Transformation (genetics)


Transformation (genetics)

In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s). Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial means in other cells. Bacteria that are capable of being transformed, whether naturally or artificially, are called competent. Transformation is one of three processes by which exogenous genetic material may be introduced into a bacterial cell, the other two being conjugation (transfer of genetic material between two bacterial cells in direct contact), and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium). Transformation may also be used to describe the insertion of new genetic material into nonbacterial cells including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the term should be avoided for animal cells when describing introduction of exogenous genetic material. Introduction of foreign DNA into eukaryotic cells is usually called "transfection".[1]

Contents

History

Transformation was first demonstrated in 1928 by British bacteriologist Frederick Griffith. 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 principle" from the heat-killed strain was responsible for making the harmless strain virulent. In 1944 this "transforming principle" was identified as being genetic by Oswald Avery, Colin MacLeod, and Maclyn McCarty. They isolated DNA from a 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 skeptically 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 Avery's experiments were accepted.[2] .

It was originally thought that Escherichia coli, a commonly-used laboratory organism, was refractory to transformation. However, in 1970, Morton Mandel and Akiko Higa showed that E. coli may be induced to take up DNA from bacteriophage λ without the use of helper phage after treatment with calcium chloride solution.[3] Two years later in 1972, Stanley Cohen, Annie Chang and Leslie Hsu showed that CaCl2 treatment is also effective for transformation of plasmid DNA.[4] The method of transformation by Mandel and Higa was later improved upon by Douglas Hanahan.[5] The discovery of artificially-induced competence in E. coli created an efficient and convenient procedure for transforming bacteria which allows for simpler molecular cloning methods in biotechnology and research, and it is now a routinely-used laboratory procedure.

Transformation using electroporation was developed in the late 1980s, increasing the efficiency of in-vitro transformation and increasing the number of bacterial strains that could be transformed.[6] Transformation of animal and plant cells was also investigated with the first transgenic mouse being created by injecting a gene for a rat growth hormone into a mouse embryo in 1982.[7] In 1907 a bacterium that caused plant tumors, Agrobacterium tumefaciens, was discovered and in the early 1970s the tumor inducing agent was found to be a DNA plasmid called the Ti plasmid.[8] By removing the genes in the plasmid that caused the tumor and adding in novel genes researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants.[9] Not all plant cells are susceptible to infection by A. tumefaciens so other methods were developed including electroporation and micro-injection.[10] Particle bombardment was made possible with the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford in 1990.[11]

Mechanisms

Bacteria

Bacterial transformation may be referred to as a stable genetic change brought about by the uptake of 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. There are two forms of competence: natural and artificial.

Natural competence

About 1% of bacterial species are capable of naturally taking up DNA under laboratory conditions; more may be able to take it up in their natural environments. DNA material can be transferred between different strains of bacteria, in a process that is called horizontal gene transfer. Some species upon cell death release their DNA to be taken up by other cells, however transformation works best with DNA from closely related species. These naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s). The transport of the exogeneous DNA into the cells may require proteins that are involved in the assembly of type IV pili and type II secretion system, as well as DNA translocase complex at the cytoplasmic membrane.[12]

Due to the differences in structure of the cell envelop between Gram-positive and Gram-negative bacteria, there are some differences in the mechanisms of DNA uptake in these cells, however most of them share common features that involve related proteins. The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the cytoplasmic membrane via DNA translocase.[13] Only single-stranded DNA may pass through, one strand is therefore degraded by nucleases in the process, and the translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a RecA-dependent process. In Gram-negitive cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by secretins on the outer membrane. Pilin may be required for competence however its role is uncertain.[14] The uptake of DNA is generally non-sequence specific, although in some species the presence of specific DNA uptake sequences may facilitate efficient DNA uptake.[15]

Artificial competence

Artificial competence can be induced in laboratory procedures that involves making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature.[16] Typically the cells are incubated in a solution containing divalent cations, most commonly calcium chloride solution under cold condition, and then exposed to a pulse of heat shock. However, the mechanism of the uptake of DNA via chemically-induced competence in this calcium chloride transformation is unclear. The surface of bacteria such as E. coli is negatively charged due to phospholipids and lipopolysaccharides on its cell surface, and the DNA is also negatively-charged. One function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface. It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure of the cells making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells either through cell pores or the damaged cell wall.

