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At least 1,045 records · Page 58Linked to original sources
PubMed1993

Thermonuclease gene as a target nucleotide sequence for specific recognition of Staphylococcus aureus.

DNA fragments, 450 bp in length, were amplified by polymerase chain reaction (PCR) from the thermonuclease gene (nuc) carried by seven epidemiologically independent Staphylococcus aureus isolates. Sequencing of the PCR products led us to characterize 210 bp strictly conserved. A 186 bp piece from within this conserved region was cloned into pUC18. The resulting recombinant plasmid, pIP1608, was used as a probe against the cellular DNA of 360 staphylococcal isolates belonging to 28 species. Only the 146 S. aureus isolates, including four which were not thermonuclease producers, had DNA that hybridized with pIP1608. Among the 214 non-S. aureus staphylococci, 55 exhibited a thermonuclease activity. For 32 of these, the enzymatic activity was inhibited by a commercially available polyclonal antiserum directed against the thermonuclease of an S. aureus strain. These results are in favour of the use of pIP1608 as a probe to specifically recognize S. aureus. Furthermore, we propose a method based on colony blot hybridization and potentially useful to enumerate S. aureus cells in biological samples.

Base Sequence↗
PubMed1995

The Arabidopsis MADS-box gene AGL3 is widely expressed and encodes a sequence-specific DNA-binding protein.

The Arabidopsis AGL3 gene was previously identified on the basis of sequence similarity to the floral homeotic gene AGAMOUS (AG), which encodes a protein with a conserved MADS domain that is also found in human and yeast transcription factors (SRF and MCM1, respectively). Analysis of newly isolated full-length cDNA clones as well as genomic clones indicates that AGL3 is indeed a MADS-box gene with a general intron-exon structure similar to other plant MADS-box genes. However, unlike the others, which are expressed specifically in flowers, AGL3 is expressed in all above-ground vegetative organs, as well as in flowers, but not in roots. Furthermore, since AGL3 is MADS-domain protein, it is likely that it is also a DNA-binding protein regulating transcription. To characterize AGL3 as a DNA-binding protein in vitro, we expressed the AGL3 protein in Escherichia coli, and characterized its DNA-binding properties. We show that AGL3 binds to sequences which resemble the target sequences of SRF and MCM1, and have determined the consensus sequence to which AGL3 binds using random oligonucleotides. These results suggest that AGL3 is a widely distributed DNA-binding protein, which may be involved the transcriptional regulation of genes in many cells.

Amino Acid Sequence↗
PubMed1996

IS431mec-mediated integration of a bleomycin-resistance gene into the chromosome of a methicillin-resistant Staphylococcus aureus strain isolated in Japan.

A methicillin-resistant Staphylococcus aureus (MRSA) strain B-26, isolated clinically in Hiroshima University Hospital, is resistant to bleomycin together with kanamycin. In the present study, we analysed the nucleotide sequence of the 5.1-kb HindIII fragment containing the bleomycin- and kanamycin-resistance genes, which were previously cloned [Bhuiyan et al. (1995) Appl Microbiol Biotechnol 43: 65-69] from the chromosomal DNA of MRSA B-26. The present study found that the DNA sequence contains the duplicated target sequence (GATTAGAT) consisting of 8 bp for transposase and the entire nucleotide sequence of the plasmid pUB110, together with the sequence of inverted repeats (16 bp), designated IR-r and IR-1 in IS431 mec. The 8-bp duplication sequence, produced by the transposable element, was first found by us. We proposed that bleomycin resistance in MRSA B-26 is attributed to the IS431 mec-mediated integration of pUB110 into the chromosome.

Bacterial Proteins↗
PubMed2001

The 15-kDa forms of the apo-peridinin-chlorophyll a protein (PCP) in dinoflagellates show high identity with the apo-32 kDa PCP forms, and have similar N-terminal leaders and gene arrangements.

Full-length genomic sequences encoding apo peridinin-chlorophyll a proteins (PCPs) from Heterocapsa pygmaea have been obtained by PCR. Two of the derived mature proteins of 150 residues have molecular masses of 15,795 and 15,780, respectively. Contrary to an earlier report, these show a high degree of identity (approximately 70%) over the whole of both domains to the mature 32-kDa PCP forms. The two genes lack introns, are arranged in tandem and separated by 526 bp. A putative N-terminal extension with three domains characteristic of a signal sequence, a chloroplast-targeting sequence and a thylakoid lumen-directing sequence, is present. Modelling of the Heterocapsa PCP amino acid sequence on to the high-resolution structure available for Amphidinium PCP shows that the main differences between two forms are in trimer contact regions.

Animals↗
PubMed1988

N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides as effectors of the sequence-specific modification of nucleic acids with reactive oligonucleotide derivatives.

It has been found that mono- and especially diphenazinium derivatives of oligonucleotides complementary to the DNA sequence adjacent to the target sequence of the addressed alkylation of DNA, significantly enhance the extent and specificity of alkylation with p-(N-2-chloroethyl-N-methylamino)benzylamide derivatives of the addressing oligonucleotides, thus playing the role of effector of the sequence-specific (complementary addressed) modification.

