Transient generation of reactive oxygen or nitrogen (ROS/RNS), detected with dihydrodichlorofluoroscein by fluorescence microscopy, occurs within minutes of exposing cells to ionizing radiation. In the 1-10 Gy dose range, the amount of ROS/RNS produced/cell is constant, but the percentage of producing cells increases with dose (20 to 80%). Reversible depolarization of the mitochondrial membrane potential () and decrease in fluorescence of a mitochondria-entrapped dye, calcein, are observed coincidentally.
View Article and Find Full Text PDFThe effects of dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 on the apoptotic response of U937 monocytic leukemia cells to 1-beta-D-arabinofuranosylcytosine (ara-C) were examined. After a 6-h exposure to 1 microM ara-C, cells stably transfected with a p21WAF1/CIP1 antisense construct were significantly more sensitive to the induction of classic apoptotic morphology, DNA fragmentation, caspase-3 activation, poly(ADP-ribose) polymerase degradation, and underphosphorylation of the retinoblastoma protein (pRb) than their empty-vector counterparts. Enhanced susceptibility of antisense-expressing cells to ara-C was accompanied by a corresponding reduction in clonogenic and suspension culture growth.
View Article and Find Full Text PDFUsing polymerase chain reaction (PCR) with back-to-back primers, 85 different mitochondrial DNA (mtDNA) rearrangements, consisting of partial duplications or mini-circles, were detected in brain, liver, and heart tissue from Fischer 344 rats. The regions around the mitochondrial tRNALeu(UUR) gene, the cluster of three tRNA genes [His, Ser(AGY), Leu(UUC)], as well as the region of the displacement loop were analyzed separately with different primer sets. Rearrangements were detected in all regions analyzed in samples taken throughout the animal life span, ranging from 1 day old to 33 months of age (senescent).
View Article and Find Full Text PDFThe gene for the rat mitochondrial single-stranded DNA-binding protein (mtSSB) was amplified by PCR and isolated as several overlapping genomic clones. The clones encompassed the 5' untranslated sequence and all intron/exon junctions. The gene contained seven exons and six introns.
View Article and Find Full Text PDFBrain mtDNA from rats ranging in age from 1 day to 33 months were analyzed for large-scale rearrangements using nested PCR. The region of the mtDNA targeted by the primers was the shorter are between the two origins of replication and encompassed the heavy (H) and light (L) strand promoters (HSP) and (LSP). Rearrangements lacking 4 to 5 kb of genomic sequence were found in animals of all ages.
View Article and Find Full Text PDFThis study identified 33 different deletions in mitochondrial DNA from four aging Fischer-344 rat brains and from a cultured rat lymphoma cell line (Nb2 cells). The deletions were located in the longer arc between the heavy and light strand origins of replication. PCR products that spanned across the deleted regions were sequenced, and deletions ranging between 6548 bp and 9977 bp in length were identified.
View Article and Find Full Text PDFNucleic Acids Res
August 1994
The 5' processing of rat pre-tRNA(Lys) and a series of mutant derivatives by rat cytosolic RNase P was examined. In standard, non-kinetic assays, mutant precursors synthesized in vitro with 5' leader sequences of 10, 17, 24, 25, and 46 nucleotides were processed to approximately equal levels and yielded precisely cleaved 5' processed intermediates with the normal 7-base pair aminoacyl stems. The construct containing the tRNA(Lys) with the 46-nucleotide leader was modified by PCR to give a series of pre-tRNA(Lys) mutants designed to measure the effect on processing by (1) substituting the nucleotide at the +1 position, (2) pairing and unpairing the +1 and +72 bases, (3) elongating the aminoacyl stem, and (4) disrupting the helix of the aminoacyl stem.
View Article and Find Full Text PDFRat liver ribonuclease P was isolated from a cytosolic fraction and shown to have optimal activity in the presence of 1 mM MgCl2 and 150-200 mM KCl using Escherchia coli pre-tRNA(Tyr) as substrate. In cesium sulfate isopycnic density gradients, the enzyme had a buoyant density of 1.36 g/ml, indicating that it is a ribonucleoprotein complex.
View Article and Find Full Text PDFArch Biochem Biophys
October 1990
The rat mitochondrial single strand DNA binding protein (SSB) P16 was purified to apparent homogeneity by elution from single strand DNA agarose with ethidium bromide. Each monomer of P16 contains two tryptophan residues, and the intrinsic fluorescence from these residues is quenched upon binding to single strand polynucleotides. From fluorescence quench titrations of ligand to fixed amounts of DNA lattice, a binding site size of 8 or 9 nucleotides per P16 monomer was found.
View Article and Find Full Text PDFThe 5'- and 3'-tRNA processing nucleases have been isolated from rat liver mitochondria. The two activities co-purified through heparin-agarose and phenyl-Sepharose columns and then efficiently separated on a DEAE-cellulose column. The 5' processing nuclease was found in the flow-through fraction, and the 3' processing activity eluted with 0.
