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Regulation of the hematopoietic transcription factor PU.1 (Spi-1) plays a critical role in the development of white cells, and abnormal expression of PU.1 can lead to leukemia. We previously reported that the PU.1 promoter cannot induce expression of a reporter gene in vivo, and cell-type-specific expression of PU.1 in stable lines was conferred by a 3.4-kb(More)
The reaper protein of Drosophila melanogaster has been shown to be a central regulator of apoptosis in that organism. However, it has not been shown to function in any vertebrate nor have the cellular components required for its action been defined. In this report we show that reaper can induce rapid apoptosis in vitro using an apoptotic reconstitution(More)
Apoptosis is essential for the development and homeostasis of multicellular organisms. Recently, a cell-free extract prepared from Xenopus eggs was shown to recapitulate intracellular apoptotic pathways in vitro. While many stimuli have been shown to trigger apoptosis in a variety of cell types, the intracellular signaling pathways involved in apoptosis(More)
Reaper is a potent apoptotic inducer critical for programmed cell death in the fly Drosophila melanogaster. While Reaper homologs from other species have not yet been reported, ectopic expression of Reaper in cells of vertebrate origin can also trigger apoptosis, suggesting that Reaper-responsive pathways are likely to be conserved. We recently reported(More)
The transcription factor C/EBP alpha (CCAAT/enhancer binding protein alpha) is critical for granulopoiesis. Gene disruption in mice blocks early granulocyte differentiation and disruption of C/EBP alpha function has been implicated in human acute myeloid leukemia (AML), but no systematic structure-function analysis has been undertaken to identify the(More)
Many of the biochemical reactions of apoptotic cell death, including mitochondrial cytochrome c release and caspase activation, can be reconstituted in cell-free extracts derived from Xenopus eggs. In addition, because caspase activation does not occur until the egg extract has been incubated for several hours on the bench, upstream signaling processes(More)
In this unit, Xenopus eggs are isolated from hormonally primed female frogs, and then the extract is treated with cyclohexamide so it remains in interphase of the cell cycle. In the presence of sperm chromatin and ATP, membrane vesicles in the extract fuse to assemble nuclei, making the extract suitable for studies of DNA replication and nuclear transport.
In the presence of a subcellular fraction enriched for mitochondria, after prolonged incubation the Xenopus egg extract can mimic biochemical aspects of apoptosis such as caspase activation, and DNA fragmentation. This unit describes preparation of an apoptotic extract from a crude interphase extract and an extract fractionated into latent and execution(More)
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