Alkylation↗
PubMed1988

Pure exogenous singlet oxygen: nonmutagenicity in bacteria.

Singlet oxygen (1 delta gO2) is the lowest energy-excited state of molecular oxygen, and more reactive than the triplet ground-state molecule. Although singlet oxygen has been implicated in a variety of biological effects, including reactions with DNA or some of its components, evidence for mutagenesis by singlet oxygen has remained unclear. We have previously described a system for bacterial exposure to pure exogenous singlet oxygen that eliminates ambiguity regarding the identity of the reactive species responsible for observed results. Despite the potent toxicity of pure singlet oxygen for several different strains of bacteria, we have found no evidence for mutagenicity of singlet oxygen in 26 Salmonella typhimurium histidine-auxotrophic strains killed to 35% survival. These strains included a variety of base-pair substitution or frameshift target sequences for reversion, including targets responsive to oxidative damage and targets rich in GC base pairs. Some strains combined histidine mutations with one or more mutations affecting DNA-repair capacity. 4 strains possessing the hisG46 mutation also were not mutated when exposed to dose ranges killing less than 28% and up to 99% of the bacteria. The relative frequency of small inphase deletions was assayed in hisG428 bacteria exposed to single oxygen and found to be the same as the spontaneous level. In addition to lack of induction of mutation in these strains, the 8-azaguanine forward mutation assay yielded no evidence of mutagenesis by singlet oxygen in strains killed to 15% survival. No induction of genetic changes by singlet oxygen was seen in an assay for duplication of approximately 1/3 of the bacterial chromosome. Tests for the ability of singlet oxygen to induce lambda prophage in E. coli K12 also proved negative. These studies collectively indicate that pure singlet oxygen generated outside the bacterial cell does not react significantly with the bacterial chromosome in ways leading to base-pair substitutions, frameshift mutations, small or large deletions, large duplications, or damage that interferes with DNA replication and induces the SOS system.

Azaguanine↗
PubMed2004

Nonbonded bivalence approach to cell-permeable molecules that target DNA sequences.

Polyamides such as the natural antibiotic distamycin A can form binary or ternary complexes with B-DNA. The driving forces and advantages for forming the ternary complexes are not fully understood. The computational studies reported herein suggest that three- and four-ring polyamides have a propensity for forming the same dimer conformations in water as those in their ternary complexes. The pre-dimerization of a polyamide in water facilitates the formation of the ternary complex, making the polyamide more selective, and tighter binding to the minor groove whose minimal width is predetermined by the B-DNA sequence. Relative to the dimer tethered with covalent bonds, the smaller, monomeric polyamide available from reversible dimerization in water makes the molecule inherently more cell permeable. A nonbonded bivalence approach that dimerizes molecules by intermolecular interactions is proposed for improving affinity, selectivity, and cell permeability.

Animals↗
PubMed2005

Structural and dynamic effects of single 7-hydro-8-oxoguanine bases located in a frameshift target DNA sequence.

DNA 7-hydro-8-oxoguanine (8-oxoG) is implicated in frameshift formation in an G(6) sequence of the HPRT gene in mismatch repair (MMR) defective cells. Using oligonucleotides based on this frameshift hotspot, we investigated how a single 8-oxoG modified the structural and dynamic properties of the G(6) tract. A 30 ns molecular dynamics (MD) simulation indicated compression of the minor groove in the immediate vicinity of the lesion. Fluorescence polarization anisotropy (FPA) and MD demonstrated that 8-oxoG increases DNA torsional rigidity and also constrains the movement of the single-stranded region at the single/double stranded DNA junction of model DNA replication template/primer. These constraints influenced the efficiency of primer extension by Klenow (exo(-)) DNA polymerase.

Base Pair Mismatch↗
PubMed2005

Quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea, using a molecular beacon.

A real-time polymerase chain reaction (PCR) assay utilizing a molecular beacon for the quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea (LYCIV), was developed, which involved the amplification of a 122bp DNA fragment from a conserved region of LYCIV ATPase gene. The specific probe consisting of two short arm and a central loop sequences complementary to the target amplicon was characterized with respect to its efficiency of quenching (E(ff)), and signal to background ratio by spectrofluorometric analysis of its hybridization with the complementary oligonucleotide target. The positive control plasmid pFHT-ATPase containing the target sequence was quantified to make the standard curve for sample detection after serial 10-fold dilution. Linear coefficient correlations between cycle threshold (C(T)) value and logarithmic positive plasmid concentration were close to one (r(2)=0.998) and the detection limit of the assay was 70 copies of positive plasmid/assay. The specificity of this real-time PCR was also demonstrated by using the genomic DNA templates from the healthy fish, white spot syndrome baculovirus (WSSV), and epizootic heamatopietic necrosis virus (EHNV), respectively. The coefficient of variation (CV) of the assay ranged from 1.16 to 4.42%, depending on the concentration of the positive plasmid. The quantitative detection of different tissues from LYCIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (6.86 x 10(6) and 4.62 x 10(6) viral genome copies/mg tissue, respectively) while no viral DNA was detected in the muscular tissue. These results suggested that the real-time PCR assay reported here could be used for rapid, sensitive, and quantitative detection of LYCIV infection.

