PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “DNA sequence analysis”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources
PubMed1993

Spontaneous mutagenesis in Escherichia coli harbouring plasmid pKM101: DNA sequence analysis of forward lacI- mutations.

To investigate the influence of plasmid pKM101 on spontaneous mutagenesis, 198 lacI- mutations generated in Escherichia coli harbouring pKM101 were characterized at the DNA sequence level. pKM101 by itself did not enhance the lacI- forward mutation frequency. In general, the resultant distribution of mutation highlights particular sequences at which a variety of mutational events repeatedly occur, including 5'-(G/C)TGG-3', 5'-CCAGG-3', 5'-GATC-3' and 5'-TCGCG-3' sequences. Specifically, the distribution of mutation within base substitution and deletion classes distinguishes the pKM101 spectrum from the wild-type distribution (i.e. absence of pKM101). An even distribution amongst base substitutions was observed which corresponds to a 2.9- to 6.3-fold increase in occurrence of low frequency events (A-->G,T,C; G-->C); high frequency events in the wild-type distribution (G-->A,T) were not influenced by the presence of pKM101. One complex event was recovered which was comprised of two base substitutions separated by 4 bp. An 11-fold increase in small deletion events (3-6 bp) was also observed. The observed pKM101 spectrum does not closely resemble the mutational consequences of SOS induction in the absence of mutagenic treatment (recA441 spectrum) but does return a distribution like that obtained in E. coli deficient in polymerase I activity (polA1 spectrum).

Amino Acid Sequence↗
PubMed1989

Cloning and DNA sequence analysis of a Lactococcus bacteriophage lysin gene.

A gene for the lysin of Lactococcus lactis bacteriphage phi vML3 was cloned using an Escherichia coli/bacteriophage lambda host-vector system. The gene was detected by its expression of antimicrobial activity against L. lactis cells in a bioassay. The cloned fragment was analysed by sub-cloning on to E. coli plasmid vectors and by restriction endonuclease and deletion mapping. Its entire DNA sequence was determined and an open reading frame for the lysin structural gene was identified. The sequenced lysin gene would express a protein of 187 amino acids with a molecular weight of 21,090, which is in good agreement with that of a protein detected after in vitro transcription and translation of DNA encoding the gene. Expression of the lysin gene in E. coli and B. subtilis from an adjacent bacteriophage promoter was readily detected but in L. lactis expression of lysin was found to be lethal. The bacteriophage phi vML3 lysin had sequence homology with protein 15 of B. subtilis bacteriophage PZA. This protein is involved in DNA packaging during bacteriophage maturation rather than in host cell lysis. The cloning and analysis of the phi vML3 lysin gene is of importance in further understanding lactic streptococcal bacteriophages, for the development of positive selection vectors and for biotechnological applications of relevance to the dairy industry.

Amino Acid Sequence↗
PubMed1984

DNA sequence analysis on the IBM-PC.

We have developed, for the IBM-PC microcomputer, a menu driven, interactive set of programs which provide the functions routinely used for DNA sequence data analyses.

Amino Acid Sequence↗
PubMed2000

16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates.

Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of > or =97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of > or =99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria.

Animals↗
PubMed1982

DNA sequence analysis of a Drosophila foldback transposable element rearrangement.

The complete nucleotide sequence of a DNA rearrangement associated with the foldback 4 (FB 4) transposable element is presented. The results demonstrate that the entire loop sequence and almost all of one of the inverted terminal repeats is absent. Moreover, the sequence of the remaining inverted repeat suggests that the FB elements might undergo inversions via recombinations between the two inverted repeats of a single element.

Base Sequence↗
PubMed1991

Plasmid vectors for selecting IS1-promoted deletions in cloned DNA: sequence analysis of the omega interposon.

We have constructed two plasmid vectors which allow selection for in vivo deletions within cloned DNA fragments. The plasmids are derivatives of pBR322 which carry the Escherichia coli rpsL (strA) gene, known to confer a dominant streptomycin (Sm)-sensitivity phenotype to the host cell, and a copy of the IS1 transposable element. Sm-resistant strains that harbor these plasmids display sensitivity to Sm. Spontaneous IS1-promoted deletions across the rpsL gene can be isolated simply by selection for Sm resistance. Hence, nested sets of deletions of a cloned DNA can be obtained and sequenced with an IS1-specific primer. Using this approach, we have determined the complete nucleotide sequence of the omega interposon [Prentki and Krisch, Gene 29 (1984) 303-313].

