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In molecular biology, transformation is the genetic alteration of a cell resulting from the uptake, genomic incorporation, and expression of environmental genetic material (DNA).[1] Transformation occurs most commonly in bacteria, both naturally and artificially, and refers to DNA taken up from the environment through their cell wall. Bacteria that are capable of being transformed are called competent. New genetic material can also be transferred to cells through conjugation or transduction. Conjugation involves cell-to-cell contact between two different bacterial cells, with the DNA being transferred from one bacterial cell to the other. In transduction, viruses called bacteriophages inject the foreign DNA into their host. Introduction of foreign DNA into eukaryotic cells is usually called "transfection".[2] Transformation is also used to describe the insertion of new genetic material into nonbacterial cells including animal and plant cells.

Contents

History

Transformation was first demonstrated in 1928 by Frederick Griffith, an English bacteriologist searching for a vaccine against bacterial pneumonia. Griffith discovered that a harmless strain of Streptococcus pneumoniae could be made virulent after being exposed to heat-killed virulent strains. Griffith hypothesized that some "transforming factor" from the heat-killed strain was responsible for making the harmless strain virulent. In 1944 this "transforming factor" was identified as being genetic by Oswald Avery, Colin MacLeod, and Maclyn McCarty. They isolated DNA from virulent strain of S. pneumoniae and using just this DNA were able to make a harmless strain virulent. They called this uptake and incorporation of DNA by bacteria "transformation." See Avery-MacLeod-McCarty experiment.

The results of Avery et al.'s experiments were at first sceptically received by the scientific community, and it was not until the development of genetic markers and the discovery of other methods of genetic transfer (conjugation in 1947 and transduction in 1953) by Joshua Lederberg that the full meaning of Avery's experiments were understood.[3] Transformation did not become routine procedure in scientific laboratories until 1972, when Stanley Cohen, Annie Chang and Leslie Hsu successfully transformed Escherichia coli by treating the bacteria with calcium chloride.[4] This created an efficient and convenient procedure for transforming DNA into bacteria and opened the way for molecular cloning in biotechnology and research.

Transformation using electroporation was developed in the late 1980's, increasing the efficiency and number of bacterial strains that could be transformed.[5] Transformation of other animal and plant cells were investigated, with the first transgenic mouse being created by injecting genetic material that included a rat growth hormone gene into a mouse embryo in 1982.[6] In 1907 a bacterium that caused plant tumors, agrobacterium tumefaciens, was discovered and in the early 1970's the Tumour Inducing agent was discovered to be a DNA plasmid, called the Ti plasmid[7] By removing the genes in the plasmid that caused the cancer and adding in novel genes, researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the plants genome. Not all plant cells are susceptible to infection by A. tumefaciens so other methods were developed, including electroporation and micro-injection.[8] Particle bombardment was made possible with the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford in 1990.[9]

Mechanisms

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Bacteria

Bacteria transformation may be referred to as 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

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 the cause of the machinery for bringing DNA across the cell's membrane or membranes.[10]

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.[11]

Calcium chloride transformation is a method of promoting competence. Chilling cells in the presence of divalent cations such as Ca2+ (in CaCl2) prepares the cell membrane 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 usually does not work well for linear molecules such as fragments of chromosomal DNA, probably because exonuclease enzymes in the cell rapidly degrade linear DNA. However, 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. [12] 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 a 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.

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) is cut into 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 Agrobacterium 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 agribacterial 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.

References

  1. ^ bacterial transformation at Dorland's Medical Dictionary
  2. ^ Alberts, Bruce; et al. (2002). Molecular Biology of the Cell. New York: Garland Science. p. G:35. ISBN 9780815340720. 
  3. ^ Lederberg, Joshua (1994). The Transformation of Genetics by DNA: An Anniversary Celebration of AVERY, MACLEOD and MCCARTY(1944) in Anecdotal, Historical and Critical Commentaries on Genetics. The Rockfeller University, New York, New York 10021-6399. http://www.ncbi.nlm.nih.gov/pubmed/8150273. 
  4. ^ Cohen, Stanley; Chang, Annie and Hsu, Leslie (1972). "Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA". Proceedings of the National Academy of Sciences. http://www.pnas.org/content/69/8/2110.abstract. 
  5. ^ Wirth, Reinhard; Friesenegger, Anita and Fiedlerand, Stefan (1989). "Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporation". Molecular and General Genetics MGG. http://www.springerlink.com/content/xx826w544343jt8l/. 
  6. ^ Palmiter, Richard; Ralph L. Brinster, Robert E. Hammer, Myrna E. Trumbauer, Michael G. Rosenfeld, Neal C. Birnberg & Ronald M. Evans (1982). "Dramatic growth of mice that develop from eggs microinjected with metallothionein−growth hormone fusion genes". Nature. http://www.nature.com/nature/journal/v300/n5893/abs/300611a0.html. 
  7. ^ Nester, Eugene. "Agrobacterium: The Natural Genetic Engineer (100 Years Later)". http://www.apsnet.org/online/feature/Agrobacterium/. Retrieved 28th January 2010. 
  8. ^ Peters, Pamela. "Transforming Plants - Basic Genetic Engineering Techniques". http://www.accessexcellence.org/RC/AB/BA/Transforming_Plants.php. Retrieved 28th January 2010. 
  9. ^ Voiland, Michael; McCandless, Linda. "DEVELOPMENT OF THE "GENE GUN" AT CORNELL". http://www.nysaes.cornell.edu/pubs/press/1999/genegun.html. Retrieved 28th january 2010. 
  10. ^ Chen I, Dubnau D (2004). "DNA uptake during bacterial transformation". Nat. Rev. Microbiol. 2 (3): 241–9. doi:10.1038/nrmicro844. PMID 15083159. 
  11. ^ Large-volume transformation with high-throughput efficiency chemically competent cells. Focus 20:2 (1998).
  12. ^ Transformation efficiency of E. coli electroporated with large plasmid DNA. Focus 20:3 (1998).

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