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Modern Genetic Analysis Chapter 8. Chromosome Mutations
Chromosomal Rearrangements

Chromosomal rearrangements encompass several different classes of events: deletions, duplications, inversions; and translocations. Each of these events can be caused by breakage of DNA double helices in the genome at two different locations, followed by a rejoining of the broken ends to produce a new chromosomal arrangement of genes, different from the gene order of the chromosomes before they were broken (Figure 8-16a). Consistent with the origin of chromosomal rearrangements by breakage, rearrangements can be induced artificially by using ionizing radiation. This kind of radiation, of which X rays and gamma rays are the most commonly used, is highly energetic and causes numerous double-stranded breaks in DNA.

To understand how chromosomal rearrangements are produced by breakage, several points should be kept in mind:

1. Each chromosome is a single double-stranded DNA molecule.
2. The first event in the production of a chromosomal rearrangement is the generation of two or more double-strand breaks in the chromosomes of a cell.
3. Double-strand breaks in a cell are potentially lethal, unless they are repaired.
4. Repair systems in the cell correct the double-stranded breaks by joining broken ends back together.
5. If the two ends of the same break are rejoined, the original DNA order is restored. If ends of two different breaks are joined together, then a chromosomal rearrangement is produced.
6. However, the only recoverable chromosomal rearrangements are those that produce DNA molecules that have one centromere and two telomeres. If a chromosome lacks a centromere, it will not be dragged to either pole at anaphase of mitosis and meiosis and will end up not being incorporated into the progeny nucleus. Such acentric chromosomes are not inherited. If a chromosome is dicentric (has two centromeres), it will often be simultaneously dragged to opposite poles at anaphase. This will cause an anaphase bridge to form. These anaphase bridge chromosomes will typically not be incorporated into either progeny cell, depending on the organism under consideration. Telo-meres are special DNA sequences at each end of the linear DNA molecule of a eukaryotic chromosome. Telomeres are needed to prime proper DNA replication at the ends. Broken (nontelomeric) ends cannot replicate properly. (This topic will be considered in more detail in Chapter 12.)
7. There cannot be "too large" a segment of DNA lost or duplicated in the rearrangement. If a rearrangement duplicates or deletes a segment of a chromosome, the rules governing gene balance apply. The larger the segment of a chromosome lost or duplicated, the more likely will it cause phenotypic abnormalities.

In organisms with repetitive DNA, homologous repetitive segments within one chromosome or on different chromosomes can act as sites for illegitimate crossing-over. Deletions, duplications, inversions, and translocations can all be produced by such crossing- over (Figure 8-16b); thus crossing-over probably constitutes a significant source of these rearrangements.

There are two general types of rearrangements, balanced and imbalanced. Balanced rearrangements change the chromosomal gene order but do not remove or duplicate any of the DNA of the chromosomes. The two simple classes of balanced rearrangements are inversions and reciprocal translocations.

An inversion is a rearrangement in which an internal segment of a chromosome has been broken twice, flipped 180 degrees, and rejoined:

A reciprocal translocation is a rearrangement in which acentric fragments of two nonhomologous chromosomes trade places, as follows:

Note that, for both inversions and translocations, no chromosomal material is gained or lostthere is simply a change in the relative locations of genes on the rearranged chromosomes. In addition to the effects of the rearrangement itself, it is important to realize that the DNA molecules are disrupted at each of the two breaks that are rejoined abnormally to produce the inversion or the translocation. Sometimes these breaks will occur within genes. When they do, they will generally disrupt gene function. In addition, the DNA sequences on either side of the translocation junction points are not normally juxtaposed. Sometimes the junction occurs in such a way that a novel gene fusion is produced. We shall consider examples of such gene fusions later in this chapter and in Chapter 14.

