scholarly journals THE SYNTHESIS OF ACIDIC CHROMOSOMAL PROTEINS DURING THE CELL CYCLE OF HELA S-3 CELLS

1972 ◽  
Vol 52 (2) ◽  
pp. 292-307 ◽  
Author(s):  
G. S. Stein ◽  
T. W. Borun
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Nuclear Proteins ◽  
Regulatory Function ◽  
Nuclear Fraction ◽  
Tightly Coupled ◽  
Acidic Proteins

The synthesis and accumulation of acidic proteins in the tightly bound residual nuclear fraction goes on throughout the cell cycle of continuously dividing populations of HeLa S-3 cells; however, during late G1 there is an increased rate of synthesis and accumulation of these proteins which precedes the onset of DNA synthesis. Unlike that of the histones, whose synthesis is tightly coupled to DNA replication, the synthesis of acidic residual nuclear proteins is insensitive to inhibitors of DNA synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of acidic residual nuclear proteins shows different profiles during the G1, S, and G2 phases of the cell cycle. These results suggest that, in contrast to histones whose synthesis appears to be highly regulated, the acidic residual proteins may have a regulatory function in the control of cell proliferation in continuously dividing mammalian cells.

Plasma Membrane and Nuclear Proteins of the Goose Erythrocyte

1973 ◽  
Vol 51 (10) ◽  
pp. 1442-1447 ◽  
Author(s):  
Keith R. Shelton
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Nuclear Proteins ◽  
Nuclear Fraction ◽  
Nonhistone Protein ◽  
Large Molecular Weight ◽  
Goose Erythrocyte

Plasma membrane and nuclear fractions have been prepared from mature goose erythrocytes. Examination of the nonhistone protein of the nuclear fraction by sodium dodecyl sulphate – polyacrylamide gel electrophoresis reveals a limited number of molecular weight species some of which are peculiar to the nuclear fraction.Electrophoretic comparison of the goose plasma membrane proteins with those of the human erythrocyte reveals many similarities. In particular, three large molecular weight species occur in both cells. Their function appears to predate the evolutionary loss of the nucleus.

scholarly journals Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

1989 ◽  
Vol 9 (5) ◽  
pp. 1940-1945 ◽  
Author(s):  
B Y Tseng ◽  
C E Prussak ◽  
M T Almazan
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Small Subunit ◽  
Mrna Levels ◽  
Proliferating Cells ◽  
Restriction Point ◽  
Serum Stimulation ◽  
G1 Cells

Expression of the small-subunit p49 mRNA of primase, the enzyme that synthesizes oligoribonucleotides for initiation of DNA replication, was examined in mouse cells stimulated to proliferate by serum and in growing cells. The level of p49 mRNA increased approximately 10-fold after serum stimulation and preceded synthesis of DNA and histone H3 mRNA by several hours. Expression of p49 mRNA was not sensitive to inhibition by low concentrations of cycloheximide, which suggested that the increase in mRNA occurred before the restriction point control for cell cycle progression described for mammalian cells and was not under its control. p49 mRNA levels were not coupled to DNA synthesis, as observed for the replication-dependent histone genes, since hydroxyurea or aphidicolin had no effect on p49 mRNA levels when added before or during S phase. These inhibitors did have an effect, however, on the stability of p49 mRNA and increased the half-life from 3.5 h to about 20 h, which suggested an interdependence of p49 mRNA degradation and DNA synthesis. When growing cells were examined after separation by centrifugal elutriation, little difference was detected for p49 mRNA levels in different phases of the cell cycle. This was also observed when elutriated G1 cells were allowed to continue growth and then were blocked in M phase with colcemid. Only a small decrease in p49 mRNA occurred, whereas H3 mRNA rapidly decreased, when cells entered G2/M. These results indicate that the level of primase p49 mRNA is not cell cycle regulated but is present constitutively in proliferating cells.

Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein

1992 ◽  
Vol 12 (1) ◽  
pp. 164-171
Author(s):  
M J Matunis ◽  
W M Michael ◽  
G Dreyfuss
Keyword(s):  
Hela Cells ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Rna Binding ◽  
Consensus Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Hnrnp Proteins

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.

Centrosomal components immunologically related to tektins from ciliary and flagellar microtubules

1994 ◽  
Vol 107 (8) ◽  
pp. 2095-2105 ◽  
Author(s):  
W. Steffen ◽  
E.A. Fajer ◽  
R.W. Linck
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Immunoblot Analysis ◽  
Surf Clam ◽  
Alpha Tubulin ◽  
Sea Urchin Sperm

Centrosomes are critical for the nucleation and organization of the microtubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule proteins, we characterize here the presence and behavior of certain components associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella and used to produce monoclonal antibodies, 16 of which were specific- or cross-specific for the major polypeptides associated with this microtubule fraction: tektins A, B and C, acetylated alpha-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylamide gel electrophoresis the tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal antibodies against a given tektin, indicating that the different polypeptide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain centrosomes of S. solidissima oocytes and CHO and HeLa cells, by immunofluorescence microscopy. In particular, the centrosome staining of one monoclonal antibody specific for tektin B (tekB3) was cell-cycle-dependent for CHO cells, i.e. staining was observed only from early prometaphase until late anaphase. By immuno-electron microscopy tekB3 specifically labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal material throughout the cell cycle. Finally, by immunoblot analysis tekB3 stained polypeptides of 48–50 kDa in isolated spindles and centrosomes from CHO cells.

scholarly journals Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein.

