edited by: MICHAEL R. SLABAUGH HELEN J. JAMES Weber State College Ogden. Utah 84408
Industrial Applications of Recombinant DNA Technology Michael D. Jones and Jeffrey T. Fayerman' Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center. Indianapolis. IN 46265 There have been many fundamental advances in recomhinant DNA technology during the last several years. The potential for industrial application of this technology has therefore increased greatly. We will descrihe some of the essential requirements for carrying out recomhinant DNA experiments. In addition, we will describe potential industrial applications of recombinant DNA technology and discuss three specific current applications. Essential Requirements
Recombinant DNA technology requires the use of, a t a minimum, endonucleases, DNA ligases, cloning vectors, and a host organism into which DNA can he introduced readily. The bacterium Escherichia coli isoften used as a host organism because its genetics and biochemistry have been studied extensively; however, other hacteria and some types of fungi, as well as plant and animal cells, can he used. Efficient expression of a cloned gene, or genes, in the host organism is also an important requirement. Restriction endonucleases (BamHI and EcoRI, for example) recognize a specific sequence in a DNA molecule and cleave the phosphodiester honds of the DNA molecule a t a specific site, usually within the recognition sequence. Many of the known restriction endonucleases cleave DNA in such a manner that cohesive ends are generated (Fig. 1). If two different DNA molecules having a recognition sequence for a particular restriction endonuclease are cleaved with that enzyme, the DNA fragments generated can he recombined by mixing the fragments together and treating them with DNA ligase, an enzyme that rejoins the phosphodiester honds. The plasmid cloning vector pBR322 ( I ) , a circular DNA molecule that can replicate independently of the chromosome in E. coli, has only one recognition site for the restriction endonuclease BamHI. If one were interested in cloning a gene from the chromosome of another E. coli strain or another organism, pBR322 could he digested with BamHI while chromosomal DNA from the donor organism could he similarly digested. The resulting DNA fragments could he mixed together in appropriate ratios and then he treated with DNA ligase. Under ideal conditions, a population of DNA molecules, composed of the cloning vector plus pieces of inserted chromosomal DNA, would result. E. coli cells could then he transformed by these recomhinant DNAmolecules using one of several available methods (2). Transformation of hacteria is a process where exogenous DNA (recombinant DNA, for example) is brought into the cell and a phenotype encoded by the exogenousDNA is selected. Thus, the recipient cell is "transformed" to a form that produces a Author to whom correspondence should be addressed.
B.
ACGCTCGATCGATCG TGCGAGCTAGCTAGCCTAG
GATCCACGTCGATCGAT GTGCAGCTAGCTA
Figure 1. A. DNA sequence containing one B a w l recognition sequence. B. Resuiting DNA fragments anar cleavage by BamHI. Ligation of these B a w l fragments would result in regeneration of sequence A.
gene product encoded by exogenous DNA. Only those recipient cells competent for uptake of DNA can be transformed. Several methods can he used to cultivate competent E. coli cells for transformation (2). pBR322 carries two genes encoding proteins that confer drug resistance, one to ampicillin and the other to tetracycline. The recognition sequence for BamHI is within the tetracyrline-resistanre gene. E. coli transformants eontaininp 11H11322with DNA inserted at the h m H l site would not he-resistant to tetracycline due to interruption of the gene (Fig. 2). In this way, insertional inactivation allows one to distinguish transformants that carry vectors with inserts (ampicillin resistant and tetracycline sensitive) from transformants which contain the vector without an insert (resisrant tu ampicillin and tetraryrline). The transformants ron. tainine \.QCt
