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At least 523 records · Page 29Linked to original sources
PubMed1985

Comparison of physical and genetic properties of palindromic DNA sequences.

Some viable palindromic DNA sequences were found to cause an increase in the recovery of genetic recombinants. Although these palindromes contained no Chi sites, their presence in cis caused apparent recA+-dependent recombination to increase severalfold. This biological property did not correlate with the physical properties of the palindromes' extrusion of cruciform structures in vitro. Thus, two unrelated palindromes with similar effects on recombination in both Escherichia coli and Pseudomonas syringae displayed quite different kinetics of cruciform formation. In plasmids of native superhelical density, one palindrome underwent rapid cruciform formation at 55 degrees C, whereas the other did not form detectable cruciforms at any temperature. A shorter palindrome with similarly rapid kinetics of cruciform formation did not affect recombination detectably. The lack of a clear relationship between physical and genetic properties was also demonstrated in the case of longer, inviable palindromes. Here we found that the degree of asymmetry required in vivo to rescue a long palindrome from inviability far exceeded that required to kinetically prohibit cruciform extrusion in vitro.

Base Sequence↗
PubMedDate not supplied

Use of biotinylated DNA probes in screening cells obtained from cervical swabs for human papillomavirus DNA sequences.

A nonradioactive DNA-detection procedure using biotinylated DNA probes in the screening of cells from cervical swabs for DNA sequences homologous to human papillomavirus (HPV) DNA was tested. This alternative DNA-detection method yielded results comparable to those obtained with radioisotope-labeled DNA probes in 32 cases tested. This procedure obviates the special precautions required for radioisotope materials. Accordingly, this technique can be made available to many laboratories, and conclusive evidence as to the relation of HPV infection to cervical cancer may thus be accumulated.

Biotin↗
PubMed1989

[Gene technology in clinical medicine--DNA sequencing].

There are two established procedures for sequencing cloned DNA fragments. In Maxam-Gilbert sequencing, or sequencing by chemical degradation, end-labeled DNA is treated with various chemicals which induce base specific chain breaks with a low frequency. In dideoxy sequencing, or sequencing by the Sanger procedure, a single-stranded DNA is used as a template for synthesis of a labeled complementary strand by a DNA polymerase. Addition of dideoxynucleoside triphosphates will induce base-specific chain termination. In both procedures the nucleotide sequence of the cloned DNA can be deduced after fractionation of the labeled products by polyacrylamide gel electrophoresis. As yet, radioactive labeling of the reaction products is most common, but fluorescence labeling and computer-assisted automated sequence interpretation has become as a powerful alternative during the last years. Further automation of the various processes involved in DNA sequencing will be necessary for the planned sequencing of large genomes, such as the Escherichia coli genome, the yeast genome, and the human genome.

Animals↗
PubMed1993

Discrimination between closely related Pacific oyster species (Crassostrea) via mitochondrial DNA sequences coding for large subunit rRNA.

Mitochondrial DNA sequence variation was characterized for the large subunit rRNA-coding gene (16SrDNA) in two closely related Pacific oyster species (Crassostrea gigas and C. sikamea) and an out group, the Olympia oyster (Ostrea lurida). Although each species was shown to have a single, fixed haplotype for the DNA sequence under study, 7 nucleotide differences were found between C. gigas and C. sikamea, and these two species differed from the O. lurida haplotype at 62 and 60 nucleotide sites, respectively. Nucleotide differences for the two Crassostrea species showed a notable transition bias (85.7%) in contrast to the marginal transversion bias (54.5%) in nucleotide differences between Crassostrea haplotypes and the more distantly related O. lurida. Conservation of primary sequence in all three oyster species as well as other published 16SrDNA sequences was noted for regions with apparent functional significance. We developed DNA sequence-specific discrimination techniques and employed sequence-specific PCR primers, dot-blot hybridization, and restriction digests as alternate techniques for rapid diagnosis of Crassostrea oyster larvae.

Animals↗
PubMed1990

Amplification of DNA sequences of Epstein-Barr and human immunodeficiency viruses using DNA-polymerase from Thermus thermophilus.

Using thermophilic DNA-polymerase from Thermus thermophilus we have amplified by polymerase chain reaction (PCR) specific DNA sequences of Epstein-Barr virus (EBV) and human immunodeficiency virus (HIV). DNA-polymerase from Thermus thermophilus (molecular mass of 80-86 kDa) differs in its physico-chemical properties from DNA-polymerase from Thermus aquaticus (molecular mass of 62-68 kDa). To amplify the specific EBV DNA sequence, oligonucleotide primers for the virus replicon region (oriP region) were used. As a result of amplification, a specific 405-bp DNA fragment was produced.

