Synthesis of soluble protein during the cell cycle of the fission yeast Schizosaccharomyces pombe

Many enzymes show a pattern of increase in activity through the cell cycle which is different from the continuous exponential pattern of total protein synthesis. A group of proteins at an intermediate level between single enzymes and total protein, the soluble proteins, was examined to resolve this anomaly. The synthesis of the pH 8.1 soluble proteins of Schizosaccharomyces pombe through the cell cycle was followed by pulse labelling with ³H-leucine in synchronous cultures. The soluble proteins were analysed by electrophoresis on acrylamide gels. Soluble proteins represent 30% of the total proteins of S. pombe and the rates of synthesis showed a continuous increase through the cell cycle. Individual groups of proteins, represented by a single band after electrophoresis, showed a similar continuous increase in synthesis through the cell cycle. Any proteins which may be synthesised discontinuously, such as some enzymes, represent such a small proportion of any one protein group in the electrophoretic separation that their effect was not detectable. These results are different from those described for mammalian cells.     

Phase-specific protein synthesis in human fibroblasts after synchronization by a double thymidine block

Kinetics of acid-insoluble non-histone protein synthesis during S and G2 cell cycle phases of diploid human fibroblasts and heteroploid transformed cells was investigated. Two distinct groups of protein with different kinetic pattern depending on the cell culture type were revealed. Export of one group of protein and turnover of the other group of acid-insoluble non-histone protein is arrested in heteroploid transformed cells.     

Regulation of cell cycle events in asymmetrically dividing cells: functions required for DNA initiation and chain elongation in Caulobacter crescentus

To study the regulation of cell cycle events after asymmetric cell division in Caulobacter crescentus, we have identified functions that are required for DNA synthesis in the stalked cell produced at division and in the new stalked cell that develops from the swarmer cell 60 min after division. The initiation of DNA synthesis in the two progeny cells is dependent upon at least two common functions. One of these is a requirement for protein synthesis and the other is a gene product identified in a temperature-sensitive cell cycle mutant. DNA chain elongation requires a third common function. The characteristic pattern of DNA synthesis in C. crescentus appears to be controlled in part by the expression of these functions in the two stalked cells at different times after cell division. The age distribution for Caulobacter cells in an exponential population has been calculated (Appendix by Robert Tax) and used to analyze some of the results.     

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.     

Synthesis of glycoprotein, glycolipid, protein, and lipid in synchronized L5178Y cells

Synthesis of four macromolecular classes found in membranes—glycoprotein, glycolipid, protein, and lipid—was measured as a function of time of the cell cycle in synchronized L5178Y cells. Incorporation of leucine, choline, fucose, glucosamine, or thymidine into the cells, protein, nucleic acid, or lipid was measured by pulse-labeling for ½ hr at ½ hr intervals after release from the mitotic block. The amount of protein, lipid, glycoprotein, or glycolipid released or secreted into the medium by the L5178Y cells was also measured as a function of time of the cell cycle. Cellular protein was found to be synthesized throughout the cell cycle, with the highest synthesis occurring in the S period; synthesis was depressed in the M period. Cellular glycoprotein was synthesized at approximately the same times as protein, except that the rates of glycoprotein synthesis in the S period relative to other periods were much greater than for protein. Secreted protein was synthesized throughout the cell cycle without any general pattern, except that secretion was elevated in the late S and G₂ periods. Secreted glycoprotein was similar to secreted protein. Cellular lipid and cellular glycolipid were synthesized almost exclusively in the G₂ and M periods; there was no synthesis in the G₁ and S periods. Release or secretion of glycolipid and lipid also occurred in the G₂ and M periods.     

The role of protein accumulation in the cell cycle control of human NHIK 3025 cells

The cell cycle kinetics of NHIK 3025 cells, synchronized by mitotic selection, was studied in the presence of cycloheximide at concentrations (0.125-1.25 μM) which inhibited protein synthesis partially and slowed down the rate of cell cycle traverse. The median cell cycle duration was equal to the protein doubling time in both the control cells and in the cycloheximide-treated cultures at all drug concentrations. This conclusion was valid whether protein synthesis was continuously depressed by cycloheximide throughout the entire cell cycle, or temporarily inhibited during shorter periods at various stages of the cell cycle. These results may indicate that cell division does not take place before the cell has reached a critical size, or has completed a protein accumulation-dependent sequence of events. When present throughout the cell cycle, cycloheximide increased the median G1 duration proportionally to the total cell cycle prolongation. However, the entry of cells into S, once initiated, proceeded at an almost unaffected rate even at cycloheximide concentrations which reduced the rate of protein synthesis 50%. The onset of DNA synthesis seemed to take place in the cycloheximide-treated cells at a time when the protein content was lower than in the control cells. This might suggest that DNA synthesis in NHIK 3025 cells is not initiated at a critical cell mass.     

