Transformation (genetics)

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.

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History

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".

Mechanisms

Bacteria

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.

Natural competence

Main article: Competence (biology)

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.^[1]

Artificial competence

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.^[2]

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. ^[3] Natural membrane-repair mechanisms will rapidly close these holes after the shock.

Plasmid transformation

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:

• Lithium acetate/single-stranded carrier DNA/polyethylene glycol method

Several variations have been described, including rapid transformation and high efficiency transformation methods.^[4]

• Frozen Yeast Protocol allows you to prepare frozen yeast cells that are competent for transformation after thawing.
• Gene Gun Transformation

Gold or tungsten nanoparticles coated with DNA can be shot into fungal cells growing on PDA, transforming them. This is described in more detail under Plants below.

• Protoplast Transformation

Fungal spores can be converted to protoplasts by removing their protective coating, and can then be soaked in DNA solution and transformed.

Plants

A number of mechanisms are available to transfer DNA into plant cells:  

• Agrobacterium mediated transformation is the easiest and most simple plant transformation. Plant tissue (often leaves) are cut in small pieces, eg. 10x10mm, and soaked for 10 minutes in a fluid containing suspended agrobacterium. Some cells along the cut will be transformed by the bacterium, that inserts its DNA into the cell. Placed on selectable rooting and shooting media, the plants will regrow. Some plants species can be transformed just by dipping the flowers into suspension of Agrobacteria and then planting the seeds in a selective medium. Unfortunately, many plants are not transformable by this method.
• Particle bombardment: Coat small gold or tungsten particles with DNA and shoot them into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. This method also allows transformation of plant plastids. The transformation efficiency is lower than in agrobacterial mediated transformation, but most plants can be transformed with this method.
• Electroporation: make transient holes in cell membranes using electric shock; this allows DNA to enter as described above for Bacteria.
• Viral transformation (transduction): Package the desired genetic material into a suitable plant virus and allow this modified virus to infect the plant. If the genetic material is DNA, it can recombine with the chromosomes to produce transformant cells. However genomes of most plant viruses consist of single stranded RNA which replicates in the cytoplasm of infected cell. For such genomes this method is a form of transfection and not a real transformation, since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome. The progeny of the infected plants is virus free and also free of the inserted gene.

Animals

Introduction of DNA into animal cells is usually called transfection, and is discussed in the corresponding article.

See also

• Competence (biology)
• Triparental mating
• Molecular Cloning

References

1. ^ Chen I, Dubnau D (2004). "DNA uptake during bacterial transformation". Nat. Rev. Microbiol. 2 (3): 241-9. doi:10.1038/nrmicro844. PMID 15083159.
2. ^ Large-volume transformation with high-throughput efficiency chemically competent cells. Focus 20:2 (1998).
3. ^ Transformation efficiency of E. coli electroporated with large plasmid DNA. Focus 20:3 (1998).
4. ^ Gietz RD, Woods RA (2002). "Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method". Meth. Enzymol. 350: 87-96. PMID 12073338.