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The hypocholesterolemic drug clofibrate raises hepatic levels of mitochondria by over 100 %, with maximum increase at 2 to 4 days after drug administration, and a slow rise continuing at 10 days. This time response was similar to that seen after partial hepatectomy. However, several clear differences exist. During the doubling in mitochondrial content, no change in particle size or specific content of DNA or RNA was observed, whereas during liver regeneration the former decreases by 50 % and the latter increases by 300 %. The half-life of 5.8 days for normal mitochondria was not altered significantly by clofibrate, using [guanidinio-14C]arginine. However, the initial rate of short term incorporation of [35S]methionine into control mitochondria in vivo was 60 % of that for drug-tested animals, while the in vitro efficiency of [14C]leucine incorporation was reversed; mitochondria from drug-treated animals were about 61 % as active as those from controls. Enzyme activity of drug-treated animals was not significantly altered for inner membrane or matrix enzymes. However, a 30 to 40 % fall in specific activity of three outer membrane enzymes was noted from 2 to 15 days. This resembles changes seen during early liver regeneration. One striking action of clofibrate was to inhibit the neutral protease activity of mitochondria. Even at 2 days on clofibrate, neutral protease activity was inhibited to 62 % of the control value of 0.334 µmole of amino acid per hour per mg of protein. Inhibition was a maximum of 50 % at 6 days, and was still at 63 % at 20 days. Clofibrate added in vitro had negligible influence on neutral protease activity. The inhibition was independent of lysosomes since the protease activity of purified lysosomes was neither inhibited, nor was acid phosphatase activity in whole homogenates or the purified mitochondrial fractions changed during drug treatment. The endogenous amino acid content of 0.032 µmole per mg protein remained constant during the stimulation in bio-genesis. The results of this study on clofibrate support a hypothesis that net levels of hepatic mitochondria may be controlled by the activity of their own neutral proteases. A possible action is to degrade newly synthesized mitochondrial proteins, which may be structural or binding, such that a lowered protease activity would raise their levels and thereby increase mitochondrial content.

Formate is utilized for mitochondrial polypeptide chain initiation, and formyl label is retained as N-formylmethionine in mitochondrial proteins. Chloramphenicol inhibits this incorporation of formate into mitochondrial proteins by more than 95% demonstrating the specificity of this labeling procedure. A second product derived from formate and appearing in mitochondrial proteins is represented by serine which is formed and incorporated in the cell sap. These two products can be distinguished either after complete enzymatic hydrolysis or, on the intact protein, by the acid lability of the formyl linkage. Proteins initiated, translated, and integrated into their final membrane sites within the mitochondria may therefore be identified by the lability of their formate label. These techniques should prove valuable in determining the site of synthesis of purified protein subunits without the use of inhibitors, as well as for studying inter-compartmental regulation of polypeptide synthesis. We have examined this question and now provide evidence for tight coupling between the mitochondrial and cell sap systems. Interruption of protein synthesis on cell sap ribosomes has no effect on polypeptide chain initiation in the mitochondria, yet the elaboration of complete membrane-associated proteins stops rapidly.

The functional distinction of membrane-bound and free polyribosomes for the synthesis of exportable and non-exportable proteins respectively is not so strict as was initially thought, and it was therefore decided to investigate their relative contribution to the elaboration of an internal protein integrated into a cell structure. Cytochrome c was chosen as an example of a soluble mitochondrial protein, and the incorporation of [14C]leucine and δ-amino[14C]laevulinate into the molecule was studied by using different ribosomal preparations from regenerating rat liver. A new procedure was devised for the purification of cytochrome c, based on ion-exchange chromatography combined with sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. In spite of cytochrome c being a non-exportable protein, the membrane-bound polyribosomes were at least as active as the free ribosomes in the synthesis in vitro of the apoprotein and the haem moiety. The detergent-treated ribosomes could also effect the synthesis of cytochrome c, although at a lower rate. Since in liver more than two-thirds of the ribosomes are bound to the endoplasmic-reticulum membranes, it is considered that in vivo they are responsible for the synthesis of most of the cytochrome c content of the cell. This suggests that in secretory tissues the endoplasmic reticulum plays a predominant role in mitochondrial biogenesis, although free ribosomes may participate in the partial turnover of some parts of the organelle. The hypothesis on the functional specialization of the different kinds of ribosomes was therefore modified to account for their parallel intervention in the synthesis of proteins associated with membranous structures.