Periodic fluctuations of nuclear high mobility group like proteins during the cell cycle of Physarum polycephalum

High mobility group like (HMG-like) nuclear proteins were isolated from plasmodia of the lower eucaryote Physarum polycephalum and characterized by different types of polyacrylamide gel electrophoresis. The synthesis of these proteins was measured during the naturally synchronous cell cycle of Physarum. The four HMG-like proteins (AS1-4) exhibit a pronounced cell cycle dependent pattern of synthesis: AS1 and AS4 have a clear maximum of synthesis in mid S phase with a basal synthesis during the entire G2 period. In contrast, AS2 and AS3 have little synthesis in S phase but a broad maximum in mid G2 period. The four HMG-like proteins have a very low synthesis in early S phase and late G2 period. In addition, other non-histone proteins, which are coextracted with the HMG proteins, exhibit distinct periodic synthesis patterns. A novel non-histone protein, which is the most abundant protein species in 0.35 M NaCl extracts, was detected. It exhibits a high rate of synthesis around the time of mitosis. In general, the results indicate that, in contrast to the main cytoplasmic proteins, most nuclear proteins are phase-specific with respect to their synthesis in the cell cycle.     

Correlation of the rhythms of protein synthesis and secretion in a hepatocyte monolayer culture

In primary monolayer culture of hepatocytes, circahoralian rhythms of protein synthesis and secretion were discovered. In one of the hepatocyte populations the rhythmic cycle of protein secretion was found to be about twice as long as that of protein synthesis. Combination of different experimental variants such as the impulse and the long-term labelling of proteins or inhibition of protein synthesis, revealed that the secretion rhythms were predetermined by the secretion of both newly formed proteins and those stored in the cell for a long time. Upon the inhibition of protein synthesis with cyclohexeimide, the protein secretion in the monolayer changes its rhythmic pattern for a constant rate secretion. Possible causes of the alteration of circahoralian variations of protein synthesis are discussed.     

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.     

Generation of asymmetry during development. Segregation of type-specific proteins in Caulobacter.

An essential event in developmental processes is the introduction of asymmetry into an otherwise undifferentiated cell population. Cell division in Caulobacter is asymmetric; the progeny cells are structurally different and follow different sequences of development, thus providing a useful model system for the study of differentiation. Because the progeny cells are different from one another, there must be a segregation of morphogenetic and informational components at some time in the cell cycle. We have examined the pattern of specific protein segregation between Caulobacter stalked and swarmer daughter cells, with the rationale that such a progeny analysis would identify both structurally and developmentally important proteins. To complement the study, we have also examined the pattern of protein synthesis during synchronous growth and in various cellular fractions. We show here, for the first time, that the association of proteins with a specific cell type may result not only from their periodicity of synthesis, but also from their pattern of distribution at the time of cell division. Several membrane-associated and soluble proteins are segregated asymmetrically between progeny stalked and swarmer cells. The data further show that a subclass of soluble proteins becomes associated with the membrane of the progeny stalked cells. Therefore, although the principal differentiated cell types possess different synthetic capabilities and characteristic proteins, the asymmetry between progeny stalked and swarmer cells is generated primarily by the preferential association of specific soluble proteins with the membrane of only one daughter cell. The majority of the proteins which exhibit this segregation behavior are synthesized during the entire cell cycle and exhibit relatively long, functional messenger RNA half-lives.     

Control of tubulin and actin gene expression in Tetrahymena pyriformis during the cell cycle

Poly(A)-containing mRNA was isolated from division synchronized populations of the ciliated protozoan, Tetrahymena pyriformis. The level of tubulin and actin mRNA at specific cell cycle stages was analyzed by hybridization to tubulin and actin cDNA probes and by gel analysis of their in vitro translation products. The pattern of fluctuation of tubulin mRNA levels was similar to that observed for the in vivo tubulin synthesis previously reported [1]. This suggests that as the cells progress through the cell cycle, tubulin synthesis is controlled at the mRNA level. There was little fluctuation of actin synthesis or actin mRNA levels during the cell cycle, which may be indicative of a different regulatory mechanism for actin than for tubulin.     

