Principles of Biochemistry, Volume 3 - A. Lehninger 1985

Molecular Mechanisms of Genetic Information Transfer
More on Genes: Repair, Mutation, Recombination, and Cloning
Plasmids and phage lambda serve as vectors for introducing foreign genes into bacteria

The next milestone in The Development of Recombinant DNA technology was devising Methods for introducing foreign genes into host Cells. Plasmids and λ phage DNA became the most widespread carriers used to introduce foreign genes into the E. coli genome, known as vectors. Plasmids (Sec. 27.15) are small, circular, double-stranded DNA molecules present in the Cytoplasm of most bacterial species. Each plasmid contains between 2,000 and 100,000 Base Pairs. Small plasmids may be present in 20 or more copies per Cell, whereas larger plasmids typically occur in no more than 1 to 2 copies. Each plasmid harbors several—and sometimes many—genes that are replicated, transcribed, and translated independently of the chromosomal genes, yet concurrently with them. Plasmids are readily isolated and separated from bacterial Chromosomes, from which they differ in size, nucleotide composition, and density. Plasmids possess two remarkable properties that make them exceptionally useful for genetic manipulation. First, they can be transferred from one cell to another, and even between different bacterial species. For instance, if Salmonella typhimurium cells are mixed with an E. coli strain resistant to penicillin, the former acquire penicillin resistance. This occurs because the penicillin-resistance Gene in the E. coli plasmid, known as the R-factor, can be transferred from E. coli to S. typhimurium cells. The second key property of plasmids is that foreign genes can be integrated into them with relative ease; these foreign genes can then be ferried as “passengers” into E. coli, where they become an integral part of the host cell genome.

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Fig. 30-20. The Use of terminal transferase to extend DNA ends, thereby providing DNA fragments with complementary sticky ends.

Phage λ DNA can also be used to transfer a foreign gene into E. coli. When recombinant λ phage DNA carrying a foreign gene is mixed with λ phage coat Proteins, infectious phage particles are formed—provided, of course, that the size of the recombinant DNA does not differ significantly from that of native phage DNA. This method of introducing a foreign gene into E. coli is superior to the previous one because phage λ infects host cells with extremely high efficiency, whereas plasmids penetrate intact E. coli cells only rarely. Phage λ is a temperate phage (Sec. 30.9), and its DNA, along with the foreign gene it carries, is capable of integrating into the E. coli chromosome. In this case, both the λ phage DNA and the foreign gene will replicate during every cycle of Cell Division.

Let us now examine in greater detail how genes are isolated, introduced into host cells, cloned, and translated to yield specific products. The word “clone” originates from Greek, meaning a twig or slip used for plant propagation, and is used in two Senses. First, cell cloning refers to the generation of a group of genetically identical cells derived from a single progenitor cell, as seen in a line of immunocytes programmed to synthesize a specific type of antibody. Second, molecular cloning, or Gene cloning, refers to The production of multiple identical copies of a gene resulting from the Replication of a single gene introduced into a host cell.