The cytochromes of mitochondria from Tetrahymena pyriformis strain ST

1. Mitochondria of the obligately aerobic ciliate protozoon, Tetrahymena pyriformis strain ST, are unusual in that they possess a cytochrome oxidase system that does not react with reduced mammalian cytochrome c; the presence of cytochromes a₆₀₃+a₃ is masked in the α-band region of spectra by the broad absorption band of cytochrome a₆₂₀. 2. Other haemoproteins present include cytochromes b₅₆₀, b₅₅₆, c₅₅₃ and c₅₄₉. 3. The reaction of reduced cytochrome a₃ with CO is reversed by flash photolysis, and in the presence of O₂ the subsequent oxidation of this cytochrome is followed by that of cytochrome a₆₀₃. 4. Cytochromes a₆₂₀ and b₅₆₀ also react with CO and with KCN; the latter cytochrome corresponds with that designated cytochrome o by other workers. 5. The contribution of cytochrome a₆₀₃ to difference spectra is revealed by making use of the fact that it does not react with KCN. 6. Cytochrome a₆₂₀ is unstable, and its α-absorption band is lost from spectra of mitochondria which have been aged or treated with ultrasound, detergents or organic solvents. 7. Possible pathways of electron transport via the several different terminal oxidases in Tetrahymena mitochondria are proposed.     

The organization of ribosomal RNA genes in the mitochondrial DNA of Tetrahymena pyriformis strain ST.

1. We have constructed a physical map of the mtDNA of Tetrahymena pyriformis strain ST using the restriction endonucleases EcoRI, PstI, SacI, HindIII and HhaI. 2. Hybridization of mitochondrial 21 S and 14 S ribosomal RNA to restriction fragments of strain ST mtDNA shows that this DNA contains two 21-S and only one 14-S ribosomal RNA genes. By S1 nuclease treatment of briefly renatured single-stranded DNA the terminal duplication-inversion previously detected in this DNA (Arnberg et al. (1975) Biochim. Biophys. Acta 383, 359--369) has been isolated and shown to contain both 21-S ribosomal RNA genes. 14 S ribosomal RNA hybridizes to a region in the central part of the DNA, about 8000 nucleotides or 20% of the total DNA length apart from the nearest 21 S ribosomal RNA gene. 3. We have confirmed this position of the three ribosomal RNA genes by electron microscopical analysis of DNA . RNA hybrid molecules and R-loop molecules. 4. Hybridization of 21 S ribosomal RNA with duplex mtDNA digested either with phage lambda-induced exonuclease or exonuclease III of Escherichia coli, shows that the 21-S ribosomal RNA genes are located on the 5'-ends of each DNA strand. Electron microscopy of denaturated mtDNA hybridized with a mixture of 14-S and 21-S ribosomal RNAs show that the 14 S ribosomal RNA gene has the same polarity as the nearest 21 S ribosomal RNA gene. 5. Tetrahymena mtDNA is (after Saccharomyces mtDNA) the second mtDNA in which the two ribosomal RNA cistrons are far apart and the first mtDNA in which one of the ribosomal RNA cistrons is duplicated.     

Identification of true thymidine kinase in Tetrahymena pyriformis ST.

Thymidine kinase is present in the cytoplasm (outside mitochondria) of Tetrahymena pyriformis. Previous workers have been unable to find a specific thymidine kinase activity in this organism. The cytoplasm of Tetrahymena contained a thymidine phosphorylating activity which was ATP dependent, was stimulated by Mg²⁺, and was inhibited by dTTP. This activity was also partly inhibited by dCTP. Although the mitochondrial fraction also exhibited ATP-dependent phosphorylation, it is not stimulated by Mg²⁺ and not significantly inhibited by dTTP. Nucleoside phosphotransferase activity is detectable both in cytoplasmic and mitochondrial fractions, although it is not clear whether they represent separate enzymes. Nucleoside phosphotransferase activity is inhibited both by NaF and by ATP. Thymidine kinase and nucleoside phosphotransferase activities were separated by polyacrylamide gel electrophoresis, establishing the presence of both enzymes in this organism. Both crude mitochondrial lysate and postmitochondrial supernatant samples exhibited similar gel electrophoretic patterns for thymidine kinase and nucleoside phosphotransferase activities. The former, however, exhibited a relatively small peak of thymidine kinase migrating at the same rate as that of the postmitochondrial supernatant. A separate peak of thymidine kinase was not found in the mitochondria of Tetrahymena.     

The distribution of DNA among dividing mitochondria of Tetrahymena pyriformis

A squash technique was developed for log phase Tetrahymena pyriformis which permitted the resolution of over 100 individual mitochondria from a single cell. Mitochondria incorporated thymidine at all stages of the cell cycle, even when nuclear DNA synthesis was not occurring. During the stage of macronuclear DNA synthesis, however, there was a significant increase in the extent of mitochondrial labeling. Low radioautograph background suggests that mitochondrial DNA is synthesized at the mitochondria themselves. All mitochondria incorporated thymidine-3H within one population-doubling time. Grain counts also showed that the amount of mitochondrial label was retained for four generations and that this label remained randomly distributed among all mitochondria during this time. The results are not consistent with any theory of de-novo or "microbody" origin of mitochondria, but do support the hypothesis that mitochondria are produced by the growth and division of preexisting mitochondria. The stability of the mitochondrial DNA and its distribution among daughter mitochondria satisfy two prerequisites for a genetic material. The possibility is discussed that some of the genetic information for the mitochondrion is contained in the DNA associated with this organelle.     

