Lecture 30 - B cells.

Question 1

How do ~20,000–25,000 protein-coding genes result in a wide variety of antibodies and TCRs?

Answer 1

Through primary diversity, which is achieved by somatic recombination, creating combinatorial and junctional diversity in antibodies and TCRs.

Question 2

What are the two types of diversity that contribute to primary diversity in antibodies and TCRs?

Answer 2

Combinatorial diversity and junctional diversity.

Question 3

What is somatic recombination in the context of antibody production?

Answer 3

Somatic recombination is the process by which gene segments are rearranged to create different antibodies. It is similar to shuffling a deck of cards and dealing out different hands.

Question 4

What is the analogy used to explain somatic recombination?

Answer 4

Shuffling a deck of cards and dealing out different hands.

Question 5

What types of gene segments are involved in light chain and heavy chain recombination?

Answer 5

1. Light chain: Variable (V) and Joining (J) segments.
2. Heavy chain: Variable (V), Diversity (D), Joining (J), and Constant (C) segments.

Question 6

Which gene segments are involved in the recombination of the light chain and heavy chain?

Answer 6

1. Light chain: V and J segments.
2. Heavy chain: V, D, J, and C segments.

Question 7

What is unique about the D segment in antibody recombination?

Answer 7

The D segment is only present in the heavy chain.

Question 8

What is CDR1 and CDR2, and where are they encoded?

Answer 8

CDR1 and CDR2 are regions of the antibody’s variable region and are encoded in the V segments of both light and heavy chains.

Question 10

Where is CDR3 encoded and why is it important?

Answer 10

CDR3 is encoded in the joining of the V-J segments (light chain) and V-D-J segments (heavy chain). It is the most variable CDR and contributes to the diversity of antibodies and TCRs.

Question 11

What is the structure of the variable region in both light and heavy chains?

Answer 11

The variable region consists of recombined V, D (only in heavy chain), and J gene segments.

Question 12

How does V(D)J recombination contribute to antibody diversity?

Answer 12

V(D)J recombination randomly selects one segment from each V, D, and J gene pool to create a unique variable region in antibodies.

Question 13

What is the role of the proteins involved in somatic recombination?

Answer 13

The recombination process is tightly regulated by machinery involving many proteins that also participate in DNA repair functions.

Question 14

What is the process of choosing V, D, and J gene segments during B cell development?

Answer 14

During B cell development, V(D)J recombination occurs, selecting one segment from each of the V, D, and J gene pools to form the variable region of the antibody.

Question 15

How many different loci are involved in light chain production, and what are they?

Answer 15

There are two different loci in the light chain: the κ (kappa) chain locus and the λ (lambda) chain locus.

Question 16

What happens to the κ and λ loci in light chain production?

Answer 16

Only one chain (either κ or λ) will be expressed, and the other chain will be silenced.

Question 17

What does each light chain locus contain?

Answer 17

Each locus contains many different Variable (V) and Joining (J) regions.

Question 18

How many loci are involved in heavy chain production?

Answer 18

There is one locus involved in heavy chain production.

Question 19

What gene segments are involved in the heavy chain recombination?

Answer 19

The heavy chain locus contains many different V (Variable), D (Diversity), and J (Joining) regions.

Question 20

How do constant regions in the heavy chain relate to different antibody isotypes?

Answer 20

Different constant regions represent the different isotypes (e.g., IgM, IgD, IgG, etc.) of antibodies.

Question 21

What is the source of the V, D, and J regions in heavy chain recombination?

Answer 21

The V, D, and J regions are inherited from the stem cell (precursor B cell).

Question 22

What is the process called by which recombinase proteins join gene segments in antibody production?

Answer 22

The process is called somatic recombination.

Question 23

What happens to the B cell receptor (BCR) in a naïve B cell?

Answer 23

The BCR of a naïve B cell is formed during somatic recombination, and this process is irreversible.

Question 24

How many V regions are available to choose from for the heavy chain?

Answer 24

There are 46 V regions to choose from (e.g., VH1, VH2, VH3, etc.).

Question 25

How many D regions are available to choose from for the heavy chain?

Answer 25

There are 23 D regions to choose from (e.g., DH1, DH2, DH3, etc.).

Question 26

How many J regions are available to choose from for the heavy chain?

Answer 26

There are 6 J regions to choose from (e.g., JH1, JH2, JH3, etc.).

Question 27

How would you create your own VH region?

Answer 27

To create a VH region, choose one V, one D, and one J region, and combine them as a single word: V#D#J# (e.g., VH1DH2JH3).

