Structure of Genes and DNA

DNA is composed of two antiparallel strands of unbranched polymer wrapped around each other to form a right-handed double helix (Fig. 3-1).³ Each strand is composed of four types of deoxyribonucleotides containing the bases adenine (A), cytosine (C), guanine (G), and thymine (T). The nucleotides are joined together by phosphodiester bonds that join the 5′carbon of one deoxyribose group to the 3′ carbon of the next. Whereas the sugar-phosphate backbone remains constant, the attached bases can vary to encode different genetic information. The nucleotide sequences of the opposing strands of DNA are complementary to each other, thus allowing the formation of hydrogen bonds that stabilize the double-helix structure. Complementary base pairs require that A always pairs with T and C always pairs with G.

FIGURE 3-1 DNA double-helix structure. The sequence of four bases (guanine, adenine, thymine, and cytosine) determines the specificity of genetic information. The bases face inward from the sugar-phosphate backbone and form pairs (dashed lines) with complementary bases on the opposing strand.

(Adapted from Rosenthal N: DNA and the genetic code. N Engl J Med 331:39–41, 1994.)

The entire human genetic information, or human genome, contains 3 × 10⁹ nucleotide pairs. However, less than 10% of the DNA sequences are copied into messenger RNA (mRNA) molecules, which encode proteins, or structural RNA, such as transfer RNA (tRNA) or ribosomal RNA (rRNA) molecules. Each nucleotide sequence in a DNA molecule that directs the synthesis of a functional RNA molecule is called a gene (Fig. 3-2). DNA sequences that do not encode genetic information may have structural or other unknown functions. Human genes commonly contain more than 100,000 nucleotide pairs, yet most mRNA molecule–encoding proteins consist of only 1000 nucleotide pairs. Most of the extra nucleotides consist of long stretches of noncoding sequences, called introns, that interrupt the relatively short segments of coding sequences called exons. For example, the thyroglobulin gene has 300,000 nucleotide bases and 36 introns, whereas its mRNA has only 8700 nucleotide bases. The processes whereby genetic information encoded in DNA is transferred to RNA and protein molecules are discussed later.

FIGURE 3-2 Gene structure. The DNA sequences that are transcribed as RNA are collectively called the gene and include exons (expressed sequences) and introns (intervening sequences). Introns invariably begin with the nucleotide sequence GT and end with AG. An AT-rich sequence in the last exon forms a signal for processing the end of the RNA transcript. Regulatory sequences that make up the promoter and include the TATA box occur close to the site where transcription starts. Additional regulatory elements are located at variable distances from the gene.

(Adapted from Rosenthal N: Regulation of gene expression. N Engl J Med 331:931–933, 1994.)

The human genome contains 24 different DNA molecules; each DNA has 10⁸ bases and is packaged in a separate chromosome. Thus, the human genome is organized into 22 different autosomes and two different sex chromosomes. Because humans are diploid organisms, each somatic cell contains two copies of each different autosome and two sex chromosomes, for a total of 46 chromosomes. One copy of chromosomes is inherited from the mother and one is inherited from the father. Germ cells contain only 22 autosomes and one sex chromosome. Each chromosome contains three types of specialized DNA sequences that are important in the replication or segregation of chromosomes during cell division (Fig. 3-3). To replicate, each chromosome contains many short, specific DNA sequences that act as replication origins. A second sequence element, called a centromere, attaches DNA to the mitotic spindle during cell division. The third sequence element is a telomere, which contains G-rich repeats located at each end of the chromosome. During DNA replication, one strand of DNA becomes a few bases shorter at its 3′ end because of limitation in the replication machinery. If this is not remedied, DNA molecules will become progressively shorter in their telomere segments with each cell division. This problem is solved by an enzyme called telomerase, which periodically extends the telomerase sequence by several bases.

FIGURE 3-3 Chromosome structure. Each chromosome has three types of specific sequences that facilitate its replication during the cell cycle. Origins of replication are located throughout each chromosome to facilitate DNA synthesis. The centromere holds the duplicated chromosome together and is attached to the mitotic spindle through a protein complex called a kinetochore. Telomere sequences are located at each end of the chromosome and are replicated in a special way to preserve chromosome integrity.

