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Modern Genetic Analysis Chapter 12. Genomics
Structural Genomics

Structural genomics depends heavily on a knowledge of DNA sequence organization in the genome, so we turn to this topic first.

Sequence Organization

In Chapter 2, genomic DNA was classified simply into two general types, single-copy genes and repetitive DNA. However, within the repetitive class, there are distinct subclasses. A more complete classification of eukaryotic DNA is shown in Figure 12-2, and we shall consider each class in turn.

Functional repetitive sequences.

Some functional genetic units are present in multiple copies. They include the following classes.

Dispersed Gene Families.

Many protein-coding genes belong to families of related genes spread throughout the genome. Such families may comprise only a few genes or very many. For example, the protein ovalbumin is encoded by a family of three genes, whereas histones are encoded by families ranging between 100 and 1000 genes. The exact DNA sequences of the genes within a family may diverge, as have, for example, the genes for the human hemoglobin family that we studied in Chapter 8, and the different homologous genes may come to have slightly different functions. Some genes within families have become nonfunctional, giving rise to untranscribed pseudogenes, as illustrated in Figure 12-3.

Tandem Gene-Family Arrays.

Cells need large amounts of the products of some genes, such as those that encode transcriptional components, and such genes have evolved families of tandem arrays. A good example is seen in the nucleolar organizer (NO), which is the site of tandem repeats of ribosomal RNA. In Drosophila, the NOs on the X and Y chromosomes contain 250 and 150 tandem copies of rRNA genes, respectively. One human NO has about 250 copies.

Another example of a tandem array is afforded by the genes for tRNA. In humans, there are about 50 chromosomal sites corresponding to the different tRNA types, and each site comprises between 10 and 100 copies.

Finally, the genes for histones are arranged in tandem arrays in some species (Figure 12-4). For histone arrays, as for the other arrays of tandem repeats, the multiple copies are identical. One might expect that mutation would lead to divergence between the copies, but there seems to be a mechanism for maintaining constancy across the members of the array.

Noncoding Functional Sequences.

Telomeres (the tips of chromosomes) have tandem arrays of simple DNA sequences that do not code for an RNA or a protein product but nevertheless have a definite function. For example, in the ciliate Tetrahymena, there is repetition of the sequence of TTGGGG, and, in humans, it is TTAGGG. The telomeric repeats are there to solve a functional problem that is inherent in the replication of linear DNA molecules. Figure 12-5 on the following page illustrates the problem: for the leading strand, the polynucleotide addition in the course of replication can always extend to the end because it is automatically primed from behind. However, at the tip, the lagging strand reaches a point where its system of RNA priming cannot work, and an unpolymerized section remains; the result would be chromosome shortening. However, an enzyme called telomerase adds the simple repeat units to the ends. The telomerase protein is a type of reverse transcriptase. It carries a small RNA molecule, part of which acts as a template for the polymerization of the telomeric repeat unit that is added to the 3 end. For example, in Tetrahymena, the RNA is 3-AACCCC-5, which acts as the template for the 5-TTGGGG-3 repeat unit (Figure 12-6). The additional DNA is able to act as a template for synthesis on the lagging strand. Figure 12-7 demonstrates the positions of the telomeric DNA through in situ hybridization. An age-dependent decline in telomere length has been found in several human somatic tissues. In addition, human fibroblasts in culture show progressive telo-mere shortening until their eventual death. Such observations have led to the telomere theory of aging, and the validity of this theory is now under test.

Sequences with no known function.

For some repetitive DNA, no function is known. This category contains DNA sequences that generally have many more copies in the genome than do the functional gene families. The combined size of this class is surprising; for example, it has been calculated that about 20 percent of the human genome consists of nonfunctional repetitive sequences of one kind or another. We shall consider three types.

Highly Repetitive DNA Flanking the Centromere.

When genomic DNA is centrifuged in a cesium chloride density gradient, the "satellite" band is often visible at a position different from that of the main DNA band. On isolation, such satellite DNA is found to consist of multiple tandem repeats of short nucleotide sequences, stretching to hundreds of kilobases in length. When probes are prepared from such simple-sequence DNA and used in chromosomal in situ labeling experiments (considered later in this chapter), the great bulk of the satellite DNA is found to reside in the regions of heterochromatin, densely staining, highly compact regions flanking the centromeres (Figure 12-8). There can be either one or several basic repeating units, but usually they are less than 10 bases long. For example, in Drosophila melanogaster, the sequence AATAACATAG is found in tandem arrays around all centromeres. Similarly, in guinea pigs, tandem arrays of the shorter sequence CCCTAA flank the centromeres. In situ labeling of a mouse satellite DNA is shown in Figure 12-9.

Chromosome-mapping experiments in Drosophila show that most of the active genes that are detectable through mutation are located in euchromatin, the nonheterochromatic areas. Euchromatin stains less densely because it is packed less tightly, and the general idea is that this is the state most compatible with transcription and gene activity. The proportion of Drosophila heterochromatin that maps as genes is only 1/100 of the proportion of euchromatin that maps as genes: there is clearly a major distinction in the gene activity of the two kinds of chromatin.

