Molecular Biology

Molecular Biology

Molecular biology, subdiscipline of BIOCHEMISTRY that studies the structure, synthesis and degradation of macromolecules (very large molecules) found in living cells, their metabolic regulation (how they are interrelated and balanced during synthesis and degradation) and their expression (how the GENETIC code operates and is controlled through structural interrelationships). Macromolecules include the nucleic acids DNA (deoxyribonucleic acid) and RNA (ribonucleic acid); proteins (including enzymes); carbohydrates; and complexes of carbohydrates and proteins and lipids (soluble cellular fats and waxes) and proteins. The term was used by Oswald T. Avery in the late 1940s and was early equated with the study of nucleic acids.

DNA

As a result of their work on BACTERIA, Canadian researchers Avery and Colin M. MacLeod and American Maclyn McCarty were the first to provide firm evidence that DNA was the genetic material in the cell (1944). The structure of DNA is a double helix, first described in 1953 by Nobel laureates (1962) J.D. Watson (an American who had been influenced by Avery's work) and British researcher Francis Crick. The double helix is composed of 2 antiparallel chains of sugar-phosphate "backbones" with complementary pairs of nucleic acid bases in the centre.

American biochemist Erwin Chargaff's observation of base pairing was important to Watson and Crick's discovery, as were the X-ray diffraction studies of British biophysicist Rosalind Franklin. Just before the elucidation of the structure of DNA by Watson and Crick, G. Wyatt (now at Queen's University) described 5-methylcytosine, the first modified base found in DNA molecules, and confirmed base pairing and the base composition of a number of DNAs.

DNA was postulated as the template for RNA synthesis by French biochemists Jacques Monod and François Jacob (NOBEL PRIZE, 1965). RNA synthesis is called transcription; protein synthesis from the RNA transcript is called translation. Once the protein is translated, depending on the type of cell, it can then undergo a series of changes (eg, addition of carbohydrate or lipid) to make the complex proteins that are part of many cells.

Viral Systems

A large part of research in molecular biology has been undertaken on VIRUSES, the simplest of life forms, because they have no complex cellular structure or cell membrane. Some of the earliest basic studies on viral systems were made by Canadian researcher Felix d'Herelle, who independently discovered bacteriophages (bacterial viruses). Viruses are composed either of DNA or RNA with associated proteins, and thus can be used as model systems for examining replication (DNA synthesis), transcription and translation.

Bacteriophages were the first to be examined in detail. DNA synthesis is best understood in these viruses mainly because of studies led by American biochemist A. Kornberg (Nobel Prize, 1959). More recently, eukaryotic viruses (those that attack nucleated cells) have been used. The virus can be considered a microcosm of the cells and tissues of animals, and its molecular biology is controlled by mechanisms analogous to those in cells. Viruses that have RNA as their genetic material are called retroviruses; some retroviruses have been implicated in human CANCER. However, most viruses that attack eukaryotic cells are DNA viruses.

Genetic Engineering

Molecular biology has expanded as a result of the application of recombinant DNA techniques (GENETIC ENGINEERING), following the isolation of restriction enzymes in 1970 by American biochemist Hamilton Smith (Nobel Prize, 1978). Restriction enzymes cut DNA molecules at specific sites (base sequences); joining enzymes can link together the DNA fragments.

In 1977 the first recombinant molecules composed of mammalian DNA inserted into bacterial elements (plasmids) were constructed. Plasmids are particles found in some bacterial cells that contain DNA, but they are not part of the chromosomal apparatus of the bacterium. The next important discovery was of "gene splicing"; ie, the RNA synthesized from the DNA of eukaryotic cells can be much larger than the final messenger RNA product, because those segments of the RNA product that are not necessary to code for the protein message are cut out, and the remaining RNA spliced together.

This work was followed by the development of powerful, fast and relatively easy methods for determining the base sequence of DNA (the order of the base components in DNA). Two laboratories, those of Walter Gilbert in Cambridge, Massachusetts, and Fred Sanger in Cambridge, England, developed different methods. Gilbert, Sanger and Californian Paul Berg (who performed the first cloning experiments) received a Nobel Prize in 1980. The automation of DNA sequencing, pioneered by Leroy Hood, has made feasible the human genome project and other megasequencing projects on bacteria, yeast and a nematode (see NEMATODA).

In the early 1960s Canadian-born Julius Marmur and American Paul Doty described experiments on DNA strand separation, renaturation and hybridization. When DNA is heated the forces holding the base pairs together, and hence the 2 antiparallel strands together break and the strands separate from each other (melt). If the DNA solution is cooled slowly, the strands reassociate again (renaturation), and base pairing is restored, as is the structure of the DNA as 2 antiparallel strands. This observation was key to the development of 2 techniques, site-directed mutagenesis and the polymerase chain reaction (PCR).

