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PubMed1996

Compact encoding strategies for DNA sequence similarity search.

Determining whether two DNA sequences are similar is an essential component of DNA sequence analysis. Dynamic programming is the algorithm of choice if computational time is not the most important consideration. Heuristic search tools, such as BLAST, are computationally more efficient, but they may miss some of the sequence similarities (Altschul et al., 1990). These tools often use common k-tuples (words) between the two sequences to determine anchor points for the alignment, and spend most of their computational time extending the alignment beyond these anchor points. We discuss and provide a DNA sequence similarity search implementation (called SENSEI) that improves upon the performance of BLASTN by almost an order of magnitude for comparable sensitivity. This improvement is a result of using compactly encoded scoring tables for k-tuples, encoding bases with a single bit, filtering the sequence to remove the simple sequence repeats using XNUN, and masking the known species-specific repeats in the query sequence. To reduce memory requirements, especially for large genomic DNA query sequences, we recommend generating the neighborhood words from the target sequence at run-time, instead of generating them by preprocessing the query sequence.

Base Sequence↗

PubMed1989

Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN function and terminal DNA sequence.

Linear retroviral DNA, the major precursor to the integrated provirus of the murine leukemia viruses, contains a mixture of two structures at its ends: some termini are full-length and blunt, and some have recessed 3' strands. A temporal study of the end structures showed that the proportion of the DNA with recessed ends increases during the course of infection, and suggests that the blunt ends are precursors to the recessed ends. We have examined the DNA structures of the ends of retroviral mutants defective in the integration (IN) function. The results show that the formation of the recessed ends requires the presence of IN. Finally, we have analyzed the structures at the ends of mutant genomes with alterations in the terminal DNA sequence. The exact position of the recessed 3' end can be recessed one, two, or four nucleotides relative to the 5' end. In all cases the position of the recessed 3' end correlates perfectly with, and thus presumably determines, the site of joining to the target DNA.

Animals↗

PubMed1995

BULLET: a computer simulation of shotgun DNA sequencing.

BULLET is a computer program that simulates shotgun sequencing of DNA. The program has been used to simulate the sequencing of DNA fragments from 2 to 30 kb. To obtain 80% single or double-stranded sequence, a doubling of the DNA fragment size necessitates twice the number of sequencing reactions. However, a similar linear relationship does not apply to the determination of 100% single or double-stranded sequence. Data from BULLET were used to derive simple linear formulae that estimate the number of sequencing reactions necessary to partially sequence a fragment of DNA irrespective of size. When 80% of the double-stranded sequence has been determined, approximately 98% of single-stranded sequence will be known. This is a reasonable point to change to a more directed strategy of DNA sequencing.

Algorithms↗

PubMed1994

Synchronous luminescence: a new detection technique for multiple fluorescent probes used for DNA sequencing.

A method fo nonradioactive DNA sequencing, which has been marketed commercially, uses four different fluorescent tags to label DNA fragments and fixed-wavelength excitation/fluorescence detection of the labels. This study presents an alternative method of detection based on synchronous luminescence (SL), in which both excitation and emission wavelengths are scanned simultaneously. This approach has proven in the past to have significant advantages over fixed-wavelength luminescence in the analysis and identification of fluorescent analytes. In this paper, the utility of synchronous excitation was investigated as a method for DNA sequencing with fluorescent tags. A laser-based SL instrument, recently developed in this laboratory, was used to resolve the spectra of a mixture of the dyes used in the fluorescence-based sequencing scheme. A preliminary limit of detection of 720 zeptomoles (10(-21) M) of fluorescein isothiocyanate dye in solution was achieved with this instrument. The results presented here suggest that the SL technique could result in an increase in sensitivity and a decrease in error rate of identification during DNA sequencing.

4-Chloro-7-nitrobenzofurazan↗

PubMed1996

Genetic similarity among Arabidopsis thaliana ecotypes estimated by DNA sequence comparison.