Because the centromeric repeats are repeats of small segments of DNA, they constitute a nonrepresentative sample of the genomic DNA and consequently the nucleotide proportions can be significantly different from the rest of the DNA. This is why the DNA forms a separate satellite band in a cesium chloride gradient. There is no demonstrable function for centromeric repetitive DNA, nor is there any understanding of its relation to heterochromatin or to genes in the heterochromatin. Some organisms have staggering amounts of this DNA; for example, as much as 50 percent of kangaroo DNA can be centromeric satellite DNA.

VNTRs.

A special class of tandem repeats shows variable number at different loci and in different individual organisms. These tandem repeats are called VNTRs or variable number tandem repeats, sometimes referred to as minisatellite DNA. The VNTR loci in humans are 1- to 5-kb sequences consisting of variable numbers of a repeating unit from 15 to 100 nucleotides long. If a VNTR probe is available and the total genomic DNA is cut (say) with a restriction enzyme that has no target sites within the VNTR arrays, then a Southern blot will reveal a large number of different-sized fragments that are bound by the probe. Because of the variability in the number of tandem repeats from person to person, the set of fragments that shows up on the Southern autoradiogram is highly individualistic In fact, these patterns are called DNA fingerprints (Figure 12-10). They are used extensively in forensic medicine. In a criminal investigation, blood or semen can be used to prepare DNA fingerprints, and the patterns are compared with those of a suspect. Minute samples of tissue such as follicle cells clinging to single hairs can be used by amplifying specific VNTR sequences through the polymerase chain reaction.

One of the first probes that detected human VNTRs was obtained from a quadruple repeat of a 33-bp sequence found within the first intron of the myoglobin gene (Figure 12-11a). When some of the VNTRs in the DNA fingerprint were cloned and sequenced, the reason for hybridization of the probe to the VNTRs was a 13-bp core sequence common to all the repeats and to the probe. The longer sequences into which the core was embedded were not necessarily similar. DNA fingerprints can be made in many different species of organisms, so VNTRs must be common, and the technique has great value in testing genetic individuality.

Another class of dispersed repetitive DNA is composed of dinucleotide repeats. This class of repetitive DNA is called microsatellite DNA. Although not normally included in the VNTR class, microsatellites are in fact dispersed regions composed of variable numbers of dinucleotides repeated in tandem. This type of DNA has been most useful in providing a dense array of molecular markers for mapping the huge expanses of the human and other genomes, as we shall see later.

Transposed Sequences.

A large proportion of the eukaryotic genome is composed of repetitive elements that have propagated within the genome by making copies of themselves, which can move into new locations. These repetitive elements are collectively called transposed sequences, and they are described in detail in Chapter 13. Those elements that move as DNA are called transposons. In Chapter 13, we shall consider examples of transposons in bacteria, corn, and Drosophila. Many genomes have multiple copies of such elements or truncated versions of them dispersed throughout the genome.

Another general class of transposed DNA sequences, called retrotransposons, propagate themselves through the action of reverse transcriptase, an enzyme that can make a DNA strand from RNA. One type within this category comprises repetitive sequences whose structures are similar to those of retroviruses (which replicate by reverse transcriptase) and may be derived from such viruses. They move by reverse transcription of their RNA transcripts into DNA, which then inserts throughout the genome. Examples of retroviral-like retrotransposons are the copia elements of Drosophila (5-kb sequences present at about 50 copies per genome), the Ty elements of yeast (6-kb elements with about 30 full-length copies per genome), and the long interpsersed elements, or LINES, of mammals (1- to 5-kb elements present in 20,000 to 40,000 copies per human genome. Figure 12-12 shows the general structures of several retrotransposons.

The Alu repetitive sequence in the human genome, so named because it contains a target site for the Alu restriction enzyme, is an example of a class of retrotransposons that are not similar to retroviruses. The human genome contains hundreds of thousands of whole and partial Alu sequences, scattered between genes and within introns and making up about 5 percent of human DNA. The full Alu sequence is about 200 nucleotides long and bears remarkable resemblance to 7SL RNA, an RNA that is part of a complex by which newly synthesized polypeptides are secreted through the endoplasmic reticulum. Presumably, the Alu sequences originate as reverse transcripts of these RNA molecules. Short interspersed repeats such as Alu sequences are collectively called SINEs, for short interspersed elements.

Other examples of this class of moderately repetitive elements are the many scattered pseudogenes that have clearly been created by the reverse transcription process, because they do not contain the introns that are found in the original functional gene.

The existence of DNA with no known function has presented a dilemma for geneticists. Previous ideas on the power of natural selection would have predicted that nonfunctional DNA should be purged by selection. It might be imagined that nonfunctional DNA is a genetic burden if only because of the additional energy that the organism needs to put into its synthesis. However, this notion seems inadequate. Perhaps the seemingly functionless DNA does have a function, possibly as a kind of genetic ballast that gives the chromosome bulk to permit efficient partitioning at cell division or perhaps to separate the functional elements (genes) for their efficient regulation. Alternatively, the repetitive elements that make up most of the nonfunctional DNA might have hit upon a way of avoiding detection by the forces of natural selection. Such DNA has been termed selfish DNA, a type of DNA that exists only for the purpose of existing and is never exposed to the rigors of phenotype.

Figure 12-13 is a stylized diagram of the overall organization of a hypothetical eukaryotic chromosome, summarizing much of the preceding discussion. There is considerable variation between species in the amounts of nonfunctional repetitive DNA; for example, there is little in yeasts and other fungi.

