MOLECULAR BIOLOGY
Molecular Biology: An Overview Edward Passaro, Jr., MD, Michael Hurwitz, MD, Ghassan Samara, MD,
Mark Sawicki, MD, Los Angeles,California
An overview of molecular biology is presented for the practicing surgeon. Definitions of the constructs and activity of DNA, RNA, and protein synthesis
are defined. These principles are illustrated in their use in reeombinant DNA technologies. A glossary is provided for the terms utilized.
PREFACE This is the lru'st in a series of articles designed to acquaint the practicing surgeon with selected but practical aspects of molecular biology. We believe that it is critical that surgeons have a basic understanding of this field since it will have important effects on their method of diagnosis, treatment, and prognostication, particularly in treating cancer. Further, it is our contention that, because of their experience with cancer and their access to tumor tissue, surgeons can make important contributions to molecular biology. We recognize that much of the language of molecular biology is unknown to surgeons and can be confusing to those who do not utilize it on a daily basis. To assist, we have prepared a simple glossary that will accompany each of the succeeding articles. To illustrate the selected topics, we will present a patient with cystic fibrosis. Our current under-
standing of this disease's pathology, transmission, diagnosis, and treatment will be related to these topics. ILLUSTRATIVE CASE DISCUSSION: CYSTIC FIBROSIS
Cystic fibrosis is the most common lethal genetic disease in whites. The apical cell membranes of epithelia in the sweat glands, pancreas, intestine, and lungs do not allow the usual transport of chloride ions. Although cellular channels for chloride transport are present in these patients, their regulation is defective. Clinically, such patients have chronic disorders of digestion, absorption, and viscid pulmonary secretions, resulting in retarded growth and development. Although formerly lethal in infancy, many patients now live into adulthood. The major cause of death is severe chronic pulmonary infections.
A n Historical C h r o n o l o g i c O v e r v i e w N o b e l L a u r e a t e (Year A w a r d e d )
9 1866-~Mendel His experiments showed that inherited traits were transmitted by discrete factors, which occurred in pairs. Further, these pairs of factors separated during reproduction (gamete formation), with one of each pair being transmitted to the resultant egg and one to the sperm. Half of the sperm and egg cell's gamete will have one factor of the pair. 9 1877--Flemming Discovered chromosomes. 9 1902--Sutton Noted the similarity between Mendel's "factors" and the separation of chromosomes during gamete formation (meiosis). Deduced that genes ("factors") were located on chromosomes. 9 1911--Wilson Noted that color blindness was transmitted through females, thus assigning a human gene for the first time to a particular chromosome (X). 9 1915--Morgan Noted a linkage of genes in studies in fruit flies. Observed that the percentage of recombination of two genes was the function of the distance between them. The centimorgan, an expression of this percentage, is approximately the distance of 1 million base pairs between genes. 9 1941--Tatum, Beadle, and Lederberg (1958) Established that each gene regulates the production of a specific protein, hence the adage "One gene, one protein." 9 19A.'! Avery Suggested that genes consist of DNA 9 1952--Hershey (1969) Proved that genes were made of DNA.
From the Surgical Service, Veterans Administration Medical Center, and the Department of Surgery, UCLA School of Medicine, Los Angeles, California. Requests for reprints should be addressed to Edward Passaro, Jr., 146
THE AMERICAN JOURNAL OF SURGERY
9 1953 Watson, Crick, and Wilkins (1962) Established the double helix structure of DNA and the codon that expresses its language. 9 1970---Smith Restriction enzymes were identified. 9 1971--Berg (1980) Successfully inserted simian virus 40 into X. bacteriophage, initiating the era of recombinant DNA technology. 9 1972--Cohen and Boyer Developed the cloning technique that permitted the multiplication in bacteria of large quantities of a DNA segment. Bacteria could be induced to elaborate human insulin. 9 1975--Southern Described a technique whereby DNA fragments could be transferred to a synthetic membrane for subsequent studies. 9 1978~--Collins Developed cosmids that allow a largeDNA fragment (40,000 bp) to be attached to a viral vector. This is used to "infect" bacteria, enabling the bacteria to begin to elaborate these large foreign DNA fragments. 9 1980--Botstein Used restriction fragment length polymorphisms (RFLP) to construct a genetic linkage map. 9 1983~Murray Created yeast artificial chromosomes (YACs) by splicing very large DNA fragments (250,000 to 1 million bp) into yeast DNA and successfully reproducing and elaborating them. 9 1984---Sinsheimer Suggested the Human Genome Project. 9 1987--Mullis Described the technique of multiplying a length of DNA: the polymerase chain reaction (PCR).
MD, Surgical Service (W112), Veterans Administration Medical Center, Los Angeles, California 90073. Manuscript submitted March 31, 1992, and accepted April 20, 1992.
