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PubMed2000

Characterization of gyrA mutations associated with fluoroquinolone resistance in Campylobacter coli by DNA sequence analysis and MAMA PCR.

Increasing numbers of fluoroquinolone-resistant Campylobacter coli isolates received at the Minnesota State Public Health Laboratory and at the Centers for Disease Control and Prevention have been a cause for concern. The gyrA quinolone resistance-determining regions of several fluoroquinolone-resistant isolates were sequenced to examine the mechanism of resistance. Ciprofloxacin-resistant C. coli isolates examined by DNA sequencing had a Thr-86 to Ile (ACT-->ATT) gyrA mutation, leading to resistance to fluoroquinolone antibiotics. A mismatch amplification mutation assay polymerase chain reaction protocol was developed to detect this gyrA mutation.

Anti-Infective Agents↗

PubMed1984

A computer package for DNA sequence analysis.

We have developed a comprehensive software-hardware package for a CP/M based or other microcomputer which can perform four major functions in a molecular biology laboratory: (1) the acquisition and analysis of DNA sequence data, (2) word processing in the preparation of scientific manuscripts and figures, (3) general lab computation and data reduction, and (4) as a data terminal for communication of data over the phone line.

Base Sequence↗

PubMed1994

Phylogenetic relationships between some members of the genera Neisseria, Acinetobacter, Moraxella, and Kingella based on partial 16S ribosomal DNA sequence analysis.

We obtained 16S ribosomal DNA (rDNA) sequence data for strains belonging to 11 species of Proteobacteria, including the type strains of Kingella kingae, Neisseria lactamica, Neisseria meningitidis, Moraxella lacunata subsp. lacunata, [Neisseria] ovis, Moraxella catarrhalis, Moraxella osloensis, [Moraxella] phenylpyruvica, and Acinetobacter lwoffii, as well as strains of Neisseria subflava and Acinetobacter calcoaceticus. The data in a distance matrix constructed by comparing the sequences supported the proposal that the genera Acinetobacter and Moraxella and [N.] ovis should be excluded from the family Neisseriaceae. Our results are consistent with hybridization data which suggest that these excluded taxa should be part of a new family, the Moraxellaceae. The strains that we studied can be divided into the following five groups: (i) M. lacunata subsp. lacunata, [N.] ovis, and M. catarrhalis; (ii) M. osloensis; (iii) [M.] phenylpyruvica; (iv) A. calcoaceticus and A. lwoffii; and (v) N. meningitidis, N. subflava, N. lactamica, and K. kingae. We agree with the previous proposal that [N.] ovis should be renamed Moraxella ovis, as this organism is closely related to Moraxella species and not to Neisseria species. The generically misnamed taxon [M.] phenylpyruvica belongs to the proposed family Moraxellaceae, but it is sufficiently different to warrant exclusion from the genus Moraxella. Further work needs to be done to investigate genetically similar species, such as Psychrobacter immobilis, before the true generic position of this organism can be determined. Automated 16S rDNA sequencing with the PCR allows workers to accurately determine phylogenetic relationships between groups of organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Acinetobacter↗

PubMed1995

DNA sequence analysis of a cyclophilin gene from maize: developmental expression and regulation by salicylic acid.

In plants, such as maize, cyclophilin (Cyp) genes are expressed at a basal level in all tissues. Amounts of Cyp mRNA above the basic level are observed in germinating seedlings, in growing tissues/organs such as roots and leaf meristematic tissue of young maize plants, nodes and embryonic female inflorescences of adult plants and also in non-proliferating tissues such as the internodes of adult plants. Salicylic acid (SA) enhances the transcription of maize Cyp genes. The possible involvement of SA in the pathway leading to defense responses induced by abiotic stresses such as mercuric chloride treatment is discussed. A maize Cyp genomic clone isolated using a maize Cyp cDNA probe contains 737 bp of the 5' upstream and the entire coding region. This Cyp gene is not interrupted by intervening sequences. In the 5' upstream region, characteristic transcription signals as well as putative regulatory sequences were identified. Two TATA boxes are found at positions -56 bp and -66 bp with respect to the transcription start site. Two putative heat shock elements were identified in the promoter region; a metal regulatory element and a third heat shock element were localized in the 5' untranslated leader. Several putative polyadenylation signals and (G)T-rich sequence motifs were identified in the 3' untranslated region.

Amino Acid Isomerases↗

PubMed2006

Genetic mutations in von Willebrand disease identified by DHPLC and DNA sequence analysis.