Some idea of the profusion of repetitive elements in the human genome can be obtained from Figure 12-14, which shows the distribution of repetitive elements in the gene for homogentisate 1,2-dioxygenase [the alkaptonuria gene (AKU or HGO), introduced in Chapter 10].

Spacer DNA.

The final category of DNA is spacer DNA, which is basically what is left after all the recognizable units have been identified. Needless to say, little is known about spacer DNA. Possibly its only function is to space, but no studies have yet been performed to delete such DNA and observe the consequences. Spacer DNA might be a category that represents our current ignorance: there may be no such thing as spacer DNA.

MESSAGE
Single-copy genes are embedded in a complex array of tandem and dispersed types of repetitive DNA, most of which have no known function.

We now return to the increasing levels of resolution of genomic analysis, starting with assigning genes and markers to chromosomes, then mapping these within a chromosome, and finally preparing a physical map culminating in sequencing.

Assigning Loci to Specific Chromosomes

Several different methods are useful in assigning genes or markers to individual chromosomes. Some of them have already been covered elsewhere in this book and are included here for completeness.

Linkage to known loci.

In well-studied organisms, it is a simple matter to cross a strain carrying the "new" unmapped allele with a set of strains carrying markers spread throughout the genome, each one of known chromosomal location. Meiotic recombinant frequencies of less than 50 percent indicate that the unmapped allele and the marker are linked and therefore reside on the same chromosome. Often such linkage data give a rough idea of chromosomal position, perhaps in a specific chromosomal arm or even a specific band.

Pulsed field gel electrophoresis.

If chromosomes are small enough to be separated by pulsed field gel electrophoresis (PFGE; Figure 12-15), the DNA bands on the gel can be used to locate new genes by hybridization. First, correlations must be made to establish which DNA band corresponds to which chromosome. Chromosome size, translocations between known chromosomes, and hybridization to probes of known location are useful for this purpose. Then a new cloned gene can be used as a probe in a Southern blot of the PFGE gel; hence its chromosomal locus can be determined.

Humanrodent somatic cell hybrids.

The technique of somatic cell hybridization is used extensively in human genome mapping, but it can in principle be used in many different animal systems. The procedure uses cells growing in culture. A virus called the Sendai virus has a useful property that makes the mapping technique possible. Each Sendai virus has several points of attachment, so it can simultaneously attach to two different cells if they happen to be close together. The membranes of the two cells may fuse together and the two cells become onea binucleate heterokaryon.

If suspensions of human and mouse cells are mixed together in the presence of Sendai viruses that have been inactivated by ultraviolet light, the virus mediates fusion of the cells from the different species (Figure 12-16). When the cells fuse, the nuclei also subsequently fuse to form a uninucleate cell line composed of both a human and a mouse chromosome set. Because the mouse and human chromosomes are recognizably different in number and shape, the two sets can be readily distinguished in the hybrid cells. However, in the course of subsequent cell divisions, for unknown reasons, the human chromosomes are gradually eliminated from the hybrid at random.

The loss of human chromosomes can be arrested to encourage the formation of a stable partial hybrid in the following way. The mouse cell line used is mutant for a specific biochemical function; so, if the cells are to grow, the missing function must be supplied by the human genome. This selective technique results in the maintenance of hybrid cells that have a complete set of mouse chromosomes and a small number of human chromosomes, which vary in number and type from hybrid to hybrid but which always include the human chromosome carrying the wild-type allele defective in the mouse genome.

Let's look at the specific genes that make the selective system work. In cells, DNA can be made either de novo ("from scratch") or through a salvage pathway that uses molecular skeletons already available. The selective technique requires the application of a chemical, aminopterin, that blocks the de novo synthetic pathway, confining DNA synthesis to the salvage pathway. Two essential salvage enzymes are relevant to the system, as shown in the following two reactions:

The mouse cell line to be fused is genetically unable to make TK because it is homozygous for the allele tk, whereas the human cell line is genetically unable to make HGPRT because it is homozygous at another locus for the allele hgprt. So the genotypes of the two fusing cell lines are

Because each is deficient for one enzyme, neither the mouse nor the human cells are able to make DNA individually. In the hybrid cells, however, the tk+ allele complements the hgprt+ allele, so the cells can make both enzymes. Therefore, DNA is synthesized and the cells can proliferate. Most human chromosomes are eliminated from the hybrid cell cultures because their loss has no effect on the cultures' ability to grow. But, to continue to grow in medium containing hypoxanthine, aminopterin, and thymidine (HAT medium), a hybrid culture must retain at least one of the human chromosomes that carries the tk+ allele.

Luckily, the progressive elimination of the human chromosomes from the fused cell lines can be followed under the microscope because mouse chromosomes can be easily distinguished from human chromosomes. Different hybrid cells are grown separately into lines, and eventually a bank of lines is produced that contains, in total, all the human chromosomes.

