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What did Herostratus do? A. He designed the Colossus of Rhodes. B. He financed the construction of the lighthouse of Alexandria. C. He burned the temple of Artemis. D. He decorated the statue of Zeus at Olympia. E. Dude, I don’t care what you say, Milla Jovovich ROCKS. Can’t wait for “Resident Evil IV: the Life of Dick Cheney” MCB 140, 09/26/07 1 Image courtesy of Prof. Jay Hollick, MCB Department MCB 140, 09/26/07 2 ?!!! R. Alexander Brink, 1950 Vicky Chandler, Jay Hollick et al MCB 140, 09/26/07 3 What is going on? 1. The green maize plant is not outcrossing, it is selfing. 2. The red allele is incompletely penetrant, except when in a homozygous state. 3. This is actually two-locus inheritance, and the green locus exhibits full dominant epistasis to the red locus. 4. This is actually two-locus inheritance, and the green locus exhibits full recessive epistasis to the red locus. 5. Mmmmm … Milla … MCB 140, 09/26/07 4 What IS going on This is a phenomenon known as “paramutation.” It involves a change in gene behavior that is not associated with a change in the DNA sequence. Hence, it is, strictly speaking, not in the realm of genetics. Thus, it is epigenetic. MCB 140, 09/26/07 5 Broadly speaking An “epigenetic” effect on the genome changes the phenotype without changing the genotype. The power of the environment and of life history MCB 140, 09/26/07 6 Technically “A mitotically or meiotically heritable change in gene expression state (or genome functional state) that is not associated with a change in the primary sequence of DNA.” MCB 140, 09/26/07 7 In other words Genetics Organism (or a cell) with a phenotype ↓ Mutation (change in DNA) ↓ Different phenotype Epigenetics Organism (or a cell) with a phenotype ↓ Something happens, but not a change in the DNA ↓ Different phenotype MCB 140, 09/26/07 8 “Cloning”: hello, Dolly, and hello again, Dolly MCB 140, 09/26/07 9 King and Briggs (1956). Serial transplantation of embryonic nuclei Cold Spring Harbor Symp. Quant. Biol. 21: 271-289. MCB 140, 09/26/07 10 McKinnell, R. G. 1978. Cloning: Nuclear Transplantation in Amphibia. University of Minnesota Press, Minneapolis. MCB 140, 09/26/07 11 ? MCB 140, 09/26/07 12 How can one explain the fact that cloning works so much better if one use a cell from an early embryo as the donor of the nucleus? MCB 140, 09/26/07 13 Two explanations 1. Alteration of the actual DNA of the cells as the embryo develops. 2. Something else. MCB 140, 09/26/07 14 MCB 140, 09/26/07 15 Reya, Clarke, and Weissman. Nature 2001. MCB 140, 09/26/07 16 MCB 140, 09/26/07 17 MCB 140, 09/26/07 18 MCB 140, 09/26/07 19 Of the cells on the previous set of slides, which have significant alterations in their DNA sequence relative to that of the original zygote? A. B. C. D. E. The neuron. The fibroblast. The muscle cell. The T cell. The macrophage. MCB 140, 09/26/07 20 King and Briggs (1956). Serial transplantation of embryonic nuclei Cold Spring Harbor Symp. Quant. Biol. 21: 271-289. MCB 140, 09/26/07 21 MCB 140, 09/26/07 22 MCB 140, 09/26/07 23 Dolly MCB 140, 09/26/07 24 Bill Ritchie Ian Wilmut Dolly MCB 140, 09/26/07 25 Semantics 1. Reproductive cloning: make new organisms. 2. Therapeutic cloning (aka “somatic cell nuclear transfer”): no organism made. MCB 140, 09/26/07 26 Extensive abnormalities in cloned animals • • • • Lung failure Liver failure Obesity Etc etc Two problems: 1. Cloning is incredibly inefficient. 2. Of the animals that are born, many have severe defects. MCB 140, 09/26/07 27 Proof that these abnormalities are entirely epigenetic Dolly’s lambs, and the offspring of all cloned animals, are normal. MCB 140, 09/26/07 28 Solter and Surani Gynogenetic embryos – very small. Androgenetic embryos – very large. MCB 140, 09/26/07 29 MCB 140, 09/26/07 30 Ah, terminology Genes for which you have your Mother’s copy turned on: Maternally expressed Genes for which you have your Dad’s copy turned on: Paternally expressed MCB 140, 09/26/07 31 What covalent posttranslational modification does genomic DNA undergo during mammalian ontogeny? A. B. C. D. E. Phosphorylation Methylation Acetylation All of the above None of the above MCB 140, 09/26/07 32 Spontaneous meCpG deamination (colon cancer) Should be 4% of all NN – in fact, is 0.8%. Methylation: C 5mC CpG 5mCpG 5mCpG TpG TpA deamination MMR CpG UpG CpG (no mutation) MCB 140, 09/26/07 33 Pl is Changed to Pl’ “paramutation”! MCB 140, 09/26/07 34 The Haig hypothesis Imprinting evolved as a manifestation of parental conflict over the allocation of maternal resources to the developing fetus: “intrauterine tug of war” over how big the fetus will be. Paternally expressed genes increase embryo size. Maternally expressed genes decrease embryo size. MCB 140, 09/26/07 35 Peromyscus polionotus (the monogamous mouse) Vrana et al. Nature Genetics 20: 362 (1998). MCB 140, 09/26/07 36 Don’t clone humans 1. Responsibility for child and his/her “developmental abnormalities.” 2. Naïve overestimation of role of DNA in shaping the human being. MCB 140, 09/26/07 37 “Therapeutic cloning” = somatic cell nuclear transfer MCB 140, 09/26/07 38 Embryonic stem cells MCB 140, 09/26/07 39 Annu. Rev. Cell Dev. Biol. 2001. 