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PubMed1993

Artificial intelligence approach in analysis of DNA sequences.

We present an approach for designing a knowledge-based system, called Sequence Acquisition In Context (SAIC), that will be able to cooperate with a biologist in the analysis of DNA sequences. The main task of the system is the acquisition of the expert knowledge that the biologist uses for solving ambiguities from gel autoradiograms, with the aim of re-using it later for solving similar ambiguities. The various types of expert knowledge constitute what we call the contextual knowledge of the sequence analysis. Contextual knowledge deals with the unavoidable problems that are common in the study of the living material (eg noise on data, difficulties of observations). Indeed, the analysis of DNA sequences from autoradiograms belongs to an emerging and promising area of investigation, namely reasoning with images. The SAIC project is developed in a theoretical framework that is shared with other applications. Not all tasks have the same importance in each application. We use this observation for designing an intelligent assistant system with three applications. In the SAIC project, we focus on knowledge acquisition, human-computer interaction and explanation. The project will benefit research in the two other applications. We also discuss our SAIC project in the context of large international projects that aim to re-use and share knowledge in a repository.

Artificial Intelligence↗
PubMed1995

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.

Bacteriophage M13↗
PubMed2002

Association of promoter variants in the alpha7 nicotinic acetylcholine receptor subunit gene with an inhibitory deficit found in schizophrenia.

BACKGROUND: The alpha7 neuronal nicotinic acetylcholine receptor subunit gene (CHRNA7) has been implicated as a candidate gene for schizophrenia, and for an auditory sensory processing deficit found in the disease, by both genetic linkage at 15q14 and biochemical data. The expression of CHRNA7 is reduced in several brain regions in schizophrenic subjects compared with control subjects. This study presents DNA sequence analysis of the core promoter region for CHRNA7 in schizophrenic and control subjects. METHODS: Single-strand conformation polymorphism analysis and DNA sequencing were used for mutation screening of the core promoter in the CHRNA7 gene. The sample included subjects from 166 schizophrenic families and 165 controls. Controls had no evidence of current or past psychosis and had auditory evoked potentials recorded. RESULTS: Multiple polymorphic patterns were identified in the CHRNA7 core promoter in both schizophrenic and control subjects. Functional analysis of polymorphisms indicated that transcription was reduced. The prevalence of functional promoter variants was statistically greater in schizophrenic subjects than in the controls. Presence of an alpha7 promoter polymorphism in controls was associated with failure to inhibit the P50 auditory evoked potential response. CONCLUSIONS: Although linkage disequilibrium with other genetic alterations cannot be excluded, the CHRNA7 core promoter variants, found in this study, may contribute to a common pathophysiologic feature of schizophrenia.

Auditory Perceptual Disorders↗
PubMed1987

Automated DNA sequencing and analysis of the human genome.

In the past few years, striking advances have been made in automating DNA sequence analysis. Currently, efforts are underway to automate and improve DNA purification, mapping, and data processing procedures. The predictable advances in these technologies should soon place us in a position to sequence the entire human genome. The information derived from this project will have profound implications for basic biology and clinical medicine alike.

Base Sequence↗
PubMed1995

Matrix-assisted laser desorption/ionization mass spectrometry of immobilized duplex DNA probes.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry was used to analyze short DNA duplex probes with one strand immobilized on solid supports (straptavidin-coated magnetic beads or controlled pore glass beads). Only the non-immobilized strand could be detected. Partial denaturation was found when the duplex probes were mixed with 3-hydroxypicolinic acid, ammonium citrate matrix. The strategy has several applications, such as fast DNA sequence analysis and DNA diagnostics.

Base Sequence↗
PubMed2003

Autosomal dominant Stargardt-like macular dystrophy: identification of a new family with a mutation in the ELOVL4 gene.

