scholarly journals RIBONUCLEIC ACID AND PROTEIN SYNTHESIS IN MITOTIC HELA CELLS

1965 ◽  
Vol 27 (3) ◽  
pp. 565-574 ◽  
Author(s):  
Terry C. Johnson ◽  
John J. Holland
Keyword(s):  
Protein Synthesis ◽  
Rna Polymerase ◽  
Hela Cells ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Mitotic Cell ◽  
Interphase Cell ◽  
Cell Extracts ◽  
Interphase Cells ◽  
Mitotic Cells

HeLa cells arrested in mitosis were obtained in large numbers, with only very slight interphase cell contamination, by employing the agitation method of Terasima and Tolmach, and Robbins and Marcus. Protein synthesis and RNA synthesis were almost completely suppressed in mitotic cells. Active polyribosomes were nearly absent in mitotic cells as compared with interphase cells treated in the same way. Cell-free protein synthesis and RNA polymerase activity were also greatly depressed in extracts of metaphase cells. The deoxyribonucleoprotein (DNP) of condensed chromosomes from mitotic cells was less efficient as a template for Escherichia coli RNA polymerase than was DNP from interphase cells, although isolated DNA from both sources was equally active as a primer. Despite very poor endogenous amino acid incorporation by extracts of metaphase cells, polyuridylate stimulated phenylalanine incorporation by a larger factor in mitotic cell extracts than it did in interphase cell extracts. These results suggest that RNA synthesis is suppressed in mitotic cells because the condensed chromosomes cannot act as a template, and that protein synthesis is depressed at least in part because messenger RNA becomes unavailable to ribosomes. This conclusion was supported by the demonstration that cells arrested in metaphase supported multiplication of normal yields of poliovirus, thereby showing that the mitotic cell is capable of considerable synthesis of RNA and protein.

scholarly journals Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells.

1992 ◽  
Vol 119 (6) ◽  
pp. 1559-1572 ◽  
Author(s):  
V M Fowler ◽  
E J Adam
Keyword(s):  
Cell Body ◽  
Hela Cells ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Beta Subunit ◽  
Actin Binding ◽  
Culture Dish ◽  
Cell Rounding ◽  
Interphase Cells ◽  
Mitotic Cells

Dramatic changes in morphology and extensive reorganization of membrane-associated actin filaments take place during mitosis in cultured cells, including rounding up; appearance of numerous actin filament-containing microvilli and filopodia on the cell surface; and disassembly of intercellular and cell-substratum adhesions. We have examined the distribution and solubility of the membrane-associated actin-binding protein, spectrin, during interphase and mitosis in cultured CHO and HeLa cells. Immunofluorescence staining of substrate-attached, well-spread interphase CHO cells reveals that spectrin is predominantly associated with both the dorsal and ventral plasma membranes and is also concentrated at the lateral margins of cells at regions of cell-cell contacts. In mitotic cells, staining for spectrin is predominantly in the cytoplasm with only faint staining at the plasma membrane on the cell body, and no discernible staining on the membranes of the microvilli and filopodia (retraction fibers) which protrude from the cell body. Biochemical analysis of spectrin solubility in Triton X-100 extracts indicates that only 10-15% of the spectrin is soluble in interphase CHO or HeLa cells growing attached to tissue culture plastic. In contrast, 60% of the spectrin is soluble in mitotic CHO and HeLa cells isolated by mechanical "shake-off" from nocodazole-arrested synchronized cultures, which represents a four- to sixfold increase in the proportion of soluble spectrin. This increase in soluble spectrin may be partly due to cell rounding and detachment during mitosis, since the amount of soluble spectrin in CHO or HeLa interphase cells detached from the culture dish by trypsin-EDTA or by growth in spinner culture is 30-38%. Furthermore, mitotic cells isolated from synchronized spinner cultures of HeLa S3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25%). The beta subunit of spectrin is phosphorylated exclusively on serine residues both in interphase and mitosis. Comparison of steady-state phosphorylation levels of spectrin in mitotic and interphase cells demonstrates that solubilization of spectrin in mitosis is correlated with a modest increase in the level of phosphorylation of the spectrin beta subunit in CHO and HeLa cells (a 40% and 70% increase, respectively). Two-dimensional phosphopeptide mapping of CHO cell spectrin indicates that this is due to mitosis-specific phosphorylation of beta-spectrin at several new sites. This is independent of cell rounding and dissociation from other cells and the substratum, since no changes in spectrin phosphorylation take place when cells are detached from culture dishes with trypsin-EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Transcription of bacteriophage T4 deoxyribonucleic acid in vitro

