scholarly journals Changes in nuclear protein during the cell cycle in cultured mast cells separated by zonal centrifugation

1972 ◽  
Vol 128 (5) ◽  
pp. 1213-1219 ◽  
Author(s):  
M E. Cross
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Mast Cells ◽  
Nuclear Protein ◽  
Specific Radioactivity ◽  
S Phase ◽  
Ficoll Gradient ◽  
Thiol Content ◽  
Histone Synthesis ◽  
Dna 2

1. Exponentially grown mouse mast cells (cell line P815, strain Y) were separated by zonal centrifugation on a Ficoll gradient. Fractions were allocated to different phases of the cell cycle according to the specific radioactivity of their DNA. 2. Histones were extracted and their thiol content was analysed. The proportion of reduced thiol increased in S phase, decreasing subsequently. 3. The phosphate content of histone F1 and of the other histones reached a peak in early and later S phase respectively. The incorporation of 32P into these fractions showed a corresponding increase. 4. The timing of histone synthesis was examined. Incorporation of 14C-labelled amino acids into the histone fractions took place at the same times as phosphorylation. 5. Acid nuclear proteins differ from the histones in incorporating labelled amino acids and 32P fairly constantly through the cell cycle.

scholarly journals Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase

2003 ◽  
Vol 23 (5) ◽  
pp. 1590-1601 ◽  
Author(s):  
Lianxing Zheng ◽  
Zbigniew Dominski ◽  
Xiao-Cui Yang ◽  
Phillip Elms ◽  
Christy S. Raska ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Posttranscriptional Regulation ◽  
Binding Protein ◽  
S Phase ◽  
Mrna Processing ◽  
Histone Mrna ◽  
Stem Loop ◽  
Histone Mrnas ◽  
Nuclear Extracts

ABSTRACT The replication-dependent histone mRNAs, the only eukaryotic mRNAs that do not have poly(A) tails, are present only in S-phase cells. Coordinate posttranscriptional regulation of histone mRNAs is mediated by the stem-loop at the 3′ end of histone mRNAs. The protein that binds the 3′ end of histone mRNA, stem-loop binding protein (SLBP), is required for histone pre-mRNA processing and is involved in multiple aspects of histone mRNA metabolism. SLBP is also regulated during the cell cycle, accumulating as cells enter S phase and being rapidly degraded as cells exit S phase. Mutation of any residues in a TTP sequence (amino acids 60 to 62) or mutation of a consensus cyclin binding site (amino acids 99 to 104) stabilizes SLBP in G2 and mitosis. These two threonines are phosphorylated in late S phase, as determined by mass spectrometry (MS) of purified SLBP from late S-phase cells, triggering SLBP degradation. Cells that express a stable SLBP still degrade histone mRNA at the end of S phase, demonstrating that degradation of SLBP is not required for histone mRNA degradation. Nuclear extracts from G1 and G2 cells are deficient in histone pre-mRNA processing, which is restored by addition of recombinant SLBP, indicating that SLBP is the only cell cycle-regulated factor required for histone pre-mRNA processing.

A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division

1994 ◽  
Vol 107 (1) ◽  
pp. 253-265 ◽  
Author(s):  
I.T. Todorov ◽  
R. Pepperkok ◽  
R.N. Philipova ◽  
S.E. Kearsey ◽  
W. Ansorge ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Amino Acid ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Nuclear Protein ◽  
Amino Acid Level ◽  
S Phase ◽  
Significant Similarity