Imbalanced rearrangements change the gene dosage of a part of the affected chromosomes. As with aneuploidy for whole chromosomes, the loss of one copy or the addition of an extra copy of a segment of a chromosome can disrupt normal gene balance. The two simple classes of imbalanced rearrangements are deletions and duplications. A deletion is the loss of a segment within one chromosome arm and the juxtaposition of the two segments on either side of the deleted segment:

A duplication is a repetition of a segment of a chromosome arm. In the simplest type of duplication, the two segments are adjacent to one another (a tandem duplication):

The following sections consider the properties of these balanced and imbalanced aberrations.

Inversions

Inversions are of two basic types. If the centromere is outside the inversion, then the inversion is said to be paracentric, whereas inversions spanning the centromere are pericentric.

Because inversions are balanced rearrangements, they do not change the overall amount of the genetic material, so they are generally viable and show no particular abnormalities at the phenotypic level. In some cases, one of the chromosome breaks is within a gene of essential function, and then that breakpoint acts as a lethal gene mutation linked to the inversion. In such a case, the inversion could not be bred to homozygosity. However, many inversions can be made homozygous, and, furthermore, inversions can be detected in haploid organisms; so, in these cases, the breakpoint is clearly not in an essential region. Some of the possible consequences of inversion at the DNA level are shown in Figure 8-17.

Most analyses of inversions are carried out on cells that contain one normal haploid chromosome set plus one set carrying the inversion. This type of cell is called an inversion heterozygote, but note that this designation does not imply that any gene locus is heterozygous, but rather the fact that the normal chromosome set and the chromosomal rearrangement are present. Microscopic observation of meioses in inversion heterozygotes reveals the location of the inverted segment because one chromosome twists once at the ends of the inversion to pair with the other, untwisted chromosome; in this way the paired homologs form an inversion loop (Figure 8-18).

At meiosis, crossing-over within the inversion loop of a heterozygous paracentric inversion connects homologous centromeres in a dicentric bridge while also producing an acentric fragmentone without a centromere. Then, as the chromosomes separate during anaphase I, the centromeres remain linked by the bridge. The acentric fragment cannot align itself or move, and consequently it is lost. Tension eventually breaks the dicentric bridge, forming two chromosomes with terminal deletions (Figure 8-19). The gametes containing such deleted chromosomes may be inviable, but, even if viable, the zygotes that they eventually form will probably be inviable. Hence, a crossover event, which normally generates the recombinant class of meiotic products, instead produces lethal products. The overall result is a lower recombinant frequency. In fact, for genes within the inversion, the RF is zero. For genes flanking the inversion, the RF is reduced in proportion to the relative size of the inversion.

Inversions affect recombination in another way, too. Inversion heterozygotes often have mechanical pairing problems in the region of the inversion, which reduces the opportunity for crossing-over in the region.

The net genetic effect of a heterozygous pericentric inversion is the same as that of a paracentriccrossover products are not recoveredbut the reasons are different. In a pericentric inversion, because the centromeres are contained within the inverted region, the chromosomes that have engaged in crossing-over separate in the normal fashion, without the creation of a bridge. However, the crossover produces chromatids that contain a duplication and a deletion for different parts of the chromosome (Figure 8-20). In this case, if a nucleus carrying a crossover chromosome is fertilized, the zygote dies because of its genetic imbalance. Again, the result is the selective recovery of noncrossover chromatids in viable progeny.

Let us consider an example of the effects of an inversion on RF. A wild-type Drosophila specimen from a natural population is crossed with a homozygous laboratory stock dp cn / dp cn. (The dp allele codes for dumpy wings and cn codes for cinnabar eyes. The two genes are known to be 45 map units apart on chromosome 2.) The F1 generation was wild type. An F1 female was backcrossed with the recessive parent and the progeny were:

In this cross, which is effectively a dihybrid testcross, 45 percent of the progeny are expected to be dumpy or cinnabar (they constitute the crossover classes), but only 12/508, about 2 percent, are obtained. Something is reducing crossing-over in this region, and a likely explanation is an inversion spanning most of the dpcn region. Because the expected RF was based on measurements made on laboratory strains, the wild-type fly from nature was the most likely source of the inverted chromosome. Hence the F1 can be represented as follows:

Pericentric inversions also can be detected through new arm ratios. Consider the following pericentric inversion:

Note that the ratio of the long to the short arm has been changed from about 4:1 to about 1:1 by the pericentric inversion. Paracentric inversions do not alter the arm ratio, but they may be detected microscopically if banding or other chromosome landmarks are available.

