History of Molecular Genetics and Biotechnology Help (page 3)
History of Molecular Genetics and Biotechnology
Prior to discovery that the chemical nature of the gene was DNA, in the early 1900s the gene was an abstract unit of heredity. This period in history is referred to as classical or formal genetics. Classical genetics was extremely successful in elucidatingmany basic biological principles without understanding the physical nature of the gene. The era of molecular genetics began with the discovery of DNA structure in 1953. In the late 1970s, yet another milestone in genetics was reached when researchers found that they could manipulate DNA molecules in a test tube (in vitro), essentially "cloning" the first gene. This discovery led to further molecular genetic advances including recombinant DNA technology (genetic engineering) and spawned the biotechnology industry known today.
The histories of most scientific disciplines are generally characterized by relatively long periods of stagnation punctuated by bursts of rapid progress. Most of these flurries of research are initiated by new technical developments. This is certainly true of biochemistry and molecular biology. At least three major areas of technology have been influential in this respect: (1) instrumentation and techniques, (2) radioactive and fluorescent labels, and (3) nucleic acid enzymology. Beyond these technological influences, the use of computer technology to process biological information (i.e., DNA nucleotide sequences or protein amino acid sequences) is driving new discoveries in molecular genetics and biotechnology. In fact, several new fields within molecular genetics—Bioinformatics, Genomics, and Proteomics—have been created. Each of these fields integrates the use of computer hardware and software to store, process and analyze biological information, such as DNA or protein sequences.
The Analytical Ultracentrifuge
The analytical ultracentrifuge was developed in the 1920s by Theodor Svedberg. The sedimentation rate of a substance during ultracentrifugation is mainly a function of its density and secondarily of its shape. The unit of sedimentation (S, in honor of Svedberg) is an expression of these parameters. The centrifuge has been modified for isolating organelles such as nuclei, ribosomes, mitochondria, and chloroplasts. It can be used for determining the minimum number of kinds of macromolecules in a biological specimen and for estimating the molecular weights of macromolecules.
The Electron Microscope
The electron microscope was invented in the 1930s, and eventually enabled the direct visualization not only of cellular substructures but also of viruses and macromolecules. Circular genetic maps of microorganisms have been shown by electron microscopy to have a corresponding circular physical structure. Multiple ribosomes attached to an mRNA molecule (polysomes) have also been visualized by this instrument.
Electrophoresis is a technique that separates molecules according to their shapes, net charges, and molecular weights in an electric field, usually on solid or semisolid support media such as paper or agarose. This technique was first used in 1949 to differentiate sickle-cell hemoglobin from normal hemoglobin. Protein sequence analysis later revealed that the difference in electrophoretic mobilities of proteins was due to a single amino acid change in the β-chains. Electrophoresis is now used in a variety of DNA and protein analysis applications. Agarose gels are usually employed to isolate and estimate the size of DNA fragments, and DNA sequencing results are obtained using similar gel matrices. Proteins are often analyzed for their size and charge using polyacrylamide gels. Electrophoresis has been extensively used to differentiate isozymes, i.e., proteins possessing the same enzymatic properties but differing in primary structure. Techniques have also been developed for two-dimensional gel electrophoresis (2-D) to further separate mixtures of proteins that might be of similar sizes (or mass) but have slightly different charges (or isoelectric points). These types of gels are first run in one dimensions using size as the separator then, using pH (an indicator of charge), proteins are separated from one another at a 90 degree angle to the size dimension. Complex images of protein "spots" are created using this technique. Researchers can physically isolate the protein from the gel matrix and subject it to mass spectrometry to determine the sequence of some of the amino acids within the spot.
X-ray-diffraction data from crystalline materials has been critical for the elucidation of three-dimensional shapes of nucleic acids (e.g., DNA, tRNAs) and proteins (e.g., myoglobins, viral capsomeres, enzymes). This technique has been expanded to include the determination of multi-subunit molecules.
