Rearrangement Flashcards
What is a theory that wasn’t supported but was used to try and explain B cell’s magnitude of variablility
Germ Line Theories: that the genetic
information for each antibody is separately encoded within the germ-line (inherited) genome
•All BCR/TCR are germ line encoded
•Not enough DNA to support this theory
What two theory in 1970’s were used to explain variability in B-cell
In 1965, Dryer and Bennett proposed that:
* heavy and light chains are in separate segments of the genome.
* gene segments encoding a variable V and the constant C region must be connected to make a heavy or light chain.
In the early 1970s, somatic hypermutation theory was suggested.
* The theory noted that maturational processes occurring only in B cells could explain the variety of antibodies
* Limited number of genes
* Shuffling.
Who demonstrated that variable and constant chains are encoded by different gene segments that are combined?
Hozuma and Tongeawa
Immunoglobulin proteins (BCR) consist of?
*2 identical heavy chains, and
*2 identical light chains, which can be either kappa(κ) or lambda (λ).
Where are the gene family encoded on the chromosome?
•Kappa (κ) genes are on human chromosome 2 and mouse chromosome 6.
•Lambda genes (λ) are on human chromosome 22 and mouse chromosome 16.
•Heavy chain genes are on human chromosome 14 and mouse chromosome 12.
B cells use recombination of gene segments to create differnet possible antibodies. What are these gene segments called?
*There are variable (V), diversity (D), joining (J), and constant (C) region gene segments
*D segments are used in antibody heavy chains only
What gene segments does the kappa(k) light chain locus include?
The kappa (κ) light-chain locus includes V, J, and C segments.
* Mice have 120–140 Vk genes.
*Coding segments are separated by noncoding gaps of 5–100 kb.
*Four functional Jk segments are downstream of the Vk segments.
*Humans have a similar arrangement but with differing numbers of Vk and Jk segments.
What gene segments does the lambda light chain locus include?
Lambda (λ) light-chain genes pair each J segment with a particular C segment.
*Only 5% of mouse Igs have lambda light chains due to a loss of most of the Vλsegments.
*There are usually only three fully functional Vλgenes.
In humans, 40% of light chains are lambda type.
*About 33 Vλgenes are used.
*There are a series of 7 Jλ-Cλpairs (four or five are functional).
What gene segments dose the heavy chain gene organize?
Heavy-chain gene organization includes VH, DH, JH, andCHsegments.
* Mice express approximately 100 VHsegments whereas humans express at least 45 functional VHsegments.
* DHsegments are downstream of the VHsegments.
* There are 14 DHsegments in mice and 23 in humans.
* There are 4 JHsegments in mice and 6 in humans.
*
Eight CH regions exist and encode different antibody isotypes.
What did Hozuma and Tongeawa do?
Who demonstrated that variable and constant chains are encoded by different gene segments that are combined?
What is the possible number of beavy and light chain immunoglobulin vairable region combinations in human?
Possible number of heavy-and light-chain immunoglobulin variable region combinations in the human = 6210 ×(205 + 165) = 2.3 ×106
What is flanked by each gene segment in V(D)J recombination?
Recombination signal sequences (RSSs) flank each gene segment.
*Each has a conserved nonamerand heptamersequence.
*Either a 12-or a 23-bp spacer sequence lies between the nonamer and heptamer.
*The spacing and arrangement dictate that a 12-bp RSS must pair with a 23-bp RSS for recombination to occur (known as the “12/23 Rule”).
What joins gene segments is V(D)J recombination?
- Gene segments are joined by the RAG1/2 recombinase
- RAG= recombination activating gene
- Both proteins are needed for recombination
- RAG1 is more important—it forms a complex with RSSs stabilized by binding RAG2
- RAG1/2complex is responsible for recognizing and cutting DNA at the immunoglobulin-encoding region and the RSS
- Numerous other proteins are required for recombination, including several that are not unique to lymphocytes
What 5 mechniams generate antibody diversity in vaive B cells?
- Multiple gene segments exist at heavy (V, D, and J) and light-chain (V and J) loci. These can be
combined with one another to provide extensive combinatorial diversity. - Heavy-chain/light-chain combinatorial diversity: The same heavy chain can combine with
different light chains, and vice versa. The combination of different heavy- and light-chain
pairs to form a complete antibody molecule provides further opportunities for increasing the
number of available antibody combining sites. - P-nucleotide addition results when the DNA hairpin at the coding joint of heavy and light
chains is cleaved asymmetrically. Filling in the single-stranded DNA piece resulting from this
asymmetric cleavage generates a short palindromic sequence. - Exonuclease trimming sometimes occurs at the V-D-J and V-J junctions, causing loss of
nucleotides. - Nontemplated (N)-nucleotide addition by TdT in heavy-chain V-D and D-J junctions and from
DNA polymerase μ in both heavy and light chains.
What is combinatorial diversity?
Heavy-chain/light-chain combinatorial diversity: The same heavy chain can combine with
different light chains, and vice versa. The combination of different heavy- and light-chain
pairs to form a complete antibody molecule provides further opportunities for increasing the
number of available antibody combining sites.
What is isotype switching?
- Occurs only after B cells have been stimulated by Ag
- Requires specialized nonhomologous DNA recombination using stretches of repetitive DNA known as switch regions
- Switch regions found in the introns upstream from C genes except Cδ= CΔ
- Details of switching process are unclear and enzymes that promote it not identified
- IgG2a or IgG3 (mice): IFN-γ
- IgM: IL-2, IL-4, IL-5
- IgE: IL-4
What chains for T-cell receptors were found first
The β chain gene setup was discovered first
*While looking for the α chain genes the γ chain genes were discovered
*A small subset of T cells express a γδ TCR rather than an αβ TCR, but the two are structurally similar
What are some similarities and differences betwwn TCR and BCR
What is the gamma/delta t cell?
