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5.2.1 The standard karyotype Chromosome number and banding patterns

All of the Mus musculus subspecies (domesticus, musculus, castaneus, and bactrianus) as well as the closely related species M. spretus, M. spicilegus and M. macedonicus have the same "standard karyotype" with 20 pairs of chromosomes, including 19 autosomal pairs and the X and Y sex chromosomes as shown in Figure 5.1. The correct chromosome number was first established by Painter in 1928 (Painter, 1928). Surprisingly, all of the 19 autosomes as well as the X chromosome appear to be telocentric, with a centromere at one end and a telomere at the other. 30 The biological explanation for this uniformity in chromosome morphology is entirely unknown; however, it makes the task of individual chromosome identification much more difficult than it is with human karyotypes. Nevertheless, trained individuals can distinguish chromosomes on the basis of reproducible banding patterns that are accentuated with the use of various staining protocols. The most common of these includes a mild trypsin treatment followed by staining with the dye Giemsa to produce dark Giemsa-stained bands — called G bands — that alternate with Giemsa-negative bands — called R bands for reverse G-bands. A variety of other staining protocols have been developed — called R, Q, and T banding — that are all based on the same principal of chromatin denaturation and/or mild enzymatic digestion followed by staining with a DNA-binding dye (Craig and Bickmore, 1993). In general, all of these different protocols produce the same pattern of bands and interbands observed with Giemsa staining, although in some cases, the dark and light regions are reversed.

The reproducibility of the alternative pattern of G and R bands observed with many different staining protocols implies an underlying difference in the structure of chromatin which, in turn, suggests an underlying heterogeneity in the long-range structure of the genome. In fact, numerous differences have been found in the DNA associated with the two types of bands. G band DNA condenses early, replicates late and is relatively A:T rich; in contrast, R band DNA condenses late, replicates early and is relatively G:C rich (Bickmore and Sumner, 1989). All housekeeping genes are located in R bands, while tissue-specific genes can be located in both G and R bands. Each band type is also associated with a different class of dispersed repetitive DNA elements: G bands contain LINE-1 elements whereas R-bands contain SINE elements (see Section 5.4 for a detailed discussion of these elements).

With all of these contrasting properties, it becomes an interesting problem to distinguish between cause and effect in the generation of the two major types of chromosomal domains. In other words, is there a particular DNA element that defines the G or R bands and somehow contributes to the preferential association, or disassociation, of all other DNA elements that contribute to the characteristics of the band type? Further research will be necessary to unravel this problem. Idiograms and band names

As a mechanism for facilitating data presentation and for comparing results obtained by different investigators, the light and dark bands observed in a raw karyotype are usually converted into idiograms, which are black and white drawings of idealized chromosomes as shown in Figure 5.2. Autosomes are numbered from 1 to 19, in descending order of length. Major bands (alternating dark and light regions) within each autosome are designated with a capital letter starting from A at the centromere, and ascending in alphabetical order. With an increase in resolution, most major bands can be resolved into a series of smaller bands, which are numbered sequentially from 1 starting at the proximal — or centromeric — side of the major band and ending at the distal — or telomeric — side. Finally, when increased resolution allows the visualization of multiple minor bands within a single previously defined sub-band, these are designated with a number (in sequence from 1) demarcated with a decimal point. As an example of the use of this nomenclature, the designation 17E1.3 represents (in reverse order), the third minor band within the first sub-band within the fifth major band (all in order from the centromere) on the mouse chromosome ranked seventeenth in size (illustrated in Figure 7.1). Chromosome length and DNA content

The amount of DNA present in each chromosome can be estimated by measuring its length — cytologically — relative to the sum of the lengths of all 20 chromosomes and multiplying this fraction by the total genome length of 3,000 mb (Evans, 1989). From these measurements, one finds that the largest chromosome (1) has a DNA length of approximately 216 mb and the smallest chromosome (19) has a DNA length of 81 mb, with all others following in a near-continuum between these two values (see Table 9.4 for estimates of the centimorgan lengths of individual chromosomes).