Summary From serial section electron micrographs, the ultrastructural cytology of whole cells of Pityrosporum ovale and P. orbiculare was analyzed volumetrically and illustrated in three-dimensional images. Cells cultured on sodium taurocholate medium were fixed in both glutaraldehyde and potassium permanganate solutions and imbedded in Vestopal for serial sectioning. The volume of the cell, mitochondria and nucleus was obtained by measurements of successive sections through whole cells. The sections of one cell were built in a solid model. The average number of mitochondria in the cells of P. ovale was 23 and in P. orbiculare 2.6 per cell. The volume of the mitochondria was from 14% to 37% of the cell volume. The mitochondria of P. orbiculare appear to increase in size with cell volume, whereas in P. ovale, there may be an increase in number rather than in size. Two types of cellular bodies, one electron light and one electron dense, present in each species, also showed differences in size and number. The volume of the nucleus was 6 to 9% of the cell volume. The serial section analysis points to differences in mitochondrial biogenesis which may be of taxonomic value in separating the two species of Pityrosporutn.

1. 1. Chromatographic and chemical analyses show that up to four major sterols may be present in blood and culture forms of trypanosomes. 2. 2. Blood and culture forms of Trypanosoma lewisi and culture forms of T. rhodesiense biosynthesize C(24)-alkyl sterols de novo. In these trypanosomes, and in blood forms of T. rhodesiense, exogenous cholesterol is incorporated and esterified, but not further metabolized. 3. 3. Changes in sterol composition and metabolism occur in T. lewisi during “maturation” in vivo, and during in vitro culture. 4. 4. The role of sterol biosynthesis in cyclical mitochondrial biogenesis in trypanosomes is discussed, and also the evolutionary significance of the phytosterols identified in this study.

Administration of allylisopropylacetamide (AIA) 1 to rats causes a rapid induction of hepatic δ-aminolevulinic acid (ALA) synthetase. Liver mitochondria obtained from animals after AIA treatment had a 50–60% greater rate of amino acid incorporation in vitro as compared to mitochondria from control animals. Similarly, pulselabeling by ( 3 H) leucine in vivo was stimulated 50% in submitochondrial fractions containing proteins insoluble in acetic acid and a membranous preparation of cytochrome oxidase. Previous studies had indicated that 15–20% of the proteins in these fractions were labeled in vivo by a cycloheximide-insensitive system, presumably that of the mitochondria ( Fed. Eur. Biol. Soc. Lett. 9, 232 1970). In time-course studies, it was observed that increased incorporation of ( 14 C)glycine into heme in vivo followed shortly after ALA synthetase induction. A slight decay occurred before the incorporation of ( 3 H)leucine into cytochrome oxidase or total mitochondrial protein was stimulated. At these latter times a 20–30% increase in the cytochrome content was also observed. Administration of aminotriazole, an inhibitor of ALA dehydratase and hence overall heme synthesis, prevented the stimulation of leucine incorporation in vivo and the increase in cytochrome content observed after AIA treatment. These results suggest that mitochondrial protein synthesis and cytochrome formation may be stimulated when heme biosynthesis is increased due to ALA synthetase induction.

Approximately 25 per cent of the mitochondria in one day old, rapidly growing, schizonts of Boderia vary considerably in their morphology from the consistently spherical organelles present in adult animals. Mitochondrial biogenesis by division is indicated and illustrated. The internal reorganisation of the mitochondrion which occurs prior to fission has not been reported previously for this organelle.

It is not known whether limitation of lifespan represents a programmed genetic event or is a result of environmental factors imposed by the conditions of culture. An investigation of the factors surrounding the limitedin vitro lifespan of human diploid fibroblasts has been undertaken. We have investigated the role of mitochondria in the finite lifespan of WI-38 human lung fibroblasts. Mitochondrial function was depressed in a controlled manner by treating cells with ethidium bromide and chloramphenicol both of which inhibit normal biogenesis. These antibiotics decrease cytochrome oxidase activity, change cell ultrastructure, and inhibit growth at high concentration. At lower concentrations the antibiotics do not affect cell proliferation for several generations. However, their effect is cumulative and after several generations the cells enlarge, stop dividing and die. Removal of antibiotics from the culture media before death restores proliferative capacity. At still lower concentrations cytochrome oxidase activity was decreased but continuous growth in the presence of the antibiotics caused no decrease inin vitro lifespan. Thus, the potential for oxidative metabolism appears to be in excess of that needed for cell proliferation at all stages of thein vitro lifespan of a culture. The importance of cytoplasmic protein synthesis was evaluated using cycloheximide, a specific inhibitor of this process. Cycloheximide was used to try to distinguish between the effects due to general inhibition and that due to specific inhibition of mitochondrial biogenesis. Exposure of cultures to concentrations of cycloheximide which inhibited growth drastically caused no decrease in cytochrome oxidase activity.

