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|Neuropsychopharmacology: The Fifth Generation of Progress|
Basic Concepts and Techniques of Molecular Genetics
Basic Concepts and Techniques of Molecular Genetics
Samuel H. Barondes
Psychopharmacology has changed a great deal in the more than three decades since this series of books was initiated. Whereas the early emphasis was on direct studies of the effects of drugs on human and animal behavior, interest has progressively turned towards the examination of the brain proteins with which drugs react and of the molecular regulatory mechanisms that control the biosynthesis and cellular function of these proteins. This trend was already apparent in several contributions (including my own) to Psychopharmacology: A Review of Progress, 1957–1967. But at that time it was hard to imagine the power of molecular biology to address the structure and function of the myriad of proteins with which drugs interact, and it was also hard to imagine the enormous importance of this work for the development of drugs that influence behavior.
The practical impact of molecular biology, which would have seemed fanciful several decades ago, is now well known even to the general public. For example, the great popularity of the novel and motion picture Jurassic Park reflects public awareness of the wide applicability of the DNA revolution. I need not, then, pay much attention, in this chapter, to restating the case for the relevance of molecular genetics to psychopharmacology.
Instead, what I will do is review briefly some of the elementary facts that are assumed in the many applications of molecular genetics that appear throughout this book. I will also call attention to a few of the critical techniques that underlie work that will be considered by other contributors. These techniques are essential for the development of molecular psychopharmacology, a major theme of this volume. For readers who wish more detailed descriptions of this information, several recent books may be consulted (1, 2, 3).
PROPERTIES OF DNA AND OF THE HUMAN GENOME
Genes are made up of deoxyribonucleic acid (DNA). DNA is a long polymer made up of four components, called deoxyribonucleotides. Each deoxyribonucleotide has one of the following constituents, called bases: adenine (A), guanine (G), thymine (T), and cytosine (C). A gene contains a few thousand to a few hundred thousand bases that are strung together in a particular sequence. The base sequence of a gene determines the structure of the gene's product, a protein. Other parts of the gene's base sequence determine the way that the gene is expressed during development of the individual and in response to various stimuli.
DNA's role in storing and transferring hereditary information depends on an inmate property of its four constituent bases. Each of these four bases has structural features which lead it to associate with one, and only one, of the other bases. Two bases that can associate with each other are said to be complementary. Guanine and cytosine are complementary to each other as are adenine and thymine.
Complementary base pairing plays an essential role in maintaining the stability of DNA and also in the transfer of its innate information. Stability is brought about because the DNA in our genes consists of two complementary strands which, by interacting with each other, shield the DNA from perturbations. Information is transferred by separating these two strands, which can then act as templates for the synthesis of new nucleic acid molecules.
DNA molecules may be used as templates in two critical ways. First the DNA is used as a template for replicating additional copies of DNA, which is essential for cell division. In this case, free deoxyribonucleotides bond with exposed complementary bases in each of the two template strands and are then linked together by an enzyme, DNA polymerase. The product is two new complementary chains which, together, reproduce the template. In DNA replication, complete DNA strands, made up of tens of millions of bases, are copied—so they can be transmitted to new cells. The other way that DNA is used as a template is more selective and has a different purpose. In this case, small bits of a strand are used as a template for the construction of molecules called messenger RNAs (mRNAs), each of which carries the message for the synthesis of a particular protein.
Messenger RNAs differ from DNA in a number of ways. First they are much shorter—generally on the order of a thousand to several thousand base pairs long. Second, they are made up of only a single strand (in contrast with double-stranded DNA) that contains all the requisite information to direct the synthesis of a particular protein. They also have a different sugar in their nucleic acid backbone—ribose rather than deoxyribose; and the thymine found in DNA is replaced with a similar base, uracil (U) in mRNA. Like thymine, uracil is complementary to adenine.
The entire human genome consists of about 100,000 genes distributed within a total DNA sequence of about 3 billion nucleotides. The DNA of the human genome is divided into 24 huge molecules, each the essential constituent of a particular chromosome (22 autosomes and two different sex chromosomes, X and Y). When we are conceived we receive 23 chromosomes from each parent, 22 autosomes and a sex chromosome.
