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10.2.1 Conservation of synteny

As cloning and mapping of both the mouse and human genomes began in earnest during the 1980s, two important evolutionary facts became clear. First, nearly all human genes have homologs in the mouse and vice versa. Second, not only are the genes themselves conserved, but so is their order — to a certain extent — along the chromosome. In 1984, Nadeau and Taylor used linkage data obtained from 83 loci that had been mapped in both species to estimate the average length of conserved autosomal segments as 8.2 cM in the mouse (Nadeau, 1984). In 1993, the same analysis was performed on linkage data obtained from 917 homologous loci mapped in both species to yield an average conserved chromosomal length of 8.8 cM which is not significantly different from the earlier estimate. 104 The major evolutionary implication of this result is that approximately 150 major rearrangements have occurred along the human or mouse lines as they diverged from a common ancestor that existed 65 million years ago.

The practical implication of conserved chromosomal segments is that the mapping of a gene in one species can provide a clue to the location of its homolog in the other species. One should be cautious, however, in not overinterpreting synteny information. There are many examples of smaller genomic segments that have popped out or into larger syntenic regions. Thus, even if a human gene maps between two human loci with demonstrated synteny in the mouse as well, there is still a small chance that it will have moved to another location in the mouse genome. Nevertheless, over 80% of the autosomal genomes of mice and humans have now been matched up at the subchromosomal level (Copeland et al., 1993). Thus, with map information for a gene in humans, it will often be possible to identify a corresponding mouse chromosomal segment of ~10 cM in length as a likely location to test first for linkage with nearby DNA markers.

10.2.2 In situ hybridization Overview

The technique of in situ hybridization was conceived of by Gall and Pardue (1969) and John and his colleagues (1969). These workers demonstrated that the DNA within preparations of chromosomes attached to microscope slides could be denatured in a gentle manner so as not to disrupt the overall morphology of the chromosomes themselves. Target sequences within these chromosomes are then available for hybridization to labeled nucleic acid probes. Thus, in situ hybridization allows the mapping of cloned DNA sequences to specific chromosomal sites that can be visualized directly by light microscopy.

In early work, probes were labeled with radioactive isotopes and target sequences were identified by autoradiography. This method of labeling and detection limited both the sensitivity of the technique and its resolution (Lawrence, 1990). In particular, the original protocol only allowed the detection of tandemly repeated sequences such as the ribosomal genes and satellite DNA. By 1981, however, investigators had optimized the in situ protocol for use in mapping single copy mammalian sequences (Harper et al., 1981), and in 1984, an improved method was developed for better resolution of chromosome banding patterns (Cannizzarro and Emanuel, 1984). Nevertheless, the technique was still not ideal because with single-copy radioactive probes, localization could not be determined within the chromosomes of a single cell; instead, it was necessary to perform a statistical analysis of silver grain distributions in 50-100 sets of metaphase chromosomes.

Two critical changes in the protocol now allow the detection of single-copy sequences and their high-resolution mapping through the direct observation of single chromosomes. The first change was in the nature of the label; with the substitution of fluorescent tags for radioactive ones, the physical resolution of the hybridization site was dramatically improved. The modified in situ protocol that utilizes fluorescent tags is referred to as FISH (for fluorescent in situ hybridization). The second change was in the nature of the hybridization cocktail. With the inclusion of a large excess of unlabeled total genomic DNA, it is possible to block dispersed repetitive sequences — present in essentially every genomic region larger than a few kilobases in length — from hybridization to their targets throughout the genome. This allows the use of whole-phage or cosmid clones as probes leading to a substantial increase in signal strength which will be proportional to the length of single-copy DNA in the clone. With these major changes in the protocol and other optimizations, it is now possible to use in situ hybridization to visualize the map position of any cloned locus within single chromosomes from any mammalian species (Lawrence, 1990; Trask, 1991).

Although in situ hybridization has played a pivotal role in the construction of the human gene map, its role in mouse gene mapping has been more limited for several reasons. First, a certain amount of specialized training and experience is required to perform this protocol, and thus, it is often not an option for independent investigators in the absence of a collaboration. Second, in humans, classical linkage analysis is not easily performed, and thus, alternative methods for human mapping are much more important. Third, whereas the human karyotype is highly amenable to direct cytogenetic analysis — chromosomes come in a variety of shapes and sizes and staining techniques yield excellent banding resolution — the mouse karyotype is a cytologist's nightmare. All 20 chromosomes have the same shape with only a single visible arm and a centromere that appears to lie at one end (see Figure 5.1). A continuum of chromosome lengths makes the identification of individual chromosomes more difficult, and finally, banding patterns are much less distinct and more difficult to resolve.

