Deoxyribonucleic Acid (DNA)

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Deoxyribonucleic acid (DNA)

Deoxyribonucleic acid (DNA), "the master molecule," is a natural polymer which encodes the genetic information required for the growth, development, and reproduction of an organism . Found in all cells, it consists of chains of units called nucleotides. Each nucleotide unit contains three components: the sugar deoxyribose, a phosphate group, and a nitrogen-containing amine or base with a ring-type structure. The base component can be any of four types: adenine, cytosine, guanine or thymine.

DNA molecules are very long and threadlike. They consist of two polymeric strands twisted about each other into a spiral shape known as a double helix , which resembles two intertwined circular staircases. DNA is found within the cell nucleus in the chromosomes, which are extremely condensed structures in which DNA is associated with proteins . Each species contains a characteristic number of chromosomes in their cells. In humans, every cell contains 46 chromosomes (except for egg and sperm cells which contain only 23). The total genetic information in a cell is called its genome .

The fundamental units of heredity are genes. A gene is a segment of a DNA molecule that encodes the information necessary to make a specific protein. Proteins are the "workhorses" of the cell. These large, versatile molecules serve as structural components: they transport molecules in and out of cells, catalyze cellular reactions, and recognize and eliminate invaders. Imagine a community in which the trash collectors, goods distributors, manufacturers, and police are all on strike, and you get an idea of the importance of proteins in the life of a cell.

DNA not only encodes the "blueprints" for cellular proteins but also the instructions for when and where they will be made. For example, the oxygen carrier hemoglobin is made in red blood cells but not in nerve cells, though both contain the same total genetic content.

Thus, DNA also contains the information necessary for regulating how its genetic messages are used.

Human cells are thought to contain between 50,000 and 100,000 genes. Except in the case of identical twins, a comparison of the genes from different individuals always reveals a number of differences. Therefore, each person is genetically unique. This is the basis of DNA "fingerprinting," a forensic procedure used to match DNA collected from a crime scene with that of a suspect.

Through the sum of their effects, genes direct the function of all organs and systems in the body. Defects in the DNA of just one gene can cause a genetic disorder which results in disease because the protein encoded by the defective gene is abnormal. The abnormal hemoglobin produced by people afflicted with sickle cell anemia is an example. Defects in certain genes called oncogenes, which regulate growth and development, give rise to cancer . Only about 100 genes are thought to be oncogenes. Therefore, defects in DNA can affect the two kinds of genetic information it carries, messages directing the manufacture of proteins and information regulating the expression, or carrying out, of these messages.

History

Prior to the discovery of the nucleic acids, the Austrian monk Gregor Mendel (1822-1884) worked out the laws of inheritance by the selective breeding of peaplants. As early as 1865 he proposed that "factors" from each parent were responsible for the inheritance of certain characteristics in plants. The Swiss biochemist Friedrich Miescher (1844-1895) discovered the nucleic acids in 1868 in nuclei isolated from pus cells scraped from surgical bandages. However, research on the chemical structure of nucleic acids lagged until new analytical techniques became available in the mid twentieth century. With the advent of these new methods came evidence that the nucleic acid we now know as DNA. DNA was present in the nuclei of all cells and evidence about the chemical structure of its nucleotide components.

Despite knowledge of the chemical structure of nucleotides and how they were linked together to form DNA, the possibility that DNA was the genetic material was regarded as unlikely. As late as the mid twentieth century, proteins were thought to be the molecules of heredity because they appeared to be the only cellular components diverse enough to account for the large variety of genes. In 1944, Oswald Avery (1877-1955) and his colleagues showed that non-pathogenic strains of pneumococcus, the bacterium that causes pneumonia, could become pathogenic (disease-causing) if treated with a DNA-containing extract from heat-killed pathogenic strains. Based on this evidence, Avery concluded that DNA was the genetic material. However, widespread acceptance of DNA as the bearer of genetic information did not come until a report by other workers in 1952 that DNA, not protein, enters a bacterial cell infected by a virus . This showed that the genetic material of the virus was contained in its DNA, confirming Avery's hypothesis.