DNA polymorphisms among Arabidopsis thaliana ecotypes are widely used as genetic markers in map-based cloning strategies. New PCR-based molecular markers do not only facilitate molecular mapping, but can also be used to obtain reliable sequence information for cladistic analyses. We have used CAPS (cleaved amplified polymorphic sequences) markers and a direct sequencing strategy to estimate genetic similarity among eighteen Arabidopsis ecotypes. Sequences at four loci, two from the nuclear and two from a non-nuclear genome, were analysed. For each ecotype more than 1000 bp of sequence information was obtained, and genetic similarity was calculated from a total of 35 polymorphic sites using a character-based approach. Divergence ranged from zero up to 50 discordant characters among the 72 characters defined by the polymorphisms. Separate calculations based on the nuclear and the non-nuclear sequences were performed and revealed a number of common features, including the existence of small clusters of very closely related ecotypes separated from each other by extensive sequence divergence. Our results provide information useful especially to investigators setting up crosses for chromosome landing strategies.

Arabidopsis↗

PubMed1999

DNA sequencing by capillary electrophoresis (review).

DNA sequencing by capillary electrophoresis has been reviewed with an emphasis on progress during the last four years. The effects of sample purification, composition of sieving matrices, electric field strength, temperature, wall coating and DNA labeling on the DNA sequencing performance are discussed. Multicapillary array instrumentation is compared with one-capillary systems. Integrated systems that perform the whole DNA sequencing operation online starting from the DNA amplification through base calling and data processing are discussed.

Bacteriophage M13↗

PubMed1977

Studies on a defective variant of simian virus 40 that is substituted with DNA sequences derived from monkey. II. Structure of DNA.

The structure of a substituted, reiterated defective variant of the simian virus 40 genome has been analyzed. The DNA of the defective variant is a closed circular duplex resistant to restriction endonuclease R-EcoRI and slightly shorter than the genome of wild type simian virus 40. Analysis of the double-stranded DNA segments produced by the action of a variety of restriction endonucleases on the defective genome allow description of the molecule as follows. (a) The full DNA molecule contains four tandem repeats of a DNA segment containing both SV40 and monkey DNA sequences. (b) Three out of the four segments are identical and are about 23% of the wild type genome in length: one segment of the four is larger and contains an additional peice of DNA about 4.3% of a wild type genome in length, but is otherwise identical with the other three segments. (c) Portions of the defective variant that contain monkey DNA can be isolated as discrete segments by restriction endonuclease digestion. (d) Some of the sequences originating from monkey DNA are derived from the highly reiterated class of monkey DNA sequences; others may represent sequences derived from infrequently reiterated or single copy monkey sequences.

Animals↗

PubMed1992

Phylogenetic position of Cowdria ruminantium (Rickettsiales) determined by analysis of amplified 16S ribosomal DNA sequences.

The 16S ribosomal DNA sequence of Cowdria ruminantium, the causative agent of heartwater disease in ruminants, was determined. An analysis of this sequence showed that C. ruminantium forms a tight phylogenetic cluster with the canine pathogen Ehrlichia canis and the human pathogen Ehrlichia chaffeensis. Although a close relationship between the genus Cowdria and several members of the tribe Ehrlichieae has been suspected previously, the tight phylogenetic cluster with E. canis and E. chaffeensis is surprising in view of known differences in host preference and target cells.

Base Sequence↗

PubMed2003

Phylogenetic systematics and evolution of primate-derived Pneumocystis based on mitochondrial or nuclear DNA sequence comparison.

