The science of genetics defines and analyzes heredity of the vast array of structural and physiologic functions that form the properties of organisms. The basic unit of heredity is the gene, a segment of deoxyribonucleic acid (DNA) that encodes in its nucleotide sequence information for a specific physiologic property. The traditional approach to genetics has been to identify genes based on their contribution to phenotype, the collective structural and physiologic properties of an organism. A phenotypic property, be it eye color in humans or resistance to antibiotics in a bacterium, is generally observed at the level of the organism. The chemical basis for variation in phenotype is change in genotype, or alteration in the DNA sequence, within a gene, or within the organization of genes.
DNA as the fundamental element of heredity was suggested in the 1930s from a seminal experiment performed by Frederick Griffith (Figure 7-1). In this experiment, killed virulent Streptococcus pneumoniae type III-S (possessing a capsule), when injected into mice along with living but nonvirulent type II-R pneumococci (lacking a capsule), resulted in a lethal infection from which viable type III-S pneumococci were recovered. The implication was that some chemical entity transformed the live, nonvirulent strain to the virulent phenotype. A decade later, Avery, MacLeod, and McCarty discovered that DNA was the transforming agent. This formed the foundation for molecular biology as we understand it today.
Griffith’s experiment demonstrating evidence for a transforming factor, later identified as DNA. In a series of experiments, mice were injected with live or killed encapsulated or nonencapsulated S. pneumoniae, as indicated in experiments A through D. The key experiment is D, showing that the killed encapsulated bacteria could supply a factor that allowed the nonencapsulated bacteria to kill mice. Besides providing key support for the importance of the capsule for pneumococcal virulence, experiment D also illustrates the principle of DNA as the fundamental basis of genetic transformation. (Reproduced by permission from McClane BA, Mietzner TA: Microbial Pathogenesis: A Principles-Oriented Approach. Fence Creek Publishing, 1999.)
Recombinant DNA technology was born in the 1960s and 1970s when investigations with bacteria revealed the presence of restriction enzymes, proteins that cleave DNA at specific sites, giving rise to DNA restriction fragments. At about the same time, plasmids were identified as small genetic elements carrying genes and capable of independent replication in bacteria and yeasts. In a single cell, as many as 1000 copies of an identical plasmid can exist.
Amplification of specific regions of DNA can be achieved with archaebacterial enzymes using polymerase chain reaction (PCR) or other enzyme-based method of nucleic acid amplification. DNA isolated from these sources and digested with appropriate restriction enzymes can be ligated into plasmids for engineered amplification. Genes can be placed under ...