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.

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 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)

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).

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.

Evidence for DNA Rearrangement During Immune Cell Differentiation

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

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

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?

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

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

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

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?

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

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

HMR a HML alpha

Evolution?

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