Andrew W. Murray and Jack W. Szostak created an invaluable tool for recombinant DNA technology when they invented a working artificial chromosome to study natural chromosome behavior.
Advancements in medicine often result from achievements in basic biology and technology. The artificial chromosome dually distinguishes itself in that it gives biologists insight into the fundamental mechanisms by which cells replicate and it also plays a role as a tool in the technology of genetic engineering. From the time of its invention in 1983 by Andrew W. Murray and Jack W. Szostak, working at the Dana-Farber Cancer Institute
In order to appreciate the benefits of an artificial chromosome, one must first be aware of the role natural chromosomes play in the living cell. Chromosomes are essentially carriers of genetic information that is, they possess the genetic code that is the blueprint for life. In higher organisms, the number and type of chromosomes that a cell contains in its nucleus is characteristic of the species. For example, each human cell has forty-six chromosomes, the garden pea has fourteen, and the guinea pig has sixty-four.
The chromosome’s job in a dividing cell is to replicate and then distribute one copy of itself into each new daughter cell. This process, referred to as mitosis or meiosis, depending on the actual mechanism by which this occurs, is of supreme importance to the continuation of life, and errors may have disastrous consequences. Indeed, most errors in chromosomal inheritance are lethal and result in the truncation of the whole cell line containing the error. This, ironically, limits the damage that would otherwise result if these abnormal cells proliferated.
More dangerous are abnormal cell lines that are not sufficiently damaged to cause cell death but rather continue to thrive and cause disease. The most famous example of this is Down syndrome,
Chromosomes are composed primarily of two raw materials, protein and the nucleic acid deoxyribonucleic acid (DNA), which is the actual carrier of genetic information. DNA is a duplex of molecular chains twisted around each other to form a spiral staircase, or double helix. Each strand of this duplex is a polymer made up of the chemical bases adenine (A), guanine (G), thymine (T), and cytosine (C) attached together chemically like links on a chain. The precise arrangement of these bases forms an elaborate code in which every group of three bases determines a specific amino acid that will be incorporated into a protein. Any group of bases large enough to code for one entire protein is called a gene, the total collection of which ultimately determines an organism’s physical characteristics. The bases on one strand of the DNA molecule are paired with the bases on the opposite strand according to strict rules: A binds with T and G binds with C; therefore, the strands are complementary and the base sequence of one strand can be deduced from the base sequence on the opposite strand.
In 1953, when James D. Watson
At left, a double-stranded DNA molecule, with its sides formed by sugar-phosphate molecules and its “rungs” formed by base pairs. Replication begins at point (a), with the separation of a base pair as a result of the action of a special initiator protein (b). The molecule splits, or “unzips,” in opposite directions (c) as each parental strand is used as a template for the daughter strand, which is formed when bases form hydrogen bonds with their appropriate “mate” bases to form new ladder “rungs.” Finally (right), one parental strand and its newly synthesized daughter strand form a new double helix, while the other parental strand and its daughter strand form the second double helix.
An artificial chromosome is a model of a natural chromosome, designed in the laboratory, that possesses only those functional elements its creators desire. In order to be a true working chromosome, however, it must, at minimum, maintain the machinery to carry out replication and segregation. The technique of studying nature by making a simplified model and subsequently refining it is an approach more common to the physicist than to the biologist. This is because the complexity of biological systems make it difficult to discern and isolate the essential features of such systems. Nevertheless, by the early 1980’s, Murray and Szostak had recognized the possible advantages of having a simple, controlled model to study chromosome behavior, as there are several inherent difficulties associated with studying chromosomes in their natural state.
Because natural chromosomes are large and have poorly defined structures, it is almost impossible to sift out for study those elements that are essential for replication and segregation and those that are dispensable. Previous methods of altering a natural chromosome and observing the effects are unsatisfactory because the cells containing the altered chromosome usually die. Furthermore, even if the cells survive, analysis is complicated by the extensive tracts of genetic information the chromosome carries. Artificial chromosomes are simple; they have known components even if the functions of those components are poorly understood. In addition, because artificial chromosomes are extra chromosomes carried around by the cell like a parasite, their alteration does not kill the cell.
Segments of DNA from any organism can be cloned by inserting the DNA segment into a plasmid a small, self-replicating circular molecule of DNA separate from chromosomal DNA. The plasmid can then act as a “cloning vector” when it is introduced into bacterial cells, which replicate the plasmid and its foreign DNA. This diagram from the Department of Energy’s Human Genome Program site illustrates the process.
