sequence↗
PubMed1979

A rapid screening for the specific DNA sequence: analysis of transforming DNA segments in adenovirus-transformed cells.

The viral DNA sequences in cells transformed by adenovirus type-12 (Ad12) DNA fragments were investigated by spot hybridization, a detection by autoradiography of nucleic acid hybrids formed between cell DNAs spotted on a membrane filter and various nick-translated Ad12 DNA fragments. In CY1 cell line, a rat cell line transformed by the EcoRI-C fragment (left hand 16%), all of the HindIII fragments included in the EcoRI-C fragment are shown to be present. In GY1 cell line, a rat cell line transformed by the HindIII-G fragment (left hand 7%), both of the BpaI-H and a part of the BpaI-J, two components consisting of the HindIII-G fragment, were found. A dominant presence of the Ad12 BpaI-H fragment (left hand end 4.5%) of the Ad12 DNA molecule, approximately 60% of the Ad12 transforming DNA sequences (Ad12 HindIII-G), was also shown in GY1. Spot hybridization used in the experiment will be of general use for detecting viral nucleic acid sequences in cells and provides a simple and useful screening method for investigating viral etiology of tumors and transformed cells.

Adenoviruses, Human↗
PubMed1996

The limiting mobility of DNA sequencing fragments for both cross-linked and noncross-linked polymers in capillary electrophoresis: DNA sequencing at 1200 V cm-1.

The mobility of DNA sequencing fragments was measured in Long-Ranger gels at an electric field ranging from 200 to 1200 V cm-1 and in noncross-linked polyacrylamide at electric fields ranging from 100 to 300 V cm-1. In both cases, N*, the fragment length that denotes the onset of biased reptation with orientation, is inversely proportional to electric field. The inverse dependence of N* is inconsistent with the original biased reptation model but is consistent with modern models of DNA migration. While separation speed increases dramatically with electric field, the number of bases determined in a separation decreases in proportion to field strength. We present a DNA sequencing run at an electric field of 1200 V cm-1. Roughly 200 bases of sequence are determined in 3.5 min.

Base Sequence↗
PubMed1997

DNA sequencing and comparative sequence analysis reveal that the Escherichia coli genomic DNA may replace the target DNA during molecular cloning: evidence for the erroneous assembly of E. coli DNA into database sequences.

DNA sequencing and similarity search of databases provide experimental evidence that portions of the host Escherichia coli genome may get ligated into the cloning vector, resulting in clones containing nontargeted inserts. Several lines of evidence suggest that this non-targeted ligation, as observed by us while subcloning troponin I cDNA, is presumably due to a recombination-mediated mechanism by which host DNA replaces the target DNA in the cloning vector. The E. coli genome mapping to 64-65 min and 92.8-00.1 min, the latter containing insertion sequences, appears to be the hotspot regions involved in this process. We examined the possibility that some sequences reported in the databases may also contain genomic sequences of E. coli. A search of current databases revealed that a rat hepatic glutathione transporter cDNA contains a 2.2-kb-long portion of the E. coli genome that has been wrongly assembled into its 5' untranslated and coding regions. In addition, about 30 sequences in databases, including a Yersinia pestis toxin gene, showed relatively high sequence identity with those portions of the E. coli genome that were present in the nonauthentic clones.

Animals↗
PubMed1984

Location of DNA sequences complementary to small nuclear repeat RNA (fr 3-RNA) in relation to DNA sequences coding for albumin and alpha-fetoprotein in rat liver cells.

Rat liver nuclei contain a 29-nucleotides-long RNA (fr 3-RNA) which is transcribed from middle repetitive DNA sequences. By Southern analysis of restriction fragments of rat albumin and alpha-fetoprotein genomic clones, DNA sequences complementary to this RNA were detected on a 4.6 kbp Eco RI fragment located 600 bp downstream from the termination exon of the albumin gene and on a 2 kbp Eco RI-HindIII fragment located 10 kbp downstream from the restriction fragment containing the alpha-fetoprotein site. No sequence complementary to this RNA was found either in the introns of exons of both genes or in the regions extending 7 kbp upstream from the first albumin exon and 10 kbp upstream of the first alpha-fetoprotein exon. We concluded that sequences complementary to fr 3-RNA are present at the 3'-end flanking regions of the rat albumin and alpha-fetoprotein gene complexes.

Animals↗
PubMed2005

Preferential binding and structural distortion by Fe2+ at RGGG-containing DNA sequences correlates with enhanced oxidative cleavage at such sequences.

Certain DNA sequences are known to be unusually sensitive to nicking via the Fe2+-mediated Fenton reaction. Most notable are a purine nucleotide followed by three or more G residues, RGGG, and purine nucleotides flanking a TG combination, RTGR. Our laboratory previously demonstrated that nicking in the RGGG sequences occurs preferentially 5' to a G residue with the nicking probability decreasing from the 5' to 3'end of these sequences. Using 1H NMR to characterize Fe2+ binding within the duplex CGAGTTAGGGTAGC/GCTACCCTAACTCG and 7-deazaguanine-containing (Z) variants of it, we show that Fe2+ binds preferentially at the GGG sequence, most strongly towards its 5' end. Substitutions of individual guanines with Z indicate that the high affinity Fe2+ binding at AGGG involves two adjacent guanine N7 moieties. Binding is accompanied by large changes in specific imino, aromatic and methyl proton chemical shifts, indicating that a locally distorted structure forms at the binding site that affects the conformation of the two base pairs 3' to the GGG sequence. The binding of Fe2+ to RGGG contrasts with that previously observed for the RTGR sequence, which binds Fe2+ with negligible structural rearrangements.

Base Sequence↗
PubMed1998

Evolution of the gibbon subgenera inferred from cytochrome b DNA sequence data.

DNA sequences for the mitochondrial cytochrome b gene from the four extant gibbon subgenera are described. The data confirm that the gibbon subgenera evolved from a common hylobatid ancestor and suggest that they diverged from each other after the divergence of the extant African great ape species. The cytochrome b gene does not resolve the evolutionary relationships between the gibbon subgenera themselves.

Animals↗