Changes in the organization of the genome during the mammalian cell cycle

By using chromosome conformation capture technology, a recent study has revealed two alternative three-dimensional folding states of the human genome during the cell cycle.     

Changes in protein phosphorylation during the cell cycle of Chinese hamster ovary cells

The phosphorylation patterns of proteins were examined during the cell cycle of Chinese hamster ovary cells. This was accomplished by labeling synchronized cells at various times with [32P]orthophosphate and separating the proteins by both isoelectric focusing and nonequilibrium pH gradient two-dimensional gel electrophoresis. The most dramatic changes occurred during late G2/M when approximately eight proteins (including vimentin, lamin B, and histones 1 and 3) showed increased phosphorylation. Ten other proteins appeared to be uniquely phosphorylated during late G2/M. Of these 10 proteins, seven were no longer phosphorylated shortly after mitosis. There is also at least one protein which showed a relative decrease in phosphorylation during late G2/M.     

Biphasic activation of two mitogen-activated protein kinases during the cell cycle in mammalian cells.

We studied mitogen-activated protein kinase (MAPK) activities during the cell cycle of Chinese hamster ovary (CHO) cells using site-specific antibodies against extracellular signal-regulated kinase-1, a 44-kDa MAPK (Boulton, T.G., Yancopoulos, G.D., Gregory, J.S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M.H. (1990) Science 249, 64-67). These antibodies detected two distinct MAPKs (44- and 42-kDa MAPKs) in CHO cells. CHO cells were arrested at metaphase in the M phase by treatment with nocodazole, and activities of MAPKs were analyzed at specific time points after release from arrest. Immune complex kinase assay and renaturation and phosphorylation assay in substrate-containing gel revealed that both 44- and 42-kDa MAPKs had activities in the G1 through S and G2/M phases and were activated biphasically, in the G1 phase and around the M phase. MAPKs were inactivated in metaphase-arrested cells. The amount of MAPKs did not change significantly in the cell cycle. In the G1, S, and G2/M phases, MAPKs were phosphorylated on both tyrosine and threonine residues and dephosphorylated in metaphase-arrested cells. Our data suggest that MAPKs may play some role in the cell cycle other than G0/G1 transition.     

Changes in epididymal protein synthesis during the sexual cycle of the lizard, Lacerta vivipara

During its annual cycle, the lizard epididymis undergoes strong modifications of the secretory epithelium. These modifications previously were classified into 10 stages. The present study gives the biochemical basis of these modifications. Several parameters, such as the quantity of soluble proteins, rates of protein synthesis, and electrophoretic profiles of newly synthesized proteins and of in vitro RNA translation products were compared at 8 stages. Two-dimensional gel electrophoresis of newly synthesized tissue proteins showed that the synthesis of about 20 proteins fluctuated during the cycle. Furthermore, it revealed that the protein band L of molecular weight 19,000 identified in one-dimensional (1-D) electrophoresis was composed of at least 10 proteins. Their rate of synthesis paralleled the concentrations of their mRNA evaluated with in vitro translation. This could indicate that in this system protein synthesis is regulated by mRNA concentrations. The present analysis has confirmed that 4 different phases characterize the annual evolution of the lizard epididymis: regeneration, onset of secretory activity, hypersecretion and involution. Well-defined, newly synthesized proteins would characterize some of these phases, and could be used as markers for future detailed analysis of epididymis control.     

Changes in the rate of synthesis of wall polysaccharides during the cell cycle of yeast

Reevaluation and comparison of seemingly contradictory literature data on the mode of synthesis of wall polysaccharides during the cell cycle ofSaccharomyces cerevisiae explained the source of discrepancies and demonstrated their general consonance in the following points: 1. The rate of synthesis of glucan and mannan is not constant and does not increase continuously throughout the entire cell cycle. 2. The rate of synthesis of both polysaccharides is considerably reduced at the time of cell division and in the prebudding phase.     

HeLa cell plasma membranes. Changes in membrane protein composition during the cell cycle.

HeLa cells grown in suspension culture were synchronized by amethopterin block and thymidine reversal. In some cases an additional Colcemid block was used to obtain mitotic cells. From the various phases of the cell cycle, cells were harvested and the plasma membranes isolated. The membrane proteins were solubilized in sodium dodecyl sulphate and separated by gel electrophoresis in the presence of sodium dodecyl sarcosinate. About 35 protein bands, five of which were stained with periodic acid-Schiff reagent, appeared. Most of the bands were identical in all membrane preparations, but a few minor bands seemed to be associated with limited periods of the cell cycle. In particular, the cells in mitosis apparently contained plasma membrane proteins which did not occur in other phases. Amino acid analyses of the plasma membranes revealed no significant cell cycle-dependent changes in the amino acid composition.     

