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At least 451 records · Page 25Linked to original sources
PubMed2006

Characterization of the Soj/Spo0J chromosome segregation proteins and identification of putative parS sequences in Helicobacter pylori.

The Soj and Spo0J proteins, together with one or more parS sequences, are crucial to chromosome segregation and the progression of cell cycle in many bacteria. In Helicobacter pylori, genes coding for Soj and a plasmid replication-partition-related protein containing a Spo0J or ParB conserved domain, together with two putative parS sites identified in this study, were found to be located within the origin-proximal 20-30% of the circular chromosome. Recombinant H. pylori Spo0J bound specifically to the two putative parS sequences and that of Bacillus subtilis. In addition, hydrolysis of ATP by H. pylori Soj was accelerated in the presence of parS and/or Spo0J. Protein-protein interactions, intracellular levels, and subcellular localization of Soj and Spo0J were analyzed through polyclonal antibodies directed against recombinant Soj and Spo0J. This study was the first implication of the existence of a functional parABS system in H. pylori.

Adenosine Triphosphatases↗
PubMed1999

MCM21 and MCM22, two novel genes of the yeast Saccharomyces cerevisiae are required for chromosome transmission.

The minichromosome maintenance genes, MCM21 and MCM22, have been cloned and are shown to code for the ORFs YDR318W and YJR135C respectively. Mutations in these genes caused a decrease in the stability of the minichromosome. This decrease in stability was associated with an increase in the copy number of the minichromosome in cells carrying it. Small circular dicentric plasmids were maintained relatively stably and structurally intact in the mutants compared with the wild-type strain. In the latter, such plasmids were mitotically unstable and, upon recovery, showed frequent rearrangements of their DNA. A centromere offered less obstruction to transcription in mutant cells than in the wild type, showing that both these mutants had a more relaxed kinetochore assembly. The mutant strains showed elevated rates of chromosome loss but not those of recombination. Both the mutations caused the cells to display a higher sensitivity towards the anti-mitotic drug benomyl. All these observations suggest that MCM21 and MCM22 are important for chromosome segregation with a potential role in kinetochore function. These genes are non-essential, as their deletions from chromosomes did not cause loss of cell viability. However, exponentially growing mutant cells carrying the deletion of the MCM21 gene had a significant population of large-budded cells with a single nucleus at the neck. Furthermore, the DNA content of these cells showed a shift towards 2N, suggesting a temporary pause of cells in G2 or in an early phase of mitosis. The mcm21 and mcm22 mutations do not show synthetic lethality or any further enhancement of growth defects, implying that they could be carrying out non-overlapping functions in chromosome segregation.

Cell Cycle Proteins↗
PubMed1999

The enteric nervous system: region and target specific projections and neurochemical codes.

The goal of this report is to summarise the current knowledge on the projection pathways of enteric neurones innervating the muscle and mucosa in different regions of the gut. Combination of neuronal tracing, immunohistochemical and electrophysiological methods has allowed researchers to gain insight into the enteric hardwiring of specific target tissue in the gut. A polarised innervation pattern of the circular muscle was demonstrated for the stomach fundus/corpus and the ileum with descending pathways being primarily nitrergic while ascending pathways were primarily cholinergic. This characteristic hardwiring is thought to set in part the functional basis for peristalsis. A similar polarised innervation pathway was found for the enteric innervation of the mucosa in the stomach and large intestine but not in the small intestine. In both the stomach (myenteric neurones) and in the proximal and distal colon (submucosal neurones), ascending pathways to the mucosa are primarily cholinergic while descending pathways are primarily non-cholinergic. In the colon, results suggest that activation of both pathways induces a cross potentiation of cholinergic and vasoactive intestinal polypeptidergic mediated secretion. Furthermore, a large population of myenteric neurone s projecting to the mucosa in the small and large intestine are probably intrinsic primary afferent neurones sensitive to mechanical as well as chemical stimuli.

Animals↗
PubMed1995

Expression of mouse uterine peptidylarginine deiminase in Escherichia coli: construction of expression plasmid and properties of the recombinant enzyme.