Shortly afterwards in 1953, James Watson (1928-) and Francis Crick (1916-) proposed their double helix model for the three-dimensional structure of DNA. They correctly deduced that the genetic information was encoded in the form of the sequence of nucleotides in the molecule. With their landmark discovery began an era of molecular genetics in biology . Eight years later investigators cracked the genetic code. They found that specific trinucleotide sequences—sequences of three nucleotides—are codes for each of 20 amino acids, the building blocks of proteins.

In 1970 scientists found that bacteria contained restriction enzymes molecular "scissors" that recognize a particular sequence of 4-8 nucleotides and will always cut DNA at or near that sequence to yield specific (rather than random ), consistently reproducible DNA fragments. Two years later it was found that the bacterial enzyme DNA ligase could be used to rejoin these fragments. This permitted scientists to construct "recombinant" DNA molecules; that is, DNA molecules composed of segments from two different sources, even from different organisms. With the availability of these tools, genetic engineering became possible and biotechnology began.

By 1984 the development of DNA fingerprinting allowed forensic chemists to compare DNA samples from a crime scene with that of suspects. The first conviction using this technique came in 1987. Three years later doctors first attempted to treat a patient unable to produce a vital immune protein using gene therapy . This technique involves inserting a portion of DNA into a patient's cells to correct a deficiency in a particular function. The Human Genome Project also began in 1990. The aim of this project is to determine the nucleotide sequence in DNA of the entire human genome, which consists of about three billion nucleotide pairs. In 2001, researchers announced the completion of the sequencing of a human genome, promising refinement by 2003.

Structure

Deoxyribose, the sugar component in each nucleotide, is so called because it has one less oxygen atom than ribose, which is present in ribonucleic acid (RNA) . Deoxyribose contains five carbon atoms, four of which lie in a ring along with one oxygen atom. The fifth carbon atom is linked to a specific carbon atom in the ring. A phosphate group is always linked to deoxyribose via a chemical bond between an oxygen atom in the phosphate group and the carbon atom in deoxyribose by a chemical bond between a nitrogen atom in the base and a specific carbon atom in the deoxyribose ring.

The nucleotide components of DNA are connected to form a linear polymer in a very specific way. A phosphate group always connects the sugar component of a nucleotide with the sugar component of the next nucleotide in the chain. Consequently, the first nucleotide bears an unattached phosphate group, and the last nucleotide has a free hydroxyl group. Therefore, DNA is not the same at both ends. This directionality plays an important role in the replication of DNA.

DNA molecules contain two polymer chains or strands of nucleotides and so are said to be double-stranded. (In contrast, RNA is typically single-stranded.) Their shape resembles two intertwined spiral staircases in which the alternating sugar and phosphate groups of the nucleotides compose the sidepieces. The steps consist of pairs of bases, each attached to the sugars on their respective strands. The bases are held together by weak attractive forces called hydrogen bonds. The two strands in DNA are antiparallel, which means that one strand goes in one direction (first to last nucleotide from top to bottom) and the other strand goes in the opposite direction (first to last nucleotide from bottom to top).

Because the sugar and phosphate components which make up the sidepieces are always attached in the same way, the same alternating phosphate-sugar sequence repeats over and over again. The bases attached to each sugar may be one of four possible types. Because of the geometry of the DNA molecule, the only possible base pairs that will fit are adenine (A) paired with thymine (T), and cytosine (C) paired with guanine (G).

The DNA in our cells is a masterpiece of packing. The double helix coils itself around protein cores to form nucleosomes. These DNA-protein structures resemble beads on a string. Flexible regains between nucleosomes allows these structures to be wound around themselves to produce an even more compact fiber. The fibers can then be coiled for even further compactness. Ultimately, DNA is paced into the highly condensed chromosomes. If the DNA in a human cell is stretched, it is approximately 6 ft (1.82 m) long. If all 46 chromosomes are laid end-to-end, their total length is still only about eight-thousandths of an inch. This means that DNA in chromosomes is condensed about 10,000 times more than that in the double helix. Why all this packing? The likely answer is that the fragile DNA molecule would get broken in its extended form. Also, if not for this painstaking compression, the cell might be mired in its own DNA.

