October 14, 2012

Unequal crossing-over rearranges gene clusters

  • Nonreciprocal recombination (unequal crossing-over) results from an error in pairing and crossing-over in which nonequivalent sites are involved in a recombination event. It produces one recombinant with a deletion of material and one with a duplication.
  • Thalassemia is disease of red blood cells resulting from lack of either α or β globin.
  • HbH disease results from a condition in which there is a disproportionate amount of the abnormal tetramer β4 relative to the amount of normal hemoglobin (a2β2).
  • Hydrops fetalis is a fatal disease resulting from the absence of the hemoglobin α gene.
  • Hb Lepore is an unusual globin protein that results from unequal crossing-over between the β and δ genes. The genes become fused together to produce a single β-like chain that consists of the N-terminal sequence of δ joined to the C-terminal sequence of β.
  • Hb anti-Lepore is a fusion gene produced by unequal crossing-over that has the N-terminal part of β globin and the C-terminal part of δ globin.
  • Hb Kenya is a fusion gene produced by unequal crossing-over between the between Aγ and β globin genes.
  • When a genome contains a cluster of genes with related sequences, mispairing between nonallelic genes can cause unequal crossing-over. This produces a deletion in one recombinant chromosome and a corresponding duplication in the other.
  • Different thalassemias are caused by various deletions that eliminate α- or β-globin genes. The severity of the disease depends on the individual deletion. 

There are frequent opportunities for rearrangement in a cluster of related or identical genes. We can see the results by comparing the mammalian β clusters included in Figure 4.5. Although the clusters serve the same function, and all have the same general organization, each is different in size, there is variation in the total number and types of β-globin genes, and the numbers and structures of pseudogenes are different. All of these changes must have occurred since the mammalian radiation, ~85 million years ago (the last point in evolution common to all the mammals).
The comparison makes the general point that gene duplication, rearrangement, and variation is as important a factor in evolution as the slow accumulation of point mutations in individual genes. What types of mechanisms are responsible for gene reorganization?

Unequal crossing-over (also known as nonreciprocal recombination) can occur as the result of pairing between two sites that are not homologous. Usually, recombination involves corresponding sequences of DNA held in exact alignment between the two homologous chromosomes. However, when there are two copies of a gene on each chromosome, an occasional misalignment allows pairing between them. (This requires some of the adjacent regions to go unpaired.) This can happen in a region of short repeats (see Figure 4.1) or in a gene cluster. Figure 4.11 shows that unequal crossing-over in a gene cluster can have two consequences, quantitative and qualitative:
  • The number of repeats increases in one chromosome and decreases in the other. In effect, one recombinant chromosome has a deletion and the other has an insertion. This happens irrespective of the exact location of the crossover. In the figure, the first recombinant has an increase in the number of gene copies from 2 to 3, while the second has a decrease from 2 to 1.
  • If the recombination event occurs within a gene (as opposed to between genes), the result depends on whether the recombining genes are identical or only related. If the noncorresponding gene copies 1 and 2 are entirely homologous, there is no change in the sequence of either gene. However, unequal crossing-over also can occur when the adjacent genes are well related (although the probability is less than when they are identical). In this case, each of the recombinant genes has a sequence that is different from either parent.
Whether the chromosome has a selective advantage or disadvantage will depend on the consequence of any change in the sequence of the gene product as well as on the change in the number of gene copies.
An obstacle to unequal crossing-over is presented by the interrupted structure of the genes. In a case such as the globins, the corresponding exons of adjacent gene copies are likely to be well enough related to support pairing; but the sequences of the introns have diverged appreciably. The restriction of pairing to the exons considerably reduces the continuous length of DNA that can be involved. This lowers the chance of unequal crossing-over. So divergence between introns could enhance the stability of gene clusters by hindering the occurrence of unequal crossing-over.
Thalassemias result from mutations that reduce or prevent synthesis of either α or β globin. The occurrence of unequal crossing-over in the human globin gene clusters is revealed by the nature of certain thalassemias.
Many of the most severe thalassemias result from deletions of part of a cluster. In at least some cases, the ends of the deletion lie in regions that are homologous, which is exactly what would be expected if it had been generated by unequal crossing-over.

