Synthesis of ribosomal proteins during the yeast cell cycle

The synthesis of ribosomal proteins during the cell division cycle of Saccharomyces cerevisiae has been examined. A technique was utilized whereby cells in unique phases of the cell cycle were selected from an asynchronous culture after the period of pulse labeling. Some of the proteins of the small and large ribosomal subunits were synthesized continuously throughout the cell cycle and there was no evidence of discontinuous synthesis for any of the ribosomal proteins.     

An autoradiographic study of the uptake of tritiated proline by osteoblasts during hibernation

Twenty-four LSH and LVG strain golden hamsters, Mesocricetus auratus, were used. Experimental animals were maintained at 5 C and allowed to hibernate. Control animals were kept at 27 C. Six animals (3 experimental, 3 control) were injected subcutaneously with 1 microCi of 3H-proline/gm body wt. (Spec. act. 3 Ci/mM) after hibernation lasting 12 hours, 1 day, 3 days, or 7 days. Animals were killed 1 hour after injection and autoradiographs were prepared from 5 microns thick decalcified sections of femurs. A greater number of endosteal cells were labeled than periosteal cells and also exhibited a greater magnitude of labeling throughout the study. Differences between endosteal and periosteal cells both in percentage of cells labeled and magnitude of labeling were maximum in control animals and progressively decreased with increasing periods of hibernation. A reduction in synthesis of matrix proteins during the early period of hibernation was seen and was attributed to a significant reduction both in average cell activity and in the number of active cells during hibernation. The latter phenomenon apparently made a large contribution to the reduced matrical synthesis. 3H-proline uptake by osteoblasts probably reflects the reduced requirements of matrical synthesis during hibernation.     

3H-fucose incorporation into glycoproteins of toad bladder epithelial cells.

Electron microscope autoradiography was used to detect the incorporation of 3H-fucose into glycoproteins of toad bladder epithelial cells. After short exposure to 3H-fucose, without a chase period, the Golgi regions of all four cell types were labeled. When exposure to 3H-fucose was followed by chase periods (1,3,4 and 6 hours) the apical and basal-lateral plasma membranes of granular cells were heavily labeled. Apical granules and the cytoplasm of granular cells were also labeled, suggesting that they both provide the means for glycoprotein transfer from the Golgi to the plasma membranes. The heaviest labeling in mitochondria-rich cells, after the 1- and 3-hour chase periods, was over the apical tubules, although the apical and basal-lateral plasma membranes were also heavily labeled. After 4- and 6-hour chases, the labeling of the apical tubules decreased, whereas the labeling of the plasma membranes increased, strongly suggesting that in these cells apical tubules play a major role in the transfer of glycoproteins from the Golgi to the plasma membrane. Our results demonstrate that the route of 3H-fucose incorporation into plasma membrane glycoproteins and the rate of glycoprotein synthesis and breakdown are not the same in the two major epithelial cell types in toad bladder.     

Comparison of Patterns of Accumulation of Ribulose Bisphosphate Carboxylase Antigen and Catalytic Activity and Measurement of Antigen Half-Life during the Cell Cycle of Chlorella sorokiniana

By use of specific immunochemical procedures, ribulose-1,5-bisphosphate carboxylase (RuBPCase), antigen and catalytic activity were shown to have coincident step-patterns of accumulation during the cell cycle of Chlorella sorokiniana. Pulse-chase studies, employing radioactive sulfate, were performed during the period of rapid accumulation of enzyme activity and during the period of constant enzyme activity in the cell cycle. No degradation of RuBPCase antigen could be detected during either of these cell cycle periods. Thus, the step-pattern of accumulation of RuBPCase activity resulted from periodic synthesis of an enzyme that was stable under steady-state cell cycle conditions. Although inhibition of protein synthesis by cycloheximide, at different times in the cell cycle in the light, resulted in rapid decay of RuBPCase activity, this loss in activity occurred without detectable loss in enzyme antigen. When synchronous cells were placed into the dark, to slow the rate of protein synthesis in the absence of cycloheximide, the levels of enzyme antigen and activity decreased by 30 and 50%, respectively, during the 10-hour dark period. Thus, in C. sorokiniana changes in RuBPCase activity do not necessarily reflect parallel changes in enzyme antigen, particularly when cell growth is perturbed by changes from steady-state cultural conditions.     

Developmental regulation of protein synthesis during Polysphondylium pallidum cyst germination: A two-dimensional gel electrophoresis study

Microcyst germination in Polysphondylium pallidum can be used as a model for studying gene expression because temporally regulated modulations in protein synthesis occur in this developmental pathway. Germinating cysts were labeled with [³⁵S]methionine for half-hourly periods during the synchronous germination sequence, and the proteins labeled in each period were resolved by two-dimensional polyacrylamide gel electrophoresis. Three major classes of proteins observed were distinguished by the time of onset and duration of their synthesis: (a) proteins made throughout germination; (b) proteins synthesized only during a portion of the germination pathway; and (c) polypeptides whose synthesis started at 1 or 1.5 h and then continued throughout germination.     