MESSAGE
The main diagnostic features of heterozygous inversions are inversion loops, reduced recombinant frequency, and reduced fertility from unbalanced or deleted meiotic products.

In some experimental systems, notably the fruit fly, Drosophila, and the nematode, Caenorhabditis elegans, inversions are used as balancers. A balancer chromosome contains multiple inversions, and so, in combination with a wild-type chromosome, there are no viable crossover products. In some analyses, it is important to keep all the alleles on one chromosome together; hence putting them in combination with a balancer achieves this goal. Balancer chromosomes are marked with a dominant morphological mutation. The marker allows the geneticist to track the segregation of the entire balancer or its normal homolog by following the presence or absence of the marker.

Reciprocal Translocations

Of several types of translocations, only the simplest reciprocal type will be illustrated here. Meiosis in heterozygotes having two translocated chromosomes and their normal counterparts causes some important genetic and cytological effects. Again, the pairing affinities of homologous regions dictate a characteristic configuration for chromosomes synapsed in meiosis. Figure 8-21 illustrates meiosis in a reciprocally translocated heterozygote and shows that the pairing configuration is cross shaped. Because the law of independent meiotic assortment is still in force, there are two common patterns of disjunction. The segregation of each of the structurally normal chromosomes with one of the translocated ones (T1·N2 and T2·N1) is called adjacent-1 segregation. Both meiotic products are duplicated and deficient for different arms of the cross. These products are inviable. On the other hand, the two normal chromosomes may segregate together, as will the reciprocal parts of the translocated ones, to produce N1·N2 and T1·T2 products. This segregation is called alternate segregation. These products are viable.

As a result of the equality of adjacent and alternate segregations, half the overall population of gametes will be nonfunctional, a condition known as semisterility or "half sterility." Semisterility is an important diagnostic tool for identifying translocation heterozygotes. However, semisterility is defined differently for plants and animals. In plants, the 50 percent unbalanced meiotic products from the adjacent-1 segregation generally abort at the gametic stage (Figure 8-22). In animals, however, the duplicationdeletion products are viable as gametes but lethal to the zygote.

Remember also that heterozygotes for the other rearrangements such as deletions and inversions may show some reduction of fertility, by an amount dependent on the size of the affected region; but the precise 50 percent reduction in viable gametes or zygotes is usually a reliable diagnostic clue for a translocation.

Genetically, markers on nonhomologous chromosomes appear to be linked if these chromosomes are involved in a translocation and the loci are close to the translocation breakpoint. Figure 8-23 shows a situation in which a translocation heterozygote has been established by crossing an a /a ;b /b individual with a translocation homozygote bearing the wild-type alleles. We shall assume that a and b are close to the translocation breakpoint. On testcrossing the heterozygote, the only viable progeny are those bearing the parental genotypes, so linkage is seen between loci that were originally on different chromosomes. Apparent linkage of genes known to be on separate nonhomologous chromosomessometimes called pseudolinkageis a genetic diagnostic clue to the presence of a translocation.

MESSAGE
Heterozygous reciprocal translocations are diagnosed genetically by semisterility and by the apparent linkage of genes whose normal loci are on separate chromosomes.

Applications of Inversions and Translocations

Translocations and inversions are useful genetic tools; some examples follow.

Gene mapping.

Translocations and inversions are useful for the mapping and subsequent isolation of human genes. The gene for neurofibromatosis was isolated in this way. The critical information came from people who not only had the disease but also carried chromosomal translocations. All the translocations had a breakpoint in chromosome 17, in a band close to the centromere. Hence it appeared that this must be the locus of the neurofibromatosis gene, which had been disrupted by the translocation breakage. Subsequent analysis showed that the chromosome 17 breakpoints were not identical but must have been within the gene, and their positions helped to map the region occupied by the neurofibromatosis gene. Isolation of DNA fragments from this region eventually led to the recovery of the gene itself.