With a complete bank of chromosome-specific cell lines, human genes or markers can be assigned to chromosomes. If the human chromosome set is homozygous for a human molecular marker, such as an allele that controls a cell-surface antigen, drug resistance, a nutritional requirement, a specific protein, or a DNA marker, then the presence or absence of this genetic marker in each line of hybrid cells can be correlated with the presence or absence of certain human chromosomes in each line. Data of this sort are presented in Table 12-1, in which a plus sign means the presence and a minus sign means the absence of the genetic marker. We can see that, in the different hybrid cell lines, genetic markers 1 and 3 are always present or absent together. We can conclude, then, that they are linked. Furthermore, the presence or absence of genes 1 and 3 is correlated with the presence or absence of chromosome 2, so we can assume that these genes are located on chromosome 2. By the same reasoning, gene 2 must be on chromosome 1, but the location of gene 4 cannot be assigned. Large numbers of human genes have now been localized to specific chromosomes in this way.

High-Resolution Chromosome Maps

The next level of increasing resolution is to determine the position of a gene or molecular marker on the chromosome. This step is important because the genetic maps that are produced will be aligned with the physical maps that we shall consider in the next section and can be used to validate the physical maps. In addition, the clones generated as parts of the physical map can be used to help identify the genomic DNA corresponding to the genes on the genetic map. Several different methods are used in localizing genes or markers.

Meiotic mapping by recombination.

Meiotic linkage mapping used in genomics is based on the principles of mapping covered in Chapter 5; in other words, on analyzing recombinant frequency in dihybrid and multihybrid crosses. In experimental organisms such as yeast, Neurospora, Drosophila, and Arabidopsis, the genes that determine qualitative phenotypic differences can be mapped in a straightforward way because of the ease with which controlled experimental crosses (such as testcrosses) can be made. Therefore, in these organisms, the chromosome maps built over the years appear full of genes with known phenotypic effect, all mapped to their respective loci.

This is not the case for humans, because, first, there is a lack of informative crosses. Second, progeny sample sizes are too small for accurate statistical determination of linkage. Third, the human genome is enormous. In fact, it was a difficult task even to assign a human disease gene to an individual autosome by linkage analysis. (Most genes with known phenotypes were assigned not by RF analysis but by human rodent cell hybrid mapping.)

Even in those organisms in which the maps appeared to be "full" of loci of known phenotypic effect, measurements showed that the chromosomal intervals between genes had to contain vast amounts of DNA. These intervals could not be mapped by linkage analysis, because there were no markers in those regions. What was needed was large numbers of additional genetic markers that could be used to fill in the gaps to provide a higher-resolution map. This need was met by the discovery of various kinds of molecular markers. A molecular marker is a site of heterozygosity for some type of neutral DNA variation. Neutral variation is that which is not associated with any measurable phenotypic variation. Such a "DNA locus," when heterozygous, can be used in mapping analysis just as with a conventional heterozygous allele pair. Because molecular markers can be easily detected and are so numerous in a genome, when they are mapped by linkage analysis they fill the voids between genes of known phenotype. Note that, in mapping, the biological significance of the DNA marker is not important in itself; the heterozygous site is merely a convenient reference point that will be useful in finding one's way around the genome. In this way, markers are being used just as milestones were used by travelers in previous centuries. Travelers were not interested in the milestones (markers) per se, but they would have been disoriented without them.

Restriction Fragment Length Polymorphisms.

RFLPs (described in Chapter 11) were the first neutral DNA markers to be applied to genome mapping by recombinant frequency.

DNA Markers Based on Variable Numbers of Short-Sequence Repeats.

Although RFLPs were the first DNA markers to have been generally used in genomic characterization, in the analysis of animal and plant genomes, they have now been largely replaced by markers based on variation in the number of short tandem repeats. These markers are collectively called simple-sequence length polymorphisms (SSLPs). They have two basic advantages over RFLPs. First, in regard to RFLPs, usually only one or two "alleles," or morphs, are found in a pedigree or population under study. This small number limits their usefulness; it would be better to have a larger number of alleles that could act as specific tags for a larger variety of homologous chromosomal regions. The SSLPs fill this need because multiple allelism is much more common, and as many as 15 alleles have been found for one locus. Second, the heterozygosity for RFLPs can be low; in other words, if one allele is relatively uncommon in relation to the other, the proportion of heterozygotes (the crucial individuals useful in mapping) will be low. However SSLPs, in addition to having more alleles, show much higher levels of heterozygosity, which makes them more useful in mapping because heterozygotes are the basis for recombination analysis. Two types of SSLPs are now routinely used in genomics.

Minisatellite markers: Minisatellite markers are based on variation of the number of VNTRs. A DNA fingerprint is an array of bands in a Southern hybridization of a restriction digest, as was shown in Figure 12-10. The individual bands of the DNA fingerprint represent different-sized DNA sequences at many different chromosomal positions. If parents differ for a particular band, then this difference becomes a heterozygous ("plus / minus") locus that can be used in mapping. A simple example is shown in Figure 12-17. This same technique can be applied in most organsms with repetitive DNA.
Microsatellite markers: Recall that microsatellite DNA is a class of repetitive DNA based on dinucleotide repeats. The most common type is a repeat of CA and its complement GT, as in the following example:

Probes for detecting these segments are made with the help of the polymerase chain reaction (PCR; see Chapter 10). Digestion of human DNA with the restriction enzyme AluI results in fragments with an average length of 400 bp, and these fragments are cloned into an M13 phage vector. Phages with (CA)n/(GT)n inserts are identified by hybridizing with a (CA)n/(GT)n probe. Positive clones are sequenced, and PCR primer pairs are designed on the basis of sequences flanking the repetitive tract:

The primers are used to amplify DNA by using genomic DNA as a substrate. An individual primer pair will amplify its own repetitive tract and any size variants of it in DNAs from different individuals. A high proportion of PCR primer pairs reveal at least three marker "alleles" of different-sized amplification products. An example of the microsatellite mapping technique is shown in Figure 12-18 on the following page. Thousands of primer pairs can be made that likewise detect thousands of marker loci. The latest microsatellite marker map of human chromosome 1 is shown in Figure 12-19, also on the following page.

