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34 Cards in this Set

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Combinationaldiversity: Light chain,Heavy chain, Immunoglobulin Loci,Control, Result, Issues with control

Class switching

Somatic hypermutation(in germinal centres) - Targeting,Mechanism, Issues

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

Combinational diversity- what is the process?

V(D)J recombination.

Where does the process occur

Only in B cells

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

Light chain, explain the gene segments

· Up to 40 variable (V) gene segments. FiveJoining (J) Segments. One Constant (C) Segment

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.

How is diversity created?

Imprecise joining

Explain the gene segments in the heavy chain

Heavychains have up to 27 additional D (diversity) region genes

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.

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.

Explain the control - overview

· Conserved DNA heptamer/nonamer sequences flankthe coding regions and form a recombination signal sequence (RSS)

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.

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.

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.

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.

location of gene segments to be recombined?

adjacentto RSS of different spacer lengths- important feature in the regulator of recombination

What repairs re-joined segments



· Highly variable antigen-binding region even whenthe same gene segments are recombined.

· Overall: costly process and is strictlyregulated and controlled.

Issues with control- syndrome

· Omenn Syndrome - Caused by a missense mutationin RAG1. Ab gene rearrangement cannot occur

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.

Class switching: what determines class

constant-regionexon next to the V(D)J exon.

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.

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.

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).

What ensures only maternal or paternal Ab genes are expressed?

· Allelic exclusion – ensures only maternal orpaternal Ab genes are expressed. Other switched off.

Where does somatic hypermutation occur?

in germinal centres

What is the reason for somatic hypermutation?

Antibodyaffinity for antigen increases during the course of an immune response.

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.

What three proteins are involved?

Activation-Induced (Cytidine) Deaminase- (AID)

uracil-DNAglycosylase (UNG) - removesuracil

APE1– excises ribose

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.

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.

Which B cell is selected?

greatest affinity will be selected todifferentiate into plasma cells producing Abs and memory B cells.


· Mistargeted somatic hypermutation is a likelymechanism in the development of B-cell lymphomas