RELATION OF PROTEIN SYNTHESIS TO THE DIVISION CYCLE IN MAMMALIAN CELL CULTURES

The distribution of the reaction product of a staining method for adenosine triphosphatase (ATPase) in rat small intestine, kidney, and liver was studied with electron microscopy. Several procedures were tried but the best results were obtained from tissue that had been quenched in liquid nitrogen, sectioned at 25 µ in a cryostat, fixed for 30 to 90 minutes at 4°C in formalin-sucrose buffered to pH 7.2, incubated with substrate, and then osmicated and prepared for electron microscopy in the usual way. This procedure enabled the localization of mitochondrial ATPase to be studied. In tissue fixed in small blocks in osmium tetroxide for 3 minutes prior to incubation with substrate, good preservation was noted, and the reaction product for ATPase was localized on the cell membrane and nuclei. The reaction product was present in abundant amount in the nuclei, and particularly within nucleoli, of all tissues studied. Because the histochemical localization of nuclear enzymes poses numerous interpretative problems at the present time, the significance of this nuclear localization is uncertain. Cell (plasma) membranes were the site of localization, especially at areas where it has been proposed that active transport mechanisms may occur, namely, on the microvilli of intestinal epithelium, endothelial lining of capillaries, glomerular epithelial cell membranes, basal infoldings of the cell membrane of renal tubules, on the microvilli of bile canaliculi, and on the microvilli of proximal convoluted tubular epithelial cells. ATPase localization on the cristae mitochondriales was also demonstrated.     

热休克蛋白代谢过程中Hela细胞热耐受性的变化

HeLa细胞受热应激后,可产生一组热休克蛋白(HSP),其中HSP73/70产量最高,其合成呈现一定的规律性,受热后4h为其合成速率高峰,10h后明显减少,24h恢复正常。随着HSP合成的消失,正常蛋白质合成逐渐恢复。HSP73/70在细胞内分解遵循指数规律,其半衰期为49.9h。HSP合成及分解规律与细胞热耐受性的增加与消退基本吻合,提示二者之间存在着伴随关系,但是否存在量效关系乃至因果关系有待今后进一步探讨。     

EFFECT OF METABOLIC INHIBITORS ON NUCLEAR PORE FORMATION DURING THE HELA S3 CELL CYCLE

The effect of various antimetabolites on nuclear pore formation was studied in synchronized HeLa S₃ cells. The nuclear size was determined by light microscopy and the pore number per unit area of nuclear surface by the freeze-etching technique and electron microscopy. It was found that the inhibition of DNA replication or ribosomal RNA synthesis has no effect on nuclear size increase or pore formation. However, the inhibition of ATP synthesis effectively stops nuclear pore formation. Cycloheximide blocks nuclear pore formation at the same time during G₁ phase of the cell cycle when nuclear size increase is blocked by high concentrations of actinomycin D. This suggests that certain proteins or other factors leading to pore formation and nuclear size increase are transcribed and synthesized at about 3–4 h after mitosis, i.e., about 1–2 h before S phase begins.     

THE ULTRASTRUCTURE OF A MAMMALIAN CELL DURING THE MITOTIC CYCLE

With a technique of preselecting the mitotic cell in the living state for subsequent electron microscopy, it has been possible to examine the ultrastructure of the various stages of mitosis with greater precision than has been reported previously. The early dissolution of the nuclear envelope has been found to be preceded by a marked undulation of this structure within the nuclear "hof." This undulation appears to be intimately related to the spindle-forming activity of the centriole at this time. Marked pericentriolar osmiophilia and extensive arrays of vesicles are also prominent at this stage, the former continuing into anaphase. Progression of the cell through prophase is accompanied by a disappearance of these vesicles. A complex that first makes its appearance in prophase but becomes most prominent in metaphase is a partially membrane-bounded cluster of dense osmiophilic bodies. These clusters which have a circumferential distribution in the mitotic cell are shown to be derived from multivesicular bodies and are acid phosphatase-positive. The precise selection of cells during the various stages of anaphase has made it possible to follow chronologically the morphological features of the initiation of nuclear membrane reformation. The nuclear membrane appears to be derived from polar aggregates of endoplasmic reticulum, and the process begins less than 2 minutes after the onset of karyokinesis. While formation of the nuclear envelope is initiated on the polar aspects of the chromatin mass, envelope elements appear on the equatorial aspect long before the polar elements fuse. Apparently interfering with this fusion are continuous spindle tubules which traverse the chromatin mass in striking density at characteristic points. Several cortical changes, also most pronounced in anaphase, have been described, as has the kinetochore which is seen to good advantage only in this stage. The Golgi complex has been found to disappear both morphologically and histochemically during mitosis and to reappear rapidly in telophase. Evidence is presented which implicates the continuous spindle tubules in certain phases of chromosome movement.     