1992 ◽  
Vol 12 (1) ◽  
pp. 164-171 ◽  
Author(s):  
M J Matunis ◽  
W M Michael ◽  
G Dreyfuss
Keyword(s):  
Hela Cells ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Rna Binding ◽  
Consensus Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Hnrnp Proteins

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.

scholarly journals Nonperiodic Activity of the Human Anaphase-Promoting Complex–Cdh1 Ubiquitin Ligase Results in Continuous DNA Synthesis Uncoupled from Mitosis

2000 ◽  
Vol 20 (20) ◽  
pp. 7613-7623 ◽  
Author(s):  
Claus Storgaard Sørensen ◽  
Claudia Lukas ◽  
Edgar R. Kramer ◽  
Jan-Michael Peters ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Cyclin B1 ◽  
S Phase ◽  
Anaphase Promoting Complex

ABSTRACT Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae andDrosophila spp., triggers exit from mitosis and during G1 prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G1/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G1/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27Kip1 cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G1/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.

scholarly journals Changes in proportion of thiol to disulphide in acid-soluble nuclear proteins of Echinus during the first cell cycle

1970 ◽  
Vol 116 (3) ◽  
pp. 415-420 ◽  
Author(s):  
Margery G. Ord ◽  
L. A. Stocken
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Acidic Protein ◽  
Nuclear Proteins ◽  
Acid Extract ◽  
Early Stages

1. DNA synthesis in Echinus esculentus eggs kept at 10°C takes place just after fusion, 0.75–1.5h after fertilization, and at telophase at about 2.67–3.33h after fertilization. 2. An increase in the thiol/thiol+disulphide ratio in acid extracts from washed nuclear fractions of the eggs is found at fusion, at early stages of mitosis and at telophase. When DNA is being synthesized, the relative amount of thiol in the extracts increases. 3. There are at least five thiol-containing histones in the acid extract together with a diffusible thiol peptide containing methyl-lysine and 3-methylhistidine and a thiol-containing acidic protein.

scholarly journals Copy Number Suppressors of the Aspergillus nidulans nimA1 Mitotic Kinase Display Distinctive and Highly Dynamic Cell Cycle-Regulated Locations

2008 ◽  
Vol 7 (12) ◽  
pp. 2087-2099 ◽  
Author(s):  
Leena Ukil ◽  
Archana Varadaraj ◽  
Meera Govindaraghavan ◽  
Hui-Lin Liu ◽  
Stephen A. Osmani
Keyword(s):  
Cell Cycle ◽  
Aspergillus Nidulans ◽  
Copy Number ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nuclear Periphery ◽  
Sodium Dodecyl ◽  
Dimorphic Fungi ◽  
Mitotic Kinase ◽  
Specific Distribution ◽  
Dynamic Cell

ABSTRACT The Aspergillus nidulans NIMA kinase is essential for mitosis and is the founding member of the conserved NIMA-related kinase (Nek) family of protein kinases. To gain insight into NIMA function, a copy number suppression screen has been completed that defines three proteins termed MCNA, MCNB, and MCNC (multi-copy-number suppressor of nimA1 A, B, and C). All display a distinctive and dynamic cell cycle-specific distribution. MCNC has weak similarity to Saccharomyces cerevisiae Def1 within a shared CUE-like domain. MCNC, like Def1, is a cytoplasmic protein with slow mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its deletion causes polarization defects and a small colony phenotype. MCNC enters nuclei during mitosis. In contrast, MCNB is a nuclear protein displaying increased nuclear levels as cells progress through interphase but is lost from nuclei at mitosis. MCNB is highly related to the Schizosaccharomyces pombe forkhead transcription factor Sep1 and is likely a transcriptional activator of nimA. Most surprisingly, MCNA, a protein restricted to the aspergilli and pathogenic systemic dimorphic fungi (the Eurotiomycetes), defines a nuclear body located near nucleoli at the nuclear periphery of G2 nuclei. During progression through mitosis, the MCNA body is excluded from nuclei. Cytoplasmic MCNA bodies then diminish during early stages of interphase, and single MCNA bodies are formed within nuclei as interphase progresses. Three sites of MCNA phosphorylation were mapped and mutated to implicate proline-directed phosphorylation in the equal segregation of MCNA during the cell cycle. The data indicate all three MCN proteins likely have cell cycle functions.

scholarly journals Characterization and purification of H1TF2, a novel CCAAT-binding protein that interacts with a histone H1 subtype-specific consensus element.

1989 ◽  
Vol 9 (4) ◽  
pp. 1566-1575 ◽  
Author(s):  
P Gallinari ◽  
F La Bella ◽  
N Heintz
Keyword(s):  
Cell Cycle ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gene Promoter ◽  
Molecular Size ◽  
Histone H1 ◽  
Nuclear Extracts ◽  
Definition Of

Definition of mechanisms regulating human histone H1 gene transcription during the cell cycle requires the isolation and biochemical characterization of protein factors which interact with specific promoter elements. Two distinct binding activities have been identified in nuclear extracts from HeLa cells and mapped within a 180-base-pair (bp) region of a cell cycle-regulated H1 gene promoter. H1TF1 bound to an H1-specific A + C-rich sequence (AC box), 100 bp upstream of the cap site; H1TF2 interacted with the H1 subtype-specific consensus element and was dependent on the presence of an intact CCAAT box for binding. H1TF2 was purified through a combination of ion-exchange and oligonucleotide affinity chromatographies. Analysis of purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV crosslinking showed that H1TF2 was a single polypeptide of 47 kilodaltons. This factor was distinct from previously characterized CCAAT-binding proteins in both molecular size and binding properties. Fractions containing H1TF2 activity activated transcription in vitro only if programmed with an H1 DNA template carrying an intact H1TF2-binding site.

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