During the mid-1940s and early 1950s, various forms of chromatography were perfected, enabling molecules to be separated by differences in solubilities in organic solvents, electrical charge, molecular weight, and specific binding properties for the support medium, or combinations of these factors. Erwin Chargaff used paper chromatography to determine the base compositions of DNAs from various sources. He found that the molecular ratio of adenine was equivalent to that of thymine and the ratio of guanine equals that of cytosine. This was a vital clue for James Watson and Francis Crick in their search for the structure of DNA.
Automated equipment is now available for doing many repetitive biochemical tasks. DNA synthesizers can be programmed to make oligonucleotide (many nucleotides) sequences of any desired composition. Automated instrumentation is now available for isolating DNA, and sequencing DNA or protein fragments, as well as for performing enzyme assays and other tasks. Computer software has been developed to interpret data from electropherograms, and to search databases for similar or identical sequences (bioinformatics).
Several techniques have been developed to separate, rejoin, synthesize, or break nucleic acid molecules. Separation of the complementary chains of a DNA molecule is known as denaturation. DNA is denatured if placed in alkali (0.2N NaOH) or when boiled. The latter process is referred to as melting. Separation of DNA strands can be detected by spectrophotometric instruments; optical density (OD) or absorbance at 260 nm increases during the melting process. The temperature at which the increase in OD260 is 50% of that attained when strand separation is complete is known as the melting temperature (Tm). Because G and C base-pair by three hydrogen bonds, whereas A and T base-pair by two hydrogen bonds, the higher the G-C content in DNA the higher the melting temperature. Melting is enhanced where there are clusters of A's and T's, and also when all the purines (A, G) are on one strand and all the pyrimidines (T, C) are on the other strand.
If DNA is boiled and then quickly cooled, the strands will remain single; if cooled slowly, complementary strands will base-pair and reform double-helical DNA molecules. This process is called renaturation or annealing. Hybrid DNA-RNA molecules can be produced by analogous processes from single strands. RNA can be totally hydrolyzed to nucleotides by exposure to high pH (alkali). This property can be used to purify DNA from amixture of DNA and RNA. Single-stranded DNA will bind to membranous filters made of nitrocellulose; RNA will pass through such filters. However, if single-stranded RNA is complementary to nitrocellulose-bound single strands of DNA, it will form DNA-RNA hybrid molecules and be retained by such a filter. This technique is used in various blotting procedures, such as Southern, Northern and Western blotting (see Section 7 later in this chapter).
There are two main methods for breaking long DNA molecules into fragments of suitable size for sequencing or for recombinant DNA techniques: (1) mechanical shearing and (2) restriction end onuclease treatment. If a solution of DNA is subjected to the stirring forces of a Waring blender or forced through a narrow tube, the ends of long DNA strands will usually move at different speeds; this stretches the DNA and tends to break it. This phenomenon is called shearing. The higher the stirring speed, or velocity, of flow through an orifice, the greater the shearing force. Shearing can also be achieved by exposure of a solution of DNA to ultrasound. The effectiveness of any shearing force increases with molecular size of the DNA, but decreases with concentration because entanglement of DNA molecules reduces the effective stretching. Restriction endonuclease treatment employs the use of enzymes that break the bonds between specific base-pair sequences of DNA. These enzymes will be discussed later in this chapter in the sections Nucleic Acid Enzymology, and Recombinant DNA Technology.
Radioactive elements have historically been used as highly sensitive labels for detecting minute amounts of specific macromolecules. However, recent advances in fluorescent molecule technology (i.e., the development of fluorescent dyes to tag molecules of interest) have led to the replacement of many radioactive tracers by these fluorescent molecules. Fluorescent molecules are often safer to use, easier to dispose of and better for the environment.