Von Willebrand disease (VWD) is a common inherited bleeding disorder caused by quantitative (types 1 and 3) and qualitative (type 2) defects in von Willebrand factor (VWF). The VWF gene is a large gene containing 52 exons; except for type 2 VWD, the majority of mutations causing VWD are not localized to specific exons. We have used denaturing high performance liquid chromatography (DHPLC) to scan the coding region of the VWF gene for sequence variations. Primers were designed to amplify all 52 exons while avoiding amplification of the VWF pseudogene. Exon-specific primers were designed with sequencing primers, allowing direct sequencing of each VWF exon. Sequence variations in 33 previously characterized von Willebrand disease (VWD) samples were all detected using DHPLC demonstrating the high sensitivity of this technique. In addition, we analyzed 42 patients or family members with VWD. Thirty-two novel sequence variations were identified (2 deletions, 2 nonsense, 15 missense, 6 silent, and 7 intronic), some with clear functional consequences. A previously described deletion in exon 18, 2435delC, was also found in two unrelated type 3 patients. This DHPLC and DNA sequencing technique will enable the full length assessment of the VWF gene necessary to detect mutations causing types 1 and 3 VWD.

Base Composition↗

PubMed1999

Organisation of the mitochondrial genome of Trichophyton rubrum III. DNA sequence analysis of the NADH dehydrogenase subunits 1, 2, 3, 4, 5 and the cytochrome b gene.

In this paper, we present the nucleotide sequence of a 9761 nt-long segment of the mitochondrial genome of the dermatophyte Trichophyton rubrum that bridges the gap between two previously published segments, making a unique contig that represents approximately 80% of the molecule. The location of all genes on the map is determined except for some tRNA genes expected to flank the LSU rRNA gene not yet sequenced. Starting from the 5' end of the present sequence, we recognized the ND5 and ND2 genes, the cytochrome b gene, an unusually long intergenic spacer of unknown function, as well as the ND3, ND1 and ND4 genes. This sequence extends and confirms the similarity with the mitochondrial genome of Aspergillus nidulans. Interestingly, two cases of partial overlaps between the terminator and initiator codons of successive genes (ND4-ND5 and ND5-ND2) are encountered.

Amino Acid Sequence↗

PubMed2004

Specific detection and typing of Mycoplasma synoviae strains in poultry with PCR and DNA sequence analysis targeting the hemagglutinin encoding gene vlhA.

Mycoplasma synoviae is a major pathogen of chickens and turkeys, causing economic losses to the poultry industry worldwide. In this study, we validated and applied polymerase chain reaction (PCR) and DNA sequence analysis on the N-terminal end of the hemagglutinin encoding gene vlhA as an alternative for the detection and initial typing of field strains of M. synoviae in commercial poultry. PCR primers were tested against isolates of M. synoviae from various sources along with other avian mycoplasma and other bacterial species. The vlhA gene-targeted PCR assay was highly specific in the identification of M. synoviae, with a detection limit of 4.7 x 10(2) color changing units/ml. DNA sequence analysis of amplified products was also conducted to validate the potential for typing M. synoviae strains using the N-terminal region of the vlhA gene. To evaluate the test, we applied the PCR assay to tracheal swabs collected from chickens challenged with M. synoviae strain K1968 and compared the results to the serologic detection. The PCR assay was also evaluated directly on tracheal samples collected from commercial layers. Overall, this vlhA gene-targeted PCR is a useful tool for detection and initial typing of M. synoviae and can be applied in the preliminary identification of M. synoviae isolates directly from clinical samples.

Agglutination Tests↗

PubMed1980

Characterisation of polyoma late mRNA leader sequences by molecular cloning and DNA sequence analysis.

The leader sequences of two of the three polyoma virus late mRNAs were characterised by molecular cloning and DNA sequence analysis. A short single-stranded DNA fragment complementary to the 5' end of the body of mVP1 was used to prime cDNA synthesis, and double-stranded cDNA was inserted into a derivative of pAT 153. Analysis of fourteen mVP1 cDNAs and two mVP3 cDNAs allowed the precise determination of the leader-body joints, and demonstrated that the majority of polyoma late leader sequences consist of exact tandem repeats of a 57 nucleotide sequence present only once in the genomic DNA at 66-67 m.u. The sequences in the genomic DNA borderline this unit are typical of those found at RNA splice points. Leader sequences contained on average three to four repeat units. Nuclease S1 mapping of total late mRNA demonstrated that most mRNA 5' ends map heterogeneously in the 50 nucleotides 5' to the repeated sequence unit. The structures of the leader sequences strongly suggest that they are generated by appropriate splicing events from a tandemly repeated transcript of the entire circular viral genome.

