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M V Simpson

Publications and source records attributed to M V Simpson.

At least 37 records · Page 2Linked to original sources
PubMed1980

Analysis of protein levels and synthesis after learning in the split-brain pigeon.

A series of experiments were performed to study the effects of learning upon proteins in the split-brain pigeon. With this preparation, one side of the brain was trained in a color discrimination task while the other side served as a naive control. Proteins from both sides of the brain were separated by one- and two-dimensional polyacrylamide gel electrophoresis and analyzed to measure protein levels or protein synthesis. No differences were found between trained and naive telencephalic hemispheres or between trained and naive optic lobes in the steady-state protein levels or in protein synthesis.

Animals↗
PubMed1980

Mitochondrial DNA polymorphism: evidence that variants detected by restriction enzymes differ in nucleotide sequence rather than in methylation.

Restriction enzyme analysis of mtDNAs for the purpose of determining sequence divergence rests on the assumption that variant recognition sites differ with respect to sequence and not methylation. This assumption was tested on two mtDNAs, A and B, which are distributed throughout the laboratory rat population and which can be distinguished by a number of restriction enzymes. The mtDNAs were cloned and the nucleotide sequences of corresponding small HindIII fragments, in which a variant EcoRI site occurs, were determined. Evidence that the fragments differ in sequence and not methylation is as follows: (i) The cloned mtDNA yielded the same fragment pattern as did native mtDNA when treated with EcoRI, Hha I, HinfI, and Hae III; (ii) three nucleotide replacements were found in the 169-base pair fragment, A.T in equilibrium G.C, A.T in equilibrium G.C, T.A in equilibrium G.C; (iii) one of these replacements, A.T in equilibrium G.C at position 80, accounts for the presence of the EcoRI site in the type A and its absence in the type B mtDNA. Examination of the sequence leads to the suggestion that these three nucleotide replacements are silent; i.e., they would not lead to amino acid substitutions in a possible encoded protein.

Animals↗
PubMed1979

Search for a DNA gyrase in mammalian mitochondria.

Incorporation of labeled deoxynucleoside triphosphates into mtDNA by isolated rat liver mitochondria has been shown previously to reflect DNA replication. We have used this system to seek evidence for a mtDNA gyrase. Coumermycin, novobiocin, nalidixic acid, and oxolinic acid are known to be inhibitors of Escherichia coli gyrase, to inhibit E. coli DNA replication, to abolish colicin E1 replication, and to depress the supercoiling of phage lambda DNA, the last two via inhibition of the DNA gyrase. Our results show that these agents inhibit [3H]dATP incorporation into bulk mtDNA at concentrations similar to those used for E. coli. Analysis by sucrose gradient sedimentation confirms the inhibition and shows further that the synthesis of the highly supercoiled form of mtDNA (i.e. 39 S DNA) is depressed relative to other mtDNA forms (i.e. 27 S DNA), suggesting an inhibition of the supercoiling process. Analysis of the DNA by CsCl/propidium diiodide centrifugation shows, in addition, that incubation with coumermycin results in the appearance of a mtDNA form shown to be relaxed mtDNA. The results are consistent with the occurrence of a mtDNA gyrase and its operation in mtDNA replication.

Animals↗
PubMed1979

Mitochondria contain a distinct DNA topoisomerase.

A topoisomerase (nicking-closing enzyme) has been isolated from rat liver mitochondria. It has purified by double-stranded DNA-cellulose chromatography approximately 50,000-fold, based on the crude mitochondrial extract. It possesses a minimum specific activity of 1.9 x 10(5) units/mg. The enzyme has been shown to be distinctly mitochondrial, differentiated from the nuclear topoisomerase by its sensitivity to the intercalating drug, ethidium bromide, and to the non-intercalating trypanocidal drug, Berenil.

Animals↗
PubMed1977

Biosynthesis of mitochondrial DNA. Is 8 S DNA an artifact?

