ZSEQ: an interactive DNA sequence analysis program designed for microcomputers.
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Base Sequence↗
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Results for “DNA sequence analysis”
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Isolation, characterization, and DNA sequence analysis of an AAC(6')-II gene from Pseudomonas aeruginosa.
The gene encoding a 6'-N-acetyltransferase, AAC(6')-II, was cloned from Pseudomonas aeruginosa plasmid pSCH884. This gene mediates resistance to gentamicin, tobramycin, and netilmicin but not amikacin or isepamicin. The DNA sequence of the gene and flanking regions was determined. The 5'- and 3'-flanking sequences showed near identity to sequences found abutting a variety of different genes encoding resistance determinants. It is likely that the current structure arose by the integration of the 572-base-pair sequence containing the AAC(6')-II gene into a Tn21-related sequence at the recombinational hot spot, AAAGTT. We have compared the sequence of the AAC(6')-II gene to genes of other 6'-N-acetyltransferases. An AAC(6')-Ib protein (encoded by the aacA4 gene; G. Tran Van Nhieu and E. Collatz, J. Bacteriol. 169:5708-5714, 1987) that results in resistance to amikacin but not gentamicin was found to share 82% sequence similarity with the AAC(6')-II protein. We speculate that these two genes arose from a common ancestor and that the processes of selection and dissemination have led to the observed differences in the spectrum of aminoglycoside resistance.
Acetyltransferases↗
Partial amino acid sequence of an L-amino acid oxidase from the cyanobacterium Synechococcus PCC6301, cloning and DNA sequence analysis of the aoxA gene.
A novel type of L-amino acid oxidase from Synechococcus PCC6301 was purified and subjected to amino acid sequence analysis. Since the N-terminus of the L-amino acid oxidase protein was not accessible for Edman degradation, the protein was partially hydrolysed and a contiguous sequence of 17 amino acid residues was obtained from an endogenous peptide fragment. Based on the partial peptide sequence two oligonucleotides were designed, which were used as probes in Southern hybridization experiments in order to identify the corresponding aoxA gene. The aoxA gene was isolated from a size-fractionated genomic library of Synechococcus PCC6301 and subsequently sequenced. From the nucleotide sequence (data base accession number Z48565) it can be deduced that the L-amino acid protein consists of 355 amino acid residues resulting in a molar mass of 39.2 kDa. The calculated isoelectric point of the protein is 9.81. The L-amino acid oxidase from Synechococcus PCC6301 shows low homologies to other flavin oxidases/dehydrogenases, especially amine oxidases, but no homologies to other so far sequenced L- or D-amino acid oxidases.
Amino Acid Oxidoreductases↗
DNA sequence analysis of the triose phosphate isomerase gene from isolates of Giardia lamblia.
OBJECTIVE: To confirm the genetic relation between Giardia lamblia (G. lamblia) isolates from different geographic regions of China and other countries. METHODS: Genomic DNA were extracted from the trophozoites or cysts of Giardia lamblia. The triose phosphate isomerase (tim) gene was amplified using polymerase chain reaction (PCR) technique. PCR products were digested with endonuclease and sequenced. The data of sequencing were analyzed with the DNAstar software and compared with that of the isolates acquired from GenBank. RESULTS: Of nine isolates of Giardia lamblia from China (C1, C2, CH2 and CH3), Cambodia (CAM), Australia (A1 and A2) and America (BP and CDC), respectively, 3 (A1, A2 and CAM) fit into Group 1 (WB), 2 (CH2 and CH3)) into Group 2, and 4 (C1, C2, BP and CDC) into Group 3 (GS). The results confirmed the genetic relatedness of G. lamblia isolates from all over the world. CONCLUSION: Genotyping isolates of G. Lamblia provides important information for establishing the phylogenetic relationship or for the epidemiological evaluation of the spreading of this organism.
Amino Acid Sequence↗
Biogenesis of mitochondria: DNA sequence analysis of mit- mutations in the mitochondrial oli2 gene coding for mitochondrial ATPase subunit 6 in Saccharomyces cerevisiae.
