Attribution: University of Michigan Medical School, Department of Microbiologyand ImmunologyLicense: Unless otherwise note...
Citation Key                          for more information see: http://open.umich.edu/wiki/CitationPolicyUse + Share + Ada...
Joining Variable Constant              Region Genes              M1 – Immunology SequenceWinter 2009
1.  How is diversity in the germline further    expanded by DNA rearrangement in B cells?2.  What are the changes in immun...
Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Note mu...
Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
Methods to generate diversity.1. Germline diversity is generated by the use of anyone of the one hundred variable region g...
3. Junctional diversity is generated during V(D)J joining byvariation in the exact point of recombination between a heavyc...
Germline sequences:CCC GGA CGA AGC TTC GTG A CACAGTG VHDJ CACTGTG GAT TAC TAC GGT AGT : TGG GAC    Cutting next to CACAGTG...
Exonuclease digestion of coding sequences from endsCCC GGA CGA AGC TTC GTG A GAT TAC TAC GGT AGT CCC GGA CGA AGC T        ...
Digestion to a different extent and ligationCCC GGA CGA AGC TTC GTG A GAT TAC TAC GGT AGTCCC GGA CGA AGC TTC GT           ...
4. N region addition occurs by addition of nucleotides by terminal deoxynucleotide transferase to variable region, D segme...
When in B cell differentiation do immunoglobulingene rearrangements take place?                                           ...
In pro B cells in the bone marrow, during antigen-independent B cell differentiation, first a D segmentis rearranged to a ...
Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
If heavy chain variable region to DJ joiningresults in expression of a µ heavy chain,the pro B cell becomes a pre B cell a...
Immature       MatureStem cell Pro B cell                             Pre B cell            B cell         B cell         ...
Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997In gene...
Each cell has four light chain loci that couldundergo variable region to J segmentrearrangement. There are several VJrearr...
If light chain variable region to J segmentjoining results in light chain productionand an assembled IgM molecule on the c...
Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
If V(D)J joining results in expression of a muheavy chain (the joining is in-frame, and doesnot use a pseudo VH), then fur...
How does a mature B cell express first themembrane form of IgM, and later, the secretedform of IgM, with the same idiotype...
SC=20 aa                                                                                                              MC=4...
Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Changes...
Summary1.  Combinatorial diversity is generated by joining of one of    many variable regions to one of a few D segments a...
Additional Source Information                  for more information see: http://open.umich.edu/wiki/CitationPolicySlide 6:...
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02.11.09(a): Joining Variable & Constant Region Genes

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02.11.09(a): Joining Variable & Constant Region Genes

  1. 1. Attribution: University of Michigan Medical School, Department of Microbiologyand ImmunologyLicense: Unless otherwise noted, this material is made available under the termsof the Creative Commons Attribution–Noncommercial–Share Alike 3.0License: http://creativecommons.org/licenses/by-nc-sa/3.0/ We have reviewed this material in accordance with U.S. Copyright Law and have tried to maximize your ability to use, share, and adapt it. The citation key on the following slide provides information about how you may share and adapt this material. Copyright holders of content included in this material should contact open.michigan@umich.edu with any questions, corrections, or clarification regarding the use of content. For more information about how to cite these materials visit http://open.umich.edu/education/about/terms-of-use. Any medical information in this material is intended to inform and educate and is not a tool for self-diagnosis or a replacement for medical evaluation, advice, diagnosis or treatment by a healthcare professional. Please speak to your physician if you have questions about your medical condition. Viewer discretion is advised: Some medical content is graphic and may not be suitable for all viewers.