1969 ◽  
Vol 115 (3) ◽  
pp. 353-361 ◽  
Author(s):  
John O. Bishop ◽  
Forbes W. Robertson
Keyword(s):  
Rna Polymerase ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Bacteriophage T4 ◽  
Rna Sequences ◽  
Experimental Conditions ◽  
E Coli ◽  
New Methods

1. RNA was synthesized in vitro from a template of bacteriophage T4 DNA, in the presence of Mn2+. A comparison was made of the RNA synthesized by purified RNA polymerase from two sources, Micrococcus lysodeikticus and Escherichia coli; these are referred to as Micrococcus cRNA and E. coli cRNA respectively (where cRNA indicates RNA synthesized in vitro by using purified RNA polymerase and a DNA primer). 2. Both types of RNA were self-complementary as judged by resistance to digestion with ribonuclease after self-annealing, Micrococcus cRNA being more self-complementary (40%) than was E. coli cRNA (30%). The cRNA was found to be much less self-complementary if Mg2+ was present during RNA synthesis instead of Mn2+. 3. Micrococcus cRNA hybridized with a larger part of bacteriophage T4 DNA than did E. coli cRNA. The E. coli cRNA competed with only part (70%) of the Micrococcus cRNA in hybridization-competition experiments. It is concluded that more sequences of bacteriophage T4 DNA are transcribed by Micrococcus polymerase than by E. coli polymerase. 4. The RNA sequences synthesized by Micrococcus RNA polymerase but not by E. coli RNA polymerase are shown by hybridization competition to compete with specifically late bacteriophage T4 messenger RNA sequences. The relevance of this finding to the control of transcription is discussed. 5. In an Appendix, new methods are described for the analysis of hybridization-saturation and -competition experiments. Particular attention is paid to the effects produced if different RNA sequences are present at different relative concentrations. 6. By using cRNA isolated from an enzymically synthesized DNA–RNA hybrid, it is estimated that, of the DNA that is complementary to cRNA, only about half can become hybridized with cRNA under the experimental conditions used.

Introduction: virus–host interactions

1980 ◽  
Vol 210 (1180) ◽  
pp. 319-320
Keyword(s):  
Protein Synthesis ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Genetic Material ◽  
Acid Synthesis ◽  
Dna Viruses ◽  
Host Interactions ◽  
Polypeptide Sequence ◽  
Form Part ◽  
Reading Frames

Viruses are among the most extreme parasites, being almost completely dependent upon their host for their growth and replication. Having no intermediary metabolism of their own they make use of the energy supply of the host, its production of nucleoside triphosphates for nucleic acid synthesis and amino acid for protein synthesis, and all of the machinery for protein synthesis. Within the infected cell the virus competes with the host for the supply of all these things and at the same time variants compete among themselves for survival and yield of progeny. It is the intensity of this competition that has produced the most subtle and intimate interactions between virus and host. The need to fit into a protective shell imposes tight limits on the size of the genome in most classes of virus. This means that additional functions can seldom be added simply by adding the necessary genetic information unless there is a compensating loss. But by making more efficient use of the genetic material, additional functions can be accommodated without altering the size of the genome significantly. This is seen to a remarkable degree in the small DNA viruses, where segments of the genome are translated in different reading frames to give different polypeptide sequences and where multiple alternative'splicing in messenger RNA synthesis allows the same polypeptide sequence to form part of two or even three proteins with different properties.

Inactivation of mitotic factors by ultraviolet irradiation of HeLa cells in mitosis

1984 ◽  
Vol 65 (1) ◽  
pp. 279-295
Author(s):  
R.C. Adlakha ◽  
Y.C. Wang ◽  
D.A. Wright ◽  
C.G. Sahasrabuddhe ◽  
H. Bigo ◽  
...  
Keyword(s):  
Hela Cells ◽  
Chromatin Condensation ◽  
Germinal Vesicle ◽  
Mitotic Cell ◽  
Xenopus Laevis Oocytes ◽  
Germinal Vesicle Breakdown ◽  
Cell Extracts ◽  
High Doses ◽  
Chromosome Decondensation ◽  
The Imf

