Organisation & Expression of Immune Genes (Antibody Diversity) Flashcards
function of heterochromatin
closed DNA structure not actively transcribed
function of Euchromatin
open structure allowing access to transcription machinery
Epigenetics (post-transcriptional) –methylation and acetylation account for…
… genome
post-transcriptional events (RNA) editing also
account for…
… differential gene expression
what do hydrophobic leader sequences allow?
(~20 amino acids) allows nascent poly
peptide transport through ER -> Golgi -> membrane (cleaved )
where is IgD expressed?
B cell surface
what does IgD engage with
engages with Ag during specific immune response activation
what receptor does IgD bind to?
B cell receptor
Ig have enormous
diversity, there are 10^ 15 to 10^ 18 B cells expressing different Ag specificities (more B cells than base pairs in the genome)
number of gene encoding B cell is in the…
… 100’s, not billions
How do you explain 10
15
to 10
18
different B cells?
genes encoding for the variable regions within the V
H
and V
L
chains rearrange within the genome of differentiating B cells (somatic
recombination)
Genetic recombination during
meiosis 1 creating…
genetically
diverse daughter (2n) cells
Meiosis 2 creates…
…four genetically distinct (1n) cells
go look at slide 10 and 11
The λ and κ light-chains are encoded by
V, J and C segments
Heavy-chain are encoded by
V, D, J and C segments
(kb denote
the genomic distance between segments)
There are two light-chains whic h are…
κ and λ
The Ig protein has…
… two major light chain domains:
1) Variable region (VL)
2) Constant region (CL)
How many amino acid residues in VL ?
VL = 108 amino acid residues
a) V (variable) = 95 residues
b) J (joining) = 13 residues
One V gene segment and one J gene segment are…
… brought together in the genome which then combine with a C (constant) gene segment.
One V gene segment and one J gene segment are brought together in the genome which then combine with a C (constant) gene segment.
This unique rearrangement if known as …
… V(D)J recombination
There are two light-chains….
κ and λ
First step of recombination is
…the joining of a V segment to a J segment (VJ complex)
any DNA in between is deleted
RNA splicing removes
introns and leftover
V and J segments
Lambda (λ) gene recombination is similar in principle to
κ gene recombination – only expressed when κ are not recombined correctly
whats the principle step of Lambda gene recombination?
Principle step involves rearranging the genomic DNA to join V and J
segment together crating a λ-VJ complex
How many Vλ and Jλ in the human λ locus?
40 Vλgene segments and 4 Jλ gene segments
Organisation of these segments in lambda genes differs from…
the κ locus
how many Cλ gene segments and J segments associate with?
There are four different Cλ gene segments that the J segment can
associate with, thus four different λ chain isotypes produced
How many gene segments involved in the heavy chain variable region?
There are three gene segments involved in the heavy-chain variable region;
VH (Variable), DH (Diversity) and JH (Joining)
The D and J segments encode for
the amino acid residues that
constitute the complementary determining region 3 (CDR3) of the heavy-chain
Heavy-chain locus (Ch. 14) includes…
50 VH gene segments
20 DH gene segments
6 JH gene segments
Heavy-chain germ line contains …
… multiple gene segments encoding for
the constant (C) region
C region determines …
… class and biological function
In the early stages of B cell differentiation, how many gene arrangement processes occur?
two gene arrangement
processes occur
In the early stages of B cell differentiation two gene arrangement processes occur
1) one D segment associates with one J segment
2) one V segment associates with the DJ complex
The new V(D)J complex is closest to…
Cμ and Cδ segments which form the primary transcript.
Alternative splicing of the primary transcript yields …
two different mature mRNA; V(D)J-μ and V(D)J-δ
A resting B cell may express…
…both IgM and IgD with identical Ag
specificity
what is the first step of recombination of heavy chains?
First step of recombination is the
joining of a D segment to a J
segment (DJ complex)
DJ complex joins with
an upstream
V segment (VDJ) complex
Primary RNA contains
μ and δ
constant segments – alternative
splicing produces two Ab classes
RSS has three elements which are…
1) a conserved heptamer 5’-CACAGTG-3’
2) a less conserved spacer (either 12 or 23 bp)
3) a second conserved nonamer5’-ACAAAAACC-3’
RAG1/2 proteins bind to…
… DNA at RSS and mediate VDJ recombination
by bringing together one 12-bp spacer with one 23-bp spacer.
V(D)J recombination mechanism steps
Step 1 RAG1/2 complexes with heptamer and RAG1 with nonamer
Step 2 RAG1/2 create single strand nicks
Step 3 V and J hairpin structures form due to RAG1/2 mutual affinity
and a blunt end cut formed
Step 4 ligation of 12 bp and 23 bp signal ends
Step 5 Hairpin cleavage
Step 6 overhang extension (palindromic hairpins)
Step 7 ligation of V and J segments
In theory any B cell has
numerous gene segments from which to select
for the synthesis
Each B cell has one set of […] and one type of […]
V(D)J genes and one type of VJ light gene
(either κ or λ)
Each B cell rearranges these segments in a unique way, therefore
each B cell produces antibodies with only one antigenic specificity
B cells are
diploid (have 2 sets of chromosomes)
In contrast to all other cells, Ig chains are coded for by…
… only one set of genes
In contrast to all other cells, Ig chains are coded for by only one set of
genes – e.g….
- Heavy-chain maybe from the paternal chromosome
- Light-chain may be from the maternal chromosome
B cell may create one single…
Ag specific Ig – VJ (light) and VDJ (heavy) are fixed
B cells can switch class to…
make different Ig’s IgA, IgG or IgE
B cells can switch class to make different Ig’s IgA, IgG or IgE while
retaining the same Ag specificity (isotype switching)
What does isotope switching invovle?
involves rearranging the VDJ gene segment complex, juxtaposing
the rearranged VDJ complex with a different C chain gene segment
In Isotype class switching,
Double stranded DNA breaks occur at consensus regions
known as switch-regions, upstream of C gene segment
Switch-regions occur …
adjacent all C genes except δ
Activation Induced (cytidine) Deaminase (AID) mediates
DNA at two selected switch regions, eliminating
unwanted C genes
Non-homologous end
joining links
VDJ with new C
Isotype class switching ocurs in…
mature B cells and is Ag stimulation dependent
Factors (CYTOKINES) that regulate this process are secreted from…
T cells
what are cytokines?
Cytokines are peptides that bind cognate cell surface receptors and
alter cell activity and function
What are the other mechanisms to create antibody diversity?
1) Presence of multiple V genes in the germ line
2) Random assortment of H and L chains
3) Junctional and insertional diversity
4) Somatic hypermutation
1) Presence of multiple V genes in the germ line
Number of different V gene segments in…
the germ line forms the baseline from which Ig is derived and represents the minimum number of different Ig that can be produced
Describe 2) Random assortment of H and L chains
Any distinct H chain may associate with any distinct L chain
Describe 3) Junctional and insertional diversity
The precise position at which the gene for the V and J or the V, D and J segments are fused together are not constant -> imprecise DNA
recombinations can cause frame shift mutations alter amino-acid
sequence at the regions -> diversifying further
Describe 4) Somatic hypermutation
After secondary Ag exposure, Ag:Ab affinity increases -> analysis
shows SNPs in VDJ region -> fine tuning of the immune system