Note some differences in the convenience of RFLP and SSLP analyses. RFLP analysis requires a specific cloned probe to be on hand in the laboratory for the detection of each individual marker locus. Microsatellite analysis requires a primer pair for each marker locus, but these primer sequences can easily be shared throughout the world, distributed by electronic mail and rapidly constructed by using a DNA synthesizer. Minisatellite analysis requires just one probe that detects the core sequence of the repetitive element at loci anywhere in the genome.

Together, the discovery of RFLP and SSLP markers has enabled the construction of a human genetic map with centimorgan (cM, or map unit, m.u.) density. Although such resolution is a remarkable achievement, a centimorgan of DNA is still a huge segment, estimated in humans as 1 megabase (1 Mb = 1 million base pairs, or 1000 kb).

MESSAGE
Meiotic recombination analysis of the loci of genes with known phenotypic effect, RFLP markers, and SSLP markers has resulted in a map of the human genome that is saturated down to the 1-centimorgan (1map unit) level.

Randomly Amplified Polymorphic DNAs (RAPDs).

A single PCR primer designed at random will often by chance amplify several different regions of the genome. The single sequence "finds" DNA bracketed by two inverted copies of the primer sequence. The result is a set of different-sized amplified bands of DNA (Figure 12-20). In a cross, some of the amplified bands may be unique to one parent, in which case they can be treated as heterozygous (plus / minus) loci and used as molecular markers in mapping analysis. Notice too that the set of amplified DNA fragments, called randomly amplified polymorphic DNA (RAPD, pronounced "rapid"), is yet another type of DNA fingerprint that can be used to characterize an individual. Such identity tags can be very useful in routine genetic analysis or in population studies.

In situ hybridization.

If a cloned gene is available, then it can be used to make a labeled probe for hybridization to chromosomes in situ. If the individual chromosomes of the genomic set are recognizable through their banding patterns, size, arm ratio, or other cytological features, then the new gene can be assigned to the chromosome to which it hybridizes. Furthermore, the locus of hybridization reveals a rough chromosomal position.

Commonly used probe labels are radioactivity and fluorescence. In the process of fluorescent in situ hybridization (FISH), the clone is labeled with a fluorescent dye, and a partly denatured chromosome preparation is bathed in the probe. The probe binds to the chromosome in situ, and the location of the cloned fragment is revealed by a bright fluorescent spot (Figure 12-21). An extension of FISH is chromosome painting. Sets of cloned DNA known to be from specific chromosomes or specific chromosome regions are labeled with different fluorescent dyes. These dyes then "paint" specific regions and identify them under the microscope (Figure 12-22). If a clone of a gene of unknown location is labeled with yet another dye, then its position can be established in the painted array.

Rearrangement breakpoints.

We learned in Chapter 8 that mutant alleles giving a new observable phenotype are sometimes caused by a chromosomal rearrangement. Usually, the new phenotype is due to the fact that the chromosome break that gives rise to the rearrangement splits the gene in two, separating vital coding or regulatory sequences. If the breakpoints can be seen or mapped to known markers by recombination analysis, then this information can be used to assign a gene to a position on a cytogenetic map of a chromosome. One helpful feature about rearrangement breakpoints is that they also serve as molecular landmarks. When cloned DNA spanning a breakpoint has been identified, the breakpoints are easily detected on Southern blots as split bands.

Radiation hybrid mapping.

The same technique that was used to localize genes to individual chromosomes can be extended to obtain map loci. An important extension is radiation hybrid mapping. This technique was designed to generate a higher-resolution map of molecular markers along a chromosome. The procedure is to irradiate human cells with 3000 rads of X rays to fragment the chromosomes, and then fuse the irradiated cells with the rodent cells to form a panel of different hybrids. In this case, the hybrids have an assortment of fragments of human chromosomes, as diagrammed in Figure 12-23. Most of the fragments are seen to be embedded in the rodent chromosomes, but truncated human chromosomes also can be found. First, the frequency of various human molecular markers in the hybrids is calculated. The next step is to calculate the frequency of the copresence of pairs of human molecular markers. Closely linked markers are assumed to be incorporated at high frequencies because the probability that a radiationinduced break will occur between the loci is low. Distant markers and markers on different chromosomes should be present at frequencies close to the product of individual frequencies. A mapping unit, cR3000, is calculated, which has been calibrated to approximately 0.1 cM (m.u.).

An advantage of this method is in sample size; a large number of radiation hybrids can be amassed relatively easily. Nevertheless, it has been estimated that a standard panel of only 100 to 200 hybrids would be enough to obtain a high-resolution cR3000 map of the human genomein other words, a tenfold increase in resolution over the centimorgan map.

MESSAGE
Correlation of human markers and chromosomes in hybrid rodenthuman cell lines allows chromosomal assignment of the markers. The copresence of different human markers in X-irradiated hybrids allows high-resolution mapping of the chromosomal loci of the markers.