To study the structure/function relationships of peptidylarginine deiminase (PAD), we constructed an Escherichia coli expression plasmid for mouse uterine PAD. First, segments of a cDNA encoding murine PAD were subcloned into a single plasmid, and the resulting plasmid, pKSPAD1, was inserted into an expression vector, pKK223-3, at the EcoRI and HindIII restriction sites. Since no detectable amount or activity of the PAD was produced by E. coli carrying that plasmid, the 5'-untranslated sequence of the cDNA was replaced with several synthetic DNAs. One of the constructed plasmids, pKKPAD4, which had a unique DNA linker containing a pair of Shine-Dalgarno sequences and a short preceding cistron inserted into the adjacent 5'-region of the coding region, produced a large quantity of mouse PAD as an unfused protein in E. coli. The purified recombinant PAD was indistinguishable from the native enzyme with respect to some structural properties, such as molecular mass, amino- and carboxyl-terminal sequences, and circular dichroism spectra. However, the alpha-amino group of the amino-terminal methionine residue of the recombinant PAD was not acetylated as was that of the native enzyme. Comparison of the recombinant PAD with the natural enzyme did not indicate significant differences in their sensitivity to activation by Ca2+ and in their substrate specificity toward arginine derivatives. The rates of modification of soybean trypsin inhibitor (Kunitz) were also similar for the recombinant and native PADs. These results indicate that the recombinant PAD has biological activities identical to those of the native enzyme and that the N alpha-acetyl group in the native PAD does not appear to have any particular role in the enzyme's catalytic function.

Amino Acid Sequence↗
PubMed2006

Genome sequence and organization of a nucleopolyhedrovirus that infects the tea looper caterpillar, Ectropis obliqua.

The complete nucleotide sequence of Ectropis obliqua nucleopolyhedrovirus (EcobNPV), which infects the tea looper caterpillar, was determined and analyzed. The double stranded circular genome is composed of 131,204 bp and is 37.6% G+C rich. The analysis predicted 126 putative, minimally overlapping open reading frames (ORFs) with 150 or more nucleotides that together compose 89.8% of the genome. The remaining 10.2% constitute non-coding and three homologous regions. Comparison with previously sequenced baculoviruses indicated that three ORFs were unique to EcobNPV, while the remaining 123 ORFs shared identity with other baculovirus genes. In addition to two bro homologues, three other repeat ORFs, including dbp, p26, and odv-e66, were identified. Phylogenetic analysis indicated that each member of the paired ORFs was acquired independently. Gene parity plot analysis and percent identity of gene homologues suggested that EcobNPV is a Group II NPV, although its genomic organization was highly distinct.

Animals↗
PubMed1999

Complete structure of the chloroplast genome of Arabidopsis thaliana.

The complete nucleotide sequence of the chloroplast genome of Arabidopsis thaliana has been determined. The genome as a circular DNA composed of 154,478 bp containing a pair of inverted repeats of 26,264 bp, which are separated by small and large single copy regions of 17,780 bp and 84,170 bp, respectively. A total of 87 potential protein-coding genes including 8 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acid species were assigned to the genome on the basis of similarity to the chloroplast genes previously reported for other species. The translated amino acid sequences from respective potential protein-coding genes showed 63.9% to 100% sequence similarity to those of the corresponding genes in the chloroplast genome of Nicotiana tabacum, indicating the occurrence of significant diversity in the chloroplast genes between two dicot plants. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.

Anticodon↗
PubMed2000

Complete structure of the chloroplast genome of a legume, Lotus japonicus.

The nucleotide sequence of the entire chloroplast genome (150,519 bp) of a legume, Lotus japonicus, has been determined. The circular double-stranded DNA contains a pair of inverted repeats of 25,156 bp which are separated by a small and a large single copy region of 18,271 bp and 81,936 bp, respectively. A total of 84 predicted protein-coding genes including 7 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids species were assigned on the genome based on similarity to genes previously identified in other chloroplasts. All the predicted genes were conserved among dicot plants except that rpl22, a gene encoding chloroplast ribosomal protein CL22, was missing in L. japonicus. Inversion of a 51-kb segment spanning rbcL to rpsl6 (positions 5161-56,176) in the large single copy region was observed in the chloroplast genome of L. japonicus. The sequence data and gene information are available on our World Wide Web database at http://www.kazusa.or.jp/en/plant/database.html.

Amino Acids↗
PubMed2004

Human glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site.

BACKGROUND: Glutaminyl cyclase (QC) forms the pyroglutamyl residue at the amino terminus of numerous secretory peptides and proteins. We previously proposed the mammalian QC has some features in common with zinc aminopeptidases. We now have generated a structural model for human QC based on the aminopeptidase fold (pdb code 1AMP) and mutated the apparent active site residues to assess their role in QC catalysis. RESULTS: The structural model proposed here for human QC, deposited in the protein databank as 1MOI, is supported by a variety of fold prediction programs, by the circular dichroism spectrum, and by the presence of the disulfide. Mutagenesis of the six active site residues present in both 1AMP and QC reveal essential roles for the two histidines (140 and 330, QC numbering) and the two glutamates (201 and 202), while the two aspartates (159 and 248) appear to play no catalytic role. ICP-MS analysis shows less than stoichiometric zinc (0.3:1) in the purified enzyme. CONCLUSIONS: We conclude that human pituitary glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site residues. In contrast to the aminopeptidase, however, QC does not appear to require zinc for enzymatic activity.

