7. Generation of lymphocyte antigen receptors Flashcards
why is the CDR3 (or HV3) region the most variable
it contains both V and J segments of the genome
what encodes the CDR1, CDR2 and CDR3 regions
1 and 2: V gene segment
3: V and beginning of J gene segments
gene rearrangement of lymphocytes
gene segments are rearranged by somatic DNA recombination to form unique variable-region coding sequences
all of the Ig H, Lk and Ly chains are enncoded by…
3 separate loci on the same gene
germline organization of the human Ig gene loci
- cluster of V gene segments at the 5’ end
- several J segments at varying distances of 3’ end
- additional D segments between V and J of heavy chains
- C regions are at the 3’ end of J segments (1 in Lk and 4 in Ly)
y Light-chain locus
V regions at 5’
4 J regions in between 4 C regions at 3’
k light chain locus
V regions at 5’
J regions cluster
1 C region at 3’
heavy chain locus
V regions at 3’
D region
J regions cluster
1 C region at 3’
construction of an antibody: light chain
somatic recombination of V and J segments
transcription of DNA to make RNA transcript
splicing makes mRNA
translation creates the polypeptide chain
construction of an antibody: heavy chain
somatic recombination of D and J segments
second round of SR of V and DJ
transcription of DNA to make RNA transcript
splicing makes mRNA
translation creates the polypeptide chain
how many V and J segments respectively are in each chains loci
heavy: 45, 6 (+23 D)
k light: 35, 5
y light: 30, 5
what is the theoretical number of combinatorial diversity
2,010,000
(from 149 gene segments)
rearrangement of V, D and J gene segments is guided by…
flanking DNA sequences
why won’t genes for y and k light chains recombine?
their RSS won’t match up (won’t follow the 12/23 rule)
what are the different Recombination signal sequences? (RSS)
heptamer - 23 bp spacer - nonamer
nonamer - 12 bp spacer - heptamer
what is the role of V(D)J recombinase
recognize 12/23 RSS and brings the exon together
recombination of V and J exons occurs by deletion of intervening DNA and ligation of the V and J segments
enzymatic steps in V(D)J recombination
- synapsis
- cleavage
- hairpin opening and end processing
- joining
step 1 of V(D)J recombination: synapsis
2 distant coding segments and their adjacent RSS are brought together
step 2 of V(D)J recombination: cleavage
V(D)J recombinase (composed of RAG 1 and 2) makes double-stranded breaks at the RSS-coding sequence junction
step 3 of V(D)J recombination: hairpin opening and end processing
Artemis opens up hairpins at coding junctions
Terminal deoxynucleotidyl transferase (Tdt) may add or remove nucleotides to create more diversity
step 4 of V(D)J recombination: joining
broken coding and signal ends are ligated by non-homologous end joining
KU70 and KU80 bind to breaks and recruit the catalytic subunit of DNA-dependent protein kinase
ligation of the processed broken ends is mediated by DNA ligase IV and XRCC4
characteristics of RAG proteins
RAG 1 and RAG2 make up V(D)J recombinase
RAG genes are only expressed in developing T and B cells
RAG protein is inactivated in proliferating cells
what is Artemis
an endonuclease that opens up the hairpins created during synapsis
what are the 2 types of diversity in antigen receptor genes
combinatorial diversity: use of different V, D and J segments
junctional diversity: different N and P nucleotides in each clone
steps in junctional diversity (insertion of P- and N- nucleotides between gene segments)
RAG complex cleaves DNA hairpin at random sires to produce ssDNA ends
hairpin opening makes palindromic P-nucleotide sequences
presence of Tdt adds random nucleotides to ss ends
2 ss ends pair together
unpaired nucleotides are trimmed
final repair via DNA synthesis and ligation, retaining P and N nucleotides
random insertion of P and N nucleotides creates unique markers useful for…
tracking individual B-cell clones (for somatic hypermutation)
how are IgM and IgD derived from the same pre-mRNA transcript
B cells produce a long primary mRNA transcript that is differentially spliced to yield 2 distinct mRNA molecules
true or false: transmembrane and secreted forms of immunoglobulin are generated from the same heavy-chain mRNA transcripts
FALSE
generated from different heavy-chain mRNA transcripts - secreted form contains a soluble component after the constant region
what parts of the TCR are homologous to the heavy and light chains of BCRs
a chain ~ light chain
B chain ~ heavy chain
difference between the B-chain and heavy chain loci
in heavy chain D regions are clustered and there is only 1 C region
in B chain D regions are separated and there are 2 C regions
