Chapter 5: Molecular biology and genetics

Molecular biology and genetics

The evolution of molecular biology  

Modern molecular biology has revolutionized research and our understanding of the molecular pathogenesis of disease in various branches of medical disciplines. In the past, the nomenclature of recombinant DNA and other techniques of molecular biology remained somewhat foreign to the practicing neuroscientist, in part because the techniques were only recently developed and their application to brain diseases became prominent only over the past decade. Here the historical development of the techniques of recombinant DNA and of molecular biology will be described. The unique features of these techniques over that of conventional scientific techniques will be discussed together with how they solve problems in a way that was previously not feasible. The historical perspective is intended to provide insight into why molecular techniques have blossomed and why they have an advantage over existing scientific techniques. It should be emphasized that the terminology and techniques are generic and are essentially the same regardless of the organ, organism or field of research to which they are applied.

Modern molecular biology is almost synonymous with the development of recombinant DNA technology. Despite the fundamental discoveries in the 1950s and 1960s, application of these techniques did not emerge until the late 1970s and early 1980s. Miescher isolated DNA for the first time in 1869 (he noted there was a substance derived from cell nuclei which differed from protein, and which dissolved in alkali but not in water; he isolated the substance from salmon sperm and bandages from surgical wounds and called it “nuclein”), and in 1944 Avery provided evidence beyond doubt that DNA, rather than protein, is responsible for transferring genetic information during bacterial transformation. In 1953, Watson and Crick deduced the double helix structure for DNA, which was based on the results of X-ray diffraction by Franklin and Gosling, and Wilkins. The work of Watson, Crick, and Wilkins was rewarded with a Nobel Prize, the first of three Nobel Prizes rewarded for advances in the understanding of molecular genetics. Marmor, Lane, and Doty showed the double helix of DNA could be separated into single strands by high temperatures (denaturation) and reannealed (double stranded) with return to lower temperatures. This is a major property of DNA enabling many processes such as DNA amplification by the polymerase chain reaction (PCR).

In 1964, Nirenberg and Matthaei, and Nishimura elucidated the genetic code by discovering the sequence of three DNA bases (the triplet codon) code for each amino acid in a protein, a discovery that irrefutably linked DNA as the molecule of life and resulted in another Nobel Prize in the field of modern genetics. In 1968, Olivera discovered DNA ligase, the enzyme used to join DNA fragments together, helping set the stage for recombinant DNA technology. However, the large size of the DNA molecule and its monotonous nature made it difficult to isolate and manipulate. The ability to isolate, manipulate, and clone DNA was, in large part, due to six further seminal contributions, as follows: (1) the discovery of restriction endonucleases; (2) the discovery of reverse transcriptase; (3) the cloning of DNA; (4) the ability to sequence the bases comprising a DNA fragment; (5) the ability to mutate specific DNA residues (altering specific amino acid codes) allowing for structure/function studies of proteins; and (6) the advent of PCR, which provided the tool to rapidly amplify exponentially selected fragments of DNA sequence (Table 1).

(1) To the molecular biologist, restriction endonucleases are what the scalpel is to the surgeon. They cut double-stranded DNA within the molecule (hence, endonucleases) at base pair sequences that are specific for each enzyme. The recognition sites for most enzymes are four to eight base pairs in length, with a few having recognition sites of only three base pairs, and even fewer recognize eight base pairs. The restriction endonucleases are isolated from bacteria where their normal function is as a defense mechanism, to digest foreign DNA, restricting it from being incorporated into the genome, and so are referred to as restriction endonucleases. It is possible to cut DNA into fragments of a desired and consistent size, knowing specifically where each cut is performed. The ability to cut DNA into fragments of specific length was absolutely essential to all of the recombinant techniques and more specifically for the development of cloning. The existence of a DNA restriction endonuclease was first discovered by Linn in 1962; however, it was not until later work, in 1970, that specific endonucleases were isolated and applied to molecular genetic techniques.

(2) The second contribution was the independent discovery of reverse transcriptase in 1970 by two laboratories, which made it possible to generate DNA complementary (cDNA) to messenger RNA (mRNA). The first stage in generating a protein from a gene requires transcribing nuclear DNA (transcription) into mRNA, which has the encoding sequence for the amino acids of the protein. Thus, isolation of an mRNA and conversion to cDNA provides one with a probe that will only bind with its complementary DNA (gene). The dogma for a long time was that DNA to RNA could not be reversed. The discovery of reverse transcription was worthy of more than a Nobel Prize for several reasons. RNA represents the expressed form of a DNA gene. Only the gene in its DNA form can be replicated such as with the cloning technique. The sequence of the DNA coding for protein is less than 1.5% of all DNA. Finding a gene is like finding a needle in a haystack. Thus, being able to convert the RNA into DNA sequences provided us with a new tool for identifying genes. The observation, that cDNA contains only the coding sequence, was all that was needed to clone or express the gene was a major revelation. The cDNA also provides a specific means to index its location on the chromosome, as well as a more stable nucleic acid structure (mRNA is easily degradable) for multiple applications, such as cloning.

