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At least 451 records · Page 25Linked to original sources
PubMed1993

Effect of salt concentration on TTF-1 HD binding to specific and non-specific DNA sequences.

The Thyroid Transcription factor 1 (TTF-1) recognizes specific DNA sequences by a Homeodomain (TTF-1 HD). The TTF-1 HD DNA-binding properties with both specific and non-specific DNA sequences were investigated. TTF-1 HD exists as a monomer in solution and as a monomer binds DNA. At 75 mM KCl, its relative binding affinity with a specific DNA sequence is about 50 fold higher than with a non-specific DNA sequence. Increase of KCl concentration reduces the apparent binding affinity both to specific and non-specific DNA sequences. However, non-specific binding is more sensitive than specific binding to the increase of salt concentration. When DNA-binding reactions are performed at temperature and salt concentration close to the intracellular environment, TTF-1 HD binds the specific sequence with an affinity at least 1000 fold higher respect to the non-specific sequence.

Animals↗
PubMed1994

DNA amplification by the polymerase chain reaction for the rapid diagnosis of tuberculous meningitis. Comparison of protocols involving three mycobacterial DNA sequences, IS6110, 65 kDa antigen, and MPB64.

DNA amplification of three Mycobacterium tuberculosis-specific DNA sequences by the polymerase chain reaction (PCR) were evaluated as a means for rapid diagnosis of tuberculous meningitis (TBM). The DNA sequences amplified were a 123 bp region of the IS6110 insertion elements which occur in multiple copies in the mycobacterial genome, a 240 bp region (nts 460-700) from the MPB 64 protein coding gene, and the 383 bp region of the 65 kDa heat shock protein (HSP) antigen. Twenty-seven cerebrospinal fluid (CSF) specimens were studied. Six were obtained from patients with TBM diagnosed by culture (4/6) or by the patients' response to anti-tuberculous therapy (2/6). The remaining 21 specimens were obtained from patients with febrile seizures (3/21), aseptic meningitis (3/21), septic meningitis (14/21), and cryptococcal meningitis (1/21), and these served as negative controls. Our results indicate that although the protocols involving the 3 DNA sequences were able to detect TB DNA in the 6 TBM specimens, the main drawback was their extreme sensitivity, thus giving rise to false positive results. In particular, the repeat copy sequence, IS6110, and the 65 kDa HSP gave unacceptably large numbers of false positive results (62% and 33%, respectively).

Antigens, Bacterial↗
PubMed2005

Identifying nonrandom occurrences of simple sequence repeats in genomic DNA sequences.

Numerous disorders, including prostate cancer and muscular dystrophy, have been associated with nonrandom occurrences of certain simple sequence repeats (SSRs) found in genomic DNA. In a previous paper, we introduced probabilistic methods for identifying such SSRs that possess nonrandom distribution profiles. Here, we apply these methods to the distribution profiles of SSRs of monomers, dimers, trimers, and tetramers occurring in the human genomic sequence data. In particular, we show that the nonrandomness of SSRs is an exponential function of SSR length. We also demonstrate the existence of threshold SSR lengths for the significant nonrandomness (specifically, under/over-representation) of SSRs. These results are consistent with previous findings and demonstrate the potential use of the previously derived probabilistic methods in the identification of (putative disease associated) SSRs that exhibit significant deviations from random expectations.

Base Sequence↗
PubMed1989

Detection of specific DNA sequences using antibodies recognizing UV-labelled DNA.

This non-isotopic method for detection of nucleic acids is based on the in situ labelling of the nucleic acid by exposure to UV-irradiation. The different UV-induced photoproducts, mainly of the thymidine dimer type, are recognized by purified rabbit antibodies specific to the lesions introduced. The UV-labelled nucleic acids can then be visualized by conventional immunostaining procedures. A major advantage of the technique is the low cost and the ease by which the DNA is specifically labelled. The purified rabbit antibodies were shown to be specific for UV-irradiated DNA, and the method was applied for detection of specific DNA sequences hybridized to homologous target DNA on membrane support. We believe that the sensitivity of the method can be improved, and the significance of using different UV-doses, immunostaining methods and membrane types is discussed.

