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Encyclopedia > 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 plasmid-encoded conjugation or by viruses (transduction). Transformation of animal cells is usually called transfection. Wikipedia does not have an article with this exact name. ... Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. ... Molecular biology is the study of biology at a molecular level. ... Drawing of the structure of cork as it appeared under the microscope to Robert Hooke from Micrographia which is the origin of the word cell being used to describe the smallest unit of a living organism Cells in culture, stained for keratin (red) and DNA (green) The cell is the... For a non-technical introduction to the topic, see Introduction to Genetics. ... The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ... Bacterial conjugation is the transfer of genetic material between bacteria through cell-to-cell contact. ... Transduction is the process by which bacterial DNA is moved from one bacterium to another by a virus. ... Introducing DNA into eukaryotic cells, such as animal cells, is 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. This article or section is in need of attention from an expert on the subject. ... Transgenic maize (corn) has been deliberately genetically modified to have agronomically desirable traits. ...


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.

Contents

Historical context

The effect 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 Streptococcus pneumoniae that had been killed with heat. That the transforming factor was genetic in nature was not demonstrated until 1944, when Oswald Avery, Colin MacLeod, and Maclyn McCarty showed gene transfer in Streptococcus pneumoniae. Avery, Macleod and McCarty call the uptake and incorporation of DNA by bacteria transformation. Year 1928 (MCMXXVIII) was a leap year starting on Sunday (link will display full calendar) of the Gregorian calendar. ... Frederick Griffith (1879 - 1941) was a British medical officer. ... Binomial name (Klein 1884) Chester 1901 Streptococcus pneumoniae, or pneumococcus, is a Gram-positive, alpha-hemolytic diplococcus bacterium and a member of the genus Streptococcus. ... Binomial name (Klein 1884) Chester 1901 Streptococcus pneumoniae, or pneumococcus, is a Gram-positive, alpha-hemolytic diplococcus bacterium and a member of the genus Streptococcus. ... 1944 (MCMXLIV) was a leap year starting on Saturday. ... Oswald Theodore Avery (October 21, 1877–1955) was a Canadian-born American physician and medical researcher. ... Colin Munro MacLeod (1909 – 1972) was a Canadian-American geneticist. ... Maclyn McCarty (June 9, 1911–January 2, 2005) was an American geneticist. ... For a non-technical introduction to the topic, see Introduction to Genetics. ... Binomial name (Klein 1884) Chester 1901 Streptococcus pneumoniae, or pneumococcus, is a Gram-positive, alpha-hemolytic diplococcus bacterium and a member of the genus Streptococcus. ...

Year 1928 (MCMXXVIII) was a leap year starting on Sunday (link will display full calendar) of the Gregorian calendar. ... Frederick Griffith (1879 - 1941) was a British medical officer. ... Binomial name Streptococcus pneumoniae Streptococcus pneumoniae is a species of Streptococcus that is a major human pathogen. ... 1944 (MCMXLIV) was a leap year starting on Saturday. ... Oswald Theodore Avery (October 21, 1877–1955) was a Canadian-born American physician and medical researcher. ... Colin Munro MacLeod (1909 – 1972) was a Canadian-American geneticist. ... Maclyn McCarty (June 9, 1911–January 2, 2005) was an American geneticist. ... The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ...

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] In microbiology and cell and molecular biology, competence is the ability of a cell to take up extracellular (naked) DNA from its environment. ...


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 introduce plasmids and genetically engineered bacteria. Artificially competent cells of standard bacterial strains may also be purchased frozen, ready to use. Figure 1: Schematic drawing of a bacterium with plasmids enclosed. ... An iconic image of genetic engineering; this autoluminograph from 1986 of a glowing transgenic tobacco plant bearing the luciferase gene, illustrating the possibilities of genetic engineering. ...


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 (42oC 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 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. Calcium chloride is a chemical compound of calcium and chlorine. ... Figure 1: Schematic drawing of a bacterium with plasmids enclosed. ...


The method does not work for linear molecules such as fragments of chromosomal DNA, probably because exonuclease enzymes in the cell rapidly degrade linear DNAs. Cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmids. Exonucleases are enzymes that cleave nucleotides one at a time from an end of a polynucleotide chain. ...