Extracts from mitotic HeLa cells, when injected into fully grown Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) indicated by germinal vesicle breakdown (GVBD) and chromosome condensation. Recently, we observed that the MPA of mitotic cell extracts is neutralized by the inhibitors of mitotic factors (IMF) in HeLa cells, which are activated at telophase and remain active throughout the G1 period. The activity of the IMF coincides with the process of chromosome decondensation, which begins at telophase and continues until the beginning of S phase, when chromatin reaches its most decondensed state. The objective of the present study was to investigate whether these two phenomena - chromosome decondensation and the activation of IMF - were related. The activity of IMF was measured in N2O-blocked mitotic HeLa cells, in which chromosome decondensation was induced by exposure to ultraviolet light, and subsequent incubation in medium containing inhibitors of DNA synthesis, hydroxyurea and arabinosylcytosine (araC). u.v. irradiation activated IMF was seen even at very high doses of X-irradiation. The IMF seemed to inactivate the mitotic factors directly by forming a complex that precipitated on heating at 60 degrees C for 15 min. Mg2+ or polyamines (i.e. spermine, spermidine, and putrescine), agents known to promote chromatin condensation partially restored the MPA of the u.v.-irradiated mitotic cell extracts. These results tend to support the conclusion that the IMF play a role in the decondensation of chromosomes.

Nuclear localization of an O-glycosylated protein phosphotyrosine phosphatase from human cells

1991 ◽  
Vol 98 (3) ◽  
pp. 303-307
Author(s):  
W. Meikrantz ◽  
D.M. Smith ◽  
M.M. Sladicka ◽  
R.A. Schlegel
Keyword(s):  
Nuclear Localization ◽  
Wheat Germ ◽  
Mitotic Cell ◽  
Histochemical Staining ◽  
Immunoaffinity Chromatography ◽  
Cell Extracts ◽  
Interphase Cells ◽  
Detergent Extraction ◽  
M Phase ◽  
Hydrolysis Of

Histochemical staining of immunoprecipitates of p65, a component of human M phase-promoting factor, identified the molecule as having phosphatase activity. The enzyme, purified 3400-fold from mitotic cell extracts by (NH4)2SO4 precipitation, DEAE chromatography, and immunoaffinity chromatography on immobilized anti-p65 IgG, was inhibited by Zn2- and Na3VO4 but not NaF or beta-glycerophosphate; 32P-labeled poly(Glu, Tyr) was more efficiently dephosphorylated than phosphorylated histone or phosphorylase a. Indirect immunofluorescence showed most of the phosphatase to be localized in the nucleus of interphase cells, with a fine, granular distribution unaltered by detergent extraction; in mitotic cells, p65 was localized on chromosomes. ELISA of subcellular fractions confirmed this localization. Immunoreactive p65 was recovered from immobilized wheat germ agglutinin (WGA) upon elution with N-acetylglucosamine; similarly, WGA recognized immunoaffinity-purified p65 on blots. Alkaline hydrolysis of blotted protein prevented WGA binding, indicating that phosphatase p65, like a small group of other nuclear proteins, contains O-linked carbohydrate terminating in N-acetylglucosamine.

Cell cycle regulation of the p34cdc2/p33cdk2-activating kinase p40MO15

1994 ◽  
Vol 107 (10) ◽  
pp. 2789-2799 ◽  
Author(s):  
R.Y. Poon ◽  
K. Yamashita ◽  
M. Howell ◽  
M.A. Ershler ◽  
A. Belyavsky ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Phosphorylation Site ◽  
Mitotic Cell ◽  
Phase Cell ◽  
Serum Starvation ◽  
Interphase Cell ◽  
Cell Extracts ◽  
M Phase ◽  
Equivalent Site ◽  
Cycle Regulation

A key component of Cdc2/Cdk2-activating kinase (CAK) is p40MO15, a protein kinase subunit that phosphorylates the T161/T160 residues of p34cdc2/p33cdk2. The level and activity of p40MO15 were essentially constant during cleavage of fertilised Xenopus eggs and in growing mouse 3T3 cells, but serum starvation of these cells reduced both the level and activity of p40MO15. Although the level and activity of endogenous p40MO15 did not vary in the cell cycle, we found that bacterially expressed p40MO15 was activated more rapidly by M-phase cell extracts than by interphase cell extracts. Bacterially expressed p40MO15 was phosphorylated mainly on serine 170 (a p34cdc2 phosphorylation site) by mitotic cell extracts, but mutation of S170 to alanine did not affect the activation of p40MO15, whereas mutation of T176 (the equivalent site to T161/T160 in p34cdc2/p33cdk2) abolished the activation of P40MO15. These studies suggest that the level and activity of p40MO15 is probably not a major determinant of p34cdc2/p33cdk2 activity in the cell cycle, and that the activation of p40MO15 may require phosphorylation on T176.

scholarly journals PROTEIN SYNTHESIS AND RNA SYNTHESIS DURING MITOSIS IN ANIMAL CELLS

