Central Dogma and antibody diversity, continued

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Central Dogma and antibody diversity, continued

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The basic mechanism of transcription
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Transcription

• RNA polymerase binds to the DNA near the
  beginning of a gene at a site called the promoter
• The polymerase "unwinds" the DNA, exposing the
  bases.
• Complementary bases are added against the
  template strand
• The resulting messenger RNA (mRNA) strand is
  called the primary transcript

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Amino acids are represented by codons
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The three roles of RNA in protein synthesis

• Messenger RNA (mRNA) carries information copied
  from DNA in the form of a series of three base
  codons
• Transfer RNA (tRNA) deciphers the code by
  delivering a specific amino acid to its
  associated codon(s)
• Ribosomal RNA (rRNA) associates with a set of
  proteins to form ribosomes, acting as some of the
  key enzymes in these machines

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Splicing

• Introns are part of the primary transcirpt, but
  not part of the functional RNA
• In mammals, the bulk of the DNA in genes is in
  the form of introns
• These are removed after transcription by splicing

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Alternative splicing saving space

• Protein domains are typically encoded in separate
  exons
• Sometimes there is more than one exon for a given
  domain.
• Thus, one gene can make more than one version of
  a protein by alternative splicing of the mRNA!

A1
A2
B
protein version 1
protein version 2
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Translation by ribosomes

• mRNA binds to the ribosome
• A complementary transfer RNA (tRNA) binds to an
  underlying codon.
• The amino acid bound to the tRNA is moved onto
  the growing protein strand.
• The ribosome movesforward

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The central dogma

• DNA is transcribed into RNA
• RNA is translated into protein
• and not really part of the central dogma, DNA
  is replicated to form more DNA

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Antibody diversity What WONT work

• Separately encode each antibody in a gene?
• No. You have millions of different antibodies yet
  there are only around 25-35,000 genes.
• Alternative splicing of mRNA?
• No, because each lymphocyte generates antibodies
  and TCRs with the same antigen specificity for
  its lifespan. How could you splice the mRNA so
  reproducibly?

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and what WILL

• The genes that code for Ig contain interchangable
  modules defining the variable and constant
  domains of the heavy and light chains.
• The GENOME ITSELF is rearranged during
  development, giving rise to slightly different Ig
  genes between individual lymphocytes!
• This occurs during antigen-independent
  differentiation of lymphocytes, early in life.

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Antibody diversity

• Both the heavy and light chains are involved in
  antigen binding, and so both contribute to
  antigen specificity. Each heavy and light chain
  has a variable region
• Within these variable regions are regions of
  hypervariability
• These hypervariable regions cluster in the folded
  protein at the antigen binding site.

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Heavy chain genes are modular

• Around 300 slightly different copies of Variable
  segments
• Around 30 Diversity segments
• Around 6 Junctional segments
• Recombine through rearrangement of the germ line
  DNA to yield somatic DNA unique to each B cell

D4
Germ line DNA
B cell DNA
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Light chain genes

• Around 100 Variable segments
• Around 4 Junctional segments
• No Diversity segments in the light chain genes

DNA splicing
RNA splicing
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Degree of diversity

• Heavy chain combinations
• 300V x 30D x 6J
• 54,000 VDJ variations
• Light chain combinations
• 100V x 4J
• 400 VJ combinations
• Heavy / Light chain combinations
• 54,000 x 400
• 21 million combinations!
• Not enough! A virus could easily adapt to avoid
  the system.

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Junctional diversity

• The joining of V, D and J segments is
  deliberately imprecise
• For example, the V sequence CCT.CCC can be joined
  with the J sequence TGG.ACG in four different
  ways
• CCT CCC ACG encodes pro.pro.thr
• CCT CCG ACG encodes pro.pro.thr
• CCT CGG ACG encodes pro.arg.thr
• CCT TGG ACG encodes pro.trp.thr
• Rearrangement / joining continues until
  successful

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Hypermutation

• T cells and cytokines stimulate B cells
  presenting an epitope
• DNA is replicated during proliferation and
  differentiation of B cells
• The V segment DNA bases are (intentionally?)
  copied with small errors (somatic mutation)
• The resulting plasma cells with slightly
  different antibodies compete for antigen
• Proliferation and survival of the fittest
  antibody producing cells.

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Hypermutation and competition
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Class switching IgM to IgD through alternative
splicing and alternative splicing
After DNA splicing
Cd
Vn
VDJ
Jn
Cm
Cd
VDJ
Jn
Cm
RNA
Alternative splicing
To make IgD
To make IgM

• Antigen specificity remains the same. Why?

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Class switching IgM and IgD to IgG through genome
rearrangement
After DNA splicing
VDJ
Cm
Cd
Cg3
Cg1
Ca1
Cg2
Cg4
Ce
Ca2
After class switch
VDJ
Cg1
Ca1
Cg2
Cg4
Ce
Ca2

• When will this IgM/IgD ? IgG switch happen?
• Since antigen specificity doesnt change here
  either, whats the advantage over alternative
  splicing?