Base Sequence↗
PubMedDate not supplied

Mixed hybridization and conventional strategies for DNA sequencing.

Oligonucleotide hybridization can be used to sequence short regions of DNA. In this report, I examine the extent to which this method can be used to sequence genomic DNA, using simulation experiments performed on known human genomic DNA sequences, and show that segments of sequence of up to 800-bp length may be unambiguously sequenced using 65,536 mixed 11-mers. Conventional sequencing or hybridization to specific, longer oligonucleotides can extend this limit in a mixed strategy that can sequence 10-kb DNA sequences with 5-7 conventional sequencing reactions and hybridization to a mixed 12-mer probe array.

Algorithms↗
PubMedDate not supplied

[Amplification of DNA sequences in Epstein-Barr virus and human immunodeficiency virus using DNA polymerase from Thermus thermophilus].

Using thermophilic DNA-polymerase from Thermus thermophilus we have amplified by polymerase chain reaction (PCR) specific DNA sequences of Epstein-Barr virus (EBV) and human immunodeficiency virus (HIV). DNA-polymerase from Thermus thermophilus (the molecular mass of 80 to 86 kDa) differs in its physico-chemical properties from DNA-polymerase from the Thermus acquaticus (the molecular mass of 62 to 68 kDa). To amplify the specific EBV DNA sequence oligonucleotide primers for the virus replicon region (oriP-region) were used. As a result of amplification, a specific 405 b.p. DNA fragment was produced. Primers for the virus Gag region were used for amplification of HIV DNA. The possibility to conduct amplification cycles under two temperature conditions was demonstrated.

Base Sequence↗
PubMed1993

Mitochondrial DNA sequence analysis of human skeletal remains: identification of remains from the Vietnam War.

Deoxyribonucleic acid (DNA) sequence analysis of the control region of the mitochondrial DNA (mtDNA) genome was used to identify human skeletal remains returned to the United States government by the Vietnamese government in 1984. The postmortem interval was thought to be 24 years at the time of testing, and the remains presumed to be an American service member. DNA typing methods using nuclear genomic DNA, HLA-DQ alpha and the variable number of tandem repeat (VNTR) locus D1S80, were unsuccessful using the polymerase chain reaction (PCR). Amplification of a portion of the mtDNA control region was performed, and the resulting PCR product subjected to DNA sequence analysis. The DNA sequence generated from the skeletal remains was identical to the maternal reference sequence, as well as the sequence generated from two siblings (sisters). The sequence was unique when compared to more than 650 DNA sequences found both in the literature and provided by personal communications. The individual sequence polymorphisms were present in only 23 of the more than 1300 nucleotide positions analyzed. These results support the observation that in cases where conventional DNA typing is unavailable, mtDNA sequencing can be used for human remains identification.

Anthropology, Physical↗
PubMed2001

DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3'-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing.

Base Sequence↗
PubMed1993

Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer.

The 16S rRNA gene sequences of 19 strains covering 97% of the molecules were determined for the members of the family Rhizobiaceae and related bacteria by PCR and DNA sequencer. The three biovars of Agrobacterium were located separately, whereas Agrobacterium rubi clustered with A. tumefaciens. Phylogenetic locations for the species of the genera Rhizobium, Sinorhizobium, Agrobacterium, Phylobacterium, Mycoplana (M. dimorpha), Ochrobactrum, Brucella and Rochalimaea (a rickettsia) were intermingled with each other with the similarity values higher than 92%. The family Rhizobiaceae should be redefined including the above-mentioned genera despite the ability for plant association and nitrogen fixation. Bradyrhizobium japonicum and Mycoplana bullata were far remote from the other species and should be excluded from this family.

Base Sequence↗
PubMed2002

Repeated DNA sequences in the microbat species Miniopterus schreibersi (Vespertilionidae; Chiroptera).

Repetitive DNA sequences represent a substantial component of eukaryotic genomes. These sequences have been described and characterized in many mammalian species. However, little information about repetitive DNA sequences is available in bat species. Here we describe an EcoRI family of repetitive DNA sequences present in the species Miniopterus schreibersi. These repetitive sequences are 57.85%, A-T rich, organized in tandem, and with a monomer unit length of 904 bp. Methylation analysis using the isoesquizomer pair MspI and HpaII indicates that the cytosines present in the sequences CCGG are partially methylated. Furthermore, Southern blot analysis demonstrated that these DNA sequences are absent in the genomes of four related microbat species and suggest that it could be specific to the M. schreibersi genome.

