Fine structural and cytochemical studies of human diploid fibroblasts arrested in an essentially nonmitotic state.

Human diploid fibroblasts can be maintained in vitro in an arrested, essentially nonmitotic state for extended periods of time by reducing the serum concentration in the medium from 10 to 0.5%. Arrested cells can be induced to re-enter the proliferative state by subcultivation in medium containing 10% serum. Fine structure, acid phosphatase, cytochrome oxidase, and extracellular carbohydrates in arrested cells were examined and compared to cultures growing in 10% serum and to cells transferred to 10% serum after 21 days in 0.5% serum. Cells in 10% serum posessed a well-developed Golgi complex, extensive rough endoplasmic reticulum, mitochondria containing transverse cristae, and many free ribosomes in the cytoplasm. In arrested cells, Golgi complexes were rarely observed, the number of both free and membrane-bound ribosomes was reduced, the number of cristae per mitochondria was decreased and the amount of demonstrable cytochrome oxidase activity was diminished. There was an accumulation of intercellular carbohydrate components. After subcultivation with medium containing 10% serum, arrested cells regained the ultrastructural characteristics of cells continuously cultured at this serum level; however, the amount of intercellular carbohydrate remained elevated. These results indicate that distinct yet reversible changes occur in the subcellular morphology and organization of cells maintained in an essentially nonmitotic state. This arrested state may be a close approximation to the situation as it occurs in vivo in expanding cell populations.     

Doubling potential, calendar time, and donor age of human diploid cells in culture

Human diploid fibroblasts from an embryonic and an adult donor were maintained in an essentially nonmitotic state for extended periods of time by reducing the serum concentration of the growth medium from 10 to 0.5%. These cells could be returned to the rapidly proliferating state by subcultivation with medium containing 10% serum. Cells treated in such a manner took a proportionately longer calendar time to reach phase III than did controls that had been continuously cultured on growth medium. The division potential of cells from the adult donor was unaffected by the arrested state; however, that of cells from the embryonic donor was extended beyond that of growth controls. The extension of division potential in cells from the embryonic donor never exceeded the maximum passage level reported for these cells. During 21 days of cultivation with medium containing 0.5% serum, cell losses ranged from 15 to 35% and the protein content of the remaining cells decreased by 15 to 20%. It was concluded that division potential and not total calendar time was the primary determinant of the in vitro lifespan of these human diploid cells and that arresting cells from younger donors increased their ability to attain the maximum limit of their lifespan.     

Cyclic AMP and serum arrest of the mitotic activity of human diploid fibroblasts.

Mitotic activity in confluent cultures of human diploid fibroblasts was arrested by the reduction of the serum concentration of the incubation medium to 0.5% or by the addition of 0.5 mM 6-N, 2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate (db cAMP). Under either of these conditions, cultures maintained a constant cell number for 14 days; cultures continuously exposed to medium containing 10% serum doubled their cell number during this 14-day period. The protein cotent per cell decreased by 20% when cells were maintained with 0.5% serum whereas that of cells exposed to db cAMP remained constant. Ultrastructural studies revealed that cells exposed to db cAMP exhibited a morphology typical of cells cultures with 10% serum alone, whereas cells incubated with 0.5% serum showed the ultrastructural changes in mitochondria, endoplasmic reticulum and Golgi complex previously identified with low-serum arrest. Cellular adenosine 3':5'-cyclic monophosphate (cAMP) levels remained constant during the 7-day growth period in which confluency was attained, as well as during the 14-day arrested period with 0.5% serum. These results indicated that the mitotic inhibition induced by reducing the serum concentration of the incubation medium was not mediated by increased intracellular levels of cAMP and differed from that induced by the addition of exogenous db cAMP.     