Previous studies have demonstrated that the agent of Pneumocystis pneumonia (PcP), Pneumocystis carinii, is actually a complex of eukaryotic organisms, and cophylogeny could explain the distribution of the hosts and parasites. In the present work, we tested the hypothesis of cophylogeny between the primate-derived Pneumocystis group and their hosts. Specific strains isolated from 20 primate species, including humans, were used to produce a phylogeny of the parasites. Aligned sequences corresponding to DNA sequences of three genes (DHPS, mtSSU-rRNA, and mtLSU-rRNA) were separately analyzed and then combined in a single data set. The resulting parasite phylogeny was compared with different controversial phylogenies for the hosts. This comparison demonstrated that, depending upon which topology is accepted for the hosts, at least 61% and perhaps 77% of the homologous nodes of the respective cladograms of the hosts and parasites may be interpreted as resulting from codivergence events. This finding and the high specificity of these parasites suggests that cophylogeny may be considered the dominant pattern of evolution for Pneumocystis organisms, representing a new example of parallel evolution between primates and their specific parasites. Because the phylogeny of Pneumocystis followed very closely the differentiation of their hosts at the species level, the study of the parasites could provide valuable information on the phylogeny of their hosts. We used this information to explore controversial hypotheses of the phylogeny of the Platyrrhini by comparison with the phylogeny of their specific Pneumocystis parasites. If these organisms were closely associated as lung parasites with primates through the ages, the hypothesis of the Pneumocystis spp. being new pathogenic agents could be refuted. However, these organisms are opportunistic symbionts, becoming pathogenic whenever the immunological defences of their hosts decline. This study also provides support for the hypothesis that the different Pneumocystis species are genetically independent organisms, helping to clarify their taxonomic status.

Animals↗

PubMed1998

Efficient identification of point mutations by automated DNA sequencing of artificial heterozygote samples.

DNA sequencing templates of individual point mutants of the lacI target gene were amplified by polymerase chain reaction (PCR). By mixing the PCR fragments from two individual mutants in a defined ratio, samples of artificial heterozygous composition were prepared. These samples were then submitted to automated DNA sequencing. The simultaneous, visual comparison of the mixed mutant traces using a graphics program efficiently revealed all heterozygous positions. Based on the individual intensities of the heterozygous base signals the identified point mutations could be assigned to the corresponding mutants. This efficient approach doubles the sample throughput for both the sequencing reactions and the gel electrophoresis using an automated DNA sequencing system.

Animals↗

PubMed1999

The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration.

After Agrobacterium-mediated plant transformation, multiple T-DNAs frequently integrate at the same position in the plant genome, resulting in the formation of inverted and direct repeats. Because these inverted repeats cannot be amplified and analyzed by PCR, Arabidopsis root cells were co-transformed with two different T-DNAs with distinct sequences adjacent to the T-DNA borders. Nine direct or inverted T-DNA border junctions were analyzed at the sequence level. Precise end-to-end fusions were found between two right border ends, whereas imprecise fusions and filler DNA were present in T-DNA linkages containing a left border end. The results suggest that end-to-end ligation of double-stranded T-DNAs occurs especially between right T-DNA ends and that illegitimate recombination on the basis of microhomology, deletions, repair activities and insertions of filler DNA is involved in the formation of left border T-DNA junctions. Therefore, a similar illegitimate recombination mechanism is proposed that is involved in the formation of complex T-DNA inserts as well as in the integration of the T-DNA in the plant genome.

Agrobacterium tumefaciens↗

PubMed2000

A locust type 1 ADP-ribosylation factor (lARF1)* is 100% identical in amino acid sequence to Drosophila ARF1 despite obvious DNA sequence divergence.

The cDNA of a type 1 ADP-ribosylation factor (ARF) from the desert locust, Locusta migratoria was cloned, sequenced and compared to ARF1 genes of other species. The locust ARF1 protein is 100% identical with the ARF1 protein of the fruit fly Drosophila melanogaster even though the DNA sequences are only 79% identical. The significance of this finding in relation to the considerable evolutionary distance between hemimetabolous and holometabolous insects is discussed.

ADP-Ribosylation Factor 1↗

PubMed1988

Fragile sites, telomeric DNA sequences, B chromosomes, and DNA content in raccoon dogs, Nyctereutes procyonoides, with comparative notes on foxes, coyote, wolf, and raccoon.

Earlier studies of the genus Nyctereutes disclosed two subspecies of differing chromosome numbers accompanied by B chromosomes. To further define the relationship of these subspecies to each other, and to other carnivores, and to learn more about the structure and function of their chromosomes, we characterized and compared the genomes in terms of DNA content by flow cytometry, fragile sites induced by aphidicolin, and telomere sequences using biotinylated DNA probes detected with fluorescence. We also characterized the B chromosomes of these two subspecies.