Function

DNA directs a cell's activities by specifying the structures of its proteins and by regulating which proteins and how much are produced, and where. In so doing, it never leaves the nucleus. Each human cell contains about 6 ft (2 m) of highly condensed DNA which encodes some 50,000–100,000 genes. If a particular protein is to be made, the DNA segment corresponding to the gene for that protein acts as a template, a pattern, for the synthesis of an RNA molecule in a process known as transcription. This messenger RNA molecule travels from the nucleus to the cytoplasm where it in turn acts as the template for the construction of the protein by the protein assembly apparatus of the cell. This latter process is known as translation and requires an adaptor molecule, transfer RNA, which translates the genetic code of DNA into the language of proteins.

Eventually, when a cell divides, its DNA must be copied so that each daughter cell will have a complete set of genetic instructions. The structure of DNA is perfectly suited to this process. The two intertwined strands unwind, exposing their bases, which then pair with bases on free nucleotides present in the cell. The bases pair only in a certain combination; adenine (A) always pairs with thymine (T) and cytosine (C) always pairs with guanine (G). The sequence of bases along one strand of DNA therefore determines the sequence of bases in the newly forming complementary strand. An enzyme then joins the free nucleotides to complete the new strand. Since the two new DNA strands that result are identical to the two originals, the cell can pass along an exact copy of its DNA to each daughter cell.

Sex cells, the eggs and sperm, contain half the number of chromosomes as other cells. When the egg and sperm fuse during fertilization , they form the first cell of a new individual with the complete complement of DNA—46 chromosomes. Each cell (except the sex cells) in the new person carries DNA identical to that in the fertilized egg cell. In this way the DNA of both parents is passed from one generation to the next. Thus, DNA plays a crucial role in the propagation of life.

Replication of DNA

DNA replication , the process by which the double-stranded DNA molecule reproduces itself, is a complicated process, even in the simplest organisms. DNA synthesis—making new DNA from old—is complex because it requires the interaction of a number of cellular components and is rigidly controlled to ensure the accuracy of the copy, upon which the very life of the organism depends. This adds several verification steps to the procedure. Though the details vary from organism to organism, DNA replication follows certain rules that are universal to all.

DNA replication (duplication, or copying) is always semi-conservative. During DNA replication the two strands of the parent molecule unwind and each becomes a template for the synthesis of the complementary strand of the daughter molecule. As a result both daughter molecules contain one new strand and one old strand (from the parent molecule), hence the term semi-conservative. The replication of DNA always requires a template, an intact strand from the parent molecule. This strand determines the sequence of nucleotides on the new strand. Wherever the nucleotide on the template strand contains the base A, then the nucleotide to be added to the daughter strand at that location must contain the base T. Conversely, every T must find an A to pair with. In the same way, Gs and Cs will pair with each other and with no other bases.

Replication begins at a specific site called the replication origin when the enzyme DNA helicase binds to a portion of the double stranded helix and "melts" the bonds between base pairs. This unwinds the helix to form a replication fork consisting of two separated strands, each serving as a template. Specific proteins then bind to these single strands to prevent them from repairing. Another enzyme, DNA polymerase, proceeds to assemble the daughter strands using a pool of free nucleotide units which are present in the cell in an "activated" form.

High fidelity in the copying of DNA is vital to the organism and, incredibly, only about one error per one trillion replications ever occurs. This high fidelity results largely because DNA polymerase is a "self-editing" enzyme. If a nucleotide added to the end of the chain mismatches the complementary nucleotide on the template, pairing does not occur. DNA polymerase then clips off the unpaired nucleotide and replaces it with the correct one.

Occasionally errors are made during DNA replication and passed along to daughter cells. Such errors are called mutations. They have serious consequences because they can cause the insertion of the wrong amino acid into a protein. For example, the substitution of a T for an A in the gene encoding hemoglobin causes an amino acid substitution which results in sickle cell anemia . To understand the significance of such mutations requires knowledge of the genetic code.

