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At least 1,657 records · Page 92Linked to original sources
PubMed1992

Sensitivity of polymerase chain reaction assay for Rickettsia tsutsugamushi in patients' blood samples.

We developed a nested polymerase chain reaction (PCR) method to detect Rickettsia tsutsugamushi (R. tsutsugamushi) DNA and determined its sensitivity. Primers were selected from the DNA sequence of the 58-kDa group-specific antigen gene of the Karp strain. The target sequence of rickettsial DNA was detectable as the band corresponding to 88 bp in 1.0 microgram of the DNA extracted from BS-C-1 cells infected with R. tsutsugamushi. Rickettsia-specific bands were observed not only for the homologous Karp strain, but also for four heterologous strains: two other reference strains (Gilliam and Kato) and two prototype strains prevalent in Miyazaki district (Irie and Hirano). The minimum copy number detectable by this method was estimated to be five rickettsiae. All of nine peripheral blood mononuclear cell samples from patients with tsutsugamushi disease who were seen 2-11 days after disease onset tested positive for rickettsial DNA. The PCR assay method presented here could be a specific diagnostic tool for tsutsugamushi disease, especially in its early acute stage.

Adult↗
PubMed1987

Reassortment of DNA recognition domains and the evolution of new specificities.

Type I restriction enzymes comprise three subunits only one of which, the S polypeptide, dictates the specificity of the DNA sequence recognized. Recombination between two different hsdS genes, SP and SB, led to the isolation of a system, SQ, which had a different specificity from that of either parent. The finding that the nucleotide sequence recognized by SQ is a hybrid containing components from both the SP and SB target sequences suggested that DNA recognition is carried out by two separable domains within each specificity polypeptide. To test this we have made the recombinant gene of reciprocal structure and demonstrate that it encodes a polypeptide whose recognition sequence, deduced in vivo, is as predicted by this model. We also report the sequence of the SB specificity gene, so that information is now available for the five known members of this family of enzymes. All show a similar organization of conserved and variable regions. Comparisons of the predicted amino acid sequences reveal large non-conserved areas which may not even be structurally similar. This is remarkable since these different S subunits are functionally identical, except for the specificity with respect to the DNA sequence with which they interact. We discuss the correlation of the variation in polypeptide sequence with recognition specificities.

Bacteriophage lambda↗
PubMed2004

GATC-specific restriction and modification systems in treponemes.

AIMS: To investigate the presence of GATC-specific modification and restriction activities in rumen isolates of Treponema sp. METHODS: The presence of N6-methyladenine within GATC (Dam) sequences was analysed using isoschizomeric restriction endonucleases having different sensitivities to the methylation of the target sequence. A fast screening method was used for testing of site-specific endonuclease activities directly in crude cell extracts. Three out of six rumen isolates of Treponema sp. showed restriction activities. Restriction endonucleases were further purified by Heparin-Sepharose chromatography. Using PCR and specific primers, no sequence homologous to the T. pallidum dam gene was found. CONCLUSIONS: Three rumen treponemal strains were documented to possess MboI isoschizomeric restriction-modification systems. SIGNIFICANCE: This is the first report on restriction activity in rumen treponemes.

Adenine↗
PubMed1996

Rapid identification of mycobacterial species by PCR amplification of hypervariable 16S rRNA gene promoter region.

A total of 0.3 to 0.4 kb of the promoter region of the 16S rRNA genes from Mycobacterium tuberculosis, M. gordonae, M. xenopi, and M. leprae was PCR amplified, cloned, and sequenced. The observed number of substitutions, insertions, and deletions exceeded those found in previously used target sequences, including the entire 16S coding region. A simple and generally applicable restriction fragment length polymorphism method that can be used to distinguish between mycobacterial species is described.

Base Sequence↗
PubMed1993

Functional organization of the Bel-1 trans activator of human foamy virus.