Animals↗
PubMed2006

The snail (Biomphalaria glabrata) genome project.

In 2001, ideas for a snail genome project were discussed at the American Society of Parasitologists meeting (New Mexico) and a snail genome consortium was subsequently established (the first consortium meeting was held in 2005). A proposal for sequencing the snail genome was submitted to the National Human Genome Research Institute, and Biomphalaria glabrata was prioritized as a non-mammalian sequencing target in 2004. The sequencing of the genome of this medically important snail is now underway.

Animals↗
PubMed1999

Identification of a silencing element in the chicken lipoprotein lipase gene promoter: characterization of the silencer-binding protein and delineation of its target nucleotide sequence.

Lipoprotein lipase (LPL) hydrolyzes triglycerides in chylomicrons and very low density lipoproteins (VLDL) and plays a central role in lipid metabolism. It is regulated tissue-specifically. By deletion analysis, a negative regulatory element was identified in the chicken LPL gene promoter at base pairs (bp) -263 to -241. This sequence contained two palindromic halves with a three nucleotide spacer. Either half was sufficient for full inhibitory function. A protein complex bound specifically to this element and a high correlation was found between binding of the complex and inhibition of transcription. Its molecular mass, evaluated by native gel electrophoresis and Ferguson plot analysis, was 120 kDa. UV cross-linking followed by SDS-PAGE revealed two protein subunits of 48 kDa and 44 kDa, respectively. This inhibitory protein complex may contribute to the tissue-specific regulation of LPL gene transcription. It was much more abundant in liver than in adipose tissue and heart. Our data showed that this negative element inhibited transcription even when placed at an upstream location (-666), but failed to function in the herpes simplex virus thymidine kinase promoter, indicating that it acted in conjunction with other element(s) in the chicken LPL gene to inhibit transcription.

Animals↗
PubMed1999

Isolation and expression analysis of a novel human homologue of the Drosophila glial cells missing (gcm) gene.

A novel human homologue (GCMB) of the Drosophila glial cells missing gene (dGCM) was isolated using RACE. GCMB contained a gcm motif sequence and a nuclear targeting sequence similar to that of dGCM and mouse GCMb. Homology searches indicated that GCMB was located within chromosome 6p24.2. Transcripts of GCMB were detected by means of RT-PCR in fetal brain, normal adult kidney, 3/3 medulloblastomas, 1/3 gliomas and 4/8 non-neuroepithelial tumor cell lines. Our data suggest that humans have two homologues of gcm like mice and that human gcm genes form a novel family which may function not only during fetal development but also in the postnatal or pathological stage.

Amino Acid Sequence↗
PubMed2001

A quantitative, internally controlled real-time PCR Assay for the detection of parvovirus B19 DNA.

Parvovirus B19 is an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild, to more severe outcomes in, for example, immune-compromised patients. B19 is spread primarily via the respiratory route, but it can also be transmitted via blood and blood products. Viral loads in blood or plasma donations amount up to 10(11) genome equivalents/ml. Therefore, screening of plasma for fractionation for the presence of B19 and removal of highly loaded donations is a way to limit considerably the input of B19 into production pools and to improve further the safety of plasma products. An assay for the quantitative detection of B19 DNA, based on real-time PCR using ABI Prism SDS7700 (TaqMan) is described here. This assay allows precise quantitation of viral loads over 7 orders of magnitude. An exogenous internal control (internal quality marker) is included in each individual sample to prevent false negative results. A linearized plasmid is used as an internal quality marker that contains the identical sequence of the B19 target sequence but with an altered probe hybridization site. This allows co-amplification of B19 and internal quality marker and co-detection of FAM (6-carboxyfluorescein) or VIC labeled probes respectively. The assay is validated according to current guidelines (of the International Conference on Harmonization, Paul Ehrlich Institute, and the Council of Europe) and is optimized for high throughput screening.

DNA, Viral↗
PubMed1997

Disruption of a behavioral sequence by targeted death of peptidergic neurons in Drosophila.

The neuropeptide eclosion hormone (EH) is a key regulator of insect ecdysis. We tested the role of the two EH-producing neurons in Drosophila by using an EH cell-specific enhancer to activate cell death genes reaper and head involution defective to ablate the EH cells. In the EH cell knockout flies, larval and adult ecdyses were disrupted, yet a third of the knockouts emerged as adults, demonstrating that EH has a significant but nonessential role in ecdysis. The EH cell knockouts had discrete behavioral deficits, including slow, uncoordinated eclosion and an insensitivity to ecdysis-triggering hormone. The knockouts lacked the lights-on eclosion response despite having a normal circadian eclosion rhythm. This study represents a novel approach to the dissection of neuropeptide regulation of a complex behavioral program.

Animals↗