As already indicated, a major function of a gene is to encode the structure of a specific protein. Translation of the information encoded in DNA (which is expressed in an alphabet of nucleotides) into a protein (which is expressed in an alphabet of amino acids) depends on a genetic code. In this code, sequences of three nucleotides, called a codon, represent one of the 20 amino acids that comprise the building blocks of all proteins. Because there are 64 possible codons that can be constructed from an alphabet consisting of four different bases, and only 20 different amino acids to be coded for, many amino acids are encoded by more than one particular codon. Three of the codons, called stop codons, are used to signal termination of translation.
Although all cells express certain genes that are required for their shared housekeeping functions, the distinctive differences between specialized cells (such as particular classes of neurons) are due to selective gene expression. For example, in the nervous system certain neurons use acetylcholine and others use dopamine as neurotransmitters. This results from selective expression of genes that encode specific proteins (in this case enzymes) that catalyze the biosynthesis of either acetylcholine or dopamine. Expression of these genes is ultimately under the control of specific regulatory proteins, called transcription factors, that bind to regions of the genes. These regulatory proteins control the transcription of mRNA from the genes they control. The regulatory proteins that control specific genes are, themselves, selectively expressed during the maturation of the particular classes of neurons.
Expression of enzymes that control biosynthesis of neurotransmitters is controlled not only by factors that operate during embryonic development, but also by factors that influence the adult organism. For example, the synthesis of certain of these enzymes depends, in part, on neuronal activation. When there is more neuronal activation, more of a critical enzyme is made and, as a result, the neuron makes more of the neurotransmitter. Complex regulation of genes involved in neurotransmitter biosynthesis (and of other genes for receptors and transporters that determine neurotransmitter function) may play important roles in the control of behavior. Regulation of genes also determines the response of the nervous system to drugs, the central concern of this volume.
The study of these genetic and cellular regulatory processes is one of the most active areas of contemporary biology. At present a great deal is being learned about the specific base sequences, called regulatory sequences, that surround the portions of the gene dedicated to encoding the sequence of a protein. These sequences are activated or inactivated by the specific transcription factors that bind to and control them. The complex interaction of regulatory sequences and transcription factors underlies adaptation of the brain to drugs. In the case of antidepressants and neuroleptics, these adaptive changes are essential for the therapeutic effect, which only develops after weeks of drug treatment. Because adaptive changes are essential, understanding them should lead to the design of new psychopharmacological agents.
One of the ultimate goals of molecular genetics is to determine the exact base sequence of all 3 billion bases that comprise the human genome. This task is very challenging because DNA molecules are gigantic, which makes them extremely difficult to deal with.
A major step toward achieving this goal came from development of methods to isolate, and then examine, the bite-size pieces of DNA that had been transcribed into mRNAs. The information encoded into mRNAs can be isolated and amplified by a technique called cDNA cloning Figure 1 . In this technique, a mixture, containing all the mRNAs from an organ, such as the brain, are first purified. The mixture of mRNAs are then treated with an enzyme, called reverse transcriptase, which transcribes the mRNAs into single complementary strands of DNA called complementary DNA (cDNA). Single-stranded cDNAs are then used as templates to make a second strand that is complementary to the first; and the double-stranded cDNAs are then inserted into bacterial plasmids to make products called recombinant DNA plasmids. The plasmids are then inserted into specially engineered bacteria in which they are replicated, along with the bacterial DNA, during the process of bacterial cell division. In this way many copies of the cDNAs are made. The bacterial population is comprised of many individual bacteria, each of which contains a particular plasmid with a particular cDNA derived from an mRNA from the original tissue sample. This mixed population is called a cDNA library.