PURPOSE: To describe the clinical features and identify the mutation responsible for an autosomal dominant macular degeneration occurring in a four-generation family. METHODS: Family members underwent clinical examination and genealogical characterization. Mutation screening of the ELOVL4 gene was performed. RESULTS: Patients reported visual loss occurring at a mean age of 20 years. Fundus examination revealed varying degrees of central macular atrophy with or without flecks in all affected individuals. DNA sequence analysis showed a 5-bp deletion in exon 6 of the ELOVL4 gene, confirming the diagnosis of autosomal dominant Stargardt-like macular dystrophy. Genealogical analysis showed that this family represents a new affected branch of a previously described 12-generation family (31 branches) with this disorder. CONCLUSIONS: We characterized a new branch of a family with autosomal dominant Stargardt-like macular dystrophy. Identification of the disease-causing gene allows for improved genetic counseling of affected individuals.

Adolescent↗
PubMed1993

Infrequency of ras, p53, WT1, or RB gene alterations in Wilms tumors.

BACKGROUND: Alteration of the ras family of oncogenes and of the tumor suppressor genes p53 and RB are the most common genetic events in human tumors. Although there have been no reports of the prevalence of these alterations in Wilms tumors, overexpression of the N-myc and insulin-like growth factor-II (IGF-II) genes have been observed, and alteration of another tumor suppressor gene (WT1) has been demonstrated. METHODS: Forty-four Wilms tumor specimens were tested for the presence of N-, K-, and H-ras mutations in codons 12, 13, and 61 by single-strand conformation polymorphism (SSCP) analysis and direct DNA sequence analysis. Sixteen tumors were tested for abnormalities of WT1 by Southern and northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). N-myc, c-myc, WT1, and IGF-II mRNA expression was measured in 16 tumors by Northern blot analysis. Thirty-eight tumors were screened for p53 mutations by SSCP analysis and direct DNA sequence analysis. Nine tumors were analyzed for loss of heterozygosity (LOH) of RB. RESULTS: Although the authors confirmed that N-myc and IGF-II are overexpressed in Wilms tumors, no mutations of ras family, p53, or RB genes were identified, and no gross alterations of WT1 were detected by Southern or Northern blot analysis. CONCLUSIONS: These findings suggest that H-ras, K-ras, N-ras, p53, and RB are not involved in the pathogenesis of Wilms tumor.

Base Sequence↗
PubMed1998

[DNA sequencing of human ciliary neurotropic factor gene by silver staining method].

OBJECTIVE: To introduce a non-radioactive protocol for DNA sequence analysis which employs a silver staining procedure METHODS: DNA template is amplified by Taq DNA polymerase to generate sequence ladder, No labeled nucleotide or primer is involved in the thermal-cycling sequencing reaction. A sensitive silver staining procedure is employed to visualize bands in sequencing gel. RESULTS: This silver staining method is used for DNA sequence analysis of human cilliary neurotrophic factor (hCNTF) gene. The resulted band resolution is comparable to radioactive sequencing method, and data can be obtain within less than 2 hours after sequencing reaction. CONCLUSION: Silver staining is a rapid, inexpensive and reliable method for DNA sequence analysis.

Ciliary Neurotrophic Factor↗
PubMed2000

Ancient DNA analysis of human populations.

The use of ancient DNA (aDNA) in the reconstruction of population origins and evolution is becoming increasingly common. The resultant increase in number of samples and polymorphic sites assayed and the number of studies published may give the impression that all technological hurdles associated with aDNA technology have been overcome. However, analysis of aDNA is still plagued by two issues that emerged at the advent of aDNA technology, namely the inability to amplify a significant number of samples and the contamination of samples with modern DNA. Herein, we analyze five well-preserved skeletal specimens from the western United States dating from 800-1600 A.D. These specimens yielded DNA samples with levels of contamination ranging from 0-100%, as determined by the presence or absence of New World-specific mitochondrial markers. All samples were analyzed by a variety of protocols intended to assay genetic variability and detect contamination, including amplification of variously sized DNA targets, direct DNA sequence analysis of amplification products and sequence analysis of cloned amplification products, analysis of restriction fragment length polymorphisms, quantitation of target DNA, amino acid racemization, and amino acid quantitation. Only the determination of DNA sequence from a cloned amplification product clearly revealed the presence of both ancient DNA and contaminating DNA in the same extract. Our results demonstrate that the analysis of aDNA is still an excruciatingly slow and meticulous process. All experiments, including stringent quality and contamination controls, must be performed in an environment as free as possible of potential sources of contaminating DNA, including modern DNA extracts. Careful selection of polymorphic markers capable of discriminating between ancient DNA and probable DNA contaminants is critical. Research strategies must be designed with a goal of identifying all DNA contaminants in order to differentiate convincingly between contamination and endogenous DNA.

