Introduction (2024)

Genomes Consist of DNA Molecules That Contain Many Genes

The genome of all living organisms consists of DNA, a very long two-stranded chemical polymer (Figure 2-1). Each DNA strand is composed of four different units, called nucleotides, that are linked end to end to form a long chain (Figure 2-2). These four nucleotides are symbolized as A, G, C, and T, which stand for the four bases—adenine, guanine, cytosine, and thymine—that are parts of the nucleotides. One DNA molecule, which together with some associated proteins constitutes a chromosome, differs from another in its length and in the order of its nucleotides. Each DNA molecule contains many genes, which are its functional units. These genes are arranged in a defined order along the DNA molecule. Most genes code for protein molecules—enzymes or structural elements—that determine the characteristics of a cell. In bacteria, the coding sequences of a gene—are continuous strings of nucleotides, but in mammals the coding segments in a gene (called exons) are generally separated from one another by noncoding segments (called introns) (Figure 2-3). Often each exon will encode a different structural region (or domain) of a larger protein molecule. Many exons have been found to be part of a family of related coding sequences that are used in the construction of many different genes (Doolittle et al., 1986). Because of the many introns in mammalian genes, a single gene is often more than than 10,000 nucleotides long, and genes that span 100,000 nucleotides are not uncommon (Table 2-1).

For the information in the coding sequences of a gene to be expressed, the DNA of a gene must first be transcribed into an RNA molecule (Figure 2-3). Before the RNA strand leaves the cell's nucleus, the intron sequences are cut out of this RNA strand by a process called RNA splicing, thereby bringing the exon sequences into contiguity. Then the RNA can be translated into a protein molecule according to the genetic code (every group of three nucleotides codes for one amino acid). Nucleotide sequences adjacent to the coding sequences in each gene encode regulatory signals for activating or inactivating transcription of the gene. Gene activity is a dynamic process; at any given time and in any given cell type, only a subset of genes is active. These active genes determine the course of embryological development and the characteristics of cells and organisms.

The Human Genome Is Composed of 24 Different Types of DNA Molecules

Human DNA is packaged into physically separate units called chromosomes. Humans are diploid organisms, containing two sets of genetic information, one set inherited from the mother and one from the father. Thus, each somatic cell has 22 pairs of chromosomes called autosomes (one member of each pair from each parent) and two sex chromosomes (an X and a Y chromosome in males and two X chromosomes in females). Each chromosome contains a single very long, linear DNA molecule. In the smallest human chromosomes this DNA molecule is composed of about 50 million nucleotide pairs; the largest chromosomes contain some 250 million nucleotide pairs.

The diploid human genome is thus composed of 46 DNA molecules of 24 distinct types. Because human chromosomes exist in pairs that are almost identical, only 3 billion nucleotide pairs (the haploid genome) need to be sequenced to gain complete information concerning a representative human genome. The human genome is thus said to contain 3 billion nucleotide pairs, even though most human cells contain 6 billion nucleotide pairs.

DNA is a double helix: Each nucleotide on a strand of DNA has a complementary nucleotide on the other strand. The information on one DNA strand is therefore redundant to that on the other (that is because of complementary base pairing (Figure 2-2A), one can in principle determine the nucleotide sequence of one strand from the other). However, it is currently necessary to determine the sequences of the nucleotides on the two DNA strands separately to achieve the desired accuracy of any DNA sequence, with the sequence of each strand being used as a check on the other. For this reason, a total of 6 billion nucleotides must actually be sequenced to order the 3 billion nucleotide pairs in the haploid human genome.

The average size of a protein molecule allows one to predict that there are approximately 1,000 nucleotide pairs of coding sequence per gene. Since humans are thought to have about 100,000 genes, a total of about 100 million nucleotide pairs of coding DNA must be present in the human genome. That this is only about 3 percent of the total size of the genome leads one to conclude that less than 5 percent of the human genome codes for proteins. The vast bulk of human DNA lies between genes and in the introns. Some of the noncoding DNA plays a role in regulating gene activity, while other portions are believed to be important for organizing the DNA into chromosomes and for chromosome replication (Alberts et al., 1983; Lewin, 1987). The function of most noncoding regions of the human genome, however, is unknown; much of this DNA may have no function at all.