Human foamy virus encodes a 300-amino-acid nuclear regulatory protein termed Bel-1 that is required for human foamy virus replication in culture. Bel-1 is a potent trans-activator of gene expression directed by the homologous HFV long terminal repeat as well as the long terminal repeat of human immunodeficiency virus type 1. We have used mutational analysis to define several discrete functional domains within Bel-1. The C-terminal approximately 50 amino acids of Bel-1 are shown to be essential for Bel-1 activity but can be effectively substituted by the C-terminal activation domain of VP16. We therefore conclude that the Bel-1 C terminus forms part of an activation domain. Mutations within a central, approximately 100-amino-acid segment of Bel-1 preclude trans-activation by either Bel-1 or the Bel-1/VP16 chimera. These sequences are therefore proposed to direct the interaction of Bel-1 with its viral DNA target sequences. A short Bel-1 segment located between the activation and binding domains is shown to mediate the nuclear localization of this regulatory protein. Although the functional organization of Bel-1 therefore appears comparable to that reported for other eukaryotic transcriptional activators, Bel-1 does not contain sequences homologous to known transcriptional activation or DNA-binding motifs.

Amino Acid Sequence↗
PubMed1999

The accessory subunit of Xenopus laevis mitochondrial DNA polymerase gamma increases processivity of the catalytic subunit of human DNA polymerase gamma and is related to class II aminoacyl-tRNA synthetases.

Peptide sequences obtained from the accessory subunit of Xenopus laevis mitochondrial DNA (mtDNA) polymerase gamma (pol gamma) were used to clone the cDNA encoding this protein. Amino-terminal sequencing of the mitochondrial protein indicated the presence of a 44-amino-acid mitochondrial targeting sequence, leaving a predicted mature protein with 419 amino acids and a molecular mass of 47.3 kDa. This protein is associated with the larger, catalytic subunit in preparations of active mtDNA polymerase. The small subunit exhibits homology to its human, mouse, and Drosophila counterparts. Interestingly, significant homology to glycyl-tRNA synthetases from prokaryotic organisms reveals a likely evolutionary relationship. Since attempts to produce an enzymatically active recombinant catalytic subunit of Xenopus DNA pol gamma have not been successful, we tested the effects of adding the small subunit of the Xenopus enzyme to the catalytic subunit of human DNA pol gamma purified from baculovirus-infected insect cells. These experiments provide the first functional evidence that the small subunit of DNA pol gamma stimulates processive DNA synthesis by the human catalytic subunit under physiological salt conditions.

Amino Acid Sequence↗
PubMed2002

Identification of a new cystic fibrosis transmembrane regulator mutation in a severely affected patient.

By using a combination of multiplex polymerase chain reaction and allele-specific labelled probes, the oligo-ligation assay is designed to detect known cystic fibrosis transmembrane regulator mutations. This study shows that this assay may also be useful to detect new mutations. The second child of a family of Bosnic origin showed all the symptoms of intestinal and pulmonary manifestations of cystic fibrosis. No signal could be obtained for the allele-specific probe 1898+1G>A. This could be explained by a nearby localized sequence change that prevented polymerase chain reaction primers or oligonucleotide probes from binding to the target sequence. Indeed, sequence analysis revealed a new 1894G>T exchange (Glu587Stop). Both parents and the healthy brother carried this mutation. Thus, the index patient was homozygous for 1894G>T, which was inherited from both parents.

Bosnia and Herzegovina↗
PubMed2004

The structure of a single unit of ribosomal RNA gene (rDNA) including intergenic subrepeats in the Australian bulldog ant Myrmecia croslandi (Hymenoptera: Formicidae).