Animals↗
PubMed1994

Identification of a point mutation in the cDNA of the catalytic subunit of DNA polymerase alpha from a temperature-sensitive mouse FM3A cell line.

DNA polymerase alpha is a key enzyme in eukaryotic chromosomal DNA replication. tsFT20 is a temperature-sensitive mutant cell line derived from mouse mammary carcinoma FM3A cells, and the cells contain heat-labile DNA polymerase alpha and are arrested at the S phase at the nonpermissive temperature. We isolated cDNA of the catalytic subunit of DNA polymerase alpha from tsFT20 cells. DNA sequence analysis revealed that the cDNA from tsFT20 has a single mutation, a cytosine to thymine substitution that changes amino acid 1180 from serine to phenylalanine. We have also shown that tsFT20 cells could be rescued by transfection with the wild-type cDNA. These results demonstrate that the point mutation in the gene of DNA polymerase alpha causes the temperature-sensitive phenotype of tsFT20 cells and provide additional evidence that DNA polymerase alpha is essential for chromosomal replication in mammalian cells. We also detected mutation sites in one spontaneous and six N-methyl-N'-nitro-N-nitrosoguanidine-induced growth revertants of tsFT20 cells by single strand conformation polymorphism analyses and direct sequencing. All revertant cell lines had a second point mutation adjacent to the first mutation site in tsFT20 cells.

Amino Acid Sequence↗
PubMed1995

Long-range correlation properties of coding and noncoding DNA sequences: GenBank analysis.

An open question in computational molecular biology is whether long-range correlations are present in both coding and noncoding DNA or only in the latter. To answer this question, we consider all 33301 coding and all 29453 noncoding eukaryotic sequences--each of length larger than 512 base pairs (bp)--in the present release of the GenBank to dtermine whether there is any statistically significant distinction in their long-range correlation properties. Standard fast Fourier transform (FFT) analysis indicates that coding sequences have practically no correlations in the range from 10 bp to 100 bp (spectral exponent beta=0.00 +/- 0.04, where the uncertainty is two standard deviations). In contrast, for noncoding sequences, the average value of the spectral exponent beta is positive (0.16 +/- 0.05) which unambiguously shows the presence of long-range correlations. We also separately analyze the 874 coding and the 1157 noncoding sequences that have more than 4096 bp and find a larger region of power-law behavior. We calculate the probability that these two data sets (coding and noncoding) were drawn from the same distribution and we find that it is less than 10(-10). We obtain independent confirmation of these findings using the method of detrended fluctuation analysis (DFA), which is designed to treat sequences with statistical heterogeneity, such as DNA's known mosaic structure ("patchiness") arising from the nonstationarity of nucleotide concentration. The near-perfect agreement between the two independent analysis methods, FFT and DFA, increases the confidence in the reliability of our conclusion.

Animals↗
PubMed1992

Determination of the coding capacity of the BamHI DNA fragment B of apathogenic Herpes simplex virus type 1 strain HFEM by DNA nucleotide sequence analysis.

