The formation of new genetic sequences by piecing together segments of previously existing ones. Classical genetics is based on the transmission of traits, attributable to particular genetic determinants or genes, through sexual generations. The genes are predominantly present in chromosomes, which are the threadlike structures that fill the cell nucleus and divide as the cells divide. The key component of the chromosomes is DNA; each chromosome has a single coiled and folded double-stranded DNA molecule running along its length, and the genes of the chromosomes occupy segments of this DNA. See also: Chromosome; Deoxyribonucleic acid (DNA); Gene; Genetics
Recombination often follows DNA transfer in bacteria and, in higher organisms, is a regular feature of sexual reproduction. The basic feature of sexual reproduction is the cyclical alternation between the diploid state, with a double set of chromosomes (two of each kind), and the haploid state, with a single set. Animals and most flowering plants are diploid, and their germ cells (eggs and sperm, or eggs and pollen grains) are haploid. In most fungi, the organism is normally haploid, but sexual fertilization creates a transient diploid cell that immediately divides to form a tetrad of four haploid spores. See also: Animal reproduction; Plant reproduction
In all sexually reproducing organisms, the transition from the diploid state to the haploid state is brought about by the process of meiosis—two rounds of cell division accompanied by only one division of the chromosomes. The two chromosomes of each kind present in the diploid meiotic mother cell are necessarily segregated into different haploid meiotic products. See also: Meiosis
Free reassortment and linkage
When the diploid cell undergoing meiosis is heterozygous, that is, it carries two alternative forms (alleles) of the same gene inherited from different parents, the meiotic segregation of chromosome pairs will ensure that half of the meiotic products (germ cells) will carry one alternative and half will carry the other. When the diploid is doubly heterozygous (A/a B/b), the two pairs of alleles segregate independently of one another at meiosis, provided the two gene differences are associated with different chromosome pairs. Consequently, four possible products—AB, Ab, aB, and ab—will occur with statistically equal frequency. See also: Allele; Mendelism
If the two genes in a double heterozygote Aa Cc are in the same chromosome and they are not very far apart, they show linkage, with the parental combinations (for example, AC and ac) tending to appear in the meiotic products more frequently than the recombinant types (Ac and aC). The fact that recombinants types Ac and aC occur at all is a result of reciprocal exchanges between chromosomes (crossing-over) that take place in the first meiotic division. The reciprocal nature of these events is shown most clearly in fungi such as yeast and the filamentous fungus Neurospora crassa, where the four products of a single meiosis can be recovered together as a tetrad of haploid cells (or, in the case of Neurospora, a tetrad of spore pairs). In such organisms, one recombinant type (Ac) is generally accompanied in the same tetrad by the reciprocal product (aC). See also: Crossing-over (genetics); Linkage (genetics)
Only two of the four tetrad members are generally recombinant. Tetrads of constitution AC, Ac, aC, ac are far more common than those containing four recombinant products, with the inference being that crossing-over between chromosome pairs occurs after the chromosomes have replicated and that each crossover involves only half of each divided chromosome (chromatid) [Fig. 1]. If more than one crossover occurs within the linkage group, it is a matter of chance which chromatid of each chromosome is involved in each chiasma (the point of contact, or crossing-over, between paired chromatids).
All the segregating genetic markers found in a given organism can be assigned to any of a number of linkage groups. Markers in different groups reassort independently, whereas markers in the same group are linked. The number of linkage groups is the same as the haploid number of chromosomes. Furthermore, through correlations of changed linkage relationships with specific chromosome aberrations, each linkage group can be assigned to a specific chromosome (Fig. 2).
