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Which DNA segments encode the light chain V region?
- VL: Variable segment
- JL: Joining segment
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Which DNA segments encode the heavy chain V region?
- VH: Variable segment
- DH: Diversity segment
- JH: Joining segment
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What is the difference between light chain and heavy chain recombination (general).
- Light chain: Joining of VL and JL region creates V region exon
- C region is encoded on a separate exon and joined by splicing
- Heavy chain: DH joins JH first, followed by then the DJH sequence joins the VH segment to form the V region exon
- C region is encoded by several exons, then joined by splicing
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What is the major difference between the heavy-chain locus and the λ and κ light chain loci (re: recombination/translation).
The heavy-chain locus has a series of C regions, each of which corresponds to a different isotype
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What is a RSS? Describe.
- Recombination signal sequence: found adjacent to the points of recombination (signal) and have three parts...
- 1. Heptamer
- 2. Spacer (either 12 or 23 bp - one or two turns)
- 3. Nonamer
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What is the 12/23 rule?
A gene segment with a 12bp spaced RSS can only join to a gene segment with a 23bp spaced RSS
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What is the major difference between CDR1, CDR2, and CDR3 origin/translation?
- CDR1 and CDR2 are encoded in the V gene segment
- CDR3 is the most important (most variable) and is formed by VJ recombination (light chain) and VDJ recombination (heavy chain)
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Describe the mechanism of DNA rearrangement in light chain and heavy chain loci re: orientation.
- two segments opposite transcriptional orientation: rearrangement results in deletion (looping out) of DNA between them
- two segments same transcriptional orientation: intervening DNA is retained in an inverted orientation
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What is the complex of enzymes that carry out VDJ recombination (molecular level) w/ brief function. What is the importance?
- VDJ recombinase
- RAG1 and RAG2: Leave hairpins adjacent to V, D, or J regions
- DNA-PK;Artemis complex: cut the hairpin, resulting in P-nucleotides (palindromes)
- terminal deoxynucleotidyl tranferase: randomly adds nucleotides to these locations
- Exonuclease: trims unpaired nucleotides, and repairs joint
- The randomness of this mechanism results in the significant diversity in CD3 regions
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What main processes generate the diversity of the immunoglobulin repertoire?
- Combinatorial diversity: VDJ recombination AND combination of heavy chain with κ or λ light chains
- Junctional diversity: introduced at joints between gene segments due to addition/subtraction of nucleotides
- Somatic hypermutation: introduces point mutations into the rearranged V-region genes of activated B cells
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How does the organization of T cell receptor gene segments compare to the organization of immunoglobulin gene segments?
- They are broadly homologous
- TCRα: contains V and J segments (Vα and Jα) like Ig light chains
- TCRβ: contains D, V, and J segments (Vβ, Dβ, and Jβ) like Ig heavy chains
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Typical associations of "class" and "type" re: immunoglobulins
- Class: typically refers to the heavy chain
- Type: typically refers to the light chain (κ or λ)
- *note: isotypes refers to types of heavy chains
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What are the greek letter associations associated with immunoglobulin isotypes?
- μ - IgM
- δ - IgD
- γ - IgG
- ε - IgE
- α - IgA
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What are the functions of the C regions (Fc) of an antibody?
- 1. Recognition by specialized Fc receptors facilitating phagocytosis and inflammation
- 2. Bind to C1q complement protein to initiate classical complement cascade (results in opsonization)
- 3. Delivery of antibodies to places they wouldn't reach without active transport
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Describe microorganism responses to the Fc region of antibodies w/ responsible organism
- Protein A: binds to Fc region of IgG, disrupting opsonization and phagocytosis (Staphylococcus aureus)
- Protein G: binds to Fab AND Fc region of IgG (Streptococcus)
- Protein D: initially thought to bind IgD, proven incorrect
- Protein L: Peptrostreptococcus
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Why are IgM and IgD expressed first during B cell development? At which stages are they most prevalent?