In this section on marker mapping we have encountered techniques based on widely differing premisesfor example, meiotic crossover frequency and radiation-induced breakage. Hence, even though these maps give the same order of markers, distances between markers on one map may not be proportional to distances between markers on another map.

Physical Mapping of Genomes

A further increase in mapping resolution is accomplished by manipulating cloned DNA fragments directly. Because DNA is the physical material of the genome, the procedures are generally called physical mapping. A goal of this procedure is to produce a set of overlapping cloned fragments that together encompass an entire chromosome or an entire genome. This goal achieves three purposes. First, the genetic markers carried on the clones can be ordered and hence contribute to the overall genome-mapping process. Second, when the contiguous clones have been obtained, they represent an ordered library of DNA sequences that can be used for future genetic analysisfor example, to intensively characterize one specific genetic region of interest or to find a previously uncharacterized gene. Third, these clones form the basis for the complete DNA sequencing of the genome.

In preparing physical maps of genomes, vectors that can carry very large inserts are naturally the most useful. Cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PACs (phage P1-based artificial chromosomes) have been the main types. Cosmids and YACs were introduced in Chapters 10 and 11, respectively. BACs (Figure 12-24) are based on the 7-kb F plasmid of E. coli. Recall that F can carry large fragments of E. coli DNA as F derivatives (Chapter 9). In a similar manner, as cloning vectors, they can also carry inserts of large fragments of foreign DNA as large as 300 kb, although the average is about 100 kb. PACs are produced by a similar type of engineering of phage P1; they carry inserts comparable to those of BACs.

Although the maximum insert sizes of BACs and PACs are not as large as those of YACs, the former types have several advantages over YACs. First, they can be amplified in bacteria and isolated and manipulated simply with basic bacterial plasmid technology. Second, BACs and PACs form fewer hybrid inserts than YACs do. Hybrid inserts are composed of several different fragments; their presence can thwart attempts to order the clones.

However, despite these useful vectors, the task of genomic cloning is a daunting one. Even so-called small genomes contain huge amounts of DNA. Consider, for example, the 100-Mb genome of the tiny nematode Caenorhabditis elegans; because an average cosmid insert is about 40 kb, at least 2500 cosmids would be required to embrace this genome, and many more to narrow the number down to such a complete set. YACs can contain on the order of 1 Mb, so here the task is somewhat simpler.

Cloning a whole genome begins by amassing a large number of randomly cloned inserts. The contents of these clones must be characterized in some way and overlaps determined. A set of overlapping clones is called a contig. In the early phases of a genome project, contigs are numerous and represent cloned "islands" of the genome. But, as more and more clones are characterized, contigs enlarge and merge into one another, and eventually the project should yield a set of contigs that equals the number of chromosomes.

Chromosome-specific libraries.

If a library of clones is prepared from total genomic DNA, then contig development is relatively slow. However, if a specific chromosome can be used to develop the library of clones, contigs emerge more rapidly. PFGE can be used to isolate individual chromosomes (if they are small) or chromosome fragments cut with "long cutter" enzymes such as NotI. Flow sorting is another option for preparing DNA of a specific chromosome. Chromosomes (such as human chromosomes) can be flow sorted by fluorescence-activated chromosome sorting (FACS; Figure 12-25). In this procedure, metaphase chromosomes are stained with two dyes, one of which binds to AT-rich regions and the other to GC-rich regions. Cells are disrupted to liberate whole chromosomes into liquid suspension. This suspension is converted into a spray in which the concentration of chromosomes is such that each spray droplet contains one chromosome. The spray passes through laser beams tuned to excite the fluorescence. Each chromosome produces its own characteristic fluorescence signal, which is recognized electronically, and two deflector plates direct the droplets containing the specific chromosome needed into a collection tube.

MESSAGE
Genomic cloning proceeds by assembling clones into overlapping groups called contigs. As more data accumulate, the contigs become equivalent to whole chromosomes.

Several different techniques are used to order genomic clones into contigs, and we shall consider some of the main ones.

Ordering by FISH.

If good chromosomal landmarks are known, FISH analysis can be used to locate the approximate positions of the large inserts. Figure 12-26 shows results of a FISH analysis that generates a rough ordering of BACs and PACs in human chromosomes.

Ordering by clone fingerprints.

The genomic insert carried by a vector has its own unique sequence that can be used to generate a DNA fingerprint. For example, a multiple-restriction-enzyme digestion can generate a set of bands whose number and positions are a unique fingerprint of that clone. The different bands generated by separate clones can be aligned either visually or by using a computer program to determine if there is any overlap between the inserted DNAs. In this way, the contig can be built up.

Ordering by sequence-tagged sites.

Unique short sequences of large cloned inserts can be used as tags to align the various clones into contigs. For example, if clone A has tags 1 and 2 and clone B has tags 2 and 3, then clones A and B must overlap in the region of tag 2. The practical procedure is to amass a large set of random clones with small genomic inserts (say, in l phage) and sequence short regions of each clone. From these sequences, pairs of PCR primers are designed to amplify each short specific sequence of DNA flanked by the primers. These short DNA sequences are known as sequence-tagged sites (STSs). Even though the locations of these STSs in the genome are not known initially, a panel of many STSs can be used to characterize clones with large genomic inserts (such as YAC clones). From this characterization, the clones that are shown to have specific STSs in common must have overlapping inserts and therefore can be aligned into contigs. An example of this process is shown in Figure 12-27.