Cyclic amp and serum arrest of the mitotic activity of human diploid fibroblasts

Summary Mitotic activity in confluent cultures of human diploid fibroblasts was arrested by the reduction of the serum concentration of the incubation medium to 0.5% or by the addition of 0.5mm 6-N, 2′-O-dibutyryl-adenosine 3′:5′-cyclic monophosphate (db cAMP). Under either of these conditions, cultures maintained a constant cell number for 14 days; cultures continuously exposed to medium containing 10% serum doubled their cell number during this 14-day period. The protein content per cell decreased by 20% when cells were maintained with 0.5% serum whereas that of cells exposed to db cAMP remained constant. Ultrastructural studies revealed that cells exposed to db cAMP exhibited a morphology typical of cells cultured with 10% serum alone, whereas cells incubated with 0.5% serum showed the ultrastructural changes in mitochondria, endoplasmic reticulum and Golgi complex previously identified with low-serum arrest. Cellular adenosine 3′:5′-cyclic monophosphate (cAMP) levels remained constant during the 7-day growth period in which confluency was attained, as well as during the 14-day arrested period with 0.5% serum. These results indicated that the mitotic inhibition induced by reducing the serum concentration of the incubation medium was not mediated by increased intracellular levels of cAMP and differed from that induced by the addition of exogenous db cAMP.     

Doubling potential, calendar time, and senescence of human diploid cells in culture

Human diploid cells (CF-1) derived from newborn foreskin tissue were maintained in a non-mitotic state for as long as 177 days by reducing the serum concentration of the incubation medium to 0.5%. The cells could be returned to the proliferative state by subcultivation with normal growth medium containing 10% serum. Cells treated in such a manner reached passage levels equivalent to controls that had been continuously cultured on growth medium, but they took a proportionately longer calendar time to achieve the equivalent passage levels. Also, by using ³H-thymidine incorporation, cells held in the non-mitotic conditions showed a longer ‘predictable life span’ than control cultures. During 21-day maintenance periods there was a 10–20% cell loss and ca 30% loss of protein per cell. The finite life span of these human diploid cells was clearly related to the number of cumulative population doublings rather than to the total calendar time in vitro.     

Coordinate regulation of protein synthesis and messenger RNA content during growth arrest of suspension Chinese hamster ovary cells

We have found Chinese Hamster Ovary cells, cultured in suspension, are subject to growth control by serum. When suspended in medium containing 0.5% serum the cells become reversibly arrested in the beginning of the G₁ phase of the cell cycle and can be maintained in this viable, nonproliferating state for several days. This system was used to examine the regulation of protein synthesis with growth rate. In particular, the experiments addressed the question whether mRNA content is the principal controlling factor determining the rate of protein synthesis. The rate of leucine incorporation in resting cells in low serum is 2- to 2.5-fold lower than that of cells growing in 10% serum. The steady-state number of cytoplasmic poly A (+) RNA molecules shows a proportional decrease, consistent with it being a determining factor controlling the rate of protein synthesis. Furthermore, the rate of production of poly A (+) and poly A (?) RNA appears to be regulated coordinately. Regulation of the rate of initiation of translation would result in fewer ribosomes bound per active message and/or a lower proportion of total mRNA's being active. Our measurements indicate that the fraction of cytoplasmic poly A (+) mRNA in polyribosomes and the relative degree of loading of each active poly A(+) mRNA with ribosomes is the same in resting and growing cells. Thus these cells resemble 3T6 and translational control does not appear to be an important part of the change in protein synthetic rate with the state of growth.     

Amino acid uptake in growing and arrested human diploid cell populations.