DNA replication in mammalian cells. Altered patterns of initiation during inhibition of protein synthesis

The effects of inhibition of protein synthesis by the antibiotics cycloheximide and puromycin on the initiation of DNA replication in mouse L cells were studied. Cellular DNA was pulse labeled with [3H]thymidine of high, then of low specific activity and prepared for fiber autoradiography. Autoradiograms containing multiple (up to four) replication units were analyzed. In control cells, the proportion of replication units that initiated during a 10-min, high specific activity pulse was approximately equal to the proportion initiating immediately before the pulse. The addition of cycloheximide or puromycin at the start of the pulse inhibited the frequency of initiation in that there was a decrease by up to one-third of units initiating during the pulse relative to controls. Replication direction was also altered. Addition of the antibiotics 2 h before the pulse reduced the proportion of bidirectional units observed from 0.98 to 0.70. Antibiotic treatment for 2 h also decreased initiation synchrony in that the proportion of multiunit autoradiograms on which neighboring units showed similar replication patterns (indicating temporally coordinated initiation) was reduced by one-half. These observations indicate that inhibition of protein synthesis alters the normal pattern of DNA initiation.     

Changes in protein synthesis and phosphorylation patterns during bovine oocyte maturation in vitro

Sequential protein synthesis and protein phosphorylation patterns were generated by radiolabelling bovine cumulus-oocyte complexes after various periods of culture with [35S]methionine and [32P]orthophosphate respectively. The radiolabelled oocytes were assessed for their nuclear status and used individually for gel electrophoresis. Marked changes in the protein synthesis patterns were observed exclusively after germinal vesicle breakdown (GVBD), whereas oocytes which remained in the germinal vesicle stage showed a consistent protein synthesis pattern. The changes were observed after 8 and 16 h or culture, shortly after GVBD and before first polar body extrusion. From 3 h of culture, dominant phosphoprotein bands with apparent molecular weights of 24,000 and two between 50,000 and 60,000 were observed. The latter bands displayed slight molecular weight changes, which were not closely time related. After GVBD, the phosphoprotein band with Mr 19,000 was no longer observed. This study demonstrates that specific changes in protein synthesis and protein phosphorylation are programmed during bovine oocyte maturation.     

Changes in membrane potential during the cell cycle

The membrane potential of isolated synchronized Chinese hamster lung cells (V79) has been determined as a function of their position in the cell cycle. During G 1 the cells exhibit a low but increasing membrane potential which rises sharply at the onset of the S phase. The elevated membrane potential is maintained throughout S and G 2 and declines again when the cells enter mitosis. Membrane potentials in an unsynchronized culture, which was recorded from both mitotic and interphase cells physically associated in groups and clusters, were similar to the plateau level obtained during S and G 2 in isolated synchronized cells, and exhibited little variation. It is concluded that although the membrane potential of isolated cells fluctuates during the cell cycle, it plays no causal role as a regulator of mitotic activity.     

The relationship between levels and rates of synthesis of polyamines during mammalian cell cycle

The objective of this study was to examine the rate of synthesis and the intracellular levels of polyamines as a function of the HeLa cell cycle. The intracellular levels of ornithine, which were high during mitosis and early G1 phase, decreased rapidly during late G1 phase when the ornithine decarboxylase activity was at its peak. The activities of ornithine decarboxylase and S-adenosyl methionine decarboxylase reached a peak during G1 and decreased rapidly during the S phase. The levels of polyamines were maximum in mitosis and S phase. In constrast, the rate of polyamine synthesis during S phase was 5–10 fold lower than that in mitosis or G1 phase. We have also observed fluctuations in diamine-oxidase activity during the cell cycle. The enzyme activity was high during mitosis and late G1 and low during S phase. Thus, the results of this study suggest an important role for the catabolic enzymes in the regulation of polyamine levels during the mammalian cell cycle.     

Changes in protein synthesis during myogenesis in a clonal cell line

Methods of quantitative two-dimensional gel electrophoresis have been used to study the changes in protein synthesis that occur during myogenic differentiation in the L6 clonal line of rat skeletal muscle cells. Pure populations of myoblasts were obtained by maintaining the cells at subconfluent densities, and virtually pure populations of fused myotubes have been obtained by sedimentation at 1 × gravity through a serum gradient. The gel analysis reveals major qualitative differences between myoblasts and myotubes, as well as numerous quantitative changes. Both the α and the β forms of tropomyosin and the LC2 myosin light chain were increased in rate of synthesis by at least 1000-fold during myogenesis. Other proteins were detectable in myoblasts but were not synthesized at a detectable rate in myotubes. One of these is a form of tropomyosin which comigrates under several electrophoretic conditions with smooth muscle tropomyosin. Another protein, which is repressed in rate of synthesis by at least 1000-fold during myogenesis, appears to be a major form of collagen. Computer analysis has been used to analyze in detail a particular region containing about 300 spots from the two-dimensional patterns representing protein synthesis in L6 myoblasts, L6 myotubes, and a rat nerve cell line. Quantiative comparisons have shown that, with respect to this set of proteins, the L6 myoblasts and myotubes are no more alike at the level of protein synthesis than are L6 myoblasts and the cells of the nerve line. Therefore, these studies show that L6 differentiation involves not only the qualitative switching on and off of major gene products but also the quantitative alteration of synthetic rates of many of the common proteins.     

