BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 31. GENE REARRANGEMENTS: RECOMBINATION, TRANSPOSITION, AND CLONING

Summary

During genetic recombination, a new DNA molecule is formed by the breakage and rejoining of DNA strands. Mutual exchange can occur between any pair of homologous sequences from parental DNA molecules, which is why this process is referred to as general genetic recombination. In E. coli, general recombination is mediated by the rec genes. The recBC protein is a nuclease that generates single-stranded DNA capable of invading a double-stranded DNA molecule. This single-strand binding and invasion reaction is catalyzed by the recA protein, which simultaneously hydrolyzes ATP.

Genetic rearrangements involving nonhomologous loci rely on Mobile Genetic Elements known as Transposons (elements capable of transposition). Plasmids (small, circular, double-stranded DNA molecules) represent an important Class of mobile genetic elements. These accessory Chromosomes carry genes responsible for antibiotic inactivation, the METABOLISM of natural compounds, and toxin production. Plasmids can replicate autonomously or integrate into the host Cell chromosome. The F factor (fertility factor) is a plasmid that confers upon Bacteria The ability to transfer genes to other bacteria via conjugation. Conjugation requires direct physical contact between the donor (F+) and recipient (F-) Cells. R factor plasmids confer resistance to

Antibiotics. The larger of these plasmids contain the resistance transfer factor (RTF), which mediates The transfer of the plasmid to another bacterium during conjugation. Some genes conferring Antibiotic Resistance can be linked to the RTF region, allowing multiple drug resistance to be transmissible. Transposons are highly mobile because they are flanked by insertion sequence (IS) elements. These terminal sequences direct the action of the Proteins involved in their integration into DNA. A transposon does not need to be homologous to the recipient DNA.

In the laboratory, novel combinations of unrelated genes can be constructed and cloned within host cells. First, a recombinant DNA molecule is synthesized by joining a DNA fragment with a vector DNA that can replicate autonomously in a suitable host. The most suitable vectors include lambda phage, SV-40 virus, and plasmids. Restriction Enzymes and DNA ligase are the primary tools for generating new DNA molecules. Cohesive ends, homopolymer tails, and chemically synthesized linkers are three Methods for joining DNA molecules. Recombinant DNA molecules can be introduced into host cells by infecting them with the reconstructed virus or by incubating them in the presence of naked DNA molecules. The final step involves selecting cells that carry the desired recombinant DNA molecule by exploiting a distinctive property of the introduced Gene (such as antibiotic resistance). Using restriction digests of genomic DNA, Specific eukaryotic genes can be cloned in E. coli cells. Many eukaryotic genes can be transcribed and translated within bacterial systems. Gene cloning is a powerful technique for investigating the Organization and expression of complex genes. Furthermore, Recombinant DNA technology holds immense promise for synthesizing large quantities of genes and proteins that are present in normal cells in minute amounts.