Animals↗
PubMed1996

The use of an improved transposon mutagenesis system for DNA sequencing leads to the characterization of a new insertion sequence of Streptomyces lividans 66.

A DNA sequencing strategy was developed based on the tetracycline resistance transposon Tn1721. A universal M13 primer binding site (UP) for DNA sequencing and restriction sites for mapping were inserted near one end of Tn1721 and the new derivative, Tn5491, introduced onto a conjugative F' plasmid. The target sequence is inserted between two inverted resolution sites (res) of Tn1721 present on the high-copy plasmid pJOE2114. Due to the inviability of long palindromic sequences in Escherichia coli insertions between the inversely orientated res sites of pJOE2114 are positively selected. Transposition of Tn5491 into the target sequence is selected by cointegrate formation of Tn5491 during transposition, mating and transfer of the nonconjugative sequencing vector. After cointegrate resolution, the additional res sites in the vector result in a second site-specific recombination removing most of the transposon (except of 136 bp) and part of the target sequence. The reduced plasmid sizes and the use of the universal primer improved the quality of the sequencing results obtained on an automated fluorescent sequencer. A 3.35-kb EcoRI fragment from the 30-kb terminal inverted repeats (TIR) of the Streptomyces lividans chromosome was sequenced by this method. A 1304-bp sequence was found on this fragment with the features of insertion elements. The element called IS1372 had 27-bp IR and two potential open reading frames. The predicted gene products had similar sizes and high similarity to gene products encoded by insertion sequences of the IS3 family. Furthermore, a potential signal stimulating ribosomal shifts and typical for members of the IS3 family was identified. Five to seven copies of IS1372 were found in different strains of S. lividans but none in other Streptomyces species tested.

Amino Acid Sequence↗
PubMed2006

MCALIGN2: faster, accurate global pairwise alignment of non-coding DNA sequences based on explicit models of indel evolution.

BACKGROUND: Non-coding DNA sequences comprise a very large proportion of the total genomic content of mammals, most other vertebrates, many invertebrates, and most plants. Unraveling the functional significance of non-coding DNA depends on how well we are able to align non-coding DNA sequences. However, the alignment of non-coding DNA sequences is more difficult than aligning protein-coding sequences. RESULTS: Here we present an improved pair-hidden-Markov-Model (pair HMM) based method for performing global pairwise alignment of non-coding DNA sequences. The method uses an explicit model of indel length frequency distribution which can be specified, and allows any time reversible model of nucleotide substitution. The method uses a deterministic global optimiser to find the alignment with the highest posterior probability. We test MCALIGN2 in simulations, and compare it to a previous Monte Carlo based method (MCALIGN), to the pair HMM method of Knudsen and Miyamoto, and to a heuristic method (AVID) that performed very well in a previous simulation study. We show that the pair HMM methods have excellent performance for all combinations of parameter values we have considered. MCALIGN2 is up to ten times faster than MCALIGN. MCALIGN2 is more accurate in resolving indels given an accurate explicit model than heuristic methods, but is computationally slower. CONCLUSION: MCALIGN2 produces better quality alignments by explicitly using biological knowledge about the indel length distribution and time reversible models of nucleotide substitution. As a result, it can outperform other available sequence alignment methods for the cases we have considered to align non-coding DNA sequences.

Algorithms↗
PubMed1975

Unique sequence DNA transcripts present in mature oocytes of Urechis caupo.

DNA - DNA reannealing of sheared Urechis caupo DNA indicates that the genome is composed of two major repetitive sequence families. The repetitive sequence DNA families compose 8 and 7% of the DNA and are repeated in the genome on an average of 2.0 - 10-6 and 6.0 - 10-3 times, respectively. The unique sequence DNA composed a minimum of 75% of the genome and the rate of reannealing indicates a complexity of 2.6 - 10-8 nucleotide pairs per genome. Hybridization of isolated 3-H-labeled unique sequence DNA with an excess of unlabeled mature oocyte RNA provides a minimum estimate of the genetic diversity of the RNA present in the mature oocytes. The apparent saturation level of unique sequence DNA with RNA indicates that about 8.6% of the unique sequence DNA is expressed. Based on the fraction of the DNA that forms a stable hybrid and the complexity of the unique sequence DNA the RNA present in the mature oocyte is a complementary with 2.2 - 10-7 nucleotide pairs of unique sequence DNA.