  2. 2. Citation Key for more information see: http://open.umich.edu/wiki/CitationPolicyUse + Share + Adapt { Content the copyright holder, author, or law permits you to use, share and adapt. } Public Domain – Government: Works that are produced by the U.S. Government. (USC 17 § 105) Public Domain – Expired: Works that are no longer protected due to an expired copyright term. Public Domain – Self Dedicated: Works that a copyright holder has dedicated to the public domain. Creative Commons – Zero Waiver Creative Commons – Attribution License Creative Commons – Attribution Share Alike License Creative Commons – Attribution Noncommercial License Creative Commons – Attribution Noncommercial Share Alike License GNU – Free Documentation LicenseMake Your Own Assessment { Content Open.Michigan believes can be used, shared, and adapted because it is ineligible for copyright. } Public Domain – Ineligible: Works that are ineligible for copyright protection in the U.S. (USC 17 § 102(b)) *laws in your jurisdiction may differ { Content Open.Michigan has used under a Fair Use determination. } Fair Use: Use of works that is determined to be Fair consistent with the U.S. Copyright Act. (USC 17 § 107) *laws in your jurisdiction may differ Our determination DOES NOT mean that all uses of this 3rd-party content are Fair Uses and we DO NOT guarantee that your use of the content is Fair. To use this content you should do your own independent analysis to determine whether or not your use will be Fair.
  3. 3. Joining Variable Constant Region Genes M1 – Immunology SequenceWinter 2009
  4. 4. 1.  How is diversity in the germline further expanded by DNA rearrangement in B cells?2.  What are the changes in immunoglobulin gene rearrangement associated with various stages of antigen-independent B cell differentiation?3.  How can IgM and IgD be expressed simultaneously on the surface of a mature, but naive, B cell?
  5. 5. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Note multiple Cλ genes, each with one J region.Heavy chain: D segments encode 2-8 aminoacids and are both preceded and followed byrecombination signal sequences. The heavychain variable region encodes amino acids 1-99and JH encodes an additional 14-20 amino acids.
  6. 6. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
  7. 7. Methods to generate diversity.1. Germline diversity is generated by the use of anyone of the one hundred variable region genes,several Ds, and four to ten Js encoded in DNA.2. Combinatorial diversity is generated by joining ofany variable region gene to any D to any J, and bycombination of any heavy chain variable region withany light chain variable region.50 V x 30 D x 6 JH = 9000 different VH 40 V x 5 Jκ = 200 different Vκ 9000 x 200 = 2x106 binding sites 9000 x 300 = 3x106 binding site (VHVλ)
  8. 8. 3. Junctional diversity is generated during V(D)J joining byvariation in the exact point of recombination between a heavychain variable region gene and a D segment, or a D segmentand a heavy chain J segment, or a light chain variable regiongene and J segment.
  9. 9. Germline sequences:CCC GGA CGA AGC TTC GTG A CACAGTG VHDJ CACTGTG GAT TAC TAC GGT AGT : TGG GAC Cutting next to CACAGTG CutCCC GGA CGA AGC TTC GTG A CACAGTG CACTGTG GAT TAC TAC GGT AGT : TGG Cut Ligation of CACAGTG to CACAGTG CACTGTG:CACAGTG
  10. 10. Exonuclease digestion of coding sequences from endsCCC GGA CGA AGC TTC GTG A GAT TAC TAC GGT AGT CCC GGA CGA AGC T AT TAC TAC GGT AGTLigation of coding ends CCC GGA CGA AGC T : AT TAC TAC GGT AGT
  11. 11. Digestion to a different extent and ligationCCC GGA CGA AGC TTC GTG A GAT TAC TAC GGT AGTCCC GGA CGA AGC TTC GT T TAC TAC GGT AGT CCC GGA CGA AGC TTC GT : T TAC TAC GGT AGT CCC GGA CGA AGC T : AT TAC TAC GGT AGT (for comparison; this is the sequence from the previous slide)Junctional diversity often results in exactly the samenumber of codons at the V(D)J junction (particularly inthe light chain variable region), but a differentsequence, and hence, a different amino acid.
