Identification of the cellular proteins whose expression is regulated during the cell cycle in normal cells is essential for understanding the mechanisms involved in the control of cell proliferation. A nuclear protein called cyclin of relative molecular mass 36,000 (Mr 36K), whose synthesis correlates with the proliferative state of the cell, has been identified in several cell types of human, mouse, hamster and avian origin. The rate of cyclin synthesis is very low in quiescent cells and increases several fold after serum stimulation shortly before DNA synthesis. Immunofluorescence and autoradiography studies have shown that the nuclear staining patterns of cyclin during S phase have a sequential order of appearance and a clear correlation can be found between DNA synthesis and cyclin positive nuclei. The proliferating cell nuclear antigen (PCNA) and cyclin have many common properties and it has been shown that these two are identical. Recently a protein which is required by DNA polymerase-delta for its catalytic activity with templates having low primer/template ratios has been isolated from calf thymus. We report here that cyclin and the auxiliary protein of DNA polymerase-delta are identical.

Abstract. Pulse-chase experiments have revealed that cyclin, the auxiliary protein of DNA polymerase-8, is stable during the transition from growth to quiescence in 3T3 cells. Immunoblotting together with immunofluorescence analysis has shown that the amount of cyclin after 24 h of quiescence is 30-40% of that of growing cells and that it presents a nucleoplasmic staining. Immunofluorescence studies show the existence of two populations of cyclin during the S phase, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent, and another that is associated to specific nuclear structures. By using antibromodeoxyuridine immunofluorescence to detect the sites of DNA synthesis, it was shown that the staining patterns of the replicon clusters and their order of appearance throughout the S phase are identical to those observed for cyclin. Two-dimensional gel analysis of Triton-extracted cells show that 20-30% of cyclin remains associated with the replicon clusters. This population of cyclin could not be released from the nucleus using high-salt extractions. This demonstrates that cyclin is tightly associated to the sites of DNA replication and that it must have a fundamental role in DNA synthesis in eukaryotic cells.T hE identification of the cellular proteins that are involved in the control of cell proliferation in normal cells is essential for understanding the mechanisms underlying growth regulation and cellular transformation. A nuclear protein, cyclin (mol wt 36,000), whose synthesis correlates with the proliferative state of the cell, may be such a candidate. This protein is present in variable amounts in normal proliferating as well as transformed cells of several species (reviewed in references 3 and 11). The level of cyclin fluctuates during the cell cycle, with a clear increase during the S phase (4). Moreover, a coordinate synthesis of cyclin and DNA has been demonstrated in quiescent cells stimulated with different mitogens (3, 7). The proliferating cell nuclear antigen (PCNA) t (18, 27-29) has been shown to be identical to cyclin (17,28). Immunofluorescence studies of the distribution of cyclin (PCNA) during the cell cycle have revealed dramatic changes in its nuclear localization during the S phase (7,10,19). It appears to be located in replicon clusters during DNA synthesis.Cyclin has been cloned and sequenced and shows homology with DNA binding proteins (1, 18). Recently, it has been proved that cyclin (PCNA) is the auxiliary protein of DNA polymerase-8 (5, 24, 30) and that it is required for SV40 replication in vitro (23).In this report we present evidence that the cyclin protein is stable during the transition from a growing to a quiescent state of the cell cycle and that a fraction of it is tightly associated with DNA replication sites during the S phase. Abbreviations used in this paper:BrdU, 5-bromodeoxyuridine; PCNA, proliferating cell nuclear antigen. Materials and Methods CellsMouse NIH 313 cells were grown in Dulbecco's modified Eagle's medium (DME) supplemented with ...

Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a A cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.A detailed knowledge of the biochemical events and the identification of the genes that are involved in the response to growth factors is currently being accumulated. A subset of these genes may play a critical role in cellular transformation. It is well established that the synthesis of new mRNA in quiescent cells is required for the cells to respond to mitogens and progress through Gl and enter the S phase (1). Furthermore, Gl seems to be a critical point of control of proliferation, where normal cells deprived of growth factors halt cycling, while transformed cells do not, suggesting that regulatory genes uncontrolled in the neoplastic phenotype are expressed during this transition. It has been estimated that in mouse fibroblasts, 3% of the mRNA species in logarithmically growing cells are absent in quiescent cells (49); thus, a set of these genes must be under the control of growth factors to allow cell proliferation. Evidence that gene expression could be controlled by the binding of a specific ligand to its receptor initially came from studies done with beta interferon (27,28) which demonstrated the transcriptional activation of two genes in human cells after interferon treatment. These findings were further supported by the interesting observation that the proto-oncogenes c-fos and c-myc present an immediate change in expression after stimulation of fibroblasts with growth factors (4,8,14,23,25,32), emphasizing also that important growth-controlling genes operate during the GO-to-Gl transition. Both genes are induced in the presence of protein synthesis inhibitors, demonstrating that their induction is a direct consequence of the growth factor-receptor interaction. Expression of c-fos is undetectable during the cell cycle (3), suggesting that its expression could be essential during the transition from GO to Gl and not for continuously growing ...