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MOLECULAR BIOLOGY: AN OVERVIEW
The nucleus of the human cell contains 22 pairs of chromosomes plus the sex chromosomes XX or XY. Each of these paired chromosomes has a long arm (q), a short arm (p), and a center (centromere). When stained with Giemsa dyes, each chromosome displays a unique pattern of light- and dark-staining bands. These bands serve as landmarks in measuring each arm, starting from the centromere, and thus help to define the location of genes on the arms (Figure 1A). A karyotype is a visual representation of an individual's chromosomes. By using a karyotype, gross chromosomal abnormalities and specific areas of interest can be located or detected. The chromosome consists of a tightly wound strand of double helical DNA. Among the 23 pairs of chromosomes, there are an estimated 50,000 to 100,000 genes. The genes are the "factors" Gregor Mendel postulated, encoded pieces of information. They regulate the production of specific proteins, one gene for each protein. Genes and their regulatory components, however, represent only a fraction (estimated 10% or less) of the total chromosomal material, with the remainder consisting of repetitive and variable DNA sequences. The importance of these sequences is not known, but they are believed to provide greater genomic protection and flexibility through evolution. DNA: The regulatory components of a cell are DNA, RNA, and the proteins that they produce. The information that regulates the cell is encoded in the DNA. The discovery of how that information is encoded represents one of the great advances of the age (see Watson): the now-familiar particular doublestranded helix of DNA (Figure 1B). The strands are composed of the nucleotide bases adenine (A), thymine (T), guanosine (G), and cytosine (C) attached to a sugar (deoxyribose) framework. The nuclcotide bases are always paired in a complementary fashion (base pairing). Adenine is always paired opposite thymine and guanine opposite cytosine (Figure 2). Thus, if the nucleotide base sequence of one strand is known, the other can be deduced. Another important feature of the double-strand arrangement is that the strands can be separated by heating and rejoined (annealed) by cooling. Because base pairing allows specific alignment, complementary strands of DNA or R N A can "find" each other. The process of breaking and rejoining is called hybridization and is widely used to find and/or localize portions of DNA and to replicate a complementary strand. RNA: RNA takes the information contained in the DNA and brings it to the organelles that are the site for protein synthesis, the ribosomes (Figure 2, A and B). R N A differs from DNA in three ways: (1) it is a single strand, (2) ribose sugar replaces deoxyribose, and (3) uracil (U) replaces thymidine (T) but remains complementary to adenine (A). R N A is formed opposite and complementary to a strand of DNA in the nucleus by the enzyme R N A polymerase. The introns are then removed, leaving the exons in the final messenger R N A (mRNA). This m R N A moves from the nucleus to the ribosomes within the cytoplasm. Within the ribosomes
is another form of RNA, ribosomal R N A (rRNA). Proteins are made by the ribosomal R N A with the assistance of the last form of RNA, transfer R N A (tRNA). The latter decodes the mRNA and thus directs the activity of the rRNA. PROTEIN SYNTHESIS Proteins are composed of amino acids arranged linearly but folded and bent into a unique shape. Genes in the DNA are encoded for specific proteins. Each gene dictates but one protein, a polypeptide, its gene product. Hence, the expression, "One gene, one protein." It is the proteins that carry out the cell's activity. They make up channels that regulate cell permeability and receptors that provide communication, and they regulate gene expression. Protein synthesis begins when the enzyme R N A polymerase copies a gene in the DNA into a linear complementary mRNA strand, a process called transcription (Figure 2A). mRNA is composed of nucleotides (A, U, G, C). A group of 3 consecutive nucleotides codes for 1 of each of the 20 amino acids or a stop signal. This three-nucleotide base is called a codon. Given the 4 nucleotides arranged in groups of 3, there are 64 (43) codes possible, more than are required to produce 20 amino acids. Several different codons, in fact, can produce the same amino acid, but all organisms use the same codons. This is referred to as the genetic code. Thus, an organism as simple as a bacterium can translate human DNA to produce a hormone protein product, for example, insulin. Because the genetic code is universal, if either the DNA or R N A sequence is known, the protein sequence will also be known. Similarly, if a protein has been sequenced, the possible DNA sequence that codes for the protein can be derived. When the sequence became known for somatostatin, a 14-amino acid protein, its gene was synthesized and then cloned into Escherichia coll. The information in mRNA is translated in ribosomes by tRNA, which are small molecules with two functional ends. One end is connected to an amino acid, and the other has an anti-codon, three consecutive nucleotides that are complementary to the codons of the mRNA (Figure 2B). The amino acids of subsequent tRNAs are aligned as a consequence of their anticodons, determined by the order of nucleotides in mRNA. This, in turn, is determined by the molecular sequence in the DNA. The process whereby mRNA is used as a template to produce proteins is called translation. Many copies of the protein product can be made by either having a single mRNA translated by multiple ribosomes or a gene can be expressed in multiple copies, or some admixture of the two. RECOMBINANT DNA TECHNIQUES A chromosome can be thought of as a long strand of DNA on which genes are strung like beads, generally in a single copy. Much of current molecular biology is directed at the identification, multiplication (cloning), and analysis of these genes. Because all genes are made of the same four basic nucleotides, they cannot be
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OtROMOSO~
C Figure 1. Karyotype of a chromosome (A).The double-stranded he/ix of DNA on a deoxyflbose framework
(B).
Figure 2. A schema of the course of
events from DNA to proteinproduction is shown. Development of mRNA (transcription) is shown in panel A, and the "reading" of the mRNA (translation) to produce a protein Is shown in panel B.
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readily identified by chemical or indirect means. Currently, the best techniques for gene identification and purification require that the gene in question be physically removed from the chromosomal DNA and that it be amplified or increased in quantity by a variety of techniques.
148
To remove a piece of DNA from a chromosome containing a gene entails the use of restriction enzymes. These enzymes are naturally generated in bacteria where they serve a protective function for the organism by breaking up foreign DNA, cutting it into pieces at specific sites. Hundreds of restriction enzymes have now been identified and isolated, each of which cleaves the DNA into distinct patterns depending on the DNA sequences. For example, an enzyme derived from E. coil, called EcoRI, cuts DNA at the site between G (guanine) and two A (adenine) molecules (Figure 3). The pattern of DNA fragments produced is characteristic for each chromosome since the restriction enzyme used always cuts the DNA at its specific site of action and nowhere else. Thus, using the restriction enzyme mapping technique, it is possible to identify both the specific restriction enzyme used, as well as the specific chromosome cleaved. Today, restriction enzyme mapping is used in forensic and clinical medicine in a variety of ways. Knowledge of the specific DNA sequences or "maps" present in clinical conditions has enabled restriction fragment length polymorphism (RFLP) to be useful in screening individuals for such genetic disorders as muscular dystrophy and sickle cell anemia. On an even smaller, finer scale, every individual has his own distinct pattern of DNA fragments (DNA fingerprinting), and much has been made of its use in homicide and rape cases. To produce quantities of a given length of DNA for
THE AMERICAN JOURNAL OF SURGERY VOLUME 164 AUGUST 1992
MOLECULAR BIOLOGY: AN OVERVIEW
Figure 4. Steps involved in cloning or duplicating in quantity a portion of DNA containing a gene.