Each chromosome, when stretched out, would span the cell nucleus thousands of times. To facilitate DNA replication and segregation, each chromosome is packaged into a compact structure with the aid of special proteins, including histones. DNA and histones form a repeated array of particles called nucleosomes; each consists of an octomeric core of histone proteins around which the DNA is wrapped twice. The condensed complex of DNA and proteins is known as chromatin. Not only does chromosome packaging facilitate DNA replication and segregation, but it also influences the activity of genes (see later).

DNA Replication and Repair

Before cell division, DNA must be duplicated precisely so that a complete set of chromosomes can be passed to each progeny. DNA replication must occur rapidly, yet with extremely high accuracy. In humans, DNA is replicated at the rate of approximately 50 nucleotides/second, with an error rate of one in every 10⁹ base pair replications. This efficient replication of genetic material requires an elaborate replication machinery consisting of several enzymes. Because each strand of DNA double helix encodes nucleotide sequences complementary to its partner strand, both strands contain identical genetic information and serve as templates for the formation of an entirely new strand.

Eventually, two complete DNA double helices are formed that contain identical genetic information. The fidelity of DNA replication is of critical importance because any mistake, called a mutation, will result in wrong DNA sequences being copied to daughter cells. Mistake in a single base pair is called a point mutation, which results in a missense mutation or nonsense mutation (Fig. 3-4). In a missense mutation, a single amino acid is changed, which can cause changes in the structure of the protein, leading to altered biologic activity. In a nonsense mutation, point mutation results in the replacement of an amino acid codon with a stop codon, leading to premature termination of translation and truncation of the encoded protein. If there is an addition or deletion of a few base pairs, it is called a frameshift mutation, which leads to the introduction of unrelated amino acids or a stop codon. Some mutations are silent and will not affect the function of the organism. Several proofreading mechanisms are used to eliminate mistakes during DNA replication.

FIGURE 3-4 Different types of mutations. Point mutations involve alteration in a single base pair. Small additions or deletions of several base pairs directly affect the sequence of only one gene. A wild-type peptide sequence and the mRNA and DNA encoding it are shown at the top. Altered nucleotides and amino acid residues are enclosed in a box. Missense mutations lead to a change in a single amino acid in the encoding protein. In a nonsense mutation, a nucleotide base change leads to the formation of a stop codon that results in premature termination of translation, thereby generating a truncated protein. Frameshift mutations involve the addition or deletion of any number of nucleotides that is not a multiple of three, thus causing a change in the reading frame.

(From Lodish HF, Baltimore D, Berk A, et al [eds]: Molecular cell biology, ed 3, New York, 1998, Scientific American, p 267.)

RNA and Protein Synthesis

In the early 1940s, geneticists demonstrated that genes specify the structure of individual proteins. The transfer of information from DNA to protein proceeds through the synthesis of an intermediate molecule known as RNA. RNA, like DNA, is made up of a linear sequence of nucleotides composed of four complementary bases. RNA differs from DNA in two respects:

RNA molecules are synthesized from DNA by a process known as DNA transcription, which uses one strand of DNA as a template. DNA transcription differs from DNA replication in that RNA is synthesized as a single-stranded molecule and is relatively short in comparison to DNA. Several classes of RNA transcripts are made, including mRNA, tRNA, and rRNA. Even though all these RNA molecules are involved in the translation of information from RNA to protein, only mRNA serves as the template. RNA synthesis is a highly selective process, with only approximately 1% of the entire human DNA nucleotide sequence transcribed into functional RNA sequences. Although each cell contains the same genetic material, only specific genes are transcribed. RNA transcription is controlled by regulatory proteins that bind to specific sites on DNA, close to the coding sequence of a gene. The complex regulation of gene transcription occurs during development and tissue differentiation and allows differential patterns of gene expression.