Animals↗
PubMed2004

A Bayesian approach to DNA sequence segmentation.

Many deoxyribonucleic acid (DNA) sequences display compositional heterogeneity in the form of segments of similar structure. This article describes a Bayesian method that identifies such segments by using a Markov chain governed by a hidden Markov model. Markov chain Monte Carlo (MCMC) techniques are employed to compute all posterior quantities of interest and, in particular, allow inferences to be made regarding the number of segment types and the order of Markov dependence in the DNA sequence. The method is applied to the segmentation of the bacteriophage lambda genome, a common benchmark sequence used for the comparison of statistical segmentation algorithms.

Algorithms↗
PubMed1999

Optimal structure for automatic processing of DNA sequences.

The faithful recovery of the base sequence in automatic DeoxyriboNucleic Acid (DNA) sequencing fundamentally depends on the underlying statistics of the DNA electrophoresis time series. Current DNA sequencing algorithms are heuristic in nature and modest in their use of statistical information. In this paper, a formal statistical model of the DNA time series is presented and then used to construct the optimal maximum-likelihood (ML) processor. The DNA-ML algorithm that is derived in this paper features Kalman prediction of peak locations, peak parameter estimation, whitened waveform comparison and multiple hypothesis processing using the M-algorithm. Properties of the algorithm are examined using both simulated and real data. Model parameters of critical importance and their impact on different types of error mechanisms, such as insertions and deletions, are pointed out. The statistical model of the DNA time-series and the structure of the DNA-ML algorithm provides a basis for future investigation and refinement of DNA sequencing techniques.

Algorithms↗
PubMed1996

High sensitivity of the single-strand conformation polymorphism method for detecting sequence variations in the low-density lipoprotein receptor gene validated by DNA sequencing.

We designed oligonucleotide primer pairs to amplify the promoter region, the translated exon sequences, and the flanking intron sequences of all 18 exons of the LDL receptor gene to compare the ability of the PCR single-strand conformation polymorphism (PCR-SSCP) method with semiautomated solid-phase genomic DNA sequencing to detect sequence variations. In 20 apparently unrelated Danish patients with a clinical diagnosis of heterozygous familial hypercholesterolemia (FH), we identified 13 different mutations in the LDL receptor gene: two silent (C331C, N494 N); five missense (W66G, E119K, T383P, W556S, T7051); one nonsense (W23X); three splice-site (313 + 1G-->A, 1061-8T-->C, 1846-1G-->A); and two frameshift (335del10, 1650delG) mutations. Four of these mutations, N494 N, T383P, 1061-8T-->C, and W556S, have not been reported earlier. The pathogenicity of the T383P, 1061-8T-->C, and W556S mutations remains to be established by in vitro mutagenesis and transfection studies. One patient had three mutations (335del10, 1061-8T-->C, and T705I) on the same allele. Further, nine well-known polymorphisms were detectable with this methodological setup. Direct DNA sequencing of the PCR products used for the SSCP analysis did not reveal any sequence variations not detected by the PCR-SSCP method. In two patients we did not detect any mutation by either method. We conclude that the PCR-SSCP analysis, performed as described here, is as sensitive and efficient as DNA sequencing in the ability to identify the sequence variations in the LDL receptor gene of the patients with heterozygous FH of this study.

Base Sequence↗
PubMed1987

Two computer programs for rapid entry of DNA sequence data.