Base Sequence↗

PubMed1995

Genomic organization of the human PAX3 gene: DNA sequence analysis of the region disrupted in alveolar rhabdomyosarcoma.

Mutations in the human PAX3 gene have previously been associated with two distinct diseases, Waardenburg syndrome and alveolar rhabdomyosarcoma. In this report we establish that the normal human PAX3 gene is encoded by 8 exons. Intron-exon boundary sequences were obtained for PAX3 exons 5, 6, 7, and 8 and together with previous work provide the complete genomic sequence organization for PAX3. Difficulties in obtaining overlapping genomic clone coverage of PAX3 were circumvented in part by RARE cleavage mapping, which showed that the entire PAX3 gene spans 100 kb of chromosome 2. Sequence analysis of the last intron of PAX3, which contains the previously mapped t(2;13)(q35;q14) translocation breakpoints of alveolar rhabdomyosarcoma, revealed the presence of a pair of inverted Alu repeats and a pair of inverted (GT)n-rich microsatellite repeats within a 5-kb region. This work establishes the complete structure of PAX3 and will permit high-resolution analyses of this locus for mutations associated with Waardenburg syndrome, alveolar rhabdomyosarcoma, and other phenotypes for which PAX3 may be a candidate locus.

Alleles↗

PubMed2004

DNA Sequence analysis of the PorB protein of nonserotypeable serogroup C ET-15 meningococci suggests a potential mutational hot spot on their serotype antigens.

The nucleotide sequences of the PorB proteins from 28 nonserotypeable serogroup C ET-15 meningococci recovered from invasive meningococcal disease cases were determined. PCR amplification of the porB genes responsible for encoding the serotype antigen was used for DNA sequence determination and identification of the nature of the serotype antigen. DNA sequencing revealed that three strains were of serotype 2a, and of the remaining 25 strains, 20 were found to have an identical single point mutation in the region of the VR3 gene, which encodes surface-exposed loop VI, where the serotype 2a epitope resides. This nonsynonymous mutation was confirmed by synthetic peptide immunochemical analysis to confer new serospecificity to these serotype 2a mutants. This finding of a potential novel mutational hot spot on the PorB proteins of meningococci may have implications for pathogenesis and vaccine development.

Amino Acid Sequence↗

PubMed1997

Physical mapping 220 kb centromeric of the human MHC and DNA sequence analysis of the 43-kb segment including the RING1, HKE6, and HKE4 genes.

A cosmid contig was constructed from a YAC clone with a 220-kb insert that spans the centromeric side of the human MHC class II region, corresponding to the mouse t complex. The gene order was identified to be HSET-HKE1.5-HKE2-HKE3-RING1-HKE6- HKE4 (RING5). The genomic sequence of a 42,801-bp long region encoded by one cosmid clone in the RING1, HKE6, and HKE4 subregions was determined by the shotgun method. The exon-intron organization of these three genes, RING1 (Ring finger protein), HKE6 (steroid dehydrogenase-like protein), and HKE4 (transmembrane protein with histidine-rich charge clusters), was determined. The previously reported RING2 gene was revealed to be identical to HKE6. Transcripts from HKE4 were detected in the placenta, lung, kidney, and pancreas. Those of HKE6 were found in the liver and pancreas. The 25-kb region proximal to the RING1 gene includes an extensive dense cluster of Alu repeats (about 1.2 Alu per kb), and no gene has been identified in this so far. The region is equivalent to part of the mouse t complex and could be of relevance to human development.

Amino Acid Sequence↗

PubMed1992

Cloning and DNA sequence analysis of an aac(3)-Vb gene from Serratia marcescens.

