Genetics of the Immunoglobulins -Thrush Flashcards
How can we produce so many antibodies with limited genes? When does this occur?
Ig gene rearrangement. the random selection of genes to make the antibodies occurs BEFORE antigen stimulation
What gene segments make up the heavy chain? Which genes make up the variable domains for the heavy chain?
V, D, C, and J region genes make up the heavy chain.
V+D+J make up the variable domain for the heavy chain.
How does the process of allelic exclusion work? What is the order of rearrangement?
a given B cell will express ONE heavy chain Ag specificity and ONE light chain Ag specificity.
during the process of Ig gene rearrangement, only one allele may be expressive at one time (because B cells only produce antibodies with ONE specificity). Therefore one allele will be expressed and one allele will be silent in the B cells.
if the first allele is successful, then the process stops.
if the first allele is non-productive, then the other allele will be re-arranged. This occurs in a specific order: H chain first, then kappa light chain then lambda light chain (if the kappa chain isn’t successful)
the heavy chain gene is transcribed into mRNA and then translated into a polypeptide. The polypeptide will associate with a surrogate light chain go the B cell surface to determine if it is good. If the heavy chain is good, the kappa light chain will be rearranged. if it is functional the new light chain +heavy chain (mIgM) will go to the surface and anchor in the lipid bilayer.
In what order are genes rearranged? What are the products?
IgH chain genes first
DH and JH join, then VH with DHJH
yielding a VDJ DNA sequence
then the VHDHJH genes are transcribed to produce mRNA through the C gene
C region selection: the mRNA is then spliced to place VHDHJH next to either C mu or C delta genes.
mRNA is translated into protein to produce wither the IgM heavy chain or the IgD heavy chain.
IgM is first, followed by co-expression of IgD only time (normally) that a B cell expresses two isotypes
When does the expression of IgM and IgD occur?
post-transcriptionally
What is responsible for causing only one V region to be selected? What is the 12/23 rule?
RSS (recombination signal sequences) flank the Ig genes and are responsible for the cutting and resealing of the DNA.
heptamer, 12 or 23 bp intervening sequence (spacer), then an AT-rich nonamer sequence
= heptamer – spacer – nonamer
12/23 bp rule – a 12 bp sequence can join with a 23 bp sequence–> ensures you get one of each gene (V and J regions have 23 bp while D regions have 12 bp)
What is the enzyme responsible for bringing the spliced DNA together?
RAG (recombination activating genes) enzymes recognize the spacer sequence and helps to bring together the 12/23 sequences
What happens to a newly mature B cells that does not come in contact with its antigen?
The B cell will die if not exposed to its antigen
What happens to a newly formed B cell that DOES come into contact with its antigen?
the B cell will proliferate and form 2 populations of B cell: memory B cells and plasma cells
memory B cells can later class switch and plasma cells produce mass amounts of secretory antibodies
What enzymes are needed for gene rearrangement and what do they do? (3)
V(D)J recombinase: recognizes and excises the intervening sequences (looping out) to make it functional
RAG1 and RAG2: recognize and bring together the RSS
Terminal deoxynucleotidyl transferase (Tdt): adds back nucleotides (N nucleotides) in the “joining region” before the DNA gets sealed –> produces more idiotypes by the addition of amino acids
What is junctional flexibility (variability)?
imprecise joining together of the V-D-J or V-J regions.
The RSS are cut out in very precise manner but the coding regions can vary in the position that they are cut and rejoined.
can yield different amino acids in this region = diversity
happens to be in CDR3–>most important contact with Ag
What are the different types of nucleotide addition that can occur? (2) What enzymes are responsible for these additions? Where do these additions take place?
- P-region nucleotide addition (P-addition): addition of bases by the repair enzymes
- N-region nucleotide addition (N-addition): addition of bases by Tdt. can add up to 15 completely random bases
the additions normally occur in the junction between the D and J region because this is where the CDR3 domain is, which is responsible for determining epitope specificity
What is somatic hypermutation? What regions of the light and heavy chain does this occur in? What does somatic hypermutation lead to?
Somatic hypermutation refers to mutations that occur in a mature B cells (after Ig gene rearrangement and exposure to an antigen). The regions of hypermutation are the hypervariable regions of the light and heavy chains (CDR1 and CDR3)
With these mutations, the antibody may be able to bind stronger to its epitope, increasing its affinity. Igs with the highest affinity for the Ag will be selected, resulting in immunodominance. These Ag specific B cells will then mutate and potentially generate higher and higher affinities in a process called affinity maturation
This is why we get better adaptive responses with each exposure to the antigen
How does a B cell produce both IgM and IgD?
the genes for Cmu and C delta are very close to each other. When the primary transcript (mRNA) is produced, it will have both sequences.
Prior to being translated, the primary transcript will be processed to cut out one of the isotopes, resulting in either IgM or IgD
What determines whether an Ig will be membrane bound or secreted? How does this process work for membrane bound and secreted Igs?
Differential RNA processing of H chains (post-transcription).
The full mRNA is produced in the primary transcript. If the cell decides that the protein will be membrane bound, it will then cut out the “secrete” signal from the mRNA before being translated. The anchor residues and the hydrophobic membrane spanning sequence will be expressed.
If the cell is going to be secreted, the sequence that codes for the hydrophobic membrane spanning region is cut out, allowing for the production of a secreted antibody.
