7. Generation of lymphocyte antigen receptors

Question 1

why is the CDR3 (or HV3) region the most variable

Answer 1

it contains both V and J segments of the genome

Question 2

what encodes the CDR1, CDR2 and CDR3 regions

Answer 2

1 and 2: V gene segment
3: V and beginning of J gene segments

Question 3

gene rearrangement of lymphocytes

Answer 3

gene segments are rearranged by somatic DNA recombination to form unique variable-region coding sequences

Question 4

all of the Ig H, Lk and Ly chains are enncoded by…

Answer 4

3 separate loci on the same gene

Question 5

germline organization of the human Ig gene loci

Answer 5

• 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)

Question 6

y Light-chain locus

Answer 6

V regions at 5’
4 J regions in between 4 C regions at 3’

Question 7

k light chain locus

Answer 7

V regions at 5’
J regions cluster
1 C region at 3’

Question 8

heavy chain locus

Answer 8

V regions at 3’
D region
J regions cluster
1 C region at 3’

Question 9

construction of an antibody: light chain

Answer 9

somatic recombination of V and J segments
transcription of DNA to make RNA transcript
splicing makes mRNA
translation creates the polypeptide chain

Question 10

construction of an antibody: heavy chain

Answer 10

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

Question 11

how many V and J segments respectively are in each chains loci

Answer 11

heavy: 45, 6 (+23 D)
k light: 35, 5
y light: 30, 5

Question 12

what is the theoretical number of combinatorial diversity

Answer 12

2,010,000
(from 149 gene segments)

Question 13

rearrangement of V, D and J gene segments is guided by…

Answer 13

flanking DNA sequences

Question 14

why won’t genes for y and k light chains recombine?

Answer 14

their RSS won’t match up (won’t follow the 12/23 rule)

Question 15

what are the different Recombination signal sequences? (RSS)

Answer 15

heptamer - 23 bp spacer - nonamer
nonamer - 12 bp spacer - heptamer

Question 16

what is the role of V(D)J recombinase

Answer 16

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

Question 17

enzymatic steps in V(D)J recombination

Answer 17

1. synapsis
2. cleavage
3. hairpin opening and end processing
4. joining

Question 18

step 1 of V(D)J recombination: synapsis

Answer 18

2 distant coding segments and their adjacent RSS are brought together

Question 19

step 2 of V(D)J recombination: cleavage

Answer 19

V(D)J recombinase (composed of RAG 1 and 2) makes double-stranded breaks at the RSS-coding sequence junction

Question 20

step 3 of V(D)J recombination: hairpin opening and end processing

Answer 20

Artemis opens up hairpins at coding junctions
Terminal deoxynucleotidyl transferase (Tdt) may add or remove nucleotides to create more diversity

Question 21

step 4 of V(D)J recombination: joining

Answer 21

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

Question 22

characteristics of RAG proteins

Answer 22

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

Question 23

what is Artemis

Answer 23

an endonuclease that opens up the hairpins created during synapsis

Question 24

what are the 2 types of diversity in antigen receptor genes

Answer 24

combinatorial diversity: use of different V, D and J segments
junctional diversity: different N and P nucleotides in each clone

Question 25

steps in junctional diversity (insertion of P- and N- nucleotides between gene segments)

Answer 25

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

Question 26

random insertion of P and N nucleotides creates unique markers useful for…

Answer 26

tracking individual B-cell clones (for somatic hypermutation)

Question 27

how are IgM and IgD derived from the same pre-mRNA transcript

Answer 27

B cells produce a long primary mRNA transcript that is differentially spliced to yield 2 distinct mRNA molecules

Question 28

true or false: transmembrane and secreted forms of immunoglobulin are generated from the same heavy-chain mRNA transcripts

Answer 28

FALSE
generated from different heavy-chain mRNA transcripts - secreted form contains a soluble component after the constant region

Question 29

what parts of the TCR are homologous to the heavy and light chains of BCRs

Answer 29

a chain ~ light chain
B chain ~ heavy chain

Question 30

difference between the B-chain and heavy chain loci

Answer 30

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