Lecture 7 - Creation of Antibody Diveristy

Question 1

Basic structure of a gene
1)
2)
3)
4)
5)

Answer 1

1) Promoter
2) Leader exon
3) Exons/introns
4) Enhancer
5) Poly A tail addition sequence

Question 2

Function of leader exon

Answer 2

Present on extracellular proteins
Encodes leader peptide
Leader peptide directs peptide towards ER for exocytosis

Question 3

Difference in shape between lambda and kappa light chain loci

Answer 3

Lambda has sequence of (J-C)n

Kappa has sequence of (J)n (C)n

Question 4

What is ‘germline configuration’?

Answer 4

Unrearranged minigenes at immunoglobulin locus.

How Ig locus is found in cells in body except for T and B lymphocytes

Question 5

What is a rosette?

Answer 5

Ig locus forms flower-like structures.

Minigenes near one another will join.

Question 6

How much of a light-chain variable-region protein is encoded by V genes?

Answer 6

~90%

Question 7

What do the three peaks in variability on the Kabat-Wu plot represent?

Answer 7

CDR1, CDR2, CDR3.

Question 8

Difference between variability of CDR1/CDR2 and CDR3?

Answer 8

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.

Question 9

What do heptamers and nonamers do?

Answer 9

Join together to form loops which are removed by V(D)J recombinase complex

Question 10

How do heptamers and nonamers join?

Answer 10

Heptamers pair and nonamers pair.

They are complementary base sequences

Question 11

In a light chain, is the 23-nucleotide loop closer to the V or J region?

Answer 11

V region

Question 12

Which minigene region is flanked by 12-nucleotide spacers?

Answer 12

D region

Question 13

Which minigene region is next to the 23-nucleotide spacer?

Answer 13

V and J regions

Question 14

Order of minigene regions and spacers

Answer 14

—V—23 spacer—12 spacer—D—12 spacer—23 spacer—J—

Question 15

Function of 12/23 rule

Answer 15

Prevents V-J recombination in heavy chain

Question 16

If a minigene lies between V(D)J cassette and C region, what happens to it?

Answer 16

It is spliced out when mRNA of Ig gene is transcribed.

Splice sites are after V(D)J cassette and C region

Question 17

What do 12 and 23 bases correspond to?

Answer 17

Length of major and minor grooves of DNA

Question 18

Excision circle

Answer 18

Loop of DNA excised from genome in V(D)J recombination

Question 19

Components of V(D)J recombinase complex
1)
2)
3)

Answer 19

1) RAG1/RAG2
2) Exonucleases
3) Terminal deoxyribnucleotidyl transferase

Question 20

Does the genome change length much with exonuclease and TdT activity?

Answer 20

Not much

Exonuclease and TdT activity are roughly in equilibrium

Question 21

Why can D region be absent in some antibodies?

Answer 21

Exonuclease function is inexact.

Can sometimes accidentally excise D region (which is small)

Question 22

Problems with TdT

Answer 22

Can add stop codons, frameshift in N regions

Question 23

What are N-regions?

Answer 23

Regions between V, D and J regions.
High diversity because of exonuclease and TdT activity.
Imprecision of V(D)J junctions

Question 24

Sources of antibody diversity
1)
2)
3)

Answer 24

1) Combinatorial diversity
2) Junctional diversity
3) Somatic hypermutation

Question 25

How are IgMs determined to be membrane-bound or secretory?
1)
2)
3)

Answer 25

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

Question 26

Can IgD and IgM be expressed by the same B cell?

Answer 26

Yes

IgM and IgD share a switch region (mu switch region)

Question 27

Is somatic hypermutation dependent on RAG function?

Answer 27

No.

Question 28

When is AID expressed in B cells?

Answer 28

In germinal centres, during somatic hypermutation and isotype switching

Question 29

What happens to antibody binding affinity as the immune response matures?

Answer 29

Kd grows lower and lower

Question 30

Can a change in framework region structure of antibodies change binding affinity?

Answer 30

Yes.

CDR loops can be moved into a more favourable or unfavourable position