Lecture 7 - Creation of Antibody Diveristy Flashcards
Basic structure of a gene 1) 2) 3) 4) 5)
1) Promoter
2) Leader exon
3) Exons/introns
4) Enhancer
5) Poly A tail addition sequence
Function of leader exon
Present on extracellular proteins
Encodes leader peptide
Leader peptide directs peptide towards ER for exocytosis
Difference in shape between lambda and kappa light chain loci
Lambda has sequence of (J-C)n
Kappa has sequence of (J)n (C)n
What is ‘germline configuration’?
Unrearranged minigenes at immunoglobulin locus.
How Ig locus is found in cells in body except for T and B lymphocytes
What is a rosette?
Ig locus forms flower-like structures.
Minigenes near one another will join.
How much of a light-chain variable-region protein is encoded by V genes?
~90%
What do the three peaks in variability on the Kabat-Wu plot represent?
CDR1, CDR2, CDR3.
Difference between variability of CDR1/CDR2 and CDR3?
CDR1 and CDR2 are germline encoded –> The variability is based on heritable gene segment sequences.
CDR3 variability is based on joining of V(D)J regions, and N region of random addition/excision of bases.
What do heptamers and nonamers do?
Join together to form loops which are removed by V(D)J recombinase complex
How do heptamers and nonamers join?
Heptamers pair and nonamers pair.
They are complementary base sequences
In a light chain, is the 23-nucleotide loop closer to the V or J region?
V region
Which minigene region is flanked by 12-nucleotide spacers?
D region
Which minigene region is next to the 23-nucleotide spacer?
V and J regions
Order of minigene regions and spacers
—V—23 spacer—12 spacer—D—12 spacer—23 spacer—J—
Function of 12/23 rule
Prevents V-J recombination in heavy chain
If a minigene lies between V(D)J cassette and C region, what happens to it?
It is spliced out when mRNA of Ig gene is transcribed.
Splice sites are after V(D)J cassette and C region
What do 12 and 23 bases correspond to?
Length of major and minor grooves of DNA
Excision circle
Loop of DNA excised from genome in V(D)J recombination
Components of V(D)J recombinase complex
1)
2)
3)
1) RAG1/RAG2
2) Exonucleases
3) Terminal deoxyribnucleotidyl transferase
Does the genome change length much with exonuclease and TdT activity?
Not much
Exonuclease and TdT activity are roughly in equilibrium
Why can D region be absent in some antibodies?
Exonuclease function is inexact.
Can sometimes accidentally excise D region (which is small)
Problems with TdT
Can add stop codons, frameshift in N regions
What are N-regions?
Regions between V, D and J regions.
High diversity because of exonuclease and TdT activity.
Imprecision of V(D)J junctions
Sources of antibody diversity
1)
2)
3)
1) Combinatorial diversity
2) Junctional diversity
3) Somatic hypermutation
How are IgMs determined to be membrane-bound or secretory?
1)
2)
3)
1) Membrane anchor exon separated from rest of gene by an occult splice site
2) Occult splice site can be removed from genome, making membrane anchor expressed in protein
3) Occult splice site can be included in protein, leading to absence of membrane anchor form protein
Can IgD and IgM be expressed by the same B cell?
Yes
IgM and IgD share a switch region (mu switch region)
Is somatic hypermutation dependent on RAG function?
No.
When is AID expressed in B cells?
In germinal centres, during somatic hypermutation and isotype switching
What happens to antibody binding affinity as the immune response matures?
Kd grows lower and lower
Can a change in framework region structure of antibodies change binding affinity?
Yes.
CDR loops can be moved into a more favourable or unfavourable position