(3) The third contribution was the birth of cloning. Cloning is a method to obtain multiple copies of a DNA fragment including a gene. In 1972, the first recombinant DNA molecule was generated at Stanford (CA, USA), and in 1973, the first foreign DNA fragment was inserted (recombined) into a plasmid (DNA vector). Plasmids are autonomously replicating DNA molecules, commonly present in bacteria, and capable of replicating in great numbers within a bacterium using the molecular machinery of the organism. This first recombined plasmid was successfully reinserted (the process of transformation) into a bacterium, which was grown in cultured media. The plasmid replicated providing millions of copies, and hence, the first successful cloning of a foreign DNA fragment. Thus, it was now possible to isolate mRNA known to code for a specific protein, and, with reverse transcriptase, convert it into a stable cDNA, which could then be recombined with a plasmid vector, transformed into a bacterium, and grown in culture, allowing for the cloning of large quantities of a specific DNA sequence or gene.

(4) The fourth contribution was made in 1977, when Sanger at Cambridge (UK) and Maxam and Gilbert at Harvard (USA) independently developed rapid nucleic acid sequencing techniques. These investigators were subsequently awarded a Nobel Prize. Thus, DNA of unknown sequence could now be cut into fragments of reasonable size, cloned into plasmid vectors, replicated into large quantities, and the specific DNA sequence determined.

(5) In 1982, Smith described the technique of site-directed mutagenesis, a technique whereby a specific DNA fragment may be manipulated or engineered, replacing a single base pair with another, resulting in an altered coding sequence, and the subsequent replacement of one amino acid for another. This powerful molecular technique enables the systematic study of various regions of a gene (protein), to identify regions or domains essential for specific functions, that is, structure–function analysis. Further, with the advances in human genetics leading to the identification of disease-causing DNA mutations, site-directed mutagenesis provided a tool to determine the physiological effect of a mutation. It also enabled the elucidation of the molecular pathogenesis of human disorders. Site-directed mutagenesis in addition to in vitro systems utilizing recombinant DNA molecules can also be performed in vivo by direct injection of the gene into the germ line. Once incorporated into the organism’s genome, it can be transferred to succeeding generations. This process referred to a transgenesis is used to generate transgenic animals as models of human disease. Genetically engineered mice have become a powerful tool to study specifically manufactured genetic changes and their resulted effects on phenotypes in vivo. It is possible to overexpress a protein of interest or eliminate a specific gene in an organ-, cell-, and time-dependent manner. These have been referred to as transgenic or knockout animals, respectively. While other mammalian species have been used for this purpose, mice have predominated because of their short breeding time, relative cost, and ease of manipulation of the germ line.

(6) The development of the PCR to amplify selected DNA or RNA fragments to several million copies instead of the need to clone provided the final tool for modern molecular biology. While cloning was a major breakthrough, PCR provides a more rapid and robust means to obtain millions of copies of a specific DNA molecule within hours. This discovery in 1985, also awarded with a Nobel Prize, is a major discovery that has markedly facilitated advances over the last decade in the study of human genetic diseases. The technique of PCR is the foundation of almost all investigations in modern molecular genetics. Although many applications exist, as will be described later, PCR was first utilized to detect the genome of pathogens responsible for human disease. For example, myocardial biopsies are obtained routinely in patients suspected of cardiomyopathy where the diagnosis is not evident and can be analyzed by PCR for the responsible pathogen. In essence, the presence of only one or two copies of RNA of DNA in a cell, which cannot be detected by conventional techniques, can be amplified by PCR to several million copies, putting particles such as viral RNA within the threshold of detection for conventional techniques.

In addition to the tremendous advances modern molecular genetic techniques have provided in the study of human health and disease, such techniques have also proven invaluable in therapeutic drug development. For example, the first cardiac drug made by recombinant DNA techniques was recombinant tissue plasminogen activator (rt-PA) in 1983, which revolutionized the therapy of acute myocardial infarction. The development of this therapeutic agent serves to illustrate the value of the techniques of recombinant DNA and molecular biology. A cDNA containing all of the coding regions of the TPA gene was mass produced in bacterial and mammalian cell-culture systems. Site-directed mutagenesis of the cDNA gene and its expression led to the identification of its functions, namely lytic activity, fibrin affinity, and fibrin-dependent enhanced lytic activity. Five domains were recognized to have specific functions that are coded by separate and autonomous portions (exons) of the gene. Hundreds of drugs have been generated by recombinant DNA technology.