Question 28

What is combinatorial diversity in antibody production?

Answer 28

Combinatorial diversity is the diversity resulting from the different combinations of V, D, and J gene segments.

Question 29

What are recombination signal sequences (RSSs)

Answer 29

RSSs are sequences that flank each antibody gene segment and direct recombination by guiding the pairing of segments.

Question 30

What is the structure of a recombination signal sequence (RSS)?

Answer 30

An RSS consists of a conserved nonamer (9 bp) and heptamer (7 bp), with either a 12-bp or 23-bp spacer sequence between them.

Question 31

What is the "12/23 Rule" in recombination?

Answer 31

The "12/23 Rule" dictates that a 12-bp RSS must pair with a 23-bp RSS for recombination to occur.

Question 32

How does recombination occur in the light chain and heavy chain?

Answer 32

1. Light chain: V-J recombination. 2. Heavy chain: D-J recombination followed by V-DJ recombination.

Question 33

What happens to the segments that are not selected during recombination?

Answer 33

The segments between the selected ones are excised and form a loop in the DNA, which is then deleted.

Question 34

How does DNA looping occur during recombination?

Answer 34

RSSs bring the regions together, creating a loop in the DNA, with the excised non-selected segments forming a signal joint.

Question 35

What proteins are involved in the recombination process, and what is their function?

Answer 35

The RAG-1 and RAG-2 proteins are necessary for recombination, responsible for recognizing and cutting DNA at the immunoglobulin-encoding region and the RSS.

Question 36

What happens to the DNA after recombination?

Answer 36

The DNA forms covalently closed hairpin ends at the excised loop, and the loop is deleted from the chromosome. The coding region of the selected V and J regions remains and forms the coding joint.

Question 37

What is a signal joint?

Answer 37

The signal joint is the excised DNA loop that is deleted after recombination, no longer present on the chromosome.

Question 38

What is the role of RAG proteins in recombination?

Answer 38

RAG-1 and RAG-2 are responsible for cutting the DNA at the immunoglobulin-encoding region and the RSS to facilitate recombination.

Question 39

What is junctional diversity in antibody production?

Answer 39

Junctional diversity is the variation that occurs when nucleotides are added or removed at the junctions between V & DJ, D & J, or V & J gene segments during recombination.

Question 40

What happens to the signal joint after recombination?

Answer 40

The signal joint is ligated together and discarded.

Question 41

What happens at the coding ends during recombination?

Answer 41

Repair proteins bind to the hairpin, and Artemis, an endonuclease, opens the DNA hairpins.

Question 42

How does Artemis contribute to recombination?

Answer 42

Artemis opens the DNA hairpins and can cleave them in three different ways, facilitating junctional diversity.

Question 43

What are palindromic (P) nucleotides, and how do they contribute to recombination?

Answer 43

P nucleotides are added at overhangs during hairpin cleavage. They are complementary nucleotides that allow DNA repair enzymes to fill in the opposite strand.

Question 44

How does exonuclease activity contribute to junctional diversity?

Answer 44

Exonuclease activity removes nucleotides on each side of the coding joint, further contributing to junctional diversity.

Question 45

What is the role of Terminal Deoxynucleotidyl Transferase (TdT) in recombination?

Answer 45

TdT adds up to 20 nontemplate-encoded nucleotides (N nucleotides) to the cleaved strands, primarily in the heavy chain, contributing to the variability of CDRs (Complementarity Determining Regions).

Question 46

How do repair enzymes contribute to recombination?

Answer 46

Repair enzymes trim off nonmatching nucleotides, fill in single-stranded gaps, and ligate the new DNA.

Question 47

What are the main mechanisms that generate B cell receptor (BCR) diversity in naïve B cells?

Answer 47

The mechanisms include: 1. Multiple gene segment combinations 2. Heavy chain/light chain combinatorial diversity 3. P nucleotide addition 4. Exonuclease trimming 5. Nontemplated N nucleotide addition by TdT

Question 48

What is the role of TdT in the heavy chain recombination?

Answer 48

TdT adds random N nucleotides between joints in the heavy chain, contributing to junctional diversity.

Question 49

What is the impact of exonuclease activity on junctions in recombination?

Answer 49

Exonuclease activity can remove nucleotides at the junctions between gene segments, leading to further diversity in the final product.

Question 50

What is the general outcome of the V(D)J recombination process?

Answer 50

The process results in a diverse range of BCRs due to the combinatorial diversity, junctional diversity, and random addition or removal of nucleotides at the junctions.