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Download Lectture 10 Biol302 Spring 2012
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The Differentiation of Vertebrate Immune Cells In the immune system, two types of cells participate directly in defense against pathogens. Plasma B cells produce and secrete immunoglobulins (antibodies), and killer T cell produce membranebound proteins that act as receptors for various substances. B cell antibodies and T cell receptors bind to specific antigens. A cell must make many varieties of these proteins because there are many potential pathogens. An Antigen-Antibody Complex Structure of an Antibody Molecule Human Antibody Genes Two light chain loci: the on chromosome 2 and on chromosome 22 One heavy chain locus on chromosome 14. Each locus consists of a long array of gene segments. Gene Segments for a Kappa Polypeptide 1. An LV gene segment, encoding a leader peptide, which is removed later, and the N-terminal 95 amino acids of the variable region of the kappa light chain. (76 gene segments in humans; 40 of these are functional) 2. A J gene segment, encoding the last 13 amino acids of the variable region of the kappa light chain. (5 gene segments in humans) 3. A C gene segment, encoding the constant region of the kappa light chain. (1 gene segment in humans) The Kappa Locus During B cell development, the kappa light chain gene that will be expressed is assembled from one LV segment, one J segment, and the C segment by somatic recombination. Segment joining is mediated by recombination signal sequences adjacent to each gene segment by a protein complex including RAG1 and RAG2 (recombination activating gene proteins 1 and 2). Many Different Antibodies Can Be Produced 40 LV segments 5 J segments 1 C segment = 200 kappa light chains. Recombination of gene segments can create 120 lambda light chains and 6600 different heavy chains. Combinatorial assembly of these allows production of 2,112,000 different antibodies. Even more antibodies are possible due to variation in recombination sites and hypermutability of the variable regions. Evidence for DNA Rearrangement During Immune Cell Differentiation http://www.youtube.com/watch?v=AxIMmNByqtM Conserved sequences in Bold CsCl centrifugation of DNA over time developed by Meselson and Stahl We will talk about this again in a later lecture: But CsCl gradients are not the same thing as Sucrose Gradients or Agarose Gel Electrophoresis. CsCl centrifugation of DNA over time N15 is heavier than N14-Can be resolved in CsCl pulse-chase Experiment: Incubator with N15 containing medium for time, then chase with N14 medium Expt 1 grows Slowly Expt 2 Bacteria Grow Faster Why? Why would they do 2 different growth rates? Experiment 1 Experiment 2 N14 N15 only N14 N15 only Fuse Results from Expt 1 and 2 Cell Divisions N14 N15 only Experiment 1 observations Watson-Crick Model N14 N15 only Does Expt 1 prove hybrid formation? N15 dsDNA N15 ssDNA Critical Experiment: Hybrid Strand Separation And CsCl centrifugation Looks like control below What about N14/N15 hybrid? N14 ssDNA N15 ssDNA Evolution? Movie time In class question (extra credit) for Quiz #4 Question 1: (0.5pts) Why does one add EtBr to CsCl gradients for the isolation of plasmid DNA? Question 2: (0.5pts-All or None credit) Is an 8kb supercoiled plasmid more dense than a 3kb supercoiled plasmid. Yes/No (circle one) Will an 8kb supercoiled plasmid have more EtBr bound to it? Yes/No (circle one)