After transcription, mRNA is processed for transport out of the nucleus (Fig. 3-5). One important step is RNA splicing, which removes noncoding sequences or introns. Once in the cytoplasm, RNA directs the synthesis of a particular protein through a process called RNA translation. The sequence of nucleotides in mRNA is translated into the amino acid sequence of a protein. Each triplet of nucleotides forms a codon that specifies one amino acid. Because RNA is composed of four types of nucleotides, there are 64 possible codon triplets (4 × 4 × 4). However, only 20 amino acids are commonly found in proteins, so most amino acids are specified by several codons. The rule whereby different codons are translated into amino acids is called the genetic code (Table 3-2).

FIGURE 3-5 Process of gene transcription. Gene expression begins with the binding of multiple protein factors to enhancer sequences and promoter sequences. These factors help form the transcription-initiation complex, which includes the enzyme RNA polymerase and multiple polymerase-associated proteins. The primary transcript (pre-mRNA) includes exon and intron sequences. Post-transcriptional processing begins with changes at both ends of the RNA transcript. At the 5′ end, enzymes add a special nucleotide cap; at the 3′ end, an enzyme clips the pre-mRNA approximately 30 base pairs after the AAUAAA sequence in the last exon. Another enzyme adds a polyadenylate (polyA) tail, which consists of as many as 200 adenine nucleotides. Next, spliceosomes remove the introns by cutting the RNA at the boundaries between exons and introns. The process of excision forms lariats of the intron sequences. The spliced mRNA is then mature and can leave the nucleus for protein translation in the cytoplasm.

(Adapted from Rosenthal N: Regulation of gene expression. N Engl J Med 331:931–933, 1994.)

Table 3-2 The Genetic Code

Protein translation requires a ribosome, which is composed of more than 50 different proteins and several rRNA molecules. Ribosomes bind an mRNA molecule at the initiation codon (AUG) and begin translation in the 5′ to 3′ direction. Protein synthesis ceases once one of the three termination codons is encountered. The rate of protein synthesis is controlled by initiation factors that respond to the external environment, such as growth factor and nutrients. These regulatory factors help coordinate cell growth and proliferation.

Control of Gene Expression

The human body is made up of millions of specialized cells, each performing predetermined functions. This is characteristic of all multicellular organisms. In general, different human cell types contain the same genetic material (i.e., DNA), yet they synthesize and accumulate different sets of RNA and protein molecules. This difference in gene expression determines whether a cell is a hepatocyte or a cholangiocyte. Gene expression can be controlled at six major steps in the synthetic pathway from DNA to RNA to protein.⁴ The first control is at the level of gene transcription, which determines when and how often a given gene is transcribed into RNA molecules. The next step is RNA processing control, which regulates how many mature mRNA molecules are produced in the nucleus. The third step is RNA transport control, which determines which mature mRNA molecules are exported into the cytoplasm where protein synthesis occurs. The fourth step involves mRNA stability control, which determines the rate of mRNA degradation. The fifth step involves translational control, which determines how often mRNA is translated by ribosomes into proteins. The final step is post-translational control, which regulates the function and fate of protein molecules.

Control of gene transcription is the best studied step of regulation for most genes. RNA synthesis begins with assembly and binding of the general transcription machinery to the promoter region of a gene (see Fig. 3-5). The promoter is located upstream of the transcription initiation site at the 5′ end of the gene and consists of a stretch of DNA sequence primarily composed of T and A nucleotides (i.e., the TATA box). The general transcription machinery is composed of several proteins, including RNA polymerase II and general transcription proteins. These general transcription factors are abundantly expressed in all cells and are required for the transcription of most mammalian genes. The rate of assembly of the general transcription machinery to the promoter determines the rate of transcription, which is regulated by gene regulatory proteins. In contrast to the small number of general transcription proteins, there are thousands of different gene regulatory proteins. Most bind to specific DNA sequences, called regulatory elements, to activate or repress transcription.

