STUART M. BROWN, PhD, is on the faculty of the New York University School of Medicine, where he is Associate Professor and Director of the Bioinfor-matics Core Facility and Director of the graduate Bioinformatics course.

Essentials of Medical Genomics 2nd Edition

By Stuart M. Brown

John Wiley & Sons

Copyright © 2009 John Wiley & Sons, Inc.
All right reserved.
ISBN: 978-0-470-14019-2

Chapter One

Introduction to Molecular Genetics

The Human Genome Project is a bold undertaking to understand, at a fundamental level, all of the genetic information required to build and maintain a human being. The human genome is the complete information content of the human cell. This information is encoded in approximately 3.2 billion base pairs of DNA contained on 46 chromosomes (22 pairs of autosomes plus the two sex chromosomes-see Figure 1.1). The completion, in 2001, of the first draft of the human genome sequence was only the first phase of this project (Venter et al. 2001; Lander et al. 2001).

To use the metaphor of a book, the draft genome sequence gives biology all of the letters, in the correct order on the pages, but without the ability to recognize words, sentences, and punctuation, or even an understanding of the language in which the book is written. The task of making sense of all of this raw biological information falls, at least initially, to bioinformatics specialists who make use of computers to find the words and decode the language. The next step is to integrate all of this information into a new form of experimental biology, known as genomics, that can ask meaningful questions about what is happening in very complex systems where tens of thousands of different genes and proteins are interacting simultaneously.

The primary justification for the considerable amount of money spent on sequencing the human genome (from governments and private corporations) is that this information will lead to dramatic medical advances. In fact, the first wave of new drugs and medical technologies derived from genome information is currently making its way through clinical trials and into the healthcare system. However, to effectively utilize these new advances, medical professionals need to understand something about genes and genomes. Just as it is important for physicians to understand how to Gram-stain and evaluate a culture of bacteria, even if they never actually perform this test themselves in their medical practices, it is important to understand how DNA technologies work in order to appreciate their strengths, weaknesses, and peculiarities.

However, before we can discuss whole genomes and genomic technologies, it is necessary to understand the basics of how genes function to control biochemical processes within the cell (molecular biology) and how hereditary information is transmitted from one generation to the next (genetics).

The Principles of Inheritance

The principles of genetics were first described by the monk Gregor Mendel in 1866 in his observations of the inheritance of traits in garden peas [“Versuche ber Pflanzen-Hybriden” (Mendel 1866)]. Mendel described “differentiating characters” (differierende Merkmale) which may come in several forms. In his monastery garden, he made crosses between strains of garden peas that had different characters, each with two alternate forms that were easily observable, such as purple or white flower color, yellow or green seed color, smooth or wrinkled seed shape, and tall or short plant height. (These alternate forms are now known as alleles.) Then he studied the distribution of these forms in several generations of offspring from his crosses.

Mendel observed the same patterns of inheritance for each of these characters. Each strain, when bred with itself, showed no changes in any of the characters. In a cross between two strains that differ for a single character, such as pink versus white flowers, the first generation of hybrid offspring (the [F.sub.1]) all resembled one parent-all pink. Mendel called this the dominant form of the character. After self-pollinating the [F.sub.1] plants, the second-generation plants (the [F.sub.2]) showed a mixture of the two parental forms (see Figure 1.2). This is known as segregation. The recessive form that was not seen in the [F.sub.1]s (white flowers) was found in one-fourth (25%) of the [F.sub.2] plants.

Mendel also made crosses between strains of peas that differed for two or more traits. He found that each trait was assorted independently in the progeny-there was no connection between whether an [F.sub.2] plant had the dominant or recessive form for one character and which form it carried for another character (see Figure 1.3).

Mendel created a theoretical model (“Mendel’s laws of genetics”) to explain his results. He proposed that each individual has two copies of the hereditary material for each character, which may determine different forms of that character. These two copies separate and are subjected to independent assortment during the formation of gametes (sex cells). When a new individual is created by the fusion of two sex cells, the two copies from the two parents combine to produce a visible trait depending on which form is dominant and which is recessive. Mendel did not propose any physical explanation for how these traits were passed from parent to progeny; his characters were purely abstract units of heredity.

Modern genetics has completely embraced Mendel’s model with some additional detail. There may be more than two different alleles for a gene in a given population, but each individual has only two, which may be the same (homozygous) or different (heterozygous). In some cases two different alleles combine to produce an intermediate form in heterozygous individuals, so that red and white flower alleles may combine to produce pink or type A and type B blood alleles, which in turn combine to produce the AB blood type.

