In this article we will discuss about the concept of gene conversion.
In eukaryotes reciprocal exchange between loci almost always produces reciprocal recombinants. In Neurospora the linear arrangement of ascospores in the ascus indicates directly which strands have exchanged segments during crossing over. Thus after meiosis in a heterozygote Aa, due to reciprocal exchange the 8 ascospores are arranged in the order 4A and 4a.
Sometimes there is non-reciprocal exchange between the paired strands so that instead of the 4:4 ratio, the ascospores are arranged in 5:3 or 2:6 arrangement. It is suggested that some A alleles are converted into a alleles and vice versa, and the phenomenon is known as gene conversion or nonreciprocal exchange (Fig. 22.6). When recombination occurs within a short chromosomal segment (intragenic) it may be sometimes nonreciprocal.
Tetrad analysis of intragenic crosses in the fungus Ascobolus in the 1960’s showed that there are special sites in the chromosome which influence recombination in their neighbourhood. Thus if the order of linked loci is a b c d e…, when a cross is made between a + x + b, wild type recombinants arise mainly due to gene conversion at site a.
When the cross b + x + c is made, recombinant wild type arises due to conversion at site b. Similar results were subsequently obtained in other materials. In yeast nonreciprocal recombination occurs in both mitosis and meiosis. When mutant loci lie very close to each other, nonreciprocal re-combinations occur more frequently.
Molecular Mechanisms of Recombination:
Studies on recombination at the molecular level have suggested two mechanisms called copy-choice and breakage-fusion. The second mechanism is generally accepted. The copy-choice hypothesis was originally proposed for higher organisms by Belling in 1931.
In this model recombination occurs at the time of chromosome replication. When homologous portions of two chromosomes are paired opposite each other, then during replication, each copies a segment of the other chromosome.
The new strand formed along one member of the pair switches to the other chromosome and becomes a template for synthesis of the new strand. If this switching over event is repeated by the other member of a pair, then two reciprocally recombinant strands are formed (Fig. 22.7).
The copy-choice model has not been accepted as it cannot account for semi-conservative replication, nor the formation of heteroduplex molecules observed in viruses and bacteria. The mechanism also requires the replication of DNA prior to crossing over, which has not been detected by any of the biochemical techniques.
The breakage-fusion mechanism provides a better understood model of recombination. The experimental work of Meselson and Weigle (1961) and Meselson (1964) on phage A gave support to the breakage-fusion model and evidence against the copy-choice model.
Meselson and Weigle took a mutant strain of lambda carrying linked mutant loci a and b labelled with heavy isotopes 13C and 15N. The label was incorporated into lambda by infecting E. coli cells growing on isotope containing medium.
The wild type phage (+ +) did not contain density labels (12C and 14N). For the genetic cross, mixed infection was done by simultaneously infecting light E. coli cells with heavy, density labelled lambda containing mutations a and b, and the wild type light phage particles (Fig. 22.8).
The progeny particles were collected and subjected to CsCl density gradient centrifugation. The different bands formed by the progeny virus particles were taken out and analysed for the presence of recombinant genotypes by infecting healthy E. coli cells.
Under the conditions of the experiment, all the newly synthesised viral DNA molecules must be light (12C and 14N). Therefore, if recombination occurs by copy-choice, then all the recombinant phages should be light. Contrarily, if recombination occurs by breakage and fusion, then some of the recombinant phage particles should contain the heavy isotopes derived from the parental chromosome.
The progeny particles from the CsCl bands showed that the recombinants contained the heavy isotopes. The experiment provided evidence for breakage and fusion as the mechanism for recombination in bacteriophage.
Site-Specific Recombination:
Besides homologous recombination which takes place at any extensive region of sequence homology, site-specific recombination occurs between specific DNA sequences which are homologous over only a short stretch of DNA. The process is mediated by proteins that recognise the specific DNA target sequences, instead of by complementary base pairing.
When bacteriophage lambda (λ) infects E. coli, it can either replicate and kill host E. coli cell (cell lysis), or it can integrate into the E. coli chromosome forming a prophage that replicates with the E. coli genome (lysogeny). Under appropriate conditions, X DNA can be excised and initiate lytic viral replication. Both integration and excision of X DNA involve site-specific recombination between viral and host cell DNA sequences.
DNA of bacteriophage λ integrates into E. coli DNA at specific sites called attachment (att) sites. The process involves recombination between att sites of phage (attP) and the bacterium (attB) that are about 240 and 25 nucleotides long, respectively. The process is mediated by the phage protein integrase (Int) which specifically binds to both attP and attB.
