PowerPoint PresentationPART IIg proteins are specified by genetic “cassettes”DNA rearrangement and alternative RNA spicingSlide 5DNA rearrangement and alternative RNA splicingAnother view….PART IICassettes rearrange…Slide 10One turn – two turn rule…Slide 12A mouse has:PART III (the HARD part…)Slide 15Junctional flexibilityThe “hairpin loop”Junctional flexibility, “P” nucleotides, and “N” nucleotides are added to CDR3Somatic hypermutationSlide 20PART IVIMMUNOLOGYBios 328a textbook-based study of immunologySpring 2003http://www.lehigh.edu/~sk08/Courses/Bios328/mainpage.htmPART I•Immnoglobulins are proteins•Proteins are specified by genes•There are too few genes to specify all the antibodies.–i. e., ~32,000 genes < 10,000,000,000 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 rearrangementandalternative RNA spicingIg 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 rearrangementandalternative RNA splicingAnother 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, 4 Jsegmentsand 2 V, 3 J segmentsThus, a mouse has: 134  13  4 = 6968 heavy chains 85 4 = 340 kappa chainand 2 3 = 6 lambda chains6968 (340 + 6) = 2,410,928 antibodiesPART III (the HARD part…) 2.4 106 < 1010 So, there must be additional mechanisms of diversity other than “fusing” “cassettes”How does a RAG enzyme work?Junctional flexibilityThe “hairpin loop”Junctional flexibility, “P” nucleotides, and “N” nucleotides are added to CDR3Somatic hypermutationOne 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 inversionPART 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