The formation of several mitochondrial enzymes during the cell cycle in heat-synchronized Tetrahymena pyriformis ST.

The mechanical properties of the endoplasm were determined in oocytes and mature eggs of the starfish, Asterina pectinifera as follows. The cell was first deformed into a dumbbell shape by aspirating it through a circular hole of 35 or 50 μm radius formed in an agar plate of about 150 μm thickness. The movement of endoplasm in the cylindrical part of the cell was determined when a definite pressure was applied between both sides of the plate. Mechanical properties were practically represented by a viscoelastic model (fig. 5a) consisting of a Voigt element and a viscous element connected in series. The strain was proportional to the 0.60 ± 0.17th power of the stress in mature eggs and to the 0.76 ± 0.17th power of the stress in primary oocytes. Viscoelastic coefficients (G, η₁ and η₂ shown in fig. 5) of endoplasm changed in parallel to one another during maturation of the oocyte. They decreased with the breakdown of the germinal vesicle, increased before the extrusion of the first polar body, decreased during and after the first polar-body formation, increased before the extrusion of the second polar body, and decreased during and after the second polar-body formation.     

Effects of chloramphenicol on the mitochondrial respiratory chain in the wild strain and in a cytoplasmic chloramphenicol-resistant mutant of Tetrahymena pyriformis

The effects of chloramphenicol (CAP) on mitochondrial respiratory activity in the wild strain (ST) and in a cytoplasmic CAP-resistant mutant (STR1) of Tetrahymena pyriformis were studied by determining oxygen consumption, by spectrophotometry, and by cytochemistry. In the absence of CAP both strains had the same respiration capacity, and the low-temperature spectra of their isolated mitochondria were similar. Furthermore, the mitochondria of both strains showed a positive reaction with diaminobenzidine, denoting a similar cytochrome oxidase activity. However, when cells were grown in CAP for 24 or 48 h, the peaks of cytochrome oxidase and cytochromb b were almost absent in the wild type. In this type the oxygen consumption was greatly decreased, and the mitochondria were no longer stained by diaminobenzidine. In the mutant, the peaks of cytochrome oxidase and cytochrome b were decreased only; respiration was less affected than in the wild type, and cytochrome oxidase activity was still disclosed by the diaminobenzidine reaction. These results show that CAP inhibits the synthesis of two cytochromes (b and oxidase) which are partially translated into the mitochrondria of T. pyriformis. In the mutant, CAP reduces only the mitochondrial translation, resulting in reduced mitochondrial activity and reduced growth rate of the cell. These results are compared with the nucleo-mitochondrial regulation mechanisms discussed in our previous works.     

The effect of temperature on unsaturated fatty acid loss in Tetrahymena pyriformis.

Cultures of Tetrahymena pyriformis W incorporate exogenous 3-[14C]-cilienic acid and gamma-[1(-14)C] linolenic acid, terminal products of unsaturated fatty acid synthesis, into glycerophosphatides without randomization of the radiolabel. There was no difference in the rate of loss of each of the two acids at 15 or 28.5 degrees C. Differential turnover of these fatty acids, therefore, does not appear to be the cause of the shift in fatty acid pattern observed with temperature reduction.     

Monotertiarybutylhydroquinone effects on Tetrahymena pyriformis.

The molecular toxicity of monotertiarybutylhydroquionone (TBHQ) was studied using $\text{Tetrahymena}$$\text{pyriformis}$ as a model cell system. TBHQ at 26 ppm in the media inhibited cell growth by 50%. TBHQ inhibited the oxidation of ¹⁴C-acetate to ¹⁴CO₂. In addition, increasing concentrations of TBHQ decreased the incorporation of ¹⁴C-acetate into lipids and protein, ¹⁴C-amino acids into protein, ³H-uridine into RNA and ³H-thymidine into DNA. The incorporation of ¹⁴C-acetate into glycogen increased with concentrations up to 20 ppm TBHQ in the media while glycogen synthesis decreased with 40 ppm TBHQ.     

The effect of cholesterol on ubiquinone and tetrahymanol biosynthesis in Tetrahymena pyriformis.

The biosynthesis of ubiquinone-8 from radioactive mevalonate by cultures of Tetrahymena pyriformis is demonstrated. Under normal conditions the incorporation of this radioactive precursor into ubiquinone and the triterpenoid alcohol tetrahymanol reflects the amounts of these two compounds in the cell. Growth of T. pyriformis in the presence of cholesterol results in a complete inhibition of incorporation of radioactive mevalonate into tetrahymanol while there is a corresponding increase of radioactive incorporation into ubiquinone. This increased incorporation of mevalonic acid into ubiquinone must reflect a reduced level of mevalonic acid in the cell under these conditions and is not due to increased ubiquinone biosynthesis, indicating tight regulation of the pathway prior to mevalonate formation.