Figure 4.12 summarizes the deletions that cause the α-thalassemias. α-thal-1 deletions are long, varying in the location of the left end, with the positions of the right ends located beyond the known genes. They eliminate both the α genes. The α-thal-2 deletions are short and eliminate only one of the two α genes. The L deletion removes 4.2 kb of DNA, including the α2 gene. It probably results from unequal crossing-over, because the ends of the deletion lie in homologous regions, just to the right of the ψα and α2 genes, respectively. The R deletion results from the removal of exactly 3.7 kb of DNA, the precise distance between the α1 and α2 genes. It appears to have been generated by unequal crossing-over between the α1 and α2 genes themselves. This is precisely the situation depicted in Figure 4.11.
Depending on the diploid combination of thalassemic chromosomes, an affected individual may have any number of α chains from zero to three. There are few differences from the wild type (four α genes) in individuals with three or two α genes. But with only one α gene, the excess β chains form the unusual tetramer β4, which causes HbH disease. The complete absence of α genes results in hydrops fetalis, which is fatal at or before birth.
The same unequal crossing-over that generated the thalassemic chromosome should also have generated a chromosome with three α genes. Individuals with such chromosomes have been identified in several populations. In some populations, the frequency of the triple α locus is about the same as that of the single α locus; in others, the triple α genes are much less common than single αgenes. This suggests that (unknown) selective factors operate in different populations to adjust the gene levels.
Variations in the number of α genes are found relatively frequently, which argues that unequal crossing-over in the cluster must be fairly common. It occurs more often in the α cluster than in the β cluster, possibly because the introns in α genes are much shorter, and therefore present less impediment to mispairing between nonhomologous genes.

The deletions that cause β-thalassemias are summarized in Figure 4.13. In some (rare) cases, only the β gene is affected. These have a deletion of 600 bp, extending from the second intron through the 3 flanking regions. In the other cases, more than one gene of the cluster is affected. Many of the deletions are very long, extending from the 5 end indicated on the map for >50 kb toward the right.
The Hb Lepore type provided the classic evidence that deletion can result from unequal crossing-over between linked genes. The β and δ genes differ only ~7% in sequence. Unequal recombination deletes the material between the genes, thus fusing them together (see Figure 4.11). The fused gene produces a single β-like chain that consists of the N-terminal sequence of δ joined to the C-terminal sequence of β.
Several types of Hb Lepore now are known, the difference between them lying in the point of transition from δ to β sequences. So when the δ and β genes pair for unequal crossing-over, the exact point of recombination determines the position at which the switch from δ to β sequence occurs in the amino acid chain.
The reciprocal of this event has been found in the form of Hb anti-Lepore, which is produced by a gene that has the N-terminal part of β and the C-terminal part of δ . The fusion gene lies between normal δ and β genes.
Evidence that unequal crossing-over can occur between more distantly related genes is provided by the identification of Hb Kenya, another fused hemoglobin. This contains the N-terminal sequence of the Aγ gene and the C-terminal sequence of the β gene. The fusion must have resulted from unequal crossing-over between Aγ and β, which differ ~20% in sequence.
From the differences between the globin gene clusters of various mammals, we see that duplication followed (sometimes) by variation has been an important feature in the evolution of each cluster. The human thalassemic deletions demonstrate that unequal crossing-over continues to occur in both globin gene clusters. Each such event generates a duplication as well as the deletion, and we must account for the fate of both recombinant loci in the population. Deletions can also occur (in principle) by recombination between homologous sequences lying on the same chromosome. This does not generate a corresponding duplication.
It is difficult to estimate the natural frequency of these events, because selective forces rapidly adjust the levels of the variant clusters in the population. Generally a contraction in gene number is likely to be deleterious and selected against. However, in some populations, there may be a balancing advantage that maintains the deleted form at a low frequency.
The structures of the present human clusters show several duplications that attest to the importance of such mechanisms. The functional sequences include two α genes coding the same protein, fairly well related β and δ genes, and two almost identical γ genes. These comparatively recent independent duplications have survived in the population, not to mention the more distant duplications that originally generated the various types of globin genes. Other duplications may have given rise to pseudogenes or have been lost. We expect continual duplication and deletion to be a feature of all gene clusters.