Amino Acid Sequence↗
PubMed2005

Efficient construction of a large collection of phage-displayed combinatorial peptide libraries.

Selections from phage-displayed combinatorial peptide libraries are an effective strategy for identifying peptide ligands to target proteins. Existing protocols for constructing phage-displayed libraries utilize either ligation into double-stranded phage DNA or Kunkel mutagenesis with single-stranded phagemid DNA. Although the Kunkel approach rapidly provides library sizes of up to 10(11), as many as 20% of the phagemids may be non-recombinant. With several modifications to current Kunkel protocols, we have generated peptide libraries with sizes of up to 10(11) clones and recombination frequencies approaching 100%. The production of phage libraries, as opposed to phagemid libraries, simplifies selection experiments by eliminating the need for helper phage. Our approach relies upon the presence of an amber stop codon in the coding region of gene III of bacteriophage M13. Oligonucleotides containing randomized stretches of DNA are annealed to the phage genome such that the randomized region forms a heteroduplex with the stop codon. The oligonucleotide is then enzymatically extended to generate covalently-closed, circular DNA, which is electroporated into a non-suppressor strain of Escherichia coli. If the amber stop codon is present in the DNA molecule, protein III is not synthesized and the phage cannot propagate itself. This method is customizable for the display of either random or focused peptide libraries. To date, we have constructed 22 different libraries ranging from 8-20 amino acids in length, utilizing complete or reduced codon sets.

Amino Acid Sequence↗
PubMed1984

Simian virus 40 T antigen is required for viral excision from chromosomes.

We describe experiments that show that simian virus 40 (SV40) T antigen is required for viral excision from host chromosomes at some point prior to or during the homologous recombination events that create circular wild-type virus. Two recombinant SV40-pBR322 plasmids were constructed such that homologous recombination across similar-sized but different duplications of SV40 would reconstitute wild-type viral DNA. One plasmid (pSVED) was constructed such that the duplication separates the viral early T-antigen promoter from the coding sequences; the other recombinant (pSVLD) contains a duplication of the late viral sequences and thus maintains a complete T-antigen gene. These plasmids were individually established in Rat 2 cells via cotransformation with the herpes virus Tk gene. Both classes of cell lines contained integrated tandem arrays of the plasmids and yielded equivalent levels of infectious virus after cell fusions with COS-7 cells; however, only the T+ lines yielded virus after cell fusion with CV-1 cells. These results are consistent with the notion that viral excision is initiated by T-antigen-mediated in situ replication of viral DNA as proposed in the "onion skin" model. In contrast, both plasmids yielded infectious virus when transiently introduced via transfection into CV-1 cells. This latter finding is discussed in terms of the possible induction of cellular repair and recombination pathways evoked by the introduction of damaged DNA into the nucleus.

Animals↗
PubMed1972

Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli.

We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40 DNA. The method involves: (a) converting circular SV40 DNA to a linear form, (b) adding single-stranded homodeoxypolymeric extensions of defined composition and length to the 3' ends of one of the DNA strands with the enzyme terminal deoxynucleotidyl transferase (c) adding complementary homodeoxypolymeric extensions to the other DNA strand, (d) annealing the two DNA molecules to form a circular duplex structure, and (e) filling the gaps and sealing nicks in this structure with E. coli DNA polymerase and DNA ligase to form a covalently closed-circular DNA molecule.

Centrifugation, Density Gradient↗
PubMed1978

BK virus DNA: cleavage map and sequence analysis.

A detailed physical map of the BK virus (MM strain) genome has been constructed with respect to the cleavage sites of 11 different restriction enzymes. The enzymes cut BKV(MM) DNA at 61 specific sites whose locations have been determined. Preliminary nucleotide sequence was carried out in the region from 0.70-0.75 map positions on BKV(MM) DNA. An 80% homology was found at 0.714-0.744 map positions on BKV(MM) DNA with 0.722-0.752 map positions on simian virus 40 DNA. This region of simian virus 40 DNA codes for the synthesis of the leader sequence of late mRNA.

Animals↗
PubMed1992

Molecular code for cooperativity in hemoglobin.

