Mouse-hamster hybrid cells were analyzed for the species of mitochondrial DNA (mtDNA) retained using Southern blotting and hybridization with highly labeled mitochondrial DNA probes. Initial analyses were performed as soon as there were 10(7) cells, which took between five and eight weeks from the time the fusion was performed (approximately 23 cell doublings). The majority of clones tested had detectable levels of both mouse and hamster mtDNA at first testing.

Animals↗

PubMed1981

In situ photochemical crosslinking of HeLa cell mitochondrial DNA by a psoralen derivative reveals a protected region near the origin of replication.

The in vivo association with proteins of HeLa cell mitochondrial DNA (mtDNA) has been investigated by analyzing the pattern of in situ crosslinking of the DNA by 4'-hydroxymethyl-4, 5',8-trimethylpsoralen (HMT). Either isolated mitochondria or whole cells were irradiated with long wavelength UV light in the presence of ths psoralen derivative, and the mtDNA was then isolated and analyzed in the electron microscope under totally denaturing conditions. No evidence of nucleosomal structure was found. The great majority of the molecules (approximately 90%) had a double-stranded DNA appearance over most of their contour length, with one to several bubbles occupying the rest of the contour, while the remaining 10% of the molecules appeared to be double-stranded over their entire length. Analysis of restriction fragments indicated the presence, in approximately 80% of the molecules, of a protected segment (300 to 1500 bp long) in a region which was centered asymmetrically around the origin of replication so as to overlap extensively the D-loop. Control experiments showed that at most 30% of the bubbles found near the origin could represent D-loops or expanded D-loops: furthermore, it could be excluded that some sequence peculiarity would account for the preferential location of bubbles near the origin of replication. The data have been interpreted to indicate that, in at least 55% of HeLa cell mtDNA molecules, the region around the origin is protected from in situ psoralen crosslinking by proteins or protein complexes which are associated in vivo with the DNA.

Chemical Phenomena↗

PubMed1976

A respiration-deficient Chinese hamster cell line with a defect in NADH-coenzyme Q reductase.

We have recently described a Chinese hamster cell line with a greatly reduced rate of respiration. In this report we conclude that the defects is in NADH-coenzyme Q reductase (NADH oxidase), the first part of the electron transport chain. The conclusion is based on the following observations. (a) In this and in the earlier report we determined that the relevant enzymes of the Krebs cycle are present and active. (b) Oxygen consumption by isolated mitochondria is normal when driven by succinate and alpha-glycerolphosphate. (c) Difference spectra between reduced and oxidized forms indicate that all cytochromes are present and functional. (d) In contrast, substrates such as malate, glutamate, alpha-ketoglutarate, and isocitrate which generate NADH do not stimulate oxygen consumption in mutant mitochondria. (e) A direct assay of the rotenone-sensitive NADH oxidase in Lubrol-treated mitochondria from mutant cells revealed less than one-tenth of the activity when compared with wild type mitochondria. (f) The treatment of wild type cells with rotenone, a specific inhibitor of NADH-CoQ reductase, yielded an exact phenocopy of the mutant by several criteria. This is the first report of a respiration-deficient mammalian cell mutant in tissue culture.

Cell Line↗

PubMed1975

Conditionally lethal mutations in chinese hamster cells. Characterization of a cell line with a possible defect in the Krebs cycle.

A variant Chinese hamster cell line has been isolated from a mutagenized population that has a markedly reduced ability to oxidize a variety of substrates via the Krebs cycle. The production of 14CO2 from 14C-labeled compounds was measured using pyruvate, acetate, beta-hydroxybutyrate, palmitate and glutamate, and in all cases it was neglibible in the mutant. In contrast to this, significant amounts of 14CO2 were produced from 14C-aspartate and 14C-succinate which suggest that some reactions of the Krebs cycle can take place and this conclusion is supported by tracer experiments with labeled compounds. The rate of respiration measured with a Clark oxygen electrode in the mutant was compared to several normal Chinese hamster cell lines and was found to be only 8%. Mitochondria appear to be present in normal numbers and with only minor differences in morphology. The measurement of difference spectra between oxidized and reduced states permits us to conclude that the cytochromes are all present and functional. These results lead us to believe that there may be a defect in the Krebs cycle between alpha-ketoglutarate and succinate. Alternatively a defect in a structural component of the mitochondria or in the electron-transport chain itself may be causing pleiotropic effects in the Krebs cycle and respiration.

Acetates↗