analytic or other purposes, DNA molecules are first cut with a restriction enzyme and the fragments sorted by chromatography or electrophoresis. Cut pieces of DNA, because of base pairing, can be made to rejoin again using the enzyme DNA ligase (Figure 4). Because of their propensity to rejoin at complementary base pairs, they are called "sticky ends" of the DNA. The DNA fragment with sticky ends can be amplified or "grown up" by inserting it into a segment of DNA capable of independent growth called a vector. Bacterial plasmids and the viral bacteriophage X are commonly used vectors. Plasmids are circular pieces of DNA, found outside the organism's chromosomes, which multiply independent of the chromosomal DNA. A bacterium may harbor a thousand copies of a plasmid. The plasmids are cut open with a restriction enzyme and then incubated with D N A fragments cut with the same enzyme. The "sticky ends," i.e., complementary base pairs, are attached to the DNA fragment, the other to the end of the opened plasmid DNA, which then join. The result is DNA combining both foreign (e.g., human) DNA with plasmid DNA, forming recombinant DNA. The latter is introduced back into bacteria where it will then make many copies of itself, resulting in a clone of a specific piece of human DNA. Although plasmids are useful to amplify a given piece of D N A containing a discrete gene, it is difficult to screen large numbers of bacteria to see which contain the desired gene. Screening of large numbers of different DNA segments that have been cloned is best done with phage ),. Pieces of the foreign (human) DNA are recombined with DNA from ~, phage using DNA ligase. An agar plate with a thin layer of bacteria over it is then infected with the phage. The phage infects the bacteria, rapidly multiplying within them, killing the
bacteria, and forming a clear area or plaque over the agar. Within each plaque are a large number of phage particles containing identical DNA fragments. Filter paper is placed over the agar plate, and phage particles corresponding to each of the plaques are absorbed onto it. The phage and the foreign DNA are denatured with a basic solution, and then the paper is treated to destroy both the phage and foreign DNA, leaving behind the piece of desired DNA. A radiolabeled DNA probe is then incubated on the paper to hybridize with the desired gene. Probes are sequences of DNA or R N A nucleotides complementary (eDNA or cRNA) to the gene of interest so that they will hybridize to it. A probe, because of its nucleotide's configuration, is highly specific and will only join a complementary piece of DNA despite the presence of overwhelming numbers of noncomplementary sequences. The probe is labeled with a radioisotope for detection. A film is placed over the DNA remnant, allowing identification of the gene. The results are traced back to the corresponding agar plate to find the DNA clone. The basic steps, thus, are (1) cutting the DNA into small pieces with restriction enzymes; (2) amplifying the pieces by inserting them into a cloning vector; and (3) identifying the desired DNA clone by using a DNA probe. The cloning of the gene allows its function and regulation to be studied. But, more importantly, its pure gene product, a protein ("one gene, one protein"), can be synthesized in large quantities, analyzed for abnormalities or used clinically, or antibodies can be raised against it for the development of a specific radioimmunoassay. The latter permits detection of overactivity of the gene.
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PASSARO ET AL
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REPEATFORAPPROXIMATELY30 CYCLES Figure 5. Schematic representationof the exponentialduplication of a specific portion of DNA by the polymerase chain reaction.
Southern and Northern blots are used to detect specific sequences of DNA and RNA, respectively. Southern blots (named after its inventor) use gel electrophoresis to separate DNA fragments cut with a restriction enzyme. Different DNA pieces of different sizes are separated out as discrete bands and then "blotted" onto a synthetic membrane (blot), which is then "probed" with a radiolabeled probe specific to the area of interest. This permits detection, in a very small amount of DNA, of the gene or DNA site of interest. "Northern" blots--a term used by laboratories since it uses the same principles as Southern blotting but identifies R N A rather than DNA--detect the specific R N A sequences from m R N A isolated from a cell. In this way one can, for example, determine whether the cell is
150
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producing a specific protein product by detecting the mRNA for it. The extraction, purification, and separation of both DNA and R N A are obviously time consuming, involved, and expensive. In an effort to circumvent these problems, investigators have developed methods of identifying both specific DNA and RNA sites by in situ hybridization of probes to properly prepared tissues. In this way, for example, the expression or elaboration of increased quantities of mRNA in a specific tissue or cell could be determined, giving information on the activity of the cell. Another important advance has been the development of the polymerase chain reaction (PCR). Much like in situ hybridization, PCR obviates the time, intricacies, and costs of extracting and purifying DNA so that it can be amplified or produced in quantity. By using PCR, it is possible to amplify minute quantities of specific portions of DNA contained within pure or impure preparations, from fresh or fixed tissues. The process is performed in a test tube that contains trace quantities of the DNA to be replicated among other bits and pieces of DNA (Figure 5). To this are added primers or short lengths of DNA bases that are complementary to both ends of the DNA sequence to be replicated (generally 50 to 2,000 base pairs in length), a heat-stable polymerase enzyme that joins DNA bases together, and a large excess of bases. The tube with its ingredients is heated, separating the DNA into single strands. The temperature is then reduced, whereupon the primers become attached to the respective ends of the specific DNA strand desired because of base pairing. Polymerase enzyme now synthesizes a single strand of DNA, beginning from each end where the primer attached itself to the specific DNA strand. The excess bases ensure that the reaction will be completed. The tube is reheated, and all newly formed double DNA is converted to single-strand DNA. The cycle is then repeated some 30 times, producing an exponential increase in specific new DNA strands. In summary, this brief review has sought to explain current techniques in molecular biology that are primarily directed at the isolation and identification of genes and their products. The technical procedures involved, although not difficult to conceptualize, are precise and demanding in practice. The molecular biology of the gene is of particular importance to all surgeons since the problems related to cancer formation and future therapy are related to a basic understanding of genes. ANNOTATED BIBLIOGRAPHY 1. Watson JD, Hopkins NH, Roberts JW, Steitz JA, Weiner AM. Molecular biology of the gene, 4th ed. Redwood City, CA: Benjamin Cummings Publishing Co, 1987. The textbook! Superbly written with unexcelled line drawings and illustrations. A delightful index (How a Frog Egg Counts Twelve Cell Divisions; Working With
VOLUME 164 AUGUST 1992
MOLECULARBIOLOGY:ANOVERVIEW
