Académique Documents
Professionnel Documents
Culture Documents
When foreign subtance called antigens (the coat protein of viruses) enter the blood
stream of mammal, they trigger a defens mechanism, the immune response, which results
in the synthesis of extremely important group of proteins called antibodies. These
antibodies bind to the antigens with exceptional specificity, thus facilitating their removal
from the circulatory system. During the last decade, scientists have discovered that the DNA
sequence that encode the vast array of antibodies produced by immune systems of
mammals are assembled during the differentiation of the antibody producing cells by the
occurrence of a novel set of genome rearrangements.
Three different types of white blood cells play central roles in the immune response
in vertebrates. These cells are:
The most remarkable aspect of the immune response, at least from a genetics
standpoint, is the seemingly infinite variety of antibodies that can be synthesized in
response to antigens that the animal has not previously encountered. How can an organism
have prepared to synthesize an antibody designed to bind very specifically to a particular
antigen without ever having made contact with the antigen? Moreover, how can an
organism store enough genetic information to code for the amino acid sequences of a
virtually unlimited variety of antibodies? These and related questions about the immune
response had puzzled geneticists for several decades. Within the last few years, however,
the main features of the answers to these questions have become clear.
We do not know many different antibodies a mouse or a human can produce, but we
do know that the number is very large, almost certainly in the millions. This presents a
paradox. The complete human genome contains about 3x109 nucleotide pairs. If all of this
DNA were in the form of uninterupted coding sequences of genes each 1000 nucleotide
pairs long, the genom would contain a maximum of about 3 million genes. Since we know
that many of these genes code for various RNA molecules, enzymes, and structural proteins,
and we know that many of these genes contain long noncoding introns, how can we account
for the genetic information needed to code for the plethora of different antibodies?
Past attempts to explain the genetic basic of antibody diversity can be roughly
grouped into three different hypotheses.
1) The germ line hypothesis stated that there is a separate germ line gene for each
antibody. (this agreed with our early knowledge about protein synthesis, but
presented the paradox of not enough DNA.)
2) The somatic mutation hypothesis stated that there is only one or a few germ line
gone specifying each major class of antibodies and that the diversity is generated by
a high frequency of somatic mutationmutation occurring in the antibody
producing somatic cells or in cell lineages leading to antibody producing cells. (there
was no precedent for a high frequency of mutation occuring in only certain genes
and in only certain types of cells. By what mechanism could this occur, and how
could it be regulated?)
3) The minigene hypothesis stated that the diversity is generated by the shuffling of
many small segments of a few genes into a multitude of possible combinations. The
shuffling would occur by recombination processes in somatic cells. (this required
totally novel mechanisms for rearranging segments of DNA).
We now know that the minigene hyothesis explains a great deal of the observed
diversity. However, we also know that somatic mutation contributes additional diversity.
Finally, we know that one segment (the constant region, see the following discussion) of
each antibody chain is specified by a gene or gene segment that is present in the
genome in only a few copies. Thus, all three hypotheses were correct in certain respects.
Figure 16.1 schematic diagram showing the major components of the immune
response in vertebrates. A foreign subtance such as the coat protein of a virus acts as an
antigen to trigger the synthesis of large amounts of antibodies that react specifically with
the antigen and remove it from the circulatory system. Two types of immune response
occur. (1) B lymphoscytes synthesize and secrete antibodies that complex with free antigens
in the bloodstream; these complexes are then ingested and destroyed by macrophages. (2)
T lymphocytes synthesize antigen receptors that remain bound to the surface of the T cells.
These antigen receptors act in concert with histocompatibility antigen receptors to
recognize and destroy cells that carry the antigens. Thus, the B cells carry out a celluler
immune response. The vast diversity of both antibodies and T cells receptors is produced by
genome rearrangements that take place during the differentiation of B and T lymphocytes
from stem cells. The histocompatibility antigens are encoded by a large cluster of genes in
the major histocompatibility complex (MHC).
Structure of Antibodies
Regions of proteins that carry out particular functions are called domains. Each
antibody has two antigen binding sites or domains, each of which is formed by the variable
regions of one light chain and one heavy chain. In addition, the constant regions of the two
heavy chains interact to form a third domain, called the effector function domain, which is
responsible for the proper interaction of the antibody with other components of the
immune system.
