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At least 811 records · Page 45Linked to original sources
PubMed2006

Common and variable contributions of Fis residues to high-affinity binding at different DNA sequences.

Fis is a nucleoid-associated protein that interacts with poorly related DNA sequences with a high degree of specificity. A difference of more than 3 orders of magnitude in apparent Kd values was observed between specific (Kd, approximately 1 to 4 nM) and nonspecific (Kd, approximately 4 microM) DNA binding. To examine the contributions of Fis residues to the high-affinity binding at different DNA sequences, 13 alanine substitutions were generated in or near the Fis helix-turn-helix DNA binding motif, and the resulting proteins were purified. In vitro binding assays at three different Fis sites (fis P II, hin distal, and lambda attR) revealed that R85, T87, R89, K90, and K91 played major roles in high-affinity DNA binding and that R85, T87, and K90 were consistently vital for binding to all three sites. Other residues made variable contributions to binding, depending on the binding site. N84 was required only for binding to the lambda attR Fis site, and the role of R89 was dramatically altered by the lambda attR DNA flanking sequence. The effects of Fis mutations on fis P II or hin distal site binding in vitro generally correlated with their abilities to mediate fis P repression or DNA inversion in vivo, demonstrating that the in vitro DNA-binding effects are relevant in vivo. The results suggest that while Fis is able to recognize a minimal common set of DNA sequence determinants at different binding sites, it is also equipped with a number of residues that contribute to the binding strength, some of which play variable roles.

Amino Acid Sequence↗
PubMed1989

Detection of DNA sequence polymorphisms by enzymatic amplification and direct genomic sequencing.

The discovery of RFLPs and their utilization as genetic markers has revolutionized research in human molecular genetics. However, only a fraction of the DNA sequence polymorphisms in the human genome affect the length of a restriction fragment and hence result in an RFLP. Polymorphisms that are not detected as RFLPs are typically passed over in the screening process though they represent a potentially important source of informative genetic markers. We have used a rapid method for the detection of naturally occurring DNA sequence variations that is based on enzymatic amplification and direct sequencing of genomic DNA. This approach can detect essentially all useful sequence variations within the region screened. We demonstrate the feasibility of the technique by applying it to the human retinoblastoma susceptibility locus. We screened 3,712 bp of genomic DNA from each of nine individuals and found four DNA sequence polymorphisms. At least one of these DNA sequence polymorphisms was informative in each of three families with hereditary retinoblastoma that were not informative with any of the known RFLPs at this locus. We believe that direct sequencing is a reasonable alternative to other methods of screening for DNA sequence polymorphisms and that it represents a step forward for obtaining informative markers at well-characterized loci that have been minimally informative in the past.

Base Sequence↗
PubMed1991

Fast Fourier transform-based correlation of DNA sequences using complex plane encoding.

The detection of similarities between DNA sequences can be accomplished using the signal-processing technique of cross-correlation. An early method used the fast Fourier transform (FFT) to perform correlations on DNA sequences in O(n log n) time for any length sequence. However, this method requires many FFTs (nine), runs no faster if one sequence is much shorter than the other, and measures only global similarity, so that significant short local matches may be missed. We report that, through the use of alternative encodings of the DNA sequence in the complex plane, the number of FFTs performed can be traded off against (i) signal-to-noise ratio, and (ii) a certain degree of filtering for local similarity via k-tuple correlation. Also, when comparing probe sequences against much longer targets, the algorithm can be sped up by decomposing the target and performing multiple small FFTs in an overlap-save arrangement. Finally, by decomposing the probe sequence as well, the detection of local similarities can be further enhanced. With current advances in extremely fast hardware implementations of signal-processing operations, this approach may prove more practical than heretofore.

Algorithms↗
PubMed1985

Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences.

The gene coding for the heat-stable and pH-stable alpha-amylase of Bacillus licheniformis 584 (ATCC 27811) was cloned in Escherichia coli and the nucleotide sequence of a DNA fragment of 1,948 base pairs containing the entire amylase gene was determined. As inferred from the DNA sequence, the B. licheniformis alpha-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55,200. The amino acid sequence of B. licheniformis alpha-amylase showed 65.4% and 80.3% homology with those of heat-stable Bacillus stearothermophilus alpha-amylase and relatively heat-unstable Bacillus amyloliquefaciens alpha-amylase, respectively. Nevertheless, several regions of the alpha-amylases appeared to be clearly distinct from one another when their hydropathy profiles were compared.

Amino Acid Sequence↗
PubMed1998

Simple miniaturized gel system for DNA sequence analysis.