The phage and the bacterium then exchange strands within a core sequence consisting of 15 nucleotides present in both attP and attB. The Int protein produces staggered cuts in the core homology region of attP and attB, catalyses exchange of strands, and ligates the broken ends, thus integrating λ DNA into E. coli chromosome. The Int protein also acts in excision of the λ prophage by a process that is reverse of integration.
Site-specific recombination plays an important role in the development of the immune system in mammalian cells, which involves recognition of foreign substances (antigens) and provides protection against infectious agents.
The two major classes of immune responses in humans and mammals are mediated by the B and T lymphocytes. The B lymphocytes secrete antibodies (immunoglobulins) that react with soluble antigens, while T lymphocytes express cell surface proteins (T cell receptors) that react with antigens expressed on the surfaces of other cells.
Both immunoglobulins and T cell receptors are characterised by enormous diversity which enables different antibody or T cell receptor molecules to recognise a large variety of foreign antigens. For example, an individual is able to produce more than 1012 different antibody molecules, which exceeds the total number of genes (about 105) in the human genome.
These diverse antibodies and T cell receptors are encoded by unique lymphocyte genes formed during the development of the immune system by site-specific recombination between distinct segments of immunoglobulin and T cell receptor genes.
The role of site-specific recombination in the formation of antibodies can be understood by first examining antibody structure. The immunoglobulins consist of pairs of identical heavy and light polypeptide chains, both composed of C-terminal constant regions and N-terminal variable regions.
The variable regions have different amino acid sequences in different immunoglobulin molecules, are responsible for antigen binding. It is the diversity of the amino acid sequences in the variable region that allows different individual antibodies to recognise unique antigens. In contrast to the vast array of different antibodies produced by an individual, each B lymphocyte produces only a single type of antibody.
An important discovery by Susumu Tonegawa in 1976 indicated that each antibody is encoded by unique genes formed by site-specific recombination during B lymphocyte development. These gene rearrangements create different immunoglobulin genes in different individual B lymphocytes, so that the population of 1012 B lymphocytes in the human body includes cells that can produce antibodies against an enormous variety of foreign antigens.
The genes that encode immunoglobulin light chains consist of three regions, a V region that encodes the 95 to 96 N- terminal amino acids of the polypeptide variable region; a joining (J) region that encodes the 12-14 C-terminal amino acids of the polypeptide variable region; and a C region that encodes the polypeptide constant region (see Figure below):
In mouse, the major class of light-chain genes are formed from combinations of approximately 250 V regions and four J regions with a single C region. Site-specific recombination during development of lymphocytes results in a gene rearrangement in which a single V region recombines with a single J region to generate a functional light-chain gene. Different V and J regions are rearranged in different B lymphocytes, so that the possible combinations of 250 V regions with 4 J regions can generate approximately 1000 (4 x 250) unique light chains.
There is a fourth region in the heavy-chain genes called the diversity or D region which codes amino acids lying between V and J. The assembly of a functional heavy-chain gene requires two recombination events.
First, a D region recombines with a J region, and next, a V region recombines with the rearranged DJ segment. In mouse there are approximately 500 heavy-chain V regions, 12 D regions, and 4 J regions, so that the total number of heavy chains that can be generated by recombination is 24,000 (500 x 12 x 4).
Combinations between the 1000 different light chains and 24,000 different heavy chains formed by site-specific recombination can give rise to approximately 2 x 107 different immunoglobulin molecules. The joining of immunoglobulin gene segments is often imprecise, so that one to several nucleotides may be lost or gained at the sites of joining, thus producing a further increase in diversity.
The mutations resulting from the loss or gain of nucleotides increase diversity of variable regions about a hundredfold, attributable to the formation of about 105 different light chains and 2 x 106 heavy chains, which can then combine to form more than 1012 distinct antibodies.
The T cell receptors consist of two chains called α and β, each of which contains variable and constant regions. The genes encoding these polypeptides are produced by recombination between V and J segments (the α chain) or between the V, D and J segments (the α chain), analogous to the formation of immunoglobulin genes.
Furthermore, site-specific recombination between these distinct segments of DNA, in combination with mutations arising during recombination, generates a similar level of diversity in T cell receptors to that in immunoglobulins.
The process of VDJ recombination that produces genes encoding polypeptides of T cell receptors is mediated by signal sequences adjacent to the coding sequences of each gene segment. These recombination signal sequences are recognised and cleaved by a complex of two proteins, RAG1 and RAG2 which are specifically expressed in lymphocytes. The subsequent joining of the coding ends of the gene segments then produces a rearranged immunoglobulin or T cell receptor gene.