A complete single unit of a ribosomal RNA gene (rDNA) of M. croslandi was sequenced. The ends of the 18S, 5.8S and 28S rRNA genes were determined by using the sequences of D. melanogaster rDNAs as references. Each of the tandemly repeated rDNA units consists of coding and non-coding regions whose arrangement is the same as that of D. melanogaster rDNA. The intergenic spacer (IGS) contains, as in other species, a region with subrepeats, of which the sequences are different from those previously reported in other insect species. The length of IGSs was estimated to be 7-12 kb by genomic Southern hybridization, showing that an rDNA repeating unit of M. croslandi is 14-19 kb-long. The sequences of the coding regions are highly conserved, whereas IGS and ITS (internal transcribed spacer) sequences are not. We obtained clones with insertions of various sizes of R2 elements, the target sequence of which was found in the 28S rRNA coding region. A short segment in the IGS that follows the 3' end of the 28S rRNA gene was predicted to form a secondary structure with long stems.

Animals↗
PubMed2001

Digital microscopy for multiparameter FISH imaging.

Fluorescence in situ hybridization (FISH) allows the direct localization of DANN and RNA sequences on chromosomes, in cells and in tissue. The technique is based on hybridization between target sequences of single-strand DNA of chromosomes or cell nuclei with marked complementary specimens. The signal is amplified using fluorochrome-marked specific antibodies and made visible under a microscope. Signals from painted chromosomes, stained subchromosomal regions or localized single probes are generally visible when an epifluorescence microsope is used. In order to view and display different fluorochromes, single filter sets, as well as double and triple bandpass filters, are in use. For multiple fluoroscence imaging, lenses with high numerical aperture, mostly oil immersion systems, are recommended. In conventional photomicrography, triple exposure on high-speed film (e.g., 400-1,000 ASA) is more or less the limitation. Opto=electronic methods using a CCD and laser summing techniques have considerably extended the application range of multiple fluorescence techniques. By means of digitized images, simultaneous detection of multiple-labelled objects and ratio imaging up to 24 colors are possible today. Current FISH approaches are based on chromosome-painting probes to distinguish all 24 chromosomes by their unique spectral signatures.

Cell Nucleus↗
PubMed2003

Activation-dependent surface expression of gC1qR/p33 on human blood platelets.

GC1qR/p33 (gC1qR) is expressed by a variety of somatic and cultured cells, including blood platelets. It interacts with several cellular, viral, bacterial, and plasma proteins, suggesting a potential role in thrombosis, inflammation, and infection. Considerable controversy has surrounded the surface membrane localization of gC1qR, however, since its cDNA sequence does not predict a traditional membrane-anchoring domain, and bears a typical mitochondrial targeting sequence. The present study examined gC1qR expression on resting and activated human blood platelets using flow cytometry and confocal microscopy with two monoclonal antibodies, 74.5.2 and 60.11, directed against gC1qR C-terminal amino acids 204-218, and N-terminal amino acids 76-93, respectively. Unstimulated platelets reacted minimally with either antibody. In contrast, platelet activation with TRAP, epinephrine, or ADP produced markedly increased gC1qR expression as reflected by 74.5.2 binding but not 60.11 binding. Platelet activation was verified using PAC-1 and anti CD 62 antibodies. Whereas PAC-1 binding to activated platelets could be reversed following platelet incubation with PGE1, 74.5.2 binding remained unchanged, suggesting the sustained expression of gC1qR following platelet stimulation. The data further demonstrate that detection of cell surface gC1qR may be dependent on antibody specificity. The ability of gC1qR to bind proteins involved in complement, coagulation, and kinin systems, as well as viral and bacterial pathogens including S. aureus protein A, supports the hypothesis that gC1qR expressed on activated platelets may contribute directly to thrombosis, inflammation, and endovascular infections.

Adenosine Diphosphate↗
PubMed2002

[Structure and regulative function of the 5' end flanking sequence of gene CCR5].

OBJECTIVE: To construct the pCAT reporter genes containing the 5'-end flanking of CC chemokine receptor 5(CCR5) gene in different sequence lengths and identify the sequence, which regulates the gene expression of CCR5 by the CAT analysis system. METHODS: The target sequences were amplified by pyrobest DNA polymerase, and were inserted into the upstream of CAT gene located in the pCAT enhancer vector by the directional clone technique respectively; the regulative sequence was identified by analyzing the CAT activities of reconstructed plasmid in Hela cells. RESULTS: The region, containing 486 bp upstreaming from exon 1, stimulated the reporter gene activity which was about 3 times that of the pCAT enhancer vector in transfected cells. CONCLUSION: There is an up-regulative element of gene transcription in the region of -1(-)-486 bp in CCR5 gene upstream.