To physically separate the individual members of the library, the bacteria are grown on a solid nutrient agar at low density. Each bacterium in the library is plated onto the agar at a large distance from the others. As each bacterium divides on the agar, it gives rise to a colony of descendants called a clone, which is physically separate from other clones derived from other bacteria that contain other cDNAs. Each member of a clone carries copies of the cDNA-containing plasmid that had been inserted into the clone's founder. Each clone may then be separately removed from the agar (without contamination with bacteria from other clones), and bacteria that all contain the particular cDNA may be grown up in large quantities. Then the cDNA within the plasmids can be excised, and its nucleotide sequence can be determined by chemical techniques. The cloned cDNA may also be used for many other purposes, a few of which are discussed later.
Of course the brain expresses many mRNAs that are also expressed by other tissues and that may have no special interest for neurobiological or pharmacological research. In most studies of brain cDNA the goal is to find the one that encodes the sequence of a particular protein of interest, such as a receptor protein for a particular neurotransmitter. There are many ways to go about searching for a specific cDNA (and its specific clone). One involves insertion of cDNA-containing plasmids (derived from bacterial clones) into cultured mammalian cells (such as fibroblasts) that can express the neurotransmitter receptor on their cell surface (in contrast with bacteria which do not process the cDNAs in the same way). The cDNA of interest is sought by reacting the mammalian cell population with a ligand (such as a neurotransmitter or a drug that binds the receptor that is being sought) and isolating the cells that bind the ligand, as shown schematically in Figure 2 .
Once a particular cDNA is isolated, it can be used to make limitless quantities of the protein whose sequence it encodes. For some proteins, this can be done in bacteria. In this case the plasmid can be induced to make mRNA that is translated by the bacterial protein-synthesizing machinery. However, in many cases the translation is done in mammalian cells, so that the protein product not only has the amino acids sequence encoded by the cDNA, but also undergoes appropriate post-translational modifications, such as glycosylation, which do not occur with expression in bacterial cells. In the case of neurotransmitter receptors the desired product may not be a pure soluble receptor protein, but may instead be a mammalian population of cells that express the receptor as a protein integrated into the plasma membrane on the cell surface. Cells with a particular receptor on their surface can then be used to screen for drugs that bind this receptor.
Cloned cDNA can also be used as a reagent in a variety of biological and medical studies. These are generally based on the innate property of nucleic acids to undergo complementary base paring, so that a single-stranded cDNA will bind to complementary nucleic acid sequences in mixtures of human nucleic acid, or even in tissue sections, by a process called hybridization. If the cDNA probe has been prepared in a radioactive form, the amount of radioactive cDNA that hybridizes to an aliquot of a tissue extract provides a measure of the amount of the mRNA that is complementary to it in the tissue extract. The radioactive cDNA probe can also be applied to brain tissue sections to localize the mRNA in specific neuronal populations in the brain, by a process called in situ hybridization. In this way the distribution of a particular protein, such as a receptor, can be inferred by determining the distribution of the mRNA that codes for this protein. The distribution of a particular receptor may have important implications for the design of drugs that are targeted to a specific brain region.
Once it became possible to isolate cDNAs that encode proteins of particular interest, a variety of techniques were developed to use the cDNAs to learn about the function of the proteins. The basic idea is to insert the cDNAs (modified by the addition of regulatory sequences that make possible their controlled expression; and, at times, also modified in other ways) into cells, then measure specific effects. Some of these studies are done in cells in tissue culture, whereas others are done by changing the genetic composition of intact organisms. This type of gene manipulation underlies very powerful approaches to the study of the function of given proteins, and it also provides cell types and animals with many applications in psychopharmacological research.
The simplest manipulation of this type is to introduce a new gene (or many additional copies of a particular gene) into a cultured cell line, a process called transfection. This is accomplished by engineering the cDNA into various vectors, such as appropriate plasmids, that will carry it into the cell and allow it to be expressed. In one form of transfection, called stable transfection, the cDNA (along with regulatory sequences) or other type of foreign DNA is stably integrated into the DNA of a chromosome. When a cell of this type replicates its DNA for cell division, the integrated cDNA is also replicated and transmitted to the daughter cells. To obtain stably transfected cells it is necessary to select them from a population that consists largely of cells that have not integrated the cDNA of interest.