Herpes simplex virus type 1 (HSV-1) strain HFEM acquired an apathogenic phenotype due to a deletion within the DNA sequences of the BamHI DNA fragment B of the viral genome. In order to investigate the coding strategy of this particular region of the genome of HSV-1 strain HFEM the DNA nucleotide sequence of the BamHI DNA fragment B was determined. This analysis revealed that the BamHI DNA fragment B of HSV-1 strain HFEM comprises 6593 bp, corresponding to the nucleotide positions (np) 113322 to 117088 and np 120643 to 123465 of the genome of HSV-1 strain 17. According to these data the deletion of the genome of HSV-1 strain HFEM occurred between the np 117089 and 120642. The promoter region of the UL56 gene of HSV-1 strain HFEM is a part of the deleted DNA sequences. Therefore, this gene of HSV-1 strain HFEM is affected and cannot be expressed. The first 35 amino acid (AA) residues of the deduced amino acid sequence of the UL56 open reading frame (ORF) were found to be identical to the amino acid sequence of the UL56 genes of HSV-1 strains 17 and F. However, due to a deletion at np 3494 of the BamHI DNA fragment B of HSV-1 strain HFEM the amino acid composition of the predicted UL56 gene of HSV-1 strain HFEM is different from HSV-1 strain 17 between amino acid positions 36 and 233. In addition the deduced amino acid sequence of the IRL (inverted repeat of the long segment) copy of the IE110 gene of HSV-1 strain HFEM was found to be about 342 amino acids shorter than the amino acid sequence of IE110 gene of HSV-1 strain 17 (775 AA). This was based on a point mutation which was detected within the DNA sequences of Exon 3 of this copy of IE110 gene of HSV-1 strain HFEM.

Amino Acid Sequence↗
PubMed1993

The Kluyveromyces gene encoding the general transcription factor IIB: structural analysis and expression in Saccharomyces cerevisiae.

The Kluyveromyces lactis gene encoding the general transcription factor IIB (TFIIB) was isolated from a genomic library by complementation of the cold-sensitive phenotype conferred by a mutation in the SUA7 gene, which encodes TFIIB in Saccharomyces cerevisiae. DNA sequence analysis of the KI-SUA7 gene revealed a 357 amino acid open reading frame that is 67% identical (81% overall similarity) to S. cerevisiae TFIIB. Comparison with other eukaryotic TFIIBs indicated that the most highly conserved sequence is located adjacent to the Zn-finger motif near the N-terminus. A plasmid shuffle system was used to replace the essential Sc-SUA7 gene with KI-SUA7 in S.cerevisiae. The resulting strain was viable and phenotypically indistinguishable from the normal strain. However, transcription start site selection at the ADH1 locus, shown previously to be affected by mutations in Sc-SUA7, was affected by K.lactis TFIIB. This result provides further evidence that TFIIB is a principal determinant of start site selection in yeast.

Amino Acid Sequence↗
PubMed2002

Complete DNA sequence and analysis of a mitochondrial plasmid in the mycoparasitic Trichoderma harzianum strain T95.

A circular plasmid called pThr1, with a monomer size of 2.6 kb, was identified in the mitochondria of a Trichoderma harzianum isolate. Hybridization studies using cloned plasmids revealed no DNA sequence similarity between the plasmid and the mitochondrial genome of the isolate. The complete sequence of the plasmid was determined, and the sequence analysis revealed that it contained a single long open reading frame of 1818 bp. Sequence comparisons indicated that the derived amino acid sequence of the ORF exhibited similarity to the reverse transcriptases of the circular Mauriceville and Varkud retroplasmids of Neurospora spp. and the linear pFOXC2 and pFOXC3 retroplasmids of Fusarium oxysporum strains. In the regions of homology all of the seven conserved amino acid blocks characteristic of RTs could be found.

Amino Acid Sequence↗
PubMed1992

Sequencing and analysis of genomic fragments from the NF1 locus.

The sequence of five non-contiguous genomic fragments encompassing 14.4 kilobases from the NF1 locus have been determined by fluorescence-based automated DNA sequence analysis. These fragments included one kilobase of the NF1 coding region, which resulted in the identification of the intron/exon boundaries of five exons. Based on these sequences, five new NF1 exon-PCR assays have been developed, that could be useful for detecting new NF1 mutations. The genomic sequences were analyzed for the presence of Alu repetitive elements and their classification is described. This analysis may provide some insight into the characterization of genetic rearrangements resulting in disruption of the NF1 gene.

