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.

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...

Trypanosoma brucei survives in the mammalian blood-stream by regularly changing its variant surface glycoprotein (VSG) coat. The active VSG gene is located in a telomeric expression site, and coat switching occurs either by replacing the transcribed VSG gene or by changing the expression site that is active. To determine whether VSG expression site control requires promoter-specific sequences, we replaced the active VSG expression site promoter in bloodstream-form T. brucei with a ribosomal DNA (rDNA) promoter. These transformants were fully infective in laboratory animals, and the rDNA promoter, which is normally constitutively active, was efficiently inactivated and reactivated in the context of the VSG gene expression site. As there is no sequence similarity between the VSG expression site promoter and the rDNA promoter, VSG expression site control does not involve sequences specific to the VSG expression site promoter. We conclude that an epigenetic mechanism, such as telomeric silencing, is involved in VSG expression site control in bloodstream-form T. brucei.

The survival of Trypanosoma brucei, the causative agent of Sleeping Sickness and Nagana, is facilitated by the expression of a dense surface coat of glycosylphosphatidylinositol (GPI)-anchored proteins in both its mammalian and tsetse fly hosts. We have characterized T. brucei GPI8, the gene encoding the catalytic subunit of the GPI:protein transamidase complex that adds preformed GPI anchors onto nascent polypeptides. Deletion of GPI8 (to give ⌬gpi8) resulted in the absence of GPI-anchored proteins from the cell surface of procyclic form trypanosomes and accumulation of a pool of non-protein-linked GPI molecules, some of which are surface located. Procyclic ⌬gpi8, while viable in culture, were unable to establish infections in the tsetse midgut, confirming that GPI-anchored proteins are essential for insect-parasite interactions. Applying specific inducible GPI8 RNAi with bloodstream form parasites resulted in accumulation of unanchored variant surface glycoprotein and cell death with a defined multinuclear, multikinetoplast, and multiflagellar phenotype indicative of a block in cytokinesis. These data show that GPI-anchored proteins are essential for the viability of bloodstream form trypanosomes even in the absence of immune challenge and imply that GPI8 is important for proper cell cycle progression. INTRODUCTIONTrypanosoma brucei is the heteroxenous, hemoflagellate protozoan parasite responsible for Sleeping Sickness in humans and Nagana in domestic animals in the tsetse belt of subSaharan Africa. All life cycle stages of the parasite utilize glycosylphosphatidylinositol (GPI) anchors as the predominant method for attaching proteins to their plasma membrane. In the mammalian host, these proteins include one subunit of the heterodimeric transferrin receptor (Schell et al., 1991), an alanine-rich protein of unknown function (Nolan et al., 2000), and the variant surface glycoprotein (VSG) (Ferguson et al., 1988) essential for evasion of the host's immune system.The bloodstream forms of T. brucei differentiate into procyclic forms once ingested by the tsetse fly vector. This differentiation involves remodeling of the surface by shedding the VSG coat and replacing it with an invariant coat of GPI-anchored proteins known as procyclins (Roditi et al., 1989). There are four types of procyclin, three bearing between 18 and 30 internal -Glu-Pro-repeats (EP1, EP2, and EP3; see Acosta-Serrano et al., 2000 for alignment), and one with a -Gly-Pro-Glu-Glu-Thr-(GPEET) repeat region . Although EP1 and EP3 both undergo N-glycosylation, EP2 and GPEET do not, although the latter is phosphorylated on the threonine residues of the repeat region (Butikofer et al., 1999). All procyclin isoforms are anchored by a GPI modified with a heterogeneous poly-Nacetyllactosamine side chain (Treumann et al., 1997), widely believed to form a glycocalyx over the cell surface. Displayed above this, both EP and GPEET procyclins are thought to adopt a rod-like conformation (Roditi et al., 1989;Treumann et al., 1997). Although the N-termini of the...