Changes in the yield of mitochondrial protein, in the incorporation of leucine into mitochondrial proteins, and in the respiratory activity of isolated mitochondria were determined in the remaining kidney (renoprival kidney) of the rat during the first 72 hr postmononephrectomy. At 24, 48, and 72 hr the yield of mitochondrial protein isolated from the renoprival kidney increased 13, 23, and 34%, respectively, whereas renal mass increased 9, 14, and 19%. Incorporation of [ 3 H]-leucine in vivo into total mitochondrial protein was increased 96 and 130% over control at 12 and 24 hr, respectively. Incorporation of leucine in vitro by mitochondria was increased 27% over control at 24 hr; chloroamphenicol, but not cycloheximide, inhibited the in vitro incorporation. At 24 hr, the rate of O 2 consumption (state 3) was decreased, but P O ratios, respiratory control values, and glutamic and succinic dehydrogenase activities were unaltered. The content of pyridine nucleotide, cytochromes a , a 3 , and c was decreased at this time; these activities approached normal values by 72 hr. The results suggest that during the 24 and 48 hr postmononephrectomy there is an increase in mitochondrial protein and specific changes in mitochondrial function which may be indicative of a proliferation of mitochondria in the renoprival kidney. This system should be valuable in investigating the events involved in mitochondrial biogenesis in mammalian cells.

Jha R. K. and Smyth J. D. 1970. Ultrastructure of the rostellar tegument of Echinococcus granulosus with special reference to biogenesis of mitochondria. International Journal for Parasitology, 1: 169–177. On the rostellum of Echinococcus granulosus the tegumental microtriches are long and thin, and often branched, hooked, barbed or curved—a pattern which probably provides firmer adhesion to the host gut and increases the absorptive area. Mitochondria are almost absent from the rostellar tegument in a freshly evaginated protoscolex, although dumb-bell shaped membranous structures are present. After 24–28 h in vitro culture, the latter structures disappeared and active mitochondrial biogenesis was seen to be taking place. It is speculated that the occurrence of this process during the early phases of the protoscolex-strobila transformation may be triggered by transfer from the environment of a tissue site (in the intermediate host) to that of an intestinal site (in the dog) and presumably results in a ‘switch’ in metabolism. This phenomenon also appears to be associated with the ‘secretory’ activity in the nuclei of the rostellar gland cells. An analogy with the biogenesis of mitochondria in trypanosomes is made. In an established and growing strobilate phase of Echinococcus, mitochondria are abundant at all levels of the distal cytoplasm. This and the occurrence of ‘concentric’ mitochondria suggests a higher metabolic activity in this region than in other parts of the worm. Elongated mitochondria with longitudinal cristae, restricted only to the rostellar tegument, were also found. In addition, unusual membrane-bound ‘mitochondrial sacs’, containing numbers of mitochondria, were present. Some of these sacs opened to the outer surface, and displayed mitochondria apparently in the process of ‘extrusion’. Such extrusion of mitochondria would explain the frequent occurrence of free ‘intact’ mitochondria on the surface of the distal cytoplasm—a phenomenon which may be related to the process of ‘membrane digestion’ believed to occur in this region. ‘Lamellar bodies’ of unknown function, were also found often in the distal cytoplasm. ‘Secretion droplets’, which appeared externally on the rostellum of in vitro cultured specimens, were found to be bubble-like outgrowths of distal cytoplasm containing vesicles, mitochondria and vacuoles of varying sizes.