In the past, in situ hybridization had the advantage that it did not require the existence of variants between parental strains for mapping to be accomplished. However, with the advent of new methods for the detection of polymorphism discussed in Chapter 8, it has become possible to quickly identify DNA variants at essentially all cloned loci. Consequently, in situ hybridization is now used most often only for specific experimental problems such as those that described below. Experimental usage

The power of in situ hybridization lies in the fact that it allows the direct localization of DNA sequences relative to all visible cytological landmarks such as centromeres, telomeres, and rearrangement breakpoints in aberrant chromosomes. In some instances, it will be important to localize a DNA marker relative to one or more of these landmarks. For example, an investigator may have a DNA marker that maps to the beginning or end of a linkage map associated with a particular chromosome. In situ hybridization can be used to determine how close to the centromere or telomere the DNA marker actually is (in physical terms); this information can serve to establish the size of the chromosomal region that is not contained within the associated linkage map. In another example, an investigator may have a DNA clone, from either a wild-type or mutant animal, that is believed to extend across a cytologically visible inversion or translocation breakpoint. If the clone was derived from a wild-type genome, the in situ results would show hybridization to two sites in the rearranged karyotype.

As discussed in Section 7.3.2, in situ hybridization is also useful in the special case of mapping transgene insertion sites. The same DNA construct that was originally injected into the embryo can often be used directly as a probe. In another instance, in situ hybridization can be combined with classical linkage analysis using the M. spretus backcross system to follow the segregation of centromeres from one parental chromosome or the other as described in Section 9.1.2 (Matsuda and Chapman, 1991; Matsuda et al., 1993). Finally, in situ hybridization is useful in experiments aimed at questions that go beyond the simple mapping of genes. For example, the technology has revealed the unexpected finding that both LINE and SINE sequences are non-randomly distributed among bands and interband regions of all chromosomes as described in Section 5.4.4.

10.2.3 Somatic cell hybrid genetics Overview of the classical approach

The ability to derive long-term cultures of mammalian cells was perfected during the 1950s. Cell cultures provided important experimental material for early biochemists and molecular biologists interested in molecules and processes that occur within mammalian cells, but they were of little use to geneticists since somatic cell genomes remain essentially unchanged during continual renewal through mitotic division. This situation changed dramatically during the early 1960s when investigators discovered and developed methods for the induction of cell fusion in culture (Ephrussi and Weiss, 1969).

Normal diploid cells from all species of mammals carry approximately the same amount of DNA in their nucleus (twice the haploid amount of 3,000 mb). Thus, after fusion between any two mammalian cells, the hybrid cell nucleus becomes, in effect, tetraploid, with a genome that is twice the normal size. The enlarged genomes of hybrid cells are inherently unstable. Presumably, the increased requirement for DNA replication acts to slow down the rate of cell division, and as a consequence, cells that lose chromosomes during mitotic segregation will divide more quickly and outgrow those cells that maintain a larger genome content. Eventually, after many events of this type, cells can reach a relatively stable genome size that is close to that normally found in diploid mammalian cells. For reasons that are not understood, hybrids formed between particular combinations of species will preferentially eliminate chromosomes from just one of the parental lines. In hybrids formed between mouse cells and either hamster or human cells, mouse chromosomes will be eliminated in a relatively random manner. This process has allowed the derivation and characterization of a number of somatic cell hybrid lines that stably maintain only one or a few mouse chromosomes.

The field of somatic cell genetics had its heyday in the 1970s and early 1980s when it provided the predominant methodology for mapping loci — albeit, often to the resolution of whole chromosomes. The major tools for gene detection in this era (before the recombinant DNA revolution was in full gear) were species-specific assays for various housekeeping enzymes. Somatic cell geneticists could type each member of a panel of hybrid cells for the presence of a particular enzyme and then use karyotypic analysis to demonstrate concordance with a particular chromosome. In a strictly formal sense, this type of analysis is analogous to classical two-locus linkage studies studies with one marker being the enzymatic activity and the other marker being the particular chromosome that contains the gene encoding the enzyme.

The somatic cell hybrid approach has always been more important to human geneticists than to mouse geneticists. This is because well-established somatic cell hybrid lines with one or a few mouse chromosomes are relatively rare compared to the large number of well-characterized hybrid lines with individual human chromosomes. There are several reasons for this state of affairs. First, the power of mouse linkage mapping has always been so great that somatic cell hybrid lines were never considered to be essential tools. Second, most mouse/hamster hybrid lines are chromosomally unstable and must be recharacterized each time they are grown in culture. With the difficulty of performing karyotypic analysis on mouse chromosomes, most investigators have shied away from this approach in the past. However, with alternative PCR-based methods for characterizing the chromosomal content of hybrids (Abbott, 1992), this problem may have been overcome so that the derivation of new hybrids for special situations may no longer be as formidable as it once was.

