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Exercise: Analysis of immunoglobolin gene rearrangements


Introduction

(From Ohm-Laursen et al., Immunology, 2006)
Diversity is a key feature of the adaptive immune system. It is partly created by combination of different heavy and light chains. Most of the diversity is, however, found within the CDR3 region that has a major influence on the binding of antigens[1]. The heavy chain CDR3 is encoded by the junction of a variability- (V), a diversity- (D), and a joining- (J) gene recombined during the B-cell development in the bone marrow. Several copies of each gene segment exist and diversity can be created by sheer recombinatorial variation. However, the molecular processes linking the genes are imprecise and involve generation of P (palindromic) nucleotides[2-4], addition of N (non-templated) nucleotides by terminal deoxynucleotidyl transferase (TdT)[4-6] and trimming of the gene ends[4] and therefore also play a major role in the generation of diversity.

Download heavy chain (H) germ line V, D, and J genes

Go to the IMGT website.

We would like to dowload the Human IG heavy chain repertoire

Under 'IMGT Web resources', select IMGT repertoire
Under '2. Proteins and alleles' select IMGT reference dirctory in FASTA format
Under IMGT/V-QUEST reference directory sets select Human under each the relevant gene versions (IGHV, IGHD, and IGHJ)
The FASTA entries can now be copy-pasted into an appropriate text editor and saved in raw text format.

  • Q1:How many germ line alleles exists for each type?

Heavy chain rearrangements

CDR region identification

We have a selection of rearrangement sequences here. (right click to open in a new window)
Open the translation tool.
Copy-paste the RAW SEQUENCE (i.e., NOT the FASTA header line) from first FASTA entry into the sequence field.
In the roll down window select compact format. The tool translate all six reading frames.
Select the reading frame with no stop codons and copy the peptide sequence to a text document for further analysis.
 
  • Q2: Identify the three CDR regions. (e.g., CDR1: res nr. X to res. nr. Y....).
To see the definition of CDR regions click Identify CDR. Note the rearrangements are all heavy chain.
  • Q3:Repeat the analyse for next two fasta sequences in the rearrangement list.

VQuest analyses

Now go to the IMGT website.
Under 'IMGT tools' select IMGT/V-QUEST.
Under 'Analyse your Immunoglobulin nucleotide sequences' click 'Human'
In a new window open the rearrangements(right click).
Copy-paste all entries into the sequence window, and push start.
  • Q4: For each of the rearrangements note which germ line genes that is identified.
  • Q5: For each of the rearrangements note the IMGT positions of the CDR regions in the peptide (from res. nr. X to res. nr. Y).

VDJsolver

Keep the rearrangement window open.
Go to the VDJsolver.
Paste in the rearrangements in the sequence window and push 'submit'.
  • Q6: For each of the rearrangements note which germ line genes that is identified.
  • Q7: What are the differences in the assignments and alignments between V-QUEST and VDJsolver?


  • This is it!