A new class of Saccharomyces cerevisiae mutants deficient in biosynthesis of all cytochromes was isolated from cultures grown in medium containing ethidium bromide. Cytochrome c synthesis may be restored to normal by growing mutant cells in medium supplemented with δ-aminolevulinic acid. Cytochrome deficiency results from mutation in two genetic determinants, one nuclear, the other mitochondrial. When cells possess normal ( ρ + ) mitochondrial DNA, expression of the abnormal nuclear determinant ( cyd -1) is largely masked, so that cells can grow on glycerol as primary carbon source and all cytochromes are present. Nevertheless, the presence of the cyd -1 mutation may be detected in ρ + strains, since synthesis of all cytochromes is enhanced to some extent by δ-aminolevulinic acid. Destruction of mitochondrial DNA unmasks the underlying defect so that cyd -1 ρ − strains are almost completely lacking in detectable cytochromes. Although spectra of cyd -1 ρ + strains resemble those of cytochrome c ( cyc ) mutants, cyd -1 mutants represent a new complementation group different from six known cyc groups. Cytochrome c biosynthesis in only one of these six types of cytochrome c mutants, cyc 4-1, was restored to normal by δ-aminolevulinic acid. Therefore, since cyc4 -1 and cyd -1 are complementary, and segregate independently, δ-aminolevulinic acid synthesis appears to be controlled by at least two nuclear genes, and by one or more genes located in mitochondrial DNA. Glycine does not replace δ-aminolevulinic acid in stimulating cytochrome biosynthesis in cyd -1 or cyc -4 mutants. A regulatory system involving exchange of information between mitochondria and the nuclear-cytosolic compartment is indicated by the results. Studies with isolated mitochondria indicate that a limitation of intra-cellular δ-aminolevulinic acid supply is reflected in mitochondrial composition, not just in numbers of organelles.

Mitochondria isolated from cysts of Artemia salina (brine shrimp) were found to be devoid of cristae and to possess a low respiratory capability. Hydration of the cysts induces marked biochemical and morphological changes in the mitochondria. Their biogenesis proceeds in two stages. The first stage is completed within 1 h and is characterized by a rapid increase in the respiratory capability of the mitochondria, their cytochrome oxidase, cytochrome b, cytochrome c and perhaps some morphological changes. In the second stage there is an increase in the protein-synthesizing capacity of the mitochondria as well as striking changes in mitochondrial morphology leading to the formation of cristae.

control of the formation of mitochondria. From these studies it has been recognized that cytoplasmic genetic determinants as well as chromosomal genes are involved in the biogenesis of yeast mitochondria., 2 Following the recognition of the occurrence of mitochondrial DNA,3 4 attention has recently been focused on the relationship between mitochondrial DNA and the cytoplasmic determinant.5 However, the information on this latter subject is limited and is derived from the study of a single class of mutant of this determinant, the respiratory-deficient cytoplasmic petite. This irreversible mutation is phenotypically characterized by the inability of the cell to form a number of components of the respiratory system, including cytochromes a, a3, b, and ci.6 A clearer understanding of the role of cytoplasmic determinants in mitochondrial biogenesis could result from the characterization of new types of cytoplasmic mutations which do not result in such extensive biochemical changes. This would thus simplify the biochemical analyses as well as providing additional cytoplasmic markers to assist further genetic studies. We have reported that the antibiotics chloramphenicol, lincomycin, and the macrolides erythromycin, carbomycin, spiramycin, and oleandomycin selectively inhibit in vitro amino acid incorporation by yeast mitochondria, while not affecting the yeast cytoplasmic ribosomal system.7' 8 Further, these antibiotics do not affect the growth of S. cerevisiae on fermentable substrates but reversibly inhibit the in vivo synthesis of cytochromes a, a3, b, and cl; the inhibited cells appear to be phenocopies of the cytoplasmic petite mutant.9, 10 This laboratory has also described the isolation of yeast mutants with varying degrees of sensitivity to the aforementioned antibiotics.1l The mutants selected for resistance to high levels of erythromycin have been shown to contain mitochondria having an amino acid-incorporating activity which is completely resistant to the drug.l2 It was also shown that the inheritance of resistance to erythromycin did not segregate as a single chromosomal gene.7 This paper establishes that erythromycin resistance is inherited extrachromosomally. In addition, it is shown that the cytoplasmic determinant for erythromycin resistance and the factor associated with the cytoplasmic petite mutation, while closely related, do not appear to be identical. Materials and Methods.-Nomenclature: The following symbols have been adopted to represent the various mutants used in this paper. The symbol p (rho) represents the cytoplasmic factor as described by Sherman,2 where p+ and p designate the ability or inability, respectively, to synthesize the respiratory enzymes cytochromes a, a3, b, and cl. 903