There are five classes of antibodies:IgM, IgD, IgG, IgE,and IgA. The class to which an
antibody belongs, and thus the functin tht it carries out, is determined by the structure of its
heavy chain constant region. For example, IgD antibodies usually remain bound to the
surface of the cells in which they are synthesized, whereas IgG antibodies are usually
secreted and circulate through the body in the bloodstream. The light chains of antibodies
are of two types, kappa and lambda, with type being determined by the structure of the
light chain constant region. As we shall see, antibodies may have the same antigen binding
specificty, as determined by the variable regions of the four chains, but different
immunological functions, as determined by the constant regions of the two heavy chains.
Thus, when we examine the structure of antibodies, we see that their diversity
resides almost entirely within the variable regions of the molecules. If these polypeptides
wee synthesized from colinear nucleotide pair sequence of genes, one gene per polypeptide
chain, the genome would have to contain a vast array of genes with highly variable
sequence at one end and essentially identical sequences at the other end. However, this is
not the case. Recombinant DNA techniques have made it possible to isolate and sequence
many of the segments of chromosomal DNA of mice and humans coding for antibidy chains.
The results of these studies have provided an elegant explanatin for the generation of
proteins with great diversity in certain regions and constancy in other regions.
Very simply, the genetic information coding for antibody chains is stored in bits and
pieces, and these bits and pieces are put together in the appropriate sequences by genome
rearrangements occurring during the development of the antibody producing cells (called B
lymphocytes) of the body. Each B lymphocte produces only a single type of antibody, that is,
all the antibodies produced by a given B lymphocyte have the same antigen binding
specificity.
Synthesis of the kappa light chain is controlled by three different gene segments
1. a Vx gene segment,coding for the N terminl 95 amino acids of the variable region
2. a Jx gene segment (J for joining segment) coding for the last (constant region
proximal) 13 amino acids of the variable region; and
3. a Cx gene segment, coding for the C terminal constant region
4. Lx segment, codes for an N terminal hydrophobic leader sequence 17-20 amino
acids long, which is essential for the transport of the antibody chain through the
cell membrane, and thus is not part of the final antibody.
The arrangement of the kappa chain gene segments in germ line cells it shown in fig.
16.4. In mice and humans, all the kappa chain gene segments are located on the same
chromosome (chromosome 2 in humans). The same is true for the lambda gene segments
(chromosome 22 in humans) and the heavy chain gene segments (chromosome 14 in
humans). There are a large number, probably about 300, of Vx gene segments, each with a
nearby Lx gene segments. On the other hand, there is only one Cx gene segment. Five Jx gene
segment (one of which is nonfunctional in the mouse) are located between the V x gene
segments and the Cx gene segment.
In germ line cells, the five Jx segment are separated from the Vx segments by a long
nonkoding sequence and from the Cx segment by an approximately 2000 nucleotide pair
long noncoding sequence. During the development of a B lymphocyte, the particular kappa
light chain gene that will be expressed in that cell is assembled from one Lx- Vx segment, one
Jx segment, and the single Cx segment by a process of somatic recombination. This process
joins any one of the approximately 300Lx-Vx segments with any one of the five Jx segments,
with the deletion of all intervening DNA. It yields a fused VxJx gene segment coding for the
entire variable region of the kappa chain. The noncoding sequence between the Jx gene
segment cluster and the Cx gene segment, and the Cx proximal Jx segments, if any, remain
between the fused VxJx segment and the Cx segment in the differentiated B lymphocytes.
This entire DNA sequence is transcribed and the noncoding sequences are removed during
RNA processing, just like that the noncoding sequences or introns of any other eukaryotic
gene.
The coding sequences (exon) of the Ch gene segments are interrupted by noncoding
sequences (introns) just like those of many other eukaryotic genes. The Ch gene segments
contain four to six exons nd three to five introns. In membrane bound antibodies, the heavy
chain constant regions are produced by splicing all six exons together. The last two exons
code for the hydrophobic tails of the membrane bound form, the fifth Ch exon is spliced to
a site 20 codons from the end of the fourth exon, thus changing the amino acid sequence of
this portion of the heavy chain constant region. In secreted antibodies, the heavy chain
constant regions are therefore the product of the first four exons.
The use of alternate pathways of transcription and RNA processing to synthesize
membrane bound and secreted forms has been firmly established for the IgM class of
antibodies. Recent evidence suggests that similar alternate pathways of transcription and
splicing are responsible for the production of the membrane bound and secreted forms of
the other classes of imunoglobulins as well.