Synthesis of cyclin in serum‐stimulated quiescent 3T3 cells increases shortly before DNA synthesis after 10 h of stimulation, reaching a maximum after 16 h. Inhibition of DNA synthesis by hydroxyurea does not affect the increase of cyclin following stimulation, as determined by quantitative two‐dimensional gel electrophoresis. The levels of cyclin decrease dramatically at the end of the S‐phase. Cells kept in the presence of hydroxyurea (G1/S boundary) do not show this decrease in cyclin, indicating that its amounts are regulated by events occurring during the S‐phase. Immunofluorescence studies of serum‐stimulated quiescent cells in the presence of hydroxyurea, using proliferating cell nuclear antigen (PCNA) autoantibodies, confirm the results obtained by protein analysis. They also reveal that there are dramatic changes in the nuclear distribution of cyclin and that these depend on DNA synthesis or events occurring during the S‐phase. Cyclin (PCNA) is no longer detectable at the end of the S‐phase. However, pulse‐chase experiments indicate that this protein is very stable, suggesting that it possibly interacts with other macromolecules rendering it inaccessible to the antibody. These results strengthen the notion that cyclin is an important component of the events leading to DNA replication and cell division.

A full-length cDNA clone for the human nuclear protein cyclin has been isolated by using polyclonal antibodies and sequenced. The sequence predicts a protein of 261 amino acids (Mr 29,261) with a high content of acidic (41, aspartic and glutamic acids) versus basic (24, lysine and arginine) amino acids. The identity of the cDNA clone was confirmed by in vitro hybrid-arrested translation of cyclin mRNA. Blot-hybridization analysis of mouse 3T3 and human MOLT-4 cell RNA revealed a mRNA species of approximately the same size as the cDNA insert. Expression of cyclin mRNA was undetectable or very low in quiescent cells, increasing after 8-10 hr of serum stimulation. Inhibition of DNA synthesis by hydroxyurea in serum-stimulated cells did not affect the increase in cyclin mRNA but inhibited 90% the expression of H3 mRNA. These results suggest that expression of cyclin and histone mRNAs are controlled by different mechanisms. A region of the cyclin sequence shows a significant homology with the putative DNA binding site of several proteins, specially with the transcriptional-regulator cAMP-binding protein of Escherichia coli, suggesting that cyclin could play a similar role in eukaryotic cells.The identification of the cellular proteins that are involved in the control of cell proliferation in normal cells is essential for understanding the mechanisms underlying growth regulation and cellular transformation. A nuclear protein, "cyclin" (Mr 36,000), whose synthesis correlates with the proliferative state of the cells, is potentially such a candidate (for reviews, see refs. 1 and 2). This protein is present in variable amounts in normal proliferating cells as well as transformed cells and tumors. It is highly conserved, as determined by onedimensional peptide mapping, and it has been identified in several cell types of human, mouse, hamster, and avian origin. The level of cyclin fluctuates during the cell cycle, with a clear increase during the S phase (3, 4). Moreover, a coordinate synthesis of cyclin and DNA has been demonstrated in serum or growth factor-induced quiescent cells (5, 6). The proliferating-cell nuclear antigen (PCNA; refs. 7-10), a human protein that shares the same properties, has been shown to be identical to cyclin (9, 11). Immunofluorescence studies of the distribution of cyclin (PCNA) during the cell cycle have revealed dramatic changes in its nuclear localization during the S phase (7,12,13). Recent studies have demonstrated that these changes are not triggered by a mechanism involving direct phosphorylation of cyclin (4) and that they depend on DNA synthesis or events during the S phase (12).To learn more about the structure and function of cyclin, we decided to isolate cDNA clones of the mRNA for cyclin. We report here the complete nucleotide sequence for human cyclin and its expression during the cell cycle. MATERIALS AND METHODSCells. Mouse 3T3 cells were routinely grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and antibiotics (penicillin, 100 units/ml...