Dna

ligase

Plasmid cloning vector cleaved with EcoRl

(c)

Similar methods were used soon after in the laboratory of Paul Berg to join DNA segments from simian virus 40 (SV40) to DNA derived from bacteriophage A, thereby creating the first recombinant DNA molecule with DNA segments from different species.

Cloning Vectors Allow Amplification of Inserted DNA Segments

The principles that govern the delivery of recombinant DNA in clonable form to a host cell, and its subsequent amplification in the host, are well illustrated by considering three popular cloning vectors commonly used in experiments with E. coli—plasmids, bacteriophages, and bacterial artificial chromosomes—and a vector used to clone large DNA segments in yeast.

Plasmids Plasmids are circular DNA molecules that replicate separately from the host chromosome. Naturally occurring bacterial plasmids range in size from 5,000 to 400,000 bp. They can be introduced into bacterial cells by a process called transformation. The cells (generally E. coli) and plasmid DNA are incubated together at 0 °C in a calcium chloride solution, then subjected to a shock by rapidly shifting the temperature to 37 to 43 °C. For reasons not well understood, some of the cells treated in this way take up the plasmid DNA. Some species of bacteria are naturally competent for DNA uptake and do not require the calcium chloride treatment. In an alternative method, cells incubated with the plasmid DNA are subjected to a high-voltage pulse. This approach, called electroporation, transiently renders the bacterial membrane permeable to large molecules.

Regardless of the approach, few cells actually take up the plasmid DNA, so a method is needed to select those that do. The usual strategy is to use a plasmid that includes a gene that the host cell requires for growth under specific conditions, such as a gene that confers resistance to an antibiotic. Only cells transformed by the recombinant plasmid can grow in the presence of that antibiotic, making any cell that contains the plasmid "selectable" under those growth conditions. Such a gene is called a selectable marker.

Investigators have developed many different plas-mid vectors suitable for cloning by modifying naturally occurring plasmids. The E. coli plasmid pBR322 offers a good example of the features useful in a cloning vector (Fig. 9-4):

1. pBR322 has an origin of replication, ori, a sequence where replication is initiated by cellular enzymes (Chapter 25). This sequence is required to propagate the plasmid and maintain it at a level of 10 to 20 copies per cell.

2. The plasmid contains two genes that confer resistance to different antibiotics (tetR, ampR),

EcoRI

Psti^y w

pBR322

Origin of replication (ori)

FIGURE 9-4 The constructed E. coli plasmid pBR322. Note the location of some important restriction sites—for PstI, fcoRI, BarnHI, Sa/I, and PvuII; ampicillin- and tetracycline-resistance genes; and the replication origin (ori). Constructed in 1977, this was one of the early plasmids designed expressly for cloning in E. co/i.

allowing the identification of cells that contain the intact plasmid or a recombinant version of the plasmid (Fig. 9-5).

3. Several unique recognition sequences in pBR322 (PstI, EcoRI, BamHI, Sail, Pvull) are targets for different restriction endonucleases, providing sites where the plasmid can later be cut to insert foreign DNA.

4. The small size of the plasmid (4,361 bp) facilitates its entry into cells and the biochemical manipulation of the DNA.

Transformation of typical bacterial cells with purified DNA (never a very efficient process) becomes less successful as plasmid size increases, and it is difficult to clone DNA segments longer than about 15,000 bp when plasmids are used as the vector.

Bacteriophages Bacteriophage À has a very efficient mechanism for delivering its 48,502 bp of DNA into a bacterium, and it can be used as a vector to clone somewhat larger DNA segments (Fig. 9-6). Two key features contribute to its utility:

1. About one-third of the À genome is nonessential and can be replaced with foreign DNA.

2. DNA is packaged into infectious phage particles only if it is between 40,000 and 53,000 bp long, a constraint that can be used to ensure packaging of recombinant DNA only.

EcoRI

Psti^y w

Sa/I

pBR322

Sa/I

Origin of replication (ori)

FIGURE 9-4 The constructed E. coli plasmid pBR322. Note the location of some important restriction sites—for PstI, fcoRI, BarnHI, Sa/I, and PvuII; ampicillin- and tetracycline-resistance genes; and the replication origin (ori). Constructed in 1977, this was one of the early plasmids designed expressly for cloning in E. co/i.

pBR322 is cleaved at the ampicillin-resistance element by PsiI.

R pBR322 ampR plasmids oH'3

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