STSs are sometimes obtained from cDNA clones and are known as expressed sequence tags (ESTs). ESTs are obtained by sequencing into the cDNA insert with the use of a primer based on the vector sequence. They can be used to align the cDNAs on the contig, thus anchoring the gene map to the physical map.

The combination of these physical methods has resulted in the cloning of whole genomes of several organisms. For example, the C. elegans genome is now available as sets of cosmid or YAC contigs. Furthermore, the DNA of the contigs has been arranged on nitrocellulose filters in ordered arrays. So, to find out where a specific piece of DNA of interest lies in the genome, that DNA is used as a probe on the contig filters, and a positive hybridization signal will announce the precise location of the DNA (Figure 12-28). Such arrays are called polytene filters because they can be used in somewhat the same way that a Drosophila polytene chromosome is used for in situ hybridization. These contigs are the basis for the sequencing of entire genomes.

An example: cloning and mapping the human Y chromosome.

Several of the smaller human chromosomes have been fully cloned as overlapping sets of YAC clones (contigs). We shall examine the cloning of the Y chromosome as an example, because it illustrates several of the techniques described so far. The STS map of the Y chromosome was in fact obtained by two different methods: YAC alignment and deletion analysis.

YAC Alignment.

Flow sorting yielded a sample of Y chromosomes, from which l clones were made. From clones that did not contain repetitive DNA, STS primers were designed. In all, 160 primer pairs were made. A Y chromosome YAC library of 10,368 clones was obtained in which the average insert size was 650 kb. From these numbers, each point on the Y chromosome was estimated to have been sampled an average of four times. The YAC clones were divided into 18 pools of 576 YACs, and the pools were screened with the STS primers. Subdivision of positive pools led rapidly to the assignment of a particular STS to specific YACs. The total STS content of each YAC was assessed, and overlaps between the YACs were determined in the same way as that shown in the generalized example in Figure 12-27.

Deletion Analysis.

Various types of Y chromosome deletions occur naturally. For example, some XX males have truncated fragments of the Y chromosome, whereas some XY females have deletions of the region containing the maleness (testis-determining) gene (see Chapter 16). These Y deletions were maintained in cell culture and formed the basis for aligning the Y chromosome STSs. Each deletion was tested for STS content. Because by nature the deletions were nested sets, the STS content could be used not only to develop an STS map, but also to map the coverage of the deletions. The principle is illustrated in Figure 12-29 on the following page. The STS maps produced by YAC alignment and by deletion analysis were identical.

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Clones can be arranged into contigs by matching DNA fingerprints, by matching short sequences within cloned segments, and by analyzing deletions.

Genome Sequencing

The highest-resolution technique in genomics is DNA sequencing. Several different strategies have been used for sequencing.

Random clone sequencing.

The first genome to be cloned was that of the bacterium Hemophilus influenzae. Genomic DNA was mechanically sheared and used to obtain a large number of random clones that were presumed to overlap each other in numerous ways. Primers based on adjacent vector DNA were used to sequence short regions at the ends of the cloned Hemophilus inserts. Then these short sequences were used (much like sequence-tagged sites) to align the genomic clones. Because so many random short sequences were obtained, together they encompassed the great majority of the Hemophilus genome. Gaps were filled in by "primer walking"; that is, by using the end of a cloned sequence as a primer to sequence into adjacent uncloned fragments.

Sequencing ordered clones.

Most genomic sequencing programs start with a set of ordered clones. We have seen that an ordered set of YAC clones has been developed for the human Y chromosome and other human chromosomes. However, YAC clones are not suitable for sequencing directly. YACs are subcloned into overlapping BACs or PACs, which are aligned into contigs by using STSs or the alignment of clone fingerprints. The BAC or PAC clones are then subcloned into smaller inserts for sequencing. At this level, multiple overlapping clones are sequenced randomly (without establishing clone alignment) so that any BAC or PAC clone is sequenced as many as five times in all.

Automation.

All stages of genomic analysis can be speeded up by automation. Preparation of clones, DNA isolation, electrophoresis, and sequencing protocols have all been adapted to machines. An example of this "high throughput" machinery is shown in Figure 12-30.

Using Genome Maps in Genetic Analysis

Genetic and physical maps are an important starting point for several types of genetic analysis, including gene isolation (including human disease genes) and functional genomics.

Isolating human disease genes by positional cloning.

Let us consider, as an example, the methods used to identify the genomic sequence of the cystic fibrosis (CF) gene. No primary biochemical defect was known at the time that the gene was isolated, so it was very much a gene in search of a function. Linkage to molecular markers had located the gene to the long arm of chromosome 7, between bands 7q22 and 7q31.1. The CF gene was thought to be inside this region, flanked by the gene met (a proto-oncogene, see Chapter 15) on one end and the molecular marker D788 at the other end. But between these markers lay 1.5 cM (map units) of DNA, a vast uncharted terrain of 1.5 million bases. Additional markers within the region were obtained by using new probes derived from a chromosome 7 library made by flow sorting.

