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 or RNA).
This is a common technique in molecular biology. The effect was first demonstrated in 1944 by Mr.L. Greenfield, Collin 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.
Historical context
- 1865 - Gregor Mendel develops a model of genetic heredity by the passing down of traits.
- 1928 - Frederick Griffith transforms nonpathogenic pneumococcus bacteria into a virulent variety by immersing them in heat-killed pathogenic material.
- 1944 - Oswald Avery and Collin Macleod announce that they have discovered the transforming factor - DNA.
Transfer mechanisms
Bacterial
In bacteria, transformation refers to a genetic change brought about by taking up and expressing DNA, and competence refers to the state of being able to take up DNA. Two different forms of competence should be distinguished, natural and artificial.
Natural competence
Natural bacterial transformation occurs only in bacterial species capable of natural competence. 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.
Most naturally transformable bacteria express their competence genes and develop competence only under specific conditions, often in response to a nutritional stress. Once the DNA has been brought into the cell's cytoplasm, it may be degraded by cellular nucleases, or, if it is very similar to the cells own DNA, enzymes that normally repair DNA may recombine it with the chromosome. Natural transformation is very efficient for linear molecules such as fragments of chromosomal DNA but not for circular plasmid DNAs.
Artificial competence
Artificial competence is not encoded in the cell's genes. Instead it is a laboratory procedure 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 can be used to genetically engineer bacteria:
Chilling cells in the presence of divalent cations such as CaCl2 prepares the cell walls to become permeable to plasmid DNA. Cells are incubated with the DNA and then briefly heat shocked (42C for 90-120 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.
Electroporation is another way to make holes in cells, by briefly shocking them with an electric field of 100-200V. Now plasmid DNA can enter the cell through these holes. Natural membrane-repair mechanisms will close these holes afterwards.
A plasmid DNA molecule will usually contain an antibiotic resistance gene which is placed in a bacterial strain that has no antibiotic resistance. Therefore, only transformed bacteria can grow on a media with the antibiotic (this is known as a selection medium).
One example of this is putting in a plasmid that contains the encoding for the protein ß-lactamase, which makes bacteria resistant to ampicillin. This is called the bla gene. The bacterial colony is then treated with ampicillin, thus weeding out those bacteria who did not take up the plasmid with the bla gene. Another selection medium is bioluminescence, using a gene taken from jellyfish.
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).
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. [link]
- 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.
Plants
A number of mechanisms are available to transfer DNA into an organism, these include:
- 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.
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.
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