Final Review. Aptamers. Making Aptamers: SELEX 6/3/2011. sirna and mirna. Central Dogma. RNAi: A translation regulation mechanism.

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1 Central Dogma Final Review Section Week 10 DNA RNA Protein DNA DNA replication DNA RNA transcription RNA Protein translation **RNA DNA reverse transcription RNAi: A translation regulation mechanism Silencing of gene expression, triggered by the doublestranded RNA homologous to portions of the gene. Effect due to cleavage and degradation of the target gene mrna and blocking translation of intact mrna Only one of the two strands, the guide strand, binds directs gene silencing. The anti-guide strand or passenger strand is degraded during RISC activation. sirna and mirna Single stranded nucleic acid molecules that bind to a specific target molecule The term "aptamer" derives from the Latin aptus to fit Antibody-like Can be selected to catalyze reactions Aptamers Making Aptamers: SELEX SELEX ("Systematic Evolution of Ligands by Exponential Enrichment") also referred to as in vitro selection or in vitro evolution Aptamers have been made against small molecules, peptides, proteins, organelles, viruses, cells 1

2 Making Aptamers: SELEX Affinity Chromatography Solid support Example: antibody affinity chromatography PCR Polymerase Chain Reaction Enzyme Linked ImmunoSorbent Assay (ELISA) Colorimetric Assay for Enzyme Activity (Measure Light Absorbance of Product) ampicons ( 0 2n N n N Figures Adapted from: Nelson & Cox, Lehninger Principles of Biochemistry, 3rd Ed., Worth Publishers, (2000) Figures Adapted from: Nelson & Cox, Lehninger Principles of Biochemistry, 3rd Ed., Worth Publishers, (2000) Western Blot An analytical technique used to detect the presence of specific proteins in a sample 1. Separation of protein (native/denaturing) using gel electrophoresis 2. Transfer of proteins to a membrane 3. Use antibodies to detected protein of interest Western Blot Use antibodies to detected protein of interest Unreacted sites can be blocked by incubating the membrane with an innocuous protein such as Bovine Serum Albumin Treat blocked membrane with primary antibody Wash Treat membrane with secondary antibody Wash Image 3. Unreacted sites are white Reacted sites are black 2

3 Flow cytometry Viral Entry Strategies A technique for counting and examining cells by suspending them in a stream of fluid and passing them by an electronic detection apparatus Simultaneous multi-parameter analysis of single cells expression of cell surface and intracellular molecules (antibody staining / cells expressing FPs) characterizing and defining different cell types in heterogeneous cell populations assessing the purity of isolated subpopulations analyzing cell size and volume Taken from Molecular Biology of the Cell. 4 th ed. Alberts et al. Influenza Lifecycle - Compilation Structure of HIV 3D reconstruction of HIV-1 virions using cryo-electron microscopy What are the red, yellow, and black objects in the capsid? Reverse transcriptase, protease, and integrase! (For exogenous retrovirus start here) A more accurate depiction of the HIV lifecycle Binding Membrane Fusion Maturation Uncoating Reverse Transcription Overview of Retroviral Life Cycle Budding A Gag-Pol protein Nuclear transport Integration Expression Membrane Targeting (For endogenous retrovirus start here) D Souza & Summers, 2005, Nature Reviews 3:

4 The HIV Genome What is an antibody? An antibody is a protein produced by the immune system that has high binding specificity for another protein*. The protein that an antibody binds to is called the antigen. When antibodies are produced and used by the adaptive immune system, they commonly recognize bacterial or viral antigens and assist in neutralizing them Biologists can now make custom antibodies that bind to almost any antigen, regardless of whether the antigen is derived from a pathogen or part of the host. This greatly facilitates our study of the function and localization of proteins. * Antibodies can also bind to DNA and carbohydrates, which you will explore later. Antibodies Making Polyclonal Antibodies Serum contains antibodies mouse Antigen Polyclonal antibodies recognize many epitopes Purify antibodies by using a tissue which doesn t have your antigen of interest. Further purification on an affinity column Multiple antibodies bind to the specific antigen, can you think of an instance where this may be beneficial? Making Monoclonal Antibodies Monoclonal antibodies recognize one epitope Hapten Carrier Effect MHC molecules can only bind peptides, so to make antibodies against non-peptide molecules (like a hapten), coupling the hapten to a protein is necessary. Antigen Why do you need to fuse antibody forming B-cells with cancer cells? 4

