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Unformatted text preview: itz undertook a genetic analysis with the goal of characterizing the mechanism and regulation of programmed cell death during C. elegans development. In the experiments reported in this 1986 paper, Hilary Ellis and Horvitz identified two genes that were required for all of the programmed cell deaths that took place during development of the nematode. The identification and characterization of these genes was a critical first step leading to our current understanding of the molecular biology of apoptosis.
H. Robert Horvitz The Experiments
Cells undergoing programmed cell death in C. elegans can readily be identified as highly refractile cells by microscopic examination, so Ellis and Horvitz were able to use this as an assay to screen for mutant animals in which the normal cell deaths did not occur. To isolate mutants that displayed abnormalities in cell death, they treated nematodes with the chemical mutagen ethyl methanesulfonate, which reacts with DNA. The progeny of approximately 4000 worms were examined to identify dying cells, and two mutant strains were found in which the expected cell deaths did not take place (see figure). Both of these mutant strains harbored recessive mutations of the same gene, which was called ced-3. Further studies indicated that the mutations in ced-3 blocked all of the 131 programmed cell deaths that would normally occur during development. Continuing studies identified an additional mutation that was similar to ced-3 in preventing programmed cell
Photomicrographs of a normal worm (A) and a ced-3 mutant (B). Dying cells are highly refractile and are indicated by arrows in panel A. These cells are not present in the mutant animal. death. However, this mutation was in a different gene, which was located on a different chromosome than ced-3. This second gene was called ced-4. Similar to mutations in ced-3, recessive mutations in ced-4 were found to block all programmed cell deaths in the worm. The Impact
The isolation of C. elegans mutants by Ellis and Horvitz provided the first identification of genes that were involved in the process of programmed cell death. The proteins encoded by the ced-3 and ced-4 genes, as well as by (A) (B) This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS
CELL DEATH AND CELL RENEWAL 5 KEY EXPERIMENT the ced-9 gene (which was subsequently identified by Horvitz and colleagues), proved to be prototypes of the central regulators and effectors of apoptosis that are highly conserved in evolution. The cloning and sequencing of ced-3 revealed that it was related to a protease that had been previously identified in mammalian cells, and which became the first member of the caspase family. The C. elegans ced-9 gene was related to the bcl-2 oncogene, first isolated from a human B cell lymphoma, which had the unusual property of inhibiting apoptosis rather than stimulating cell proliferation. And ced-4 was found to encode an adaptor protein related to mammalian Apaf-1, which is required for caspase activation. The identification of these genes in C. elegans thus led the way to understanding the molecular basis of apoptosis, with broad implications both for development and for the maintenance of normal adult tissues. Since abnormalities of apoptosis contribute to a wide variety of diseases, including cancer, autoimmune disease, and neurodegenerative disorders, the seminal findings of Ellis and Horvitz have impacted a wide range of areas in biology and medicine. tion, the normal programmed cell deaths did not take place. A third gene, ced-9, functioned as a negative regulator of apoptosis. If ced-9 was inactivated by mutation, the cells that would normally survive failed to do so. Instead, they also underwent apoptosis, leading to death of the developing animal. Conversely, if ced-9 was expressed at an abnormally high level, the normal programmed cell deaths failed to occur. Further studies indicated that the proteins encoded by these genes acted in a pathway with Ced-4 acting to stimulate Ced-3, and Ced-9 inhibiting Ced-4 (Figure 17.3). Genes related to ced-3, ced-4, and ced-9 have also been identified in Drosophila and mammals and found to encode proteins that represent conserved effectors and regulators of apoptosis induced by a variety of stimuli. Caspases: The Executioners of Apoptosis
The molecular cloning and nucleotide sequencing of the ced-3 gene indicated that it encoded a protease, providing the first insight into the molecular mechanism of apoptosis. We now know that Ced-3 is the prototype of a family of more than a dozen proteases, known as caspases because they have cysteine (C) residues at their active sites and cleave after aspartic acid (Asp) residues in their substrate proteins. The caspases are the ultimate effectors or executioners of programmed cell death, bringing about the events of apoptosis by cleaving more than 100 different cell target proteins (Figure 17.4). One key target of the caspases is an inhibitor o...
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