17:435-462. EMBRYO-DERIVED STEM CELLS: Of Mice and Men Austin G. Smith MCB 140, 09/26/07 40 ES cells – status quo • Limited number of human ES cell lines available for research with federal funds • Growth on mouse feeders makes them unsuitable for use as therapeutics • The indications being considered are, among others, cardiovascular and neurological MCB 140, 09/26/07 41 Why ES cells and not adult stem cells? For the simple reason that ES cells are incomparably easier to grow to large numbers in a dedifferentiated state, and then drive them – in a controlled fashion! – to differentiate into a specific cell type. Note: in this context, “incomparably” means “the difference between essentially impossible and feasible.” MCB 140, 09/26/07 42 “Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease” – Ron McKay et al Parkinson's disease is a widespread condition caused by the loss of midbrain neurons that synthesize the neurotransmitter dopamine. Cells derived from the fetal midbrain can modify the course of the disease, but they are an inadequate source of dopaminesynthesizing neurons because their ability to generate these neurons is unstable. In contrast, embryonic stem (ES) cells proliferate extensively and can generate dopamine neurons. If ES cells are to become the basis for cell therapies, we must develop methods of enriching for the cell of interest and demonstrate that these cells show functions that will assist in treating the disease. Here we show that a highly enriched population of midbrain neural stem cells can be derived from mouse ES cells. The dopamine neurons generated by these stem cells show electrophysiological and behavioural properties expected of neurons from the midbrain. Our results encourage the use of ES cells in cell-replacement therapy for Parkinson's disease. Nature. 2002 Jul 4;418(6893):50-6 MCB 140, 09/26/07 43 The problem In order to generate ES cells, one has to destroy an early human embryo • Twenty eight thousand IVF births in the US in 1998 • Six to fourteen embryos per birth – healthy ones frozen, and then discarded (=flushed down a sink) MCB 140, 09/26/07 44 MCB 140, 09/26/07 45 Reading Life's Dominion: An Argument about Abortion, Euthanasia, and Individual Freedom Ronald Dworkin MCB 140, 09/26/07 46 A way to overcome this entire issue? 1. Patient with failing organ. 2. Take nucleus from patient’s cell. 3. Do somatic cell nuclear transfer to generate ES line from that patient. 4. Transdifferentiate that line ex vivo into cell type relevant to disease. 5. Reimplant in patient. MCB 140, 09/26/07 47 Correction of a Genetic Defect by Nuclear Transplantation and Combined Cell and Gene Therapy Rideout et al. Cell (2002) 109, 17-27 MCB 140, 09/26/07 48 MCB 140, 09/26/07 49 Pigmented retinal epithelium Neuroepithelial rosset http://www.nature.com/news/specials/hwang/index.html Bone Cartilage Glandular epithelium with smooth muscle and connective tissue MCB 140, 09/26/07 50 What’s next with “therapeutic cloning”? (“nuclear transfer”) I don’t know. There is likely to be a complex polemic between patient advocacy groups (on the one hand) and groups opposed to somatic cell nuclear transfer on various grounds. MCB 140, 09/26/07 51 Mice cloned from olfactory sensory neurons Eggan et al. (Rudolf Jaenisch and Richard Axel) Nature (2004) 428(6978):44-9 MCB 140, 09/26/07 52 Natalie Angier Unnatural Obsessions “The adjective that scientists use to describe a well-wrought experiment is “elegant” – which means not only that it worked, but it worked in style.” MCB 140, 09/26/07 53 MCB 140, 09/26/07 54 Sense of smell “One particularly clear example of neuronal diversity is provided by the olfactory sensory epithelium. In the mouse, each of the 2,000,000 cells in the olfactory epithelium expresses only one of about 1,500 odorant receptor genes, such that the functional identity of a neuron is defined by the nature of the receptor it expresses. Thus, the sensory epithelium consists of at least 1,500 neuronal types. The pattern of receptor expression is apparently random within one of four zones in the epithelium, suggesting that the choice of receptor gene may be stochastic. One mechanism to permit the stochastic choice of a single receptor could involve DNA rearrangements.” Eggan et al. Nature. 2004 Mar 4;428(6978):44-9 MCB 140, 09/26/07 55 Allelic inactivation regulates olfactory receptor gene expression We suggest a model in which a hierarchy of controls is exerted on the family of odorant receptor genes to assure that a sensory neuron expresses a single receptor from a family of 1000 genes. We propose that a cis-regulatory element directs the stochastic expression of only one gene from a large array of linked receptor genes. Moreover, only one allelic array encoding multiple receptor genes is active in an individual neuron. We demonstrate that in a neuron expressing a given receptor, expression derives exclusively from one allele. Chess et al. Cell. 1994 Sep 9;78(5):823-34 MCB 140, 09/26/07 56 Eggan et al. Nature. 2004 Mar 4;428(6978):44-9 MCB 140, 09/26/07 57 Eggan et al. Nature. 2004 Mar 4;428(6978):44-9 MCB 140, 09/26/07 58 MCB 140, 09/26/07 59 Question answer “The regulation of gene expression by DNA rearrangement is rare, but this mechanism has nonetheless been suggested to explain the diversity inherent in complex nervous systems.” Well, we now know that it is NOT how neuronal diversity in olfactory epithelium is created. The difference between the individual neurons expressing different receptors is not at the level of DNA – it’s epigenetic. Eggan et al. Nature. 2004 Mar 4;428(6978):44-9 MCB 140, 09/26/07 60