Molecular cloning and characterisation of a human nuclear protein designated BM28 is reported. On the amino acid level this 892 amino acid protein, migrating on SDS-gels as a 125 kDa polypeptide, shares areas of significant similarity with a recently defined family of early S phase proteins. The members of this family, the Saccharomyces cerevisiae Mcm2p, Mcm3p, Cdc46p/Mcm5p, the Schizosaccharomyces pombe Cdc21p and the mouse protein P1 are considered to be involved in the onset of DNA replication. The highest similarity was found with Mcm2p (42% identity over the whole length and higher than 75% over a conservative region of 215 amino acid residues), suggesting that BM28 could represent the human homologue of the S. cerevisiae MCM2. Using antibodies raised against the recombinant BM28 the corresponding antigen was found to be localised in the nuclei of various mammalian cells. Microinjection of anti-BM28 antibody into synchronised mouse NIH3T3 or human HeLa cells presents evidence for the involvement of the protein in cell cycle progression. When injected in G1 phase the anti-BM28 antibody inhibits the onset of subsequent DNA synthesis as tested by the incorporation of bromodeoxyuridine. Microinjection during the S phase had no effect on DNA synthesis, but inhibits cell division. The data suggest that the nuclear protein BM28 is required for two events of the cell cycle, for the onset of DNA replication and for cell division.

scholarly journals THE SYNTHESES OF TOTAL MACRONUCLEAR PROTEIN, HISTONE, AND DNA DURING THE CELL CYCLE IN EUPLOTES EURYSTOMUS

1966 ◽  
Vol 31 (1) ◽  
pp. 1-9 ◽  
Author(s):  
David M. Prescott
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Total Protein ◽  
Tritiated Thymidine ◽  
S Phase ◽  
Protein Labeling ◽  
Constant Rate ◽  
Tritiated Amino Acids ◽  
Histone Synthesis ◽  
Macronuclear Division

The syntheses of histone, total protein, and DNA during the cell cycle were measured in the macronucleus of Euplotes eurystomus by assaying the incorporation of tritiated amino acids and tritiated thymidine in groups of 800 to 1000 synchronized cells. The synthesis of DNA begins at 30% completion of the cell cycle, proceeds at a constant rate, and ends very shortly before the beginning of macronuclear division. Histone labeling is absent during G1, begins in phase with DNA synthesis, continues at an unchanging rate during the S phase, and ends with the completion of DNA synthesis. The results support the view that the syntheses of histone and DNA are closely coupled events. Label in total protein accumulates at a constant rate during G1 and appears to shift to a slightly higher rate when histone synthesis begins. At division, radioactive DNA, histone, and total protein are distributed equally between the daughter macronuclei without loss of radioactivity. Radioautographic analysis showed that protein labeling occurs throughout the macronucleus during the entire life cycle. There was no clear difference in the degree of protein labeling between replicated and unreplicated regions of the macronucleus. The distribution of label suggests that most of macronuclear protein labeling during the cell cycle is concerned with the events of transcription rather than replication.

Control of histone gene expression in Physarum polycephalum. I. Protein synthesis during the cell cycle

1982 ◽  
Vol 57 (1) ◽  
pp. 139-150
Author(s):  
P.N. Schofield ◽  
I.O. Walker
Keyword(s):  
Cell Cycle ◽  
Physarum Polycephalum ◽  
Specific Activity ◽  
S Phase ◽  
Histone Gene ◽  
Synchronous Cultures ◽  
Histone Gene Expression ◽  
Core Histones ◽  
G2 Phase ◽  
Histone Synthesis

Synchronous cultures of Physarum polycephalum were pulsed with [3H]lysine hydrochloride in S and G2 phases of the cell cycle. Plasmodial extracts were separated into nuclear, ribosomal and acid-soluble post-ribosomal cytoplasmic fractions. Core histones could be detected by staining in the nuclear fractions of both S and G2 phases, but were not detected by staining in the cytoplasmic fractions. Newly synthesized histone was present in S-phase nuclei but not in S-phase cytoplasm. The specific activity of newly synthesized histone in G2-phase nuclei decreased by at least 95% compared to S phase and no newly synthesized histone was observed in G2-phase cytoplasmic fractions. Thus histone synthesis is restricted to S phase. There are no free pools of histone in the cytoplasm of Physarum in either S or G2 phases of the cell cycle.

scholarly journals Identification of a rel-related protein in the nucleus during the S phase of the cell cycle.