5.2.2 Robertsonian translocations Presence in natural populations

Since 1967, there have been numerous reports of wild-caught house mice with karyotypes containing fewer than 20 sets of chromosomes. The first report described a karyotype with 13 sets of chromosomes (seven metacentrics and 6 telocentrics) in mice captured from the "Valle di Poschiavo" in southeastern Switzerland (Gropp et al., 1972). The assumption was made that animals with such a grossly different karyotype could not possibly be members of the M. musculus species, and as a consequence, these Swiss mice were classified as belonging to a separate species named Mus poschiavinus and informally referred to as the "tobacco mouse." In subsequent years, additional populations of animals from the alpine regions of both Switzerland and Italy were found with a variety of non-standard karyotypes having anywhere from one to nine metacentrics. Further studies of wild house mice by other investigators have led to the discovery of additional non-standard karyotypes in house mice from other regions of Europe as well as South America and Northern Africa (Adolph and Klein, 1981; Wallace, 1981; Searle, 1982).

When the "M. poschiavinus" animals and others with non-standard karyotypes were subjected to a variety of tests — both morphological and genetic — to determine their relatedness to M. m. domesticus, investigators were surprised to find that no characteristics, other than karyotype, distinguished these populations from each other. In particular, phylogenetic studies place the "M. poschiavinus" animals securely within the M. m. domesticus fold; thus the M. poschiavinus species name is inappropriate and should not be used.

How can animals within the same species (and even sub-species) have karyotypes that have diverged apart so radically in what has to be a very short period of evolutionary time? The first point to consider is that the karyotypes are actually not as different from each other as they might appear to be at first glance. When subjected to staining and banding analysis, each arm of every metacentric chromosome uncovered to date has been found to be identical to one of the chromosomes present in the standard M. musculus karyotype. Thus, it would appear that all of the non-standard karyotypes have arisen by simple fusion events, each of which resulted in the attachment of two standard mouse chromosomes at their centromeres. These centromeric fusions, also referred to as whole arm translocations, have been given the formal name of Robertsonian translocations, because W. R. B. Robertson was the first to identify such chromosomes in the grasshopper.

A three part nomenclature is used to describe each individually isolated Robertsonian chromosome. First, the "Rb" symbol indicates a Robertsonian; second, the two chromosomes that have fused together are separated by a dot and listed within parenthesis (with the lower numbered chromosome first); and third, the laboratory number and symbol are indicated. Thus, the twenty-third Robertsonian uncovered at the Institute for Pathology in Lubeck, Germany, that resulted from a fusion between chromosomes 10 and 15, would be designated Rb(10.15)23Lub.

Why does the standard mouse karyotype contain no metacentric chromosomes, and at the same time, why do multiple metacentric chromosomes become fixed so rapidly in unrelated populations from isolated geographical regions? In the latter case, genetic drift alone does not appear to provide a satisfactory answer since metacentric fixation requires an intermediate stage during which animals must be karyotypically heterozygous; heterozygosity for one or more metacentric chromosomes will usually result in decreased fertility as a consequence of non-disjunction. 31 Thus, spontaneously arising Robertsonians cannot be expected to survive in a population (let alone reach fixation) unless they engender a selective advantage to the animal within which they reside. Based on the limited, but scattered, occurrence of populations that contain Robertsonians, it would appear that they can only provide a selective advantage under certain environmental conditions, whereas under other conditions, mice are better served with only telocentric chromosomes. The mechanisms by which such selective pressures would operate on chromosome structure remain totally obscure at the present time. Experimental applications

Robertsonian translocations are useful as genetic tools in two types of experimental applications. Like other translocations, they can catalyze nondisjunction events in heterozygous meiotic cells which — in this case — can lead to the genetic transmission of both homologs of the affected chromosome through individual gametes. This phenomenon will be discussed in the following section. In addition, Robertsonians are especially valuable as visible genetic markers in somatic cells. This usefulness is peculiar to the mouse where all chromosomes other than the Robertsonians will be telocentric. Under microscopic examination, Robertsonians can be easily distinguished as the only chromosomes with two arms.