- The gene encoding the μC region is closest to the assembled VH region (VDJ exon), so IgM is the first Ig to be produced
- The gene encoding the δC region is directly adjacent to the μC region
- Differential processing of the long primary mRNA transcript determines whether IgM or IgD is expressed
- Immature B cells: mostly μ transcript (IgM)
- Mature naive B cells: mostly δ transcript (IgD)
- Activated B cells cease to co-express IgD with IgM
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How are transmembrane and secreted forms generated from DNA if they are not identical?
- The last 2 exons of each CH gene contain sequences encoding secreted and transmembrane regions
- The translated form is determined by location of primary transcript cleavage/polyadenylation
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Which Igs form polymers? How? What is the role?
- IgM and IgA form multimers through a "tailpiece" that contains a cysteine residue
- The J chain (separate) links to the tailpiece (found only in secreted forms)
- IgM role: pentamer provides higher avidity for antibody binding (being created before somatic hypermatutation, IgM is less specific and thus has a weak affinity)
- IgA role: dimerization required for transport through epithelia (tend to be monomer in plasma)
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What are the 3 secondary diversification mechanisms? What is the enzyme causes them?
- RAG-mediated VDJ recombination takes place in the bone marrow BEFORE antigen interaction, further diversification occurs only for B cells after antigen interaction
- 1. somatic hyptermutation: induces point mutations into V regions which alters the affinity of Ab
- 2. Class switching: replaces Cμ with an alternative region
- 3. Gene conversion: replaces blocks of sequence in the V regions with sequences derived from V regions of pseudogenes
- They are initiated by activation-induced cytidine deaminase (AID) which is only expressed in activated B cells
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What is the mechanism of AID?
- Activation-induced cytidine deaminase
- binds to and deaminates ssDNA (not dsDNA) ∴ only targets genes during transcription
- Only expressed in activated B cells
- AID converts cytidine to uridine
- Uridine in DNA can trigger several types of DNA repair (various mutational outcomes)
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What mutational outcomes are initiated by the presence of uridine in DNA? Describe them
- 1. (somatic hypermutation) Mismatch recognized by repair proteins: remove the uridine along with adjacent nucleotides from damaged DNA strand
- Error-prone DNA polymerase fills in random nucleotides
- 2. (somatic hypermutation) Mismatch detected by base excision repair pathway: removal of uracil leaves blank space
- DNA polymerase inserts random nucleotide in that space during next round of replication
- 3. (gene conversion) APE1: removal of uracil resulting in ssDNA nick
- Repair by homologous recombination (pseudogenes)
- 4. (class switching) APE1: removal of uracil resulting in dsDNA nick
- Occur as staggered breaks in specific locations of Ig C-region genes, repair leads to class switching
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What are the two requirements for somatic hypermutation to occur?
- mature B cells must be activated by their corresponding antigen AND activated by signals from activated T cells
- (T cells do not undergo hypermutation)
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What is affinity maturation?
- During somatic hypermutation some of the mutant IgG bind antigen better than the original receptors
- These mutants are preferentially selected to mature into antibody-secreting cells
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What is the purpose of class switching, and what causes it (not the mechanism)
- Class switching allows the same V region that was originally associated with IgM or IgD to be expressed in the form of IgG, IgA, or IgE antibodies (irreversible)
- It is stimulated by external signals such as cytokines released by T cells or signals delivered by pathogens
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What is the mechanism of class switching? (detailed response :()
- Switch regions (S) (repetitive DNA sequences that guide class switching) are found upstream of EACH Ig C-region gene (except δ)
- Switching is initiated by transcription from promoters upstream of each S
- Transcription through S regions generates R-loops which serve as substrates for AID, then UNG and APE1
- These enzymes introduce ss nicks into both strands at staggered locations, but are converted to ds breaks
- The ds breaks are repaired by bringing the s regions together (μ and ___) **note Sμ no longer has an assocated C region, it has been excised)
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