Study Questions: CHAPTER 5.

1. Indicate whether each of the following statements is true or false. If you think a statement is false, explain why.

a. Vk gene segments sometimes join to C gene segments.

b. With the exception of a switch to IgD, immunoglobulin class switching is mediated by DNA rearrangements.

c. Separate exons encode the transmembrane portion of each membrane immunoglobulin.

d. Although each B cell carries two alleles encoding the immunoglobulin heavy and light chains, only one allele is expressed.

e. Primary transcripts are processed into functional mRNA by removal of introns, capping, and addition of a poly-A tail.

f. The primary transcript is an RNA complement of the coding strand of the DNA and includes both introns and exons.

2. Explain why aVH segment cannot join directly with a JH seg ment in heavy-chain gene rearrangement.

3. Considering only combinatorial joining of gene segments and association of light and heavy chains, how many differ ent antibody molecules potentially could be generated from germ-line DNA containing 500 VL and 4 JL gene segments and 300 VH, 15 DH, and 4 gene segments?

 

4. For each incomplete statement below (a-g), select the phrase(s) that correctly completes the statement. More than one choice may be correct.

___ a. Recombination of immunoglobulin gene segments serves to

(1) promote Ig diversification

(2) assemble a complete Ig coding sequence

(3) allow changes in coding information during B-cell maturation

(4) increase the affinity of immunoglobulin for antibody

(5) all of the above

 

___ b. Somatic mutation of immunoglobulin genes accounts

(1) allelic exclusion                (2) class switching from IgM to IgG

(3) affinity maturation            (4) all of the above            (5) none of the above

 

____ c. The frequency of somatic mutation in Ig genes is greatest during

(1) differentiation of pre-B cells into mature B cells    (2) differentiation of pre-T cells into mature T cells

(3) generation of memory B cells                               (4) antibody secretion by plasma cells

(5) none of the above

___ d. Kappa and lambda light-chain genes

(1) are located on the same chromosome            (2) associate with only one type of heavy chain

(3) can be expressed by the same B cell              (4) all of the above

(5) none of the above

 

___ e. Generation of combinatorial diversity munoglobulins involves

(1) mRNA splicing                (2) DNA rearrangement        (3) recombination signal sequences

(4) one-turn/two-turn joining rule        (5) switch sites

 

___ f. A B cell becomes immunocompetent

(1) following productive rearrangement of variable- region heavy-chain gene segments in germ-line DNA

(2) following productive rearrangement of variable- region heavy-chain and light-chain gene segments in germ-line DNA

(3) following class switching

(4) during affinity maturation

(5) following binding of TH cytokines to their receptors on the B cell

 

___ g. The mechanism that permits immunoglobulins to be synthesized in either a membrane-bound or secreted form is

(1) allelic exclusion                (2) codominant expression            (3) class switching

(4) the one-turn/two-turn joining rule             (5) differential RNA processing

 

5. What mechanisms generate the three hypervariable regions (complementarity-determining regions) of immunoglobulin heavy and light chains? Why is the third hypervariable region (CDR3) more variable than the other two (CDR1 and CDR2)?

6. You have a B-cell lymphoma that has made nonproductive rearrangements for both heavy-chain alleles. What is the arrangement of its light-chain DNA? Why?

7. Indicate whether each of the class switches indicated below can occur (Yes) or cannot occur (No).

a) IgM to IgD                           b) IgM to IgA                           c) IgE to IgG

d) IgAtoIgG                             e) IgM to IgG

 

8. Describe one advantage and one disadvantage of N nucleotide addition during the rearrangement of immunoglobulin heavy-chain gene segments.

9. X-ray crystallographic analyses of many antibody molecules bound to their respective antigens have revealed that the CDR3 of both the heavy and light chains make contact with the epitope. Moreover, sequence analyses reveal that the variability of CDR3 is greater than that of either CDR1 or CDR2. What mechanisms account for the greater diversity in CDR3?

10. How many chances does a developing B cell have to generate a functional immunoglobulin light-chain gene?

11. Match the terms below (a-h) to the description(s) that follow (1-11). Each description may be used once, more than once, or not at all; more than one description may apply to some terms.

Terms:

a. ______ RAG-1 and RAG-2

b. ______ Double-strand break repair (DSBR) enzymes

c. ______ Coding joints

d. ______ RSSs

e. ______ P-nucleotides

f. ______ N-nucleotides

g. ______ Promoters

h. ______ Enhancers

 

Descriptions:

(1) Junctions between immunoglobulin gene segments formed during rearrangement

(2) Source of diversity in antibody heavy chains

(3) DNA regulatory sequences

(4) Conserved DNA sequences, located adjacent to V, D, and J segments, that help direct gene rearrangement

(5) Enzymes expressed in developing B cells

(6) Enzymes expressed in mature B cells

(7) Nucleotide sequences located close to each leader segment in immunoglobulin genes to which RNA polymerase binds

(8) Product of endonuclease cleavage of hairpin intermediates in Ig-gene rearrangement

(9) Enzymes that are defective in SCID mice

(10) Nucleotide sequences that greatly increase the rate of transcription of rearranged immunoglobulin genes compared with germ-line DNA

(11) Nucleotides added by TdT enzyme


 

CHAPTER 6: Antigen-Antibody Interactions: Principles and Applications

1. Indicate whether each of the following statements is true or false. If you think a statement is false, explain why.

___ a. Indirect immunofluorescence is a more sensitive tech nique than direct immunofluorescence.

___ b. Most antigens induce a polyclonal response.

___ c. A papain digest of anti-SRBC antibodies can agglutinate sheep red blood cells (SRBCs).

___ d. A pepsin digest of anti-SRBC antibodies can agglutinate SRBCs.

___ e. Indirect immunofluorescence can be performed using a Fab fragment as the primary, non-labeled
        antibody.

___ f. For precipitation to occur, both antigen and antibody must be multivalent.

___ g. Analysis of a cell population by flow cytometry can simultaneously provide information on both the
        size distribution and antigen profile of cell populations containing several different cell types.

