Apoptosis Is Induced in BHK Cells by the tsBN462/13 Mutation in the CCG1/TAFII250 Subunit of the TFIID Basal Transcription Factor

A temperature-sensitive (ts) mutant of the BHK21 cell line derived from golden hamsters, tsBN462 has a mutation in the gene encoding the largest subunit of the TFIID complex, TAF_II250/p230/CCG1, and arrests in the G1 phase at the nonpermissive temperature, 39.5°C. We found that tsBN462 cells underwent apoptosis following growth arrest at 39.5°C, suggesting a role for CCG1 as a repressor of apoptosis. By electron microscopic observation, tsBN462 cells at 39.5°C showed characteristic features of apoptosis. Apoptosis was not suppressed by expression of Bc1-2 or the adenovirus E1B 19 kDa protein. Cell death was suppressed completely by expression of wild-type CCG1 and partially by wild-type p53, a growth suppressor protein. Cell cycle arrest induced by p53 may help survival of tsBN462 cells at 39.5°C. Apoptosis was accelerated in SV40 large T antigen-transformed tsBN462 cells at 39.5°C where SV40 large T antigen formed a complex with p53, implying that the apoptosis of tsBN462 cells at 39.5°C occurred in a p53-independent manner. Our results suggest that CCG1/TAF_II250 is required for the expression of factors regulating apoptosis.     

The cytoplasmic appearance of three functions expressed during the G0→G1→S transition is nucleus-dependent

tsAF8, ts13, tsHJ-4, and TK^?ts13 cells are G₁-specific temperature-sensitive (ts) mutants of BHK cells that do not enter S phase when serumstimulated from quiescence at nonpermissive temperature (39.6°-40.6°). TK^?ts13 are, in addition, defective in thymidine kinase. Different G₁ functions must be involved in these cells, since the first three cell lines complement each other when forming heterokaryons. We have used these cells to study the role of the nucleus in the cytoplasmic expression of these G₁ functions during the transition of cells from the non-proliferating to the proliferating state. We fused cytoplasts from either serumstarved (G₀) or serum-stimulated (S) tsAF8 cells with G₀-ts13, G₀-tsHJ-4, and G₀-TK^?ts13 recipient cells and determined, after serum stimulation of the fusion products, which type of cytoplasts could complement the defective G₁ functions. Cytoplasts from S-tsAF8 cells complemented all three functions, i.e., cybridoids between S phase cytoplasts and ts13 or tsHJ-4 recipient cells entered S at the nonpermissive temperature, and TK^?ts13 recipient cells incorporated exogenous thymidine. Cytoplasts isolated from G₀-tsAF8 cells (3 days of serum starvation) complemented ts13 cells but not tsHJ-4 and TK^?ts13 cells. Cytoplasts from 6-day starved tsAF8 cells lost the complementing capacity for ts13 cells. However, when the 6-day starved tsAF8 cells were fused with G₀-ts13 cells, the heterokaryons entered S phase at the nonpermissive temperature. Also, cytoplasts isolated from the 6-day starved cells that were serum stimulated for 40 hr before enucleation regained the capacity to complement ts13 cells. These results demonstrate that three functions required in G₁ cannot be detected in the cytoplasm of serum-starved cells, although they are present in the cytoplasm of S-phase cells. These results suggest that a functional nucleus is required for the cytoplasmic appearance of certain G₁ functions in serumstimulated cells.     

Molecular characterization of two newDrosophila melanogaster homologs of human TAFII30

Two new homologs of human (h) TAF_II30, dTAF_II16 and dTAF_II24 were revealed inDrosophila melanogaster. The proteins are encoded by neighboring genes and bind with the TATA-binding protein and other dTAF_II proteins involved in TFIID. Only dTAF_II24 interacts with GCN5 histone acetyltransferase (HAT), which is the first demonstration of the TAF_II-GCN5-HAT complex inD. melanogaster. The two proteins have both common and individual location sites on polytene chromosomes. Possibly, the functions of dTAF_II16 and dTAF_II24 are similar but not identical.     

Transient reduction in secreted 32 kD chitinase prevents somatic embryogenesis in the carrot (Daucus carota L.) variant ts11

At the nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot (Daucus carota L.) cell variant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type cells. The causative component in the conditioned medium has been identified as an acidic 32 kD endochitinase. An antiserum raised against the 32 kD chitinase detected this protein in culture medium from ts11 embryo cultures grown at the permissive temperature as well as at the nonpermissive temperature. No difference in biochemical characteristics or in effect on ts11 embryo development could be detected between the 32 kD chitinase purified from wild-type cultures and the chitinase from ts11 cultures grown at the permissive or at the nonpermissive temperature. Compared to the amount present in a ts11 embryo culture at the permissive temperature, a reduction in the amount of 32 kD chitinase was observed during the temperature-sensitive period at the nonpermissive temperature. These results imply that the arrested embryo phenotype of ts11 is not the result of a structural difference in its 32 kD chitinase, but is the result of a transient decrease in the amount of 32 kD chitinase present. Morphological observations indicate that the ts11 phenotype is pleiotropic and also affects the cell wall of nonembryogenic cells. © 1995 Wiley-Liss, Inc.