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At least 1,369 records · Page 76Linked to original sources
PubMed1995

Aromatic amino acid biosynthesis in Buchnera aphidicola (endosymbiont of aphids): cloning and sequencing of a DNA fragment containing aroH-thrS-infC-rpmI-rplT.

A 4.5-kilobase DNA fragment from Buchnera aphidicola, the endosymbiont of the aphid Schizaphis graminum, was cloned and sequenced. On the basis of homology to Escherichia coli, the following genes were found in the order listed: aroH-thrS-infC-rpmI-rplT. AroH corresponds to the E. coli tryptophan-inhibited 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. Evidence was presented indicating that this is the sole gene for DAHP synthase in the B. aphidicola genome. This enzyme initiates the complex branched pathway leading to aromatic amino acid biosynthesis. The presence of aroH is consistent with past observations indicating that aphid endosymbionts are able to synthesize tryptophan for the aphid host. thrS, infC, rpmI, and rplT correspond to genes for threonine tRNA synthase, initiation factor-3, and large ribosome subunit proteins L35 and L20, respectively. Sequence comparisons indicate some differences and similarities between E. coli and B. aphidicola with respect to the possible regulation of synthesis of these proteins.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1982

Chicken oviduct progesterone receptor: location of specific regions of high-affinity binding in cloned DNA fragments of hormone-responsive genes.

We have used a DNA-cellulose competitive binding assay to measure the extent of displacement of the chicken oviduct progesterone-receptor complex from calf thymus DNA-cellulose by purified cloned fragments of genomic DNA. Several DNA fragments from hormonally responsive genes coding for egg-white proteins were found to be efficient competitors for either crude or partially purified receptor complexes when compared with calf thymus DNA. Data obtained with deletion mutations constructed in vitro allowed delineation of a specific region necessary for strong competition, located 250--300 bp upstream from the mRNA startsite of the ovalbumin gene. Sequence homologies with this 5'-upstream region were observed in other fragments of the ovalbumin, conalbumin, ovomucoid, X and Y genes, which were also efficient competitors. Based on a comparison of such sequences of homology, a consensus sequence that may constitute a region binding progesterone-receptor complex has been constructed: ATCCCTTATTATTCTGGTTTGTA. The results suggest that specific double-stranded DNA sequences are recognized by the oviduct progesterone-receptor complex in vitro, and are relevant to the question of whether specific DNA sequences are directly involved as genomic binding sites for steroid receptors.

Animals↗
PubMed1999

Neural network input representations that produce accurate consensus sequences from DNA fragment assemblies.

MOTIVATION: Given inputs extracted from an aligned column of DNA bases and the underlying Perkin Elmer Applied Biosystems (ABI) fluorescent traces, our goal is to train a neural network to determine correctly the consensus base for the column. Choosing an appropriate network input representation is critical to success in this task. We empirically compare five representations; one uses only base calls and the others include trace information. RESULTS: We attained the most accurate results from networks that incorporate trace information into their input representations. Based on estimates derived from using 10-fold cross-validation, the best network topology produces consensus accuracies ranging from 99.26% to >99.98% for coverages from two to six aligned sequences. With a coverage of six, it makes only three errors in 20 000 consensus calls. In contrast, the network that only uses base calls in its input representation has over double that error rate: eight errors in 20 000 consensus calls. CONTACT: allex@cs.wisc.edu

Base Sequence↗
PubMed2001

Neuronal, but not microglial, accumulation of extravasated serum proteins after intracerebral hemolysate exposure is accompanied by cytochrome c release and DNA fragmentation.

Vasogenic edema after oxidative injury has been accompanied by intracellular accumulation of serum proteins and nuclear damage. This study sought to determine whether serum protein accumulation, along with other markers of brain injury, was present after exposure to intracerebral hemolysate, an oxidant model of intracerebral hemorrhage (ICH). Saline (n = 24) or hemolysate (n = 30) was injected into the caudate-putamen of adult Sprague-Dawley rats. Compared with saline, hemolysate deposition was associated with intracellular accumulation of serum proteins as evidenced by Evans blue uptake in neurons and microglia at 4 and 24 hours. Intracellular Evans blue colocalized with DNA fragmentation detected by nick end-labeling and whose presence was confirmed by gel electrophoresis. Immunoblots of cytosolic fractions confirmed cytochrome c release. Immunostaining established colocalization of cytosolic cytochrome c and intracellular Evans blue at 4 hours. At 24 hours, cytosolic cytochrome c was evident in astrocytes surrounding Evans blue-positive cells. Immunoblot analysis and immunostaining revealed HSP70 induction at 24 hours in regions adjacent to intracellular serum accumulation. Neuronal accumulation of extravasated serum proteins in this model of ICH was associated with cytochrome c release, DNA fragmentation, and cell death. Stress protein induction in adjacent regions suggested that vasogenic edema might have exacerbated cellular dysfunction and cell death after ICH.