  12. 12. 4. N region addition occurs by addition of nucleotides by terminal deoxynucleotide transferase to variable region, D segment, or J segment ends. These nucleotides are not encoded by a template.CCC GGA CGA AGC TTC GTG A GAT TAC TAC GGT AGTCCC GGA CGA AGC TT T TAC TAC GGT AGTCCC GGA CGA AGC TT : GCC T TAC TAC GGT AGT CCC GGA CGA AGC TT : GCC : T TAC TAC GGT AGT
  13. 13. When in B cell differentiation do immunoglobulingene rearrangements take place? Immature MatureStem cell Pro B cell Pre B cell B cell B cell Y Y Y µ IgD and IgM µ chain in IgM on on cell Absent Absent cytoplasm cell surface surface University of Michigan Department of Microbiology and Immunology
  14. 14. In pro B cells in the bone marrow, during antigen-independent B cell differentiation, first a D segmentis rearranged to a heavy chain J segment on bothchromosomes.Then, a heavy chain variable region is rearranged tothe DJ on one chromosome.If this V(D)J rearrangement does not result inmu heavy chain expression (because a heavy chainvariable region pseudogene is used, or the VDJexon is out of frame), the pro B cell attempts torearrange a heavy chain variable region to DJ onthe other chromosome.
  15. 15. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
  16. 16. If heavy chain variable region to DJ joiningresults in expression of a µ heavy chain,the pro B cell becomes a pre B cell and alsoattempts light chain variable region to Jsegment rearrangement on onechromosome
  17. 17. Immature MatureStem cell Pro B cell Pre B cell B cell B cell Y Y Y µ IgD and IgM µ chain in IgM on on cellAbsent Absent cytoplasm cell surface surface University of Michigan Department of Microbiology and Immunology
  18. 18. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997In general Vκ to Jκ precedes Vλ to Jλ,because κ rearrangement is favored20 to 1 over λ rearrangement.
  19. 19. Each cell has four light chain loci that couldundergo variable region to J segmentrearrangement. There are several VJrearrangements possible in each light chainlocus.---Vκ20---Vκ21---Vκ22---Vκ23Jκ2---Jκ3---Jκ4---Jκ5---Cκ---- ---Vκ20---Vκ21Jκ4---Jκ5---Cκ----
  20. 20. If light chain variable region to J segmentjoining results in light chain productionand an assembled IgM molecule on the cellsurface, the pre B cell goes on to be animmature B cell.
  21. 21. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
  22. 22. If V(D)J joining results in expression of a muheavy chain (the joining is in-frame, and doesnot use a pseudo VH), then further VH to DJjoining is shut off.If VL to JL joining results in expression of a lightchain, then further VL to JL joining is shut off forboth the kappa and lambda genes.This feed-back regulation of V(D)J joining is thebasis of allelic exclusion. The shut-off of furtherV(D)J joining prevents a B cell from expressingtwo heavy chains or two light chains.
  23. 23. How does a mature B cell express first themembrane form of IgM, and later, the secretedform of IgM, with the same idiotype (sameheavy chain variable region)?Secreted mu: P V 1 2 3 4 20 aaMembrane mu: P V 1 2 3 4 41 aa 6/12-;26 uncharged;2/3+P = the hydrophobic, 26 amino acid, region that is cleaved offas the translated protein crosses the endoplasmic reticulum.
  24. 24. SC=20 aa MC=41 aaJaneway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
  25. 25. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
  26. 26. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
  27. 27. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Changes in membrane µ, secreted µ, andmembrane δ expression are mediated by RNAsplicing; the genes themselves do not change.Hence, these changes in expression arereversible.
  28. 28. Summary1.  Combinatorial diversity is generated by joining of one of many variable regions to one of a few D segments and J segments, independently of antigenic stimulation.2.  Junctional and N region diversity are generated at V-D, D-J, and VL-JL junctions.3.  V(D)J joining is regulated in that it occurs only in the B cell lineage, the gene segments are recombined in specific order, and V(D)J joining is stopped by productive expression of heavy and light chain protein.4.  Membrane µ, secreted µ, and δ expression are mediated by RNA splicing.
  29. 29. Additional Source Information for more information see: http://open.umich.edu/wiki/CitationPolicySlide 6: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Slide 7: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Slide 13: University of Michigan Department of Microbiology and ImmunologySlide 15: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Slide 17: University of Michigan Department of Microbiology and ImmunologySlide 18: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Slide 21: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Slide 24: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Slide 25: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Slide 26: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Slide 27: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997

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