Electroporation is another method of promoting competence. In this method the cells are briefly shocked with an electric field of 10-20 kV/cm which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock the holes are rapidly closed by the cell's membrane-repair mechanisms.

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 into small pieces, e.g. 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: Particles of gold or tungsten are coated with DNA and then shot 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 Agrobacterium 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.

Practical aspects of transformation in molecular biology

The discovery of artificially-induced competence in bacteria allow bacteria such as Escherichia coli to be used as a convenient host for the manipulation of DNA as well as expressing proteins. Typically plasmids are used for transformation in E. coli. In order to 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 replication of the cell's own chromosome.

The efficiency with which a competent culture can take up exogenous DNA and express its genes is known as Transformation efficiency and is measured in colony forming unit (cfu) per μg DNA used. A transformation efficiency of 1x108 cfu/μg for a small plasmid like pUC19 is roughly equivalent to 1 in 2000 molecules of the plasmid used being transformed.

In calcium chloride transformation, the cells are prepared by chilling cells in the presence of Ca2+ (in CaCl2 solution) making the cell become permeable to plasmid DNA. The cells are incubated on ice with the DNA, and then briefly heat-shocked (e.g., at 42°C for 30–120 seconds). This method works very well for circular plasmid DNA. Non-commercial preparations should normally give 106 to 107 transformants per microgram of plasmid; a poor preparation will be about 104/μg or less, but a good preparation of competent cells can give up to ~108 colonies per microgram of plasmid.[17] Protocols however exist for making supercompetent cells that may yield a transformation efficiency of over 109.[18] The chemical method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native exonuclease enzymes rapidly degrade linear DNA. In contrast, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA.

The transformation efficiency using the CaCl2 method decreases with plasmid size, and electroporation therefore may be a more effective method for the uptake of large plasmid DNA.[19] Cells used in electroporation should be prepared first by washing in cold double-distilled water to remove charged particles that may create sparks during the electroporation process.

Selection and screening in plasmid transformation

Because transformation usually produces a mixture of relatively few transformed cells and an abundance of non-transformed cells, a method is necessary to select for the cells that have acquired the plasmid. The plasmid therefore requires a selectable marker such that those cells without the plasmid may be killed or their growth arrested. Antibiotic resistance is the most commonly used marker for prokaryotes. The transforming plasmid contains a gene that confers resistance to an antibiotic that the bacteria are otherwise sensitive to. The mixture of treated cells is cultured on media that contain the antibiotic so that only transformed cells are able to grow. Another method of selection is the use of certain auxotrophic markers that can compensate for an inability to metabolise certain amino acids and sugars. This method requires the use of suitably mutated strains that are deficient in the synthesis of a particular biomolecule.

The transformed cells with plasmid however need not contain the desired recombinant DNA inserted in cloning experiment. Various techniques may be employed to screen for those containing the insert. Reporter genes can be used as markers, such as the lacZ gene which codes for β-galactosidase used in blue-white screening. This method of screening relies on the principle of α-complementation, where a fragment of the lacZ gene (lacZα) in the plasmid can complement another mutant lacZ gene (lacZΔM15) in the cell. Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing lacZ-α is transformed into a lacZΔM15 cells, they form a functional β-galactosidase. The presence of an active β-galactosidase may be detected when cells are grown in plates containing X-gal, forming characteristic blue colonies. However, the multiple cloning site, where a gene of interest may be ligated into the plasmid vector, is located within the lacZα gene. Successful ligation therefore disrupts the lacZα gene, and no functional β-galactosidase can form, resulting in white colonies. Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones.

