Ch. 7 Organization and Expression of Lymphocyte Receptor Genes

Question 1

Classical Germ-Line Model

Answer 1

o Genetic information for each Ab is separately encoded within the germ-line genome
o Easily disproved with the power of MATH
 If there are 107 or more antibodies possible (VERY conservative estimate), & each needs 2000 nucleotides in the genome to encode, our genomes would need to be HUGE to accommodate
 ≈6.7 times larger than the human genome is…in just BCR genes…

Question 2

Dreyer and Bennett’s Model

Answer 2

• Ab heavy and light chains are encoded in two separate segments in the germ-line genome
• to complete these peptides, a V and a C region would be brought together by recombination in the DNA of B cells

Question 3

Somatic Hypermutation Model

Answer 3

• Mutation process occurred only in B cells to alter the BCRS/antibodies of an individual animal in somatic cells after antigen stimulation.
• not passed to offspring

Question 4

Which model was ultimately supported by data to explain how B cells and T cells create their unique BCRs and TCRs?

Answer 4

Dreyer and Bennett model

Question 5

Which model was ultimately supported by data to explain how B cells improve their receptors following their initial interactions with their cognate antigen?

Answer 5

Somatic Hypermutation Model

Question 6

What are the names of the two types of light chains that can be included in BCRs?

Answer 6

• kappa light chain

- lambda light chain

Question 7

Kappa light-chain locus segment organization

Answer 7

o 76 V gene segments (41 appear functionally)
o 5 J gene segments
o 1 C gene segment

Question 8

Lambda light-chain locus segment organization

Answer 8

three functional constant regions each associated with its own J segment
o Around V 33 appear functional
o 7 J-C paired gene segments, but only 4 or 5 are functional

Question 9

Heavy chain locus segment organization

Answer 9

V, D, J, and C segments

o 45 functional V, 23 D, 6 functional J

Question 10

What sequences are critical for allowing a cell to determine where the genome can be recombined?

Answer 10

• Recombination signal sequences (RSSs) flank each Ig gene segment
o Each has a conserved nonamer and heptamer sequence
o In between the nonamer/heptamer lies either a 12 or a 23 bp spacer sequence

Question 11

Heavy chain constant regions determine the antibody classes that B cell can secrete. What antibody classes/constant regions are possible?

Answer 11

o µ, δ, γ3, γ1, γ2b, γ2a, ε, α

Question 12

What is the 12/23 rule?

Answer 12

One RSS bearing the 12-bp spacer is paired with an RSS bearing the 23-bp spacer

Question 13

Where does RAG1 cut the DNA?

Answer 13

Single-stranded nick between the heptameric RSS and the gene segment, which leads to hairpin formation on the coding end and a blunt end on the RSS signal end; this occurs at the 12 bp RSS and also the 23 bp RSS

Question 14

Which enzymes are needed to piece together the blunt ends of the cut out DNA to form an episome?

Answer 14

• DNA ligase IV ligates free blunt ends together to make a signal joint upon activation by XRCC4

Question 15

What does the enzyme artemis do?

Answer 15

endonuclease opens up hairpin

Question 16

What are P nucleotides?

Answer 16

DNA repair enzymes which create palindromic sequences at the ends

Question 17

Once the hairpins are cut by Artemis & filled in to be blunt ends, which enzymes put the two pieces of chromosome back together?

Answer 17

After ends are made blunt again, DNA ligase IV complexed with XRCC4 repairs coding joints in light chains just like it did with the episome that was cut out

Question 18

What three things must a developing B cell ensure to become a productive B cell?

Answer 18

1. That heavy- and light-chain loci are productively rearranged
2. Only one heavy-chain allele and one light-chain allele is expressed
3. That the produced BCR does not bind self-antigens

Question 19

What is the process of allelic exclusion that ensures that B cells only express one type of BCR? Which alleles are recombined first?

Answer 19

• One heavy chain locus is recombined and expressed first
o Expression of a functional heavy chain leads to formation of pre-BCR
 This allows signaling that tells the cell to move forward with development
o Pre-BCR signaling triggers several cell divisions
 Maximizes use of successful heavy-chain rearrangements
 Not unusual to see multiple B cells with the same heavy chain, but different light chains

Question 20

How many chances do B cells have to recombine a heavy chain? What percentage tend to fail this step in development?

Answer 20

2 opportunities to do the heavy chain. After two failed attempts, apoptosis
55% heavy chain success rate

Question 21

How many chances do B cells have to recombine a light chain?

Answer 21

4 times to make light chain, after 4 failed attempts, cell death

Question 22

Explain what occurs if B cells are found to be autoreactive during development.

Answer 22

Cell death apoptosis

Question 23

What constant region do immature B cell include in their BCR?

Answer 23

express only membrane IgM receptors

Question 24

What constant region do mature B cell include in their BCR?

Answer 24

placement of IgD as well

Question 25

What constant region do naive B cells include in their BCR?

Answer 25

o Ig heavy-chain RNA transcript in mature, naïve B cells encodes the rearranged V region and both the Cµ & the Cδ constant regions

Question 26

Describe the overall structure of TCRs. What two types of TCRs exist?

Answer 26

TCRs are either αβ or δγ & are heterodimeric receptors

- antigen binding site
- variable region (v)
- Constant region ©
- transmembrane region
- cytoplasmic tail

Question 27

What are the three notable differences between T cell & B cell gene recombination?

Answer 27

1. TdT is downregulated in B cells following pre-BCR expression (and so light-chains don’t have the N nucleotide additions that heavy-chains have)
   a. This is not the case in T cells, and so TCR chains can have this variability
2. The locations of the 12-bp and 23-bp RSS spacers in TCR genes technically allow for skipping or adding D region segments in β & δ chains
3. Allelic exclusion at the TCR β- & γ-chain loci occurs almost as efficiently as among Ig heavy- & light-chain genes
   a. It is unlikely that a T cell with a 2 a chains can recognize more than one antigen, as T-cell antigen recognition is complexed with MHC binding.