Identification of proteins whose synthesis is modulated during the cell cycle of Saccharomyces cerevisiae

We examined the synthesis and turnover of individual proteins in the Saccharomyces cerevisiae cell cycle. Proteins were pulse-labeled with radioactive isotope (35S or 14C) in cells at discrete cycle stages and then resolved on two-dimensional gels and analyzed by a semiautomatic procedure for quantitating gel electropherogram-autoradiographs. The cells were obtained by one of three methods: (i) isolation of synchronous subpopulations of growing cells by zonal centrifugation.; (ii) fractionation of pulse-labeled steady-state cultures according to cell age; and (iii) synchronization of cells with the mating pheromone, alpha-factor. In confirmation of previous studies, we found that the histones H4, H2A, and H2B were synthesized almost exclusively in the late G1 and early S phases. In addition, we identified eight proteins whose rates of synthesis were modulated in the cell cycle, and nine proteins (of which five, which may well be related, were unstable, with half-lives of 10 to 15 min) that might be regulated in the cell cycle by periodic synthesis, modification, or degradation. Based on the time of maximal labeling in the cell cycle and on experiments with alpha-factor and hydroxyurea, we assigned the cell cycle proteins to two classes: proteins in class I were labeled principally in early G1 phase and at a late stage of the cycle, whereas those in class II were primarily synthesized at times ranging from late G1 to mid S phase. At least one major control point for the cell cycle proteins occurred between "start" and early S phase. A set of stress-responsive proteins was also identified and analyzed. The rates of synthesis of these proteins were affected by certain perturbations that resulted during selection of synchronous cell populations and by heat shock.     

Synthesis and Accumulation of Calmodulin in Suspension Cultures of Carrot (Daucus carota L.) : Evidence for Posttranslational Control of Calmodulin Expression

The expression of calmodulin mRNA and protein were measured during a growth cycle of carrot (Daucus carota L.) cells grown in suspension culture. A full-length carrot calmodulin cDNA clone isolated from a λgt10 library was used to measure steady-state calmodulin mRNA levels. During the exponential phase of culture growth when mitotic activity and oxidative respiration rates were maximal, calmodulin mRNA levels were 4- to 5-fold higher than they were during the later stages of culture growth, when respiration rates were lower and growth was primarily by cell expansion. Net calmodulin polypeptide synthesis, as measured by pulse-labeling in vivo with [³⁵S]methionine, paralleled the changes in calmodulin steady-state mRNA level during culture growth. As a consequence, net calmodulin polypeptide synthesis declined 5- to 10-fold during the later stages of culture growth. The qualitative spectrum of polypeptides synthesized and accumulated by the carrot cells during the course of a culture cycle, however, remained largely unchanged. Calmodulin polypeptide levels, in contrast to its net synthesis, remained relatively constant during the exponential phases of the culture growth cycle and increased during the later stages of culture growth. Our data are consistent with increased calmodulin polypeptide turnover associated with periods of rapid cell proliferation and high levels of respiration.     

DNA replication and the nuclear membrane

To investigate the relationship between the nuclear membrane and DNA replication, Chinese hamster cells were labeled with tritiated thymidine and examined by electron microscope autoradiography. Unsynchronized cells were labeled for periods ranging from 0.5 to 20 minutes. There was no relative increase in the frequency of membrane-associated grains with the shorter labeling times, indicating that the replication point is not necessarily close to the nuclear membrane. When cells were synchronized to the beginning of the S period with mitotic selection and hydroxyurea, the percentage of membrane-associated grains was very low, indicating that DNA synthesis is not initiated at the nuclear membrane. When cells synchronized by mitotic selection were labeled at various times throughout the cell cycle, the percentage of peripheral grains was low in early S period and became progressively higher toward late S period as heterochromatin began to replicate. The labeling of Unsynchronized Microtus agrestis cells indicated that much of the peripheral labeling is due to the replication of intercallary heterochromatin. The results indicate that there is no association between the nuclear membrane and DNA replication.     

Synthesis of the inner mitochondrial membrane and the intercalation of respiratory enzymes during the cell cycle of Chlorella.

The ratio of inner to outer mitochondrial membrane area remains close to 1-8 throughout the cell cycle in synchronized cells of Chlorella fusca var, vacuolata 211-8p. Using estimates of this ratio, together with our previous estimates of mitochondrial surface area, to calculate the absolute area of inner mitochondrial membrane, it is demonstrated that growth of the inner mitochondrial membrane during the cell cycle occupies an extended period and parallels the growth of the whole cell. In contrast, the synthesis of succinate dehydrogenase and cytochrome oxidase is restricted to the last third of the cell cycle. It is concluded that mitochondrial growth involves the intercalation of periodically synthesized respiratory enzymes into membranes made earlier in the cycle, with consequent 5-fold changes in the density of active enzyme molecules in the membrane. These observations are discussed in relation to the control of mitochondiral membrane synthesis, membrane assembly and respiration rate during the cell cycle.     