A simple miniaturized gel system suitable for DNA sequencing is described. Small ultrathin polyacrylamide gels are cast, eight or more at a time, using standard microscope slides. Gels, ready to use, can be stored for approximately 2 weeks. Gels are run horizontally in a standard mini-agarose gel apparatus. Typical run times are 6-8 min. A novel sample loading system permits volumes of standard sequencing reactions as small as 0.1 microl to be analyzed. Sequencing ladders were visualized using 35S-labeled DNA by autoradiography and by colorimetric detection. Band resolution compares favorably with that of large gels. The methods introduced here serve as a step toward the miniaturization of DNA sequencing and are amenable to automated sample loading and detection.

Autoradiography↗
PubMed2003

DNA sequencing of a plasmid with virulence from marine fish pathogen Vibrio anguillarum.

DNA sequence of a plasmid pEIB1 associated with virulence from the marine fish pathogen Vibrio anguillarum was determined using the methods of restriction endonuclease digestion, subcloning, and primer walking. The whole length of obtained pEIB1 DNA sequence was 66 164 bp, and the overall G+C content of DNA sequence is 42.7%. This sequence encodes 44 open reading frames containing the genes of DNA replication, biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes.

Animals↗
PubMed2002

Comparison of the complete DNA sequences of the Oka varicella vaccine and its parental virus.

The DNA sequences of the Oka varicella vaccine virus (V-Oka) and its parental virus (P-Oka) were completed. Comparison of the sequences revealed 42 base substitutions, which led to 20 amino acid conversions and length differences in tandem repeat regions (R1, R3, and R4) and in an origin of DNA replication. Amino acid substitutions existed in open reading frames (ORFs) 6, 9A, 10, 21, 31, 39, 50, 52, 55, 59, 62, and 64. Of these, 15 base substitutions, leading to eight amino acid substitutions, were in the gene 62 region alone. Further DNA sequence analysis showed that these substitutions were specific for V-Oka and were not present in nine clinical isolates. The immediate-early gene 62 product (IE62) of P-Oka had stronger transactivational activity than the mutant IE62 contained in V-Oka in 293 and CV-1 cells. An infectious center assay of a plaque-purified clone (S7-01) from the V-Oka with 8 amino acid substitutions in ORF 62 showed smaller plaque formation and less-efficient virus-spreading activity than did P-Oka in human embryonic lung cells. Another clone (S-13) with only five substitutions in ORF 62 spread slightly faster than S7-01 but not as effectively as P-Oka. Moreover, transient luciferase assay in 293 cells showed that transactivational activities of IE62s of S7-01 and S7-13 were lower than that of P-Oka. Based on these results, it appears that amino acid substitutions in ORF 62 are responsible for virus growth and spreading from infected to uninfected cells. Furthermore, the Oka vaccine virus was completely distinguishable from P-Oka and 54 clinical isolates by seven restriction-enzyme fragment length polymorphisms that detected differences in the DNA sequence.

Amino Acid Sequence↗
PubMed2004

Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry.

DNA sequencing by synthesis on a solid surface offers new paradigms to overcome limitations of electrophoresis-based sequencing methods. Here we report DNA sequencing by synthesis using photocleavable (PC) fluorescent nucleotides [dUTP-PC-4,4-difluoro-4-bora-3 alpha,4 alpha-diaza-s-indacene (Bodipy)-FL-510, dCTP-PC-Bodipy-650, and dUTP-PC-6-carboxy-X-rhodamine (ROX)] on a glass chip constructed by 1,3-dipolar azide-alkyne cycloaddition coupling chemistry. Each nucleotide analogue consists of a different fluorophore attached to the base through a PC 2-nitrobenzyl linker. We constructed a DNA microarray by using the 1,3-dipolar cycloaddition chemistry to site-specifically attach azido-modified DNA onto an alkyne-functionalized glass chip at room temperature under aqueous conditions. After verifying that the polymerase reaction could be carried out successfully on the above-described DNA array, we then performed a sequencing reaction on the chip by using a self-primed DNA template. In the first step, we extended the primer using DNA polymerase and dUTP-PC-Bodipy-FL-510, detected the fluorescent signal from the fluorophore Bodipy-FL-510, and then cleaved the fluorophore using 340 nm UV irradiation. This process was followed by extension of the primer with dCTP-PC-Bodipy-650 and the subsequent detection of the fluorescent signal from Bodipy-650 and its photocleavage. The same procedure was also performed by using dUTP-PC-ROX. The entire process was repeated five times by using the three fluorescent nucleotides to identify 7 bases in the DNA template. These results demonstrate that the PC nucleotide analogues can be incorporated accurately into a growing DNA strand during polymerase reaction on a chip, and the fluorophore can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing the continuous identification of the template sequence.