A series of yeast mitochondrial mit- mutants with defects in the oli2 gene, coding for subunit 6 of the mitochondrial ATPase complex, has been analyzed at the DNA sequence level. Fifteen of sixteen primary mit- mutants were shown to contain frameshift or nonsense mutations predicting truncated subunit 6 polypeptides, in various strains ranging from about 20% to 95% of the wild-type length of 259 amino acids. In only one strain could the defect in subunit 6 function be assigned to amino acid substitution in an otherwise full-length subunit 6. Many mutants carried multiple base substitutions or insertions/deletions, presumably arising from the manganese chloride mutagenesis treatment. Revertants from three of the mit- mutants were analyzed: all contained full-length subunit 6 proteins with one or more amino acid substitutions. The preponderance of truncated proteins as opposed to substituted full-length proteins in oli2 mit- mutants is suggested to reflect the ability of subunit 6 to accommodate amino acid substitutions at many locations, with little or no change in its functional properties in the membrane FO-sector of the ATPase complex.
Adenosine Triphosphatases↗
PCR detection and DNA sequence analysis of the regulatory region of lymphotropic papovavirus in peripheral blood mononuclear cells of an immunocompromised rhesus macaque.
A lymphotropic papovavirus (LPV) archetypal regulatory region was amplified from DNA from the blood of an immunocompromised rhesus monkey. We believe this is the first nonserological evidence of LPV infection in rhesus monkeys.
Animals↗
DNA sequence analysis of spontaneous and beta-methoxy-acrolein-induced mutations in Salmonella typhimurium hisD3052.
The histidine auxotroph hisD3052 results from a single base-pair deletion (C:G) from the 298th codon (alanine) in the D gene of the histidine operon of S. typhimurium LT-2. Bacteria carrying this mutation revert to histidine prototrophy by frameshift mutations (additions or deletions) that restore the correct reading frame. Among the compounds that revert hisD3052 are the naturally occurring dicarbonyl compound malondialdehyde and a structural analog, beta-methoxy-acrolein. To determine the sequence changes responsible for reversion, spontaneous and beta-methoxy-acrolein induced revertants were isolated, male derivatives constructed, and infected with M13Ho167.18, a phage carrying partial O, D, and C genes of the histidine operon. M13hisD+ recombinants were selected by complementation in TA2890, single-stranded DNA was isolated, and the reverted D genes were sequenced using Sanger dideoxy chain-termination sequencing with a synthetic oligonucleotide primer. Analysis of 37 spontaneous revertants revealed 41% additions and 59% deletions with 22% of the mutations occurring as two base-pair (-CG-) deletions in a -CG- rich 'hot spot'. The profile of additions was +1 (30%), +4 (8%), +7 (3%); the profile of deletions was -2 (32%), -5 (11%), -8 (8%), and -11 (8%). Analysis of 27 beta-methoxy-acrolein-induced revertants revealed 96% additions and 4% deletions. The profile of induced additions was +1 (70%), +4 (22%), +7 (4%). No additions occurred in the -CG- rich hot-spot. Frameshift reversions of the hisD3052 gene demonstrate a surprising degree of sequence diversity and reveal the ability of the hisD3052 tester strain to detect a wide variety of frameshift reversion events. In addition, the results demonstrate that beta-methoxy-acrolein induces a high percentage of additions in this reversion system.
Acrolein↗
DNA sequence analysis of the ade6 gene of Schizosaccharomyces pombe. Wild-type and mutant alleles including the recombination host spot allele ade6-M26.
