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In molecular biology, transformation is the genetic alteration of a cell resulting from the uptake and expression of foreign genetic material (DNA). Separate terms are used for genetic alterations resulting from introduction of DNA by viruses ("transduction") or by cell-cell contact between bacteria ("conjugation"). Transformation of animal cells is usually called transfection.
The term transformation is also used more generally to describe mechanisms of DNA and RNA transfer in molecular biology (i.e. not only the genetic consequences). For example the production of transgenic plants such as transgenic maize requires the insertion of new genetic information into the maize genome using an appropriate mechanism for DNA transfer; the process is commonly referred to as transformation.
RNA may also be transferred into cells using similar methods, but this does not normally produce heritable change and so is not true transformation.
Additional recommended knowledge
Transformation was first demonstrated in 1928 by Frederick Griffith, an English bacteriologist searching for a vaccine against bacterial pneumonia. Griffith discovered that a non virulent strain of Streptococcus pneumoniae could be transformed into a virulent one by exposure to strains of virulent S. pneumoniae that had been killed with heat. In 1944 it was demonstrated that the transforming factor was genetic, when Oswald Avery, Colin MacLeod, and Maclyn McCarty showed gene transfer in S. pneumoniae. Avery, Macleod and McCarty called the uptake and incorporation of DNA by bacteria "transformation".
In bacteria transformation refers to a stable genetic change brought about by taking up naked DNA (DNA without associated cells or proteins), and competence refers to the state of being able to take up exogenous DNA from the environment. Two different forms of competence should be distinguished: natural and artificial.
Some bacteria (around 1% of all species) are naturally capable of taking up DNA under laboratory conditions; many more may be able to take it up in their natural environments. Such species carry sets of genes specifying machinery for bringing DNA across the cell's membrane or membranes.
Artificial competence is not encoded in the cell's genes. Instead it is induced by laboratory procedures in which cells are passively made permeable to DNA, using conditions that do not normally occur in nature.
Chilling cells in the presence of divalent cations such as Ca2+ (in CaCl2) prepares the cell walls to become permeable to plasmid DNA. Cells are incubated on ice with the DNA and then briefly heat shocked (eg 42 °C for 30–120 seconds), which causes the DNA to enter the cell. This method works very well for circular plasmid DNAs. An excellent preparation of competent cells will give ~108 colonies per microgram of plasmid. A poor preparation will be about 104/μg or less. Good non-commercial preps should give 105 to 106 transformants per microgram of plasmid.
The method does not work well for linear molecules such as fragments of chromosomal DNA, probably because exonuclease enzymes in the cell rapidly degrade linear DNA. Cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmids.
Electroporation is another way to make holes in bacterial (and other) cells, by briefly shocking them with an electric field of 10-20kV/cm. Plasmid DNA can enter the cell through these holes. This method is amenable to use with large plasmid DNA.  Natural membrane-repair mechanisms will rapidly close these holes after the shock.
In order to persist and be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the chromosome. Because transformation usually produces a mixture of rare transformed cells and abundant non-transformed cells, a method is needed to identify the cells that have acquired the plasmid. Plasmids used in transformation experiments will usually also contain a gene giving resistance to an antibiotic that the intended recipient strain of bacteria is sensitive to. Cells able to grow on media containing this antibiotic will have been transformed by the plasmid, as cells lacking the plasmid will be unable to grow.
Another marker, used for identifying E. coli cells that have acquired recombinant plasmids, is the lacZ gene, which codes for β-galactosidase. Because β-galactosidase is a homo-tetramer, with each monomer made up of one lacZ-α and one lacZ-ω protein, if only one of the two requisite proteins is expressed in the resulting cell, no functional enzyme will be formed. Thus, if a strain of E. coli without lacZ-α in its genome is transformed using as plasmid containing the missing gene fragment, transformed cells will produce β-galactosidase, while untransformed cells will not, as they are only able to produce the omega half of the monomer. In this type of transformation, the polylinker region of the plasmid lies in the lacZ-α gene fragment, meaning that successfully produced recombinant plasmids will have the desired gene inserted somewhere within lacZ-α. When this disrupted gene fragment is expressed by E. coli, no usable lacZ-α protein is produced, and therefore no usable β-galactosidase is formed. When grown on media containing the colorless, modified galactose sugar X-gal, colonies that are able to metabolize the substrate (and that have therefore been transformed, but not by recombinant plasmids) will appear blue in color; colonies that are not able to metabolize the substrate (and that have therefore been transformed by recombinant plasmids) will appear white.
Yeasts and other fungi
These methods are currently known to transform yeasts:
A number of mechanisms are available to transfer DNA into plant cells:
Introduction of DNA into animal cells is usually called transfection, and is discussed in the corresponding article.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Transformation_(genetics)". A list of authors is available in Wikipedia.|