Immunoglobulin questions
Gilbert Chu
Advanced Immunology (I), January, 2004
1.
V(D)J recombination is directed by recombination signal sequences (RSS).
A. Draw a diagram showing how RSS can direct a deletion in the genomic DNA.
B. Draw a diagram showing how RSS can direct an inversion in the genomic
DNA.
In answering parts A and B, show all of the DNA products.
2.
V(D)J recombination is directed by recombination signal sequences (RSS).
A. How do RSS prevent V to J recombination in the heavy chain locus?
B. Explain how cleavage at the RSS is catalyzed. What are the enzymes?
Draw the
DNA intermediates and the recruitment of the enzymes.
3.
The formation of coding joints during V(D)J recombination sometimes involves
the addition of palindromic (P) nucleotides.
A. What is the mechanism for P-nucleotide addition?
B. List the enzymes that would be required for this process.
4.
The formation of coding joints during V(D)J recombination sometimes involves
the addition of random (N) nucleotides.
A. What is the enzymatic mechanism for N-nucleotide addition? Does this
mechanism operate at signal joints? What types of cells does it operate in?
B. How would you use a genetically engineered mouse to determine whether this
enzymatic mechanism accounts for all N-nucleotide addition?
5.
Review the evidence that class switch recombination requires the same nonhomologous end-joining pathway that is used for V(D)J recombination.
6.
AID is required for both somatic hypermutation and class switch recombination.
A. Describe the proposed enzymatic activity for AID
B. Explain how this activity can lead to a DNA nick during class switch
recombination.
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Immunoglobulin answers
Gilbert Chu
Advanced Immunology (I), January, 2003
1.
A. Recombination between two RSS in head to head orientation with respect to
each other. There is deletion of that part of the chromosome containing the
recombining RSS and the formation of a circular episome.
B. Recombination between two RSS in head to tail orientation with respect to
each other. There is a chromosomal inversion without deletion.
2.
A. The 12-23 rule. In heavy chain locus, the RSS adjacent to V and J segments
have spacers of 23 nucleotides. The RSS adjacent to the D segments have
spacers of 12 nucleotides. Recombination only occurs between RSS with
different spacers.
B. RAG1 and RAG2.
RAG1 is recruited to the nonamer. RAG2 is then recruited.
First step: nick adjacent to the RSS leaving a 3' OH at the coding end.
Second step: formation of a hairpin coding end and blunt signal end.
3.
A. Formation of a hairpin, asymmetric cleavage of the hairpin, and end joining.
B. The enzymes: RAG1, RAG2 for cleavage and a presumed hairpin
endonuclease.
4.
A. TdT. By some unknown mechanism, it must be prevented from operating at
signal ends, since signal joints are formed without addition or subtraction of
nucleotides. TdT is expressed only in lymphoid cells.
B. Make a mouse knocked out for the TdT gene and analyze coding joints for Nnucleotide addition.
5.
Both processes require Ku and DNA-PK. The junctions formed during both processes
involve deletions back to regions of microhomology.
6.
A. AID acts on DNA as a deoxycytidine deaminase, converting C to U
B. After conversion of C to U, the combined activity of uracil DNA glycosylase
and AP endonuclease produces a nick in the DNA.
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