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Questions (outlines):
1. Do yyou reallyy understand V(D)J
( ) recombination?
Functions of RAG?
2 Wh
2. Whatt are the
th differences
diff bbetween
t th
the IIg and
d TCR
gene recombination?
Dryer and Bennett 2-gene model, 1965 Tonegawa gene recombination model, 1976
Ig genes are rearranged
in B cells
(different probes)
Probe: mRNA encoding light chain ( ___ )
The germline organization of the Ig heavy
heavy-
and light-chain loci in the human genome
(Ch #22)
(8 families or clans)
(Ch #2)
(7 families or clans)
(Ch #14)
(3 families or clans)
V-region genes are constructed from gene segments
(95-101 aa) (< 13 aa) (90-95 aa) (2-3 aa) (10-15 aa)
CDR 3
V region sequences are constructed from gene segments
V-region
page 809-811
but naive
Conserved heptamer and nonamer sequences (recombination
signal sequences,
sequences RSSs) flank the gene segments encoding
the V regions of heavy (H) and light ( and ) chains
(recombination signal sequences)
2 1
1 2
RAG: recombination-activating
genes
Ku70:Ku80 + DNA-PKcs
DNA PKcs to form
DNA-PK
Artemis (with nuclease activity)
TdT: terminal deoxynucleotidyl
y y
transferase
DNA ligase IV (form complex with
XRCC4) as DSBR: double-
strand
t d break
b k repairi
3OH
P-nucleotide addition 5 P
(palindrome)
N-nucleotide addition
TdT: terminal deoxynucleotidyl
transferase
IgM
allelic
exclusion
T-cell receptor gene rearrangement
The germline organization of the human T-cell receptor and loci
CDR1CDR2CDR3
The organization of the human T-cell receptor and loci
: pseudogene
2 forms of Igs
Transmembrane and secreted forms of Ig are derived from the same
heavy-chain sequence by alternative RNA processing
Coexpression of IgD and IgM is regulated by RNA processing
Synthesis, assembly, and
secretion of the Igs
IgM: H+L
HL
(HL)2
IgG: 2H
H2+L
H2L+L
H2L2
(rough
endoplasmic
reticulum)
Immunoglobulin isotype (class) switch
IgM IgG
The formation of a gene for the heavy chain of an Ab molecule
page
p g 809-811
(recombination)
150 X (GAGCT)n(GGGGGT),
(GAGCT)n(GGGGGT) n=3-7
Enzyme: unknown
E k
AID (activation-induced cytidine deaminase)
UNG, APE1, Ku70:Ku80
AID (activation-induced cytidine deaminase) in class
switching and somatic hypermutation
The sequence
seq ence of AID is related to that of a
protein known as APOBEC1 (apolipoprotein
B mRNA editing catalytic polypeptide 1),
which convert cytosine to uracil by
deamination in apolipoprotein B mRNA.
Generation of single
strand nicks in DAN by
sequential
ti l action
ti off AID,
AID
UNG, and APE1
UNG: uracil-DNA-glycosylase
(to form abasic site in the DNA)
APE1: apurinic/apyrimidinic
endonuclease
d l 1
(to generate a single-strand nick)
AID initiates processes that lead to
somatic hypermutation, gene conversion
and
d class
l switch
it h recombination
bi ti
(
(through
g homologous
g
recombination with pseudogenes)
The primary antibody
repertoire is diversified
b th
by three processes that
th t
modify the rearranged
Ig gene
Class switching involves
recombination between
specific switch signals
S : 150 repeats of
(GAGCT)3-7(GGGGGT)
- form R-loop
V(D)J recombination
(page 186)