Animals↗
PubMed1997

Phylogenetic position of mammoth and Steller's sea cow within Tethytheria demonstrated by mitochondrial DNA sequences.

Here we report DNA sequences from mitochondrial cytochrome b gene segments (1,005 base pairs per species) for the extinct woolly mammoth (Mammuthus primigenius) and Steller's sea cow (Hydrodamalis gigas) and the extant Asian elephant (Elephas maximus), the Western Indian manatee (Trichechus manatus), and the hyrax (Procavia capensis). These molecular data have allowed us to construct the phylogeny for the Tethytheria. Our molecular data resolve the trichotomy between the two species of living elephants and the mammoth and confirm that the mammoth was more closely related to the Asian elephant than to the African elephant. Our data also suggest that the sea cow-dugong divergence was likely as ancient as the dugong-manatee split, and it appears to have been much earlier (22 million years ago) than had been previously estimated (4-8 million years ago) by immunological comparison.

Animals↗
PubMed1993

Entropic profiles of DNA sequences through chaos-game-derived images.

A new method to determine entropic profiles in DNA sequences is presented. It is based on the chaos-game representation (CGR) of gene structure, a technique which produces a fractal-like picture of DNA sequences. First, the CGR image was divided into squares 4-m in size (m being the desired resolution), and the point density counted. Second, appropriate intervals were adjusted, and then a histogram of densities was prepared. Third, Shannon's formula was applied to the probability-distribution histogram, thus obtaining a new entropic estimate for DNA sequences, the histogram entropy, a measurement that goes with the level of constraints on the DNA sequence. Lastly, the entropic profile for the sequence was drawn, by considering the entropies at each resolution level, thus providing a way to summarize the complexity of large genomic regions or even entire genomes at different resolution levels. The application of the method to DNA sequences reveals that entropic profiles obtained in this way, as opposed to previously published ones, clearly discriminate between random and natural DNA sequences. Entropic profiles also show a different degree of variability within and between genomes. The results of these analyses are discussed in relation both to the genome compartmentalization in vertebrates and to the differential action of compositional and/or functional constraints on DNA sequences.

Animals↗
PubMed1987

Induction of DNA strand breaks in transcriptionally active DNA sequences of mouse cells by low doses of ionizing radiation.

The efficiency of DNA single-strand break induction was measured in transcriptionally active DNA, transcriptionally inert satellite DNA, and bulk DNA sequences of mouse L929 cells using the alkaline filter elution assay. The cells were exposed to increasing doses of X-radiation up to 1000 rad. DNA which either eluted from or was retained on polycarbonate filters during the assays was collected onto nitrocellulose filters and hybridized against radiolabeled poly(A+)RNA (to probe transcribing DNA sequences) or mouse satellite DNA. The increasing rate and extent of elution of bulk DNA or specific DNA sequences after increasing radiation doses was taken as a measure of the increased frequency of radiation-induced DNA strand breaks. The results indicate that a significant fraction of transcriptionally active DNA contains endogenous strand breaks. With increasing dose, the efficiency of radiation-induced DNA strand breakage in bulk, transcriptionally active and satellite DNA sequences was observed to be the same when the sum of all eluted DNA was considered. However, the early eluting fractions contained DNA which was enriched in active sequences. Since DNA elutes as a function of size, the early fractions contain smaller DNA than later fractions. Therefore, our results indicate that the fraction of active sequences which elutes early resides on smaller fragments on the average than the later eluting DNA, and that even low doses of radiation preferentially cause breaks in regions of DNA containing active sequences.

Animals↗
PubMed1986

Monkey B-lymphotropic papovavirus genome: the entire DNA sequence and variable regions.

DNA from monkey B-lymphotropic papovavirus (LPV) adapted to growth in human B-lymphoblastoid cell line BJA-B consisted of three classes of molecules of 5.1, 5.0, and 4.9 kilobases (kb), and the entire DNA sequence of 5.1-kb molecule was determined. The three types of LPV DNA yielded infectious virus upon transfection to BJA-B cells. Comparison of the structures among these clones and clone K38 sequenced by Pawlita et al. (1985) suggests that the transcriptional control region, the small-T antigen gene, and the VP-1 gene are variable in the LPV genome, and that the 5.0-kb LPV resembles the parent virus, from which all the others had evolved by duplication and deletion during passages in BJA-B cells, probably for adaptation to better growth in human cells.

Animals↗