Genes Are on Chromosomes

In 1902, Walter Sutton, a microscopist, proposed that Mendel’s heritable characters resided on the chromosomes which he observed inside the cell nucleus (see Figure 1.4). Sutton observed that “the association of paternal and maternal chromosomes in pairs and their subsequent separation during cell division … may constitute the physical basis of the Mendelian law of heredity” (Sutton 1903).

In 1909, the Danish botanist Wilhelm Johanssen coined the term “gene” to describe Mendel’s heritable characters. In 1910, Thomas Hunt Morgan found that a trait for white eye color was located on the X chromosome of the fruitfly and was inherited together with a factor that determines sex (Morgan 1910). A number of subsequent studies by Morgan (1919) and others showed that each gene for a particular trait was located at a specific spot or locus on a chromosome in all individuals of a species. The chromosome was perceived as a linear organization of genes, like beads on a string. Throughout the early part of the twentieth century, a gene was considered to be a single, fundamental, indivisible unit of heredity, in much the same way as an atom was considered to be the fundamental unit of matter.

Each individual has two copies of each type of chromosome, having received one copy from each parent. The two copies of each chromosome in the parent are randomly divided into the sex cells (sperm and egg) in a process called segregation. It is possible to observe the segregation of chromosomes during meiosis using only a moderately powerful microscope. It is an aesthetically satisfying triumph of biology that this observed segregation of chromosomes in cells exactly corresponds to the segregation of traits that Mendel observed in his peas.

Recombination and Linkage

In the early twentieth century, Mendel’s concepts of inherited characters were broadly adopted by practical plant and animal breeders as well as experimental geneticists. It rapidly became clear that Mendel’s experiments represented an oversimplified view of inheritance. He must have intentionally chosen characters in his peas that were inherited independently. In the breeding experiments where many traits differ between parents, it is commonly observed that progeny inherit pairs or groups of traits together from one parent far more frequently than would be expected by chance alone. This observation fits nicely into the chromosome model of inheritance-if two genes are located on the same chromosome, then they will be inherited together when that chromosome segregates into a gamete, and that gamete becomes part of a new individual.

However, it was also observed that “linked” genes do occasionally separate. A theory of recombination was developed to explain these events. During the process of meiosis, it was proposed that the homologous chromosome pairs line up and exchange segments in a process called crossing over. This theory was supported by microscopic evidence of X-shaped structures called chiasmata forming between paired homologous chromosomes in meiotic cells (see Figure 1.5).

If a parent cell contains two different alleles for two different genes, then after the crossover, the chromosomes will contain new combinations of alleles. For example, if one chromosome contains alleles A and B for two genes, and the other chromosome contains alleles a and b, then without crossovers, all progeny must inherit a chromosome from that parent with either an A-B or an a-b allele combination. If a crossover occurs between the two genes, then the resulting chromosomes will contain the A-b and a-B allele combinations (see Figure 1.6).

Morgan, continuing his work with fruitflies, demonstrated that the chance of a crossover occurring between any two linked genes is proportional to the distance between them on the chromosome. Therefore, by counting the frequency of crossovers between alleles of a given pair of genes, it is possible to create genetic maps of chromosomes. Morgan was awarded the 1933 Nobel Prize in Medicine for this work. In fact, it is generally observed that on average there is more than one crossover between every pair of homologous chromosomes in every meiosis, so that two genes located on opposite ends of a chromosome do not appear linked at all. On the other hand, alleles of genes that are located very close together are very rarely separated by recombination (see Figure 1.7).

The relationship between the frequency of recombination between alleles and the distance between genes on a chromosome has been used to construct genetic maps for many different organisms, including humans. It has been a fundamental assumption of genetics for almost a hundred years that recombinations occur randomly along the chromosome at any location, even within genes. However, more recent data from DNA sequencing of genes in human populations suggest that there are recombination hotspots and regions where recombination almost never occurs. This creates groups of alleles from neighboring genes on a chromosome, known as haplotypes, that remain linked together across hundreds of generations.

