Exam #2 - Microbio 540 - 3/7/00 (300 points). WRITE ALL ANSWERS IN A BLUEBOOK. Keep your answers brief and focussed. Points will be lost for excessively long and diffuse answers
All answers have the same value. Some numbered items have multiple answers. Items 5, 19, 22, and 34 have ten or more answers each; items 1, 13, 26 have five or more answers each; all other items have one to four answers each. In all, there are 123 answers. Therefore, each answer is worth (300/123) = 2.44 points.

  1. For each immunoglobulin gene, mRNA, or protein below, select the corresponding schematic diagram in the figure.
    1. Immunoglobulin heavy chain DNA in a dendritic cell.
    2. Immunoglobulin light chain DNA in a kidney cell.
    3. Heavy chain DNA in a mature, naive B cell.
    4. Primary RNA transcript encoding heavy chain.
    5. Mature, processed mRNA encoding heavy chain.
    6. Heavy chain proteins observed on the membrane of a mature B cell before any class switching.
    7. Heavy chain DNA in a plasma cell secreting IgE.
    8. Heavy chain protein observed on the membrane of a B cell after class switching.

    a=3   b=1   c=4   d=2   e=7   f=5   g=10   h=8   (not using 6, 9)

  2. Which of the items in the previous question occurs only after foreign antigen has entered the system?

    G, H only.

  3. Which of the following pairs is it possible for a single B cell to synthesize simultaneously, adjusting the ratio as appropriate? Note: anything controlled at the level of mRNA splicing allows simultaneous/adjustable synthesis. Answer YES for simultaneous/adjustable synthesis, and NO if the switch is irreversible.
    1. IgM vs IgD?
    2. IgM vs. IgG?
    3. Membrane-bound vs. secreted immunoglobulin?
    4. Kappa light chain to lambda light chain?

    Y N Y N

  4. In the immunoglobulin heavy chain DNA "D" and "J" regions, what do the "D" and "J" stand for?

    Diversity, Joining.

  5. List the full names of the immunoglobulin light chain gene sequence diversification mechanisms which occur during B cell maturation and immune responses (including what any single letter abbreviations stand for).
    For each, indicate whether the DNA sequence it produces in the spliced gene is coded for in any way in the genome by adding CODED or NONCODED.
    Also indicate whether it occurs BEFORE ANTIGEN or AFTER ANTIGEN. 

    Selection of V and J segments, CODED, before antigen.
Junctional flexibility, CODED, before antigen.
Palindromic addition, CODED, before antigen.
Somatic hypermutation, NONCODED, after antigen.
(The book implies that N-addition does not occur in light chains.)

  6. When the exact same light chain genomic V and J segments are spliced together during DNA rearrangement, can the final spliced DNA have different nucleotide sequences in different B cells? Explain briefly.

    Yes. The above-mentioned junctional flexibility, P and N addition produce different nucleotide sequences in each rearrangement.

  7. Where, in the final heavy chain, do the amino acids coded for by the D and J segments end up? (Full name please.)

    Complementarity-determining region 3.

  8. Very briefly explain how productive rearrangement induces transcription of the gene.

    The distance between promoter and enhancer is reduced from several hundred thousand base pairs to several thousand.

  9. What causes nonproductive rearrangement?
    What is the next step which happens after a nonproductive rearrangement of a heavy chain gene? (There are two possible next steps -- mention both and what decides which one is taken.)

    The introduction of a stop codon, or a reading frame shift. If it is the first attempt, an attempt is made to rearrange the second allele. If both alleles have been rearranged nonproductively, the B cell undergoes apoptosis.

  10. Name a gene whose expression shows allelic exclusion.

    Immunoglobulin (or T cell antigen receptor).

  11. Name a gene which has codominant expression.

    Major histocompatibility genes (or most other genes).

  12. What does "ELISA" stand for?

    Enzyme-linked immunosorbent assay.

  13. List in outline form the steps in an indirect ELISA assay.
    Which step introduces the material of unknown concentration?
    How is the reaction measured quantitatively at the end?

    - Bind antigen to bottom of well, rinse.
    - Block bare well surface, rinse.
    - Bind standard or UNKNOWN antibody concentration, rinse.
    - Bind enzyme-conjugated anti-Ig antibody, rinse.
    - Add colorogenic substrate.
    - Read the absorbance (QUANTITATIVE MEASUREMENT).

  14. Both flow cytometry and microscopy can use fluorescent antibodies. What is the key difference between what each reports about the fluorescence?

    - Flow cytometry precisely measures the amount of fluorescence per cell, but not where that fluorescence is located in the cell.
    - Microscopy shows where the fluorescence is located in a cell or tissue.

  15. What does "MHC" stand for?

    Major histocompatibility complex (of genes).

  16. What does the term "polymorphic" mean as applied to the MHC?

    That there are more alleles per locus than for any other known group of loci.

  17. Why is the MHC polymorphic?

    (The ability of T cells to respond depends on peptide epitopes being bound to and presented in MHC grooves.)
    The existence of many alleles improves the ability to bind and present diverse foreign peptides, and hence the immunocompetence at both the individual and the population level.

