Question 1: Combinatorial diversity

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• Question 1 Combinatorial diversity
• Given the following numbers of gene segments
  (below), what are the numbers of possible Ig
  heavy chains and Ig light chains that an
  individual can make based on combinatorial
  diversity alone (ignoring junctional diversity)?
• IgH 6480 Ig k 200 Ig l 120 (just multiply the
  numbers together)
• Assuming that any light chain can successfully
  pair with any heavy chain (probably slightly
  inaccurate), what is the number of antibodies
  that can be generated by combinatorial diversity?
  
• 2.1 x 10E6 (add the Ig k and Ig l numbers
  together and multiply that number times the
  number of heavy chain combinations)
• IgH 40 V, 27 D and 6 J
• Ig k 40 V and 5 J
• Ig l 30 V and 4 J

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Question 2 Junctional diversity Which
rearrangement(s) would be in frame (assuming the
Ns dont equal a stop codon)? B and E because
they are an integral number of codons (3 bp per
codon) Calculate the junctional diversity
possible for rearrangements with this number of
base additions. In this simplified example, B has
20 possibilities, because that is the number of
amino acids used in proteins E has 400
possibilities (20 x 20). If we added to this
removal of bases, then the CCC codon or the TGG
codon could also change, providing additional 20X
multiples. Obviously, one gets very large
numbers this way, particularly if there are two
junctions, as in IgH and TCR b. In antibody and
TCR structures, these amino acids are in the part
of the structures that binds antigens.
A CCCNNTGG B CCCNNNTGG IN FRAME
20 possibilities C CCCNNNNTGG D
CCCNNNNNTGG E CCCNNNNNNTGG IN FRAME 400
possibilities
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Question 3 Analysis of diversity of antigen
receptor genes A) On the following diagram of
the rearranged Ig k locus, indicate where this
primer would sit and which location you would use
to place a second primer.
PCR primers are indicated by arrows, showing
direction of DNA synthesis during the PCR
reaction. What is amplified is a DNA fragment
with the primers at the ends. The length of the
fragment will be affected by the junctional
diversity reactions. Or could use different
leftward primers next to each J.
B) Illustrate schematically what the PCR result
would look like
With a diverse lymphocyte pool, you would get a
ladder of bands in the gel, each band
representing a different size of fragment. If
mRNA is used for PCR, the ladder will have a
spacing of 3bp, because it will be primarily the
functional rearrangements, whereas if DNA is
used, the fragments will include non-functional
rearrangements. For lymphoma, one band will be
stronger and the ladder will be weaker,
representing expansion of one clone.
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Question 4 Control of V(D)J recombination

• Do these rearrangements obey the 12/23 rule
  which is enforced by Rag1 and Rag2 action? YES
• Does the 12/23 rule determine the order of these
  two rearrangements)? NO, that is thought to be
  controlled by accessibility, see Question 5.

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• Question 5 Transcriptional control of Ig genes
  and V(D)J recombination
• On the diagram of a rearranged IgH locus shown
  below, draw in the positions of the promoters and
  the enhancer.
• How does this organization of transcriptional
  control elements provide for strong activity of
  the promoter for the VH that is rearranged to the
  DJH as compared to the other VH genes upstream of
  VDJ (which are not deleted during V(D)J
  recombination)?

enhancer
promoters
The enhancer works preferentially on the closest
promoter, so it will increase transcription from
the VH promoter that is adjacent to the VDJ
element and not increase transcription from the
upstream VH promoters that act on VH elements
that are not rearranged to DJ.
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• Question 5 continued
• Develop a hypothesis to describe how
  accessibility control could result in
  rearrangement at the IgH locus preceding
  rearrangment at the Ig light chain loci.
• Transcription factors acting on the IgH locus are
  expressed in pro-B cells whereas transcription
  factors acting on the Igk and Igl loci are turned
  on at the pre-B cell stage of development, so the
  light chain loci are not accessible when
  recombination at the IgH locus is occurring.
• Describe how the enhancer of these antigen
  receptor loci could alter the expression of the
  oncogene.
• The Ig or TCR enhancer can drive expression of
  the oncogene, which is now expressed at a higher
  than normal level, which can promote unregulated
  growth or survival of the cell with the
  translocation

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• Question 6 Errors in V(D)J recombination and
  human disease
• In what way is lymphocyte development dependent
  on Rag1, Rag2 and Artemis? (Artemis plays a role
  immediately after Rag1 and Rag2 in V(D)J
  recombination)
• In people lacking one of these proteins, there
  are no Ig or TCR rearrangements, so no T or B
  cells develop,
• Describe the immunological phenotype of
  individuals with defects in genes encoding any of
  these three proteins.
• No Ig (beyond maternal IgG early in life), No B
  cells or T cells upon analysis of blood. The
  blood contains roughly normal numbers of NK
  cells.