The advantages of using Robertsonians as genetic markers can be best exploited within animals that contain a single pair of such homologs on a standard karyotypic background. Numerous strains carrying single Robertsonian pairs have been generated through selective breeding between wild and laboratory animals. Each of the standard mouse autosomes is available within the context of a Robertsonian in one or more strains of this type, which can be purchased from the Jackson Laboratory.

The most useful Robertsonians are those with two chromosome arms that differ significantly in length. For example, if one is interested in the analysis of chromosome 2, it would make sense to work with a strain that carries the Rb(2.18)6Rma fusion in which the longer arm of the metacentric (Chr 2) will be easily distinguished under the microscope from the shorter arm (Chr 18).

Robertsonians can be used as somatic markers for both analytical and preparative purposes. Analysis of a particular chromosome by in situ hybridization or other staining protocols is greatly aided by the ability to easily identify the relevant chromosome in all metaphase plates. Preparative microdissection for the purpose of generating subchromosome-specific DNA libraries (Section 8.4) is less tedious and more rapidly accomplished by this easy identification (Röhme et al., 1984). Finally, Robertsonians can be more easily distinguished from all other mouse chromosomes by fluorescence-activated flow sorting (FACS) methods for chromosome identification and purification (Bahary et al., 1992).

5.2.3 Reciprocal translocations Derivation and genetics

Although crossovers normally occur between homologous sequences present on sister chromatids, in rare instances, aberrant crossovers will occur between sequences that are non-allelic. When the two non-allelic sequences that partake in a crossover event of this type come from different chromatids in the same pair of homologs (as illustrated in Figure 5.5), the result is a pair of reciprocal recombinant products that are "unequal" with one having a duplication and the other having a deletion of the material located between the two breakpoints. Intra-chromosomal unequal crossover events are discussed at length in Section 5.3.2.

When crossing over occurs between sequences located on entirely different chromosomes, the result is even more dramatic. As shown in Figure 5.3, inter-chromosomal crossing over results in the production of two reciprocal translocation chromosomes. Although inter-chromosomal crossover events occur even less often than intrachromosomal events, the former are more much readily detected (in most cases) for two reasons. First, reciprocal translocations cause the swapping of entire distal portions of two different chromosomes. Since the portions being swapped are usually not equal in size and are always associated with different banding patterns, each resultant translocation chromosome will usually look quite different from any normal chromosome. So long as the breakpoints are not exceedingly close to the centromeres or telomeres, these aberrant chromosomes will be easily recognized through karyotypic analysis. Second, reciprocal translocations usually cause a significant reduction in fertility as a consequence of the unusual pairing that must occur during synapsis and the production of a high frequency of unbalanced gametes through adjacent-1 segregation discussed in more detail below and illustrated in Figure 5.3. Unbalanced gametes derived from reciprocal translocation heterozygotes give rise to embryos that are partially trisomic or monosomic, and in some cases, these do not survive to birth.

Unlike Robertsonian fusions, reciprocal translocations are not found in wild populations of mice. They can arise spontaneously in laboratory animals and they are recovered at a higher frequency in offspring of males that have been subjected to chemical mutagenesis or irradiation treatment (discussed in Section 6.1). A large number of translocations have been recovered to date (Searle, 1989) and strains homozygous for many can be purchased from the Jackson Laboratory.

As shown in Figure 5.3, translocations will cause genetic linkage between chromosomal regions that assort independently in animals with normal karyotypes. Eva Eicher (1971) was the first to use this correlation between genetic linkage and karyotypic linkage to make a specific chromosomal assignment for a particular linkage group and by the end of the 1970s, all nineteen autosomal linkage groups and chromosomes had been paired together (Miller and Miller, 1975). Higher resolution studies that compared genetic and cytological breakpoint positions provided a means for further mapping of genes to particular chromosome bands; these data also provided a means for determining the centromeric and telomeric ends of each linkage map (Searle, 1989). Chromosome segregation

The main contemporary use of reciprocal translocations is as a tool to generate animals that receive both homologs of a chromosomal region from a single parent. To understand the genetic basis for this outcome, you can follow the process of chromosome segregation during meiosis for the fictitious reciprocal translocation heterozygote shown in Figure 5.3. In this example, mouse chromosomes 2 and 8 have exchanged material.