Synthesizing specific duplications or deletions.

Translocations and inversions are routinely used to delete and duplicate specific chromosomal segments. For example, recall that both translocations and pericentric inversions generate products of meiosis that contain a duplication and a deletion. If the dimensions of the parental rearrangement are such that the duplicated segment or the deleted segment is very small, then the duplicationdeletion meiotic products are effectively duplications or deletions. Unidirectional insertional translocations are those in which a segment of one chromosome is inserted into another. In an insertional translocation heterozygote, if an inserted chromosome segregates along with the normal copy, then a duplication results.

Duplications and deletions are useful for a variety of experimental applications, including the mapping of genes by deletion and duplication coverage (see the following sections on deletions and duplications) and the varying of gene dose for the study of regulation.

Position-effect variegation.

Gene action can be affected by proximity to the densely staining chromosomal regions called heterochromatin, and translocations or inversions can be used to study this effect. The locus for white eye color in Drosophila is near the tip of the X chromosome. Consider a translocation in which the tip of an X chromosome carrying w+ is relocated next to the heterochromatic region of, say, chromosome 4. Position-effect variegation is observed in flies that are heterozygotes for such a translocation and that have the normal X chromosome carrying the recessive allele w. The eye phenotype is expected to be red because the wild-type allele is dominant to w. However, in such cases, the observed phenotype is a variegated mixture of red and white eye facets (Figure 8-24). How can we explain the white areas? The w+ allele is not expressed in some cells, because it is occasionally engulfed and inactivated by the margin of the heterochromatin, thereby allowing the expression of w. Position-effect variegation can be used to study the regulatory effects of heterochromatin and thereby the effects of chromosome condensation (coiling), a key feature of chromosome structure.

Deletions

A deletion is simply the loss of a part of one chromosome arm. The process of deletion requires two chromosome breaks to cut out the intervening segment. The deleted fragment has no centromere; consequently, it cannot be pulled to a spindle pole in cell division and will be lost. The effects of deletions depend on their size. A small deletion within a gene, called an intragenic deletion, inactivates the gene and has the same effect as other null mutations of that gene. If the homozygous null phenotype is viable (as, for example, in human albinism), then the homozygous deletion also will be viable. Intragenic deletions can be distinguished from single nucleotide changes because they are nonrevertible.

For most of this section, we shall be dealing with multigenic deletions, which have more severe consequences than do intragenic deletions. If by inbreeding such a deletion is made homozygous (that is, if both homologs have the same deletion), then the combination is always lethal. This fact suggests that most regions of the chromosomes are essential for normal viability and that complete elimination of any segment from the genome is deleterious. Even an individual organism heterozygous for a multigenic deletionthat is, having one normal homolog and one that carries the deletionmay not survive. Principally, this lethal outcome is due to disruption of normal gene balance. Another cause is the expression of deleterious recessive alleles uncovered by the deletion. (Most diploid organisms carry a load of such deleterious alleles.)

MESSAGE
The lethality of large heterozygous deletions can be explained by genome imbalance and expression of deleterious recessives.

Small deletions are sometimes viable in combination with a normal homolog. The deletion may be identified by cytogenetic analysis. If meiotic chromosomes are examined, the region of the deletion can be determined by the failure of the corresponding segment on the normal homolog to pair, resulting in a deletion loop (Figure 8-25a). In dipteran insects, deletion loops are also detected in the polytene chromosomes, in which the homologs are tightly paired and aligned (Figure 8-25b). A deletion can be assigned to a specific chromosome location by determining which chromosome shows the deletion loop, as well as the position of the loop along the chromosome.

Another criterion for inferring the presence of a deletion is that deletion of a segment on one homolog sometimes unmasks recessive alleles present on the other homolog, leading to their unexpected expression. Consider, for example, the deletion shown in the following diagram:

In this case, none of the six recessive alleles is expected to be expressed; but, if b and c are expressed, then a deletion that spans the b+ and c+ genes has probably occurred on the other homolog. Because, in such cases, it seems as if recessive alleles are showing dominance, the effect is called pseudodominance.

