The key experiment of Nobumichi Hozumi and Susumu Tonegawa

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Presentation transcript:

The key experiment of Nobumichi Hozumi and Susumu Tonegawa

EXPRESSION OF THE KAPPA CHAIN Vκ-Jκ Vκ P pA Cκ E J Primary RNA transcript Cκ E J Vκ Leader mRNA Cκ J Vκ AAAA Translation Cκ J Vκ Protein Efficiency of somatic gene rearrangement?

Ig light chain rearrangement: Rescue pathway There is only a 1:3 chance of the join between the V and J region being in frame Vk Jk Ck Non-productive rearrangement Light chain has a second chance to make a productive join using new V and J elements Spliced mRNA transcript

Further diversity in the Ig heavy chain L VH DH JH CH The heavy chain was found to have further amino acids (0 – 8) between the JH és CH genes D (DIVERSITY) region Each heavy chain requires 2 recombination events JH to DH and VH to JHDH, VL JL CL L Each light chain requires 1 recombination events VL to JL

SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS 120 VH 12 D 4 JH VH1 VH2 VH3 D D D D JH JH JH JH D VH1 VH2 VH3 During B-cell development JH VH1 VH2 JH D

IMMUNOGLOBULIN CHAINS ARE ENCODED BY MULTIPLE GENE SEGMENTS ORGANIZATION OF IMMUNOGLOBULIN GENE SEGMENTS Chromosome 2 kappa light chain gene segments Chromosome 22 lambda light chain gene segments Chromosome 14 heavy chain gene segments HOW MANY IMMUNOGLOBULIN GENE SEGMENTS Variable (V) 132/40 105/30 123/65 Diversity (D) 0 0 27 Joining (J) 5 4 9 Gene segments Light chain Heavy chain kappa lambda

VH-D-JH VL-JL VARIABILITY OF B-CELL ANTIGEN RECEPTORS AND ANTIBODIES B cells of one individual 1 2 3 4 VH D JH VL JL V-Domains C-Domains VH-D-JH VL-JL

How does somatic gene rearrangement (recombination) work? How is an infinite diversity of specificity generated from finite amounts of DNA? Combinatorial diversity

Estimates of combinatorial diversity Taking account of functional V D and J genes: 65 VH x 27 DH x 6JH = 10,530 combinations 40 Vk x 5 Jk = 200 combinations 30 Vl x 4 Jl = 120 combinations = 320 different light chains If H and L chains pair randomly as H2L2 i.e. 10,530 x 320 = 3,369600 possibilities Due only to COMBINATORIAL diversity In practice, some H + L combinations do not occur as they are unstable Certain V and J genes are also used more frequently than others. GENERATES A POTENTIAL B-CELL REPERTOIRE

How does somatic gene rearrangement (recombination) work? How is an infinite diversity of specificity generated from finite amounts of DNA? Combinatorial diversity 2. How do V region find J regions and why don’t they join to C regions? 12-23 rule -Special - Recobnitation Signal Sequences (RSS) - Recognized by Recombination Activation Gene coded proteins (RAGs) PALINDROMIC SEQUENCES HEPTAMER CACAGTG CACAGTG GTGACAC GTGACAC NONAMER ACAAAAACC GGTTTTTGT TGTTTTTGG CCAAAAACA

Somatic recombination to generate antibody diversity Immunoglobulin genes are composed of separated segments of DNA that become joined together by a process called somatic recombination to make a functional gene. In heavy chain genes there are three gene segments, the variable or V segment, a diversity or D gene segment, and the joining or J segment. Light chain genes, such as those shown here, have only two gene segments - the V and the J segments. Gene segments that can be recombined have specific sequence motifs adjacent to them, called recombination signal sequence, or RSS motifs. A protein complex containing the products of the recombination activator genes, RAG1 and RAG2, binds specifically to the RSS motifs, in this example flanking a V gene segment and a J gene segment. The individual gene segments, to whose flanking RSS motifs the RAG protein complexes bind, are selected at random from a number of copies present at each gene locus. The Rag protein complexes bring together the gene segments to be recombined, and cleave the DNA exactly at the junction of the gene segment and it's adjoining RSS motif. The cleavage creates a hairpin of DNA at the ends of the gene segments and double stranded breaks at the ends of the RSS motifs. Additional proteins, DNA-dependent protein kinase, Ku, Artemis and a dimer of DNA ligase and XRCC4, are incorporated into a large complex with the RAG proteins. These RSS ends are joined, forming what is called the "signal joint", to create a closed circle of DNA that plays no further role in the recombination process. The DNA hairpins at the ends of the gene segments are then cleaved. An additional enzyme, terminal deoxynucleotidyl transferase or TdT, is recruited and adds additional nucleotides to the ends of the DNA strands The other enzymes in the complex ligate together the two ends of the gene segments, completing the recombination process.

