Transformation (genetics)

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Transformation is the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material (DNA) in molecular biology. The effect was first demonstrated in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty, who showed gene transfer in Streptococcus pneumoniae. Avery, Macleod and McCarty call the uptake and incorporation of DNA by bacteria transformation.

More generally the term is used to describe mechanisms of DNA and RNA transfer in molecular biology. For example the production of transgenic plants like transgenic maize requires the insertion of new genetic information into the maize genome using an appropriate mechanism for DNA transfer.

RNA molecules may also be transferred into cells using similar methods, but this does not normally produce heritable change and so is not true transformation.

1 Historical context
2 Mechanisms
3 External links

[edit] Historical context

1928 - Frederick Griffith transforms nonpathogenic pneumococcus bacteria into a virulent variety by mixing them with heat-killed pathogenic bacteria.
1944 - Oswald Avery, Colin MacLeod, and Maclyn McCarty discover that the transforming factor is pure DNA.

[edit] Mechanisms

[edit] Bacterial

In bacteria, transformation refers to a genetic change brought about by picking up naked strands of DNA and expressing it, and competence refers to the state of being able to take up DNA. Two different forms of competence should be distinguished, natural and artificial.

[edit] Natural competence

Some bacteria (around 1% of all species) are naturally capable of taking up DNA. Such species carry sets of genes specifying machinery for bringing DNA across the cell's membrane or membranes. The evolutionary function of these genes is controversial. Although most textbooks and researchers have assumed that cells take up DNA to acquire new versions of genes, a simpler explanation that fits most of the observations is that cells take up DNA mainly as a source of nucleotides, which can be used directly or broken down and used for other purposes. ^{[original research?]}^{[citation needed]}

[edit] 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. These procedures are comparatively easy and simple, and are widely used to genetically engineer bacteria. Artificially competent cells of standard bacterial strains may also be purchased frozen, ready to use.

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 with the DNA and then briefly heat shocked (42^oC for 30-120 seconds), which causes the DNA to enter the cell. This method works well for circular plasmid DNAs but not for linear molecules such as fragments of chromosomal DNA. An excellent preparation of competent cells will give ~10⁸ colonies per μg 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.

Electroporation is another way to make holes in cells, by briefly shocking them with an electric field of 100-200V/cm. Now plasmid DNA can enter the cell through these holes. Natural membrane-repair mechanisms will close these holes afterwards.

Lipofection can be used to transform cells via vescicles filled containing the desired plasmid. The vescicle fuses with the cell membrane (similar to how two oil spots at the top of a broth will fuse) and the contents of the vescicle & the cell are combined.

A plasmid DNA molecule contains sequences allowing it to be replicated in the cell independently of the chromosome. Plasmids used in experiments will usually also contain an antibiotic resistance gene which is placed in a bacterial strain that has no antibiotic resistance. Therefore, only transformed bacteria will grow on a media containing the antibiotic.

Another marker, useful when selecting for recombinant plasmids, is the lacZ gene of the lac operon. This gene codes for ß-galactosidase, which allows bacteria to metabolize media containing X-gal (a colorless, modified galactose sugar), the metabolites of which are blue in color. Because the polylinker region of the plasmid lies in the lacZ gene, bacteria transformed by recombinant plasmids will produce a non-functional ß-galactosidase, leaving those colonies colorless.

In bacteria the term transformation is not normally applied to genetic changes arising by Transduction or Conjugation, in which transfer of DNA is mediated by genetic parasites (phages and conjugative plasmids respectively).

[edit] Yeasts and Fungi

These methods are currently known to transform yeasts:

High Efficiency Transformation according to Gietz, R. D. and R. A. Woods. 2002 TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.
Two-hybrid System Protocol: The two-hybrid system involve the use of two different plasmids in a single yeast cell. One plasmid contains a cloned gene or DNA sequence of interest while the other plasmid contains a library of genomic or cDNA. [1]
Rapid Transformation Protocol allows for transformation with any yeast cell source. See Gietz/Wood above.
Frozen Yeast Protocol allows you to prepare frozen yeast cells that are competent for transformation after thawing.
Gene Gun Transformation Gold or tungsten nanoparticles can be shot at fungal cells growing on PDA, transforming them.
Protoplast Transformation Fungal spores can be turned into protoplasts which can then be soaked in DNA solution and transformed.

[edit] Plants

A number of mechanisms are available to transfer DNA into an organism, these include:

Plant (S. chacoense) transformed using Agrobacterium. Transformed cells start forming calluses on the side the leaf pieces

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 holes in cell walls using electricity, that allows DNA to enter.
Viral transformation: Package your genetic material into a suitable plant virus and then use the modified virus for infection of the plant. Genomes of most plant viruses consist of single stranded RNA which replicates in the cytoplasm of infected cell. So this method is 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.

[edit] Animals

Microinjection: use a thin needle and inject the DNA directly in the core of embryonic cells.
Viral transformation: Package genetic material into a virus, which delivers the genetic material to target host cells.