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34 Cards in this Set
- Front
- Back
Structure
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Introduction Combinationaldiversity: Light chain,Heavy chain, Immunoglobulin Loci,Control, Result, Issues with control Class switching Somatic hypermutation(in germinal centres) - Targeting,Mechanism, Issues |
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introduction - what is the antibody repertoire and what are hypervariable regions |
· The total number of antibody specificitiesavailable to an individual is known as the antibody repertoire and in humans isat least 10^11. · Hypervariable regions - Three CDRs from eachthe heavy and light chains |
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Combinational diversity- what is the process? |
V(D)J recombination. |
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Where does the process occur |
Only in B cells
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At what stage does Ab gene rearrangements start? |
Early pro-B cell stage: D-J in heavy chain Late pro-B V-Dj in heavy chain |
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Light chain, explain the gene segments |
· Up to 40 variable (V) gene segments. FiveJoining (J) Segments. One Constant (C) Segment |
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What are the steps |
· Step 1: Somatic Recombination (occurs at the DNAlevel). · V-J joining results in the excision of a whole seriesof V and J genes to create a new V-J combination · Step 2: Transcription and Splicing. Thesomatically recombined DNA is transcribed into pre mRNA (primary mRNA). Theintrons are spliced out, combining the V,J and C regions. · The primary RNA is processes to add apolyadenylated (poly-A) tail after Cu chain. · The message is translated into the light chainprotein. |
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How is diversity created? |
Imprecise joining |
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Explain the gene segments in the heavy chain |
Heavychains have up to 27 additional D (diversity) region genes |
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Explain somatic recombination in the heavy chain |
· Somatic recombination of heavy chain genesinvolves the D-J regions, followed by V-DJ joining. The rest of the process issimilar to the kappa chain. All other genes are deleted from genome. |
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What are the Immunoglobulin Loci? |
· kgenes are on chr 2 - gene segments for light chain. · lgenes are on chr 22 – light chain · The heavy chain genes are on chr 14. |
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Explain the control - overview |
· Conserved DNA heptamer/nonamer sequences flankthe coding regions and form a recombination signal sequence (RSS) |
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How is specificity of recombination maintained? |
Rag1and Rag2 recombinase enzymes binds an RSSs flanking a V, D and J gene segments– creates a single-strand nick in DNA between the first base of RSS and thecoding segment. This is essentially energetically neutral – no ATP and resultsin the formation of a free 3’ OH and 5’ phosphate group on the same strand. |
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Explain the next step with the OH group |
· The reactive OH group is positioned by therecombinase to attack the phosphodiester bond of opposite strand, forming twoDNA ends: a hairpin on coding segment and a blunt end of the signal segment.Current model – DNA nicking and hairpin formation occur on both strands simultaneously. |
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Next step |
Additional proteins (DNA-dependent protein kinase, Ku, Artemis and a dimer of DNA ligase and XRCC4) are incorporated into a large complex with the Rag proteins. These RSS ends are joined, forming what is called the Signal Joint, to create a closed circular DNA, that plays no further role in the recombination process. |
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Explain RSS |
· RSS composed of three elements: heptamera of 7conserved nts, spacer region (12/23 bps) and nonamer – 9 conserved nts. Whilethe majority of RSSs vary in sequence, the consensus heptamer and nonamersequences are similar. Although the sequence of the spacer region is poorlyconserved, the length is highly conserved – corresponds to 1 or 2 turns of DNAhelix. |
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location of gene segments to be recombined? |
adjacentto RSS of different spacer lengths- important feature in the regulator of recombination |
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What repairs re-joined segments
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DNAligase |
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Result |
· Highly variable antigen-binding region even whenthe same gene segments are recombined. · Overall: costly process and is strictlyregulated and controlled. |
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Issues with control- syndrome |
· Omenn Syndrome - Caused by a missense mutationin RAG1. Ab gene rearrangement cannot occur |
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Other issues |
· Evidence that signal joints may re-enter thegenome and lead to pathologies by activing oncogenes or interrupting tumoursuppressor gene functions. · Major caveat: the DNA sequence must remainin-frame in order to maintain correct aa sequence in the final protein product.Out-of-frame – development of cell will be arrested. |
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Class switching: what determines class |
constant-regionexon next to the V(D)J exon. |
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What determines which Ab to switch to |
· If activated B cells encounter specificsignalling molecules via CD40 and cytokine receptors – Ab class switching toIgG, IgA or IgE.
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What is the class switching recombination? |
· This 'class switch recombination' process resultsin the replacement of the Cmexon by Cg, andthe deletion of the intervening sequence in the heavy chain.
· IgG protein is then produced by the cell,instead of IgM. |
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How is the variable domain rejoined? |
· Thevariable domain exon is rejoined through a process called non-homologous endjoining (NHEJ) to the desired constant region (γ, α or ε).
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What ensures only maternal or paternal Ab genes are expressed? |
· Allelic exclusion – ensures only maternal orpaternal Ab genes are expressed. Other switched off.
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Where does somatic hypermutation occur? |
in germinal centres |
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What is the reason for somatic hypermutation? |
Antibodyaffinity for antigen increases during the course of an immune response. |
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What is result ofsomatic hypermutation |
· BCR locus undergoes high rate of somaticmutation – 106 times greater than normal · Variation mainly in the form of single basesubstitutions. Indels are less common · Mutations occur mostly at hotspots in DNA –concentrated in hypervariable regions. |
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What three proteins are involved? |
Activation-Induced (Cytidine) Deaminase- (AID) uracil-DNAglycosylase (UNG) - removesuracil APE1– excises ribose |
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Explain the mechanism |
· Activated B cells make (AID) which converts c to u in Ig DNA variableregions. · Hypermutation occurs when cells try to fix theerror. · AID works on ssDNA so is restricted to activeloci that are being transcribed. · (UNG)and APE1 remove the Uracil. Other nucleic acids can get inserted in the gap. |
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What fills in gap? |
· Error-proneDNAP fills the gap. Replacement of U at deaminated cytosine or neighboring bp leadsto a new codon resulting a new aa at the Ab antigen recognition site. |
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Which B cell is selected?
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greatest affinity will be selected todifferentiate into plasma cells producing Abs and memory B cells. |
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Issues |
· Mistargeted somatic hypermutation is a likelymechanism in the development of B-cell lymphomas |