抗体多样性是如何产生的_精品文档.ppt

1、How is antibody diversity generated?,Two early theories:Germline hypothesisThe genome contains many loci encoding antibody molecules.B cells express one of these loci.Different B cells express different loci.,Somatic mutation hypothesisThere are a small number of antibody genes which undergo mutatio

2、n as theB cell matures-thus giving rise to B cells expressing antibody ofdifferent specificity.,Three genetic loci encode immunoglobulin molecules:-two loci encoding the light chains-kappa locus-lambda locus-one locus encoding the heavy chainThese three loci are located on different chromosomes.,The

3、 loci encoding immunoglobulins have a unique structure.-composed of gene segmentsThe heavy chain locus has multiple V(variable)segments,multiple D(diversity)segments,multiple J(joining)segments and multiple C(constant)segments.During maturation,one of each V,D and J segment is randomly“chosen”and us

4、ed to encode the final antibody molecule.,Germline configuration of the heavy chain locus(mice),Each gene segment may have its own intron-exon structure-e.g.the Cm gene segment,Kuby Figure 5-3,The kappa locus has a similar structure-BUT-does not have Dsegments.A kappa chain is encoded by one V segme

5、nt,one J segment and one Csegment.,Kuby Figure 5-3,The lambda locus also has only V,J and C segments.,Kuby Figure 5-3,Read Kuby pages 109-110:Multigene Organization of Ig Genes,Kuby Figure 5-3,In heavy chains,the V,D and J segments encode the variable domain while the C segment encodes the constant

6、domain.In light chains,the V and J segments encode the variable domain whilethe C segment encodes the constant domain.,During B cell maturation,immunoglobulin genes undergo rearrangement.,During B cell maturation,immunoglobulin genes undergo rearrangement.,During B cell maturation,immunoglobulin gen

7、es undergo rearrangement.,During B cell maturation,immunoglobulin genes undergo rearrangement.,The kappa and lambda loci undergo similar rearrangement.Since there are no D segments,there is a single V-J rearrangement.In both heavy and light chain rearrangement,a number of J segments mayremain betwee

8、n the V(D)and C segments.These are included in the RNA transcript but are spliced out during RNAprocessing.The final light chain mRNA contains one VJC unit.,Kuby Figure 5-4,Kuby Figure 5-4,In the case of the heavy chainIn naive,mature B cells,the primary mRNA transcript contains VDJ andBOTH Cm and C

9、d.This primary transcript can be differentially processed to give rise tomRNA encoding either IgM or IgD.This explains why naive B cells express both IgM and IgD.The variable region of both are encoded by the same VDJ-so they havethe same antigen specificity.,Kuby Figure 5-5,Read Kuby pages 110-112:

10、Variable-Region Gene Rearrangements,What mechanism ensures correct joining of gene segments duringrearrangement of the heavy and light chain loci?,Kuby Figure 5-3,Recombination signal sequences-conserved sequences in regions justupstream or downstream of gene segments.Consist of a conserved heptamer

11、 and nonamer with a 12 or 23 bp spacer.,Kuby Figure 5-3,What mechanism ensures correct joining of gene segments duringrearrangement of the heavy and light chain loci?Recombination signal sequences-conserved sequences in regions justupstream or downstream of gene segments.Consist of a conserved hepta

12、mer and nonamer with a 12 or 23 bp spacer.The one-turn/two-turn rule(12/23 rule)-recombination occurs onlybetween a segment with a 12 bp spacer and a segment with a 23 bp spacer.,The one-turn/two-turn rule(12/23 rule)-recombination occurs onlybetween a segment with a 12 bp spacer and a segment with

13、a 23 bp spacer.,Read Kuby page 113:Recombination Signal Sequences Direct Recombination,Kuby Figure 5-6,Recombination Activating Genes(encode RAG-1 and RAG-2)RAG-1 and RAG-2 mediate recognition of signal sequences and rearrangement of DNA segments.Mice deficient in RAG-1 or RAG-2 are unable to rearra

14、nge heavy or lightchain genes.These mice have no mature B cells.-B cells will not mature if they cannot express a BCR.,RAG-deficient mice also lack mature T cells.-The T cell receptor is also encoded by loci containing gene segmentsthat must be rearranged before the TCR can be expressed.T cells will

15、 not mature if they cannot express a TCR.SCID mice also have a defect that affects rearrangement of BCR and TCR loci.They also have no mature T or B cells.,Flow cytometry of normal vs.RAG-1 deficient mice:Lymph node cellsFITC anti-CD19(B cell marker)and PE anti-CD3(T cell marker),Normal mice,RAG-1-d

16、eficient mice,5,3,3,5,5,3,A G C T,T A T A,Terminal deoxynucleotidyl transferase(Tdt)An enzyme that randomly adds in nucleotides during joining of heavychain(NOT light chain)segments.,Read Kuby pages 113-115:Gene Segments Are Joined by Recombinases,Productive and nonproductive rearrangementsJoining of segments is not precise and may result in loss of the correctreading frame.This may lead to introduction of stop codons-nonproductiverearrangements.Read Kuby page 115:Ig-Gene Rearrangements May Be P