TIME SEQUENCE OF NUCLEAR PORE FORMATION IN PHYTOHEMAGGLUTININ-STIMULATED LYMPHOCYTES AND IN HELA CELLS DURING THE CELL CYCLE

The time sequence of nuclear pore frequency changes was determined for phytohemagglutinin (PHA)-stimulated human lymphocytes and for HeLa S-3 cells during the cell cycle. The number of nuclear pores/nucleus was calculated from the experimentally determined values of nuclear pores/µ² and the nuclear surface. In the lymphocyte system the number of pores/nucleus approximately doubles during the 48 hr after PHA stimulation. The increase in pore frequency is biphasic and the first increase seems to be related to an increase in the rate of protein synthesis. The second increase in pores/nucleus appears to be correlated with the onset of DNA synthesis. In the HeLa cell system, we could also observe a biphasic change in pore formation. Nuclear pores are formed at the highest rate during the first hour after mitosis. A second increase in the rate of pore formation corresponds in time with an increase in the rate of nuclear acidic protein synthesis shortly before S phase. The total number of nuclear pores in HeLa cells doubles from ~2000 in G₁ to ~4000 at the end of the cell cycle. The doubling of the nuclear volume and the number of nuclear pores might be correlated to the doubling of DNA content. Another correspondence with the nuclear pore number in S phase is found in the number of simultaneously replicating replication sites. This number may be fortuitous but leads to the rather speculative possibility that the nuclear pore might be the site of initiation and/or replication of DNA as well as the site of nucleocytoplasmic exchange. That is, the nuclear pore complex may have multiple functions.     

NUCLEOLAR AND NUCLEAR RNA SYNTHESIS DURING THE CELL LIFE CYCLE IN MONKEY AND PIG KIDNEY CELLS IN VITRO

The incorporation of 5-³H-uridine and 5-³H-cytidine into nucleolar and nonnucleolar RNA in the nucleus of monkey and pig kidney cells was measured in vitro during the cell life cycle. Time-lapse cinematographic records were made of cells during asynchronous exponential proliferation, in order to identify the temporal position of individual cells in relation to the preceding mitosis. Immediately following cinematography, cells were labeled with uridine-³H and cytidine-³H for a short period, fixed, and analyzed by radioautography. Since the data permit correlation of the rate of RNA labeling with the position of a cell within the cycle, curves could be constructed describing the rate of RNA synthesis over the average cell cycle. RNA synthesis was absent in early telophase, and rose very abruptly in rate in late telophase and in very early G₁ in both the nucleus and the reconstituting nucleolus. Thereafter, through the G₁ and S periods the rate of nuclear RNA synthesis rose gradually. When we used a 10-min pulse, there was no detectable change in the rate for nucleolar RNA labeling in monkey kidney cells during G₁ or S. When we used a 30-min labeling time, the rate of nucleolar RNA labeling rose gradually in pig kidney cells. With increasing time after mitosis, the data became more variable, which may, in part, be related to the variation in generation times for individual cells.     

ULTRASTRUCTURE OF THE NUCLEOLUS DURING THE CHINESE HAMSTER CELL CYCLE

Changes in the structure of the nucleolus during the cell cycle of the Chinese hamster cell in vitro were studied. Quantitative electron microscopic techniques were used to establish the size and volume changes in nucleolar structures. In mitosis, nucleolar remnants, "persistent nucleoli," consisting predominantly of ribosome-like granular material, and a granular coating on the chromosomes were observed. Persistent nucleoli were also observed in some daughter nuclei as they were leaving telophase and entering G₁. During very early G₁, a dense, fibrous material characteristic of interphase nucleoli was noted in the nucleoplasm of the cells. As the cells progressed through G₁, a granular component appeared which was intimately associated with the fibrous material. By the middle of G₁, complete, mature nucleoli were present. The nucleolar volume enlarged by a factor of two from the beginning of G₁ to the middle of S primarily due to the accumulation of the granular component. During the G₂ period, there was a dissolution or breakdown of the nucleolus prior to the entry of the cells into mitosis. Correlations between the quantitative aspects of this study and biochemical and cytochemical data available in the literature suggest the following: nucleolar reformation following division results from the activation of the nucleolar organizer regions which transcribe for RNA first appearing in association with protein as a fibrous component (45S RNA) and then later as a granular component (28S and 32S RNA).     