The AAC(3)-V resistance mechanism is characterized by high-level resistance to the aminoglycosides gentamicin, netilmicin, 2'-N-ethylnetilmicin, and 6'-N-ethylnetilmicin and moderate resistance levels to tobramycin. Serratia marcescens 82041944 contains an AA(3)-V resistance mechanism as determined from aminoglycoside resistance profiles. This strain, however, does not exhibit hybridization with a probe derived from the previously cloned aac(3)-Va gene, (R. Allmansberger, B. Bräu, and W. Piepersberg, Mol. Gen. Genet. 198:514-520, 1985). High-pressure liquid chromatography analysis of the acetylation products of sisomicin carried out by extracts of S. marcescens 82041944 have demonstrated the presence of an AAC(3) enzyme. We have cloned the gene encoding this acetyltransferase and have designated it aac(3)-Vb. Nucleotide sequence comparisons show that the aac(3)-Va and aac(3)-Vb genes are 72% identical. The predicted AAC(3)-Vb protein is 28,782 Da. Comparisons of the deduced amino acid sequences show 75% identity and 84% similarity between the AAC(3)-Va and AAC(3)-Vb proteins. The use of a DNA fragment internal to the aac(3)-Vb as a hybridization probe demonstrated that the aac(3)-Vb gene is very rare in clinical isolates possessing an AAC(3)-V mechanism.

Amino Acid Sequence↗

PubMed2006

Microbial source tracking by DNA sequence analysis of the Escherichia coli malate dehydrogenase gene.

Criteria for sub-typing of microbial organisms by DNA sequencing proposed by Olive and Bean were applied to several genes in Escherichia coli to identify targets for the development of microbial source tracking assays. Based on the aforementioned criteria, the icd (isocitrate dehydrogenase), and putP (proline permease) genes were excluded as potential targets due to their high rates of horizontal gene transfer; the rrs (16S rRNA) gene was excluded as a target due to the presence of multiple gene copies, with different sequences in a single genome. Based on the above criteria, the mdh (malate dehydrogenase) gene was selected as a target for development of a microbial source tracking assay. The mdh assay was optimized to analyze a 150 bp fragment corresponding to residues G191 to R240 (helices H10 and H11) of the Mdh catalytic domain. 295 fecal isolates (52 horse, 50 deer, 72 dog, 52 seagull and 69 human isolates) were sequenced and analyzed. Target DNA sequences for isolates from horse, dog plus deer, and seagull formed identifiable groupings. Sequences from human isolates, aside from a low level (ca. 15%) human specific sequence, did not group; nevertheless, other hosts could be distinguished from human. Positive and negative predictive values for two- and three-way host comparisons ranged from 60% to 90% depending on the focus host. False positive rates were below 10%. Multiple E. coli isolates from individual fecal samples exhibited high levels of sequence homogeneity, i.e. typically only one to two mdh sequences were observed per up to five E. coli isolates from a single fecal sample. Among all isolates sequenced from fecal samples from each host, sequence homogeneity decreased in the following order: horse>dog>deer>human and gull. For in-library isolates, blind analysis of fecal isolates (n=12) from four hosts known to contain host specific target sequences was 100% accurate and 100% reproducible for both DNA sequence and host identification. For blind analysis of non-library isolates, 18/19 isolates (94.7%) matched one or more library sequences for the corresponding host. Ten of eleven geographical outlier fecal isolates from Florida had mdh sequences that were identical to in-library sequences for the corresponding host from California. The mdh assay was successfully applied to environmental isolates from an underground telephone vault in California, with 4 of 5 isolates matching sequences in the mdh library. 146 sequences of the 645bp mdh fragment from five host sources were translated into protein sequence and aligned. Seven unique Mdh protein sequences, which contained eight polymorphic sites, were identified. Six of the polymorphic sites were in the NAD+ binding domain and two were in the catalytic domain. All of the polymorphic sites were located in surface exposed regions of the protein. None of the non-silent mutations of the Mdh protein were in the 150bp mdh target. The advantages and disadvantages of the assay compared to established source tracking methods are discussed.

Animals↗

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↗

PubMed2000

Identification of coryneform bacterial isolates by ribosomal DNA sequence analysis.

Identification of coryneform bacteria to the species level is important in certain circumstances for differentiating contamination and/or colonization from infection, which influences decisions regarding clinical intervention. However, methods currently used in clinical microbiology laboratories for the species identification of coryneform bacteria are often inadequate. We evaluated the MicroSeq 500 16S bacterial sequencing kit (Perkin-Elmer Biosystems, Foster City, Calif.), which is designed to sequence the first 527 bp of the 16S rRNA gene for bacterial identification, by using 52 coryneform gram-positive bacilli from clinical specimens isolated from January through June 1993 at the Mayo Clinic. Compared to conventional and supplemented phenotypic methods, MicroSeq provided concordant results for identification to the genus level for all isolates. At the species level, MicroSeq provided concordant results for 27 of 42 (64.3%) Corynebacterium isolates and 5 of 6 (83.3%) Corynebacterium-related isolates, respectively. Within the Corynebacterium genus, MicroSeq gave identical species-level identifications for the clinically significant Corynebacterium diphtheriae (4 of 4) and Corynebacterium jeikeium (8 of 8), but it identified only 50.0% (15 of 30) of other species (P < 0.01). Four isolates from the genera Arthrobacter, Brevibacterium, and Microbacterium, which could not be identified to the species level by conventional methods, were assigned a species-level identification by MicroSeq. The total elapsed time for running a MicroSeq identification was 15.5 to 18.5 h. These data demonstrate that the MicroSeq 500 16S bacterial sequencing kit provides a potentially powerful method for the definitive identification of clinical coryneform bacterium isolates.