5 Different ways antibodies neutralize pathogens The Mammalian Immune System Timeline of the Immune Response These antibodies circulate through the bloodstream and lymphatic vessels. Phagocytes: white blood cells that protect the body by ingesting harmful foreign particles, bacteria, and dead or dying cells Macrophages (big eaters) A large phagocytic cell of the mononuclear series found within tissues. Properties include phagocytosis, and antigen presentation to T cells Dendritic cells Their main function is to process antigen material and present it on the surface to other cells of the immune system. That is, they function as antigen-presenting cells. They act as messengers between the innate and adaptive immunity Major Players Cellular Lymphocytes: a type of white blood cell in the vertebrate immune system Helper T cells A lymphocyte which undergoes a developmental stage in the thymus Cytotoxic T cells Cell that kills target cells bearing appropriate antigen within the groove of an MHC class I molecule that is identical to that of the T cell B cells The precursors of antibody-forming plasma cells; these cells carry immunoglobulin and class II MHC (major histocompatibility complex) antigens on their surfaces Major Players Molecular Major histocompatibility complex (MHC class I and class II) A cluster of genes on chromosome 6 in humans, encoding cell surface molecules that are polymorphic and that code for antigens which lead to rapid graft rejection between members of a single species which differ at these loci. Several classes of protein such as MHC class I and II proteins are encoded in this region. These in humans, are known as 'Human leukocyte antigens' (HLA) CD4 A protein on the surface of certain leukocytes. Helper T cells are among the cell types that possess CD4. CD4 is involved in the binding of HIV to its host cells CD8 A protein that binds to a major histocompatibility complex (MHC) molecule, but is specific for the class I MHC protein. There are two isoforms of the protein, alpha and beta, each encoded by a different gene. CD8 is found on cytotoxic T lymphocytes. Complement system A series of serum proteins involved in the mediation of immune reactions. The complement cascade is triggered classically by the interaction of antibody with specific antigen. Antibodies Serum protein formed in response to immunization; antibodies are generally defined in terms of their specific binding to the immunizing antigen. 63_2.html B-cells presenting a compatible class II MHC:antigen complex to viral TCR leads to clonally expansion and to produce antibody Generation of Antibody Diversity This can take place in the lymph nodes or in the spleen. The antibodies produced are specific to molecules associated with the bacterial pathogen. Multiple V, D, J gene segments Junctional diversity -- random pairing of a V and a J gene segment (light chain) or a V, D, and a J gene segment (heavy chain). Addition or deletion of bases during joining of V to D and/or V to J and/or D to J creates even more diversity within CDR3 Combinatorial pairing of H and L chains Each antibody molecule has either the κ or λ light chain, but not both. Somatic hypermutation -- high rate of mutation in antibody variable region genes during clonal expansion of a B cell. Result: some B cell descendents produce antibodies that bind more tightly to an antigen (these are stimulated to divide further); others produce antibodies that don t bind as tightly (these are not stimulated to divide further). 5

6 Antibody diversity: VDJ recombination Generation of antibody diversity involves somatic gene rearrangements and hypermutation Vaccination For most viruses, you are immune to reinfection by the same virus (e.g., chicken pox). Immunization or vaccination: Process by which one is exposed to a live or inactivated virus, or to components of the virus, in order to establish a state of immunity. Immunizations against smallpox introduced >1000 years ago. Variolation: introduce dried smallpox scabs into nose of an uninfected person, who then contracted a mild form of the disease but was immune to smallpox. 1-2% died after variolation compared with 30% after smallpox. Vaccination against smallpox introduced by Edward Jenner in Jenner infected a boy with cowpox (a live-attenuated virus), then exposed him to smallpox, which he failed to contract. Jenner later experimented on other children, including his son. These sorts of experiments are illegal today. HIV vaccines? Pantello and Koup, 2004, Nature Medicine 10: HIV has a very fast mutation rate which can effectively overthrow many antibodies produced against it. The sheer diversity of strains of HIV in the population also prove to be a problem for vaccine makers. Broadly neutralizing HIV antibodies are rare but real. Good luck! Figures taken from Pamela s lectures 6

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