Principles of Biochemistry Volume 3 - A. Lehninger 1985

Molecular Mechanisms of Genetic Information Transmission
More About Genes: Repair, Mutation, Recombination, and Cloning
Genes from different organisms can be artificially combined

We have examined several Variants of the genetic recombination process that occur naturally in various Cell types. Gene recombination, or the recombination of gene sets, can also be performed in vitro to yield novel gene combinations that do not exist in nature. For example, genes encoding two different Proteins can be isolated from two distinct organisms, joined together, and configured into a new genetic combination. Such artificial recombinant DNAs serve as an invaluable tool in genetic research and, as we will see later, can also be applied for practical purposes.

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Fig. 30-18. Schematic diagram illustrating how an antibody heavy-chain gene can be assembled by the translocation of V, D, and J gene segments from different Regions of the genome; ultimately, a complete V_H gene is formed, joined to a C_H gene. The mRNA transcript then undergoes Processing, during which spacer regions are removed to yield a mature mRNA encoding the heavy chain.

The Development of Methods for Gene Isolation and their recombination into novel combinations has been a major biochemical breakthrough, ushering in a new era in genetic research.

The discovery of Restriction Endonucleases marked the first step toward solving Structure/149.html">The problem of artificial gene recombination. Suppose we wish to join together two double-stranded DNA molecules isolated from different organisms (Fig. 30-19). Each is treated individually with the same restriction endonuclease, which cleaves both DNA strands to generate protruding ends at the site of the break (Section 27.24). Assuming there is only a single site recognized by this restriction enzyme in each DNA molecule, The nucleotide sequences of the protruding ends of the two cleaved DNAs will be complementary. If these DNAs are then mixed, heated, and slowly cooled, their sticky ends will form complementary Base Pairs, resulting in a novel recombinant DNA whose strands contain single breaks (Fig. 30-19). Subsequent Treatment of these DNAs with DNA ligase in the presence of Energy Sources yields a newly created, covalently sealed recombinant DNA.

Fig. 30-19. The Use of a specific restriction endonuclease that generates protruding ends to cleave two different DNAs (DNA₁ and DNA₂) and obtain fragments for subsequent recombination. Because many restriction endonucleases cleave only specific DNA sites possessing twofold rotational Symmetry, the resulting sticky ends interact complementarily with the ends of any other DNA cut by the same enzyme. Upon annealing, the appropriate fragments of DNA₁ and DNA₂ can re-form the original molecules or generate new recombinant DNAs, as shown in the figure. Covalent joining of the fragments is catalyzed by DNA ligase.

Another key enzyme widely used for joining DNA fragments is terminal transferase, which is capable of adding A large number of consecutive deoxyribonucleotide residues to the 3' ends of DNA strands. This enzyme is non-specific and can utilize dATP, dTTP, dGTP, or dCTP as precursors. Because terminal transferase does not require a template, it can synthesize 3'-terminal sequences consisting of a single type of nucleotide. Consequently, poly(G) tails can be added to the 3' ends of one double-stranded DNA, and poly(C) tails to the 3' ends of another. Since such tails are complementary to each other, they can be used to join the two DNA molecules through The formation of complementary base pairs between their sticky ends (Fig. 30-20); covalent sealing of the thus-joined DNAs is then accomplished by DNA ligase.

Using these and Other Enzymes, numerous DNA molecules from diverse sources have been successfully joined. One of the earliest achievements in this direction was the insertion of an rRNA gene isolated from the African clawed frog Xenopus laevis into an E. coli plasmid. In another early experiment, the DNA of the simian virus SV40 (Section 27.29) was inserted into phage λ DNA, thereby combining the Chromosomes of animal and bacterial Viruses. Since then, hundreds of different artificial recombinant DNAs have been generated in laboratories.