Generation of antibody diversity

Question 1

immunoglobulin variation types

Answer 1

isotopic -> variations in heavy chain types + either kappa or lambda chain
allotypic -> smal variation in the constant region
idiotypic -> variability in the variable chain of an antibody = the variation that generates antibody specificity

Question 2

what is germline DNA

Answer 2

information acquired from sex cells (egg and sperm)

Question 2

somatic recombination?

Answer 2

somatic = alterations in DNA after conception –> somatic mutations can occur in any of the cells of the body except germ cells (sperm and egg)

Question 3

“structure” of the immunoglobulin genes

Answer 3

germline genes are a large cluster of gene segment -> which are non functional (until somatic recombination)

Question 4

what are the gene segments that code for immunoglobulins

Answer 4

variable region (V) segments
diversity (D) segments -> only Ig heavy and TCR beta chains
Joining (J) segments
constant (C) segments

Question 5

RSS? where are they located

Answer 5

recombination signal sequence, and either right before or after a gene segment in the loci

Question 6

where are the RSSs located (with specific segments)

Answer 6

each V segment has an RSS right after it
each J segment has an RSS immediately preceeding it
D segment has an RSS on each side

Question 7

what are the two different types RSSs

Answer 7

RSS with a 23-base-pair-spacer and RSS with a 12-base-pair-spacer

Question 8

structure of a RSS

Answer 8

an RSS contains a heptamer and a nonamer of a conserved sequence (a base sequence in a DNA molecule that has remained relatively unchange throughout evolution) + either the 23 or 12 bp spacer

Question 9

RSS function

Answer 9

critical in somatic recombination -> bringing different segments of DNA together while losing the loop in between them (changing the DNA sequence of the Ig)

Question 10

what is the “12-23 rule”

Answer 10

RSS with a 23-base-pair spacer can be only joined an RSS with a 12-base-pair sequence

Question 11

RAG?

Answer 11

recombination activating gene enzymes

Question 12

function of RAG enzymes

Answer 12

RAG-1/2 bind to the RSSs and bring them together = forming a synapsis. then RAG induces DNA cleavage exactly at the junction of the gene segment and the RSS. this leaves a hairpin of DNA at the end of the gene segments

Question 13

Ku70:Ku80 ?

Answer 13

protein complex which holds the two hairpin DNAs together after RSSs are cleaved

Question 14

the formation of the signal joint

Answer 14

after RSS cleavage, Ku70:Ku80 protein complex holds together the DNA ends. DNA ligase then binds the DNA ends together which generates a precise signal joint (RSSs are bound together)

Question 15

how is the coding joint produced?

Answer 15

the hairpin DNA needs to be cut open so the two strands can be joined together -> facilitated by a comnination of proteins. DNA protein kinase and artemis open the haipin. then a bunch of enzymes process the DNA ends and facilitate their ligation

Question 16

TdT ? function?

Answer 16

terminal deoxynucleotide transferase -> one of the enzymes that facilitate coding joint ligation

Question 17

SCID?

Answer 17

severe combined immunodeficiency

Question 18

gene rearrangement can result into additional nucleotides added to the joint, what are different types of additions?

Answer 18

• non-templated addition -> when the TdT adds extra random bases
• palindromic additions -> by DNA polymerase (dont know the mechanism)

Question 19

what is the importance of nucleotide additions in the coding joint?

Answer 19

they alter the potential peptide reading frame –> basically, it is essential for producing the diversity of antibodies

Question 20

allelic exclusion ?

Answer 20

a single T or B cell will only express the product of a single allele for each of its relevant antigen receptor genes = aka a single BCR will express only one heavy chain even though it has two receptors for it)

Question 21

why is monospecificity important?

Answer 21

it ensures that a cell will only be able to interact with one antigen –> important because during clonal expansion, the clones produced will be identical as the primary B cell (meaning they will al have the same specificity)

Question 22

location of somatic hypermutation

Answer 22

germinal centres of secondary lymphoid organs (where B cells are interacting with antigens)

Question 23

what is the process of somatic hypermutation

Answer 23

the accummulation of mutations in the V region of immunoglobulins -> both heavy and light chains

Question 24

what is the purpose of somatic hypermutation

Answer 24

some mutations might improve binding to the antigen -> those B cells will keep proliferating

Question 25

another way of describing somatic hypermutation

Answer 25

affinity maturation -> the end result will improve the antibody affinity of the Ig to the specific antigen

Question 26

which enzyme is key in facilitating somatic hypermutation and what is its function

Answer 26

activation induced cytidine deaminase (AID) -> AID induces point mutation during the replication process

Question 27

what happens when a body has a lack of AID

Answer 27

antibodies are unable to class switch -> the body produces only IgM antibodies = hyper IgM syndrome type 2

Question 28

what are the initial Ig isotypes produced and how are they generated

Answer 28

IgD and IgM and they are produced by RNA splicing

Question 29

when do antibodies class switch?

Answer 29

after antigen stimulation (in the germinal centres) at the same time as somatic hypermutation

Question 30

what are the 2 signals which induce class switching

Answer 30

1. activation of B cells in the germinal centre
2. CD40-CD40L signalling

Question 31

mechanism of Ab class switching

Answer 31

looks similar to VDJ recombination