320회 Heterozygous disruption of the TATA-binding protein gene in DT40 …

320회

연사 : 엄 문 경 , Department of  Biology, Columbia University

제목:  Heterozygous disruption of the TATA-binding protein gene in DT40 cells causes multiple growth defects, including reduced cdc25B phosphatase expression and  delayed mitosis

Abstract

 TATA-binding protein (TBP) is a key general transcription factor required for transcription by all three nuclear RNA polymerases.  Although it has been intensively analyzed in vitro and in S. cerevisiae, in vivo studies in vertebrates have been limited.  We applied gene targeting techniques using chicken DT 40 cells to generate heterozygous cells with one copy of the TBP gene disrupted.  Such TBP-heterozygous (TBP-Het) cells show unexpected phenotypic abnormalities, resembling those of cells with delayed mitosis: a significantly slower growth rate and larger size, more G2-M than G1 phase cells, and a high proportion of sub-G1, presumably apoptotic, cells.  Further evidence for delayed mitosis in TBP-Het cells was provided by the differential effects of several cell-cycle arresting drugs.  To determine the cause of these defects, we first examined the status of cdc2 kinase, which regulates the G2/M transition, and strikingly observed more hyperphosphorylated, inactive cdc2 in TBP-Het cells.  Offering an explanation for this, mRNA and protein levels of cdc25B, the trigger cdc2 phosphatase, were significantly lower in TBP-Het cells.  These properties of TBP-Het cells are all due to the TBP haploid state, as they could be rescued by expression of exogeneous TBP, including, in most but not all cases, a mutant lacking the species-specific N-terminal domain.  These results emphasize the importance of maintaining proper levels of TBP in vertebrate cells.