1993 ◽  
Vol 13 (10) ◽  
pp. 6147-6156 ◽  
Author(s):  
R B Evans ◽  
P D Gottlieb ◽  
H R Bose
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
S Phase ◽  
Cellular Proteins ◽  
Related Protein ◽  
Carboxy Terminus ◽  
Nf Kappa B ◽  
Western Immunoblot ◽  
Molecular Masses ◽  
Carboxy Terminal

The c-rel proto-oncogene encodes a 75-kDa protein (p75c-rel) which is present in the cytosol of chick embryo fibroblasts (CEF) associated with a distinct set of cellular proteins with molecular masses of 40, 115, and 124 kDa. CEF cultures arrested in S phase of the cell cycle, or enriched for G2 or mitotic cells, were examined to determine whether the expression of c-rel was altered during the cell cycle. Levels of p75c-rel remained constant in all portions of the cell cycle examined; however, a Rel-related protein with an apparent molecular mass of 64 kDa was detected in nuclei of S-phase cells. As cells enter G2, the level of this protein in the nucleus decreases. This protein reacts with antiserum generated against the carboxy terminus of p75c-rel in radioimmunoprecipitations and Western immunoblot experiments and was also detected in a Western immunoblot with antiserum generated against the first 161 amino acids of pp59v-rel. Thus, unlike other Rel/NF-kappa B family members, p64 has carboxy-terminal homology with c-Rel. The majority of peptides generated by partial proteolytic cleavage of p64 are shared with peptides generated by digestion of p75c-rel and/or pp59v-rel. We suggest that this protein represents a new member of the Rel family of transcription factors and is located in the nucleus of avian fibroblasts during S phase of the cell cycle.

scholarly journals Replication-dependent histone biosynthesis is coupled to cell-cycle commitment

2021 ◽  
Vol 118 (31) ◽  
pp. e2100178118
Author(s):  
Claire Armstrong ◽  
Sabrina L. Spencer
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Live Cell Imaging ◽  
Current Model ◽  
S Phase ◽  
Strong Increase ◽  
Key Factors ◽  
Histone Gene Expression ◽  
Small Pool ◽  
Histone Synthesis

The current model of replication-dependent (RD) histone biosynthesis posits that RD histone gene expression is coupled to DNA replication, occurring only in S phase of the cell cycle once DNA synthesis has begun. However, several key factors in the RD histone biosynthesis pathway are up-regulated by E2F or phosphorylated by CDK2, suggesting these processes may instead begin much earlier, at the point of cell-cycle commitment. In this study, we use both fixed- and live-cell imaging of human cells to address this question, revealing a hybrid model in which RD histone biosynthesis is first initiated in G1, followed by a strong increase in histone production in S phase of the cell cycle. This suggests a mechanism by which cells that have committed to the cell cycle build up an initial small pool of RD histones to be available for the start of DNA replication, before producing most of the necessary histones required in S phase. Thus, a clear distinction exists at completion of mitosis between cells that are born with the intention of proceeding through the cell cycle and replicating their DNA and cells that have chosen to exit the cell cycle and have no immediate need for histone synthesis.

Identification of a rel-related protein in the nucleus during the S phase of the cell cycle

1993 ◽  
Vol 13 (10) ◽  
pp. 6147-6156
Author(s):  
R B Evans ◽  
P D Gottlieb ◽  
H R Bose
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
S Phase ◽  
Cellular Proteins ◽  
Related Protein ◽  
Carboxy Terminus ◽  
Nf Kappa B ◽  
Western Immunoblot ◽  
Molecular Masses ◽  
Carboxy Terminal