Gene regulatory proteins are expressed in small amounts in a cell, and different selections of proteins are expressed in different cell types. Similarly, different combinations of regulatory elements are present in each gene to allow differential control of gene transcription. Many human genes have more than 20 regulatory elements; some bind transcriptional activators, whereas others bind transcriptional repressors. Ultimately, the balance between transcriptional activators and repressors determines the rate of transcription, which can vary by a factor of more than 10⁶ between genes that are expressed and those that are repressed. Most regulatory elements are located at a distance (i.e., thousands of nucleotide bases) away from the promoter. These distant regulatory elements are brought into the proximity of the promoter through DNA bending, thus enabling control of promoter activity. In summary, the combination of regulatory elements and the types of gene regulatory proteins expressed determines where and when a gene is transcribed.

Post-translational control is another important step in the regulation of gene expression because most proteins are modified in one form or another.⁵ Modifications such as proteolytic cleavage, disulfide formation, glycosylation, lipidation, and biotinylation allow the protein to achieve the proper structural conformation essential for its biologic activity. The complexity of regulation is greatly increased by additional amino acid modifications that can occur at multiple sites of a protein. Examples of amino acid modification include phosphorylation, acetylation, methylation, ubiquitination, and sumoylation.

Restriction Nucleases

Restriction nucleases are bacterial enzymes that cut the DNA double helix at specific sequences of four to eight nucleotides. More than 400 restriction nucleases have been isolated from different species of bacteria and they recognize over 100 different specific sequences. Commonly used restriction enzymes often recognize a six–base pair palindromic sequence, such as GAATTC. Each restriction nuclease will cut a DNA molecule into a series of specific fragments, which can be joined to other DNA fragments with compatible ends (Fig. 3-6A). By using a combination of different restriction enzymes, a restriction map of each DNA can be created, thus facilitating the isolation of individual genes.

FIGURE 3-6 Amplification of recombinant DNA and amplification by PCR. A, The DNA segment to be amplified is separated from surrounding genomic DNA by cleavage with a restriction enzyme. The enzymatic cuts often produce staggered, or sticky, ends. In the example shown, the restriction enzyme EcoRI recognizes the sequence GAATTC and cuts each strand between G and A; the two strands of genomic DNA are shown as black. The same restriction enzyme cuts the circular plasmid DNA (gray) at a single site, thereby generating sticky ends that are complementary to the sticky ends of the genomic DNA fragment. The cut genomic DNA and the remainder of the plasmid, when mixed together in the presence of a ligase enzyme, form smooth joints on each side of the plasmid–genomic DNA junction. This new molecule, recombinant DNA, is carried into bacteria, which replicate the plasmid as they grow in culture. B, The DNA sequence to be amplified is selected by primers, which are short synthetic oligonucleotides that correspond to sequences flanking the DNA to be amplified. After an excess of primers is added to the DNA, together with a heat-stable DNA polymerase, the strands of both the genomic DNA and primers are separated by heating and allowed to cool. A heat-stable polymerase elongates the primers on either strand, thus generating two new, identical, double-stranded DNA molecules and doubling the number of DNA fragments. Each cycle takes just a few minutes and doubles the number of copies of the original DNA fragment.

(From Rosenthal N: Tools of the trade—recombinant DNA. N Engl J Med 331:315–317, 1994.)

Polymerase Chain Reaction

An ingenious technique to amplify a segment of a DNA sequence in vitro rapidly was developed in 1985 by Saiki and coworkers.⁷ This method, called the polymerase chain reaction (PCR), can enzymatically amplify a segment of DNA a billion-fold. The principle of the PCR technique is illustrated in Figure 3-6B.