EXAMPLE 12.1 A. D. Hershey and M. Chase differentially labeled the nucleic acid and the protein components of T2 phages. They used radioactive 32P in place of normal 32P to label DNA; radioactive 35S was used in place of normal 35S to label protein (cysteine and methionine are two amino acids that contain sulfur). Since there is no phosphorus in phage proteins and no sulfur in nucleic acids, the fate of both viral components could be followed during the viral life cycle. After allowing the phages to become attached to sensitive Escherichia coli host cells, the mixture was subjected to the shearing forces of a Waring blender. The mixture was centrifuged to sediment the cells and then activity characteristic of each radionuclide was assayed in the pellet and in the supernatant fluid. All of the 32P activity was found in the bacterial pellet and virtually all of the 35S was found in the supernatant. 32P was found in some progeny phages, but no 35S was found. The inference is that phages inject their DNA into host cells. Blender treatment shears the phage tail fibers from receptor sites on host cells; the empty phage protein capsids (ghosts) are therefore left free in the supernate. Semiconservative replication from the infecting 32P-labeled DNA caused some progeny phages to be released with one of the original radioactively labeled infecting strands. This experiment was the first to demonstrate that DNA, and not protein, is the genetic material in phages.
DNA labeled with radioactive elements can be revealed in a photographic technique called autoradiography. A preparation of DNA on a filter paper can be covered with a photographic film. As the radioactive elements undergo radioactive decay, they release charged particles that cause a chemical reaction on the film. After development of the X-ray film, the location of the labeled DNA is revealed by dark spots.
A radioactive isotope of phosphorus (32P) is also widely used to label nucleic acids; it emits a strong beta particle and has a half-life of 14.3 days. An instrument called a scintillation counter is used to detect radioactive disintegrations.
Any organic substance can be labeled with radioactive carbon (14C). This isotope emits a weak beta particle and has a relatively long half-life of 5730 years. All living organisms incorporate a predictable amount of 14C while alive. After death, 14C decays to 14N at the predictable rate of its half-life. This knowledge allows the dating of organic remains from the time of death up to about 40,000 years before the present.
Radioactive iodine (125I) has a half-life of about 60 days, emits γ-rays, and is used to label proteins. Highenergy γ-rays (gamma) can be detected by a crystal scintillation counter. A liquid scintillation counter must be used to detect weaker beta particles, although it can also detect γ-rays. This isotope is easily coupled to the amino acid tyrosine. Radioactive sulfur (35S) is used to label the amino acids cysteine and methionine; 35S can also be used to label nucleic acids. 35S is more desirable than 32P for most autoradiography because it has a half-life of 87.1 days and emits a much weaker beta particle that gives sharper bands. It is much less hazardous to handle than 32P and poses less waste-disposal problems.
Even nonradioactive isotopes (e.g., 15N) have been useful in solving fundamental problems in molecular biology.
Fluorescent molecules contain a fluorophore that absorbs and emits light at particular wavelengths characteristic to the specific fluorophore. This emitted light can be detected and recorded by a number of different instruments (microscopes, scanners, spectrophotometers, etc). Fluorescent molecules (probes) can be attached to a variety of biological molecules in order to detect and study them. DNA, proteins, and lipids, as well as specific molecules, such as actin and tubulin, can be labeled with a fluorescent molecule. It is possible to use two or more fluorescent probes in one experiment if their emitted light occurs at different wavelengths. For example, different molecules within a cell can each be labeled with a different fluorescent molecule and the activities and amounts of each molecule can be detected and compared at the same time. During DNA sequencing reactions, each nucleotide is labeled with a different fluorescent molecule, allowing strands terminating with a different nucleotide to be differentiated yet detected simultaneously (see section on DNA Sequencing later in this chapter).
