Chapter 21 The Genetic Control of Animal Development

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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 membrane-bound 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.

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An Antigen-Antibody Complex
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Structure of an Antibody Molecule
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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.

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Gene Segments for a Kappa Polypeptide

1. An L?V? 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)

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The Kappa Locus

• During B cell development, the kappa light chain
  gene that will be expressed is assembled from one
  L?V? 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).

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Many Different Antibodies Can Be Produced

• 40 L?V? 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.

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Evidence for DNA Rearrangement During Immune Cell
Differentiation
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http//www.youtube.com/watch?vAxIMmNByqtM
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Conserved sequences in Bold
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CsCl centrifugation of DNA over time developed by
Meselson and Stahl
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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.
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CsCl centrifugation of DNA over time
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N15 is heavier than N14-Can be resolved in CsCl
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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?
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Why would they do 2 different growth rates?
Experiment 1
Experiment 2
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Fuse Results from Expt 1 and 2
Cell Divisions
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Experiment 1 observations
Watson-Crick Model
Does Expt 1 prove hybrid formation?
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N15 dsDNA
N15 ssDNA
Critical Experiment Hybrid Strand Separation And
CsCl centrifugation
Looks like control below
N15 ssDNA
N14 ssDNA
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Evolution?
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Movie time
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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)