Animals↗

PubMed1984

Detection of tumor-inducing plasmid DNA sequence in Agrobacterium tumefaciens by DNA-DNA hybridization.

Absence of plasmid DNA sequences in non-tumorigenic (crown gall tumor) strains of Agrobacterium tumefaciens was confirmed by DNA-DNA hybridization between purified tumor-inducing (Ti) plasmid DNAs isolated from the progenitor tumorigenic strains and 3H-labeled whole cell DNAs from tumorigenic strains and their non-tumorigenic derivatives. The genes controlling tumorigenicity in plant and utilizability of nopaline as their sole nitrogen source were confirmed to locate on Ti-plasmids.

Arginine↗

PubMed2005

A likelihood ratio test for species membership based on DNA sequence data.

DNA barcoding as an approach for species identification is rapidly increasing in popularity. However, it remains unclear which statistical procedures should accompany the technique to provide a measure of uncertainty. Here we describe a likelihood ratio test which can be used to test if a sampled sequence is a member of an a priori specified species. We investigate the performance of the test using coalescence simulations, as well as using the real data from butterflies and frogs representing two kinds of challenge for DNA barcoding: extremely low and extremely high levels of sequence variability.

Animals↗

PubMed1983

Genetic instability of an artificial palindrom DNA sequence.

A short DNA palindrom, produced by head to head ligation of a 29 bp DNA fragment, was inserted into a 27,000 bp plasmid DNA element composed of two functional replicons (R6K, ColE1). Several plasmid types containing a single copy of this palindrom in different locations of insertion on the R6K sequence were obtained. The palindrom was engineered to possess a unique EcoRI recognition sequence at its axis of symmetry. The presence of this restriction site allowed to monitor the genetic stability of the artificial palindrom at their different insertion loci. Out of 5 different insertion locations, one (in pAS807) was found to lead to a significant destabilization of the palindrom. This insertion site lies within the replication control region of R6K. We have shown that the inserted palindrom in pAS8O7 does not affect the functionality of the R6K replication origins. Excission of the palindrom sequences from pAS8O7 was not accompanied by loss of the adjacent R6K DNA sequences. Different deletion derivatives of pAS807 were generated in-vitro in order to determine the driving unit of DNA sequences around the palindrom that are involved in its excision. The results imply that large DNA structure(s) around the palindrom are involved in its excission. Complete deletion of R6K sequences from either the left or the right side of the palindrom resulted in new configurations which stabilized the palindrom. A configuration of R6K DNA sequences exceeding 270 bp long sequence from both sides of the palindrom are necessary for the transition from a palindrom stable to palindrom unstable state. In addition evidence is presented to show that the excision process of palindrom sequences requires a functional polymerase I but not the gene product of recA.

Base Sequence↗

PubMed1976

DNA sequence organization in the lepidopteran Antheraea pernyi.

A large fraction of the genome of the lepidopteran Antheraea pernyi consists of interspersed single-copy sequences and repetitive DNA sequences of 300 nucleotide average length. Most of the single-copy sequences are about 800 nucleotides long and a minority are considerably longer. Thus, the organization of the DNA sequences of this insect is similar to that of most higher organisms and different from that of Drosophila.

Animals↗

PubMed1992

Dideoxy linear PCR on a commercial fluorescent automated DNA sequencer.

The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods play an important role in the actual effort to improve the efficiency of large-scale DNA analysis. Here we show the application of the linear PCR using a single fluorescent primer and dideoxynucleotide terminators in four separate sequencing reactions on the EMBL/Pharmacia's fluorescent automated DNA sequencer. We have used dideoxy/deoxynucleoside triphosphate ratios and linear amplification cycle conditions to obtain an accurate sequencing response of up to, and over, 500 bases from just 400 ng of double-stranded DNA template without chemical denaturation. The sequencing protocol described in this paper is effectively suited for enhancement of sensitivity and performance of the automated DNA sequencing system.

Autoanalysis↗