a Mammal Takes Time; Why RNA and Not DNA Was the First Living Molecule; etc.) invites you to look deeper and further. A must for the interested student (about $70) and should be known by all as a place to start to look things up. 2. Wong RS, Passaro E Jr. DNA technology. Am J Surg 1990; 159: 610-4. A simple explanation of many of the basic technologies dealing with DNA and how they are applied clinically. 3. Green ED, Waterson RH. The Human Genome Project. JAMA 1991; 266: 1966-75. An excellent description of the Project and how DNA mapping is done. 4. Watson JD. The double helix. New York: Norton Publishing; 1968. A candid personal narrative of the elucidation of the structure of DNA. GLOSSARY Allele: One member of a pair of genes for a given trait. Each member is of maternal or paternal origin. Amplify: To increase the quantity of a specific gone by a variety of techniques. Aneuploid: A cell containing extra chromosomes. Band: A pattern of light and dark regions by Giemsa staining that can serve as landmarks on chromosomes. Base pairing: The pairing of specific nitrogenous bases between complementary strands of DNA. For example, adenine is always paired with thymine and guanine with cytosine. eDNA (complementary DNA): Single- or doublestranded DNA made from an RNA template using the enzyme reverse transcriptase. Centimorgan (eM): A measure of the statistical probability of recombination between alleles. One cM represents a 1% chance of recombination per meiotic event. Centromere: The constricted region of a chromosome separating the short and long arms from one another. Chromosome: A single, linear, highly condensed DNA molecule. Clone (noun) : One of a collection of cells or vectors containing identical genetic material. Clone (verb) : The act of duplicating genetic material within a vector. Codon: A group of three consecutive nucleotides within messenger RNA (mRNA) that encodes one of 20 amino acids or encodes a message to stop translation (see Translation). Cosmid: A vector that incorporates components of plasmids and phage to carry larger clones (up to 40 kilobases). Diploid: Cells containing copies of both the maternal and paternal chromosomes. DNA (deoxyribonueleie acid): The molecule responsible for storing and transmitting genetic information; composed of two strands of nucleotides twisted around each other in the shape of a double helix.
Double helix: The twisted double-strand shape assumed by DNA. Exon: A contiguous segment of genomic DNA that is translated into polypeptide (see Intron). Familial: An inherited trait. Flow cytometry: Method used to measure nuclear DNA quantity in order to determine ploidy status. Gone: A segment of DNA within a chromosome encoding a single protein. Genome: The entire complement of genetic material in the form of DNA for a given organism. Genetic map: The ordering of genes by the statistical determination of recombination events between them. Genes separated by greater distances are more likely to recombine. Heterozygous: An individual containing dissimilar alleles for a given gone or locus. Homozygous: An individual containing identical alleles for a given gone or locus. Hybridization: The alignment of complementary strands of DNA (or RNA) via base pairing, widely used to identify portions of DNA on a Southern (or Northern) blot, using labeled probes. lntron: A noncoding sequence of DNA within the gone (see Exon). Karyotype: The physical appearance of the full complement of stained chromosomes for an individual. Locus: A site on a segment of DNA. Long arm (g) : One of the two prominent segments of a chromosome; the short or "p" arm is the other. The arms of a given chromosome join at its centromere. Library: A collection of recombinant genes cloned into a vector. Linkage: A measure of proximity between two alleles determined by recombination events. If they are not linked, they arc on separate chromosomes; if loosely linked, they are distant to each other on the same chromosome. The closer they are to one another, the more tightly they are linked. mRNA (messenger RNA) : The single-stranded edited copy of a gcnc ultimately translated into protein. Northern blot: The transfer of size-separated RNA fragments to a synthetic membrane for further studies. Nueleotide: One of the four building blocks of DNA (dATP, dGTP, dCTP, or dTTP) or RNA (ATP, CTP, GTP, or ATP) that are combined to form the nucleic acids. Oncogene: A cancer-inducing gone. Phage: A virus that infects a bacterial host, used in the laboratory as a cloning vector. Physical map: Analysis of the distance, in base pairs, between loci. Plasmid: Circularized DNA fragment, distinct from genomic DNA, found within bacteria, used as a cloning vector or to alter characteristics of the bacteria. Polymerase chain reaction (PCR): An efficient, simple, and rapid technique to multiply a length of DNA in a test tube. Promoter site: Region of a DNA molecule found in front of a gone that controls the expression of the gone.
THE AMERICANJOURNALOF SURGERY VOLUME164 AUGUST1992 151
PASSAROET AL
Prnto-oneogene: Normal gene that may become an oncogene; also called cellular oncogene. Recombinant DNA- The combination of foreign DNA inserts with vector DNA (e.g., plasmid, phage, or cosmid) to produce a clone within a host. Recombination: The rearrangement of DNA by breaking and re-ligations of the DNA strands; also called crossovers. Replieon: A sequence in the D N A that initiatesreplication. Restrictionmapping: The creationof a physicalmap by ordering enzymatically cut D N A fragments, Restriction endonuelease: Enzyme, isolated from bacteria, that recognizes specific base-pair sequences to cut DNA. The sites vary from frequent to rare cutting, depending upon the length of the restriction site. Restriction f r a g m e n t length polymorphism (RFLP): Variation in the distance between restriction enzyme cleavage sites that exist within a population producing unique DNA fingerprint patterns. RNA (ribonueleie acid): Single-strand nucleic acid found mainly in the nucleolus and ribosomes; contains ribose sugar and uracil, whereas DNA contains thymine. Short arm (p): One of the two prominent segments of a chromosome; the long or "q" arm is the other. The arms of a given chromosome join at its centromere. Southern blot: The transfer of size-separated DNA fragments to a synthetic membrane for further studies, initially described by E.N. Southern.
152
Sporadic: Not familial. Sticky ends: The cut pieces of a single strand of DNA, which, because of base pairing, can be made to rejoin again at complementary base pairs, using the enzyme DNA ligase. Telomere: The distal ends of the chromosome. Transcription: The copying of DNA into messenger RNA. Transduction: The incorporation of a cellular gene in a viral genome that can then be introduced into other cells. Transfeetion: The process of placing foreign DNA into mammalian cells. Transformation: The cancerous alteration of mammalian cells; also the act of putting foreign DNA into bacteria. Translation: The process of converting the genetic code into polypeptides, mRNA codons are recognized by tRNA anti-codons. Each tRNA codes for a single amino acid. Tumor suppressor: A gene that prevents tumor formation until deleted or mutated. Vector: A construct used to propagate DNA in a host (bacteria, yeast, or cultured cells) (see Plasmid, Phage, and/or Yeast Artificial Chromosome). Virion: A replication virus particle. Western analysis: Protein electrophoresis characterizing size of unknown protein. Yeast artificial ehromosome (YAC) : A vector used in yeast that can propagate large fragments of DNA.