Animals↗
PubMed1994

DNA fragmentation as a consequence of cell cycle traverse in doxorubicin- and idarubicin-treated human lymphoma cells.

We have studied some of the factors involved in the cytotoxic actions of the anticancer anthracycline antibiotics doxorubicin (DOX) and idarubicin (IDA) towards human B-cell lymphoma cells in vitro. IDA was found to accumulate within cells to a greater degree than the related drug DOX for both short (1 h) and long-term (24 h) exposures. Both agents showed a similar capacity for trapping topoisomerase II in intact cells, but cross-linking activity was significantly lower than that induced by the specific poison VP16. IDA was four- to eight fold more potent for the induction of cytostasis and cell cycle arrest and for the instigation of DNA breakdown as a prelude to the full expression of apoptosis. Inhibition of DNA fragmentation at higher drug doses was linked closely with the inhibition of S-phase traverse. The findings suggest that DOX and IDA act in a similar fashion, the latter agent being more effective due to enhanced intracellular accumulation. We conclude that the presence of drug and topoisomerase II-associated DNA damage is not sufficient to induce DNA fragmentation; rather, unregulated commitment to S-phase traverse is an important factor in the activation of programmed cell death.

Apoptosis↗
PubMed1985

Transduction of virulence in herpes simplex virus type 1 from a pathogenic to an apathogenic strain by a cloned viral DNA fragment.

Herpes simplex virus (HSV) type 1 strain HFEM, the genome of which contains a deletion of about 4 kilobasepairs (kbp) between 0.7 and 0.8 viral map units, is apathogenic in the tree shrew when the animals are inoculated intravenously, intraperitoneally and/or subcutaneously. Similar results were obtained using Balb/c mice. Studies of the state of viral latency in animals infected with HSV-1 strain HFEM revealed that this strain was unable to colonize the ganglia of tree shrews. Infectious virus could be recovered only from the spleens of latently infected tree shrews. Thus, this system offers new opportunities for investigating the gene functions responsible for the virulence of HSV-1. Marker rescue experiments were performed by the contransfection technique using native DNA of HSV-1 strain HFEM and Bam H I DNA fragment B derived from the pathogenic HSV-1 strain F and cloned in a bacterial vector. A number of different intratypic recombinants were established in which the deleted region of HSV-1 strain HFEM had been repaired. The pathogenicity of these recombinants was examined in vivo. One of the recombinants (HSV-R-HFehx-C19) caused generalized and lethal herpes virus infection in juvenile and adult tupaias, indicating that the virulence of the pathogenic HSV-1 strain F can be transduced by the cloned Bam H I DNA fragment B to the apathogenic HSV-1 strain HFEM.

Animals↗
PubMed1981

Characterization of nascent DNA fragments produced by excision of uracil residues in DNA.

Nascent short DNA chains could result from repair of incorporated uracil residues or be intermediates in discontinuous replication. We have characterized short DNA chains having apyrimidinic/apurinic-sites at 5' ends, the expected intermediates of repair, to distinguish them from RNA-linked replication intermediates. We have synthesized model substrates for the repair products; d(pRib[32P]poly(T)) and d(Rib[32P]poly(T)). Alkaline hydrolysis of both substrates has produced [5'-32P]poly(dT). Nascent short DNA was prepared from an Escherichia coli sof (dut) mutant, in this strain fragments from excision repair of uracil residues accumulate. The products of alkaline treatment are hardly digested by spleen exonuclease which selectively degrades 5'-hydroxyl-terminated DNA. These two results show that alkaline hydrolysis of the uracil repair fragments produces 5'-phosphoryl-terminated DNA, whereas it is known that 5'-hydroxyl-terminated DNA is generated from RNA-linked DNA molecules. The two types of nascent fragments thus can be distinguished by the 5'-terminal structure produced by an alkaline hydrolysis.

Animals↗
PubMed2000

Sequence and organization analyses of a Zygosaccharomyces rouxii DNA fragment containing the HIS3 gene.

