human immune cells must respond to a staggering variety of antigens
more than 108 different antigen recognition sequences are necessary, theoretically requiring 104 light and 104 heavy chains
if all of these were separately encoded, 1/5 of the genome would be devoted exclusively to antibody (Ig) production (to say nothing of TCR and MHC)
So how is the variability produced?
– there are only a few genes, but these are recombined to produce incredible variety
deletions of DNA between variable and constant regions during development (Nobel Prize: variable and constant regions are on different restriction fragments in embryonic B-cell cDNA, but the same in adult)
V-J and V-D-J are rearranged during B-cell development
Example of Genetic Basis of Variation: Antibody (Ig) Light and Heavy Chains
Light Chain
– there are two types (l and k ) with different constant regions, different structures, and different gene loci
l
is on chromosome 22, k is on chromosome 2
each
l gene consists of one variable region (V), a few thousand base pairs, then a short, 4 amino acid joining region (J)
V is joined to J by DNA recombination; since there is just one J site, all lambda short chains are similar
each
k gene has 300 V regions (each with an independent promotor and leader sequence) followed by 4 J regions
one of the Vs is randomly joined to one of the Js, producing 4x300 different sequences
allelic exclusion – once a particular VJ joint is produced, it is the only one expressed in that cell
Heavy Chain
– encoded on chromosome 14
three regions: many V, 4 J as before, and 12 D (for "diversity")
two joinings take place: (1) any D with any J (12x4), then (2) any V with any J (like
k light chain)
the resultant VDJ system is transcribed, spliced, and joined up with the constant region
class switching occurs by RNA splicing; DNA recombination
Þ VDJ; RNA splicing Þ class switching in constant region
Additional Diversity
V-J heptamer – 7 base pair sequence in DNA to be removed (after V and before J) which binds to itself forming a loop
joining enzyme catalyzes a recombination event, producing one of three different amino acids
D-J mutations – hexamer and nonomer are removed from intervening DNA and random nucleotides are added
potentially disrupts reading frame, further increasing variability
Net Result (in mouse)
18
l (2 V x 3 J x 3 genes)
3000
k (250 V x 4 J x 3 genes)
90,000 heavy (250 V x 100 D x 4 J x 9 (two potential D-J reading frame disruptions each increase variability by 3x))
total of 2.7x10^8 variant Ig molecules, potentially sufficient to bind any antigen