Actinomycetales↗

PubMed1995

35H, a sequence isolated as a protein kinase C binding protein, is a novel member of the adducin family.

We recently cloned a partial cDNA (35H) for a protein kinase C (PKC) binding protein from a rat kidney cDNA library and demonstrated that it is a PKC substrate in vitro (Chapline, C., Ramsay, K., Klauck, T., and Jaken, S. (1993) J. Biol. Chem. 268, 6858-6861). Additional library screening and 5' rapid amplification of cDNA ends were used to obtain the complete open reading frame. Amino acid sequence analysis, DNA sequence analysis, and Northern analysis indicate that 35H is a unique cDNA related to alpha-and beta-adducins. Antisera prepared to the 35H bacterial fusion protein recognized two polypeptides of 80 and 90 kDa on immunoblots of kidney homogenates and cultured renal proximal tubule epithelial cell extracts. The 35H-related proteins were similar to alpha- and beta-adducins in that they were preferentially recovered in the Triton X-100-insoluble (cytoskeletal, CSK) fraction of cell extracts and were predominantly localized to cell borders. Phorbol esters stimulated phosphorylation of CSK 35H proteins, thus emphasizing that sequences isolated according to PKC binding activity in vitro are also PKC substrates in vivo. The phosphorylated forms of the 35H proteins were preferentially recovered in the soluble fraction, thus demonstrating that phosphorylation regulates their CSK association and, thereby, their function in regulating cytoskeletal assemblies. We have isolated another PKC binding protein partial cDNA (clone 45) from a rat fibroblast library with substantial homology to alpha-adducin. Antisera raised against this expressed sequence recognized a protein of 120 kDa, the reported size of alpha-adducin, on immunoblots of renal proximal tubule epithelial cell extracts. A 120-kDa protein that cross-reacts with the clone 45 (alpha-adducin) antisera coprecipitated with 35H immunecomplexes, indicating that alpha-adducin associates with 35H proteins in vivo. Taken together, these results indicate that 35H is a new, widely expressed form of adducin capable of forming heterodimers with alpha-adducin. We propose naming this adducin homologue gamma-adducin.

Amino Acid Sequence↗

PubMed1974

DNA sequence analysis: a formula to predict electrophoretic mobilities of oligonucleotides on cellulose acetate.

A simple method has recently become available for sequence analysis of large oligonucleotide fragments. Sequences are derived from the characteristic mobility shifts of the sequential partial degradation products of the oligonucleotide on two dimensional homochromatography. We have now developed an empirical formula for predicting the relative mobilities of each of the partial products in the first dimension (electrophoresis on cellulose acetate gel). The formula allows a more precise interpretation of the sequence of the oligonucleotide. It eliminates the ambiguities present in the method previously reported for sequence analysis by simple inspection of the mobility shifts. In order to amplify the mobility shifts so that they may be more easily and accurately measured, methods have been developed for preparing and fractionating the oligonucleotides on 40 x 40 cm DEAE-cellulose plates. Both improvements have proven valuable for direct sequence analysis by mapping.

Algorithms↗

PubMed1984

Isolation and characterization of genomic clones corresponding to the human type II procollagen gene.

A recombinant human DNA library was screened using probes corresponding to the chick alpha 1 (II) procollagen gene. This resulted in the isolation of 2 different genomic clones, LgHCol(II)a and LgHCol(II)b. LgHCol(II)a was identified as corresponding to the alpha 1(II) gene by comparative hybridization and DNA sequence analysis. DNA sequence established that LgHCol(II)a extends at least from amino acid 694 of the triple helix through 54 amino acids of the COOH-propeptide. Hybridization with a probe containing only the exon at the 3' end of the chicken gene suggests that the clone contains the 3' end of the human gene. Thus LgHCol(II)a contains approximately 40% of the coding sequences of the human type II collagen gene.

Amino Acid Sequence↗