Question

(i) Describe the phenomenon of functional sequence variation using

antibodies that can bind to a range of different antigens. (ii) Explain the process of domain shuffling in

protein evolution and give an example of how this process can generate proteins with novel functio

Answer 1

(i) With the passage of time after immunization, there is usually a progressive increase in the affinity of the antibodies produced against the immunizing antigen. This phenomenon, known as affinity maturation, is due to the accumulation of point mutations specifically in both heavy-chain and light-chain V-region coding sequences. The mutations occur long after the coding regions have been assembled, when B cells are stimulated by antigen and helper T cells to generate memory cells in a lymphoid follicle in a peripheral lymphoid organ . They occur at the rate of about one per V-region coding sequence per cell generation. Because this is about a million times greater than the spontaneous mutation rate in other genes, the process is called somatic hypermutation. The molecular mechanism is still uncertain, but it is believed to involve some form of error-prone DNA repair process targeted to the rearranged V-region coding sequence by specific regions of DNA brought together by V(D)J joining. Surprisingly, an enzyme involved in RNA editing is required, but its function in the hypermutation process is unknown.

Only a small minority of the altered antigen receptors generated by hypermutation have an increased affinity for the antigen. The few B cells expressing these higher-affinity receptors, however, are preferentially stimulated by the antigen to survive and proliferate, whereas most other B cells die by apoptosis. Thus, as a result of repeated cycles of somatic hypermutation, followed by antigen-driven proliferation of selected clones of memory B cells, antibodies of increasingly higher affinity become abundant during an immune response, providing progressively better protection against the pathogen.

(ii)

Domain shuffling, which refers to the duplication of a domain or the insertion of a domain from one gene into another , may have been a major factor in the evolution of human phenotypic complexity. Domain shuffling is often mediated by intronic recombination of exons encoding the domain.

Transduction of genome sequences mediated by retrotransposons likely represents a frequent mechanism to shuffle exons. . Phase combinations of flanking introns are useful indicators of exon shuffling. Intron phase is a parameter that determines the intron position relative to the translational reading frame. Introns that interrupt the reading frame between codons are known as phase 0 introns; those that split codons between the first and second nucleotides are known as phase 1 introns; and those that split codons between the second and the third nucleotides are known as phase 2 introns. Successful shuffling requires that the domain in question is bordered by introns that are of the same phase, that is, that the domain is symmetrical in accordance with the phase-compatibility rules of exon shuffling, because shuffling of asymmetrical exons/domains will result in a shift of the reading frame in the downstream exons of recipient genes. The abundance of genomic data from humans now permits a comprehensive study of protein domains and their coding structure.

Exon shuffling is a molecular mechanism for the formation of new genes. It is a process through which two or more exons from different genes can be brought together ectopically, or the same exon can be duplicated, to create a new exon-intron structure

In order for exon shuffling to start to play a major role in protein evolution the appearance of spliceosomal introns had to take place. This was due to the fact that the self-splicing introns of the RNA world were unsuitable for exon-shuffling by intronic recombination. These introns had an essential function and therefore could not be recombined. Additionally there is strong evidence that spliceosomal introns evolved fairly recently and are restricted in their evolutionary distribution. Therefore, exon shuffling became a major role in the construction of younger proteins.