THE AMERICANJOURNALOF SURGERY VOLUME164 AUGUST1992
Molecular Biology: An Overview Edward Passaro, Jr., MD, Michael Hurwitz, MD, Ghassan Samara, MD,
Mark Sawicki, MD, Los Angeles,California
An overview of molecular biology is presented for the practicing surgeon. Definitions of the constructs and activity of DNA, RNA, and protein synthesis
are defined. These principles are illustrated in their use in reeombinant DNA technologies. A glossary is provided for the terms utilized.
PREFACE This is the lru'st in a series of articles designed to acquaint the practicing surgeon with selected but practical aspects of molecular biology. We believe that it is critical that surgeons have a basic understanding of this field since it will have important effects on their method of diagnosis, treatment, and prognostication, particularly in treating cancer. Further, it is our contention that, because of their experience with cancer and their access to tumor tissue, surgeons can make important contributions to molecular biology. We recognize that much of the language of molecular biology is unknown to surgeons and can be confusing to those who do not utilize it on a daily basis. To assist, we have prepared a simple glossary that will accompany each of the succeeding articles. To illustrate the selected topics, we will present a patient with cystic fibrosis. Our current under-
standing of this disease's pathology, transmission, diagnosis, and treatment will be related to these topics. ILLUSTRATIVE CASE DISCUSSION: CYSTIC FIBROSIS
Cystic fibrosis is the most common lethal genetic disease in whites. The apical cell membranes of epithelia in the sweat glands, pancreas, intestine, and lungs do not allow the usual transport of chloride ions. Although cellular channels for chloride transport are present in these patients, their regulation is defective. Clinically, such patients have chronic disorders of digestion, absorption, and viscid pulmonary secretions, resulting in retarded growth and development. Although formerly lethal in infancy, many patients now live into adulthood. The major cause of death is severe chronic pulmonary infections.
A n Historical C h r o n o l o g i c O v e r v i e w N o b e l L a u r e a t e (Year A w a r d e d )
9 1866-~Mendel His experiments showed that inherited traits were transmitted by discrete factors, which occurred in pairs. Further, these pairs of factors separated during reproduction (gamete formation), with one of each pair being transmitted to the resultant egg and one to the sperm. Half of the sperm and egg cell's gamete will have one factor of the pair. 9 1877--Flemming Discovered chromosomes. 9 1902--Sutton Noted the similarity between Mendel's "factors" and the separation of chromosomes during gamete formation (meiosis). Deduced that genes ("factors") were located on chromosomes. 9 1911--Wilson Noted that color blindness was transmitted through females, thus assigning a human gene for the first time to a particular chromosome (X). 9 1915--Morgan Noted a linkage of genes in studies in fruit flies. Observed that the percentage of recombination of two genes was the function of the distance between them. The centimorgan, an expression of this percentage, is approximately the distance of 1 million base pairs between genes. 9 1941--Tatum, Beadle, and Lederberg (1958) Established that each gene regulates the production of a specific protein, hence the adage "One gene, one protein." 9 19A.'! Avery Suggested that genes consist of DNA 9 1952--Hershey (1969) Proved that genes were made of DNA.
From the Surgical Service, Veterans Administration Medical Center, and the Department of Surgery, UCLA School of Medicine, Los Angeles, California. Requests for reprints should be addressed to Edward Passaro, Jr., 146
THE AMERICAN JOURNAL OF SURGERY
9 1953 Watson, Crick, and Wilkins (1962) Established the double helix structure of DNA and the codon that expresses its language. 9 1970---Smith Restriction enzymes were identified. 9 1971--Berg (1980) Successfully inserted simian virus 40 into X. bacteriophage, initiating the era of recombinant DNA technology. 9 1972--Cohen and Boyer Developed the cloning technique that permitted the multiplication in bacteria of large quantities of a DNA segment. Bacteria could be induced to elaborate human insulin. 9 1975--Southern Described a technique whereby DNA fragments could be transferred to a synthetic membrane for subsequent studies. 9 1978~--Collins Developed cosmids that allow a largeDNA fragment (40,000 bp) to be attached to a viral vector. This is used to "infect" bacteria, enabling the bacteria to begin to elaborate these large foreign DNA fragments. 9 1980--Botstein Used restriction fragment length polymorphisms (RFLP) to construct a genetic linkage map. 9 1983~Murray Created yeast artificial chromosomes (YACs) by splicing very large DNA fragments (250,000 to 1 million bp) into yeast DNA and successfully reproducing and elaborating them. 9 1984---Sinsheimer Suggested the Human Genome Project. 9 1987--Mullis Described the technique of multiplying a length of DNA: the polymerase chain reaction (PCR).
MD, Surgical Service (W112), Veterans Administration Medical Center, Los Angeles, California 90073. Manuscript submitted March 31, 1992, and accepted April 20, 1992.
