• Variable region genes are encoded by multiple exons on the same chromosome
• Light chain variable region genes are produced by DNA rearrangements that join a V_L and a J_L gene from a pool of many such genes that are variable in sequence
• Heavy chain variable regions are similarly produced but use a different set of V_H and J_H genes with an additional gene, the D gene.
• Sources of Ig diversity

1. Recombinational diversity
2. Imprecision of V_L-J_L and V_H-D-J_­H joins
3. N region addition ( extra nucleotides added during recombination)
4. Somatic Mutation (mutations in variable genes with B cell proliferation)

B-Cell Ontogeny

stem cell®B stem cell®Pro-B cell®Pre-B cell®Immature B cell®B cell

Pro-B cell- TDT + DJ_H joining

Pre-B cell- V_HDJ_H joining (also surrogate light chain and surface m chain)

Immature B cell- V_HDJ_H joining and V_LJ_L joining (surface IgM)

B cell- surface IgM + IgD

Recombinational Diversity

• The human genome is currently estimated to contain from 70,000-100,000 genes (10^5)
• How can these genes code for 10^8 B and T cells with different antigen specificities?
• Answer: Each receptor chain is encoded by several different gene segments; Random assortment of these gene segments allows for the receptor diversity

Review

Exons- base pairs transcribed into mRNA

Introns- those that are excised

• Preceeding each gene that codes for a protein expressed at the cell surface is a leader sequence, which codes for a signal peptide that is about 200 a.a. in length
• This hydrophobic amino terminus is used to transport the protein through the membrane of the ER and into the G.A where the signal peptide is cleaved off and the remaining polypeptide is inserted into the newly forming cell membrane.

Genetic Events in the synthesis of Immunoglobulin chains

I.                 Organization and rearrangement of k Light chains

A.     V_k consists of approx. 108 residues coded by 2 separate gene segments, a V segment that codes for the amino terminal 95 residues and a J segment that codes for 13 residues at the carboxy terminal end of the variable region.

B.     k genes are located on chromosome 2.

C.    gene rearrangement occurs

D.    A k chain consists of one of a possible 100-200 different Vk genes and one of the five different Jk genes.  Both types of genes are separated by introns.  Another long section of an intron exists between the Jk genes and the single gene which codes for the constant region.

E.     From the rearranged DNA, a primary RNA transcript is made, then spliced to remove non-coding sequences yielding a Vk, Jk, and Ck mature mRNA. This mRNA can then be translated into the k polypeptide chain on rER.

F.     After transport, the leader sequence is cleaved off and the k chain can then be joined with an H chain to form an Ab molecule.

G.    The family of enzymes called reombinases catalyze the joining of the segments.  The steps of rearrangement are common to both B & T cells and the two genes whose products are required to activate the recombinases are active in both B & T cells.

II.               l Chain synthesis

A.     l genes on human chromosome 22

B.     The human l locus contains about 100 variable l genes and 6 possible J_l gene segments.

C.    With l chains it is possible for the V_l and J_l to be associated with one of six different types of C_l polypeptides.

D.    During the differentiation of the B cell, and before any apparent encounter with antigen, the DNA rearrangement in B cells, relative to a single antigenic specificity, becomes fixed.

III.              Organization and Rearrangement of Heavy Chain genes

A.     Chromosome 14

B.     3 variable gene segments

C.    The D and J segments code for the amino acids located in the third hypervariable region.

D.    There are about 200 V_H genes, 12 D_H genes, and 6 J_H genes.

E.     In the germ line, there are multiple genes coding for the C region of the Ig. The C region determines the class and also the biological function of a particular Ab. The C regions are flanked by introns.  The order of C genes of humans (Fig 6.4 of text).  The C genes closest to the V region genes are m and d which are transcribed first during B-cell development.  Once the polypeptides are transcribed,a B cell will express both m and d Antibodies with identical antigenic specificity.

IV.             Allelic Exclusion

A.     Maternal or Paternal genes

B.     Allelic exclusion- the unused chromosome ceases to rearrange its VDJ genes

V.               Class Isotype switching

A.     Although one B cell forms antibody of one antigenic specificity, it can switch from one class to another.  It requires an adjustment of juxtapositioning the VDJ segment to another C region heavy chain segment.

B.     Class switching occurs in mature B cells and occurs following an antigen stimulation of the B cell and the presence of certain cytokines released by T cells

C.    The B cell is evidently able to do this because of a switch region at the 5′ end, a stretch of repeating base sequences in front of the different classes of antibodies.  IgD has no switch region.  Once the change is made, this B cell removes the intervening C region DNA and it can no longer synthesize any other class of Ab.  The specific cytokine released is responsible for this control.

VI.             Generation of Antibody Diversity

A.     Presence of multiple V genes in the germ line

• The number of different genes represent the minimum # of different kinds of Ab that could be synthesized

B.     DJ-VDJ Combinational Association (Recombinational diversity)

• If there are 200 Vk and 5 Jk genes coding for a k chain, then there would be 200 x 5 = 1000 different kinds of chains that could be formed

C.    Random Assortment of H & L chains

• Remember any H chain may associate with any L chain

D.    Junctional & Insertional Diversity

• The apparent absence of precision in joining during DNA rearrangement would lead to deletions (junctional diversity) or insertions(insertional diversity) affecting the Ag binding site. This additional diversity is called N region diversity.

E.     Somatic Cell Mutation

• Mutations in the germ line can affect the VJ or VDJ regions variability, thereby further increasing the possible Ab that could be made available.

T Cell Receptor Genes

• The genes that code for the TCR and mechanisms are similar to those of Ig
• Structure of the TCR
• Ag-recognizing part- disulfide linked two-chain molecule, very similar in genetic organization and structure to an immunoglobulin.
• Two different types of TcRs are found
• ab and gd
• These are all distinct proteins with molecular weights varying between 30-45 kDa.
• A T cell will NEVER have both as its receptor

Organization of the Human genes

1.     a and g use V and J gene segments like Ig Light chains

2.     b and d use V,D, and J gene segments like Ig heavy chains

3.     There are more aand b genes than g and d.

• Also show allelic exclusion
• Do not somatically mutate
• Thymus

Notable differences between the TcR genes and Ig genes

1.     No somatic mutation in TcR

2.     No isotype switching in TcR

3.     Greater Junctional diversity in TcR genes due to the greater number of J segments

Exon-shuffling occurs when the Ig domain is recruited by other genes and moves in the genome.

The following molecules resemble the Ig domain so much that it could not have occurred by chance.

NCAMs- neural adhesion molecules

Thy-1 differentiation antigen expressed in neural tissue

CEA- carcinoembryonic Ag

PDGFR- platelet derived growth factor receptor

Tags: Allelic Exclusion, Cell Ontogeny, chromosome 22, Class Isotype switching, DNA rearrangements, Exons, Generation of Antibody Diversity, Heavy Chain genes, human genes, Introns, junctional diversity, Somatic Cell Mutation, Somatic Mutation, T Cell Receptor Genes

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