Two computer programs for the IBM personal computer are described for rapid and accurate entry of DNA sequence data. The DNA sequence files produced can be used directly by the DNA sequence manipulation programs by R. Staden (the DataBase system), the University of Wisconsin Genetics Computer Group, DNASTAR, or D.Mount. The first program, DIGISEQ, utilizes a sonic digitizer for semi-automation of sequence entry. To enter the DNA sequence each band of a gel reading is touched by the stylus of the sonic digitizer. DIGISEQ corrects for both changes in lane width and lane curvature. The algorithm is extremely efficient and rarely requires re-entering the centers of the lanes. The second program, TYPESEQ, uses only the keyboard for input. The keyboard is reconfigured to place nucleotides and ambiguity codes under the fingers of one hand, corresponding to the order of the nucleotides on the gel defined by the user. Both programs produce individual tones for each nucleotide, and certain ambiguity codes. This verifies input of the correct nucleotide or ambiguity code, and thus eliminates the need to visually check the screen display during sequence entry.

Base Sequence↗
PubMed1995

Recognition and alignment of homologous DNA sequences between minichromosomes and single-stranded DNA promoted by RecA protein.

The incorporation of DNA into nucleosomes and higher-order forms of chromatin in vivo creates difficulties with respect to its accessibility for cellular functions such as transcription, replication, repair and recombination. To understand the role of chromatin structure in the process of homologous recombination, we have studied the interaction of nucleoprotein filaments, comprised of RecA protein and ssDNA, with minichromosomes. Using this paradigm, we have addressed how chromatin structure affects the search for homologous DNA sequences, and attempted to distinguish between two mutually exclusive models of DNA-DNA pairing mechanisms. Paradoxically, we found that the search for homologous sequences, as monitored by unwinding of homologous or heterologous duplex DNA, was facilitated by nucleosomes, with no discernible effect on homologous pairing. More importantly, unwinding of minichromosomes required the interaction of nucleoprotein filaments and led to the accumulation of circular duplex DNA sensitive to nuclease P1. Competition experiments indicated that chromatin templates and naked DNA served as equally efficient targets for homologous pairing. These and other findings suggest that nucleosomes do not impede but rather facilitate the search for homologous sequences and establish, in accordance with one proposed model, that unwinding of duplex DNA precedes alignment of homologous sequences at the level of chromatin. The potential application of this model to investigate the role of chromosomal proteins in the alignment of homologous sequences in the context of cellular recombination is considered.

Adenosine Diphosphate↗
PubMed1984

Localization and sequence analysis of chloroplast DNA sequences of Chlamydomonas reinhardii that promote autonomous replication in yeast.

Four distinct chloroplast DNA segments from Chlamydomonas reinhardii of 400, 415, 730 and 2300 bp which promote autonomous replication in yeast have been mapped on the chloroplast genome. Plasmids carrying these chloroplast DNA fragments are unstable in yeast when the cells are grown under non-selective conditions. Sequence analysis of three of these chloroplast ARS regions (autonomously replicating sequences in yeast) reveals a high AT content, numerous short direct and inverted repeats and the presence of at least one element in each region that is related to the yeast ARS consensus sequence. A/T TTTATPuTTT A/T. These three chloroplast regions share, in addition, two common elements of 10 and 11 bp which may play a role in promoting autonomous replication.

Journal Article↗
PubMed2000

Detection of mutation of the p53 gene with high sensitivity by fluorescence-based PCR-SSCP analysis using low-pH buffer and an automated DNA sequencer in a large number of DNA samples.

Detection of mutations in genes responsible for hereditary diseases or tumors is important clinically. It is necessary to establish a simple technique for screening mutations in large numbers of samples. The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method has proved to be a useful technique for analyzing mutations or DNA polymorphisms. Non-radioisotopic versions using fluorescent dye and an automated DNA sequencer have also been exploited to extend this technique into the clinical field. We have examined mutations of exons 5-9 of the p53 gene in 112 colorectal, 28 esophageal and 33 hepatocellular carcinomas by fluorescence-based PCR-SSCP (F-SSCP) under various conditions. We found 64 types of mutations in 63, 17 and 12 cases of colon, esophageal and hepatocellular carcinomas by F-SSCP. We determined the sequence of all samples, and confirmed that all mutations were successfully detected by F-SSCP. With the low-pH buffer system, 61 types of mutants were detected, while 51 types were detected by TBE and 57 types were detected by TBE with glycerol gel. The polyacrylamide gel in TME or TBE without glycerol was tough and could be used repeatedly, but the glycerol containing gel was fragile and could not stand repeated use. Thus, use of a low-pH buffer in the electrophoresis of F-SSCP is simpler and better at detecting mutations than the conventional TBE buffer system. We believe that low-pH F-SSCP analysis is an efficient and powerful technique for examination of a large number of samples, in particular clinical specimens obtained by biopsy or surgery.