The nucleotide sequence of a 3.4 kb fragment containing the HIS3 gene of the osmotolerant yeast Zygosaccharomyces rouxii has been determined. The fragment was cloned from a Z. rouxii genomic DNA library by complementation of a Saccharomyces cerevisiae his3 mutant strain. The sequenced DNA fragment contained three open reading frames; the middle one (678 bp long, predicting a protein of 226 amino acids) shared a high degree of similarity with HIS3 genes of other yeast species. In the promoter region of the putative ZrHIS3 gene, a T(c) element required for constitutive transcription was found. The GenBank Accession No. of the sequenced DNA region is Y18561.

Amino Acid Sequence↗
PubMed1993

Analysis of Epstein-Barr virus BamHI F DNA fragment in nude mouse-passaged nasopharyngeal carcinoma tissues.

Two nude mouse-passaged nasopharyngeal carcinoma (NPC) tissues, NPC-295 and NPC-306, were different in ability to transform cord blood lymphocytes and contained Epstein-Barr virus (EBV) genomes with difference in BamHI F DNA fragment. Four clones containing DNA sequences of the BamHI F fragment (54,853-62,249) were obtained from genomic libraries of NPC-295 and NPC-306 and their partial restriction enzyme maps and sequences were determined. The restriction enzyme maps of EBV DNA at the BamHI F fragment in NPC-295 and NPC-306 appeared to be similar to that of EBV B95-8 strain. However, 8 nucleotide differences were revealed between NPC-295 and the B95-8 strain when 566 nucleotides (55,405-55,970) were compared, and 15 out of 677 nucleotides analyzed (55,410-56,086) were different between NPC-306 and B95-8.

Animals↗
PubMed1991

Genetic and sequence analyses of a Pseudomonas denitrificans DNA fragment containing two cob genes.

A genetic analysis of a 12-kb DNA fragment containing Pseudomonas denitrificans cob genes was performed by transposon-mediated insertional mutagenesis. The nucleotide sequence and genetic analysis have shown that a 4.8-kb DNA subfragment carried two cob genes (cobS and cobT). Biochemical data concerning the complemented cobS and cobT mutants suggested that the cobS product was involved in cobalt insertion-mediating reactions and that the cobT product was involved in the transformation of precorrin-3 into cobyrinic acid.

Amino Acid Sequence↗
PubMed1999

Telomere shortening triggers a p53-dependent cell cycle arrest via accumulation of G-rich single stranded DNA fragments.

It has been repeatedly suspected that telomere shortening might be one possible trigger of the p53-dependent cell cycle arrest, although the mechanism of this arrest remained unclear. Telomeres in human cells under mild oxidative stress accumulate single-strand damage faster than interstitial repetitive sequences. In MRC-5 fibroblasts and U87 glioblastoma cells, which both express wild-type p53, oxidative stress-mediated production of single-strand damage in telomeres is concomitant to the accumulation of p53 and p21 and to cell cycle arrest. This response can be modeled by treatment of cells with short single stranded telomeric G-rich DNA fragments. The arrest is transient in U87 cells. Recovery from it is accompanied by up-regulation of telomerase activity and elongation of telomeres. Overexpression of mutated p53 is sufficient to reverse the phenotype of inhibition as well as the delayed activation of telomerase. These data suggest that the production of G-rich single stranded fragments during the course of telomere shortening is sufficient to trigger a p53 dependent cell cycle arrest.

Adenocarcinoma↗
PubMed1987

A simple and rapid procedure for sequencing long (40-kb) DNA fragments.

A simple procedure has been developed for sequencing long (40-kb) DNA fragments by the dideoxy method. The fragment is cloned in the sequencing cosmid pAA113X by in vitro packaging, and subdivided by a series of overlapping IS1-promoted deletions. The deletions are isolated by positive selection for galactose resistance. Plasmids from several thousand galactose-resistant colonies are fractionated on an agarose gel, and DNA from each fraction is restricted with enzymes (such as SphI and SalI) to shorten each deletion from the opposite end. As a result, a series of short overlapping segments, spread across the entire length of the fragment, are fused to IS1. The plasmids are extracted by a rapid method, arranged according to size, and used for supercoil sequencing with an IS1 primer. Sequences of IS1-promoted deletions contain extensive overlaps that are connected further by restriction enzyme-generated deletions to give the complete 40-kb sequence.

Base Sequence↗
PubMed1998

Identification of a DNA fragment encoding a putative membrane transporter protein expressed in Sporothrix schenckii.