- Gamma/Delta T cells can recognize antigen in an MHC-independent manner
- Gamma/Delta T cells play a role in responses to certain viral and bacterial pathogens
What is the difference between a/b and g/d in TCR
*TCR genes are located somewhat differently between mice and humans.
*The βand δchain genes have V, D, and J segments, whereas α and γchain genes have only V and J segments.
*TCR genes undergo a process of rearrangement very similar to that of Ig genes.
*Note the general arrangement of the TCR genes in the last diagram.
*The machinery for their rearrangement has already been discussed.
What are the steps for V(D)J recombinatio?
- RAG 1/2 and HMGB 1/2 proteins bind to the RSS and catalyze synapse formation between a V and a J gene segment (called synapses)
- RAG 1/2 performs a single-stranded nick at the exact 5’ border of the heptameric RSSs bordering both the V and J segments
- The hydroxyl group that was liberated by the nick at the 3’ end of the coding strand attacks the corresponding phsophate group on the noncoding strands of both the V and the J segments to yield a covalently sealed hairpin coding end and a blunt signal end (creates coding end and signal end)
- signal end joining ligates the ends of the two RSS heptameric sequences that were originally in contact with the V and J coding sequences
- Opening of the hairpin can resilt in a 5’ overhand, a 3’ overhand or a blunt end. The most common result generated by Artemis is a 3’ overhand
- Cleavage of the hairpin generates sites for P nucleutide addition
- Ligation of the light chain V and J region
What are steps 8-10 for Ig heavy chain only?
** Steps 8-10 only occur in Ig heavy chains **
- exonuclease cleavage can result in the loss of nucleotides on either or both sides of the coding joint
- Nontenplated nucleotides (in red) are added to the coding join by TdT or occasionally by pol u
- Ligation of heavy chain by NHEJ DAN ligase complex
How is V(D)J recombination regulated
Regulation of V(D)J gene recombination involves alterations to chromatin.
- The catalytic activity of RAG1/2 occurs in an extraordinarily complex nuclear environment.
- RAG1/2 binding is affected by epigenetic modifications on histones associated with the target sequences.
*Histone acetylation or methylation affects sequence accessibility to enzymatic activity.
- Because the V, D, and J gene segments are so spread out along the chromosome, higher-order chromatin structure must also play a role.
What form does chromatin take for V,D,J
Chromatin
visualization techniques have shown that chromatin folds extensively into loops of various lengths
that cluster into the form of rosettes (Figure 6-13a).
Detailed analysis of the three-dimensional structure of the Ig heavy-chain gene locus has
indicated that it initially appears to be arranged in space into three rosette-containing chromatin
regions. One of these regions contains the distal V genes (those farthest from the D region); a
second contains the proximal V genes (those nearest to the D region); and the third contains the
gene segments of the D, J , and C regions. Since recombination events are topologically limited
to include only genes within a rosette, the rosette loop that contains the D, J , and C regions
defines the scope of RAG activity in the earliest B lymphoid precursors, pre-pro-B cells. Once D -J
recombination has occurred, the loop structure is altered to allow V -D recombination at the pro-
B-cell stage. Figure 6-13a illustrates the change in the structure of the Ig loci as development
proceeds.
What occurs within the nucleus as the B-cell developes
Within the nucleus:
* inactive chromatin is near the nuclear lamina.
* active chromatin is near the nucleus interior.
* Chromosomes relocate within the nucleus during B-cell development.
Thus, colocalization with the nuclear lamina as well as the physical nature of the chromatin loops ensures that the only transcriptional and associated recombinational events that can occur are restricted.
What are the steps for Ig Gene recombination?
- Germline DNA
- Rearranged DNA
- Primary RNA transcript
- Messenger RNA (mRNA)
- Nascent polypeptide
- Mature polypeptide
- assembled Ig molecule
What does allelic exclusion ensure?
- Allelic exclusion ensures that each B cell synthesizes only one heavy and one light chain
- Heavy chains are recombined and expressed first
- Expression of a functional heavy chain shuts down recombination machinery temporarily
- The heavy chain is paired with a surrogate light chain (SLC) to form a pre-BCR
- If the SLC will pair with the heavy chain, the machinery is started up again
- Light-chain recombination then takes place
- Nonproductive arrangements lead to programmed cell death (apoptosis) during development
What is receptor editing?
Receptor editing of potentially autoreactive receptors occurs in light chains.
* A functional antibody in an immature B cell may bind to self-antigens.
- The recombination machinery can be turned back on to edit the original rearrangement or at least inactivate it.
Why do mature B cells express both IgM and IgD
•Mature B cells express both IgM and IgD antibodies due to differential use of polyadenylation sites and mRNA splicing.
•Heavy-chain transcripts have VDJ put together, but there is a spacer RNA sequence between the VDJ and C regions.
•mRNA splicing removes the intervening spacer, leaving VDJC mRNA ready to be translated.
•Primary transcripts for IgM heavy chains may result from RNA polymerase transcribing through both the IgM and IgD constant regions.
•Depending on which C segment becomes polyadenylated, IgM or IgD could end up being produced.
How are other classes of antibodies produced
Production of antibodies containing other heavy-chain classes requires class switch recombination(CSR).
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What happens if no Rag 1 or 2
- Partial or complete loss of activity (in either RAG 1 or 2) causes severe combined immunodeficiency (SCID) due to arrested lymphocyte development
- Null -> T-B _SCID
- Partial –Omenn syndrome (No B cells, variable poorly functioning T cells)
- Antigen receptor assembly uses the same genetic machinery in both T and B cells.
- Defects in V(D)J recombination affect generation of both T and B cells, leading to severe combined immunodeficiency (SCID).