The genetic code

Genetic information is stored as nucleotide sequences in DNA (or RNA) molecules. This sequence specifies the identity and position of the amino acids in a particular protein. Amino acids are the building blocks of proteins in the same way that nucleotides are the building blocks of DNA. However, though there are only four possible bases in DNA (or RNA), there are 20 possible amino acids in proteins. The genetic code is a sort of "bilingual dictionary" which translates the language of DNA into the language of proteins. In the genetic code the letters are the four bases A, C, G, and T (or U instead of T in RNA). Obviously, the four bases of DNA are not enough to code for 20 amino acids. A sequence of two bases is also insufficient, because this permits coding for only 16 of the 20 amino acids in proteins. Therefore, a sequence of three bases is required to ensure enough combinations or "words" to code for all 20 amino acids. Since all words in this DNA language, called codons , consist of three letters, the genetic code is often referred to as the triplet code.

Each codon specifies a particular amino acid. Because there are 64 possible codons (for example 43 = 64 different 3-letter "words" can be generated from a 4-letter "alphabet") and only 20 amino acids, several different codons specify the same amino acid, so the genetic code is said to be degenerate. However, the code is unambiguous because each codon specifies only one amino acid. The sequence of codons are not interrupted by "commas" and are always read in the same frame of reference, starting with the same base every time. So the "words" never overlap.

Since DNA never leaves the nucleus, the information it stores is not transferred to the cell directly. Instead, a DNA sequence must first be copied into a messenger RNA molecule, which carries the genetic information from the nucleus to protein assembly sites in the cytoplasm. There it serves as the template for protein construction. The sequences of nucleotide triplets in messenger RNA are also referred to as codons.

Four codons serve special functions. Three are stop codons that signal the end of protein synthesis. The fourth is a start codon which establishes the "reading frame" in which the message is to be read. For example, suppose the message is PAT SAW THE FAT RAT. If we overshoot the reading frame by one "nucleotide," we obtain ATS AWT HEF ATR AT, which is meaningless.

The genetic code is essentially universal. This means that a codon which specifies the amino acid tryptophan in bacteria also codes for it in man. The only exceptions occur in mitochondria and chloroplasts and in some protozoa . (Mitochondria and chloroplasts are sub-cellular compartments which are the sites of respiration in animals and plants, respectively, and contain some DNA.) The structure of the genetic code has evolved to minimize the effect of mutations. Changes in the third base of a codon do not necessarily result in a change in the specified amino acid during protein synthesis. Furthermore, changes in the first base in a codon generally result in the same or at least a similar amino acid. Studies of amino acid changes resulting from mutations have shown that they are consistent with the genetic code. That is, amino acid changes resulting from mutations are consistent with expected base changes in the corresponding codon. These studies have confirmed that the genetic code has been deduced correctly by demonstrating its relevance in actual living organisms.

Expression of genetic information

Genetic information flows from DNA to RNA to protein. Ultimately, the linear sequence of nucleotides in DNA directs the production of a protein molecule with a characteristic three dimensional structure essential to its proper function. Initially, information is transcribed from DNA to RNA. The information in the resulting messenger RNA is then translated from RNA into protein by small transfer RNA molecules.

In some exceptional cases the flow of genetic information from DNA to RNA is reversed. In retroviruses, such as the AIDS virus, RNA is the hereditary material. An enzyme known as reverse transcriptase makes a copy of DNA using the virus' RNA as a template. In still other viruses which use RNA as the hereditary material, DNA is not involved in the flow of information at all.

Most cells in the body contain the same DNA as that in the fertilized egg. (Some exceptions to this are the sex cells, which contain only half of the normal complement of DNA, as well as red blood cells which lose their nucleus when fully developed.) Some "housekeeping" genes are expressed in all cells because they are involved in the fundamental processes required for normal function. (A gene is said to be expressed when its product, the protein it codes for, is actively produced in a cell.) For example, since all cells require ribosomes , structures which function as protein assembly lines, the genes for ribosomal proteins and ribosomal RNA are expressed in all cells. Other genes are only expressed in certain cell types, such as genes for antibodies in certain cells of the immune system . Some are expressed only during certain times in development. How is it that some cells express certain genes while others do not, even though all contain the same DNA? A complete answer to this question is still in the works. However, the main way is by controlling the start of transcription. This is accomplished by the interaction of proteins called transcription factors with DNA sequences near the gene. By binding to these sequences transcription factors may turn a gene on or off.