Sucrose 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. The evidence that 8 S DNA is not an artifact resulting from DNA breakdown during mitochondrial incubation or DNA isolation is as follows: (a) 8 S DNA can be isolated from unincubated mitochondrial; (b) 8 S DNA becomes labeled when labeled DNA precursors are administered in vivo; (c) 8 S DNA biosynthesis continues in the complete absence of labeled 39 S or 27 S DNA (whose synthesis is repressed by ethidium bromide), making it unlikely that 8 S DNA is formed from the breakdown of 39 S or 27 S DNA; (d) substitution of milder methods of DNA extraction does not decrease 8 S DNA labeling; moreover, the usual conditions of extraction, when applied to purified 39 S and 27 S DNA, do not generate 8 S DNA, nor does an additional mitochondrial washing cycle; (e) the specific radioactivity of 8 S DNA is higher than that of 39 S or 27 S DNA, making it improbable that the latter forms are precursors of 8 S DNA. Since 8 S DNA is double-stranded, it is not identical to the 7 S fragment of D loop DNA. The hypothesis that the artifactual nicking of those DNA molecules which contain opposing D loops leads to the release of double-stranded fragments was tested. The DNA which was released was predominantly (and probably completely) single-stranded. We conclude that 8 S DNA is probably not an artifact and studies are in progress on its function.

Animals↗
PubMed1971

A translocation-associated ribosomal conformational change detected by hydrogen exchange and sedimentation velocity.

Translocation in ribosomes consists of transposition of peptidyl-tRNA from the aminoacyl to the peptidyl site and, probably concomitantly, the movement of ribosomes on mRNA. Does a conformational change in the ribosome provide the motive force for this process? Hydrogen exchange and sedimentation velocity experiments indicate that the Escherichia coli ribosome does undergo a conformational change associated with translocation. When pretranslocational ribosomes carrying acetyldiphenylalanyl-tRNA in the aminoacyl site were incubated with G factor and GTP, translocation occurred, with a concomitant increase in hydrogen exchange rate and a decrease in sedimentation constant. These changes did not occur when GTP was replaced by a nonhydrolyzable analogue, GDP-CH(2)-P, and they were blocked by the antibiotics fusidic acid and thiostrepton. When posttranslocational ribosomes were cycled back to the pretranslocational state by T factor, GTP, and phenylalanyl-tRNA, the sedimentation constant reverted to the original value. Whether or not this conformation change drives translocation requires further study.

Anti-Bacterial Agents↗
PubMed1969

The role of ribosomal conformation in protein biosynthesis: the streptomycin-ribosome interaction.

The role played by ribosomal conformation in codon-anticodon recognition has been studied using streptomycin as a probe, inasmuch as streptomycin is known to cause misreading of the genetic code. Changes in ribosomal structure have been followed by the method of hydrogen-tritium exchange. The results show that streptomycin induces two types of change in the hydrogen exchange pattern. At low molar ratios of streptomycin to ribosomes, a stimulation of the hydrogen exchange rate ("loosening" of ribosomal structure) is observed, with a small inhibition of polypeptide synthesis. As the streptomycin: ribosome ratio is increased, a maximum exchange rate is reached, after which the rate decreases ("tightening" of structure); in this region, inhibition of peptide synthesis increases sharply, and misreading of the code begins. None of these effects is observed with streptomycin-resistant ribosomes.

Escherichia coli↗
PubMed1968

DNA biosynthesis by isolated mitochondria: a replicative rather than a repair process.

The previously observed incorporation of deoxynucleoside triphosphate precursors into DNA by isolated rat liver mitochondria could reflect either replication of DNA or a repair process. Density labeling experiments in cesium chloride demonstrate that DNA synthesized in the presence of 5-bromodeoxyuridine triphosphate instead of thymidine triphosphate shows an appreciable increase in density. In some of the molecules undergoing synthesis, the amount of the density increase indicated a replacement of thymine by bromouracil to the extent of 33 percent. This extensive replacement, which would compute to twice this amount if only a single strand of the duplex is labeled, provides evidence for the synthesis of fairly long pieces of DNA. Such synthesis is characteristic of replication rather than repair, and the results thus suggest that mitochondria are able to replicate their own DNA.

Animals↗