To amplify a segment of DNA, two single-stranded oligonucleotides, or primers, must be synthesized, each designed to complement one strand of the DNA double helix and lying on opposite sides of the region to be amplified. The PCR reaction mixture consists of the double-stranded DNA sequence (the template), two DNA oligonucleotide primers (heat stable), DNA polymerase, and four types of deoxynucleotide triphosphate. Each round of amplification involves separation of the DNA template into two single strands, hybridization of the two DNA primers to complementary sequences on each strand of the DNA template, and DNA synthesis downstream of each primer. Each round of PCR requires only approximately 5 minutes and results in a doubling of the double-stranded DNA molecules, which serve as templates for subsequent reactions. After only 32 cycles, more than 1 billion copies of the desired DNA segment are produced. Not only is the PCR technique extremely powerful, but it is also the most sensitive technique to detect a single copy of a DNA or RNA molecule in a sample. To detect RNA molecules, they must first be transcribed into complementary DNA sequences with the enzyme reverse transcriptase. The number of research and clinical applications for PCR continues to grow. In molecular laboratories, PCR has been used for cloning of DNA, engineering of DNA, analysis of allelic sequence variations, and sequencing of DNA. PCR techniques have many clinical applications, including the diagnosis of genetic diseases, assay of infectious agents, and genetic fingerprinting for forensic samples.

DNA Sequencing

DNA encodes information for proteins and, ultimately, the phenotype of a human being. Each gene may contain over 3000 nucleotide bases. Identification of the nucleotide sequences of a fragment of DNA has been made possible through the development of rapid techniques that take advantage of the ability to separate DNA molecules of different lengths, even those differing by only a single nucleotide. Currently, the standard method for sequencing DNA is based on an enzymatic method requiring in vitro DNA synthesis. This method is rapid and can be automated to allow sequencing of large segments of DNA. With these techniques, it is possible to determine the boundaries of a gene and the amino acid sequence of the protein that it codes. Sequencing techniques have enabled the identification and in vitro synthesis of important proteins such as insulin, interferon, hemoglobin, and growth hormones.

DNA Cloning

DNA cloning techniques allow identification of a gene of interest from the human genome. First, the entire DNA content of a cell is cut with a restriction nuclease to generate DNA fragments, which are joined to a self-replicating genetic element (a virus or plasmid). Viruses or plasmids are small circular DNA molecules that occur naturally and can replicate rapidly when introduced into bacterial cells. They are extremely useful tools for amplifying a segment of unknown DNA. With this method, a collection of bacteria plasmids containing the entire human genome can be created. This human DNA library can then be used to identify genes of interest.

DNA Engineering

One of the most important outcomes of recombinant DNA technologies is the ability to generate new DNA molecules of any sequence through DNA engineering. New DNA molecules can be synthesized by the PCR method or by using automated oligonucleotide synthesizers. The PCR method can be used to amplify any known segment of the human genome and to redesign its two ends. Automated oligonucleotide synthesizers enable the rapid production of DNA molecules, up to approximately 100 nucleotides in length. The sequence of such synthetic DNA molecules is entirely determined by the experimenter. Larger DNA molecules are formed by combining two or more DNA molecules that have complementary cohesive ends created by restriction enzyme digestion. One powerful application of DNA engineering is the synthesis of large quantities of cellular proteins for medical applications. Most cellular proteins are produced in small amounts in human cells, making it difficult to purify and study these proteins. However, with DNA engineering, it is possible to place a human gene into an expression vector that is introduced into bacterial, yeast, insect, or mammalian cells to produce a large quantity of protein. The protein can easily be purified and used for scientific studies or clinical applications. Medically useful proteins, such as human insulin, growth hormone, interferon, and viral antigens for vaccines, have been produced by engineering expression vectors containing these genes of interest.

DNA engineering techniques are also important for solving problems in cell biology. One of the fundamental challenges of cell biology is to identify the biologic functions of the protein product of a gene. With the use of DNA engineering techniques, it is now possible to alter the coding sequence of a gene to alter the functional properties of its protein product or the regulatory region of a gene and thus produce an altered pattern of its expression in the cell. The coding sequence of a gene can be changed in such subtle ways that the protein encoded by the gene has only one or a few alterations in its amino acid sequence. The modified gene is then inserted into an expression vector and transfected into the appropriate cell type to examine the function of the redesigned protein. With this strategy, one can analyze which parts of the protein are important for fundamental processes, such as protein folding, enzyme activity, and protein-ligand interactions.