Although tetrameric hemoglobin has been studied extensively as a prototype for understanding mechanisms of allosteric regulation, the functional and structural properties of its eight intermediate ligation forms have remained elusive. Recent experiments on the energetics of cooperativity of these intermediates, along with assignments of their quaternary structures, have revealed that the allosteric mechanism is controlled by a previously unrecognized symmetry feature: quaternary switching from form T to form R occurs whenever heme-site binding creates a tetramer with at least one ligated subunit on each dimeric half-molecule. This "symmetry rule" translates the configurational isomers of heme-site ligation into six observed switchpoints of quaternary transition. Cooperativity arises from both "concerted" quaternary switching and "sequential" modulation of binding within each quaternary form, T and R. Binding affinity is regulated through a hierarchical code of tertiary-quaternary coupling that includes the classical allosteric models as limiting cases.

Allosteric Regulation↗
PubMed1976

An enzyme system for replication of duplex circular DNA: the replicative form of phage phi X174.

Viral single strands (SS) are converted to the duplex from (RF) by a soluble enzyme fraction uninfected Escherichia coli [Schekman et al. (1975) J. Biol. Chem. 250, 5859-5865]. When reactions were supplemented with a soluble enzyme fraction from phi X174-infected cells, replication of phi X174 superhelical RF I DNA was observed. The activity supplied by infected cells was absent in cells treated with chloramphenicol or in cells infected with a phi X174 phage mutant in cistron A (cis A). A host function coded by the rep gene, essential in vivo for RF replication (but not for SS leads to RF), was supplied by enzyme fractions from either infected or uninfected cells. Based on complementation assays, the cisA-dependent and the rep-dependent proteins have each been purified about 1000-fold. The synthetic products of the enzymatic reaction were identified as RF I and RF II in which viral (+) and complementary (-) strands were newly synthesized.

Bacterial Proteins↗
PubMed1985

Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors.

We have previously shown linear and circular splicing intermediates resembling intermediates that result from self-splicing of ribosomal precursor RNA of Tetrahymena to be present in mitochondrial RNA. Here we show that splicing of yeast mitochondrial precursor RNA also occurs in vitro in the absence of mitochondrial proteins. The large ribosomal RNA gene, consisting of the intron and part of the flanking exon regions, was inserted behind the SP6 promoter in a recombinant plasmid and was transcribed in vitro. The resulting RNA shows self-catalyzed splicing via incorporation of GTP at the 5'-end of the excised intron, 5'- to 3'-exon ligation, and intron circularization. When purified mitochondrial RNA is incubated under similar conditions with alpha-32P-GTP, the excised ribosomal intron RNA is also labeled, as well as several other RNA species. Some of these RNAs are derived from excised introns from the multiply split gene coding for cytochrome oxidase subunit I.

Base Sequence↗
PubMed2003

The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins.

The complete genomic sequence of Corynebacterium glutamicum ATCC 13032, well-known in industry for the production of amino acids, e.g. of L-glutamate and L-lysine was determined. The C. glutamicum genome was found to consist of a single circular chromosome comprising 3282708 base pairs. Several DNA regions of unusual composition were identified that were potentially acquired by horizontal gene transfer, e.g. a segment of DNA from C. diphtheriae and a prophage-containing region. After automated and manual annotation, 3002 protein-coding genes have been identified, and to 2489 of these, functions were assigned by homologies to known proteins. These analyses confirm the taxonomic position of C. glutamicum as related to Mycobacteria and show a broad metabolic diversity as expected for a bacterium living in the soil. As an example for biotechnological application the complete genome sequence was used to reconstruct the metabolic flow of carbon into a number of industrially important products derived from the amino acid L-aspartate.

Amino Acid Sequence↗
PubMed2002

Molecular understanding of aluminum-induced topological changes in (CCG)12 triplet repeats: relevance to neurological disorders.

Recent studies have shown that gene mutations are involved in the pathology of neurological disorders. CCG repeats cause genetic instability and are localized at the 5' end of the non-coding regions of the FMR1 gene in fragile X syndrome. Our studies for the first time showed that aluminum (Al) levels were elevated in the serum samples of fragile X syndrome and also provide evidence for the interaction of aluminum with (CCG)12-repeats. Circular dichroism spectroscopic studies of (CCG)12 indicated B-DNA conformation and in the presence of Al (10(-5) M) CCG repeats attained Z-DNA conformation. Further spectroscopic studies, which included melting profiles, ethidium bromide binding patterns and interaction of Z-DNA specific polyclonal antibodies confirmed the Z-conformation in (CCG)12-repeats in the presence of Al (10(-5) M). It is interesting to mention that Al-induced Z-conformation is stable even after the total removal of Al from CCG by desferoximine, a chelating drug. This is the first report to proof the role of Al in modulating the DNA (CCG repeats) topology and this information provides a clue about the possible involvement of Al at a molecular level in neurological/neurodegenerative disorders.

Aluminum↗