Amino Acid Sequence↗
PubMed2002

DNA sequence analysis of interlocus recombination between the human T-cell receptor gamma variable (GV) and beta diversity-joining (BD/BJ) sequences on chromosome 7 (inversion 7).

V(D)J recombinase-mediated recombination between the T-cell receptor (TCR) gamma variable (GV) genes at chromosome 7p15 and the TCR beta joining (BJ) genes at 7q35 leads to the formation of a hybrid TCR gene. These TCR gamma/beta interlocus rearrangements occur at classic V(D)J recombination signal sequences (RSS) and, because the loci are in an inverted orientation, result in inversion events that are detectable in the chromosome structure as inv(7)(p15;q35). Similar rearrangements involving oncogenes and either TCR or immunoglobulin genes mediated by the V(D)J recombinase are found in lymphoid malignancies. Oligonucleotide primers that allow polymerase chain reaction (PCR) amplification across the inv(7) genomic recombination junction sequence have been described. Southern blot analysis has been primarily used to confirm the GV/BJ hybrid nature of the product, with limited information on the DNA sequence of these recombinations. We have modified this PCR method using total genomic DNA from the mononuclear cells in peripheral blood samples to increase specificity and to allow direct sequencing of the translocation junction that results from the recombination between the GV1 and BJ1 families of TCR genes in 25 examples from 11 individuals (three adults, one child, six newborns, and one ataxia telangiectasia (AT) patient). We focused on samples from newborns based on previous studies indicating that the predominant hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutations in newborns are V(D)J recombinase-mediated deletion events and that the frequency of these mutations decreases with increasing age. Although the dilution series-based PCR assay utilized does not yield sharply defined quantitative endpoints, results of this study strongly suggest that inv(7) recombinations in newborns occur at equal or lower frequencies than those seen in adults. Consistent with the PCR primer pairs, all sequenced products contain a GV1 and a BJ1 segment and most also contain a BD1 segment. GV1s2 and 1s4 were the most frequently found GV1 genes (8 and 9 examples, respectively) and BJ1s5 and 1s6 were the most frequently found BJ1 genes (9 and 10 examples, respectively). These results demonstrate the effectiveness of this methodology for assessing GV/BJ interlocus rearrangements mediated by V(D)J recombinase.

Adult↗
PubMed1995

Cloning and DNA sequence analysis of pepQ, a prolidase gene from Lactobacillus delbrueckii subsp. lactis DSM7290 and partial characterization of its product.

From a genomic library of Lactobacillus delbrueckii subsp. lactis (DSM7290) DNA, in the low-copy-number vector pLG339, a recombinant clone was selected, which complemented a mutation in the prolidase gene (pepQ) of Escherichia coli UK173. Nucleotide sequence analysis revealed an open reading frame of 1104 nucleotides corresponding to a protein of 368 amino acids with a calculated pI of 4.64 and a molecular mass of 41,087 Da. The start site of pepQ transcription was determined by primer extension analysis with mRNA prepared from L. delbrueckii. Based on homology of the gene product to various peptidases and on the substrate specificity determined, the peptidase was designated PepQ. The influence of various protease inhibitors and cations on peptidase activity indicated that PepQ is a metalloprotease. The absence of a membrane-spanning domain and a signal peptide sequence argues for a cytoplasmic localization of the enzyme.

Amino Acid Sequence↗
PubMed1993

Molecular cloning, expression, and DNA sequence analysis of the gene that encodes the 16-kilodalton outer membrane lipoprotein of Serpulina hyodysenteriae.

The gene (smpA) that encodes the 16-kDa outer membrane lipoprotein of Serpulina hyodysenteriae was cloned in Escherichia coli, and its primary structure was determined by nucleotide sequencing. The putative open reading frame encodes a prolipoprotein of 16.8 kDa which in its fully acylated and cleaved form is 15.1 kDa. Analysis of the N-terminal amino acid sequence derived from the DNA sequence revealed the presence of a signal sequence and a putative acylation and signal peptidase II cleavage site (Phe-Ala-Val-Ser-Cys). In E. coli, processing of the prolipoprotein was less efficient than that observed in S. hyodysenteriae, and globomycin, an inhibitor of signal peptidase II, inhibited cleavage of the lipoprotein expressed in E. coli but did not inhibit cleavage in S. hyodysenteriae.