Human diploid fibroblasts cultured in vitro weremonitored for amino acid uptake in preconfluent and confluent cultures under conditions amenable to growth and in confluent cultures arrested in an essentially nonmitotic state. Preconfluent and confluent cultures in growth medium showed similar uptake patterns for leucine and alpha-aminoisobutyrate; arrested cultures exhibited a reduced uptake of both amino acids. Kinetic measurements revealed a 4-fold reduction in apparent Vmax for alpha-aminoisobutyrate influx in arrested cultures. These results suggest that the culture conditions used in this study to produce restrictions in mitotic activity likewise influence amino acid accumulation.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactylium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, and extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (holoenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (less than 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 micrometer, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 micrometer medium copper, holoenzyme secretion is maintained throughout cell growth. The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN(-)-insensitive, manganese form of this enzyme. Cells grown at 10 micrometer copper show 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Studies on a possible molecular basis for the structure of mitochondrial cristae

We have investigated a possible molecular basis for mitochondrial cristae formation. Proteoliposomes containing electron transport proteins, cytochrome oxidase, or complex III in their proper orientation bind to pig heart mitoplasts but not pig heart mitochondria. Using Leydig tumor cells, we have confirmed earlier reports that chloramphenicol causes a diminution in cristae content and a change in its characteristic lamellar form. We show that the proteoliposomes containing cytochrome oxidase or complex III in the proper orientation bind to mitoplasts from Leydig tumor cells but do not bind as well to mitoplasts from chloramphenicol-treated Leydig tumor cells. These experiments provide a possible mechanism to explain cristae formation.     

epsilon-(gamma-Glutamyl)lysine isopeptide bonds in normal and virus transformed human fibroblasts.

Normal human lung cells (WI-38) possessed 20–40 times more ε-(γ-glutamyl)lysine isopeptide bonds than Simian virus transformed counterparts, WI-38 VA13A and WI-38 VA13-2RA. Normal cells arrested in an essentially nonmitotic state had more isopeptide bonds than proliferating cells. Isopeptide content paralleled the transglutaminase activity of these cells. The results suggest that isopeptide crosslinks contribute to a cellular architecture conducive to a nonproliferating state.     

Structure of taste buds in foliate papillae of the rhesus monkey, Macaca mulatta

Taste buds in foliate papillae of the rhesus monkey were examined by electron microscopy. Three distinct cell types were identified. Type I cells were narrow elongated cells containing an oval nucleus, bundles of intermediate filaments, several Golgi bodies, and characteristic apical membrane-bounded dense granules. These cells exhibited morphological variations: some had a moderately dense cytoplasm, perinuclear free ribosomes, and flattened sacs of rough endoplasmic reticulum; others had a more lucent cytoplasm, dilated irregular rough endoplasmic reticulum, lysosome-like dense bodies, and lipid droplets. Type II cells typically contained a spherical, pale nucleus, a prominent nucleolus, supranuclear and infranuclear Golgi bodies, mitochondria with tubular cristae, and one or two centrioles. This cell type, too, showed some variation in the relative amounts of ribosomes and smooth endoplasmic reticulum, which varied inversely with each other. Type III cells were characterized by a clear apical cytoplasm essentially devoid of ribosomes and containing microtubules. In a few type III cells, the peri- and infranuclear regions contained many ribosomes and some rough endoplasmic reticulum. In most Type III cells, there were large numbers of dense and clear vesicles in the peri- and infranuclear regions; some of the vesicles were grouped in synapse-like arrangements with adjacent nerves. The morphological variations exhibited by all three cell types could be accounted for by age differences in each of the cells. This would be consistent with the notion that cell renewal occurs in each of the three cell populations.     

Induction and expression of mutations in mammalian cells in the absence of DNA synthesis and cell division