Prior to the synthesis of the first artificial chromosome, the essential functional elements necessary to accomplish replication and segregation had to be identified and harvested. One of the three chromosomal elements thought to be required is the origin of replication, the site at which the synthesis of new DNA begins. The relatively weak interaction between DNA strands at this site facilitates their separation, making possible with the help of appropriate enzymes the subsequent replication of the strands into sister chromatids. The second essential element is the centromere, a thinner segment of the chromosome that serves as the attachment site for the mitotic spindle. Sister chromatids are pulled into diametric ends of the dividing cell by the spindle apparatus, thus forming two identical daughter cells. The final functional element is a repetitive sequence of DNA located at each end of the chromosome, called a telomere,
In the late 1970’s, a group of researchers at Stanford University Kevin Struhl,
With all the functional elements at their disposal, Murray and Szostak proceeded to construct their first artificial chromosome. Once made, this chromosome would be inserted into yeast cells to replicate, as these cells are relatively simple and well characterized but otherwise resemble cells of higher organisms. In addition, inheritance errors are rare in yeast. Construction begins with a commonly used bacterial plasmid, a small, circular, autonomously replicating span of DNA. Enzymes are then called on to create a gap in this cloning vector into which the three chromosomal elements are spliced. In addition, genes that confer some distinct trait such as color to yeast cells are also inserted, thus allowing the detection of which cells have actually taken up the new chromosome subsequent to their incubation together. Although their first attempt resulted in a chromosome that failed to segregate properly, by September, 1983, Murray and Szostak had published in the prestigious British journal Nature a report on their success in creating the first artificial chromosome.
The artificial chromosome had a tremendous impact on the understanding of chromosome behavior and the mechanism of heredity. Murray and Szostak set out to answer three questions. First, they wanted to test the validity of the commonly held belief that replication origins, centromeres, and telomeres are the only essential items necessary for competent chromosome segregation. They proved this by demonstrating competent chromosomes after the removal of all segments of DNA except for the functional elements. Second, they wanted to learn more about how the mitotic spindle attaches to the centromere. Third, they wanted to learn how the errors in segregation that give rise to diseases such as Down syndrome arise.
One of their discoveries was that chromosomes must be greater than a certain minimum length, about 100,000 base pairs; otherwise, they will segregate randomly. A possible explanation for this finding is that chromosomes will not remain attached to the mitotic spindle unless they are under tension. This tension is created when sister chromatids are entwined around each other as they are being pulled to opposite sides of the cell. The shorter the chromosome is, the fewer molecular interactions it has with its homologue and the less avidly it will bind to it. The implication is that anything that breaks a chromosome, such as a mutagen, might shorten it enough to cause problems in segregation and genetic disease.
One of the most exciting aspects of the artificial chromosome is its application to recombinant DNA technology, which is the creation of novel genetic materials through the combination of segments of DNA from various sources. For example, the yeast artificial chromosome can be used as a cloning vector. That is, a segment of DNA containing some desired gene is inserted into an artificial chromosome and is then allowed to replicate in yeast until large yields of the gene are produced. David T. Burke,
-
Berg, Jeremy, John L. Tymoczko, and Lubert Stryer. Biochemistry. 6th ed. New York: W. H. Freeman, 2006. Excellent introductory textbook considered a standard for students of biology, biochemistry, or medicine presents clear discussion of DNA’s role in the mechanism of heredity and of recombinant DNA technology. -
Murray, Andrew W., and Jack Szostak. “Artificial Chromosomes.” Scientific American 257 (November, 1987): 62-68. Brief account of artificial chromosomes and their applications to biological problems. Written by the scientists who created the first artificial chromosome. Informative article for nonscientists; includes several clarifying illustrations as well as a bibliography. -
_______. “Construction of Artificial Chromosomes in Yeast.” Nature 305 (September, 1983): 189-193. The original, landmark paper that started it all. Intended for readers with scientific background. Explains in detail how to construct artificial chromosomes and provides original data and discussion of results. -
Perbal, Bernard. A Practical Guide to Molecular Cloning. 3d ed. New York: John Wiley & Sons, 2008. Detailed and functional guide on all aspects of molecular cloning. Intended for advanced readers who are interested in the practical details of genetic engineering and recombinant DNA technology. -
Watson, James D. The Double Helix: A Personal Account of the Discovery of the Structure of DNA. 1968. Reprint. New York: Simon & Schuster, 2001. Watson’s account of the origin of the Watson-Crick model of DNA, which revolutionized biology and earned Watson and Crick the 1962 Nobel Prize in Physiology or Medicine. Classic work, written for the lay reader, shows how scientific discoveries occur. Filled with anecdotes. Includes photographs of several eminent scientists involved in the discovery.
Cohen and Boyer Develop Recombinant DNA Technology
Berg, Gilbert, and Sanger Develop Techniques for Genetic Engineering
Geneticists Create Giant Mice
Jeffreys Discovers the Technique of Genetic Fingerprinting
FDA Approves a Genetically Engineered Vaccine for Hepatitis B
Genetically Altered Bacteria Are Released into the Environment
Patent Is Granted for Genetically Engineered Mice
Genetically Engineered Food Reaches Supermarkets
Completion of the Sequencing of the Human Genome Is Announced