Amino Acid Sequence↗
PubMed1985

Statistical methods of DNA sequence analysis: detection of intragenic recombination or gene conversion.

Simple but exact statistical tests for detecting a cluster of associated nucleotide changes in DNA are presented. The tests are based on the linear distribution of a set of s sites among a total of n sites, where the s sites may be the variable sites, sites of insertion/deletion, or categorized in some other way. These tests are especially useful for detecting gene conversion and intragenic recombination in a sample of DNA sequences. In this case, the sites of interest are those that correspond to particular ways of splitting the sequences into two groups (e.g., sequences A and D vs. sequences B, C, and E-J). Each such split is termed a phylogenetic partition. Application of these methods to a well-documented case of gene conversion in human gamma-globin genes shows that sites corresponding to two of the three observed partitions are significantly clustered, whereas application to hominoid mitochondrial DNA sequences--among which no recombination is expected to occur--shows no evidence of such clustering. This indicates that clustering of partition-specific sites is largely due to intragenic recombination or gene conversion. Alternative hypotheses explaining the observed clustering of sites, such as biased selection or mutation, are discussed.

Animals↗
PubMed1995

Molecular cloning and DNA sequence analysis of pepL, a leucyl aminopeptidase gene from Lactobacillus delbrueckii subsp. lactis DSM7290.

A genomic library of Lactobacillus delbrueckii subsp. lactis DSM7290 DNA fragments from a Sau3A partial digestion in the low-copy-number vector pLG339, was used to screen Escherichia coli for the presence of peptidases. Using the chromogenic substrate leucine-beta-naphthylamide (Leu-NH-Nap) and E. coli strain CM89 lacking the corresponding enzyme activity in an enzymic plate assay, allowed the isolation of two peptidase genes; the newly described pepL and the recently cloned and sequenced pepN. Clones could be distinguished not only by the restriction pattern of isolated plasmids but also by the rate and intensity of their colour reaction with Leu-NH-Nap. Three out of five clones were identified to express the Lactobacillus pepN gene; the others were shown to express a second aminopeptidase gene, designated pepL. This gene, together with 200 bp upstream of the proposed AUG initiation codon, was further subcloned and sequenced. The corresponding open reading frame of 897 nucleotides is predicted to encode a protein of 299 amino acids (34,541 Da). Searching the EMBL database revealed similarity to the prolinase of Lactobacillus helveticus (45.8% identity), to the iminopeptidases of Lb. delbrueckii subsp. lactis and Lb. delbrueckii subsp. bulgaricus (25.5%), and to the Bacillus coagulans prolinase (21.5%). Minor similarities were detected for hydrolytic enzymes with serine active sites. The product encoded by the pepL gene was functional but could not be visualized on Coomassie-blue-stained polyacrylamide gels. High level expression of peptidase L in E. coli was achieved by placing the gene under the control of the T7 promoter.

Amino Acid Sequence↗
PubMed1980

A subcloning strategy for DNA sequence analysis.

We describe here a new strategy of fragment preparation for sequencing procedures using endlabelled DNA fragments as substrates (2,3) which is directly applicable to DNA fragments cloned into the Pst I site of pBR322, or in modified form, to inserts into the BamH I or Sal I site of the same plasmid. Ordered sets of subclones of predetermined overlap are are generated. These can be sequenced directly without further strand- or fragment separation steps.

Base Sequence↗
PubMed1975

DNA sequence analysis. Terminal sequences of bacteriophage phi80.

