Transcript Lectture 10 Biol302 Spring 2012

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 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
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)