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Which DNA segments encode the light chain V region?
- VL: Variable segment
- JL: Joining segment
Which DNA segments encode the heavy chain V region?
- VH: Variable segment
- DH: Diversity segment
- JH: Joining segment
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
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
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
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
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)
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
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
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
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
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
What are the greek letter associations associated with immunoglobulin isotypes?
- μ - IgM
- δ - IgD
- γ - IgG
- ε - IgE
- α - IgA
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
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
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
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
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)
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
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)
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
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)
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
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
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)