1963 ◽  
Vol 19 (2) ◽  
pp. 267-277 ◽  
Author(s):  
Carol G. Konrad
Keyword(s):  
Protein Synthesis ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Animal Cells ◽  
Drastic Reduction ◽  
Human Amnion ◽  
Culture Cells ◽  
Cent Increase ◽  
Protein And Rna Synthesis ◽  
Synthetic Capacity

Protein synthesis and RNA synthesis during mitosis were studied by autoradiography on mammalian tissue culture cells. Protein synthesis was followed by incubating hamster epithelial and human amnion cells for 10 or 15 minutes with phenylalanine-C14. To study RNA synthesis the hamster cells were incubated for 10 minutes with uridine-C14. Comparisons of the synthetic capacity of the interphase and mitotic cells were then made using whole cell grain counts. The rate of RNA synthesis decreased during prophase and reached a low of 13 to 16 per cent of the average interphase rate during metaphase-anaphase. Protein synthesis in the hamster cells showed a 42 per cent increase during prophase with a subsequent return to the average interphase value during metaphase-anaphase. The human amnion cells showed no significant change at prophase but there was a 52 to 56 per cent drop in phenylalanine incorporation at metaphase-anaphase as compared to the average interphase rate. Colcemide was used on the hamster cells to study the effect of a prolonged mitotic condition on protein and RNA synthesis. Under this condition, uridine incorporation was extremely low whereas phenylalanine incorporation was still relatively high. The drastic reduction of RNA synthesis observed under mitotic conditions is believed to be due to the coiled condition of the chromosomes. The lack of a comparable reduction in protein synthesis during mitosis is interpreted as evidence for the presence in these cells of a relatively stable messenger RNA.

scholarly journals Regulation of Ca2+ influx during mitosis: Ca2+ influx and depletion of intracellular Ca2+ stores are coupled in interphase but not mitosis.

1991 ◽  
Vol 2 (11) ◽  
pp. 915-925 ◽  
Author(s):  
S F Preston ◽  
R I Sha'afi ◽  
R D Berlin
Keyword(s):  
Hela Cells ◽  
Membrane Receptors ◽  
Rapid Rise ◽  
Cell Responses ◽  
Initial Release ◽  
Interphase Cells ◽  
Strong Stimulation ◽  
Two Phases ◽  
Stimulation Of ◽  
Mitotic Cells

Activation of a wide variety of membrane receptors leads to a sustained elevation of intracellular Ca2+ ([Ca2+]i) that is pivotal to subsequent cell responses. In general, in nonexcitable cells this elevation of [Ca2+]i results from two sources: an initial release of Ca2+ from intracellular stores followed by an influx of extracellular Ca2+. These two phases, release from intracellular stores and Ca2+ influx, are generally coupled: stimulation of influx is coordinated with depletion of Ca2+ from stores, although the mechanism of coupling is unclear. We have previously shown that histamine effects a typical [Ca2+]i response in interphase HeLa cells: a rapid rise in [Ca2+]i followed by a sustained elevation, the latter dependent entirely on extracellular Ca2+. In mitotic cells only the initial elevation, derived by Ca2+ release from intracellular stores, occurs. Thus, in mitotic cells the coupling of stores to influx may be specifically broken. In this report we first provide additional evidence that histamine-stimulated Ca2+ influx is strongly inhibited in mitotic cells. We show that efflux is also strongly stimulated by histamine in interphase cells but not in mitotics. It is possible, thus, that in mitotics intracellular stores are only very briefly depleted of Ca2+, being replenished by reuptake of Ca2+ that is retained within the cell. To ensure the depletion of Ca2+ stores in mitotic cells, we employed the sesquiterpenelactone, thapsigargin, that is known to affect the selective release of Ca2+ from intracellular stores by inhibition of a specific Ca(2+)-ATPase; reuptake is inhibited. In most cells, and in accord with Putney's capacitative model (1990), thapsigargin, presumably by depleting intracellular Ca2+ stores, stimulates Ca2+ influx. This is the case for interphase HeLa cells. Thapsigargin induces an increase in [Ca2+]i that is dependent on extracellular Ca2+ and is associated with a strong stimulation of 45Ca2+ influx. In mitotic cells thapsigargin also induces a [Ca2+]i elevation that is initially comparable in magnitude and largely independent of extracellular Ca2+. However, unlike interphase cells, in mitotic cells the elevation of [Ca2+]i is not sustained and 45Ca2+ influx is not stimulated by thapsigargin. Thus, the coupling between depletion of intracellular stores and Ca2+ influx is specifically broken in mitotic cells. Uncoupling could account for the failure of histamine to stimulate Ca2+ influx during mitosis and would effectively block all stimuli whose effects are mediated by Ca2+ influx and sustained elevations of [Ca2+]i.

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