The induction of mutations by the alkylating agent ethyl methanesulfonate (EMS) was determined with Chinese hamster ovary cells maintained in serum-free medium to arrest DNA synthesis and cell division. The arrested cultures were treated with EMS and maintained in serum-free medium for various time intervals post-treatment before serum containing medium was added to initiate DNA synthesis and cell division. The concentration-dependent increase in 6-thioguanine-resistant mutants in the arrested cultures was similar to that found with exponentially dividing cultures when serum was added to the arrested cultures immediately after the EMS treatment; the time course of phenotypic expression was also similar with both cultures. In addition, maintenance of the arrested cultures in serum-free medium for up to 18 days post-treatment resulted in no change in the mutant frequency. This suggests that the mutagenic damage is not removed in these arrested cultures. Furthermore, maintenance of the arrested state for increasing time intervals before serum addition results in decreases in the time necessary for maximum phenotypic expression. Cultures maintained in serum-free medium for 16 days after mutation treatment show complete expression of the mutations with no need for subculture. This last result suggests that the mutagenic damage induced by EMS in Chinese hamster ovary cells is not removed and that this damage results in both the induction and expression of mutation in the absence of DNA replication.     

In vitro Proliferation of Human Bone Marrow Mesenchymal Stem Cells Employing Donor Serum and Basic Fibroblast Growth Factor

A method for the in vitro proliferation of human bone marrow mesenchymal stem cells (MSCs) employing a medium not containing fetal calf serum (FCS) was developed for a regenerative medicine of cartilage using MSCs. Without using density-gradient centrifugation, the bone marrow aspirate was poured into a dish (6.0 \times 10⁵ nucleated cells/cm²) with DMEM medium containing 10% serum (FCS or donor serum) and basic fibroblast growth factor, and incubated at 37 °C under a 5% CO₂ atmosphere. The density of adhesive cells incubated with the medium containing human serum and basic fibroblast growth factor (10 ng/ml) almost reached confluence at 19d and was 1.4-2.7 times that in the medium containing only FCS. The density of cells incubated with the medium containing only human serum was 0.1-0.6 times that in the medium containing only FCS. The content of CD45^- CD105⁺ cells among the cells harvested after a 19-d incubation in the medium containing human serum and basic fibroblast growth factor was higher than 90%. This high content and chondrogenic activity, which was confirmed by pellet cultivation and staining with Safranine O, were maintained even after further subcultivation in the medium to 17 population doubling levels. Consequently, this method might be applicable to in vitro proliferation of MSCs for the regeneration of cartilage.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactytium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, an extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (haloenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (< 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 μM, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 μM medium copper, holoenzyme secretion is maintained throughout cell growth.The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN^?-insensitive, manganese form of this enzyme. Cells grown at 10 μM copper shown 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Late G1 amino acid restriction point in human dermal fibroblasts

Human dermal fibroblasts arrested in G0 by maintenance in medium supplemented with 0.1% serum were not restimulated to divide when fresh medium containing 10% dialyzed serum but lacking group B amino acids (cystine, isoleucine, lysine, phenylalanine and tyrosine) was added. Unlike rodent cells, the addition of fresh serum-supplemented medium lacking only isoleucine did not cause a growth arrest. The amino acid sensitive growth arrest in human fibroblasts was dependent both on presynchronization in G0 as well as a prestarvation for amino acids prior to stimulation with high serum. When cells were restimulated in the absence of amino acids, they arrested predominantly in G1, although a small percentage of cells entered early S phase. When medium containing a complete complement of amino acids was then added, cells initiated DNA synthesis following a minimum lag of 2-3 hr. Growth arrested cells initiated DNA synthesis even when complete unsupplemented medium was added, although the addition of high concentrations of insulin or 10% serum increased the rate of entry.     

Secretion of alpha-L-fucosidase by human embryonal skin fibroblasts

The amount of alpha-L-fucosidase secreted by normal human fibroblasts was higher in the medium containing 10% bovine serum than in the medium containing 0.1% bovine serum. Glycosidase secretion was twice higher at the advanced than at the initial stage of subcultivation. Extracellular activity of alpha-L-fucosidase from 3 different fibroblast strains differed insignificantly in the medium containing 0.1% bovine serum, while intracellular activity of the enzyme in these strains was altogether different. The results suggest that the lysosomal glycosidase secretion is determined by the level of cellular endocytosis.     

