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IMIN 371 > V(D)J Recombination > Flashcards

V(D)J Recombination Flashcards

1
Q

What are the two theories of how a genome of finite size can generate 10^14 Ab.s of different specificities?

A
  1. germ line theory
    - entire genetic info to generate Abs is present in the germ-line genome
    - Ab diversity generated by recombining separate DNA segments in the germline genome of a B cell
    - applies to B and T cells
  2. somatic mutation theory
    - Ab variable regions undergo mutations in B cells to generate a diverse repertoire of Ab.s
    - applies to only B cells
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2
Q

What is the Dreyer and Bennett hypothesis?

A

Regions coding for the V and C regions of Ab.s are brought together through recombination of DNA in B cells - somatic recombination

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3
Q

What did Hozumi and Tonegawa show experimentally?

A

That DNA encoding for Lv and Lc regions are far apart in non-B cells and brough together in developing B cells - evidence of somatic recombinations

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4
Q

What are a couple of characterisitics of light and heavy chain gene segments?

A
  1. segments are quite far apart
  2. each variable gene has a leader sequence in front of it
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5
Q

What is the purpose of a leader sequence?

A

ER localization signal

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6
Q

What segments do light chains have and which segments do heavy chains have?

A

light = V and J
heavy = V, D and J

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7
Q

Which segment is the major region of Ag-binding

A

V segment

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8
Q

What is V(D)J recombination and what kind of diversity does it create?

A

V(D)J = combining different V and J (light chain) or V, D and J (heavy chain) segments allow different heavy and light chains to be generated

diversity = combinatorial diversity

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9
Q

What two factors contribute to combinatorial diversity?

A
  1. V(D)J recombination
  2. combining of heavy and light chains
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10
Q

What is the recombinational signal sequence (RSS)

A
  • at least one RSS flanks each V, D, and J segment
  • critical for recombination
  • conserved heptamer-spacer-nonamer sequence
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11
Q

How are heptamers, nonamers, and spacers conserved in the RSS?

A

heptamers and nonamers = conserved with respect to size and sequence

spacers = only size is conserved, either 12bp or 23bp

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12
Q

What is the 12/23 rule?

A

RSS sequences with 12bp spacers only recombine with RSS sequences with 23bp spacers (also known as one turn/two turn rule)

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13
Q

How is the kappa light chain recombined?

A
  1. 3’ of each Vkappa segment is a 12bp RSS, each Jkappa segment has a 5’ 23bp RSS
  2. one V segment recombines with one J segment to generate to V(L) of the kappa light chain
  3. intervening DNA is removed
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14
Q

How is the heavy chain recombined?

A
  1. 3’ of each V segment is as 23bp RSS, each J segment as a 5’ 23bp RSS, D segments have a 5’ and 3’ 12bp RSS
  2. one D segment recombines with one J segment
  3. one V segment recombines with the recombined DJ segment to generate the V(H) region
  4. intervening DNA is removed in both cases
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15
Q

What is recombinase activating genes - 1/2 (RAG-1/2)?

A
  1. lymphoid specific proteins that catalyze DNA strand breakage
  2. required for V(D)J recombination (including TCR gene rearrangement)
  3. high mobility group box (HNGB) proteins stabilize Rag-1/2 binding and help facilitate bending of DNA
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16
Q

What is the first step in V(D)J recombination?

A

Rag-1/2 and HNGB proteins bind to the RSS and catalyze synapse formation between a V and a J segment (bends the DNA)

17
Q

What is the second step in V(D)J recombination?

A

Rag-1/2 proteins nick 5’ of the sequence of the heptameric RSS sequence bordering both the V and J segments

18
Q

What is the third step in V(D)J recombination?

A

Free hydroxyl group created by nick attacks phosphate on opposite strand and creates a convalently sealed hairpin coding end and a blunt signal end. This happens at both the V and J segments.

19
Q

What is the fourth step in V(D)J recombination?

A

Ligation of the heptamer sequences by DNA ligase IV to create a signal joint. The circular episome (intervening DNA) is lost as a B cell divides during development

20
Q

What is the fifth step in V(D)J recombination?

A

Artemis (endonuclease) with the help of other proteins nicks hairpin at one spot, either creating a 5’ overhang, 3’ overhang (most common) or a blunt end. Where the strand is nicked affects the nucleotides that are added -> diff a.a -> variability

21
Q

What is the sixth step in V(D)J recombination?

A

cleavage events that generate overhangs can be filled in with P (palindromic) nucleotides -> junctional diversity

22
Q

What is the seventh step in V(D)J recombination?

A

Ligation of the light chain V and J regions

23
Q

What is the eighth step in V(D)J recombination (note this only happens in the heavy chain)?

A

Deletion of nucleotides by an exonuclease - exonuclease trimming -> junctional diversity

24
Q

What is the ninth step in V(D)J recombination (note this only happens in the heavy chain)?

A

addition of up to 20 N (non-templated) nucleotides by TdT (terminal deoxynucleotidyl transferase) which is not expressed during L chain reassortments -> junctional diversity

25
Q

What is the tenth step in V(D)J recombination (note this only happens in the heavy chain)?

A

ligation of heavy chain

26
Q

How does V(D)J recombination effect Ag binding sites of Ab.s?

A

most diversity (combinatorial/junctional) occurs in changing CDR3, CDR1 and 2 are within the V segments

27
Q

What processes result in changing CDR3?

A
  1. V segment diversity
  2. V(D)J recombination
  3. exonuclease trimming
  4. P-NT addition
  5. N-NT addition
  6. somatic hypermutation
28
Q

What process result in changing CDR1 and 2?

A
  1. V segment diversity
  2. somatic hypermutations
29
Q

Briefly describe V(D)J recombination in TCRs

A
  • alpha (gamma) is similar to light chain and has V and J segments
  • beta (delta) is similar to heavy chain and has V, D, and J segments
  • TCR alpha and delta chains are on the same chromosome (delta completely within alpha region) which is important in development -> lineage differences -> differentiation
30
Q

Describe how TCRs follow the 12/23 rule

A
  • V region segments contain a 3’ 23bp RSS
  • D region segments contain a 5’ 12bp RSS and a 3’ 23bp RSS
  • J region segments contain a 5’ 12bp RSS
31
Q

Where does V(D)J recombination generate the most diversity in a TCR?

A

CDR3 in both the alpha and beta chain, CDR1 and 2 are within the V segments

32
Q

Where does junctional diversity arise from in V(D)J recombination?

A
  1. P- NT addition
  2. exonuclease trimming
  3. N-NT addition

IMIN 371 (20 decks)

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