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 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 developedby Meselson and Stahl

We will talk about this again in a later lecture:But CsCl gradients are not the same thing as SucroseGradients 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 2BacteriaGrow FasterWhy?

Experiment 1

Experiment 2

N14 N15

only

Why would they do 2 different growth rates?

N14 N15

only

Fuse ResultsfromExpt 1 and 2

Cell Divisions

N14 N15

only

Experiment 1 observations

Watson-Crick Model

Does Expt 1 prove hybrid formation?

N14 N15

only

CriticalExperiment:

Hybrid StrandSeparationAnd CsCl centrifugation N15

ssDNAN14

ssDNA

N15

ssDNA

N15

dsDNA

Looks like control below

What aboutN14/N15 hybrid?

Question 1: Why does one add EtBr to CsCl gradients for the isolationof plasmid DNA?

Question 2: Is an 8kb supercoiled plasmid more dense than a3kb supercoiled plasmid. Yes/No (circle one)

Will an 8kb supercoiled plasmid have more EtBrbound to it? Yes/No

Figure 19.22

Copyright © 2010 Academic Press Inc.

GeneConversionof HML alphaor HMR a Loci into MAT locus

HMR a HMR a

HML alpha HML alpha

Evolution?

Movie time