scholarly journals Localization of the ribonucleotide sites in rat liver mitochondrial deoxyribonucleic acid

1978 ◽  
Vol 169 (1) ◽  
pp. 79-85 ◽  
Author(s):  
D M Lonsdale ◽  
I G Jones
Keyword(s):  
Mitochondrial Dna ◽  
Electron Microscope ◽  
Rat Liver ◽  
Deoxyribonucleic Acid ◽  
Ribonuclease A ◽  
Single Strand ◽  
Ribonuclease H ◽  
Ribonuclease T1 ◽  
Dna Endonuclease ◽  
Calf Thymus

Supercoiled rat liver mitochondrial DNA is relaxed by treatment with ribonucleases A, T1 or H. All the supercoiled mitochondrial DNA is sensitive to ribonuclease H and ribonuclease A, but only 35% of the supercoiled population is sensitive to ribonuclease T1. Removal of the ribonucleotides with calf thymus ribonuclease H, followed by denaturation of the mitochondrial DNA and analysis of the single-strand fragment lengths in the electron microscope, showed that the ribonucleotides were randomly located on both strands of the DNA. Endonuclease-S1 digestion of mitochondrial DNA after removal of the ribonucleotides reveals that no unique fragments are produced and ribonucleotides are randomly distributed with respect to one another. The average number of ribonucleotide sites per molecule was estimated to be between 8 and 13. Two possible mechanisms for the origin of ribonucleotide sites are discussed.

scholarly journals HEAT DENATURATION STUDIES OF RAT LIVER MITOCHONDRIAL DNA

1972 ◽  
Vol 53 (2) ◽  
pp. 393-406 ◽  
Author(s):  
David R. Wolstenholme ◽  
Robert G. Kirschner ◽  
Nicholas J. Gross
Keyword(s):  
Mitochondrial Dna ◽  
Electron Microscope ◽  
High Temperatures ◽  
Rat Liver ◽  
Heat Denaturation ◽  
Similar Length ◽  
Patterns Of Distribution ◽  
Supercoiled Form ◽  
Strand Separation ◽  
Open Circular

The effect of progressive denaturation of open circular molecules (component II) and supercoiled covalently closed circular molecules (component I) of rat liver mitochondrial DNA has been followed by heating in the presence of formaldehyde and examination in the electron microscope. After heating at 49°C, two, three, or four regions of strand separation were visible in 25% of the component II molecules. Comparisons of the patterns of distribution of these regions in individual molecules indicated that they occurred at at least three specific positions around the molecule. Also, these regions, which were assumed to be rich in adenine and thymine, were within a segment which was less than 50% of the length of the molecule. After heating at 50°C, up to 14 regions of strand separation were observed, but when comparisons were made no clear groupings were found. At 51°C, component II molecules were completely separated into a single-stranded circle and a single-stranded linear piece of similar length. Strand separation was accompanied by shortening of the molecule. At 70°C, single-stranded circles had a mean length of 2.7 µ, compared with 5.0 µ for native molecules. Progressive heating of component I molecules resulted first in conversion to an open circle (I') and then to a second supercoiled form (I''). Visualization of further denaturation products of component I was prevented by crosslinking of the molecule by formaldehyde at high temperatures.

scholarly journals Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria.

1985 ◽  
Vol 100 (1) ◽  
pp. 258-264 ◽  
Author(s):  
P A Pavco ◽  
G C Van Tuyle
Keyword(s):  
Mitochondrial Dna ◽  
Dna Binding ◽  
Rat Liver ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Liver Mitochondria ◽  
Single Strand ◽  
Proteinase K ◽  
Rat Liver Mitochondria