The ability to synthesize DNA in a test tube, pioneered by Gobind KHORANA, was another key tool in the development of the techniques. If a piece of synthetic DNA of similar but not exact base sequence is added to a solution of melted DNA, it can compete in the reassociation reaction during cooling (hybridization), and a different 2-stranded molecule results, with one strand from the melted DNA, the other synthetic DNA. The base difference introduced is considered a mutation and can be made into a permanent change by use of DNA replication enzymes.

Canadian Michael SMITH (Nobel Prize, 1993) developed site-directed mutagenesis while working at UBC. He shared the prize with Kary Mullis, an American who invented the PCR method that allows the copying and amplification of any selected piece of DNA. The method utilizes the hybridization property of DNA, the ability to synthesize small pieces of DNA in the test tube and the availability of thermally stable enzymes (DNA polymerases that are active even at temperatures as high as 95° C) from bacteria found in hot SPRINGS and deep sea vents.

Two small synthetic pieces of DNA that can hybridize specifically to the sequences flanking the area of DNA in a chromosome that is to be studied are used as primers (starting points) for the synthesis of the DNA. The reaction mixture containing the DNA to be studied, the DNA polymerase and the primers is heated; the primers hybridize to the melted DNA during the cooling time. The enzyme synthesizes the DNA wanted, starting at the primers, one for each strand, the temperature is raised quickly, the newly synthesized DNA melted off the template and the solution cooled quickly. Since the enzyme used for the synthesis is heat-stable up to 95° C, the process can be repeated many times. A special apparatus, a thermal cycler, is used to raise and lower the temperature rapidly and to time the synthetic reaction precisely. At the end of 25-30 cycles, there is usually enough of the desired DNA for sequence analysis or use in other studies.

Changes in Classical and Molecular Genetics

The combination of all of these technologies has led to a complete change in classical and molecular genetics. The ability to identify and sequence genes has led to explanations of clinical observations at the level of the DNA molecule and a better understanding of many GENETIC DISEASES. One example is the identification of the gene and mutation(s) for the majority of the cases of cystic fibrosis by Canadians Lap-Chee TSUI and Jack Riordan and American Frances Collins. Changes in the DNA at the base sequence level cause differences in the messenger RNA sequence which, when translated into protein, result in the wrong protein being made or in an altered protein, leading to a dysfunction characteristic of the hereditary disease.

Many genetic concepts have been revised; eg, multiple gene copies have been proven to exist for many proteins. The combination of site-directed mutagenesis and the PCR methodology provides a powerful means of targeting genetic defects, developing screening tests and ultimately repair of the defects. A different application of the properties of restriction enzymes, DNA hybridization and the PCR, first described by Alec Jeffries of England, is DNA footprinting used for the identification of the DNA from different individuals, be they humans, animals or plants.

Site-directed mutagenesis provides the means to change an amino acid in a protein by altering the DNA sequence that codes for the protein. This allows identification of the role of specific amino acids in proteins; eg, at the substrate binding site of an enzyme. This can be correlated with other structural data such as X-ray crystallographic analysis of the shape and conformation of the protein, leading to understanding of the function of the individual amino acids in the protein.

Evolution is the focus of a new part of molecular biology, the theory that life on Earth started off in an RNA world and DNA came much later. The theory has gained credence from the discovery of RNA molecules (ribozymes) that act as enzymes that can cleave RNA molecules. Canadian-born Sidney ALTMAN and American Thomas Cech (Nobel Prize, 1989) independently discovered RNA self-cleavage and ribozyme function.

Role of Canadians

Canadians have played an important part in the development of molecular biology. Gobind Khorana (Nobel Prize, 1968), the first to chemically synthesize a nucleic acid molecule, started his work at the BC Fisheries Research Laboratories in Vancouver. Khorana's many students include Gordon Tener, who developed a chromatographic column for separating nucleic acids over a range of sizes, an innovation that has had tremendous methodological impact on molecular biology, and Michael Smith, who was a member of Sanger's laboratory when the first DNA virus molecule was sequenced in 1977.

Groups are now undertaking research in molecular biology in all major Canadian universities (within laboratories in faculties of medicine and science), in the BIOTECHNOLOGY companies located in most provinces, and in many federal and provincial laboratories.

See alsoNATIONAL RESEARCH COUNCIL OF CANADA; RESEARCH, PROVINCIAL ORGANIZATIONS.