We have isolated two DNA fragments from Sporothrix schenckii. These fragments were nonspecifically amplified from the whole-cell DNA using polymerase chain reaction (PCR) primers originally designed in the gene encoding heat shock protein 70 of Mycobacterium leprae. Reverse transcription PCR demonstrated that the smaller (sp-2) fragment was expressed, and a database search indicated that the deduced amino acids sequence from the sp-2 fragment contained a region homologous to the conserved sequence of the membrane transporter protein family. This is the first report of partial cloning of the gene encoding the putative membrane transporter in S. schenckii.

Amino Acid Sequence↗
PubMed1992

Characterization of Pseudomonas putida mutants unable to catabolize benzoate: cloning and characterization of Pseudomonas genes involved in benzoate catabolism and isolation of a chromosomal DNA fragment able to substitute for xylS in activation of the TOL lower-pathway promoter.

Mutants of Pseudomonas putida mt-2 that are unable to convert benzoate to catechol were isolated and grouped into two classes: those that did not initiate attack on benzoate and those that accumulated 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (benzoate diol). The latter mutants, represents by strain PP0201, were shown to lack benzoate diol dehydrogenase (benD) activity. Mutants from the former class were presumed either to carry lesions in one or more subunit structural genes of benzoate dioxygenase (benABC) or the regulatory gene (benR) or to contain multiple mutations. Previous work in this laboratory suggested that benR can substitute for the TOL plasmid-encoded xylS regulatory gene, which promotes gene expression from the OP2 region of the lower or meta pathway operon. Accordingly, structural and regulatory gene mutations were distinguished by the ability of benzoate-grown mutant strains to induce expression from OP2 without xylS by using the TOL plasmid xylE gene (encoding catechol 2,3-dioxygenase) as a reporter. A cloned 12-kb BamHI chromosomal DNA fragment from the P. aeruginosa PAO1 chromosome complemented all of the mutations, as shown by restoration of growth on benzoate minimal medium. Subcloning and deletion analyses allowed identification of DNA fragments carrying benD, benABC, and the region possessing xylS substitution activity, benR. Expression of these genes was examined in a strain devoid of benzoate-utilizing ability, Pseudomonas fluorescens PFO15. The disappearance of benzoate and the production of catechol were determined by chromatographic analysis of supernatants from cultures grown with casamino acids. When P. fluorescens PFO15 was transformed with plasmids containing only benABCD, no loss of benzoate was observed. When either benR or xylS was cloned into plasmids compatible with those plasmids containing only the benABCD regions, benzoate was removed from the medium and catechol was produced. Regulation of expression of the chromosomal structural genes by benR and xylS was quantified by benzoate diol dehydrogenase enzyme assays. The results obtained when xylS was substituted for benR strongly suggest an isofunctional regulatory mechanism between the TOL plasmid lower-pathway genes (via the OP2 promoter) and chromosomal benABC. Southern hybridizations demonstrated that DNA encoding the benzoate dioxygenase structural genes showed homology to DNA encoding toluate dioxygenase from the TOL plasmid pWW0, but benR did not show homology to xylS. Evolutionary relationships between the regulatory systems of chromosomal and plasmid-encoded genes for the catabolism of benzoate and related compounds are suggested.

Benzoates↗
PubMed1998

Fas-induced DNA fragmentation and proteolysis of nuclear proteins.

BACKGROUND: Fas is a member of the tumour necrosis factor (TNF) receptor family. Activation of Fas by its ligand or an agonistic anti-Fas antibody causes apoptosis in Fas-bearing cells, by activating various members of the caspase family. RESULTS: Specific fluorogenic substrates (MCA-DEVDAPK[dnp] and MCA-VEVDAPK[dnp]) for caspases 3 and 6 were prepared. Using these substrates, a gradual increase of the caspase 3-and 6-like proteases were detected during the Fas engagement in human Jurkat. This activation of caspases correlated well with the cleavage of poly(ADP-ribose) polymerase and lamin B1, as well as with DNA fragmentation. When the recombinant caspases were added to the extracts from Jurkat cells, caspase 3 produced active caspase 6-like protease, while caspase 6 activated the caspase 3 protease, suggesting that these proteases can activate each other. The caspase-treated cell extracts, as well as the extracts from the Fas-activated cells, caused the proteolysis of nuclear proteins and DNA degradation. The cleavage of nuclear proteins was inhibited by caspase inhibitors, while the same inhibitors had no effect on DNA degradation. CONCLUSIONS: At one stage of the caspase cascade, caspases activate each other, and amplify the apoptotic signal. Caspases downstream of the cascade then cause the proteolysis of nuclear proteins and DNA degradation.