The measure of linkage is recombination frequency [(Ac + aC)/(AC + Ac + aC + ac)], which is often expressed as a percentage, and it must be significantly less than 50% to be distinguishable from random reassortment. The map distance, in map units or centimorgans (cM), between two markers is 100 times the mean number of crossovers per chromatid, or 50 times the mean number per meiotic tetrad. If there is never more than one crossover between the markers, recombination frequency equals map distance. However, if there is a significant number of double or multiple crossovers, recombination frequency is an underestimate of map distance because each additional crossover within a marked interval has as much chance of canceling recombination as of causing it. Above a certain distance, recombination frequency approaches a limiting value of 50%. In practice, the linkage map of each chromosome is built up from the summing of a series of intervals, with each being too short for double crossovers to have a significant effect. A corollary of the theory of chromosome mapping is that the total map length of a linkage group should be 50 times the mean chiasma frequency of the corresponding chromosome pair at meiosis. This has been confirmed in organisms, including corn (Zea mays), where both relatively complete linkage maps and accurate chiasma counts are available. See also: Genetic mapping
The average amount of DNA that corresponds to 1% recombination (one map unit) varies enormously between species, from less than 10,000 DNA base pairs in the yeast Saccharomyces cerevisiae to about one million base pairs in humans and even higher values in some amphibians and flowering plants. Furthermore, the value varies from one part of the genome to another within a species because not all chromosome regions have the same probability of crossing-over.
A standard method of determining the sequence of three linked markers is the three-point test cross. This measures the frequencies of the eight possible kinds of meiotic product formed by a diploid segregating with respect to three linked markers, say ACD/acd. If the markers are in the order written, recombinant meiotic products of constitution AcD or aCd will be relatively infrequent compared with Acd, aCD, ACd, and acD because, unlike the latter classes, which arise from single crossovers, they require relatively rare double crossovers for their formation. The method would be seriously undermined if crossovers tended to occur in clusters, such that doubles and multiples were as probable as singles. In fact, the occurrence of a crossover in one chromosome interval tends to reduce the likelihood of a crossover in an adjacent interval, which is an effect called crossover or chiasma interference. A decreased probability of occurrence of crossovers beyond the first has also been inferred from chiasma counts under the microscope. Whereas the chance of at least one chiasma is nearly 100% (very few chromosome pairs are left unjoined by chiasmata at the first meiotic division), shorter chromosomes commonly have no more than one and even the longest may never have more than three or four.
Recombination was once thought to occur only between genes, never within them. Indeed, the supposed indivisibility of the gene was regarded as one of its defining features, with the other being that it was a single unit of function. The functional criterion was failure of alleles to complement one another. If two defective mutants of independent origin failed to compensate for each other's defects to determine a more normal phenotype when present together in a heterozygote, then neither was thought capable of supplying the normal function that was defective in the other; hence, both must be defective in the same gene, defined as a unit of function. The two definitions agreed to the extent that mutations placed in the same gene by the complementation criterion were always at least very closely linked. However, examination of very large progenies shows that, in all organisms that were studied, nearly all functionally allelic mutations of independent origin can recombine with each other to give nonmutant products, generally at frequencies ranging from a few percent (the exceptionally high frequency found in Saccharomyces) down to 0.001% or less. The indivisible unit of recombination is no larger than a single DNA base pair.
Mutations within a gene can be mapped in a linear order either by using the criterion of recombination frequency or, more accurately, by deletion mapping, which uses deletions of extended segments of the gene. Overlapping deletion mutations cannot recombine to give normal recombinants since neither has all of the sequence in which the other is defective. For the same reason, a deletion mutation gives no recombinants with any single-base-pair mutation falling within the region defined by the deletion. Given a series of overlapping deletions, any point mutation can be positioned within one or any other of the segments defined by the deletions and their overlaps.
Tetrad analysis, especially in S. cerevisiae, shows that recombination within genes is most frequently nonreciprocal. In a cross between two allelic mutants, both nonmutant and double-mutant products are formed, but seldom in the same tetrad (Fig. 3). This phenomenon is termed gene conversion and must represent the nonreciprocal transfer of DNA sequence between chromatids of paired chromosomes. Gene conversion also is found in crosses that involve only a single mutational difference. In addition to the expected 2:2 tetrad segregation pattern, up to several percent 3:1 and 1:3 ratios can be found in yeast; frequency in other experimental organisms is generally very much lower. In both Saccharomyces and other fungi, genes often show a gradient of conversion frequency from one end of a gene to the other, which is an effect called conversion polarity.