Alkynes↗
PubMed2005

Complete mitochondrial DNA sequence of the eastern oyster Crassostrea virginica.

The complete mitochondrial genome of the eastern oyster Crassostrea virginica (GenBank accession number AY905542) is 17,243 bp in length and contains 2 ribosomal genes, 12 protein-coding genes, and 23 transfer RNAs. The arrangement of protein-coding genes is identical to that of the congeneric Pacific oyster C. gigas, but tRNA genes show several duplications and extensive rearrangements between the species. Unique features in C. virginica include an additional trnM gene, the absence of an ATPase subunit 8 (atp8) gene, and an inferred translational frameshift within the cytochrome b (cob) gene. In both species the large subunit ribosomal RNA gene is encoded by 2 separate regions of the mitochondrial genome, the first reported case of a split ribosomal RNA gene in a metazoan. Translation of protein-coding genes in both species is initiated with methionine, with the exception of cob, which uses leucine. In C. virginica translation of all protein-coding genes (except possibly cob) terminates with TAA, with polyadenylation completing the primary transcript in cytochrome oxidase subunit III (cox3) and NADH dehydrogenase subunit 4L (nad4L), whereas C. gigas employs stop codons TAA and TAG equally. Interspecific divergence of mitochondrially encoded proteins is considerable, with amino acid identities ranging from 47% to 92%. A single major noncoding region representing the putative control region is found in both species.

Amino Acid Sequence↗
PubMed2001

Downstream DNA sequences are required to modulate Pvlea-18 gene expression in response to dehydration.

We have previously shown that mRNA and protein encoded by the Pvlea-18 gene from Phaseolus vulgaris L., a member of a new family of late embryogenesis-abundant (LEA) proteins, accumulate in dark-grown bean seedlings not only in response to water deficit but also during optimal irrigation. In this work, we studied Pvlea-18 gene transcriptional regulation by using transgenic Arabidopsis thaliana plants containing a chimeric gene consisting of the Pvlea-18 promoter region and the 3'-nos terminator fused to the GUS gene-coding region. We demonstrate that the chimeric gene is active during Arabidopsis normal development under well-irrigated conditions, and that it is further induced in response to ABA and dehydration treatments. Replacing the 3'-nos terminator with the Pvlea-18 3' region led to an additional increase in expression during development and in response to dehydration, but not in response to exogenous ABA. These results reveal an enhancer effect of the Pvlea-18 3' region, which showed to be higher specifically under dehydration. The small decrease in Pvlea-18 promoter expression observed when transgenic plants treated with fluridone (an ABA biosynthesis inhibitor) were subjected to dehydration suggests that the Pvlea-18 gene dehydration response is predominantly ABA-independent. Finally, we present evidence indicating that Pvlea-18 gene expression is negatively regulated during etiolated growth, particularly in roots, in contrast to the expression pattern observed during normal development.

Abscisic Acid↗
PubMed1998

DNA sequence diversity in a 9.7-kb region of the human lipoprotein lipase gene.

Lipoprotein lipase plays a central role in lipid metabolism and the gene that encodes this enzyme (LPL) is a candidate susceptibility gene for cardiovascular disease. Here we report the complete sequence of a fraction of the LPL gene for 71 individuals (142 chromosomes) from three populations that may have different histories affecting the organization of the sequence variation. Eighty-eight sites in this 9.7 kb vary among individuals from these three populations. Of these, 79 were single nucleotide substitutions and 9 sites involved insertion-deletion variations. The average nucleotide diversity across the region was 0.2% (or on average 1 variable site every 500 bp). At 34 of these sites, the variation was found in only one of the populations, reflecting the differing population and mutational histories. If LPL is a typical human gene, the pattern of sequence variation that exists in introns as well as exons, even for the small number of samples considered here, will present challenges for the identification of sites, or combinations of sites, that influence variation in risk of disease in the population at large.

Base Sequence↗
PubMed1994

Determining the DNA sequence elements required for binding integration host factor to two different target sites.