Thymidine as synchronizing agent. 3. Persistence of cell cycle patterns of phosphatase activities and elevation of nuclease activity during inhibition of DNA synthesis

Synchronous cultures of HeLa cells were obtained by selective detachment of cells in mitosis and fluctuations in enzyme activity were followed during the subsequent cell cycle. The enzymes measured were alkaline and acid phosphatases and a nuclease active on denatured DNA at alkaline pH (alkaline DNase). Each of these enzymes showed a different pattern of activity in the cell cycle, but a temporal relationship to the DNA synthetic phase was apparent in each case. Treatment of the cultures at the beginning of the cell cycle with 15 mM thymidine did not alter the subsequent pattern of fluctuations in activity of alkaline phosphatase or of acid phosphatase, although DNA synthesis was fully inhibited by this treatment. This indicates that the pattern of activity of some enzymes is not linked to DNA replication. On the other hand, the pattern of fluctuations in the activity of alkaline DNase was abolished by thymidine treatment, and elevation of the activity of this enzyme was observed. These results suggest complex and variable relationships between phases of the cell cycle and enzyme activity, and show that inhibition of DNA synthesis is not a suitable procedure for induction of culture synchrony if enzyme activities are to be studied.     

Cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase/uracil DNA glycosylase gene in normal human cells.

The cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/uracil DNA glycosylase (UDG) gene was examined in normal human cells. Steady state RNA levels were monitored by Northern blot analysis using a plasmid (pChug 20.1) which contained the 1.3 kb GAPDH/UDG cDNA. The biosynthesis of the 37 kDa GAPDH/UDG protein was determined using an anti-human placental GAPDH/UDG monoclonal antibody to immunoprecipitate the radiolabeled protein. Increases in steady state GAPDH/UDG mRNA levels were cell cycle specific. A biphasic pattern was observed resulting in a 19-fold increase in the amount of GAPDH/UDG mRNA. The biosynthesis of the 37 kDa GAPDH/UDG protein displayed a similar biphasic regulation with a 7-fold increase. Pulse-chase experiments revealed a remarkably short half life of less than 1 hr. for the newly synthesized 37 kDa protein, comparable to that previously documented for a number of oncogenes. GAPDH/UDG mRNA levels were markedly reduced at 24 hr. when DNA synthesis was maximal. These results define the GAPDH/UDG gene as cell cycle regulated with a characteristic temporal sequence of expression in relation to DNA synthesis. The cell cycle synthesis of a labile 37 kDa monomer suggests a possible regulatory function for this multidimensional protein. Further, modulation of the GAPDH/UDG gene in the cell cycle may preclude its use as a reporter gene when the proliferative state of the cell is not kept constant.     

Regulation of proliferating cell nuclear antigen during the cell cycle

The proliferating cell nuclear antigen (PCNA), also known as cyclin and DNA polymerase delta auxiliary factor, is present in reduced amounts in nongrowing cells and is synthesized at a greater rate in the S phase of growing cells. The recently discovered involvement of PCNA in DNA replication suggested that this pattern of expression functions to regulate DNA synthesis. We have investigated this possibility further by examining the synthesis, stability, and accumulation of PCNA in HeLa cells fractionated by centrifugal elutriation into nearly synchronous populations of cells at various positions in the cell cycle. In these fractionated cells we found that there is an increase in the rate of PCNA synthesis with a peak in early S phase of the cell cycle, but the magnitude of the increase is only 2-3-fold. This change reflects similar changes in the amount of PCNA mRNA. The fluctuating synthesis of PCNA maintains this protein at a roughly constant proportion of the total cell protein, although the amount doubles/cell in the cell cycle. Consistent with this observation, the stability of PCNA does not differ significantly from that of total cellular protein in synchronized HeLa cells. We also observed that a maximum of one-third of the total PCNA is tightly associated with the nucleus, presumably in replication complexes, at the peak of S phase. We conclude that the cyclic synthesis of PCNA in cycling HeLa cells maintains PCNA in excess of the amount involved directly in DNA replication and the amount of the protein neither fluctuates significantly with the cell cycle nor is limiting for DNA synthesis.     