The desired cells may be selected from a mixed population that also consists of many cells that do not contain the desired cDNA by a trick of genetic engineering. The trick is to transfect the cDNA of interest along with other DNA that makes possible the survival of transfected cells under experimentally induced toxic conditions. For example, the transfected DNA may include a sequence for a protein that renders the recipient cell resistant to a toxic compound so that only cells containing the transfected DNA (including the cDNA of interest) will survive if this toxic compound is added to the culture medium. In this way, clones of cells that contain the cDNA of interest can be isolated and used for various purposes.
To make animals that express a particular segment of foreign DNA, it is possible to inject this particular DNA into one-celled embryos. The DNA is then integrated into the genome of the recipient embryo and into all its cells, including its germ cells, so that it will be transmitted to future generations. Most of these experiments are done with mice, and a single mouse with a segment of foreign DNA incorporated into its genome can give rise to a line of mice, each of which has the foreign DNA, which is called a transgene. Such mice are called transgenic mice. Regulatory sequences surrounding the coding sequence of the transgene may bring it under specific control. For example, certain regulatory sequences will direct expression of the transgene only in a particular cell type, such as muscle cells.
A particularly interesting variety of transgenic mice has a foreign gene inserted not in addition to, but in place of, a normal gene. This is accomplished by a technique in which a normal gene is removed from a chromosome in the same process in which the foreign gene is inserted. One common application of this approach is to replace a normal gene in the germ line with one that is inactive or defective, thereby giving rise to progeny that lack the normal gene and its function. By mating brothers and sisters each with one defective gene copy, progeny can be raised that have two defective genes (i.e., no normal copies of the gene). Such "knockout experiments" are one way of examining the normal biological role of the gene in question.
In some cases the results are not very informative because absence of the functional gene during early embryonic development results in the death of the embryo. In other cases, loss of a particular gene has no obvious effect, presumably because other genes take over for the one that was inactivated. In many cases, however, mice that lack a particular gene have proved useful for determining a particular gene's function. In the context of the present volume, mice lacking a particular receptor for a neurotransmitter may provide clues to this receptor's function in the cells that express it, and in the animal as a whole. Animals lacking a particular receptor may also prove useful for certain approaches to drug development.
IMPLICATIONS FOR PSYCHOPHARMACOLOGY
It should be apparent from this brief review that molecular genetics is already providing tools that will facilitate the design of new drugs that influence behavior. Just the simple availability of a specific cell line that expresses only one receptor for a neurotransmitter (in contrast with many, in usual neuronal cell lines) represents an important technical advance in drug screening procedures. The ability to screen brain sections by in situ hybridization with specific cDNAs allows for precise localization of neurotransmitter receptors and receptor subtypes (e.g., see Cytology and Circuitry and Dopamine Receptor Expression in the Central Nervous System) with an accuracy and detail not achievable by other approaches. There are also many potential applications of transgenic mice in drug design (see Genetic Stategies in Preclinical Subtsance Abuse Research and New Drug Design in Psychopharmacology: The Impact of Molecular Biology).
There may also come a time when genes themselves will be used as drugs (see Towards an Understanding of the Genetics of Alzheimer’s Disease, Amyotrophic Lateral Sclerosis, Glutamate, and Oxidative Stress, Tic Disorders, Genetic Influences in Drug Abuse, and Ethical Issues in Genetic Screening and Testing, Gene Therapy, and Scientific Conduct). Already attempts are being made to treat certain diseases, caused by the absence of a specific enzyme, by removing the patient's cells, transfecting them with the cDNA that directs the synthesis of the enzyme, and then injecting the cells back into the patient. In this way, sustained enzyme expression can be achieved. Applications of such cellular engineering to psychopharmacology are presently being considered. Molecular genetic techniques are also being used in human genome screening designed to search for genes that may be responsible for psychiatric disorders such as bipolar disorder and schizophrenia. Should such genes be found, their identity may point the way to specific psychopharmacological treatment.
Given the increasing power of molecular genetics, it is safe to predict that its impact on psychopharmacology will become progressively more significant in the years to come. A decade from now, with the publication of the next volume in the series, there should be many new and interesting applications to consider.