The gene ade6 is located on chromosome III of the fission yeast Schizosaccharomyces pombe. It codes for the enzyme phosphoribosylaminoimidazole carboxylase involved in purine biosynthesis. A DNA fragment of 3043 nucleotides has been sequenced. It complements ade6 mutations when present on plasmids. An uninterrupted open reading frame of 552 amino acid residues was identified. A method for the cloning of chromosomal mutations by repair of gapped replication vectors in vivo has been developed. Twelve ade6 mutant alleles have been isolated. The sequence alterations of four mutant alleles have been determined. Among them are the ade6-M26 recombination hot spot mutation and the nearby ade6-M375 control mutation. Both are G to T base substitutions, converting adjacent glycine codons to TGA termination codons. They are suppressed by defined tRNA nonsense suppressors of the UGA type. The ade6-M26 mutation leads to a tenfold increase of the occurrence of conversion tetrads in comparison with other ade6 mutations. Possible explanations for the M26-induced increase of recombination frequency are discussed in relation to specific features of the nucleotide sequence identified in the region of the M26 mutation.
Alleles↗
DNA sequence analysis of the dye gene of Escherichia coli reveals amino acid homology between the dye and OmpR proteins.
Mutation of the dye gene of Escherichia coli results in sensitivity of dyes, envelope protein changes, loss of expression of alkaline phosphatase, and reduced transcription of sex factor F genes. We have determined the DNA sequence of a 1.4-kilobase pair fragment encompassing the dye gene. The coding sequence of dye was identified as an open reading frame coding for a protein of Mr 27,346. A sequence of 54 residues at the amino terminus was extremely acidic, with 12 aspartic plus glutamic acid residues and only 2 lysine plus arginine residues. A sequence of 19 adjacent residues near the center of the protein was identical, except for one mismatch, with a sequence in the OmpR protein, involved in controlling the amounts of the major outer membrane proteins OmpF and OmpC at the level of transcription. 28% of the Dye protein was homologous with OmpR. The positions of dye and ompR on the genetic map were indicative of a gene duplication. It seems likely, therefore, that the Dye and OmpR proteins are related, and Dye may thus be involved in the osmoregulation of envelope protein genes as well as being required for sex factor gene expression. The Dye protein itself, like OmpR, was shown not to be an envelope protein. A second open reading frame on the other DNA strand may use the same transcription termination site as dye.
Amino Acid Sequence↗
DNA sequence analysis using hierarchical ART-based Classification Networks.
Adaptive resonance theory (ART) describes a class of artificial neural network architectures that act as classification tools which self-organize, work in realtime, and require no retraining to classify novel sequences. We have adapted ART networks to provide support to scientists attempting to categorize tandem repeat DNA fragments from Onchocerca volvulus. In this approach, sequences of DNA fragments are presented to multiple ART-based networks which are linked together into two (or more) tiers; the first provides coarse sequence classification while the subsequent tiers refine the classifications as needed. The overall rating of the resulting classification of fragments is measured using statistical techniques based on those introduced by Zimmerman, et al. (1994) to validate results from traditional phylogenetic analysis. Tests of the Hierarchical ART-based Classification Network, or HABclass network, indicate its value as a fast, easy-to-use classification tool which adapts to new data without retraining on previously classified data.
Algorithms↗
DNA sequence analysis of the C4 antigen WH: evidence for two mechanisms of expression.
Amino acid and protein analyses have allowed the construction of a model for the C4-based Rodgers and Chido blood group antigens. The single low-frequency allele (WH) in this blood group system, however, has not been characterized at the molecular level. Two WH+ donors were studied by C4 agarose gel electrophoreses, immunoblot studies using monoclonal anti-Rg: 1 or anti-Ch: 1, serological phenotyping, polymerase chain reaction-restriction fragment length polymorphism of their C4 genes, and DNA sequencing of the WH allele. The first donor had the C4A1, A3 phenotype; the C4A1 carried Ch: 1, 3, 6 (thus exhibiting reversed antigenicity) and the C4A3 carried the WH antigen. The amino acid sequence of the WH allele was PCPVLD at positions 1101 - 1106, S at position 1157, and VDLL at positions 1188 - 1191. A second donor typed as C4A2, A4, B1 and was also WH+. Immunoblot analysis showed that a C4B1 protein expressed Rg: 1. Sequence analysis of the C4B genes showed the amino acids LSPVIH at positions 1101 - 1106, S at position 1157, and ADLR at positions 1188 - 1191. Thus, the WH antigen is a conformational epitope that can arise through different mechanisms on either a C4A or C4B gene.
