1Lecture 17: C. elegansSome facts about C. elegans3 days life cycleInvariant cell lineage959 cells in hermaphrodite1031 cells in male Small genome 97 MB19,000 genes (1 gene per 5 kb)Two sexes: hermaphrodite and maleSix pairs of chromosomes2Fig. C.13XX: hermaphroditeXO: male4Fig. C.5bLife cycle5DNA transformation• Inject DNA into distalsyncytial gonad ofhermaphrodites• Irradiation promotesintegration oftransgenes into genome• Reporter constructsshow transgenesFig. C.86Fig. C.7b7Fig. C.5a8Fig. C.7aInvariant cell lineages(959 somatic cells in hermaphrodite)9Sydney BrennerJohn SulstonBob Horvits2002 Nobel Prize Winners10TUNEL: TdT-mediated dUTP digoxigenin nick end labelingApoptosis patterns in developing Xenopus embryos detected via TUNELApoptosis is important in development11Fig. C.1512Programmed Cell Death Naturally occurring, or programmed,cell death (apoptosis) is common during animal development,and abnormalities in programmed cell death are associatedwith many human diseases, including certain cancers andneurodegenerative disorders. Our laboratory has defined amolecular genetic pathway for programmed cell death. Wehave characterized genes that cause cells to die, that protectcells from dying, that function in the engulfment of dying cellsby their neighbors, and that are involved in destroying thedebris generated by cell corpses. Most of these genes havehuman counterparts. For example, the killer gene ced-3encodes a caspase (cysteine aspartate protease);mammalian caspases similarly cause programmed cell death.The action of ced-3 is facilitated by ced-4, which is similar tohuman APAF1, identified because it promotes caspaseactivation in a biochemical system. The function of ced-4 isblocked by ced-9, which protects cells against programmedcell death and is similar to the human proto-oncogene BCL2,which also protects against cell death. The activity of ced-9 isinhibited by the worm killer gene egl-1, which is similar to anumber of mammalian killer genes. The activity of egl-1 iscontrolled in a cell-specific fashion by genes that specifywhich cells are to live and which are to dieBob Horvitz13 Figure 13.48. Regulators and effectors of apoptosis Many cell death signalsinduce apoptosis via a conserved pathway of regulators, adaptors, and caspases. InC. elegans, the negative regulator Ced-9 inhibits apoptosis by binding to the adaptorCed-4. In the absence of inhibition by Ced-9, Ced-4 binds two molecules of thecaspase Ced-3, resulting in autocleavage and caspase activation. In mammals,regulators of the Bcl-2 family (Ced-9 homologs) act at the mitochondria to controlrelease of cytochrome c, which is required for the binding of caspase-9 to the adaptorApaf-1 (the Ced-4 homolog). Release of cytochrome c from mitochondria thus signalsthe activation of caspase-9, which then activates downstream caspases to induceapoptosis14 Image using confocal microscopy of a C. elegans embryo in which allcells have been caused to initiate programmed cell death(apoptosis). The cell-death killer protein CED-4 (red) and the nuclearenvelope protein lamin (green) are both seen at the nuclearenvelope (overlap is yellow). By contrast, in normal embryos, CED-4is instead localized to mitochondria. This experiment helped revealthat CED-4 translocates from mitochondria to the nuclear envelopeduring programmed cell death.Programmed cell death in c. elegans15http://chronic.dartmouth.edu/VRA/ambroslab.htmlThe C. elegans Lin-4 was the first microRNA discoveredIn Dr. Victor Ambros’ labLee RC, Feinbaum RL, Ambros V. (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell 75(5):843-54.Lee RC, Ambros V. (2001) An extensive class of small RNAs in Caenorhabditis elegans.Science. 2001 Oct 26;294(5543):862-4.16micro RNA (miRNA)• Gene expression regulation• Created by similar process to siRNA• Generally prevents binding of ribosome• Or degrade target mRNA17Genetic pathway that regulates developmental timing181920RNAi was first discovered in C. elegansAndy FireFire et al., (1998) Potent and specific genetic interference bydouble stranded RNA in C. elegans. Nature vol 391, 806-810Nobel prize-200621Fig.
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