PART I Immnoglobulins are proteins Proteins are specified

PART I • Immnoglobulins are proteins • Proteins are specified by genes • There are too few genes to specify all the antibodies. – i. e. , ~32, 000 genes < 10, 000, 000 Ab’s • How is Ig diversity specified genetically?

Ig proteins are specified by genetic “cassettes” • Light chains are specified by “variable” (V), “joining (J), and “constant” (C) gene segments (aka “cassettes”).

DNA rearrangement alternative RNA spicing and

Ig proteins are specified by genetic “cassettes” • Heavy chains are specified by “variable” (V), “diversity” (D), “joining (J), and “constant” (C) gene segments (aka “cassettes”).

DNA rearrangement alternative RNA splicing and

Another view….

PART II • Cassettes rearrange… • How does this happen? • How do you get one “V” fusing to one “J” (in a light chain)? • In a heavy chain, a “D” fuses with a “J”; then the fused DJ cassette fuses with a “V” cassette… • The orderliness of this process implies that there are genetic instructions. What are they?

Cassettes rearrange… • The heptamer is a palindrome – (i. e. , it exhibits two-fold rotational symmetry. ) • The nonamer is AT-rich – “Turns” refer to the DNA helix…

Cassettes rearrange…

One turn – two turn rule… • one turn and two turn are “recombination signal sequences” • one turn only reacts with a two turn • Recombination signal sequences are the substrates of enzymes RAG-1 and RAG-2 (“RAG” = recombination-activating gene)

So… cassettes are marked by RSS (i. e. , they are substrates for recombination. ) Thus, cassettes can be fused. What is the consequence? Look at mouse:

A mouse has: 134 VH, 13 DH, 4 JH segments 85 V 6, 4 J 6 segments and 2 V 8, 3 J 8 segments Thus, a mouse has: 134 C 13 C 4 = 6968 heavy chains 85 C 4 = 340 kappa chain and 2 C 3= 6 lambda chains

PART III 2. 4 C (the HARD part…) 106 < 1010 So, there must be additional mechanisms of diversity other than “fusing” “cassettes” How does a RAG enzyme work?

Junctional flexibility

The “hairpin loop”

Junctional flexibility, “P” nucleotides, and “N” nucleotides are added to CDR 3

Somatic hypermutation

One turn – two turn rule… • one turn only reacts with a two turn • crossover between direct repeats (same transcriptional orientation) leads to deletion • crossover between indirect repeats leads to inversion

PART IV • How do immunoglobulins assemble? • Some immunoglobulins are in the surface membrane of immature B-cells while other immunoglobulins of the same idiotype are secreted by mature B-cells. What’s the difference? • Similarly, identical variable regions can be shared among different isotypes. How? • B-cells are diploids with two sets of genetic instructions. How does just one set get expressed?