5' Flanking Region↗
PubMed2005

[Cloning and characterization of a novel mouse aging-related gene Arzc].

OBJECTIVE: To clone and identify novel mouse aging-related genes. METHODS: The improved differential display reverse transcription-polymerase chain reaction method was used to analyze the differential expression of old and young BALB/C mouse cortex tissues. The novel expression sequence targets (EST) with differential expression were further confirmed by Northern blot and used as original sequence to clone the full-length cDNA sequence by bioinformatics technique. The reverse transcription products of BALB/C mouse cortex total RNA was used as the amplification template. RESULTS: A total of 42 differentially expressed EST were obtained. Homologic analysis showed that 29 EST represented known cDNAs and 13 were probably new EST. One of the EST, which were confirmed to have down regulative expression in the old mouse cortex tissues by Northern blot analysis, was used to obtain the full-length cDNA sequences from reverse transcription products of total RNA of BALB/C mice cortex by bioinformation method. A 1 382 bp of cDNA sequence including the EST was obtained and designed as Arzc (GenBank accession number: AY344585). The Arzc cDNA contains an open reading frame of 261 bp and encodes a peptide of 86 amino acids residuals. FASTA analysis showed that the deduced peptide had a homology with a portion of integrase/recombinase, a member of a phage integrase family. CONCLUSION: A novel mouse gene that is probably related to aging was identified and cloned.

Aging↗
PubMed1994

Production of a vector to facilitate DNA mutagenesis and recombination.

Most methods for the generation of site-specific mutants and recombinant constructs require primer extension in vitro. These methods include the Kunkel method and PCR-based methods. Such methods to manipulate DNA are prone to sequence error because they take place outside the complex in vivo mechanisms that increase sequence fidelity during plasmid replication in Escherichia coli. Sequence errors are of particular concern when using PCR-based methods. We have constructed two new plasmids that facilitate the generation of site-specific mutants and recombinant constructs. The plasmids we have constructed are designed to maximize the number of unique restriction enzyme recognition sites in inserts that have been cloned into either plasmid. This was accomplished by eliminating extraneous sequence and many restriction enzyme recognition sites. New recombinant circle and recombination PCR protocols for multiplex site-specific plasmid mutagenesis were used to make these plasmids. These plasmids permit small portions of an insert sequence to be readily removed by restriction enzyme digestion. A small DNA segment, containing the targeted sequence alteration, can subsequently be ligated into a plasmid construct that has not been subjected to primer extension in vitro, diminishing the probability of encountering a sequence error and reducing the amount of DNA sequencing necessary to assess for errors.

Base Sequence↗
PubMed1996

The targeting and assembly of peroxisomal proteins: some old rules do not apply.

Several proteins have been identified that catalyze the import of proteins into peroxisomes. Some recognize specific peroxisomal targeting sequences, but most probably work further downstream the import pathway. Recent evidence suggests that peroxisomal targeting and assembly do not follow the same rules as those for targeting and import into other organelles, such as the mitochondria and the endoplasmic reticulum, i.e. the import of unfolded proteins and subsequent folding within the organelle. Specifically, proteins may be translocated into the peroxisomal matrix in a folded or oligomerized state.

Biological Transport↗
PubMed1987

Use of oligodeoxynucleotide probes for quantitative in situ hybridization to actin mRNA.