Alleles↗
DNA sequence analysis of three Lactococcus lactis plasmids encoding phage resistance mechanisms.
The three Lactococcus lactis plasmids pSRQ700, pSRQ800, and pSRQ900 encode the previously described anti-phage resistance mechanisms LlaDCHI, AbiK, and AbiQ, respectively. Since these plasmids are likely to be introduced into industrial Lactococcus lactis strains used to manufacture commercial fermented dairy products, their complete DNA sequences were determined and analyzed. The plasmids pSRQ700 (7784 bp), pSRQ800 (7858 bp), and pSRQ900 (10,836 bp) showed a similar genetic organization including a common lactococcal theta-type replicon. A second replication module showing features of the pMV158 family of rolling circle replicons was also found on pSRQ700. The theta replication regions of the three plasmids were associated with two additional coding regions, one of which encodes for HsdS, the specificity subunit of the type I restriction/modification system. When introduced into L. lactis IL1403, the HsdS of pSRQ800 and pSRQ900 conferred a weak resistance against phage P008 (936 species). These results indicated that both HsdS subunits can complement the chromosomally encoded type I restriction/modification system in IL1403. The genes involved in the phage resistance systems LlaDCHI, AbiK, and AbiQ were found in close proximity to and downstream of the replication modules. In pSRQ800 and pSRQ900, transfer origins and putative tyrosine recombinases were found upstream of the theta replicons. Genes encoding recombination proteins were also found on pSRQ700. Finally, open reading frames associated with bacteriocin production were found on pSRQ900, but no anti-lactococcal activity was detected. Based on our current knowledge, these three plasmids are safe and suitable for food-grade applications.
Amino Acid Sequence↗
DNA sequence analysis of the C3H H-2Kk and H-2Dk loci. Evolutionary relationships to H-2 genes from four other mouse strains.
We generated nucleotide sequences for H-2Kk and H-2Dk from the C3H mouse, as well as for a genomic clone of H-2Db, in order to conduct an evolutionary analysis of the H-2 genes from three haplotypes, k, d, and b. H-2Kk from both the C3H and AKR strains, H-2Kd, H-2Kb, H-2Dk, H-2Ld, H-2Dd, H-2Db, and H-2Dp DNA sequences were aligned, and the alignments used to construct phylogenetic trees inferring the evolutionary relationships among the nine genes by two independent methods. Both approaches yielded trees with similar topologies. In addition, the sequence alignments revealed patterns of nucleotide substitutions which implicate both point mutation and recombination in the divergence of the H-2 genes. Future considerations for evolutionary analysis of class I genes are discussed.
Amino Acid Sequence↗
Exonuclease III: use for DNA sequence analysis and in specific deletions of nucleotides.
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Base Sequence↗
DNA sequence analysis of spontaneous histidine mutations in a polA1 strain of Escherichia coli K12 suggests a specific role of the GTGG sequence.
Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level. We screened approximately 150,000 colonies and isolated 106 histidine auxotrophs. Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5'-GCTGGCTGGCTGGCTG-3'. Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA). A single hisA point mutation was found to be a missense mutation. Two extended deletions, covering the his operon were not analysed. We could not identify the hisC deletion by sequencing. We conclude that polA1 is a strong mutator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences. Finally, the possible role of a 5'-GTGG-3' sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed.
Base Sequence↗
DNA sequence analysis of 66 kb of the human MHC class II region encoding a cluster of genes for antigen processing.