Patterns of protein synthesis during the cell cycle of Chinese hamster ovary cells

The protein synthesis patterns at various stages of the cell cycle of Chinese hamster ovary cells were examined by labelling cells with [35S]methionine and then separating the proteins by isoelectric focussing and two-dimensional, nonequilibrium pH gradient gel electrophoresis. We have observed a number of proteins which display quantitative differences in synthesis at specific cell cycle stages and of these the alpha- and beta-tubulins have been identified. A few proteins appear to be uniquely synthesized at specific times during the cell cycle. These include the histones and a modified version of them, which are synthesized only in S phase, and a pair of 21 kilodalton (kDa), pI 5.5 proteins, which appear only in late G2 and mitosis. We have also identified a 58-kDa, pI 7.5 protein which is present at all cell cycle stages except during late G2. This protein appears to have the same temporal properties as a 57-kDa protein called "cyclin" originally described in sea urchin embryos.     

Changes in phosphorylation of an F-actin capping protein of Physarum polycephalum during the cell cycle

The Cap 42(b), a Ca2+-dependent F-actin capping phosphoprotein of 42,000 daltons, was shown to be localized in the cytosol of Physarum polycephalum by measurements of phosphorylatability in the absence of Ca2+. The phosphorylation of Cap 42(b) in the cytosol changed during the cell cycle: it was high in the S and G2 phase, and low in the M phase and boundary phase between S and G2 phase. When the isolated Cap 42(b) was added to M phase cytosol, the phosphorylation of Cap 42(b) was significantly increased by at least 6-fold. Compared with this result, about 2-fold increase in the phosphorylation of Cap 42(b) was observed when the Cap 42(b) kinase was added to M phase cytosol. Therefore, it is likely that the low level of Cap 42(b) phosphorylation in M phase cytosol is mostly due to the decreased amount of phosphorylatable Cap 42(b) and to a lesser extent due to a low level of the Cap 42(b) kinase activity.     

Phosphorylation of ribosome-associated proteins during the mammalian cell cycle. Unique phosphorylation of a specific protein during mitosis

Chinese hamster ovary cells in monolayer culture were incubated with [^{3 2}P] phosphate. Ribosome-associated proteins, including both structural proteins and those tightly bound to washed, centrifuged ribosomes, were isolated and separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The electrophoretic pattern showed five major regions or peaks of ^{3 2}P radioactivity which represented phosphorylated ribosome-associated proteins with molecular weights of 17 500, 23 000, 30 000, 38 000, and 57 000. When asynchronous cells were pulse-labeled with [^{3 2}P] phosphate, the predominant peak of ^{3 2}P radioactivity was associated with the protein of 38 000 daltons. Similar results were obtained with cells synchronized in the G₁, S, or G₂ phase of the mammalian cell cycle. Conversely, proteins isolated from the ribosomes of mitotic cells, collected and labeled with [^{3 2}P] phosphate in the presence of colcemid, showed a new and predominant peak of ^{3 2}P radioactivity migrating with a protein of 45 000 daltons. When cells labeled in mitosis were allowed to progress into G₁ phase, this peak of ^{3 2}P radioactivity rapidly disappeared from the electrophoretic pattern. These results suggest that a specific protein associated with the ribosomes was phosphorylated uniquely during the mitotic phase of the cell cycle.     

Changes in L-ornithine decarboxylase activity during the cell cycle

The activity of L-ornithine decarboxylase (L-ornithine carboxy-lyase; EC 4.1.1.17), the enzyme that catalyzes the initial and rate-limiting step in polyamine biosynthesis, has been studied in Chinese hamster ovary fibroblasts synchronized by selective detachment of mitotic cells. At various times after plating the distribution of cells among the G1, S and G2+M phases of the cell cycle was calculated from DNA distributions obtained by high-speed flow cytometric analysis. At these same times determination of the cellular L-ornithine decarboxylase activity showed that polyamine (putrescine) synthesis was initiated in mid-G1, that the rate of synthesis was maximal prior to DNA synthesis, and that it decreased during the S phase. A second increase in enzyme activity occurred before mitosis.     

The cell cycle dependence of protein synthesis during Xenopus laevis development.

The regulation of early embryonic development in the amphibian Xenopus laevis depends largely upon translational and post-translational regulatory mechanisms to direct the complex cytodifferentiations that take place during early cleavage and blastula formation. The cell cycle dependence of protein synthesis was examined in developing Xenopus embryos as well as in cycling cell-free lysates from Xenopus eggs. In both cases M-phase and the activation of the M-phase kinase were found to be correlated with an inhibition of translation. Translation in both the rough endoplasmic reticulum and cytosolic-free ribosomes were affected by this inhibition. Since elongation was found to be unaffected by M-phase, shifts in the polysome profiles during M-phase indicated that the inhibition affected initiation processes. The activity of the M-phase kinase may inhibit initiation through the modification of initiation factors or some other component during this process. The cell cycle dependence of translation may affect developmental mechanisms controlled by the titration of regulatory proteins.