We have employed an analytical approach for the development of an in situ hybridization methodology using synthetic oligodeoxynucleotide probes for actin messenger RNA detection in cultures of chicken fibroblasts and myoblasts. The methodology developed shows that oligonucleotides can complement the use of nick-translated probes in specific situations. Since they can be made to specific nucleic acid regions independent of restriction enzyme sites, they may be the most convenient approach for analysis of gene families among which sequences are highly conserved. However, it was found that oligonucleotides synthesized to different regions of a messenger RNA behave in situ with differing efficiencies, indicating that not all target sequences are equivalent. Therefore it was necessary to screen several oligonucleotide probes to a target molecule to find the optimal one. The convenience of using synthetic DNA probes makes it worthwhile to explore some of these characteristic properties so as to increase the sensitivity of this approach beyond its application to targets in high abundance.

Actins↗
PubMed2005

Localization and function of three monothiol glutaredoxins in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe contains two dithiol glutaredoxins (Grx1 and Grx2) and genes for three putative monothiol glutaredoxins (grx3, 4, and 5). We investigated the expression, sub-cellular localization, and functions of the three monothiol glutaredoxins. Fluorescence microscopy revealed that Grx3 is targeted to nuclear rim and endoplasmic reticulum, Grx4 primarily to the nucleus, and Grx5 to mitochondria. Null mutation of grx3 did not significantly affect growth and resistance against various oxidants, whereas grx5 mutation caused slow growth and sensitivity toward oxidants such as hydrogen peroxide, paraquat, and diamide. The grx2grx5 double mutation, deficient in all mitochondrial glutaredoxins, caused further retardation in growth and severe sensitivity toward all the oxidants tested. The grx4 mutation was not viable, suggesting a critical role of Grx4 for the physiology of S. pombe. Overproduction of Grx3 and Grx5, but not the truncated form of Grx5 without mitochondrial target sequence, severely retarded growth as Grx2 did, supporting the idea that Grx2, 3, and 5 are targeted to organellar compartments. Our results propose a distinct role for each glutaredoxin to maintain thiol redox balance, and hence the growth and stress resistance, of the fission yeast.

Amino Acid Sequence↗
PubMed2000

Reinforced variability and operant learning.

Reinforcement of variability may help to explain operant learning. Three groups of rats were reinforced, in different phases, whenever the following target sequences of left (L) and right (R) lever presses occurred: LR, RLL, LLR, RRLR, RLLRL, and in Experiment 2, LLRRL. One group (variability [VAR]) was concurrently reinforced once per minute for sequence variations, a second group also once per minute but independently of variations, that is, for any sequences (ANY), and a control group (CON) received no additional reinforcers. The 3 groups learned the easiest targets equally. For the most difficult targets, CON animals' responding extinguished whereas both VAR and ANY responded at high rates. Only the VAR animals learned, however. Thus, concurrent reinforcers--contingent on variability or not--helped to maintain responding when difficult sequences were reinforced, but learning those sequences depended on reinforcement of variations.

Animals↗
PubMedDate not supplied

Design of oligonucleotide arrays to detect point mutations: molecular typing of antibiotic resistant strains of Neisseria gonorrhoeae and hantavirus infected deer mice.

Microarrays are promising tools for use in molecular diagnostics due to their ability to perform a multitude of tests simultaneously. In the case of genotyping many such tests will require discrimination of sequence at the single nucleotide level. A number of challenges exist including binding of optimal quantities of probe to the chip surface, the use of uniform hybridization conditions across the chip and the generation of labeled target. We investigated two model systems to test out the efficacy and ease with which probes can be designed for this purpose. In the first of these we designed primers to identify five mutations found in two genes from N. gonohorroeae, gyrA and parC that have been implicated in ciprofloxacin resistance. In the second system we used a similar strategy to identify four mutations in AT rich mitochondrial DNA from deer mice. These mutations are associated with deer mice subspecies that originate from different geographical regions of Canada and harbor different hantavirus strains. In every case we were able to design probes that could discriminate mutations in the target sequences under uniform hybridization conditions, even when targets were fairly long in length, up to 400 bp. Our results suggest that microarray analysis of point mutations might be very useful for automated identification and characterization of pathogens and their hosts.

Animals↗