CHANGES IN CELL CHEMICAL COMPOSITION DURING THE LIFE CYCLE OF SCRIPPSIELLA TROCHOIDEA (DINOPHYCEAE)1

The cellular content of carbon, nitrogen, amino acids, polysaccharides, phosphorus and adenosine trtphosphate (ATP) was determined at several stages during the life cycle of the dinoflagellate Scrippsiella trochoidea (Stein) Loeblich. Carbon per cell decreased slightly between exponential and stationary phase growth in vegetative cells whereas nitrogen per cell did not change. Both of these cellular components increased markedly on encystment and then decreased to vegetative cell levels during dormancy and germination. C/N ratios increased gradually during cyst dormancy and activation, reflecting a more rapid decrease in N than in C pools, even though both decreased through time. Amino acid composition was relatively constant during the vegetative cell stages; glutamic acid was the dominant component. Arginine was notably higher in cysts than in vegetative cells but decreased significantly during germination, suggesting a role in nitrogen storage. The ratio of neutral ammo acids to total ammo acids (NAA/TAA) decreased as cysts were formed and then gradually increased during storage and germination. The ratio of basic ammo acids to total ammo acids (BAA/TAA) changed in the opposite direction of NAA/TAA, whereas the ratio of acidic acids to total amino adds (AAA/TAA) was generally invariant. Ammo acid pools were not static during the resting slate in the cysts: there was degradation or biosynthesis of certain, but not all, classes of these compounds. The monosacchande composition of cold and hot water extracted polysaccharides was quite different between cells and cysts. A high percentage of glucose in cysts suggests that the storage carbohydrate is probably in the form of glucan. Total cellular phosphorus was higher in all cyst stages than in vegetative cells. However, ATP-cell^?1 decreased as vegetative cells entered stationary phase and encysted, and continued to decrease in cysts during dark cold storage. ATP increased only as the cysts were activated at warm temperatures in the light and began to germinate. The above data demonstrate that dormancy and quiescence are not periods of inactive metabolism but instead are times when numerous biochemical transformations are occurring that permit prolonged survival in a resting state.     

A COMPARISON OF THE HETEROGENEOUS NUCLEAR RNA OF HELA CELLS IN DIFFERENT PERIODS OF THE CELL GROWTH CYCLE

HeLa cells synthesize heterogeneous nuclear RNA (HnRNA) in the G₁, S, and G₂ portions of the cell cycle. HnRNA prepared from these various periods was compared by RNA-DNA hybridization experiments. The results indicated that some of the HnRNA molecules were equivalent at all times in the cell cycle, but limitations in the sensitivity of the hydridization reactions, as well as in the spectrum of hybridizing molecules, restrict the conclusions that can be drawn from these comparisons.     

THE SYNTHESES OF TOTAL MACRONUCLEAR PROTEIN, HISTONE, AND DNA DURING THE CELL CYCLE IN EUPLOTES EURYSTOMUS

Increased alkaline phosphatase activity is induced in certain epithelial cell cultures by hormones with adrenal glucocorticoid activity or their analogues such as prednisolone (Δ^I-hydrocortisone). Enzyme induction occurs in two distinct phases. During the first 12 hr after the addition of prednisolone, there is a small increase in alkaline phosphatase levels. After 15 to 24 hr, the enzyme activity shows a sudden, marked linear rise, reaching a maximum at 60 to 80 hr. Puromycin blocks enzyme induction immediately, even when added during the period of rapid increase of enzyme. Actinomycin D blocks induction when added no later than 8 hr after the addition of prednisolone. On the other hand, Actinomycin D added during the phase of rapid enzyme induction has no effect for at least 12 hr. These findings suggest that de novo protein synthesis is involved in prednisolone induction of alkaline phosphatase and that the RNA messenger for this enzyme is relatively stable.     

PERIODICITY OF DNA SYNTHETIC ENZYMES DURING THE HeLa CELL CYCLE

The levels of the deoxynucleoside phosphokinases have been determined during the cell cycle in synchronous cultures of HeLa cells. Deoxycytidine and thymidine phosphokinases showed temporally similar periodic fluctuations while the purine deoxynucleoside phosphokinases showed a constant high activity. Experiments with metabolic inhibitors suggest that the observed rise in thymidine phosphokinase activity is directly due to the enzyme's biosynthesis.     