DNA Mutational Analysis↗
PubMed2005

Optimal word sizes for dissimilarity measures and estimation of the degree of dissimilarity between DNA sequences.

MOTIVATION: Several measures of DNA sequence dissimilarity have been developed. The purpose of this paper is 3-fold. Firstly, we compare the performance of several word-based or alignment-based methods. Secondly, we give a general guideline for choosing the window size and determining the optimal word sizes for several word-based measures at different window sizes. Thirdly, we use a large-scale simulation method to simulate data from the distribution of SK-LD (symmetric Kullback-Leibler discrepancy). These simulated data can be used to estimate the degree of dissimilarity beta between any pair of DNA sequences. RESULTS: Our study shows (1) for whole sequence similiarity/dissimilarity identification the window size taken should be as large as possible, but probably not >3000, as restricted by CPU time in practice, (2) for each measure the optimal word size increases with window size, (3) when the optimal word size is used, SK-LD performance is superior in both simulation and real data analysis, (4) the estimate beta of beta based on SK-LD can be used to filter out quickly a large number of dissimilar sequences and speed alignment-based database search for similar sequences and (5) beta is also applicable in local similarity comparison situations. For example, it can help in selecting oligo probes with high specificity and, therefore, has potential in probe design for microarrays. AVAILABILITY: The algorithm SK-LD, estimate beta and simulation software are implemented in MATLAB code, and are available at http://www.stat.ncku.edu.tw/tjwu

Algorithms↗
PubMed1989

SDSE: a software package to simulate the evolution of a pair of DNA sequences.

An algorithm to simulate DNA sequence evolution under a general stochastic model, including as particular cases all the previously used schemes of nucleotide substitution, is described. The stimulation is carried out on finite, variable length, DNA sequences through a strict stochastic process, according to the particular substitution rates imposed by each scheme. Five FORTRAN programs, running on an IBM PC and compatibles, carry out all the tasks needed for the simulation. They are menu driven and interfaced to the system through a principal menu. All sequence data files used and generated by the SDSE package conform to the standard GenBank database format, thus allowing the use of any sequence retrieved from this databank, as well as the application of other packages to analyse, manipulate or retrieve stimulated sequences.

Algorithms↗
PubMed2004

Yeast DNA sequences initiating gene expression in Escherichia coli.

DNA transfer between pro- and eukaryotes occurs either during natural horizontal gene transfer or as a result of the employment of gene technology. We analysed the capacity of DNA sequences from a eukaryotic donor organism (Saccharomyces cerevisiae) to serve as promoter region in a prokaryotic recipient (Escherichia coli) by creating fusions between promoterless luxAB genes from Vibrio harveyi and random DNA sequences from S. cerevisiae and measuring the luminescence of transformed E. coli. Fifty-four out of 100 randomly analysed S. cerevisiae DNA sequences caused considerable gene expression in E. coli. Determination of transcription start sites within six selected yeast sequences in E. coli confirmed the existence of bacterial -10 and -35 consensus sequences at appropriate distances upstream from transcription initiation sites. Our results demonstrate that the probability of transcription of transferred eukaryotic DNA in bacteria is extremely high and does not require the insertion of the transferred DNA behind a promoter of the recipient genome.

Artificial Gene Fusion↗
PubMed1992

Rapid surveying of DNA sequence variation in natural populations.

DNA sequencing can be costly and time consuming for population studies because of the relative rarity of variation along exons. These problems can be substantially reduced by the use of the polymerase chain reaction on introns using primers from the exon region. These problems can be further reduced by the use of denaturing gradient gel electrophoresis to identify those alleles in need of sequencing.

