The human CCG1 gene, essential for progression of the G1 phase, encodes a 210-kilodalton nuclear DNA-binding protein.

Abstract

The human CCGI gene complements tsBN462, a temperature-sensitive G1 mutant of the BHK21 cell line. The previously cloned cDNA turned out to be a truncated form of the actual CCGI cDNA. The newly cloned CCGI cDNA was 6.0 kb and encoded a protein with a molecular mass of 210 kDa. Using an antibody to a predicted peptide from the CCG1 protein, a protein with a molecular mass of over 200 kDa was identified in human, monkey, and hamster cell lines. In the newly defined C-terminal region, an acidic domain was found. It contained four consensus target sequences for casein kinase II and was phosphorylated by this enzyme in vitro. However, this C-terminal region was not required to complement tsBN462 mutation since the region encoding the C-terminal part was frequently missing in complemented clones derived by DNA-mediated gene transfer. CCG1 contains a sequence similar to the putative DNA-binding domain of HMG1 in addition to the previously detected amino acid sequences common in nuclear proteins, such as a proline cluster and a nuclear translocation signal. Consistent with these predictions, CCG1 was present in nuclei, possessed DNA-binding activity, and was eluted with similar concentrations of salt, 0.3 to 0.4 M NaCl either from isolated nuclei or from a DNA-cellulose column.The cell cycle of eucaryotic cells is composed of the consecutive phases Gl, S, G2, and M. In the G, phase, depending on the stimulus of growth factors, protein and RNA molecules are produced and the new cell cycle initiates (25). Unresolved problems concerning the start of the cell cycle include how the stimulus of external growth factors reaches the nucleus and how cells recognize the accumulation of materials required to enter the new cell cycle. To investigate the initiation of the cell cycle at the molecular level, it is essential to identify the genes involved. In both budding and fission yeasts, various temperature-sensitive (ts) mutants defective in the progression of the cell cycle have been isolated and have proven to be most useful for identifying the genes required for cell cycle progression (12,22). Various ts+ cell cycle mutants have also been isolated from cultured animal cells (3). The FT210 cell line, a ts G2 mutant of FM3A, has a ts cdc2 gene product; thus, the cdc2 gene is apparently essential for the cell cycle of animal cells (36).We isolated ts cell cycle mutants from the BHK21 cell line derived from the Syrian hamster and then classified them into complementation groups (21). One of these ts mutants, tsBN462, has a defect in progression of the G1 phase, which is that after release from the G, block, it is unable to enter the S phase at a nonpermissive temperature. But once it enters the S phase, DNA replication progresses normally. By using this mutant as the recipient of the DNA-mediated gene transfer, a human gene located between qll and q13 on the X chromosome has been cloned and designated the cell cycle gene 1 (CCGI) (5,31,32). Its cDNA complements tsi3 in addition to tsBN462, both of which were independently isolated ...

The cloning, mapping and expression of a novel gene, BRL, related to the AF10 leukaemia gene

“…This motif was ®rst described following the cloning of the brahma (brm) gene in drosophila (Haynes et al, 1992;Tamkun et al, 1992) and has since been identi®ed in other transcriptional co-activators including the human brahma protein, BRM (Muchardt and Yaniv, 1993) and the yeast protein SNF2/SWI2 (Laurent et al, 1993). The cell cycle gene 1 (CCG1) (Sekiguchi et al, 1991) which was later identi®ed as hTAF II 250 (Hisatake et al, 1993;Ruppert et al, 1993) and shown to be necessary for G1 progression in mammalian cells also contains a bromodomain. More than one bromodomain can be present within a protein: polybromo, a gene of unknown function isolated from chicken monoblasts, encodes a putative protein with ®ve bromodomains (Nicolas and Goodwin, 1996).…”

Overexpression of D-type cyclins, E2F-1, SV40 large T antigen and HPV16 E7 rescue cell cycle arrest of tsBN462 cells caused by the CCG1/TAFII250 mutation

“…Recombinant plasmid DNAs were transfected into cultures of the tsBN462 and BHK21 cells (2610 5 cells per 100-mm diameter dish) by the calcium phosphate precipitation method as previously described (Sekiguchi et al, 1991). Transfected cells were grown at 33.58C for 17 h, washed once with isotonic bu er, and then grown either at 33.58C, the permissive temperature, or shifted to 39.58C, the nonpermissive temperature for 24 h. At this time transiently transfected cells were suspended in 40 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA and lysed by freezing and thawing three times.…”

Induction of growth arrest and cell death by overexpression of the cyclin-Cdk inhibitor p21 in hamster BHK21 cells

“…The Tet-p21, E2F-1 (Kaelin et al, 1992), SV40 large T antigen (Ariga and Sugano, 1983) (20 mg/dish) and CD20 (2 mg/dish) vectors were transfected into cultures of BHK21 cells (2610 5 cells per 100 mm diameter dish) by the calcium phosphate precipitation method as previously described (Sekiguchi et al, 1991). Transfected cells were grown for 17 h, washed once with isotonic bu er, and then grown for 48 h. For stable transformation of tetracycline vectors, Tet-p21 and Tetrepressible vector into BHK21 cells, cells were exposed to hygromycin-containing medium until colonies appeared.…”

“…Protein samples were electrophoresed in an 12.5% SDS-polyacrylamide slab gel and analysed by immunobloting, as described previously (Sekiguchi et al, 1991). Immunoblotting was performed using an ECL kit (Amersham, Arlington Heights, IL) as recommended by the supplier.…”