MITOCHONDRIAL PROTEIN SYNTHESIS IN HELA CELLS

HeLa cell mitochondrial proteins have been shown to be the products of two separate protein-synthesizing systems; one, the general cellular mechanism, sensitive to inhibition by cycloheximide, the other, a specific mitochondrial system subject to inhibition by low concentrations of chloramphenicol (Galper, J. B., and J. E. Darnell. 1971. J. Mol. Biol 57:363). Preliminary data have suggested that a mitochondrial N-formyl-methionyl-tRNA (f-Met-tRNA) might be the initiator tRNA in the latter (Galper, J. B., and J. E. Darnell. 1969. Biochem. Biophys. Res. Commun. 34:205; 1971. J. Mol. Biol. 57:363). It is demonstrated here that the synthesis of these endogenous mitochondrial proteins is also subject to inhibition by ethidium bromide and decays with a half-life of 1½–2 h in cultures incubated with low concentrations of this dye. The role of formylated f-Met-tRNA as the initiator tRNA in the synthesis of mitochondrial proteins is supported by data from several experiments. The rates of ethidium bromide inhibition of both the charging of f-Met-tRNA and of the synthesis of mitochondrial proteins are strikingly similar. Inhibition by aminopterin of the formylation of f-Met-tRNA greatly depresses the rate of mitochondrial-specific protein synthesis. In the absence of the synthesis of these proteins, respiration, the levels of cytochromes a–a₃ and b, and the number of mitochondrial cristae are decreased. The implications of these findings as they relate to mitochondrial biogenesis are discussed.     

小麦核仁在细胞周期中的变化

运用Bernhard染色方法研究了小麦根端分生组织细胞核仁在细胞周期中的变化。结果显示,间期核仁染色很深,能够区分出纤维中心（FC）、致密纤维组分（DFC）和颗粒组分（G）,而染色质被漂白,在染色质间可以观察到细小的RNP颗粒。进入前期,在染色质的边缘有小的RNP颗粒分布。中期,染色体周边分布着类似于间期核仁的深染的大RNP颗粒,形成一个不完全连续的“鞘”状结构;在染色体内部看不到类似核仁的深染颗粒。到了后期时,仍可见RNP“鞘”状结构的存在。进入末期,这些RNP植物逐渐由“鞘”脱离,最后参与新核仁的形成。这些结果表明,核仁解体后的物质直接转移到了中期染色的表面,并形成不连续的表层,没有进入染色体的内部。     

REGULATION OF PROTEIN SYNTHESIS DURING POSTNATAL MATURATION OF THE BRAIN

—The activities of sRNA-aminoacyl synthetases (EC 6.1.1) were investigated in the cerebral white and grey matter of rabbits subjected during their prenatal life to a single X-ray dose of 150 rad. The results of investigations have shown that ionizing radiation acting during intrauterine development of the experimental animal brings about a distinct depression of all sRNA-aminoacyl synthetase activities in the newborn irradiated litter. During the postnatal development of these animals the activities of some of the synthetases further decreased and even at adulthood, where they are normally very low, their activities were below the control values. The activities of some other synthetases, after the initial depression, showed no further decrease and at adulthood had values comparable to controls. Our results indicate clearly that prenatal exposure to ionizing radiation also affects the step of protein biosynthesis which depends on the activity of sRNA-aminoacyl synthetases.     

THE TIMING OF DEOXYRIBONUCLEIC ACID SYNTHESIS IN THE CELL CYCLE OF SACCHAROMYCES CEREVISIAE

Randomly dividing cultures of Saccharomyces cerevisiae were briefly exposed to radioactive adenine and then treated successively with dilute acid, ribonuclease, buffered formaldehyde, and NaOH. This treatment was shown to remove virtually all the radioactivity of the labelled cells other than that in DNA. Thus, in subsequent autoradiographs, only cells which had been synthesizing DNA during exposure to the precursor were labelled. The ages of these individuals within the cell cycle were estimated by measuring their sizes. This revealed that incorporation into DNA occurred almost exclusively during the first quarter of the cell cycle, starting with the initial appearance of the bud. This behaviour agreed closely with that of cells growing in artificially synchronized cultures.