2. 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.

3. An Antigen-Antibody Complex

4. Structure of an Antibody Molecule

5. 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.

6. 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)

7. 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).

11. 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.

12. Evidence for DNA Rearrangement During Immune Cell Differentiation

13. http://www.youtube.com/watch?v=AxIMmNByqtM

17. Conserved sequences in Bold

25. CsCl centrifugation of DNA over time developed by Meselson and Stahl

27. 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.

29. CsCl centrifugation of DNA over time

30. N 15 is heavier than N 14 -Can be resolved in CsCl

31. pulse-chase Experiment: Incubator with N 15 containing medium for time, then chase with N 14 medium Expt 1 grows Slowly Expt 2 Bacteria Grow Faster Why?

32. Experiment 1 Experiment 2 N 14 N 15 only Why would they do 2 different growth rates? N 14 N 15 only

33. Fuse Results from Expt 1 and 2 Cell Divisions N 14 N 15 only

34. Experiment 1 observations Watson-Crick Model Does Expt 1 prove hybrid formation? N 14 N 15 only

35. Critical Experiment: Hybrid Strand Separation And CsCl centrifugation N 15 ssDNA N 14 ssDNA N 15 ssDNA N 15 dsDNA Looks like control below What about N 14 /N 15 hybrid?

36. Question 1: Why does one add EtBr to CsCl gradients for the isolation of plasmid DNA? Question 2: 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

40. Figure 19.22 Copyright © 2010 Academic Press Inc. Gene Conversion of HML alpha or HMR a Loci into MAT locus

41. HMR a HML alpha

44. Evolution?

46. Movie time