The isolation of a new class of mutants permitting facultative anaerobiosis in Neurospora crassa is described. Backcross analyses to the obligate aerobe prototroph (An-) indicate single nuclear gene inheritance (An-/An+). An+ and An- are indistinguishable in morphology and growth rates under aerobic conditions. Anaerobic growth requires nutritional supplements that are dispensable for aerobic growth. Conidiogenesis, conidial germination, and vegetative growth rate are suppressed by anaerobiosis. An+ mutants produce substantial quantities of ethanol under anaerobic conditions. Anaerobiosis and chloramphenicol both affect mitochondrial enzyme activity and morphology. Chloramphenicol inhibition leads to reduction in cytochrome oxidase and swollen mitochondria with few cristae. Anaerobiosis leads to reduction in both cytochrome oxidase and malate dehydrogenase activities, enlarged mitochondria with fewer cristae, enlarged nuclei, and other alterations in cellular morphology. The fine structure of anaerobically grown cells changes with the time of anaerobic growth. We conclude that either inhibition of mitochondrial membrane synthesis or inhibition of respiration might lead to the observed alterations in mitochondria.

1. The heavy, light and fluffy mitochondrial fractions obtained by differential centrifugation were further characterized with respect to their protein synthesizing ability in vitro, their nucleic acid content, buoyant density of their DNA and ultrastructure. 2. The light mitochondrial fraction synthesized proteins in vitro at a rate 4–5 times as high as heavy and fluffy mitochondria. The incorporation ability of this fraction was also maximally affected by the thyroid status of the animal. The radioactivity in leucyl-tRNA of the light mitochondrial fraction was about 3–4 times as high as that of the other two fractions. 3. The heavy, light and fluffy mitochondrial fractions contained small but consistent amounts of RNA and DNA. Although the DNA content was the same in all mitochondria fractions, the light mitochondria contained relatively more RNA. The buoyant density of DNA from all the fractions was 1.701g/cm3. 4. Electron microscopy revealed that the heavy mitochondria have a typical mitochondrial architecture, with densely packed cristae and a well developed double membrane. Light mitochondria were also surrounded by double membranes, but were smaller in size and contained less cristae. The fluffy fraction consisted of a mixture of well formed mitochondria and those in the process of degradation. 5. The significance of these findings in relation to mammalian mitochondrial genesis is discussed.

Cellular organization of Polytomella agilis changes when the trophic form undergoes encystment: 1) initially a thin capsule is secreted about the organism which in the mature cyst develops into a thick 3-layered wall; 2) starch bodies increase in number and volume; and 3) mitochondria, ribosomes and other organelles disappear. These data point to the need for metabolic studies on homogeneous populations and suggest that Polytomella agilis may serve as a useful tool for investigation of mitochondrial biogenesis.

Neurospora crassa is an organism well suited for the study of mitochondrial biogenesis. It has a relatively prolonged logarithmic growth phase, with mass doubling times of about two hours. During the exponential growth period the mitochondrial mass doubles at the same rate as does the culture. Highly purified mitochondrial fractions can be isolated from the cells using equilibrium centrifugation in sucrose gradients (Luck, 1963a). A mutant strain requiring choline incorporates radioactive choline into the lecithin of mitochondria, and when the choline of the growth medium is changed to the non-radioactive compound, the radioactive choline of the mitochondria is retained there and can serve as a stable marker. We made use of these features of choline requiring Neurospora to find out how pre-existing membrane material is distributed among individual mitochondria in a growing population (Luck, 1963a,b). Cultures labeled with tritiated choline were transferred to unlabeled medium and permitted to grow for three doubling cycles. The distribution of label among individual organelles was measured by quantitative radioautography. The results showed that throughout three doubling cycles of the mitochondrial mass the label was uniformly distributed among all mitochondria. These studies gave support to the hypothesis that in exponentially growing cells the mitochondrial mass grows by addition of new material to an existing framework and the mitochondrial population increases by division. Recent studies with the choline requiring mutant, a leucine requiring mutant, and a wild-type strain, indicate that exponentially growing cells may vary the chemical composition of their mitochondria within substantial limits (Luck, 1965a). The variation affects the phospholipid to protein ratio, and may be brought about by change in the available level of amino acids or choline present in the growth medium. In a cholineless strain, for example, two levels of choline supplementation can be defined: one which is just adequate to support exponential growth at maximal rates, and another, ten times higher, which gives maximal incorporation of choline into cellular phospholipids but does not alter the growth rate. Mitochondria produced by the culture at these two choline levels differ in their density. Figure 1 is a photograph of centrifuge tubes containing continuous sucrose gradients. Mitochondria have been sedimented to their equilibrium position. Cultures growing at low choline conditions produce mitochondria (tube A)