Conversion sometimes affects only half a chromatid, equivalent to a single DNA strand, rather than both strands of the double helix. In this case, formation of a completely converted gene is delayed until after the first replication of the DNA following meiosis. When donor and recipient genes differ in more than one mutational marker, the two markers often are transferred together (coconverted). The frequency of coconversion, compared with single-marker conversion, diminishes as the distance between the markers increases. The relationship between frequency and distance indicates that conversion involves the transfer of segments of DNA commonly of the order of several hundred to a few thousand base pairs in length. Coconversion does not result in any marker recombination. In order to recombine two markers within a gene, a conversion segment has to include one but not the other. See also: Mutation
Recombination between and within genes may seem to be two distinct processes that involve reciprocal crossing-over and nonreciprocal conversion, respectively. However, it is attractive to postulate that both follow from the same kind of initiating event. Conversion within a gene is accompanied often (with a frequency of 40–50%) by a closely adjacent crossover, which is detectable through the reciprocal recombination of markers that flank the gene undergoing conversion (Fig. 3). Both conversions and crossovers are formed probably from precursor structures that involve local unilateral transfer of DNA segments between chromatids. Such structures, which are called conversion tracts, will lead to observable gene conversion only in the rare event that the transferred segment happens to include a genetic marker. If the structure develops into a crossover (which is presumed to happen in yeast with a probability of 40–50%), it always will lead to reciprocal crossing-over between flanking markers, regardless of how distant they may be from the initiating event. In the case of recombination between extremely close markers (particularly those within the same gene), the initiating event is necessarily very close to the markers being recombined, which thus will have a high probability of being included in the conversion tract.
There are two prevailing hypotheses as to the nature of the event that initiates recombination. In the first model, a chromatid involved in meiotic pairing is cut in one strand of its DNA, probably at one of a large number of special sites distributed along the chromosome. A stretch of single-stranded DNA then unwinds from one side of the cut and invades the DNA of the other chromatid, displacing the corresponding single-strand segment from its duplex. In the simplest sequence of events, the invading single strand becomes sealed into the recipient duplex, creating a mismatch between the complementary strands at any point at which the recipient and donor duplexes differ. Such a mismatch can be left unrepaired, resulting in half-chromatid conversion and postmeiotic segregation; or (more frequently, at least in yeast) it is repaired by excision and replacement of the mismatch in one strand either to restore the original arrangement or to convert the recipient whole chromatid to the type of the donor. To explain the association between conversion and crossing-over, it is supposed that the single-strand bridge can lead to a reciprocal single-strand exchange (a Holliday junction) and then to a reciprocal double-strand exchange, that is, a regular crossover. The chance of a single-strand transfer leading to a crossover is variable, but it seldom exceeds 50% (Fig. 4a).
A radically different hypothesis proposes that the initiating event is a double-stranded break, which may be enlarged subsequently to a gap, in one chromatid. The gap is healed by copying of both strands of the undamaged partner chromatid. The repair process involves the formation of Holliday junctions on both sides of the gap and, depending on the way that these are resolved, crossing-over may take place. The double-strand break–repair hypothesis accounts for conversion either with or without adjacent crossing-over, but it does not readily explain the occurrence of conversion events that affect only half a chromatid, leading to postmeiotic segregation. However, this too can be explained if the regions of heteroduplex that would be formed on each side of the gap (from annealing of single-strand ends into the undamaged duplex) are sufficiently extensive (Fig. 4b).