Binding sites for the Escherichia coli protein integration host factor (IHF) include a set of conserved bases that can be summarized by the consensus sequence WATCAANNNNTTR (W is dA or dT, R is dA or dG, and N is any nucleotide). However, additional 5'-proximal bases, whose common feature is a high dA+dT content, are also thought to be required for binding at some sites. We examine the relative contribution of these two sequence elements to IHF binding to the H' and H1 sites in attP of bacteriophage lambda by using the bacteriophage P22-based challenge-phage system. IHF was unable to act as a repressor in the challenge-phage assay at H' sites containing the core consensus element but lacking the dA+dT-rich element. This indicates that both elements are required for IHF to bind to the H' site. In contrast, the core consensus determinant alone is sufficient for IHF binding to the H1 site, which lacks an upstream dA+dT-rich region. Fifty mutants that decreased or eliminated IHF binding to the H1 site were isolated. Sequence analysis showed changes in the bases in the core consensus element only, further indicating that this determinant is sufficient for IHF binding to the H1 site. We found that placement of a dA+dT-rich element upstream of the H1 core consensus element significantly increased the affinity, suggesting that the presence of a dA+dT-rich element enhances IHF binding.

Bacterial Proteins↗
PubMed2006

Limitations of DNA sequencing for diagnosis of a mixed infection by two fungi, Phaeoacremonium venezuelense and a Plectophomella sp., in a transplant recipient.

We describe a rare case of a subcutaneous infection by both Phaeoacremonium venezuelense and Plectophomella sp. in a Brazilian male. Sequencing of a beta-tubulin gene fragment allowed us to confirm the identification of the former. However, a similar procedure of sequencing rRNA gene fragments was not useful for the identification of the latter fungus.

Adult↗
PubMed2003

Ribosomal DNA sequencing and reinstatement of the genus Arthroascus von Arx.

Sequence analysis of the D1/D2 domain of 26S rDNA was conducted upon seven Arthroascus strains from different geographic localities. The European and Asian species Arthroascus schoenii was documented from the North-American continent and from the Island of Hawaii. We discuss the heterogeneity of the genus Saccharomycopsis sensu Kurtzman and Robnett 1995. On the basis of molecular and genetic data the genus Arthroascus von Arx is reinstated.

Base Sequence↗
PubMed1993

Fluorescent labeling of a DNA sequencing primer.

Several oligonucleotides containing one to four fluorescein labels in various positions were synthesized and the fluorescence intensity and thermal stability of the duplex forms with their complementary sequences were measured. Oligomers that contain two fluorescein molecules, one attached at the 5' terminus and the other at an internal phosphate, were hybridized with less stability than that containing fluorescein at only the 5' terminus, but it formed more efficient primers for dideoxy sequencing with an automated sequencer.

Base Sequence↗
PubMed1983

DNA sequence of two linked actin genes of sea urchin.

DNA sequences have been determined for two actin genes which are closely linked in the genome of the sea urchin Strongylocentrotus purpuratus. The two genes have the same 5'-3' orientation; they were apparently formed originally by tandem gene duplication. The amino acids encoded by the two genes closely resemble those of cytoplasmic actins of mammals and slime molds and differ somewhat from those of mammalian muscle actin. Actin gene 1 had been tentatively identified earlier as the gene for an embryonic cytoplasmic actin by the homology of the 3' noncoding region with that of the cDNA of an embryonic actin mRNA from S. purpuratus. The DNA sequence of gene 1 shows presumptive signals for the initiation and termination of transcription which would govern the formation of a mature mRNA of 1.9 kilobases. Both actin genes 1 and 2 have introns in their coding regions at codons 121/122 and 204. These positions for actin introns have been reported so far only in the rat, not in lower organisms. The divergence of the sequences of these coding-region introns in the two actin genes is 66%, suggesting that the genes diverged about 90 million years ago. By contrast to the introns, the coding regions have been highly conserved; the amino acids of the two genes differ by only 1.3%, and the silent sites of the codons differ by only 12%.

Actins↗
PubMed1996

[Distribution of motifs of functional sites in the MDG2 DNA sequence].

Computer context analysis of the MDG2 (Dm412) DNA sequence was performed, and 530 motifs similar to functional sites from the database were identified. These motifs were found to be nonrandomly distributed along the DNA sequence. Clustering of motifs was revealed in both LTR sequences, in the regions upstream of sORF2, ORF1, and ORF2, and in the spacers between the main polypeptide domains corresponding to ORF2. The total number of clusters found is highly significant due to the presence of a group of the largest "peaks." Internal regions of some ORF contained a decreased number of site motifs (sORF1, ORF1, and ORF2.) In other words, clustering of site motifs was strongly correlated with positions of the regulatory zones in LTR and in the vicinity of the start sites of ORF and their domains. Thus, MGE of Drosophila and other organisms are considered as mobile batteries of functional sites with the increased content of half-finished motifs easily transformed into functional sites. We suggest that these sequences are involved in the adaptive rearrangements of regulatory relationships within molecular genetic regulatory systems.

Animals↗