Electroporation is another way to make holes in bacterial (and other) cells, by briefly shocking them with an electric field of 10-20kV/cm. Provided plasmid DNA is present when the shock is applied, it can enter the cell through these holes. Natural membrane-repair mechanisms will rapidly close these holes after the shock. Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by externally applied electrical field. ... In physics, the space surrounding an electric charge or in the presence of a time-varying magnetic field has a property called an electric field. ... Josephson junction array chip developed by NIST as a standard volt. ...


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. Selection for cells able to grow on media containing this antibiotic can then select the cells that have acquired the plasmid by transformation, as cells lacking the plasmid will be unable to grow. The origin of replication (also called the replication origin) is a particular DNA sequence at which DNA replication is initiated. ...


Another marker, useful when screening for recombinant plasmids in E. coli, is the lacZ gene fragment encoding the α fragment of ß-galactosidase, which by α complementation allows bacteria expressing the omega fragment of ß-galactosidase to metabolize media containing the colorless chemical X-gal, the metabolites of which are blue in color. Thus colonies of cells containing the intact lacZα gene fragment are blue when cultured on media containing X-gal. Because the polylinker region of the plasmid lies in lacZα, bacteria transformed by recombinant plasmids whose polylinker contains inserted DNA will be unable to express the α fragment of ß-galactosidase, leaving those colonies white. Binomial name Escherichia coli T. Escherich, 1885 Escherichia coli (usually abbreviated to E. coli) is one of the main species of bacteria that live in the lower intestines of warm-blooded animals (including birds and mammals) and are necessary for the proper digestion of food. ... Lactase (or β-galactosidase) is the enzyme involved in the hydrolysis of lactose to galactose and glucose. ... In gene cloning X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) is used to indicate whether a bacterium expresses the Beta-galactosidase enzyme, which is encoded by the lacZ gene. ... A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (usually 20+) restriction sites - a standard feature of engineered plasmids. ...


Yeasts and 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.[2]
  • 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.

Look up PDA in Wiktionary, the free dictionary. ... Initially protoplast (in Greek: proton = first and platho = mould) referred to the first organized body of a species this meaning is similar to the non-biological definition, the first from from which all subsequent forms are derived. ...

Plants

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

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 transient holes in cell membranes using electric shock; this allows DNA to enter as described above for Bacteria.
  • Viral transformation (transduction): Packages the desired genetic material into a suitable plant virus and is 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.

Image File history File links Download high-resolution version (869x717, 120 KB) I (seb951) took this image. ... Image File history File links Download high-resolution version (869x717, 120 KB) I (seb951) took this image. ... Species Agrobacterium tumefaciens Agrobacterium rhizogenes áAgrobacterium is a genus of bacteria that causes tumors in plants. ... The gene gun is a device for injecting cells with genetic information, originally designed for plant transformation. ... Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by externally applied electrical field. ... This page is a candidate for speedy deletion. ... The word transduction has several meanings: In developmental psychology, transduction is reasoning from specific cases to specific cases, typically employed by children. ... Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. ...

Animals

Introduction of DNA into animal cells is usually called Transfection, and is discussed in detail under that topic. Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. ...


See also

Figure 1: Schematic drawing of a bacterium with plasmids enclosed. ... In microbiology and cell and molecular biology, competence is the ability of a cell to take up extracellular (naked) DNA from its environment. ... Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. ... Triparental mating is a form of Bacterial conjugation where a conjugative plasmid present in one bacterial strain assists the transfer of a mobilizable plasmid present in a second bacterial strain into a third bacterial strain. ... Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vivo. ...

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. ^ 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. 

A digital object identifier (or DOI) is a standard for persistently identifying a piece of intellectual property on a digital network and associating it with related data, the metadata, in a structured extensible way. ...

External links


  Results from FactBites:
 
Transformation (genetics) Summary (3588 words)
Transformation may be either a natural process—that is, one that has evolved in certain bacteria—or it may be an artificial process whereby the recipient cells are forced to take up DNA by a physical, chemical, or enzymatic treatment.
Transformation is the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material (DNA or RNA).
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).
Genetics in Medicine - Vol 1 (5013 words)
Genetics is relevant in the areas of identification of suspects and victims, identification of illegal goods (for example, items that involve the killing of endangered animals), environmental monitoring for harmful microorganisms, parent…
Medical genetics is the application of genetics to the study of human health and diseases.
The other primary mechanisms are transformation, in which free DNA is transported across the cell membrane, and transduction, in which DNA is carried into the recipient cell by a bacterial virus.
  More results at FactBites »

 
 

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