12.1: Transformation alters the phenotype of a cell
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Transformation refers to the uptake of DNA by a cell, causing a change in its phenotype. Naturally-occurring transformation was first described in 1928 by Frederick Griffith, who described a heat-stable transforming principle from virulent Streptococcus pneumoniae that could transform non-virulent S. pneumoniae to an encapsulated, virulent form. The transforming principle was subsequently identified as DNA by Avery and colleagues in 1944. Since then, transformation has become an indispensable tool in the molecular biology laboratory. The physical basis for yeast transformation is still incompletely understood, but researchers have empirically developed conditions that give fairly consistent transformation in the lab. Reliable transformation techniques have been developed for bacteria and many eukaryotes, ranging from yeast to mammalian cells.
Transformation conditions have been developed empirically
The challenge in laboratory transformation is to devise conditions under which
DNA will pass across the cell wall and plasma membrane of living cells, which are normally impermeable to DNA. Very few cells are naturally competent, or able to take up DNA on their own. Consequently, researchers use a variety of chemical treatments to render cells competent. In general, these chemical treatments have some kind of destabilizing effect on the plasma membrane. The introduction of DNA into these competent cells can be further encouraged by a physical stress, such as a pulse of electric current or temperature elevation. Transformation is not a very efficient process, but because large numbers of microorganisms can be cultured in the laboratory, useful numbers of transformants can be obtained with most microorganisms.
Techniques for yeast transformation are now standard in the laboratory. Depending on the details of the experimental procedure, reactions can yield as many as 106 transformants per μg DNA. The structure of the DNA used for transformation greatly affects the transformation efficiency. Transformation efficiencies are considerably higher with supercoiled plasmid DNA than with linear pieces of DNA, possibly because plasmids enter the cell more readily and/or plasmids are less susceptible to endonuclease digestion.
The most commonly used yeast transformation methods use a combination of lithium acetate, single-stranded carrier DNA and polyethylene glycol (PEG). Although no one knows exactly how these components promote transformation, a number of hypotheses have been advanced. Lithium ions neutralize the negative charges on DNA molecules to be transformed and the phospholipid bilayer of the yeast cell, and they may also generate small holes in the plasma membrane that allow the passage of nucleic acids. Single-stranded DNA acts as a carrier for the plasmid DNA to be transferred into the cell and it may help to protect the latter from endonucleases. The source of the carrier DNA is unimportant. Since the carrier DNA concentration is considerably higher than that of the DNA to be introduced into the cell,
the carrier DNA is usually isolated from an inexpensive source, such as salmon sperm. It is imperative that the carrier DNA for transformations be single-stranded. In our experiments, we will boil the carrier DNA for 5 minutes and then rapidly chill it to prevent reanneling of the DNA helix. PEG may help bring the DNA into closer apposition with the membrane. PEG is often used to promote membrane fusion and is thought to alter water structure around plasma membranes.