Genes Encode Proteins

Beadle and Tatum (1941) showed that a single mutation, caused by exposing the fungus Neurospora crassa to X rays, destroyed the function of a single enzyme, which interrupted a biochemical pathway at a specific step due to the loss of function of a particular enzyme. This mutation segregated among the progeny exactly as Mendel’s traits did in peas. The X-ray-induced damage to a specific region of one chromosome destroyed the instructions for the synthesis of a specific enzyme. Thus a gene is a spot on a chromosome that codes for a single enzyme. In subsequent years, a number of other researchers broadened this concept by showing that genes code for all types of proteins, not just enzymes, leading to the one gene-one protein model, which is the core of modern molecular biology. Beadle and Tatum shared the 1958 Nobel Prize in Medicine.

Genes Are Made of DNA

The next step in understanding the nature of the gene was to dissect the chemical structure of the chromosome. Crude biochemical purification had shown that chromosomes are composed of both protein and DNA. Avery et al. (1944) conducted the classic experiment on the “transforming principle.” They found that DNA purified from a lethal S (smooth) form of Streptococcus pneumoniae could transform a harmless R (rough) strain into the S form (see Figure 1.8). Treatment of the DNA with protease to destroy all of the protein had no effect, but treatment with DNA-degrading enzymes blocked the transformation. Therefore, the information that transforms the bacteria from R to S must be contained in the DNA (McCarty 1985).

Hershey and Chase (1952) confirmed the role of DNA with their classic “blender experiment” on bacteriophage viruses. The phage were radioactively labeled with either [sup.35]S in their proteins or [sup.32]P in their DNA. They used a blender to interrupt the process of infection of Escherichia coli bacteria by the phage. Then they separated the phage from the infected bacteria by centrifugation and collected the phage and the bacteria separately. They observed that the [sup.35]S-labeled protein remained with the phage while the [sup.32]P-labeled DNA was found inside the infected bacteria (see Figure 1.9). This proved that it is the DNA portion of the virus that enters the bacteria and contains the genetic instructions for producing new phage, not the proteins, which remain outside. Hershey was awarded the 1969 Nobel Prize for this work.

DNA Structure

Now it was clear that genes are made of DNA, but how does this chemically simple molecule contain so much information? DNA is a long polymer molecule that contains a mixture of four different chemical subunits: adenine, cytosine, guanosine, and thymine (abbreviated as A, C, G, and T). These subunits, known as nucleotide bases, have similar two-part chemical structures that contain a deoxyribose sugar and a nitrogen ring (see Figure 1.10), hence the name deoxyribose nucleic acid. The real challenge is to understand how the nucleotides fit together in a way that can contain a lot of information.

Chargaff (1950) discovered that there was a consistent one-to-one ratio of adenine to thymine and guanine to cytosine in any sample of DNA from any organism. In 1951, Linus Pauling and R. B. Corey described the [alpha]-helical structure of a protein (Pauling and Corey 1951). Shortly thereafter, Rosalind Franklin (Sayre 1975) provided X-ray crystallographic images of DNA to James Watson and Francis Crick (see Figure 1.11); this form of DNA was very similar to the -helix described by Pauling. Watson and Crick’s crucial insight (1953) was to realize that DNA formed a double helix with complementary bonds between adenine-thymine and guanine-cytosine pairs.

The Wastson-Crick model of the DNA structure resembles a twisted ladder. The two sides of the ladder are formed by strong covalent bonds between the phosphate on the 5′ carbon of one deoxyribose sugar and the methyl side groups of the 3′ carbon of the next (a phosphodiester bond). Thus, the deoxyribose sugar part of each nucleotide is bonded to the one above and below it, forming a chain that forms the backbone of the DNA molecule (see Figure 1.12). The phosphate-to-methyl linkage of the deoxyribose sugars give the DNA chain a direction or polarity, generally referred to as 5′ to 3′. Each DNA molecule contains two parallel chains that run in opposite directions forming the sides of the ladder.

The rungs of the ladder are formed by weaker hydrogen bonds between the nitrogen ring parts of pairs of nucleotide bases. There are only two types of base pair bonds: adenine bonds with thymine, and guanine bonds with cytosine. The order of nucleotide bases on both sides of the ladder always reflects this complementary base pairing-so that wherever there is an A on one side, there is always a T on the other side, and vice versa. Since the A-T and G-C units always occur together, they are often referred to as base pairs. The G-C base pair has three hydrogen bonds, while the A-T pair only has two (see Figure 1.13), so the bonds between G-C bases are more stable at high temperatures than are A-T bonds. The nucleotide bases are strung together on the polydeoxyribose backbone-like beads on a string. It is the particular order of the four different bases as they occur along the string that contains all of the biological information.

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