Animals↗
PubMed1982

Molecular cloning of the endogenous rat C-type helper virus DNA sequence: structural organization and functional analysis of some restricted DNA fragments.

Recently, we have identified and purified the integrated and proviral DNA sequences specific for two endogenous rat type C leukaemia helper viruses: WR-RaLV which originated from a fibrosarcoma induced in a feral rat and RHHV from the cell line HTC-H1 which originated from a Buffalo rat hepatoma. The rat leukaemia helper virus DNA sequences have previously been shown to be 8.4 to 8.8 kilobases (kb) in size. In this communication, we report the molecular cloning of the 8.8 kb DNA of RHHV by ligation at the BamHI site of the vector pBR322, cultured in an Escherichia coli RR1 host. After screening 5750 clones for ampicillin resistance and tetracycline sensitivity and testing by colony hybridization using 32P-labelled RHHV cDNA, four clones were isolated, two of which carried the total 8.8 kb DNA. A detailed restriction endonuclease map of the cloned RHHV DNA was deduced by sequential digestions of either 3'- or 5'-labelled DNA. Of the 14 restriction enzymes tested, EcoRI, BamHI, PstI, KpnI, TaqI, PvuII and SmaI gave informative cleavage patterns. At least two copies of long terminal repeated sequences (LTR) flanking the 3' and 5' termini of the proviral DNA were identified by TaqI and PstI cleavages. LTR in the rat endogenous leukaemia helper virus DNA measured 780 +/- 20 nucleotides in length. The genetic information encoded by the cloned DNA was also analysed by hybridization selection of RHHV mRNA, which was then used in cell-free protein synthesis in a rabbit reticulocyte lysate system. Essentially all major RaLV-specific proteins precipitable by anti-RaLV serum were synthesized in vitro, confirming that the RHHV genomic DNA was successfully cloned with little fidelity loss or scrambling of the genetic information.

Animals↗
PubMed1997

Control of methylation spreading in synthetic DNA sequences by the murine DNA methyltransferase.

Methylation spreading, which involves a propensity for the mammalian DNA-(cytosine-5)-methyltransferase to de novo methylate cytosine-guanine dinucleotides (CpGs) near pre-existing 5-methylcytosine bases, has been implicated in the control of numerous biological processes. We have assessed methylation spreading by the murine DNA methyltransferase in vitro using synthetic copolymers and oligonucleotides which differ only in their methylation state. Double-stranded oligonucleotides were found to undergo higher levels of de novo methylation overall than otherwise identical single-stranded oligonucleotides. This difference reflects the greater number of de novo methylatable cytosine bases in double-stranded than single-stranded sequences. All tested oligonucleotides containing pre-existing 5-methyl-cytosine(s) were de novo methylated at several fold the rates of non-methylated controls. No mammalian proteins besides the DNA methyltransferase were required for this observed enhancement of de novo methylation. Studies using oligonucleotides differing in patterns of pre-methylation showed that methylation spreading can be initiated by hemimethylated or duplex methylated CpGs indicating that recognition of 5-methylcytosine by the enzyme is sufficient to stimulate methylation spreading. Double and single-stranded oligonucleotides with several bases between CpGs underwent considerably more de novo methylation per CpG than sequences containing sequential uninterrupted methylatable sites. Spacing preferences by the DNA methyltransferase were also observed in hemimethylated oligonucleotides, suggesting that this is a general property of the enzyme. Although methylation spreading outside of CpG dinucleotides was relatively rare, single-stranded DNA incurred higher levels of de novo methylation at sites other than CpG as compared to double-stranded DNA. This indicates less specificity of methylation spreading in single-stranded sequences. Finally, enhanced de novo methylation in the presence of fully methylated CpG sites in double-stranded oligonucleotides was not as high as the rates of methylation of hemimethylated CpGs in otherwise identical oligonucleotides. These studies provide further elucidation of the mechanisms and regulation of the methylation spreading process and its potential role in the biological processes it influences.

5-Methylcytosine↗