The use of somatic cell hybrid panels as a general approach to gene mapping has now been superseded by in situ hybridization — which resolves map positions to chromosome bands rather than whole chromosomes — and, of course, classical linkage analysis. However, there are two special cases where somatic cell hybrid lines can provide unique tools for mouse geneticists. First, their DNA can be used as a source of material for the rapid derivation of panels of DNA markers to saturate particular chromosomes or subchromosomal regions as described in Section 8.4.4 (Herman et al., 1991; Simmler et al., 1991). Second, their DNA can also be used for the rapid screening of new clones obtained from other sources for their presence in a particular interval of interest as described in Section 7.3.3. This can be accomplished with the use of duplicate blots containing just three lanes of restriction digested and fractionated DNA from: (1) the somatic cell hybrid line containing the chromosome of interest; (2) mouse tissue (a positive control); and (3) the host cell line without mouse chromosomes (a negative control). Each blot can be subjected to repeated probing with different potential markers. A negative result allows one to discard a particular probe immediately; a positive result can be followed-up by higher resolution linkage analysis. Radiation hybrid analysis

In 1990, Cox, Meyers, and their colleagues described a novel technique for determining gene order and distance which is as highly resolving as traditional linkage analysis but does not depend upon breeding. The approach used has similarities to, as well as differences from, both recombinational mapping and physical mapping. Radiation hybrid mapping was originally developed for use with the human genome, but with appropriate starting material and a sufficient number of chromosome-specific DNA markers, it can be used in the analysis of any species (Cox et al., 1990).

The starting material is a somatic cell hybrid line that contains only the chromosome of interest within a host background derived from another species. As indicated above, a common host species used for mouse chromosomes is the hamster. A well-established, stable hamster cell hybrid line containing a single mouse chromosome can be subjected to irradiation with X-rays that shatter each chromosome into multiple fragments. The irradiated cells are then placed together with pure hamster cells under conditions that promote fusion. Approximately 100 new hybrid clones are recovered that contain fragments of the mouse chromosome present in the original hybrid line. Finally, each of these lines are analyzed for the presence of various DNA markers that had been mapped previously into the chromosomal region of interest.

The order and distance of loci from each other can be determined according to the premise that X-rays will break the chromosome at random locations. Thus, the closer two loci are together, the less likely it is that a break will occur between them. If two loci are side by side, they will either both be present or both be absent from all 100 cells with 100% concordance. If two loci are at opposite ends of the chromosome, there will still be cells that have neither or both, but there will also be a large number that have only one or the other. (A cell can carry both loci even if the frequency of breakage between the two is 100% since it is possible for a hybrid cell to pick-up more than one chromosomal fragment.) As the probability of chromosome breakage varies between 0% and 100% for various pairs of loci under analysis, the fraction of hybrid cells that carry both loci will vary from 100% down to a control value obtained for unlinked loci. Thus, by typing each of the "radiation hybrid cells" in the set of 100 for a series of DNA markers, it becomes possible to construct a linkage map that is highly analogous to traditional recombinational maps.

It is possible to obtain linkage maps at different levels of resolution through the use of different intensities of radiation to break chromosomes. For example, with high levels of radiation that break chromosomes once every 100 kb, on average, one could map loci from 10 kb to 500 kb; with lower levels of radiation, mapping could be performed over a window from 500 kb to 5 mb.

The analogy to classical recombination mapping is striking in that a determination of linkage distance in both cases is based on the probability with which chromosomes will break followed either by recombination (in the classical case) or by segregation upon cell fusion (in the radiation hybrid case). In both cases, linkage distances are determined by counting the ratio of offspring (pups or cells) that do or do not carry particular sets of DNA markers (alleles or genes). However, linkage distances obtained through radiation hybrid analysis are much more likely to be indicative of actual physical distances.

Although radiation hybrid analysis has provided a crucial tool for genetic analysis in humans, once again, it has not been as widely used by the mouse community because classical linkage analysis is so much more powerful. Nevertheless, the resolution of this protocol has been validated in a study of the region of mouse chromosome 2 surrounding the agouti locus (Ollmann et al., 1992). In this study, the radiation hybrid map that was obtained corresponded exactly with that predicted from linkage analysis, with a level of resolution that was approximately 40-fold higher. Thus, radiation hybrid mapping could serve to fill in the gap between linkage maps and physical maps, especially in "cold" regions between hotspots where distantly spaced markers cannot be separated by recombination (Section 7.2.3).

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