Lecture 16: Generating Immunoglobulin Variation and Diversity Flashcards
What is an isotype? What does this determine?
variation in constant regions determines class or sub-class of Ig
What is an allotype?
inherited variation in constant regions due to sequence variation in the alleles of Ig loci e.g. IgG1 will have minor sequence differences between different individuals
What is an idiotype?
variation in variable regions
individuals may have up to 10^7 different encoded idiotypes i.e. different specificities
How is variability determined?
by comparing sequences of different antibody molecules
What is the formula for variability?
number of different aa at a given position / frequency of most common aa at position
Where is the highest variability of amino acids in immunoglobulins?
in the hypervariable regions (CDRs)
What is the genetic basis for Ig variability?
there are at least 10^7 different antibody specificities but there are only 10^4 different mammalian genes (total)
What does one gene allow for the transcription / translation of?
one protein
What is antibody diversity generated by?
somatic diversification
How does somatic diversification generate antibody diversity?
Ig genes are rearranged in the progenitors of antibody-producing cells
rearranged segments generate complex Ig H+L chain genes as well as unique Ig H+L chain protein sequences
unique genes generate unique idiotype or specificity for each B cell
What is the light chain composed of?
V (variable), J (joining) and constant (C) gene segments
What is the heavy chain composed of?
V (variable), D (diversity), J (joining) and constant (C) gene segments
What are complete genes that encode a variable region generated by?
the somatic recombination of separate gene segments
What is the key to diversity?
that there are multiple contiguous variable gene segments present at each immunoglobulin locus
What do heavy and light chain genes undergo?
random rearrangement of V, (D) and J segments
rearrangement is unique for each cell
Which antibodies are always produced first in the humoral response?
IgM
Most eukaryotic genes are encoded in a set of exons that are brought together to form a contiguous protein coding sequence by the process of mRNA splicing. In contrast, immunoglobulin genes only use somatic recombination of gene segments and not mRNA splicing to generate the final mRNA that is translated into protein. (true / false)
false
Igs do still require mRNA splicing to remove the intron between the V region exon and the C region exon to make a complete light / heavy chain protein coding mRNA sequence
the step that is different is that immunoglobulin genes use somatic recombination to create the V region exon by joining V-J or V-D-J gene segments in the DNA before transcription occurs
What do the V gene segments form and why?
“rosettes” to bring different V segments into proximity with J or rearranged DJ segments
What is rearrangement of germline V, D and J gene segments guided by?
flanking DNA sequences = recombination signal sequences (RSS)
What rule does joining of V, D and J gene segments follow?
there can only be joining of signal sequences with 23-base-pair spacers to signal sequences with 12-base-pair spacers
What do RSSes form?
loops which are removed by the V(D)J recombinase enzyme complex
What is the V(D)J recombinase enzyme complex made up of?
both lymphocyte-specific and ubiquitous DNA-modifying factors to facilitate the recombination of V, D, and J gene segments
What is the role of recombination-activating genes (RAG-1 / RAG-2)
involved with breaking / joining DNA
What is the role of nucleases?
involved in nicking hairpin sequences or the removal of nucleotides generated by RAGs
e.g. artemis complex, exonucleases
What is the role of terminal deoxynucleotide transferases (TdT)?
involved in insertion of random, non-template-encoded nucleotides
-> N-nucleotides / N-regions
What is the role of ligases?
involved in ligating DNA ends together
e.g. DNA ligase IV
What gives rise to N-regions?
inserted nucleotides
What do N-regions contribute to? What is their downfall?
contribute to diversity i.e. almost any sequence is possible in the N-regions
may lead to failure of arrangement e.g. insertion of stop codons or frameshifts
What is the process of joining in Ig gene rearrangement?
RSSs brought together -> RAG complex generates DNA hairpin at coding ends -> Artemis:DNA-PK complex opens DNA hairpins, generating palindromic P-nucleotides -> N-nucleotide additions by TdT -> pairing of strands -> unpaired nucleotides are removed by an exonuclease -> the gaps are filled by DNA synthesis and ligation to form coding joint
What does variable addition and subtraction of nucleotides at the junctions between gene segments contribute to?
diversity of the third hypervariable region
What are the sources of Ig diversity?
combinatorial diversity, junctional diversity and somatic mutation
What are the two components of combinatorial diversity?
combinatorial assembly = different combinations of V, D and J for a particular chain
combinatorial association = different combinations of heavy and light chains
What is junctional diversity contributed by?
random N-region sequences
How does somatic mutation create diversity?
as immune response progresses or “matures”
mostly found in the secondary response