Another way is to change the rate of messenger RNA synthesis. Sometimes the stability of the messenger RNA is altered. The protein product itself may be altered, as well as its transport or stability. Finally, gene expression can be altered by DNA rearrangements. Such programmed reshuffling of DNA is the means of generating the huge assortment of antibody proteins found in immune cells.

Genetic engineering and recombinant DNA

Restriction enzymes come from microorganisms . Recall that they recognize and cut DNA at specific base pair sequences. They cleave large DNA molecules into an assortment of smaller fragments ranging in size from a few to thousands of base pairs long, depending on how often and where the cleavage sequence appears in the original DNA molecule. The resulting fragments can be separated by their size using a technique known as electrophoresis . The fragments are placed at the top of a porous gel surrounded by a solution which conducts electricity . When a voltage is applied, the DNA fragments move towards the bottom of the gel due to the negative charge on their phosphate groups. Because it is more difficult for the large fragments to pass through the pores in the gel, they move more slowly than the smaller fragments.

DNA fragments isolated from a gel in this way can be joined with DNA from another source, either of the same or a different species, into a new, recombinant DNA molecule by enzymes. Usually, such DNA fragments are joined with DNA from subcellular organisms—"parasites" that live inside another organism but have their own DNA. Plasmids and viruses are two such examples. Viruses consist only of nucleic acids encapsulated in a protein coat. Though they can exist outside the cell, they are inactive. Inside the cell, they take over its metabolic machinery to manufacture more virus particles, eventually destroying their host. Plasmids are simpler than viruses in that they never exist outside the cell and have no protein coat. They consist only of circular double-stranded DNA. Plasmids replicate their DNA independently of their hosts. They are passed on to daughter cells in a controlled way as the host cell divides.

Cells that contain the same recombinant DNA fragment are clones. A clone harboring a recombinant DNA molecule that contains a specific gene can be isolated and identified by a number of techniques, depending upon the particular experiment. Thus, recombinant DNA molecules can be introduced into rapidly growing microorganisms, such as bacteria or yeast , to produce large quantities of medically or commercially important proteins normally present only in scant amounts in the cell. For example, human insulin and interferon have been produced in this manner.

In recent years a technique has been developed which permits analysis of very small samples of DNA without repeated cloning, which is laborious. Known as the polymerase chain reaction, this technique involves "amplifying" a particular fragment of DNA by repeated synthesis using the enzyme DNA polymerase. This method can increase the amount of the desired DNA fragment by a million-fold or more.

Resources

books

Berg, Paul, and Maxine Singer. Dealing with Genes—The Language of Heredity. Mill Valley, CA: University Science Press, 1992.

Blueprint for Life. Journey Through the Mind and Body series. Alexandria, VA: Time-Life Books, 1993.

Lee, Thomas F. Gene Future. New York: Plenum Publishing Corporation, 1993.

Rosenfeld, Israel, Edward Ziff, and Borin Van Loon. DNA forBeginners. New York: Writers and Readers Publishing Cooperative Limited, 1983.

Sofer, William H. Introduction to Genetic Engineering. Stoneham, MA: Butterwoth-Heineman, 1991.

Patricia V. Racenis

KEY TERMS

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Codon: —The base sequence of three consecutive nucleotides on DNA (or RNA) that codes for a particular amino acid or signals the beginning or end of a messenger RNA molecule.
Cytoplasm: —All the protoplasm in a living cell that is located outside of the nucleus, as distinguished from nucleoplasm, which is the protoplasm in the nucleus.
Gene: —A discrete unit of inheritance, represented by a portion of DNA located on a chromosome. The gene is a code for the production of a specific kind of protein or RNA molecule, and therefore for a specific inherited characteristic.
Genetic code: —The blueprint for all structures and functions in a cell as encoded in DNA.
Genetic engineering: —The manipulation of the genetic content of an organism for the sake of genetic analysis or to produce or improve a product.
Genome: —The complete set of genes an organism carries.
Nucleotide: —The basic unit of DNA. It consists of deoxyribose, phosphate, and a ring-like, nitrogen-containing base.
Nucleus: —A compartment in the cell which is enclosed by a membrane and which contains its genetic information.
Replication: —The synthesis of a new DNA molecule from a pre-existing one.
Transcription: —The process of synthesizing RNA from DNA.
Translation: —The process of protein synthesis.