Severe combined immunodeficiency syndrome (SCID). SCID is characterized by a lack of functional T cells and B cells and the inability to make an adaptive immune response. Infants with SCID typically show infections with opportunistic pathogens. Panel a shows chronic Candida albicans infection in the mouth of an infant with SCID. SCID can be caused by various genetic defects, one of which is complete loss of RAG function. Panel b shows an infant with Omenn syndrome, a similar immunodeficiency which is due to a genetic defect that results in 80% loss of RAG activity. The bright red rash on the face and shoulders, which is due to chronic inflammation, is a characteristic of this condition. Unless an immune system can be reconstituted by bone marrow transplantation from a healthy donor, babies with SCID or Omenn syndrome die in infancy. Panel a courtesy of Fred Rosen; panel b courtesy of Luigi Notarangelo. Omen syndrome RAG deficiency Early onset loose bowel movements Red scaly rashes all over the body Opportunistic infections (Candida albicans, Pneumocystis carnii pneumonia) No palpable lymph nodes

V, D, J flanking sequences Sequencing upstream and downstream of V, D and J elements revealed conserved sequences of 7, 23, 9 and 12 nucleotides in an arrangement that depended upon the locus Vl 7 23 9 Jl 7 12 9 Vk 7 12 9 Jk 7 23 9 D 7 12 9 VH 7 23 9 JH

Recombination signal sequences (RSS) HEPTAMER - Always contiguous with coding sequence NONAMER - Separated from the heptamer by a 12 or 23 nucleotide spacer VH 7 23 9 D 12 JH   VH 7 23 9 D 12 JH 12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS

Molecular explanation of the 12-23 rule 23-mer = two turns 12-mer = one turn Intervening DNA of any length 23 V 9 7 12 D J 7 9

Molecular explanation of the 12-23 rule V1 D J V2 V3 V4 V8 V7 V6 V5 V9 V1 V2 V3 V4 Loop of intervening DNA is excised V8 V7 V6 V5 Heptamers and nonamers align back-to-back The shape generated by the RSS’s acts as a target for recombinases 7 9 23-mer 12-mer V9 D J An appropriate shape can not be formed if two 23-mer flanked elements attempted to join (i.e. the 12-23 rule)

V1 D J 9 7 CONSEQUENCES OF RECOMBINATION Generation of P-nucleotides 23-mer 12-mer

V1 D J 9 7 Generation of N-nucleotides V4 V5 Terminal deoxynucleotidyl Transferase (TdT) Loop of intervening DNA is excised 7 9 23-mer 12-mer

How does somatic gene rearrangement (recombination) work? How is an infinite diversity of specificity generated from finite amounts of DNA? Combinatorial diversity 2. How do V region find J regions and why don’t they join to C regions? 12-23 rule How does the DNA break and rejoin? Imprecisely, with the random removal and addition of nucleotides to generate sequence diversity Junctional diversity (P- and N- nucleotides, see above)

Mini-circle of DNA is permanently lost from the genome Junctional diversity 7 12 9 23 7 12 9 23 Mini-circle of DNA is permanently lost from the genome Signal joint Coding joint V D J V D J Imprecise and random events that occur when the DNA breaks and rejoins allows new nucleotides to be inserted or lost from the sequence at and around the coding joint.

V D J TCGACGTTATAT AGCTGCAATATA TTTTT Junctional Diversity Germline-encoded nucleotides Palindromic (P) nucleotides - not in the germline Non-template (N) encoded nucleotides - not in the germline Creates an essentially random sequence between the V region, D region and J region in heavy chains and the V region and J region in light chains

GGGACAGGGGGC GlyThrGlyGly GlyGlnGly AspArgGly Reading D segment in 3 frames Analysis of D region from different antibodies show that the same D region can be translated in all three frames to make different protein sequences and hence antibody specificities GGGACAGGGGGC GlyThrGlyGly GlyGlnGly AspArgGly Frame 1 Frame 2 Frame 3

RESULT OF SOMATIC GENE REARRANGEMENT AND ALLELIC EXCLUSION Somatic rearrangement of Ig gene segments in a highly controlled manner Single B-cells become committed to the synthesis of one unique H-chain and one unique L-chain variable domain, which determine their specificities In each of us huge B-cell repertoire is generated consisting of B-cell clones with different H- and L-chain variable domains This potential B-cell repertoire is able to recognize a wide array of various antigens INDEPENDENT ON ANTIGEN OCCURS IN THE BONE MARROW

How does somatic gene rearrangement (recombination) work? How is an infinite diversity of specificity generated from finite amounts of DNA? Combinatorial diversity 2. How do V region find J regions and why don’t they join to C regions? 12-23 rule How does the DNA break and rejoin? Imprecisely, with the random removal and addition of nucleotides to generate sequence diversity Junctional diversity How B cells express one light chain species and one heavy chain species even though every B cell possesses a maternal and paternal locus of both genes. Since all other genes known at the time appeared to be expressed co-dominantly, how could B cells shut down the genes on one of their chromosomes? Allelic exclusion