The Human Genome Can Be Mapped in Many Different Ways

It would be enormously useful to determine the order and spacing of all the genes that make up the genome. Such information is said to constitute a gene or genome map. Since there are 24 different DNA molecules in the human genome, a complete human gene map consists of 24 maps, each in the linear form of the DNA molecule itself.

One type of useful genome map is the messenger RNA (mRNA) or exon map. Cellular enzymes transcribe or copy all of an organism's genes into mRNAs so that the functions of the genes can be expressed. Complementary DNA (cDNA) of all the mRNAs present in an organism can be synthesized enzymatically with reverse transcriptase. These cDNAs can then be cloned and used to locate the corresponding genes on a chromosome map. In this way, the genes can be mapped in the absence of knowledge of their function. Another type of genome map would consist of an ordered set of the overlapping DNA clones that constitute an entire chromosome. Both the exon map and the ordered set of DNA clones are usually referred to as physical maps. Alternatively, the position of a gene can be mapped by following the effect of the expression of the gene on the cells containing it. Here, a map is constructed on the basis of the frequencies of co-inheritance of two or more genetic markers. This type of map is referred to as a genetic linkage map. The distinction between physical and genetic linkage maps is discussed in detail in Chapter 4.

Maps of the human genome can be made at many different scales, or levels of resolution. Low-resolution physical maps have been derived from the distinctive patterns of bands that are observed along each chromosome by light microscopy of stained chromosomes. Genes have been physically associated with particular bands or clusters of bands in a number of ways. These associations permit genes to be mapped only approximately since a given gene might be assigned to a region of about 10 million nucleotides containing several hundred genes. Its exact position on the chromosome must be determined by more precise methods.

Maps of higher resolution are based on sites in the DNA cut by special proteins called restriction enzymes. Each enzyme recognizes a specific short sequence of four to eight nucleotides (a restriction site) and cuts the DNA chain at one point within the sequence (Watson et al., 1983). Since dozens of different sequences are recognized by one or another enzyme, and these sequences are closely spaced throughout the genome, high-resolution physical maps can be constructed by determining the relative location of different restriction sites precisely. Of particular value in human gene mapping are restriction sites that are highly variable (or polymorphic) in the population. DNA lacking a specific restriction site yields a larger restriction fragment when cut by the enzyme than DNA containing the site; hence, the designation restriction fragment length polymorphism (RFLP). Hundreds of polymorphic restriction sites have so far been identified and mapped in the human genome. Some disease-related genes have already been localized by determining the frequency of co-inheritance of RFLPs and genetic diseases (Gusella et al., 1983). Examples of these diseases include cystic fibrosis, duch*enne muscular dystrophy, Alzheimer's disease, and neurofibromatosis. Identifying a much larger number of useful polymorphic restriction sites should make it possible to map disease-related genes precisely enough to greatly facilitate the isolation of any human gene.

The map based on a collection of ordered clones of genomic fragments has a special value (see Chapter 4). In such a map, not only are the genomic positions of restriction fragments known, but each fragment is available as a clone that can be propagated and distributed to interested researchers. Such clones are immensely valuable because they serve as the starting point for gene isolation, for functional analyses, and for the determination of nucleotide sequences.

The ultimate, highest resolution map of the human genome is the nucleotide sequence, in which the identity and location of each of 3 billion nucleotide pairs is known (see Chapter 5). Only such a sequence reveals all or nearly all the information in the human genome. A number of specific regions of human DNA have already been analyzed in this way, providing information about the structure of genes and their encoded proteins in both normal and abnormal individuals and about sequences that regulate gene expression (Figure 2-4). At present, however, the nucleotide sequence of substantially less than 0.1 percent of the human genome is known. This includes the sequence containing 0.5 percent of our genes.