During the anaphase I stage of the first meiotic division, the two homologs of every chromosome "disjoin" from each other and are pulled to opposite poles by spindles that attach to the centromeric regions. This disjunction of chromosomes is the physical basis for the genetically observed segregation of alleles according to Mendel's first law. In mice with a normal karyotype, the segregation of any one pair of homologs will not affect the segregation of any other pair of homologs. Thus, individual homologs of different chromosomes that came into the animal together from one parent will go out into the offspring in an independent manner. This is the physical basis for Mendel's second law of independent assortment.

In animals with a normal karyotype, chromosome disjunction will always lead to the production of gametes that are "balanced" with a complete haploid genome — no more, no less. However, the same is not true with animals heterozygous for a reciprocal translocation. As shown in Figure 5.3, there are two equally-likely outcomes called "alternate segregation" and "adjacent-1 segregation." With the alternate segregation pathway, one gamete class will receive one chromosome (Chr 2) homolog and one Chr 8 homolog {2,8}, just like all gametes produced by mice with a normal karyotype. The other gamete class will receive both translocated chromosomes called 2' and 8' in this example {2',8'}; although the genetic material is rearranged, one complete haploid genome is present, and thus these gametes are considered to be "balanced". If a balanced {2', 8'} gamete joins together with a normal gamete during fertilization, the resulting animal will be a balanced, reciprocal translocation heterozygote just like the original parent.

With the adjacent-1 segregation pathway, the two gamete classes are unbalanced in a reciprocal fashion. One will have a normal Chr 2 and a translocated Chr 8' {2, 8'}; this gamete is deleted for sequences at the distal end of the normal Chr 8 (8d) and duplicated with both homolog copies of sequences from the distal end of Chr 2 (2d). The other {2',8} will be deleted for distal Chr 2 sequences (2d) and duplicated for distal Chr 8 sequences (8d). Partial trisomies and uniparental disomies

The special consequences of chromosome segregation from reciprocal translocation heterozygotes have been exploited with two types of breeding protocols. In the first, translocation heterozygotes are bred to animals with a normal karyotype. Adjacent-1 segregation will give rise to animals that are partially trisomic (for the distal end of one translocated chromosome) and partially monosomic (for the distal end of the other). Thus, by choosing appropriate translocations, it becomes possible to construct animals that are deleted or duplicated for particular genes of interest. By breeding in mutations at these loci, it becomes possible to construct genotypes of the {+/+/m} and {+/m/m} variety (where + and m are wild-type and mutant alleles respectively) as a means toward a better understanding of gene dosage effects and dominance and recessive relationships (Agulnik et al., 1991; Ruvinsky et al., 1991).

In the second type of breeding protocol, animals heterozygous for the same pair of reciprocal translocations are mated to each other. The most interesting offspring to emerge from such unions are those formed through the fusion of complementary unbalanced gametes that represent the two different products of adjacent-1 segregation (Figure 5.3). Although the resulting zygotes have fully balanced genomes — they are not deleted nor duplicated for any sequences — they carry two subchromosomal regions in which both homologs came from only one or the other parent respectively. In other words, for one chromosomal region, these animals are maternally disomic and paternally nullisomic; for the other chromosomal region, the opposite holds true.

Uniparental disomy can also be obtained, albeit with lower frequency, in the offspring of matings between animals heterozygous for the same Robertsonian translocation. Pairing between the Robertsonian and the homologous acrocentric chromosomes can lead to non-disjunction with gametes that contain either two copies or no copies of one homolog represented within the Robertsonian. Once again, the fusion of two complementary nondisjunction gametes will lead to zygotes with fully balanced genomes but with whole chromosome uniparental disomy. Both whole and partial chromosome disomy provide powerful genetic tools for the analysis of genomic imprinting which is discussed later in this chapter (Cattanach and Kirk, 1985).

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