View Article and Find Full Text PDFA DNA primase was partially purified from rat liver mitochondria and separated from the bulk of DNA polymerase gamma and mtRNA polymerase by heparin-agarose chromatography. The primase was distinguished from mtRNA polymerase by its response to pH, monoand divalent cations, and ATP concentrations. In the absence of an active DNA polymerase and using poly(dT) as template, primase synthesized mixed polynucleotide products consisting of units of oligo(A) 1-12 alternating with units of oligo(dA)25-40.
View Article and Find Full Text PDFThree types of Amy-2-related DNA sequences, Amy-2a I, Amy-2a II and Amy-X, exist in the genome of mice of the inbred strain A/J. Amy-2a I and Amy-X are single copy sequences. Amy-2a II occurs as three copies per haploid genome.
View Article and Find Full Text PDFThe mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCl isopycnic gradients. The top fraction of the gradients contained a single polypeptide species (Mr approximately equal to 15,200) based upon SDS PAGE. Digestion of single strand DNA-bound P16 with proteinase K produced a protease-insensitive, DNA-binding fragment (Mr approximately equal to 6,000) that has been purified by essentially the same procedures used for intact P16.
View Article and Find Full Text PDFMitochondrial DNA (mtDNA)-protein complexes were released from the organelles by sodium dodecyl sulfate-lysis and purified by Phenyl-Sepharose CL-4B chromatography. The mitochondrial DNA-binding protein P16 was the only detectable protein in the complex. Treatment of the complex with proteinase K, or subtilisin, revealed the presence of a protease-insensitive, submolecular domain (Mr approximately equal to 6,000) that retained the capacity to bind tenaciously to the DNA.
View Article and Find Full Text PDFRes Commun Chem Pathol Pharmacol
October 1983
The incubation of non-proliferating primary cultures of rat hepatocytes with benzo[a]Pyrene for 24 or 48 hours produced a concentration-dependent stimulation of nuclear DNA synthesis (measured as [3H]thymidine incorporation into isolated, purified DNA) whereas the normal processes of D-loop formation and expansion synthesis on mitochondrial DNA were inhibited by as much as 62% of that in control cells. Incubation of cultures with [3H]benzo[a]pyrene showed that highly purified mitochondrial DNA retained 4-15 times more covalently bound adducts per unit length of DNA than did nuclear DNA. We suggest, therefore, that this carcinogenic, environmental pollutant may exert its most profound effects on the mitochondrial, rather than the nuclear, genetic apparatus.
View Article and Find Full Text PDFA stable DNA-protein complex was released from rat liver mitochondria by sodium dodecyl sulfate-lysis and isolated by sedimentation velocity in sucrose density gradients. The mtDNA-protein complex was washed with 0.5 M NaCl and any unbound contaminants were removed by hydroxyapatite column chromatography.
View Article and Find Full Text PDFA highly folded, rapidly sedimenting form of rat liver mitochondrial DNA has been released from the organelles wiht BRIJ 58 and sodium deoxycholate in the presence of 0.5 M NaCl and isolated by sedimentation velocity in sucrose gradients. Under these conditions a majority of the mitochondrial DNA labeled in vitro sedimented beyond 39 S, the sedimentation coefficient of a highly purified mitochondrial DNA supercoil, and appeared as a stable, heterogeneous population of species ranging in s values between 42 S and about 70 S.
View Article and Find Full Text PDFRes Commun Chem Pathol Pharmacol
August 1978
The acute and subchronic effects of aflatoxin B1 and dimethylnitrosamine (DMN) on in vitro incorporation of thymidine-3H into DNA (mt-DNA) from mouse liver mitochondria were studied after single injection or after one month or three months of weekly injection. Both carcinogens induced 50% decreases in mt-DNA synthesis acutely, with a 50% decrease in synthesis of high molecular weight mt-DNA. Subchronic administration of aflatoxin B1 inhibited mt-DNA synthesis by 54% with elution profiles similar to the controls.
View Article and Find Full Text PDFSucrose density gradient fractionation of isolated rat liver mitochondrial DNA ordinarily yields two peaks, one at 39 S, the other at 27 S. However, when these mitochondria are first incubated with a labeled DNA precursor, a labeled peak at about 8 S is also observed. Is this low molecular weight 8 S DNA merely an artifact of contamination or breakdown, or is it a functioning part of the mitochondrial genome? That it is not a nuclear contaminant is shown by: (a) the absence of nuclei or nuclear fragments in active mitochondrial preparations; (b) the insensitivity of 8 S DNA synthesis to treatment of mitochondria with DNase and RNase; (c) the ability of inner membrane preparations to synthesize this DNA; (d) the ability of atractyloside to inhibit incorporation of [3H]dATP into 8 S and 39 S or 27 S DNA equally; (e) the labeling of 8 S DNA (as well as 39 S and 27 S DNA) but not of nuclear DNA after the administration in vivo of [3H]thymidine.
View Article and Find Full Text PDFArch Biochem Biophys
April 1972