The 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. The partial amino acid compositions for P16 and the DNA-binding fragment were obtained by conventional methods. Analysis of subcellular fractions revealed that nearly all of the cellular P16 was located in the mitochondria and that only trace amounts of protein of comparable electrophoretic mobility could be isolated from the nuclear or cytoplasmic fractions. The labeling of P16 with [35S]methionine in primary rat hepatocyte cultures was inhibited by more than 90% by the cytoplasmic translation inhibitor cycloheximide, but unaffected by the mitochondrial-specific agent chloramphenicol. These results indicate that P16 is synthesized on cytoplasmic ribosomes and imported into the mitochondria. The addition of purified P16 to deproteinized mitochondrial DNA resulted in the complete protection of the labeled nascent strands of displacement loops against branch migrational loss during cleavage of parental DNA with SstI, thus providing strong evidence that P16 is the single entity required for this in vitro function. Incubation of P16 with single strand phi X174 DNA, double strand (RF) phi X174 DNA, or Escherichia coli ribosomal RNA and subsequent analysis of the nucleic acid species for bound protein indicated a strong preference of P16 for single strand DNA and no detectable affinity for RNA or double strand DNA. Examination of P16-single strand phi X174 DNA complexes by direct electron microscopy revealed thickened, irregular fibers characteristic of protein-associated single strand DNA.

scholarly journals Ribonuclease-sensitivity of covalently closed rat liver mitochondrial deoxyribonucleic acid

1974 ◽  
Vol 141 (1) ◽  
pp. 155-158 ◽  
Author(s):  
David M. Lonsdale ◽  
I. Gwyn Jones
Keyword(s):  
Mitochondrial Dna ◽  
Rat Liver ◽  
Deoxyribonucleic Acid ◽  
Reducing Agents

Preparations of covalently closed mitochondrial DNA of rat liver contain 10–30% of molecules that are converted into relaxed circular molecules after treatment with ribonuclease. Control experiments, with covalently closed bacteriophage PM2 DNA, indicate that ribonuclease-sensitivity cannot be induced either by depurination or by incubation with reducing agents.

scholarly journals High total histone/deoxyribonucleic acid ratios for rat liver nuclei

1973 ◽  
Vol 135 (1) ◽  
pp. 115-123 ◽  
Author(s):  
Subir K. Chanda ◽  
Regina Ickowicz ◽  
Alexander L. Dounce
Keyword(s):  
Amino Acid ◽  
Direct Measurement ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Deoxyribonucleic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Total Histone ◽  
Rat Liver Nuclei ◽  
Calf Thymus ◽  
Liver Nuclei

The ratios of total histone to DNA for rat liver nuclei isolated by four methods as well as for calf liver nuclei isolated by one method were determined by obtaining the ratios of the total areas of the electrophoretic histone peaks for the liver nuclei to the corresponding total area given by a known amount of standard calf thymus histone. Ratios of total histone to DNA of approx. 2 for rat liver nuclei isolated at pH3.8 or 5.8 and for calf liver nuclei isolated at pH3.8 were confirmed twice by the above procedure and also by direct measurement, by the method of Lowry et al. (1951), of histone extracted in 0.2m-H2SO4. The histones of calf thymus, calf liver and rat liver were characterized by their amino acid compositions and by polyacrylamide-gel electrophoresis.

scholarly journals Multiple forms of mammalian deoxyribonucleic acid polymerase. An attempt to relate their interactions with nuclei and free deoxyribonucleic acid in vitro with their possible functions in vivo

1971 ◽  
Vol 125 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Patricia G. Wallace ◽  
D. R. Hewish ◽  
M. M. Venning ◽  
L. A. Burgoyne
Keyword(s):  
Rat Liver ◽  
Deoxyribonucleic Acid ◽  
Ionic Composition ◽  
Dna Polymerases ◽  
Calf Thymus Dna ◽  
Type I ◽  
Isolation Medium ◽  
Calf Thymus ◽  
Ionic Effects

The DNA polymerases of the following eukaryotic tissues were studied: regenerating rat liver, normal rat liver, rat thymus, normal mouse liver and Ehrlich ascites-tumour cells. In all cases two main polymerase forms are observed, one of mol.wt. 200000, preferring denatured DNA to native calf thymus DNA primer, designated type I, and the other, designated type II, of mol.wt. 100000, showing a variable and slight preference for native calf thymus DNA primer. Some catalytic properties of these polymerases are described. Nuclei have been isolated from some of these tissues by using two different buffer systems. The ionic composition of the isolation medium is found to affect greatly the amounts and types of polymerase that bind to the nuclei, and also affects the kinetic properties of the polymerases. The way the polymerases and nuclei change properties as the ionic composition of the buffers is changed suggests that ionic effects may be a significant factor in the control of DNA synthesis in vivo. These ionic effects also explain much of the previous confusion over the localization of specific DNA polymerases.

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