Nucleic Acid Enzymology
Nucleases are enzymes that hydrolyze, or break, the phosphodiester bonds that hold the nucleotides together. Those that remove terminal nucleotides one at a time are called exonucleases; those that break the sugar-phosphate backbone at nonterminal sites are called endonucleases.Adeoxyribonuclease (DNase) degrades DNA molecules; a ribonuclease (RNase) degrades RNA molecules. Some endonucleases act nonspecifically, cleaving the phosphodiester bonds at different unspecified nucleotide sequences. Others, such as the restriction endonucleases, break the bonds only at specific DNA sequences, called recognition sites. There are three classes of restriction endonucleases: Types I, II and III. Types I and III do not have qualities useful for recombinant DNA technology (i.e., they cut DNA at random sites). Type II enzymes recognize and bind to a specific double-stranded DNA sequence. Once bound, they cleave the phosphodiester backbone of each strand at or near (within 20 bp) the site. The recognition sites of these enzymes are symmetrical and commonly consist of 4–8 bp that are either continuous (e.g.,GAATTC) or interrupted (e.g., CANTG, where N is any base). The symmetry occurs around a midpoint, or axis of symmetry, formed on opposite DNA strands by inverted base sequences called palindromes. These enzymes occur naturally in bacteria, and hundreds of enzymes have been isolated and characterized and are available for use by genetic engineers. Thus, Type II enzymes are the most commonly used in recombinant DNA technology today.
Restriction enzymes play a role in defending bacteria against invasion by foreign nucleic acids, such as viruses. The host bacterial cell restriction enzyme systems can recognize the invading DNA as foreign and destroy it (see Example 11.1). Host DNA may be protected by specific base-pair modifications carried out by host-specific methylase enzymes. Thus, the restriction enzyme works in conjunction with the modifying enzyme and together the system is referred to a restriction-modification system. Foreign DNA is recognized as such because its restriction sites are unmodified. Restriction endonucleases are named after the bacterial species or strain from which they were derived. For example, an enzyme from Providentia stuartii, will have the name Pst derived from the first letter of the genus (P) and the first two letters of the specific epithet (st). This name is italicized to honor the scientific name of the bacterium and can be followed by roman numerals to indicate that it is one of several enzymes isolated from that particular bacterial strain, e.g. PstI, PstII (the numerals are not italicized). Occasionally, a letter derived from the specific bacterial strain follows the name. For example, the enzyme EcoRI was derived from E. coli strain RY13 and Hind III was derived from Haemophilus influenzae strain Rd. If this is the case neither the strain letter nor the numerals are italicized.
EXAMPLE 12.2 The EcoRI restriction enzyme cuts bonds on the upper and lower strands within the palindromic DNA sequence at the arrows shown below.
Notice that the 5' to 3' nucleotide sequence within the palindrome is the same starting from the 5' (pronounced five prime) ends on both strands of the DNA (the palindrome). EcoRI cuts the DNA molecule in a staggered fashion, leaving "overhang,'' "cohesive'' or "sticky'' ends. In the above example, the overhangs are called 5' overhangs because the unbonded nucleotides have a 5' end. Other restriction enzymes can leave a 3' overhanging end. Another restriction endonuclease (HaeIII), derived from the bacterium Haemophilus aegypticus, snips DNA as shown below. Note that this enzyme cuts both strands at opposite bonds, leaving "blunt" ends.
Restriction enzyme maps for any given DNA segment (linear or circular) can be constructed. Such maps show the location of various restriction endonuclease recognition sites on the DNA fragment. The sizes of the various restriction fragments can be expressed by molecular weight, but more commonly they are given in terms of number of base pairs (bp) or thousands of base pairs (kilobases, kb).
EXAMPLE 12.3 A linear 10,000-bp fragment of DNA has an unknown number of EcoRI restriction enzyme sites. You perform a restriction digestion of this fragment with EcoRI and obtain the following result: three fragments of sizes 5, 3, and 2 kb. These results suggest that there are two EcoRI sites present in this molecule. One possible arrangement of sites (arrows) is shown below. There are two other possible arrangements (not shown).
If this molecule were circular, as are bacterial plasmids commonly used in recombinant DNA technology, only two fragments (3 and 7 kb) would be obtained. The 7 kb fragment is a result of the joining of the 5 and 2 kb segments (see diagram below).
Many other enzymes that are involved in the replication, recombination, repair, modification, transcription, and translation of nucleic acids are utilized in recombinant DNA technology. In particular, DNA synthesis enzymes, known as DNA polymerases, have become very useful for synthesizing copies of DNA molecules in vitro (in a test tube) in a process called the polymerase chain reaction, or PCR (see later section).
Practice problems for these concepts can be found at:
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