Membrane adenosine triphosphatase in synchronous cultures of Rhodobacter sphaeroides

Studies of intracytoplasmic membrane biogenesis utilizing synchronized cultures of Rhodobacter sphaeroides have revealed that most intracytoplasmic membrane proteins accumulate continuously throughout the cell cycle while new phospholipid appears discontinuously within the intracytoplasmic membrane. The resulting changes in the structure of the membrane lipids was proposed to influence the activities of enzymes associated with the intracytoplasmic membranes (Wraight, C.A., Leuking, D.R., Fraley, R.T. and Kaplan, S. (1978) J. Biol. Chem. 253, 465-471). We have extended the study of intracytoplasmic membrane biogenesis in R. sphaeroides to include the membrane adenosine triphosphatase. The membrane bound Mg2+-dependent, oligomycin-sensitive adenosine triphosphatase activity was measured throughout the cell cycle for steady-state synchronized cells of R. sphaeroides and found to accumulate discontinuously. Following treatment with an uncoupling reagent (2,4-dinitrophenol) the intracytoplasmic membrane associated adenosine triphosphatase activity was stimulated uniformly in membranes isolated at different stages of the cell cycle. The adenosine triphosphatase was also measured by quantitative immunoblots utilizing specific antibody to compare the enzyme activity and enzyme protein mass. Immunologic measurement of the adenosine triphosphatase in isolated membranes indicated a constant ratio of enzyme to chromatophore protein exists during the cell cycle in contrast to the discontinuous accumulation of adenosine triphosphatase activity. These results are discussed in light of the cell-cycle specific synthesis of the intracytoplasmic membrane.     

Envelope synthesis during the cell cycle in Escherichia coli B/r

The rate of synthesis of envelope proteins and phospholipids during the cell cycle of Escherichia coli B/r has been studied using both synchronous cultures and random cultures, first labelled and then subsequently fractionated on an age basis by the membrane elution technique. The rate of total protein synthesis and of phospholipid synthesis, measured by incorporation of [2-³H]glycerol into whole cells, was found to increase exponentially throughout the cell cycle. Total envelope protein was also synthesized continuously throughout the cycle, but the rate of synthesis showed a stepwise pattern with a discrete doubling in rate in the first half of the cycle. Analysis of the pattern of synthesis of about 29 individual envelope polypeptides by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography revealed that the great majority followed the pattern of the bulk measurements, with a discrete increase in rate of synthesis early in the cycle. One envelope polypeptide, molecular weight 76,000, was, however, only synthesized during a brief period, near the time of division of the bacteria. Pulse-chase studies of envelope polypeptide synthesis in synchronous cultures demonstrated that (1) synthesis and insertion of polypeptide into the envelope was always completed within the pulse period; (2) no post-synthetic modification of polypeptides was detected; (3) one group of polypeptides, including a major outer membrane protein, maintained a stable association with the envelope, whilst a second group displayed considerable “turnover”; (4) about 70% of newly synthesized 76,000 molecular weight protein was lost from the envelope during the succeeding generation.     

Determining degradation and synthesis rates of arabidopsis proteins using the kinetics of progressive 15N labeling of two-dimensional gel-separated protein spots

The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a (15)N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (K(S)) and degradation (K(D)) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify K(S) and K(D) for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. K(S) and K(D) correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive (15)N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability.     

Light-dependent regulation of the synthesis of soluble and intracytoplasmic membrane proteins of Rhodopseudomonas sphaeroides.

Cells of Rhodopseudomonas sphaeroides grown under saturating light conditions (30 W/m2) and then shifted to low light intensity (3 W/m2) required 2.5 h to adapt to the new lower light conditions. After the shift, cell growth, whole cell protein accumulation, and bacteriochlorophyll accumulation ceased immediately. Approximately midway into the adaptation period, bacteriochlorophyll synthesis commenced at a new, higher rate, which continued through the beginning of the low-light growth period until new steady-state levels were reached. Immediately after the downshift, the rate of cellular protein synthesis declined to 22% of its preshift rate. Pulse-labeling of protein throughout the adaptation period and comparison with a steady-state prelabel culture revealed that synthesis of two of the three light-harvesting proteins, as well as two additional high-molecular-weight photosynthetic membrane proteins, was derepressed three- to fivefold compared with bulk cellular protein. Finally, the synthesis of at least three soluble proteins showed light-dependent regulation after the light downshift. These results are discussed in terms of the light-dependent regulation of synthesis of the photosynthetic membrane macromolecular components and the division of protein synthesis between the photosynthetic membranes and the soluble cell phase.     