The genomic sequence of a 66,109 bp long region within the human MHC has been determined by manual and automated DNA sequencing. From cDNA mapping and sequencing data it is known that this region contains a cluster of at least four genes that are believed to be involved in antigen processing. Here, we describe the genomic organization of these genes, which comprise two proteasome-related genes (LMP2 and LMP7), thought to be involved in the proteolytic degradation of cytoplasmic antigens and two ABC transporter genes (TAP1 and TAP2), thought to be involved in pumping of the degraded peptides across the endoplasmic reticulum membrane. Analysis of the sequence homology and the intron/exon structures of the corresponding genes suggests that one gene pair arose by duplication from the other. Comparison of the available sequence data from other organisms shows striking conservation (70 to 84%) of this gene cluster in human, mouse and rat. The presence of several potential interferon stimulated response elements (ISREs) is in agreement with the experimentally observed up-regulation of these genes with gamma-interferon.
Amino Acid Sequence↗
Characterization of Borrelia lusitaniae sp. nov. by 16S ribosomal DNA sequence analysis.
We determined the complete sequence of the rrs gene from five strains of genomic species PotiB2. Both distance and parsimony methods were used to infer the evolutionary relationships of the rrs gene sequence of this genomic species in comparison with the rrs gene sequence of Borrelia valaisiana and the rrs gene sequences of Borrelia burgdorferi sensu lato species obtained from sequence databases. The phylogenetic analysis revealed that the genomic species PotiB2 strains clustered in a separate lineage, which was consistent with data from previous DNA-DNA hybridization experiments (D. Postic, M. V. Assous, P. A. D. Grimont, and G. Baranton, Int. J. Syst. Bacteriol. 44:743-752, 1994). A PCR-restriction fragment length polymorphism analysis was used to identify genomic species PotiB2 and to differentiate it from B. burgdorferi sensu lato species. Moreover, signature nucleotide positions were identified for each B. burgdorferi sensu lato species. In accordance with DNA relatedness values, our findings suggest that genomic species PotiB2 can be more clearly defined and identified, and we propose that it should be referred to as a new species, Borrelia lusitaniae. The type strain is PotiB2.
Bacterial Proteins↗
Section-level relationships of North American Agalinis (Orobanchaceae) based on DNA sequence analysis of three chloroplast gene regions.
BACKGROUND: The North American Agalinis are representatives of a taxonomically difficult group that has been subject to extensive taxonomic revision from species level through higher sub-generic designations (e.g., subsections and sections). Previous presentations of relationships have been ambiguous and have not conformed to modern phylogenetic standards (e.g., were not presented as phylogenetic trees). Agalinis contains a large number of putatively rare taxa that have some degree of taxonomic uncertainty. We used DNA sequence data from three chloroplast genes to examine phylogenetic relationships among sections within the genus Agalinis Raf. (=Gerardia), and between Agalinis and closely related genera within Orobanchaceae. RESULTS: Maximum likelihood analysis of sequences data from rbcL, ndhF, and matK gene regions (total aligned length 7323 bp) yielded a phylogenetic tree with high bootstrap values for most branches. Likelihood ratio tests showed that all but a few branch lengths were significantly greater than zero, and an additional likelihood ratio test rejected the molecular clock hypothesis. Comparisons of substitution rates between gene regions based on linear models of pairwise distance estimates between taxa show both ndhF and matK evolve more rapidly than rbcL, although the there is substantial rate heterogeneity within gene regions due in part to rate differences among codon positions. CONCLUSIONS: Phylogenetic analysis supports the monophyly of Agalinis, including species formerly in Tomanthera, and this group is sister to a group formed by the genera Aureolaria, Brachystigma, Dasistoma, and Seymeria. Many of the previously described sections within Agalinis are polyphyletic, although many of the subsections appear to form natural groups. The analysis reveals a single evolutionary event leading to a reduction in chromosome number from n = 14 to n = 13 based on the sister group relationship of section Erectae and section Purpureae subsection Pedunculares. Our results establish the evolutionary distinctiveness of A. tenella from the more widespread and common A. obtusifolia. However, further data are required to clearly resolve the relationship between A. acuta and A. tenella.
Chloroplasts↗