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The nucleus of the human cell contains 22 pairs of chromosomes plus the sex chromosomes XX or XY. Each of these paired chromosomes has a long arm (q), a short arm (p), and a center (centromere). When stained with Giemsa dyes, each chromosome displays a unique pattern of light- and dark-staining bands. These bands serve as landmarks in measuring each arm, starting from the centromere, and thus help to define the location of genes on the arms (Figure 1A). A karyotype is a visual representation of an individual's chromosomes. By using a karyotype, gross chromosomal abnormalities and specific areas of interest can be located or detected. The chromosome consists of a tightly wound strand of double helical DNA. Among the 23 pairs of chromosomes, there are an estimated 50,000 to 100,000 genes. The genes are the "factors" Gregor Mendel postulated, encoded pieces of information. They regulate the production of specific proteins, one gene for each protein. Genes and their regulatory components, however, represent only a fraction (estimated 10% or less) of the total chromosomal material, with the remainder consisting of repetitive and variable DNA sequences. The importance of these sequences is not known, but they are believed to provide greater genomic protection and flexibility through evolution. DNA: The regulatory components of a cell are DNA, RNA, and the proteins that they produce. The information that regulates the cell is encoded in the DNA. The discovery of how that information is encoded represents one of the great advances of the age (see Watson): the now-familiar particular doublestranded helix of DNA (Figure 1B). The strands are composed of the nucleotide bases adenine (A), thymine (T), guanosine (G), and cytosine (C) attached to a sugar (deoxyribose) framework. The nuclcotide bases are always paired in a complementary fashion (base pairing). Adenine is always paired opposite thymine and guanine opposite cytosine (Figure 2). Thus, if the nucleotide base sequence of one strand is known, the other can be deduced. Another important feature of the double-strand arrangement is that the strands can be separated by heating and rejoined (annealed) by cooling. Because base pairing allows specific alignment, complementary strands of DNA or R N A can "find" each other. The process of breaking and rejoining is called hybridization and is widely used to find and/or localize portions of DNA and to replicate a complementary strand. RNA: RNA takes the information contained in the DNA and brings it to the organelles that are the site for protein synthesis, the ribosomes (Figure 2, A and B). R N A differs from DNA in three ways: (1) it is a single strand, (2) ribose sugar replaces deoxyribose, and (3) uracil (U) replaces thymidine (T) but remains complementary to adenine (A). R N A is formed opposite and complementary to a strand of DNA in the nucleus by the enzyme R N A polymerase. The introns are then removed, leaving the exons in the final messenger R N A (mRNA). This m R N A moves from the nucleus to the ribosomes within the cytoplasm. Within the ribosomes
is another form of RNA, ribosomal R N A (rRNA). Proteins are made by the ribosomal R N A with the assistance of the last form of RNA, transfer R N A (tRNA). The latter decodes the mRNA and thus directs the activity of the rRNA. PROTEIN SYNTHESIS Proteins are composed of amino acids arranged linearly but folded and bent into a unique shape. Genes in the DNA are encoded for specific proteins. Each gene dictates but one protein, a polypeptide, its gene product. Hence, the expression, "One gene, one protein." It is the proteins that carry out the cell's activity. They make up channels that regulate cell permeability and receptors that provide communication, and they regulate gene expression. Protein synthesis begins when the enzyme R N A polymerase copies a gene in the DNA into a linear complementary mRNA strand, a process called transcription (Figure 2A). mRNA is composed of nucleotides (A, U, G, C). A group of 3 consecutive nucleotides codes for 1 of each of the 20 amino acids or a stop signal. This three-nucleotide base is called a codon. Given the 4 nucleotides arranged in groups of 3, there are 64 (43) codes possible, more than are required to produce 20 amino acids. Several different codons, in fact, can produce the same amino acid, but all organisms use the same codons. This is referred to as the genetic code. Thus, an organism as simple as a bacterium can translate human DNA to produce a hormone protein product, for example, insulin. Because the genetic code is universal, if either the DNA or R N A sequence is known, the protein sequence will also be known. Similarly, if a protein has been sequenced, the possible DNA sequence that codes for the protein can be derived. When the sequence became known for somatostatin, a 14-amino acid protein, its gene was synthesized and then cloned into Escherichia coll. The information in mRNA is translated in ribosomes by tRNA, which are small molecules with two functional ends. One end is connected to an amino acid, and the other has an anti-codon, three consecutive nucleotides that are complementary to the codons of the mRNA (Figure 2B). The amino acids of subsequent tRNAs are aligned as a consequence of their anticodons, determined by the order of nucleotides in mRNA. This, in turn, is determined by the molecular sequence in the DNA. The process whereby mRNA is used as a template to produce proteins is called translation. Many copies of the protein product can be made by either having a single mRNA translated by multiple ribosomes or a gene can be expressed in multiple copies, or some admixture of the two. RECOMBINANT DNA TECHNIQUES A chromosome can be thought of as a long strand of DNA on which genes are strung like beads, generally in a single copy. Much of current molecular biology is directed at the identification, multiplication (cloning), and analysis of these genes. Because all genes are made of the same four basic nucleotides, they cannot be
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readily identified by chemical or indirect means. Currently, the best techniques for gene identification and purification require that the gene in question be physically removed from the chromosomal DNA and that it be amplified or increased in quantity by a variety of techniques.
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To remove a piece of DNA from a chromosome containing a gene entails the use of restriction enzymes. These enzymes are naturally generated in bacteria where they serve a protective function for the organism by breaking up foreign DNA, cutting it into pieces at specific sites. Hundreds of restriction enzymes have now been identified and isolated, each of which cleaves the DNA into distinct patterns depending on the DNA sequences. For example, an enzyme derived from E. coil, called EcoRI, cuts DNA at the site between G (guanine) and two A (adenine) molecules (Figure 3). The pattern of DNA fragments produced is characteristic for each chromosome since the restriction enzyme used always cuts the DNA at its specific site of action and nowhere else. Thus, using the restriction enzyme mapping technique, it is possible to identify both the specific restriction enzyme used, as well as the specific chromosome cleaved. Today, restriction enzyme mapping is used in forensic and clinical medicine in a variety of ways. Knowledge of the specific DNA sequences or "maps" present in clinical conditions has enabled restriction fragment length polymorphism (RFLP) to be useful in screening individuals for such genetic disorders as muscular dystrophy and sickle cell anemia. On an even smaller, finer scale, every individual has his own distinct pattern of DNA fragments (DNA fingerprinting), and much has been made of its use in homicide and rape cases. To produce quantities of a given length of DNA for
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Figure 4. Steps involved in cloning or duplicating in quantity a portion of DNA containing a gene.