Sequences of the cohesive ends and the 3'-terminal regions of phi80 DNA have been determined. Sequences of the cohesive ends were obtained through the use of two standard methods. The first method involved the incorporation of all four labeled deoxyribonucleotides into the phi80 cohesive ends using DNA polymerase I. The DNA was then partially digested with micrococcal nuclease or pancreatic DNase. The products were separated by two-dimensional electrophoresis and characterized by composition, 3'-terminal, and nearest neighbor analyses. The second method involved partial incorporation using one, two, or three labeled deoxyribonucleotides followed by similar analyses. Sequences of the double-stranded regions adjacent to the cohesive ends were determined by three new methods. These methods were: (a) the DNA was specifically labeled at the 3' terminus and then partially degraded. Labeled oligonucleotide products were sequenced by their mobilities on various separation systems. (b) The cohesive ends were enlarged by limited degradation with exonuclease III. After this treatment, the DNA was partially repaired with labeled nucleotides, digested, and the products were analyzed. (c) A synthetic ologonucleotide primer was bound to phi80 DNA which had been repaired with DNA polymerase I, and then partially digested with lambda-exonuclease. The primer was extended into the region of interest by partial repair with labeled nucleotides. The extended primer was isolated and analyzed.

Base Sequence↗
PubMed1988

DNA sequence analysis of spontaneous mutations in the SUP4-o gene of Saccharomyces cerevisiae.

A collection of 196 spontaneous mutations in the SUP4-o gene of the yeast Saccharomyces cerevisiae was analyzed by DNA sequencing. The classes of mutation identified included all possible types of base-pair substitution, deletions of various lengths, complex alterations involving multiple changes, and insertions of transposable elements. Our findings demonstrate that at least several different mechanisms are responsible for spontaneous mutagenesis in S. cerevisiae.

Base Composition↗
PubMed1993

Cloning of the rat insulin- like growth factor binding protein-5 gene and DNA sequence analysis of its promoter region.

To understand the regulation of the insulin-like growth factor binding protein-5 (IGFBP-5) gene expression, we have cloned the IGFBP-5 gene from rat genomic libraries and determined its genomic organization as well as the DNA sequence at the 5' flanking region of the gene. The rat IGFBP-5 gene spans at least 17 kilobases (kb) of the genome and contains 4 exons interrupted by 3 introns of approximately 10, 0.6 and 0.7 kb, respectively. Southern blot analysis of the rat chromosome DNA revealed a single copy gene for IGFBP-5 in the haploid genome. Primer extension experiments demonstrated a single transcriptional start site located at 772 nucleotides 5' of the ATG translational start codon. In addition to a TATA box and a CAAT box, multiple putative cis-regulatory elements, including an AP-1, an AP-2 and a binding site for progesterone receptor are present in the promoter region. This finding suggests that the IGFBP-5 gene is one of those that are expressed in a tissue-specific manner.

Amino Acid Sequence↗
PubMed1977

On the mechanism of oligonucleotide-primed RNA synthesis. II. Synthesis of specific primer-initiated RNA copies suitable for DNA sequence analysis.

The effect of temperature and primer concentration on oligonucleotide-primed transcription has been studied using the separated strands of a well-defined natural DNA as template. Results were similar to those obtained in the homopolymer-directed model systems. At high temperature and excess primer concentration mainly primer-initiated RNA copies are synthesized. Omission of one ribonucleoside triphosphate also makes the termination specific. The unique RNA fragments thus obtained have been used to determine the perfectly-repeated sequence of 68 base pairs in this DNA.

Base Sequence↗
PubMed1987

Mobilization of the non-conjugative plasmid RSF1010: a genetic and DNA sequence analysis of the mobilization region.

The entire region required for mobilization of the non-conjugative plasmid RSF1010 has been cloned into a mobilization-deficient pBR322 derivative. The segment of DNA cloned was approximately 1.8 kb and included the origin of conjugal DNA transfer (oriT). The DNA sequence of the mobilization region has been determined, and revealed the presence of several overlapping reading frames. The isolation and mapping of both Tn1725 and BamH1-linker insertions and comparison with the DNA sequence data has allowed the identification of three genes required for mobilization. Two of these genes are overlapping and encode proteins of 16 kDa and greater than 65 kDa (although the truncated protein is functional, the gene extends outside the region cloned). The third gene is transcribed in the opposite direction. Promoters capable of transcribing these genes were located by S1 mapping in the inter-cistronic region between these divergently transcribed genes. The oriT site is located in this region, and the transcriptional patterns observed for mob+ and mob- plasmids implied that the promoters may be regulated by two of the mobilization proteins binding to the oriT site.

Base Sequence↗