Organization and

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Chapter 5
Organization and Expression of
Immunoglobulin Genes

• Genetic Model Compatible with lg Structure
• Multigene Organization of lg Genes
• Variable-Region Gene Rearrangements
• Mechanism of Variable-Region DNA Rearrangements
• Generation of Antibody Diversity
• Class Switching among Constant-Region Genes
• Expression of lg Genes
• Synthesis, Assembly, and Secretion of
  Immunoglobulins
• Regulation of lg-Gene Transcription
• Antibody Genes and Antibody Engineering

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FIGURE 5-1
Overview of B-cell development in bone marrow.
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FIGURE 5-1
Overview of B-cell development in peripheral
lymphoid organs.
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Three properties of antibodies
? The vast diversity of Ab specificities ? A
variable region a constant region in each
chain ? The existence of isotypes with the same
antigenic specificity
Hypotheses
- The germ-line theories the entire variable
region repertoire is transmitted through the germ
cells - The somatic-variation theories (germ
line contains a limited of variable genes a
large of Ab specificities are generated by
mutation or recombination in the somatic cells) -
Two gene model in 1965 proposed by Dreyer
Bennett (one or several genes for the V region
the other for the C region) (lt-gt one
gene-one polypeptide principle) gtgtgt
Immunoglobulin genes rearrange, in 1976
(In 1987, Tonegawa was awarded the Nobel prize)
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1. Genetic Model Compatible with lg Structure
FIGURE 5-2
Experimental basis for diagnosis of rearrangement
at an immunoglobulin locus.
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2. Multigene Organization of lg Genes
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2. Multigene Organization of lg Genes
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3. Variable-Region Gene Rearrangements
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3. Variable-Region Gene Rearrangements
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4. Mechanism of Variable-Region DNA Rearrangements
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4. Mechanism of Variable-Region DNA Rearrangements
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4. Mechanism of Variable-Region DNA Rearrangements
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4. Mechanism of Variable-Region DNA Rearrangements
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4. Mechanism of Variable-Region DNA Rearrangements
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4. Mechanism of Variable-Region DNA Rearrangements
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5. Generation of Antibody Diversity
Seven means of Ab diversification
? Multiple germ-line gene segments (Table 5-2)
(functional gene segments many
pseudogenes) ? combinational V-(D)-J joining
(Table 5-2) (due to the random rearrangement
of these segments in somatic cells) ?
combinational association of light heavy
chains. -gt minimal calculations of potential
Ab diversity gt 106 ? Junctional flexibility
(the RSS joined the signal sequences precisely
but not the coding sequences (Fig. 5-12)
leading to many nonproductive rearragement) ?
P-region nucleotide addition, producing
palindromic sequence in the coding joint ?
N-region nucleotide addition, (upto 15 N-nts can
be added by TdT) ? Somatic hypermutation -
occurs only within germinal centers at a
frequency approaching 10-3 / bp /
generation gtgtgt B cells with higher-affinity
receptors will be preferentially selected
for survival affinity maturation - The
potential for Ab diversity in humans gt10 B
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5. Generation of Antibody Diversity
FIGURE 5-12
Experimental evidence for junctional flexibility
in immunoglobulin-gene rearrangement.
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5. Generation of Antibody Diversity
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5. Generation of Antibody Diversity
FIGURE 5-13
P-nucleotide and N-nucleotide addition during
joining.
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5. Generation of Antibody Diversity
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5. Generation of Antibody Diversity
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7. Expression of lg Genes
(a)
FIGURE 5-16 (a)
Secreted membrane forms of the heavy chain by
alternative ( differential ) RNA processing of
primary transcript.
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7. Expression of lg Genes
(b)
cleavage
In naïve mature cells
In differentiated plasma cells
FIGURE 5-16
Expression of secreted and membrane forms of the
heavy chain by alternative RNA processing.
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7. Expression of lg Genes
5kb
FIGURE 5-17
Simultaneous expression of membrane forms of µ
and d heavy chain by alternative RNA processing.
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8. Synthesis, Assembly, and Secretion of Ig
FIGURE 5-18
Synthesis, assembly, and secretion of the
immunoglobulin molecule.
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9. Regulation of lg-Gene Transcription
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10. Antibody Genes Engineering
FIGURE 5-20
Production of chimeric mouse-human mAb By
transfection of the chimeric vector
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10. Antibody Genes Engineering
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10. Antibody Genes Engineering
Technology Improvement
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10. Antibody Genes Engineering
FIGURE 5-22
General procedure for producing gene libraries
containing an enormous of H-L constructs ( Fab
fragments ).
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10. Antibody Genes Engineering
Library technology (Phage display)
????
CHO CELL
Antibody library 1011 different phage pool
gene
??
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10. Antibody Genes Engineering
( 80 kb )
FIGURE 5-23
Grafting human heavy- and light-chain miniloci
into mice producing human mAb
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10. Antibody Genes Engineering
Some monoclonal antibodies in clinical use
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