The c-rel proto-oncogene encodes a 75-kDa protein (p75c-rel) which is present in the cytosol of chick embryo fibroblasts (CEF) associated with a distinct set of cellular proteins with molecular masses of 40, 115, and 124 kDa. CEF cultures arrested in S phase of the cell cycle, or enriched for G2 or mitotic cells, were examined to determine whether the expression of c-rel was altered during the cell cycle. Levels of p75c-rel remained constant in all portions of the cell cycle examined; however, a Rel-related protein with an apparent molecular mass of 64 kDa was detected in nuclei of S-phase cells. As cells enter G2, the level of this protein in the nucleus decreases. This protein reacts with antiserum generated against the carboxy terminus of p75c-rel in radioimmunoprecipitations and Western immunoblot experiments and was also detected in a Western immunoblot with antiserum generated against the first 161 amino acids of pp59v-rel. Thus, unlike other Rel/NF-kappa B family members, p64 has carboxy-terminal homology with c-Rel. The majority of peptides generated by partial proteolytic cleavage of p64 are shared with peptides generated by digestion of p75c-rel and/or pp59v-rel. We suggest that this protein represents a new member of the Rel family of transcription factors and is located in the nucleus of avian fibroblasts during S phase of the cell cycle.

Supercoiling of DNA and nuclear conformation during the cell-cycle

1978 ◽  
Vol 30 (1) ◽  
pp. 211-226
Author(s):  
A.C. Warren ◽  
P.R. Cook
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Sedimentation Rate ◽  
Nuclear Protein ◽  
S Phase ◽  
Dna Conformation ◽  
Nuclear Morphology ◽  
Ionic Detergent ◽  
High Concentrations ◽  
The Individual

When cells are lysed in solutions containing high concentrations of salt and a non-ionic detergent, structures are released which retain many of the morphological features of nuclei. These nucleoids contain superhelical DNA but are depleted of nuclear protein. We have analysed DNA conformation in nucleoids derived from HeLa cells synchronized at different stages in the cell cycle. The gross differences in nuclear morphology seen during the cell cycle are reflected in the morphology of the nucleoids; for example, the individual chromosomes of mitotic cells remain identifiable and aggregated within the mitotic nucleoid. The sedimentation rate of nucleoids in sucrose gradients reflects the gross nuclear morphology; the small S-phase nucleoids sediment 9 times faster than the large mitotic nucleoids. Despite these large differences at the gross level of organization, both the degree of supercoiling and the size of the units in which supercoiling is maintained are roughly similar in the nucleoids derived from cells in the different phases. The protein content of the various nucleoids is also very similar. Like the nucleoids made from randomly growing cultures of cells, mitotic nucleoids are excellent templates for the RNA polymerase of Escherichia coli.

A mutant E2F-1 transcription factor that affects the phenotype of NIH3T3 fibroblasts inefficiently associates with cyclin A - cdk2

1998 ◽  
Vol 76 (1) ◽  
pp. 37-44 ◽  
Author(s):  
Kelly L Jordan-Sciutto ◽  
David J Hall
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Transcription Factor ◽  
Cyclin A ◽  
Chemotherapeutic Agents ◽  
S Phase ◽  
Full Length ◽  
Vitro Assay ◽  
Amino Terminal

The amino-terminal domain of the E2F1 transcription factor is the site of association with cyclin A - cdk2, mapping to residues 87-94. A mutant of E2F1 lacking the first 87 amino acids (termed E2F1d87) has a number of potent effects on cellular phenotype when constitutively expressed in NIH3T3 fibroblasts. For example, in these fibroblasts the duration of S phase and the sensitivity to S phase chemotherapeutic agents are both increased. Since E2F1d87 only partially truncates the cyclin A - cdk2 binding domain, it was important to determine the level of cyclin A - cdk2 association with this mutant to correlate any reduction in association with the observed effects on the cell cycle. It was found that cyclin A - cdk2 binds E2F1d87 in an in vitro assay but that this binding is reduced approximately 8 fold compared with binding to full-length E2F1, whereas no detectable binding was seen to a mutant E2F1 that lacks the first 117 amino acids. Correspondingly, H1 kinase activity in E2F1d87 immunoprecipitates from E2F1d87-expressing cells was significantly reduced compared with that seen for full-length E2F1. From these data it appears that E2F1 with reduced cyclin A - cdk2 binding activity mediates the alteration in cell cycle parameters seen in these cells.Key words: E2F1, apoptosis, cyclin A, cell cycle.

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