analytic or other purposes, DNA molecules are first cut with a restriction enzyme and the fragments sorted by chromatography or electrophoresis. Cut pieces of DNA, because of base pairing, can be made to rejoin again using the enzyme DNA ligase (Figure 4). Because of their propensity to rejoin at complementary base pairs, they are called "sticky ends" of the DNA. The DNA fragment with sticky ends can be amplified or "grown up" by inserting it into a segment of DNA capable of independent growth called a vector. Bacterial plasmids and the viral bacteriophage X are commonly used vectors. Plasmids are circular pieces of DNA, found outside the organism's chromosomes, which multiply independent of the chromosomal DNA. A bacterium may harbor a thousand copies of a plasmid. The plasmids are cut open with a restriction enzyme and then incubated with D N A fragments cut with the same enzyme. The "sticky ends," i.e., complementary base pairs, are attached to the DNA fragment, the other to the end of the opened plasmid DNA, which then join. The result is DNA combining both foreign (e.g., human) DNA with plasmid DNA, forming recombinant DNA. The latter is introduced back into bacteria where it will then make many copies of itself, resulting in a clone of a specific piece of human DNA. Although plasmids are useful to amplify a given piece of D N A containing a discrete gene, it is difficult to screen large numbers of bacteria to see which contain the desired gene. Screening of large numbers of different DNA segments that have been cloned is best done with phage ),. Pieces of the foreign (human) DNA are recombined with DNA from ~, phage using DNA ligase. An agar plate with a thin layer of bacteria over it is then infected with the phage. The phage infects the bacteria, rapidly multiplying within them, killing the
bacteria, and forming a clear area or plaque over the agar. Within each plaque are a large number of phage particles containing identical DNA fragments. Filter paper is placed over the agar plate, and phage particles corresponding to each of the plaques are absorbed onto it. The phage and the foreign DNA are denatured with a basic solution, and then the paper is treated to destroy both the phage and foreign DNA, leaving behind the piece of desired DNA. A radiolabeled DNA probe is then incubated on the paper to hybridize with the desired gene. Probes are sequences of DNA or R N A nucleotides complementary (eDNA or cRNA) to the gene of interest so that they will hybridize to it. A probe, because of its nucleotide's configuration, is highly specific and will only join a complementary piece of DNA despite the presence of overwhelming numbers of noncomplementary sequences. The probe is labeled with a radioisotope for detection. A film is placed over the DNA remnant, allowing identification of the gene. The results are traced back to the corresponding agar plate to find the DNA clone. The basic steps, thus, are (1) cutting the DNA into small pieces with restriction enzymes; (2) amplifying the pieces by inserting them into a cloning vector; and (3) identifying the desired DNA clone by using a DNA probe. The cloning of the gene allows its function and regulation to be studied. But, more importantly, its pure gene product, a protein ("one gene, one protein"), can be synthesized in large quantities, analyzed for abnormalities or used clinically, or antibodies can be raised against it for the development of a specific radioimmunoassay. The latter permits detection of overactivity of the gene.
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Southern and Northern blots are used to detect specific sequences of DNA and RNA, respectively. Southern blots (named after its inventor) use gel electrophoresis to separate DNA fragments cut with a restriction enzyme. Different DNA pieces of different sizes are separated out as discrete bands and then "blotted" onto a synthetic membrane (blot), which is then "probed" with a radiolabeled probe specific to the area of interest. This permits detection, in a very small amount of DNA, of the gene or DNA site of interest. "Northern" blots--a term used by laboratories since it uses the same principles as Southern blotting but identifies R N A rather than DNA--detect the specific R N A sequences from m R N A isolated from a cell. In this way one can, for example, determine whether the cell is
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producing a specific protein product by detecting the mRNA for it. The extraction, purification, and separation of both DNA and R N A are obviously time consuming, involved, and expensive. In an effort to circumvent these problems, investigators have developed methods of identifying both specific DNA and RNA sites by in situ hybridization of probes to properly prepared tissues. In this way, for example, the expression or elaboration of increased quantities of mRNA in a specific tissue or cell could be determined, giving information on the activity of the cell. Another important advance has been the development of the polymerase chain reaction (PCR). Much like in situ hybridization, PCR obviates the time, intricacies, and costs of extracting and purifying DNA so that it can be amplified or produced in quantity. By using PCR, it is possible to amplify minute quantities of specific portions of DNA contained within pure or impure preparations, from fresh or fixed tissues. The process is performed in a test tube that contains trace quantities of the DNA to be replicated among other bits and pieces of DNA (Figure 5). To this are added primers or short lengths of DNA bases that are complementary to both ends of the DNA sequence to be replicated (generally 50 to 2,000 base pairs in length), a heat-stable polymerase enzyme that joins DNA bases together, and a large excess of bases. The tube with its ingredients is heated, separating the DNA into single strands. The temperature is then reduced, whereupon the primers become attached to the respective ends of the specific DNA strand desired because of base pairing. Polymerase enzyme now synthesizes a single strand of DNA, beginning from each end where the primer attached itself to the specific DNA strand. The excess bases ensure that the reaction will be completed. The tube is reheated, and all newly formed double DNA is converted to single-strand DNA. The cycle is then repeated some 30 times, producing an exponential increase in specific new DNA strands. In summary, this brief review has sought to explain current techniques in molecular biology that are primarily directed at the isolation and identification of genes and their products. The technical procedures involved, although not difficult to conceptualize, are precise and demanding in practice. The molecular biology of the gene is of particular importance to all surgeons since the problems related to cancer formation and future therapy are related to a basic understanding of genes. ANNOTATED BIBLIOGRAPHY 1. Watson JD, Hopkins NH, Roberts JW, Steitz JA, Weiner AM. Molecular biology of the gene, 4th ed. Redwood City, CA: Benjamin Cummings Publishing Co, 1987. The textbook! Superbly written with unexcelled line drawings and illustrations. A delightful index (How a Frog Egg Counts Twelve Cell Divisions; Working With
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a Mammal Takes Time; Why RNA and Not DNA Was the First Living Molecule; etc.) invites you to look deeper and further. A must for the interested student (about $70) and should be known by all as a place to start to look things up. 2. Wong RS, Passaro E Jr. DNA technology. Am J Surg 1990; 159: 610-4. A simple explanation of many of the basic technologies dealing with DNA and how they are applied clinically. 3. Green ED, Waterson RH. The Human Genome Project. JAMA 1991; 266: 1966-75. An excellent description of the Project and how DNA mapping is done. 4. Watson JD. The double helix. New York: Norton Publishing; 1968. A candid personal narrative of the elucidation of the structure of DNA. GLOSSARY Allele: One member of a pair of genes for a given trait. Each member is of maternal or paternal origin. Amplify: To increase the quantity of a specific gone by a variety of techniques. Aneuploid: A cell containing extra chromosomes. Band: A pattern of light and dark regions by Giemsa staining that can serve as landmarks on chromosomes. Base pairing: The pairing of specific nitrogenous bases between complementary strands of DNA. For example, adenine is always paired with thymine and guanine with cytosine. eDNA (complementary DNA): Single- or doublestranded DNA made from an RNA template using the enzyme reverse transcriptase. Centimorgan (eM): A measure of the statistical probability of recombination between alleles. One cM represents a 1% chance of recombination per meiotic event. Centromere: The constricted region of a chromosome separating the short and long arms from one another. Chromosome: A single, linear, highly condensed DNA molecule. Clone (noun) : One of a collection of cells or vectors containing identical genetic material. Clone (verb) : The act of duplicating genetic material within a vector. Codon: A group of three consecutive nucleotides within messenger RNA (mRNA) that encodes one of 20 amino acids or encodes a message to stop translation (see Translation). Cosmid: A vector that incorporates components of plasmids and phage to carry larger clones (up to 40 kilobases). Diploid: Cells containing copies of both the maternal and paternal chromosomes. DNA (deoxyribonueleie acid): The molecule responsible for storing and transmitting genetic information; composed of two strands of nucleotides twisted around each other in the shape of a double helix.