___ h. ELISA tests using chemiluminescence are more sensitive than chromogenic ones and precipitation
        tests are more sensitive than agglutination tests.

___ i. Western blotting and immunoprecipitation assays are useful quantitative assays for measuring the
        levels of pro teins in cells or tissues.

___ j. Assume antibody A and antibody B both react with an epitope C. Furthermore, assume that
        antibody A has a Ka 5 times greater than that of antibody B. The strength of the monovalent
        reaction of antibody A with epitope C will always be greater than the avidity of antibody B for an
        antigen with multiple copies of epitope C.

 

2.  You have obtained a preparation of purified bovine serum albumin (BSA) from normal bovine serum. To determine whether any other serum proteins remain in this preparation of BSA, you decide to use immunoelectrophoresis.

a. What antigen would you use to prepare the antiserum needed to detect impurities in the BSA preparation?

b. Assuming that the BSA preparation is pure, draw the immunoelectrophoretic pattern you would expect if the assay was performed with bovine serum in a well above a trough containing the antiserum you prepared in (a) and the BSA sample in a well below the trough as shown below:

 

___ 3. Which of the following does not participate in the formation of antigen-antibody complexes?

a. Hydrophobic bonds        b. Covalent bonds            c. Electrostatic interactions

d. Hydrogen bonds             e. Van der Waals forces

 

4. Explain the difference between antibody affinity and anti body avidity. Which of these properties of an antibody better reflects its ability to contribute to the humoral immune response to invading bacteria?

5. In preparing a demonstration for her immunology class, an instructor purified IgG antibodies to sheep red blood cells (SRBCs) and digested some of the antibodies into Fab, Fc, and F(ab)2 fragments. She placed each preparation in a separate tube, labeled the tubes with a water- soluble marker, and left them in an ice bucket. When the instructor returned for her class period, she discovered that the labels had smeared and were unreadable. Deter mined to salvage the demonstration, she relabeled the tubes 1, 2, 3, and 4 and proceeded. Based on the test results described below, indicate which preparation was con tained in each tube and explain how you identified the contents.

a. The preparation in tube 1 agglutinated SRBCs but did not lyse them in the presence of complement.

b. The preparation in tube 2 did not agglutinate SRBCs or lyse them in the presence of complement. However, when this preparation was added to SRBCs before the addition of whole anti-SRBC, it prevented agglutination of the cells by the whole anti-SRBC antiserum.

c. The preparation in tube 3 agglutinated SRBCs and also lysed the cells in the presence of complement.

d. The preparation in tube 4 did not agglutinate or lyse SRBCs and did not inhibit agglutination of SRBCs by whole anti-SRBC antiserum.

 

6. You are given two solutions, one containing protein X and the other containing antibody to protein X. When you add 1 ml of anti-X to 1 ml of protein X, a precipitate forms. But when you dilute the antibody solution 100-fold and then mix 1 ml of the diluted anti-X with 1 ml of protein X, no precipitate forms.

a. Explain why no precipitate formed with the diluted anti body.

b. Which species (protein X or anti-X) would likely be pre sent in the supernatant of the antibody-antigen mixture in each case?


 

CHAPTER 7.  Major Histocompatibility Complex.

1. Indicate whether each of the following statements is true or false. If you think a statement is false, explain why.

___ a). A monoclonal antibody specific for 13 can be used to detect both class I MHC K and D
    molecules on the surface of cells.

___ b). Antigen-presenting cells express both class I and class II MHC molecules on their membranes.

___ c). Class III MHC genes encode membrane-bound proteins.

___ d). In outbred populations, an individual is more likely to be histocompatible with one of its parents
    than with its siblings.

___ e). Class II MHC molecules typically bind to longer peptides than do class I molecules.

___ f). All cells express class I MHC molecules.

___ g). The majority of the peptides displayed by class land class II MHC molecules on cells are derived
     from self-proteins.

 

2. You cross a BALB/c (H-2d) mouse with a CBA (H-2k) mouse. What MHC molecules will the progeny express on (a) its liver cells and (b) its macrophages?

 

3. The SJL mouse strain, which has the H-2k haplotype, has a deletion of the IEα locus.

a. List the classical MHC molecules that are expressed on the membrane of macrophages from SJL mice.

 

4. Draw diagrams illustrating the general structure, including the domains, of class I MHC molecules, class II MHC molecules, and membrane-bound antibody on B cells. Label each chain and the domains within it, the antigen-binding regions, and regions that have the immunoglobulin-fold structure.

5. One of the characteristic features of the MHC is the large number of different alleles at each locus.

a.  How is MHC polymorphism thought to be generated?

 

6. How can you determine if two different inbred mouse strains have identical MHC haplotypes?