A respiration-deficient nuclear mutant of Saccharomyces cerevisiae was isolated that lacked mitochondrial adenosine triphosphatase (F1). The absence of this enzyme was established by adenosine triphosphatase assays, by antibody-binding studies, and by radioimmunochemical tests for F1 subunits. The mutant also lacked mitochondrial 32Pi-ATP exchange activity and exhibited greatly reduced amounts of cytochromes aa3, b, and c1. It possessed a mitochondrial protein-synthesizing system, but several products of this system were either abnormal or missing. Thus, the level of the three mitochondrially synthesized subunits of cytochrome c oxidase was at least 10 times lower than in the wild type. The mutant was unable to grow anaerobically even if the growth medium contained a fermentable substrate and unsaturated lipids. This property has not yet been reported for any S. cerevisiae strain. The mutant had a great tendency to lose its mitochondrial genome. The conversion to the double mutant state decreased the growth rate by a factor of 20 to 30. These results suggest that mitochondrial adenosine triphosphatase has an essential function even in nonrespiring cells. The mutant described here offers new possibilities for studying the assembly of F1 from its subunits and the role of F1 in mitochondrial biogenesis.

Studies of mitochondrial biogenesis in yeast have been hampered by a lack of suitable membrane markers in anaerobically grown cells subsequently grown in air. Cytochrome c peroxidase activity and subcellular location was studied to determine whether it would be a useful marker for an analysis of mitochondrial formation. Cytochemical tests revealed enzyme reaction product on all mitochondrial membranes in aerobically grown wild-type cells. Anaerobically grown wild-type and all petite cultures contained cytochrome c peroxidase cytochemical reaction deposits on abundant cytoplasmic membranes and on the few mitochondrial profiles which also were seen in the electron photomicrographs. Biochemical studies corroborated the cytochemistry because mitochondrial fractions were greatly enriched in cytochrome c peroxidase activity for aerobically grown wild-type cultures, but petite and anaerobically grown wild-type cultures showed higher enzyme activities in supernatant fractions than was present in the corresponding particulate fractions after differential centrifugation. Evidence from low-temperature microspectroscopy, spectrophotometric assays of mitochondrial enzyme activities, and electron microscopy showed mitochondrial formation during the time required for preparation and lysis of spheroplasts from anaerobically grown cultures. The data were interpreted as indicating that cytochrome c peroxidase was an oxygen-inducible enzyme, and that there was a developmental relationship between enzyme-reactive membranes of mitochondria and cytoplasm during the period of respiratory adaptation.

Cytochemical peroxidase activity in a haploid yeast ( Saccharomyces cerevisiae, strain D243) was studied in glutaraldehyde-fixed cells with a diaminobenzidine (DAB) procedure. In early stationary phase cells the predominant sites of peroxidatic activity were the mitochondrial cristae, with activity of lesser intensity on other mitochondrial membranes. The reaction required H2O2, was insensitive to aminotriazole but was blocked by cyanide and azide, by high peroxide concentration or by a decrease in medium pH from 8.5 to 6.0. The enzyme visualized was probably cytochrome c peroxidase, an enzyme known to be highly active in yeast mitochondria, although the possibility that H2O2 may somehow activate DAB oxidation via other mitochondrial heme proteins cannot be completely eliminated. The value of the DAB procedure as a means of specifically staining yeast mitochondria is discussed in relation to studies of mitochondrial biogenesis. No conclusive evidence of the presence of nonmitochondrial peroxisomal organelles (microbodies) was seen in cells grown on either glucose or galactose, but there was an indication of their presence in very low numbers in glycerol-grown yeast. Although catalase was clearly present, as evidenced by the rapid evolution of O2 from the H2O2-containing reaction media, it showed little apparent localized peroxidatic activity toward DAB. We were unable to demonstrate cytochrome oxidase activity in these cells with the usual DAB-cytochrome oxidase procedure.