However, the two key techniques that were used to traverse the huge genetic distances were chromosome walking (page 319) and a related technique called chromosome jumping. The latter technique not only provides a way of jumping across potentially unclonable areas of DNA, but also generates widely spaced landmarks along the sequence that can be used as initiation points for multiple bidirectional chromosomal walks.

Chromosome jumping is illustrated in Figure 12-31. In this procedure, large fragments are created by partial restriction cleavage of the DNA of the region believed to contain the gene of interest. Each DNA fragment is then circularized, thus bringing the beginning and end of the fragment together. This junction is cut out and cloned into a phage vector, which, together with the other junction segments, makes up a jumping library. A probe from the beginning of the stretch of DNA under investigation can be used to screen the jumping library to find the clone that contains the beginning sequence. Once the clone is found, the other end of the junction sequence is excised and used to screen the library again to make a second jump. From each jump position, chromosome walks can be made in both directions to search for genelike sequences.

A restriction map of the overall region thought to contrain the CF gene was obtained with rare-cutting restriction enzymes, and the restriction sites were used to position and orient the sequences obtained from jumping and walking. When enough sequencing had been done to cover representative parts of the overall region, the hunt for any genes along this stretch began. Genes were sought by using several clues. First, it was known that genes in humans are generally preceded at the 5 end by clusters of cytosines and guanines, called CpG islands, and several of these clusters were found. Second, it was reasoned that a gene would show homology to the DNA of other animals, because of evolutionary conservation, so candidate sequences were used to probe what were called zoo blots of genomic DNA from a range of animals. Third, genes should have appropriate start and stop signals. Fourth, genes should be transcribed, and transcripts should be found.

Ultimately, a strong candidate gene spanning 250 kb of the region was found. Some CF symptoms are expressed in sweat glands; so, from cultured sweat gland cells, cDNA was prepared, and a 6500-nucleotide cDNA homologous to the candidate gene was detected. When this cDNA from normal persons and from CF patients was sequenced, the cDNA of the patients showed a deletion of three base pairs (Chapter 3), eliminating a phenylalanine from the protein encoded by the candidate gene. Therefore it was very likely that this was the CF coding sequence. Thus the CF gene had been found. From its sequence, an amino acid sequence was inferred; and, from the inferred amino acid sequence, the three-dimensional structure of the protein was predicted. This protein was structurally similar to ion-transport proteins in other systems, suggesting that a transport defect is the primary cause of CF. When used to transform mutant cell lines from CF patients, the wild-type gene restored normal functionthe final confirmation that the isolated sequence was in fact the CF gene.

The candidate-gene approach.

Inevitably, intensive cloning and characterization of a region reveal the presence of genes of unknown function. If a gene of interest such as a disease gene is mapped to that general area, then these genelike sequences become candidate genes for the disease gene without the need for extensive chromosome walking and jumping. This procedure is termed the candidate-gene approach to gene isolation. Knowledge about the gene's phenotype such as biochemical defect and pattern of tissue expression can be matched to the sequence domains and tissue expression of the candidate gene.

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Cloning is made easier by the availability of a set of overlapping genomic clones.

Genes underlying complex inheritance patterns.

Most of the contrasting phenotypes analyzed in this book are determined simply by alleles of a single gene. However, there are many phenotypes that are determined in a complex manner. Here two situations can be distinguished.

In the first situation, the phenotypic variation may be quantitative (Chapters 1 and 18), and the characters (traits) are called quantititative traits. Examples are metric characters such as height and weight. This type of variation is thought to be based on the cumulative interaction of "+ and " alleles of several genes and the environment. The availability of thousands of molecular markers such as SSLPs ranged along all the chromosomes of a genome has made it possible to map some of the genes that contribute to quantitative variation, whose loci are called quantitative trait loci, abbreviated QTLs. The approach is to interbreed two lines that show widely contrasting phenotypes for a quantitative trait, so as to generate homozygous descendants that contain only one segment or a small number of segments from one line, as shown in Figure 12-32. (These segments can be identified by the SSLP alleles that they carry.) Such hybrid organisms are then assessed for their quantitative phenotype, and estimates made of the contributions (or lack of contribution) of specific segments to the observed variation. The average phenotype of lines with, say, region A is compared with the average of lines lacking region A, and, if there is a difference, region A becomes a candidate to contain a QTL. Ideally, a derived pure line would carry only one QTL; then, in backcrosses with the appropriate parent, this QTL would segregate in a monohybrid manner. The QTL can then be mapped precisely by recombination with SSLP markers.

The second type of complex inheritance involves discrete variants that do not show single gene inheritance patterns. These are called complex traits. This type of inheritance is found in many human diseases such as epilepsy, heart disease, diabetes, and Alzheimer's disease. Such phenotypes "run in families" but appear in the pedigree sporadically. Here the model is that alleles of one to several genes interact with each other and with the environment. In certain combinations a cellular threshold is attained beyond which the disease is expressed. The genes that contribute to complex traits can be identified using an extension of the approach in Figure 12-32. Obviously pure human lines are not available, but nevertheless the approach of correlating phenotype with specific molecular markers can be applied to any groups of related individuals of mixed ancestry. The statistical analysis is more complex. In humans the genomes of affected siblings are "scanned" for molecular markers and correlations sought. Data must be combined from many families. Alternatively correlations of phenotype with specific markers may be attempted in the population at large; this is most effective in relatively homogeneous populations.

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