Apoptosis↗
PubMed1996

[Structure analysis of a DNA fragment containing strong promoter activity from Streptomyces mycarofaciens 1748].

A HindIII-HindIII 2.0kb DNA fragment containing promoter activity has been cloned from medicamycin producing strain (S. mycarofaciens 1748), using promoter -probe plasmid vector pIJ486. Transformants of recombinant plasmid p4H2 containing this fragment were resistant to Km in the level above 500 micrograms/ml in MM medium. The result of subcloning deletion analysis indicated that different part of deletion of this fragment would show various extent of effect on the promoter activity. A rather complicated transcriptional regulatory mechanism was suggested. DNA sequence analysis revealed that this fragment consisted of 1984 nucleotides with 47.7% of G+C content. No typical ORF of Streptomyces gene was found. Certain regions showed some homologies with promoters of Streptomyces genes. In 520-570bp region, a sequence similar to the upstream activator sequence (UAS) of E.coli tRNA was discovered. This implied that a possible "enhancer" element was involved in Streptomyces gene transcription.

Anti-Bacterial Agents↗
PubMed1995

Tumor necrosis factor-induced hepatic DNA fragmentation as an early marker of T cell-dependent liver injury in mice.

BACKGROUND & AIMS: The injection of mice with anti-CD3 monoclonal antibody causes activation of T lymphocytes and leads to a lethal shock syndrome. The aim of this study was to investigate T cell-dependent, cytokine-mediated target cell death that leads to organ injury. METHODS: Anti-CD3 monoclonal antibody or staphylococcal enterotoxin B was injected into mice sensitized by D-galactosamine. Liver injury was assessed biochemically and histologically, and circulating cytokines were determined. RESULTS: Mice sensitized with D-galactosamine developed severe liver injury within 8 hours after injection of anti-CD3 monoclonal antibody or staphylococcal enterotoxin B. Apoptotic bodies and chromatin condensation were detectable 5 hours after anti-CD3 monoclonal antibody challenge. DNA fragmentation in the liver preceded the increase in plasma alanine aminotransferase activity. Anti-CD3 monoclonal antibody induced the release of tumor necrosis factor and other cytokines. Passive immunization against tumor necrosis factor or pretreatment with immunosuppressive drugs protected mice from liver injury. Liver injury associated with apoptotic cell death and DNA fragmentation was also noted in D-galactosamine-sensitized mice injected with staphylococcal enterotoxin B. CONCLUSIONS: Tumor necrosis factor-induced hepatic apoptosis followed by necrosis may represent a general pathomechanism of T-cell shock models using D-galactosamine-sensitized mice.

Alanine Transaminase↗
PubMedDate not supplied

The cytoprotective effects of exogenous DNA fragments.

Ultraviolet irradiation of normal human keratinocytes induces a cytotoxic effect. The chromophore for this effect is believed to be genomic DNA. However, DNA damage is known to be repaired in UVB irradiated keratinocytes. The trigger for this DNA repair is potentially damaged DNA itself. To test the hypothesis that damaged DNA can induce the host cell's own DNA repair mechanism, we treated the keratinocytes with the damaged DNA and evaluated its cytoprotective effects. We have observed that fragmented calf thymus DNA irradiated and damaged with a UVC light can induce a protective effect in cultured human keratinocytes. Keratinocytes treated with UVC damaged DNA fragments are less susceptible to UVB irradiation-induced cell death as measured by neutral red uptake. Unirradiated exogenous DNA did not induce this protective effect. Similar protective effects can be seen with irradiated salmon sperm DNA. UVC damaged DNA fragments induced 60% increase in protection in human HaCaT keratinocyte in culture to the cell death induced by UVB. Similar protection was observed with UVC irradiated oligothymidylic acid (dT3-dT5) which increased the survival of human HaCaT keratinocytes after UVB irradiated by 50%. Isolated mononucleotides, irradiated or not, do not increase UVB survivability. Cellular DNA synthesis was greatly inhibited by UVB, becoming undetectable at 40 mJ/cm(2). Exogenous treatment with damaged fragments causes immediate and significant inhibition of total cellular DNA synthesis. This inhibition was dose dependent. Cells that undergo damage to their DNA are known to inhibit endogenous DNA synthesis via p53 suppressor gene activation. This is believed to allow them sufficient time to repair the host DNA. The cellular response to exogenous damaged DNA may be a similar mechanism.

Animals↗