The pseudodominance effect can also be used in the opposite direction. A set of defined overlapping deletions is used to locate the map positions of new mutant alleles. This procedure is called deletion mapping. An example from the fruit fly Drosophila is shown in Figure 8-26. In this diagram, the recombination map is shown at the top, marked with distances in map units from the left end. The horizontal bars below the chromosome show the extent of the deletions identified at the left. The mutation prune (pn), for example, shows pseudodominance only with deletion 26438, which determines its location in the 2D-4 to 3A-2 region. However, fa shows pseudodominance with all but two deletions, so its position can be pinpointed to band 3C-7.

MESSAGE
Deletions are recognized by deletion loops and pseudodominance.

Clinicians regularly find deletions in human chromosomes. In most cases, the deletions are relatively small, but they nevertheless have an adverse phenotypic effect, even though heterozygous. Deletions of specific human chromosome regions cause unique syndromes of phenotypic abnormalities. An example is the cri du chat syndrome, caused by a heterozygous deletion of the tip of the short arm of chromosome 5 (Figure 8-27). The specific bands deleted in cri du chat syndrome are 5p15.2 and 5p15.3, the two most distal bands identifiable on 5p. The most characteristic phenotype in the syndrome is the one that gives it its name, the distinctive catlike mewing cries made by infants with this deletion. Other phenotypic manifestations of the syndrome are microencephaly (abnormally small head) and a moonlike face. Like syndromes caused by other deletions, the cri du chat syndrome also includes mental retardation.

Most human deletions, such as those that we have just considered, arise spontaneously in the germ line of a normal parent of an affected person; thus no signs of the deletions are found in the somatic chromosomes of the parents. In rarer cases, deletion-bearing offspring can arise through adjacent segregation of a reciprocal translocation heterozygote or recombination within a pericentric inversion heterozygote. Cri du chat syndrome, for example, can result from a parent heterozygous for a translocation.

Animals and plants show differences regarding survival of deletions. A male animal that is heterozygous for a deletion and a normal chromosome produces functional sperm carrying one or the other of the two chromosomes in approximately equal numbers. In other words, sperm seem to function to some extent regardless of their genetic content. In diploid plants, on the other hand, the pollen produced by a deletion heterozygote is of two types: functional pollen carrying the normal chromosome, and nonfunctional (aborted) pollen carrying the deficient homolog. Thus, pollen cells seem to be sensitive to changes in amount of chromosomal material, and this sensitivity might act to weed out deletions. This effect is analogous to the sensitivity of pollen to whole-chromosome aneuploidy, described earlier in this chapter. Ovules in either diploid or polyploid plants, in contrast, are quite tolerant of deletions, presumably because of the nurturing effect of the surrounding maternal tissues.

Duplications

The processes of chromosome mutation sometimes produce an extra copy of some chromosome region. In considering a haploid organism, we can easily see why such a product is called a duplicationbecause the region is now present in duplicate. The duplicate regions can be located adjacent to each other, called a tandem duplication, or one duplicated region can be in its normal location and the other in a novel location on a different part of the same chromosome or even on another chromosome, called an insertional duplication. In a diploid organism, the chromosome set containing the duplication is generally present together with a standard chromosome set. The cells of such an organism will have three copies of the chromosome region in question, but nevertheless such duplication heterozygotes are generally referred to as duplications because they carry the product of one duplication event. In meiotic prophase, tandem duplication heterozygotes show a loop representing the unpaired extra region.

Synthetic duplications can be used for mapping genes by duplication coverage. For example, in haploids, by crossing to a number of duplicationgenerating rearrangements (for example, translocations and pericentric inversions), various wild-type segments can be added to a genome bearing a new recessive mutant allele "m." If the duplication strain is "m" in phenotype, then the duplication does not span gene m, but, if the strain is wild type, then m must be in that equivalent segment.

© 1999 by W. H. Freeman and Company. All rights reserved.