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At least 145 records · Page 8Linked to original sources
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↗
PubMed1979

DNA sequence analysis of Tn10 insertions: origin and role of 9 bp flanking repetitions during Tn10 translocation.

The sequences of insertions of the translocatable tetracycline-resistance element Tn10 into the repressor (cl) gene of bacteriophage lambda have been analyzed. Each insertion contains the same discrete set of Tn10 sequences flanked by a direct repetition of a 9 bp cl-gene sequence. The flanking repititions are generated by duplication of information present only in the target DNA molecule rather than by a Campbell-type recombination event between one 9 bp sequence on the target DNA and a second one provided on the incoming element. The repetitions do not contain genetic or structural information important for translocation. A genetically constructed Tn10 insertion which lacks flanking repetitions is fully functional in translocation to a new position. Tn10 insertions cluster at preferred positions along a target DNA (Kleckner et al., 1979). Sequence analysis shows that four independently isolated cl::Tn10 insertions occur at identical positions in the cl gene. We speculate that homology between Tn10 and its target, at some distance from the site of the actual recombination event, could be relevant to the preference of Tn10 for particular insertion sites.

Base Sequence↗
PubMed1993

An adaptive, object oriented strategy for base calling in DNA sequence analysis.

An algorithm has been developed for the determination of nucleotide sequence from data produced in fluorescence-based automated DNA sequencing instruments employing the four-color strategy. This algorithm takes advantage of object oriented programming techniques for modularity and extensibility. The algorithm is adaptive in that data sets from a wide variety of instruments and sequencing conditions can be used with good results. Confidence values are provided on the base calls as an estimate of accuracy. The algorithm iteratively employs confidence determinations from several different modules, each of which examines a different feature of the data for accurate peak identification. Modules within this system can be added or removed for increased performance or for application to a different task. In comparisons with commercial software, the algorithm performed well.

Algorithms↗
PubMed1989

Molecular cloning and DNA sequence analysis of Escherichia coli topB, the gene encoding topoisomerase III.

Escherichia coli contains two type 1 topoisomerases, topoisomerase I and III. Although topoisomerase III can be purified as a potent decatenase, its role in DNA metabolism is unclear. In order to address this issue, the gene encoding topoisomerase III from E. coli has been molecularly cloned and its DNA sequence determined. The cloned fragment of DNA contains an open reading frame that can encode a polypeptide of 73.2 kDa. The first 20 amino acids of this open reading frame are identical to those of topoisomerase III as determined by amino-terminal gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame, using a bacteriophage T7 transient expression system, results in the accumulation of a 74-kDa polypeptide. Soluble extracts prepared from cells overexpressing this gene product show a dramatic increase in topoisomerase activity when compared with control extracts. We propose that this gene be designated topB.

Amino Acid Sequence↗
PubMed1989

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↗
PubMed1995

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↗
PubMed1986

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↗