Other commonly used reporter genes are green fluorescent protein (GFP), which produces cells that glow green under blue light, and the enzyme luciferase, which catalyzes a reaction with luciferin to emit light. The recombinant DNA may also be detected using other methods such as nucleic acid hybridization with radioactive RNA probe, while cells that expressed the desired protein from the plasmid may also be detected using immunological methods.

References

  1. ^ Alberts, Bruce; et al. (2002). Molecular Biology of the Cell. New York: Garland Science. p. G:35. ISBN 9780815340720. 
  2. ^ 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. PMID 8150273. 
  3. ^ Mandel, Morton; Higa, Akiko (1970). "Calcium-dependent bacteriophage DNA infection". Journal of Molecular Biology 53 (1): 159–162. doi:10.1016/0022-2836(70)90051-3. PMID 4922220. http://www.sciencedirect.com/science/article/pii/0022283670900513. 
  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 69 (8): 2110–4. doi:10.1073/pnas.69.8.2110. PMC 426879. PMID 4559594. http://www.pnas.org/content/69/8/2110.abstract. 
  5. ^ Hanahan, D. (1983). "Studies on transformation of Escherichia coli with plasmids". Journal of molecular biology 166 (4): 557–580. doi:10.1016/S0022-2836(83)80284-8. PMID 6345791.  edit
  6. ^ 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/. 
  7. ^ 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 300 (5893): 611–5. doi:10.1038/300611a0. PMID 6958982. http://www.nature.com/nature/journal/v300/n5893/abs/300611a0.html. 
  8. ^ Nester, Eugene. "Agrobacterium: The Natural Genetic Engineer (100 Years Later)". http://www.apsnet.org/publications/apsnetfeatures/Pages/Agrobacterium.aspx. Retrieved 14 January 2011. 
  9. ^ Zambryski, P.; Joos, H.; Genetello, C.; Leemans, J.; Montagu, M. V.; Schell, J. (1983). "Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity". The EMBO journal 2 (12): 2143–2150. PMC 555426. PMID 16453482. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=555426.  edit
  10. ^ Peters, Pamela. "Transforming Plants - Basic Genetic Engineering Techniques". http://www.accessexcellence.org/RC/AB/BA/Transforming_Plants.php. Retrieved 28 January 2010. 
  11. ^ 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. 
  12. ^ Chen I, Dubnau D (2004). "DNA uptake during bacterial transformation". Nat. Rev. Microbiol. 2 (3): 241–9. doi:10.1038/nrmicro844. PMID 15083159. 
  13. ^ Lacks, S.; Greenberg, B.; Neuberger, M. (1974). "Role of a Deoxyribonuclease in the Genetic Transformation of Diplococcus pneumoniae". Proceedings of the National Academy of Sciences of the United States of America 71 (6): 2305–2309. PMC 388441. PMID 4152205. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=388441.  edit
  14. ^ Long, C. D.; Tobiason, D. M.; Lazio, M. P.; Kline, K. A.; Seifert, H. S. (2003). "Low-Level Pilin Expression Allows for Substantial DNA Transformation Competence in Neisseria gonorrhoeae". Infection and immunity 71 (11): 6279–6291. PMC 219589. PMID 14573647. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=219589.  edit
  15. ^ Sisco, K. L.; Smith, H. O. (1979). "Sequence-specific DNA uptake in Haemophilus transformation". Proceedings of the National Academy of Sciences of the United States of America 76 (2): 972–976. PMC 383110. PMID 311478. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=383110.  editFull text at PMC: / 383110
  16. ^ Large-volume transformation with high-throughput efficiency chemically competent cells. Focus 20:2 (1998).
  17. ^ Bacterial Transformation
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  19. ^ Transformation efficiency of "'E. coli'" electroporated with large plasmid DNA. Focus 20:3 (1998).

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