Regulation of the synthesis of surface protein in the cell cycle of E. coli B/r.

We have studied the biogenesis of the envelope of E. coli B/r by measuring the synthesis of protein in separated inner and outer membranes during the cell cycle. While total protein and bulk inner membrane protein were synthesized continuously and at an exponentially increasing rate throughout the cycle, bulk outer membrane protein was synthesized at a constant rate throughout the cycle with an abrupt doubling in rate occurring 10–15 min before division. A similar pattern was observed when the rate of synthesis of an individual protein, the 36.5K outer membrane protein, was measured directly in total cell lysates. Neither thymine starvation nor changes in gene dosage of exponential cultures affected the synthesis of outer membrane protein, indicating that the doubling in rate is not controlled by a gene duplication mechanism. Other findings, however, further indicate that outer membrane protein synthesis is regulated in some way. Thus the concentration of 36.5K porin per unit surface area remained constant as the surface area/volume ratio varied widely with growth rate. We also obtained direct evidence for an overall limitation on the rate of synthesis of bulk outer membrane proteins; when a new class of outer membrane proteins was induced, the rate of synthesis of other surface proteins was correspondingly reduced. On the basis of these results, we discuss a model in which the linear growth of outer membrane protein results from a limitation of outer membrane polypeptide synthesis at the translational level, reflecting the linear expansion of the underlying peptidoglycan layer in the envelope.     

Detection of G1 proteins in Chinese hamster cells synchronized by isoleucine deprivation or mitotic selection.

Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle. Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals. Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations. Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm. Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h. A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection. This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.     

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.     

RNA synthesis in embryo axes of germinating pea seeds

RNA synthesis during germination was investigated by labelingpea embryo axes or seedling roots with radioactive uridine oradenosine. The results indicated that all RNA species of pre-rRNAs(ribosomal precursor RNAs), rRNAs, heterodisperse-type RNA and4–5S low molecular weight RNA were synthesized from the6th to 64th hour of the period examined. At the very early stageof germination, some conspicuous labeling of the heterodisperse-typeRNA was observed after pulse-labeling. There was no great differencein the labeling patterns of various RNA species with regardto other later stages. When embryo axes were labeled for 1 hrwith ³H-adenosine from the 16th hour, about 25% of the labeledwhole cell RNA was retained on the membrane filter. The ratioof labeled poly(A)-containing RNA, however, decreased as germinationproceeded. The poly (A)-containing RNA sedimented heterodisperselywith a mean value of about 20S in a sucrose density gradient;this size-distribution did not vary throughout germination. (Received January 16, 1979; )     

Does protein secretion activity vary during the cell cycle of Escherichia coli?

It is not known whether the activity of the Escherichia coli protein export system changes during the division cycle. To address and answer this question, we took two approaches. First, we pulse-labeled a random culture and size-fractionated the labeled cells in the presence of inhibitors of secretion. Second, we pulse-labeled synchronously growing cells at different phases in the cell cycle. In the latter experiment, we used a new method of synchronization in which a random culture was simply filtered through glass fiber filters. In both cases, the proportions of unprocessed precursor molecules were measured by immunoprecipitation and gel electrophoresis for some representative periplasmic and outer membrane proteins. Within the sensitivity limits of these methods, we could not detect any significant variation in the precursor labeling for cells of different ages. Thus, E. coli cells appear to secrete proteins continuously throughout the division cycle.     

Entry of Vesicular Stomatitis Virus into L Cells

Early stages of the entry of vesicular stomatitis (VS) virus into L cells were followed by electron microscopy with the aid of ferritin antibody labeling. Cells which were infected at 0 C and incubated for 10 min at 37 C were reacted first with antiviral-antiferritin hybrid antibody and then with ferritin or fluorescein-labeled apoferritin. Extensive ferritin labeling of the cell surface was detected by both electron and fluorescence microscopy. The labeled regions of the cell surface were continuous with and indistinguishable from the rest of the host cell membrane, suggesting incorporation of viral antigens into the cell surface during viral penetration. Fusion of parental viral membrane with host cell membrane was further demonstrated by examining the localization of (3)H-labeled viral structural proteins in cells infected at 0 C and incubated for short periods at 37 C. Viral nucleoprotein was found in a soluble fraction of the cells which was derived primarily from the cytoplasm, whereas a particulate fraction from the cells was enriched in viral envelope proteins. Cytoplasmic membrane was isolated from these cells, and this membrane contained viral envelope proteins. These results suggest that penetration by VS virus occurs by fusion of the viral and cellular membranes followed by release of nucleo-protein into the cytoplasm.