The products of mitochondrial protein synthesis in yeast have been studied by analytical gel electrophoresis. Over 60% of the total counts incorporated into mitochondria when cytoplasmic protein synthesis is blocked are associated with five proteins which are separated on acrylamide gels in the presence of sodium dodecyl sulfate. The mitochondrial products have been found to be extracted from the membrane with acidic chloroform-methanol. Under neutral conditions chloroform-methanol removes predominantly a protein which has been estimated to have a molecular weight of 7,800. This protein has been found to be present in the membrane in a polymeric form with an apparent molecular weight of 45,000. Conversion of the polymer to the low molecular weight form is achieved by treatment of mitochondrial membranes with chloroform-methanol or by depolymerization in sodium dodecyl sulfate at alkaline pH. The low molecular weight protein appears to be the major product of mitochondrial protein synthesis. Its possible role in mitochondrial biogenesis is discussed.

and for their biogenesis. Either the energy generated by the mitochondria themselves may be necessary to maintain their integrity and to drive the synthesis of new mitochondrial components or the energy may be derived from extramitochondrial sources. Synthesis of the respiratory chain components during respiratory adaptation of anaerobically-grown yeast was found not to be inhibited by cyanide [l] , antimycin A [2] or by oligomycin [3] indicating that the energy generated by mitochondria is not re- quired for respiratory adaptation. The respiratory adaptation, however, appears to reflect mainly a transformation of preformed “promitochondria” into aerobic mitochondria [4] and may not represent a true biogenesis of new mitochondria. The present paper indicates that even in aerobically growing cells the formation of mitochondria is not obligatorily dependent upon energy generated by the organelles. 2. Experimental Diploid strains of wild-type yeast

The quantitative comparison of oligomycin sensitivity of ATPase from two mitochondrially determined oligomycin-resistant mutants (locus OI and OII) and the wild-type strain (all three isonuclear), as a function of mitochondrial biogenesis, gave the following results: 1 The mutations at the loci OI and OII confer distinctly different oligomycin resistance on mitochondrial ATPase, OI being more resistant than OII, and both more resistant than the ATPase from the wild-type strain. 2 In spite of these differences in oligomycin resistance, the binding of the ATPase to the mitochondrial membranes does not appear to be modified. 3 The phenotypic oligomycin resistance of mitochondrial ATPase, produced by growth of the wild type strain in the presence of chloramphenicol, is notably higher than the highest genotypic oligomycin resistance. 4 The oligomycin sensitivity or resistance of ATPase is strictly identical in anaerobic promitochondria and aerobic mitochondria, i.e. the difference between the oligomycin sensitivity of the ATPase from these strains is maintained in such extremely different physiological states. 5 In contrast, the ATPase isolated during respiratory adaptation of the wild-type strain had distinctly higher oligomycin resistance than the promitochondria or the aerobic mitochondria. This “transient” resistance displayed by the wild-type ATPase during adaptation (in the absence of growth) was very similar to the genotypic resistance of the ATPase from the mutant at locus OI.

The specific rates of respiration of cells of wild type and four extrachromosomal mutants of Neurospora crassa were measured throughout the vegetative growth cycle. Two forms of respiration were observed: (i) cyanide sensitive; and (ii) cyanide resistant, salicyl hydroxamate sensitive. These two forms are called terminal and alternate, respectively. The former proceeds by the mitochondrial electron transfer chain and involves the cytochromes; the latter apparently proceeds by the initial portion of the electron transfer chain and does not involve cytochromes. Large and rapid changes of both the terminal and alternate respiratory activities occurred during the vegetative growth cycle. The kinetics of these changes in wild type were compared under some conditions which inhibit protein synthesis and others in which the nitrogen source was varied. The kinetics of the changes of the two forms of respiration of mutants differed from those normally exhibited by wild type, but with varied experimental conditions wild type could be made to resemble the mutants. The results of these studies are discussed in terms of a dynamic model of regulation of mitochondrial biogenesis in the coordination of the synthesis of mitochondrial proteins encoded by nuclear and mitochondrial genomes.

Although mitochondria have been studied for nearly a century, and their role as the “power houses” of the aerobic cell has been recognised for twenty years, the mode of replication of these very important subcellular organelles is by no means clearly understood. Research effort in the area of mitochondrial biogenesis is at present intense and is increasing rapidly along the interconnected lines of biochemistry, cytology, genetics and molecular biology. Numerous review articles have recently appeared on the subject (e.g. Work, Coote, and Ashwell, 1968; Roodyn, 1968; Lloyd, 1969; Wagner, 1969) as well as at least two books (Roodyn and Wilkie, 1968; and Slater, Tager, Papa, and Quagliariello, 1968) and the reader is referred to these for detail and discussion in depth of the several aspects of the subject. The intention in this chapter is to trace the outline of our knowledge of mitochondrial genesis and continuity and to indicate how evidence from recent experiments is causing a continual modification of our understanding of how mitochondria are formed, and how they multiply.