A set of proteins showing cell cycle dependent modification in the early mouse embryo

The pattern of protein synthesis in the mouse egg shows several changes at fertilization and during first mitosis. Three groups of newly synthesized proteins, with molecular weights of about 30,000, 35,000, and 46,000, show variations in mobility on one- and two-dimensional gels that correlate with the cell cycle. Each group is composed of a polypeptide that is synthesized in unmodified form during interphase but is modified reversibly during meiosis or mitosis, by a process involving phosphorylation. Although these proteins cease to be synthesized during the second cell cycle, those made earlier persist and continue to show the same modifications during the next cell cycle. Like other eggs, fertilized mouse eggs show a requirement for protein synthesis in order to enter mitosis.     

Synthesis and secretion of albumin by a synchronized rat hepatoma cell line.

The rat hepatoma cell H4-12 which synthesizes and secretes albumin was synchronized by growth in isoleucine-deficient medium followed by a second block with excess thymidine. Albumin synthesis and secretion was measured in the synchronized cells at different time intervals representative of early S, late S, G2, mitosis, early G1 and late G1 phases of the cell cycle. Maximal albumin synthesis occurred during G1 although significant synthesis also occurred during the other cell cyle phases. Most (75--80%) of the radioactive albumin produced during a 15 min pulse incubation with L-[4,5-3H] leucine was found in the microsomal cell fraction and this nascent albumin was secreted into the incubation medium during a 160 min chase period. Fifty percent of the nascent albumin was secreted by 50--55 min and this pattern of secretion did not change during the cell cycle. These data indicate that albumin synthesis occurs throughout the cell cycle but that it is preferred during G1. The rate of intracellular transport and secretion of albumin does not vary during the different phase of the cell cycle.     

Self-cycling operation increases productivity of recombinant protein in Escherichia coli

Self-cycling fermentation (SCF), a cyclical, semi-continuous process that induces cell synchrony, was incorporated into a recombinant protein production scheme. Escherichia coli CY15050, a lac(-) mutant lysogenized with temperature-sensitive phage λ modified to over-express β-galactosidase, was used as a model system. The production scheme was divided into two de-coupled stages. The host cells were cultured under SCF operation in the first stage before being brought to a second stage where protein production was induced. In the first stage, the host strain demonstrated a stable cycling pattern immediately following the first cycle. This reproducible pattern was maintained over the course of the experiments and a significant degree of cell synchrony was obtained. By growing cells using SCF, productivity increased 50% and production time decreased by 40% compared to a batch culture under similar conditions. In addition, synchronized cultures induced from the end of a SCF cycle displayed shorter lysis times and a more complete culture-wide lysis than unsynchronized cultures. Finally, protein synthesis was influenced by the time at which the lytic phase was induced in the cell life cycle. For example, induction of a synchronized culture immediately prior to cell division resulted in the maximum protein productivity, suggesting protein production can be optimized with respect to the cell life cycle using SCF.     

Topography of the insertion of LamB protein into the outer membrane of Escherichia coli wild-type and lac-lamB cells

The appearance of newly induced LamB protein at the cell surface of Escherichia coli was followed topographically by immuno-electron microscopy. LamB protein was induced in E. coli wild-type or lac-lamB cells for a short period of time (4 to 6 min), such that the overall level of LamB protein in induced cells was at least twofold higher than that in uninduced cells. Antibodies bound to LamB protein exposed at the cell surface were labeled with a protein A-gold probe, and the probe distribution in briefly induced cells was compared to that in uninduced cells. Analysis of large numbers of cells showed that newly inserted LamB protein appeared homogeneously over the entire cell surface, both in wild-type cells and in lac-lamB cells. A peak of insertion which was observed at the division site of the cell was also observed in the absence of induction and in control experiments in which a nonspecific probe was used. It is concluded therefore that insertion of LamB protein into the cell envelope of E. coli occurs at multiple sites over the entire cell surface. The average amount of LamB protein which appeared at the cell surface after induction was determined for various cell size classes. It was found that cells of various size classes all synthesized LamB protein after induction, indicating that synthesis of the protein was not restricted to cells in a particular stage of the cell cycle. However, the rate of LamB synthesis was found to vary during the cell cycle: this rate was constant regardless of cell size in nondividing cells, whereas it increased in dividing cells. It is concluded that the accumulation of newly induced LamB protein follows a linear pattern.