Base Sequence↗
PubMed2000

Mosaic plasmids and mosaic replicons: evolutionary lessons from the analysis of genetic diversity in IncFII-related replicons.

The alpha replicons of the multi-replicon plasmids pGSH500 and pLV1402 have been characterized by DNA sequence analysis. Analysis of the DNA sequence of a 3672 bp HIN:dIII fragment from pFDT100, which contains the pGSH500 alpha replicon, revealed similarity to a number of replicons belonging to, or related to, those of the IncFII family. The replicon region contains copA, tapA, repA and oriR, and replication initiation and termination sites are related to those from the IncFII replicon of R1. A copB gene was found to lie upstream of the HIN:dIII site in the parental plasmid pGSH500. Downstream of oriR, a 707 bp region shows 72.6% identity to a region of the Escherichia coli chromosome at 43.3', suggesting this region of pGSH500 may have been incorporated into the plasmid during a past chromosomal recombination event. Oligonucleotide primers homologous to consensus regions in the copB and repA genes, and the oriR regions from a number of IncFII-related replicons were used to amplify replication regions from pLV1402. Analysis of the amplified regions has shown the presence of copB, copA, tapA and repA genes. Phylogenetic analysis of Rep protein sequences from the RepFIIA family of antisense-control-regulated replicons revealed the presence of three distinct subgroups of Rep proteins. Comparative analysis of DNA and protein sequences from members of the RepFIIA family provides evidence supporting the roles of both non-selective divergence in co-integrate (multi-replicon) plasmids and Chi-mediated-recombination in replicon evolution, and in particular, that such processes may have been widespread in the evolution of the RepFIIA family.

Base Sequence↗
PubMed1989

1-Tuple DNA sequencing: computer analysis.

A new method of DNA reading was proposed at the end of 1988 by Lysov et al. According to the authors' claims it has certain advantages as compared to the Maxam-Gilbert and Sanger methods, which are revealed by automation and rapidity of DNA sequencing. Nevertheless its employment is hampered by a number of biological and mathematical problems. The present study proposes an algorithm that allows to overcome the computational difficulties occurring in the course of the method during reconstruction of the DNA sequence by its l-tuple composition. It is shown also that the biochemical problems connected with the loss of information about the l-tuple DNA composition during hybridization are not crucial and can be overcome by finding the maximal flow of minimal cost in the special graph.

Algorithms↗
PubMed1995

Transcriptional analysis of the Pseudomonas aeruginosa exoenzyme S structural gene.

The transcriptional regulation of the Pseudomonas aeruginosa exoS gene was investigated. Expression of exoS in P. aeruginosa PA103 was dependent upon growth in a low-cation environment and the presence of a functional exsA gene. Promoter fusion analysis indicated that a 285-bp PstI-NsiI fragment, located 5' of the exoS coding region, contained a functional promoter for exoS. Expression of the reporter gene was inducible in a low-cation growth environment and required a functional copy of exsA. Divergent promoters, coordinately regulated with exoS transcription, were identified within the PstI-NsiI fragment. A fusion derivative of ExsA, MALA3A2, was shown to bind directly to the PstI-NsiI probe. DNase I protection analysis demonstrated that MALA3A2 bound to the intergenic region between the postulated -35 boxes of each promoter region. Northern (RNA) blot analysis with probes internal to and upstream of exoS demonstrated that separate, coordinately regulated mRNAs were expressed in P. aeruginosa. These data suggested that a locus, coregulated with exoS transcription, was located upstream of exoS. DNA sequence analysis of the exoS upstream region revealed three open reading frames, ORF 1, ORF 2, and ORF 3. ORF 1 demonstrated significant homology to the SycE/YerA protein of Yersinia sp. SycE/YerA is postulated to function as a chaperone for the YopE cytotoxin. The loci encoding YopE and ExoS show similarities in genetic organization, protein composition, and regulation.

ADP Ribose Transferases↗