Genetic Engineering Or How to Build a Better (at Least Different) Mousetrap Charles E. Canaher, Jr. and Deborah W. Siegmann Florida Atlantic University. Boca Raton. FL 33431 One of the scientific areas captivating and permeating our society today is molecular biology. Molecular biology deals with the macromolecules found in living things and the interactions of these molecules. Within molecular biology, the area that has attracted the most attention is genetic engineering. In its broadest sense, genetic engineering refers to any artificial process that alters the genetic composition of an organism. Such alterations can be accomplished by chemicals, by radiation, or through selective breeding. Today, the term usually refers to the process whereby genes or portions of chromosomes are chemically manipulated and rearranged. The potential applications of genetic engineering often appear in fiction where the possibility of cloning human beings has fascinated novelists, playwrights, and comic hook writers. Captain America had to deal with "mad scientists" who cloned their attack personnel. Legions of mindless auto beings were cloned in Huxley'sBraue New World and infant "Hitlers" were created from the genes of the Fuhrer in Levin's The Boys from Brazil. Actually, the process of cloning is far less sinister and dramatic. The term clone comes from the Greek word klon meaning a cutting used to propagate a plant. Cell cloning is the production of identical cells from asingle cell. In like manner, gene cloningis the production of identical genes from a single gene. During gene cloning, genes from different organisms are often joined to form one artificial molecule known as a recombinant DNA. Before explaining the process of gene cloning, it is advantageous to discuss briefly the type of chemical reactions involved and to describe the structure of genes. The chemical reactions used in gene cloning are analogous to those encountered in elementary organic chemistry. The two major chemical reactions employed are actually the reverse of one another. The first of these, "cutting" a gene, is the hydrolysis of DNA as shown below.
--9.--
0
0
H20
-lzz endonvcleasc
-o-pH + HO-
0-
This is analogous to the hydrolysis of esters as fats t o form glycerol and fatty acids. H
0
II
R-C-O-
I
O11 R'-C-O-C-H
I
+ 30H-
-
R
R'-C-O-
R
R"-C-O-
H-0-CH2 t H-O-CH
I I
H-O-CHZ
The second type of reaction involves joining DNA molecules together, and this is accomplished by the dehydration reaction shown below. 854
Journal of Chemical Education
This reaction is analogous to esterification reactions including the sequence employed in the synthesis of polyesters such as Dacron.
~ H $ - M ~ - M H ,
I1
0 0 dimethyl terephthalate
+ nHO(CHJ,OH
+
ethylene glycol
polyethylene terephthalate T o understand how these reactions apply t o cloning, it is necessary first to understand the structure of genes. A gene is a segment of DNA that codes for a single RNA or polypeptide molecule. Thus, genes contain the information the cell needs for constructing molecules required for the cell's basic operation. The genes of a cell are joined together to form one long DNA molecule called a chromosome. Chemically, DNA is composed of smaller units known as nucleotides. Each nucleotide contains a phosphate group, deoxyribose, and one of four nitrogen-containing bases (adenine, cytosine, guanine, or thymine) (Fig. 1). The nucleotides are joined t o ~ e t h e through r the ~ h o s ~ h a tand e s the deoxvriboses with t b i haws nttnrhed toihe deoxyribose moiety. Most DNA molecules are douhle-stranded with two DNA chains wrapped around each other forming a double helix (Fig. 1). The double helix is held together by interactions between the bases in the two strands. The bases form hydrogen bonds in a specific fashion, taking advantage of steric and electronic factors, with an adenine in one chain pairing with a thymine in the other chain, and with a cytosine in the first chain pairing with a guanine in the second chain (Fig. A,
61.
These DNA chains can be manipulated in a variety of ways, but the one of major interest here is the production of recombinant DNA during gene cloning. T o make a recombinant DNA molecule requires that genes he "cut" and then rejoined, and this in turn involves the use of enzymes. Enzymes are proteins that catalyze chemical reactions. One group of enzymes, known as restriction endonucleases, recognize specific series of base pairs in DNA. Within this series, which can be up to six base pair sin length, theenzyme then cuts the DNA at a specific point. The sequence of base pairs that the enzyme recognizes often forms a palindrome; that is, the base sequence read from left to right in one strand is the same as the sequence read from right to left in the other strand. The restriction endonuclease usually breaks the DNA asymmetrically, with the cut occurring in a differentplace in each strand. This leaves asmall segment of
single-stranded DNA a t the cleavage site (Fig. 3). The biological function of restrictionenzymes is to recognize foreign, invading DNA in a cell and t o render it harmless bv deeradine it. The cell's own DNA is not deeraded to because i t i s moiified by attachment of methyl snecific sites on the bases and is therefore not susce~tibleto the restriction enzymes. Various microorganisms pnniuce different restrirtivn enzvmes with different sperificitie~that can be used in gene cloiing. With this background in DNA structure and restriction enzymes, we now summarize the processof genecloning. The gene to be cloned is excised from the chromosome using a restriction endonuclease. I t is then joined to a piece of bacterial DNA, and the resulting recombinant DNA molecule is introduced into a bacterial cell. As the hacteria erow and reproduce, the recombinant DNA molecule is replicated, nroducine -c o.~ i e ofs the orieinal -eene. Each s t e in~ this process will be examined in greater detail. In isolating the eene to be cloned. an a~propriaterestriction enzymeUmus