Double helix: The twisted double-strand shape assumed by DNA. Exon: A contiguous segment of genomic DNA that is translated into polypeptide (see Intron). Familial: An inherited trait. Flow cytometry: Method used to measure nuclear DNA quantity in order to determine ploidy status. Gone: A segment of DNA within a chromosome encoding a single protein. Genome: The entire complement of genetic material in the form of DNA for a given organism. Genetic map: The ordering of genes by the statistical determination of recombination events between them. Genes separated by greater distances are more likely to recombine. Heterozygous: An individual containing dissimilar alleles for a given gone or locus. Homozygous: An individual containing identical alleles for a given gone or locus. Hybridization: The alignment of complementary strands of DNA (or RNA) via base pairing, widely used to identify portions of DNA on a Southern (or Northern) blot, using labeled probes. lntron: A noncoding sequence of DNA within the gone (see Exon). Karyotype: The physical appearance of the full complement of stained chromosomes for an individual. Locus: A site on a segment of DNA. Long arm (g) : One of the two prominent segments of a chromosome; the short or "p" arm is the other. The arms of a given chromosome join at its centromere. Library: A collection of recombinant genes cloned into a vector. Linkage: A measure of proximity between two alleles determined by recombination events. If they are not linked, they arc on separate chromosomes; if loosely linked, they are distant to each other on the same chromosome. The closer they are to one another, the more tightly they are linked. mRNA (messenger RNA) : The single-stranded edited copy of a gcnc ultimately translated into protein. Northern blot: The transfer of size-separated RNA fragments to a synthetic membrane for further studies. Nueleotide: One of the four building blocks of DNA (dATP, dGTP, dCTP, or dTTP) or RNA (ATP, CTP, GTP, or ATP) that are combined to form the nucleic acids. Oncogene: A cancer-inducing gone. Phage: A virus that infects a bacterial host, used in the laboratory as a cloning vector. Physical map: Analysis of the distance, in base pairs, between loci. Plasmid: Circularized DNA fragment, distinct from genomic DNA, found within bacteria, used as a cloning vector or to alter characteristics of the bacteria. Polymerase chain reaction (PCR): An efficient, simple, and rapid technique to multiply a length of DNA in a test tube. Promoter site: Region of a DNA molecule found in front of a gone that controls the expression of the gone.
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Prnto-oneogene: Normal gene that may become an oncogene; also called cellular oncogene. Recombinant DNA- The combination of foreign DNA inserts with vector DNA (e.g., plasmid, phage, or cosmid) to produce a clone within a host. Recombination: The rearrangement of DNA by breaking and re-ligations of the DNA strands; also called crossovers. Replieon: A sequence in the D N A that initiatesreplication. Restrictionmapping: The creationof a physicalmap by ordering enzymatically cut D N A fragments, Restriction endonuelease: Enzyme, isolated from bacteria, that recognizes specific base-pair sequences to cut DNA. The sites vary from frequent to rare cutting, depending upon the length of the restriction site. Restriction f r a g m e n t length polymorphism (RFLP): Variation in the distance between restriction enzyme cleavage sites that exist within a population producing unique DNA fingerprint patterns. RNA (ribonueleie acid): Single-strand nucleic acid found mainly in the nucleolus and ribosomes; contains ribose sugar and uracil, whereas DNA contains thymine. Short arm (p): One of the two prominent segments of a chromosome; the long or "q" arm is the other. The arms of a given chromosome join at its centromere. Southern blot: The transfer of size-separated DNA fragments to a synthetic membrane for further studies, initially described by E.N. Southern.
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Sporadic: Not familial. Sticky ends: The cut pieces of a single strand of DNA, which, because of base pairing, can be made to rejoin again at complementary base pairs, using the enzyme DNA ligase. Telomere: The distal ends of the chromosome. Transcription: The copying of DNA into messenger RNA. Transduction: The incorporation of a cellular gene in a viral genome that can then be introduced into other cells. Transfeetion: The process of placing foreign DNA into mammalian cells. Transformation: The cancerous alteration of mammalian cells; also the act of putting foreign DNA into bacteria. Translation: The process of converting the genetic code into polypeptides, mRNA codons are recognized by tRNA anti-codons. Each tRNA codes for a single amino acid. Tumor suppressor: A gene that prevents tumor formation until deleted or mutated. Vector: A construct used to propagate DNA in a host (bacteria, yeast, or cultured cells) (see Plasmid, Phage, and/or Yeast Artificial Chromosome). Virion: A replication virus particle. Western analysis: Protein electrophoresis characterizing size of unknown protein. Yeast artificial ehromosome (YAC) : A vector used in yeast that can propagate large fragments of DNA.
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