Both hypotheses provide for a scattering of conversion segments in all chromosome pairs, with some being associated with crossovers. Both explain polarity in gene conversion as a result of the initial DNA cuts, whether single- or double-stranded, occurring at preferred sites in the DNA; the probability of heteroduplex formation or gapping, and thus the probability of a marker undergoing conversion, should decrease with increasing distance from such a site. The double-strand break–repair model appears more probable, especially as a site at the high-conversion end of at least one yeast gene appears to undergo double-strand breakage at meiosis. However, elements of both proposed mechanisms may have to be included in a more refined model.
Most speculation on recombination mechanisms has focused on recombining chromosomes as DNA molecules, which is an oversimplification. Little is yet known about how the higher-order DNA protein structure of chromosomes affects their recombination. In addition, the role of the synaptonemal complex, across which the exchanged strands of DNA may need to pass, is obscure. See also: Molecular biology
Crossing-over between homologous chromosome pairs also can occur during the prophase of mitotic nuclear division. The frequency is very much lower than that in meiosis, presumably because the mitotic cell does not form the synaptic apparatus for efficient pairing of homologs. Mitotic crossing-over has been studied in the fruit fly Drosophila melanogaster, the filamentous fungus Aspergillus nidulans, and Saccharomyces yeast. In these species, it is detected through the formation of homozygous clones of cells in an initially heterozygous diploid. There is a 50% chance of homozygosity in daughter cells whenever a crossover occurs between chromatids in the interval between the marker and the centromere (the chromosomal site of attachment to the mitotic spindle) [Fig. 5]. The frequency of mitotic crossing-over is increased to a great extent by radiation. See also: Mitosis
Integration of bacterial DNA fragments
Bacteria have no sexual reproduction in the true sense, but many or most of them are capable of transferring fragments of DNA from cell to cell by one of three mechanisms: (1) Fragments of the bacterial genome can become joined to plasmid DNA and transferred by cell conjugation by the same mechanism that secures the transfer of the DNA of transmissible plasmids. (2) Genomic fragments can be carried from cell to cell in the infective coats of bacterial viruses (phages); this process is called transduction. (3) Many bacteria have the capacity to assimilate fragments of DNA from solution and thus may acquire genes from disrupted cells. See also: Bacteriophage; Plasmid; Transduction (bacteria)
Fragments of DNA acquired by any of these methods can be integrated into the DNA of the genome in place of homologous sequences previously present. If the incoming DNA has no homology with that of the recipient cell, it usually cannot be integrated and is lost for lack of ability to replicate autonomously. Homologous integration in bacteria is similar, in its nonreciprocal nature and perhaps also in its mechanism, to gene conversion in eukaryotic organisms. See also: Bacterial genetics
Bacteriophages, plasmids, bacteria, and unicellular eukaryotes provide many examples of differentiation through controlled and site-specific recombination of DNA segments. In vertebrates, a controlled series of deletions leads to the generation of the great diversity of gene sequences that encode the antibodies and T-cell receptors that are necessary for immune defense against pathogens. All these processes depend on interaction and recombination between specific DNA sequences (generally but not always with some sequence similarity) that are catalyzed by site-specific recombinase enzymes. The molecular mechanisms may have some similarities with those responsible for general meiotic recombination. However, the latter does not depend on any specific sequence; instead, it is dependent only on similarity (homology) of the sequence recombined.
Techniques have been devised for the artificial transfer of DNA fragments from any source into cells of many different species, thus conferring new properties on them (transformation). In bacteria and the yeast S. cerevisiae, integration of such DNA into the genome (on which the stability of transformation generally depends) requires substantial sequence similarity between incoming DNA and the recipient site. However, cells of other fungi, higher plants, and animals are able to integrate foreign DNA into their chromosomes with little or no sequence similarity. These organisms appear to have some unidentified system that recombines the free ends of DNA fragments into chromosomes, regardless of their sequences. It may have something in common with the mechanism, equally obscure, whereby broken ends of chromosomes can heal by nonspecific mutual joining. See also: Genetic engineering; Transformation (bacteria)