Selective cytochemical localization of peroxidase,cytochrome oxidase and catalase in rat liver with 3,3′-diaminobenzidine

Summary In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specifity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15–30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylate buffer pH 6.5 and 0.02% H₂O₂. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) butffer at pH 10.5 and 0.15% H₂O₂. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

Selective cytochemical localization of peroxidase, cytochrome oxidase and catalase in rat liver with 3,3'-diaminobenzidine

In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specificity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15-30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylte buffer pH 6.5 and 0.02% H2O2. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) buffer at pH 10.5 and 0.15% H2O2. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

The effects of division rate-limiting amounts of fetal bovine serum on the proliferation and aging of cultured chick cells

Chick embryo fibroblasts serially propagated in media containing division ratelimiting amounts of fetal bovine serum underwent premature culture senescence as illustrated by accelerated declines in the number of cells incorporating ³H-thymidine, increased population doubling times, reduced cell densities at subcultivation, and reduced replicative life-spans compared to cells grown in medium containing non-rate-limiting amounts of serum. Low serum serially propagated “senescent” cultures returned to 10% serum containing medium had proliferative rates, incorporated ³H-thymidine, and attained saturation densities at confluency similar to younger cells. “Senescent” cells serially propagated in low serum and returned to 10% serum achieved life-spans similar to cells continuously grown in the presence of 10% serum. The results of these and other studies show that cells serially propagated in the presence of division rate-limiting amounts of fetal bovine serum, or at high inoculation densities, accumulate a substantial number of cells in the population during exponential growth conditions that are not senescent but are prevented from entering DNA synthesis becuase of mitogen limitations. Our results indicate that the amount of serum mitogen in the growth medium affects only the rate at which cells express their genetically predetermined replicative potential and not the replicative lifespan per se. These results are discussed in relation to the techniques that should be employed for studying cellular aging and the mechanism of senescent cell formation.     

Formation of the yeast mitochondrial membrane. 3. Accumulation of mitochondrial proteins synthesized in both the cytoplasm and mitochondria in yeast undergoing glucose depression

The inhibitors of protein synthesis, chloramphenicol and cycloheximide, were added to cultures of yeast undergoing glucose derepression at different times during the growth cycle. Both inhibitors blocked the increase in activity of coenzyme QH₂-cytochrome c reductase, suggesting that the formation of complex III of the respiratory chain requires products of both mitochondrial and cytoplasmic protein synthesis.The possibility that precursor proteins synthesized by either cytoplasmic or mitochondrial ribosomes may accumulate was investigated by the sequential addition of cycloheximide and chloramphenicol (or the reverse order) to cultures of yeast undergoing glucose derepression. When yeast cells were grown for 3 hr in medium containing cycloheximide and then transferred to medium containing chloramphenicol, the activity of cytochrome oxidase increased at the same rate as the control during the first hour in chloramphenicol. These results suggest that some accumulation of precursor proteins synthesized in the mitochondria had occurred when cytoplasmic protein synthesis was blocked during the growth phase in cycloheximide. In contrast, essentially no products of mitochondrial protein synthesis accumulated as precursors for either oligomycin-sensitive ATPase or complex III of the respiratory chain during growth of the cells in cycloheximide.When yeast were grown for 3 hr in medium containing chloramphenicol followed by 1 hr in cycloheximide, the activities of cytochrome oxidase and succinate-cytochrome c reductase increased at the same rate as the control, while the activities of oligomycin-sensitive ATPase and NADH or coenzyme QH₂-cytochrome c reductase were nearly double that of the control. These data suggest that a significant accumulation of mitochondrial proteins synthesized in the cytoplasm had occurred when the yeast cells were grown in medium containing sufficient chloramphenicol to block mitochondrial protein synthesis. The possibility that proteins synthesized in the cytoplasm may act to control the synthesis of mitochondrial proteins for both oligomycin-sensitive ATPase and complex III of the respiratory chain is discussed.