  18. List the name of the human MHC and the names of the human class I and II loci.

    Human MHC is HLA, Human Leukocyte Antigens.
    Human Class I: HLA-A, HLA-B, HLA-C
    Human Class II: HLA-DP, HLA-DQ, HLA-DR

  19. For each, indicate whether the term designates class I or II or both, and whether the genetic region designated by the term codes for a single protein chain (1C) or more than one protein chain (>1C).
    1. H-2
    2. H-2D
    3. H-2 I-E
    4. H-2Kb
    5. H-2b

    1. Both, >1C
    2. I, 1C
    3. II, >1C
    4. I, 1C
    5. Both, >1C

  20. In the previous question, which if any of the terms designates a single allele?

    Only item d. (e designates a group of alleles or haplotype; b and c designate loci.)

  21. In each case, what percentage of the cells in the named tissue express the listed protein alleles?
    1. MHC class I alleles inherited from your father, in the thyroxin-producing gland cells in your thyroid?
    2. MHC class I alleles inherited from your father, in the naive T lymphocytes in your spleen?
    3. MHC class II alleles inherited from your mother, in the naive T lymphocytes in your spleen?
    4. MHC class II alleles inherited from your mother, in the dendritic cells in your spleen?

    1. 100%
    2. 100%
    3. 0%
    4. 100%

  22. Copy this table into your bluebook and fill it in.
    MHC
    Number of polypeptide chains PER MOLECULE
    Number of interchain disulfide bridges
    Coded within MHC
    Coded outside MHC
    Forming the peptide-binding groove
    Containing at least one immunoglobulin domain
    Containing transmembrane domains
    Class I
    1
    1
    1
    2
    1
    0
    Class II
    2
    0
    2
    2
    2
    0

  23. What is the probability that any two human siblings will have identical MHC genes? (Disregard the possibility of crossovers.)

    25%.

  24. What is the probability that any two sibling mice, in the first generation resulting from a cross between two different inbred strains, will have identical MHC genes? (Disregard the possibility of crossovers.)

    100%.

  25. Both human identical twins, and members of an inbred strain of mice, are genetically identical. How do these humans and mice differ regarding their alleles?

    Human identical twins have as many heterozygous loci as nontwins.
    Mice in an inbred strain are homozygous at all loci.

  26. Copy this table into your bluebook and fill it in.
    Antigen
    Responding T Cell
    MHC Presenting
    Typical source
    CD4 or CD8?
    Function
    Class I
    endogenous
    CD8
    Majority CTL; minority Th
    Class II
    exogenous
    CD4
    Majority Th; minority CTL

  27. Briefly describe the differences in the peptides, and the way they are bound, for MHC class I vs. II.

    MHC I: 8-10 amino acids, bound largely at ends which are enclosed in the MHC groove. Arches up in the middle towards the T cell.
    MHC II: 12-20 amino acids, bound with ends hanging out open ends of the MHC groove. No arch; most binding is to the protein backbone (a constant feature of all peptides).

  28. How variable in sequence are peptides eluted from purified HLA-A2?
    Is the variation, if any, greater near the ends or the center of the peptide?

    They are extremely variable except for one or two positions.
    Variation is greater near the center.

  29. All peptides eluted from HLA-A2 have glutamic acid at position 2. What is glutamic acid called in this situation?

    An anchor residue.

  30. Cheetahs have low polymorphism in their MHC.
    What is likely to be responsible for this during their evolution?
    What selective advantage or disadvantage results?

    - Reduction of the population to a small number with limited alleles at some point in their recent evolution.
    - This is disadvantageous for their ability to present diverse peptides, and makes them more susceptable to viruses.

  31. Briefly explain what "restriction of recognition of antigen by T cells" means.

    T cell recognition of antigen is restricted by the MHC; A clone of T cells will recognize the antigen epitope which selected it only when the epitope is presented in the same MHC allele which selected the clone in the first place.

  32. What did Zinkernagel and Doherty discover in the 1970's which resulted in their winning the Nobel Prize in Physiology or Medicine in the 1990's?

    MHC restriction of T cell (CTL) recognition of (virus) antigen by T cells (CTL). (Page 202.)

  33. Which class of MHC can present antigens from viruses which have been rendered noninfectious by heating?
    Why?
    Why can't the other class?

    - Class II.
    - It presents exogenous protein antigens, regardless of whether the microorganism is alive.
    - Presentation via class I MHC requires that the protein enter the cytoplasm. This usually requires that the protein be synthesized by an active virus infection.

  34. For each case, indicate whether it has a direct role in antigen processing and presentation by class I or class II, both or neither.
    1. Lysosomes.
    2. RAG-1/2.
    3. Phagocytosis or endocytosis.
    4. TAP.
    5. Heptamer/nonamer.
    6. Invariant chain.
    7. Proteolysis.
    8. Chaperonins.
    9. Low pH.
    10. Proteasome.

    1. II
    2. N
    3. II
    4. I
    5. N
    6. II
    7. B
    8. B
    9. II
    10. I

  35. What does "TAP" stand for?
    What do TAP's do?

    - Transporter of antigenic peptides.
    - Transport peptides from the cytoplasm into the endoplasmic reticulum.

  36. If an antibody and antigen have an association constant of 3 x 109 liters/mole, what is the value of the dissociation constant? (Give units too!)

    Kd = 3.3 x 10-10 moles/liter.

  37. Briefly explain the practical meaning of the value of Kd.

    It is the molar concentration of free antibody when, at equilibrium, the antigen is half-saturated with antibody.