Mitochondria in sections of liver samples from newborn and weanling rats were analyzed by quantitative electron microscopy. Specific activities of the mitochondrial enzymes, NADH-cytochrome c reductase, succinate-cytochrome c reductase and cytochrome oxidase, were determined in similar samples. In the liver parenchymal cell of the weanling, compared to that of the newborn, the number of mitochondria per unit volume of cytoplasm was 2.6 times larger, while the volume of the average mitochondrion was 0.58 times smaller, resulting in a net increase of 1.4 in the relative size of the mitochondrial compartment. The decrease in volume of the average mitochondrion was the result of a decrease in the number of large mitochondria and an increase in the number of small mitochondria. These data indicate a mitochondrial biogenesis in rat liver during postnatal development, the magnitude of which exceeds the overall growth of the organ parenchyma during this period. The increase in the size of the mitochondrial compartment was paralleled by increases of 18–21 times in the specific activities of the mitochondrial enzymes.

In the 75 years since the publication of Altmann’s “bioblast theory” the question of how mitochondria are formed by growing cells has been a continuing point of active speculation and discussion. During this period all possible mechanisms for mitochondrial biogenesis have been advanced, but in recent years greater attention has been given in speculations as well as in experimental work to the possibility that new mitochondria are derived through growth and division of pre-existing organelles. The observation that mitochondria contain a DNA of distinct base composition [6, 9] has provided an additional support for such a mechanism, and for some biologists it is now “established” that mitochondria are “self replicating” cell organs, just like Altmann’s bioblasts. The term “self replication” does not properly describe our current understanding of the problem: it fails to acknowledge some important uncertainties concerning mitochondrial biogenesis, and if applied in its strictest sense, it is incorrect.

Publisher Summary This chapter discusses the physicochemical properties, replication, and genetic function of mitochondrial DNA. The biosynthesis of functional mitochondria requires the cooperation of two genetic systems: the nuclear system, involving nuclear mRNA's, probably translated on extramitochondrial ribosomes, and a mitochondrial system localized in the mitochondrial matrix space. The genetic continuity and expression of the mitochondrial system appear to be ensured by the presence within the mitochondrial inner membrane of the enzymes required for DNA and RNA synthesis and the complete machinery required for protein synthesis. Since several vital parts of this machinery differ from their extramitochondrial counterparts (e.g., the ribosomes), the mitochondrial system for the synthesis of macromolecules is in part unique and not merely a copy of the extramitochondrial system stationed in the mitochondrial matrix space for the benefit of translating the information present in mitochondrial DNA. The chapter also discusses several aspects of mitochondrial biogenesis.

Intramitochondrial loci for δ-aminolaevulate synthetase and ferrochelatase, the initial and final enzymes in haem synthesis, have been found in rat liver. Two different methods of fractionation were applied to mitochondria: (a) sonication and density-gradient centrifugation; (b) treatment with digitonin and differential centrifugation. Similar results were obtained with each technique. δ-Aminolaevulate synthetase is distributed similarly to two known matrix enzymes, malate dehydrogenase and glutamate dehydrogenase. Ferrochelatase is firmly bound to the the inner mitochondrial membrane. These results are considered in terms of the regulation of haem synthesis and in relation to mitochondrial biogenesis.

Using yeast-cell metabolism as a Summary model, it is suggested that certain fetal cells may be especially sensitive to exogenous glucose levels. In the embryos of diabetic women high blood-glucose levels might switch such sensitive cells to a glycolytic (low energy) pattern of metabolism and, more significantly, repress mitochondrial biogenesis. This impairment of energy production could mean that some cells would be unable to meet their spatiotemporal commitments, resulting in a disruption of orderly embryonic development and congenital defects. Reversion to normal blood-glucose might further widen the energy gap (or decrease the energy supply) of sensitive cells by reducing the rate of glycolysis before the mitochondrial complement is significantly restored. Thus fluctuating glucose levels could carry a greater risk of fetal deformity than consistently high levels.