WHAT IS TRANSFORMATION?
By definition, transformation is the bacterial mechanism for the transfer of genetic material in which free DNA of one genotype is taken in through the cell surface of bacteria of another genotype and is incorporated into the recipient cell chromosome. In eukaryotic cells, the term transformation refers to the conversion of normal eukaryotic cells into a cancer-like state (Purves et al., 1998). The term transfection is used to describe uptake, incorporation, and expression of foreign DNA into eukaryotic cells (Unknown, 2003).
HISTORY OF TRANSFORMATION
Griffith was the first to observe transformation. In his classic experiment, Griffith transformed nonvirulent pneumococcus to virulence with DNA (Purves et al., 1998).
Fig. 1. Griffith's experiment. This figure illustrates that when mice were infected with heat-killed pathogenic (S) strain of Pneumococcus the mice lived, as opposed to being infected with the live pathogenic strain, which caused death. Infection with the live nonpathogenic (R) strain did not cause the mice to die. Mixing the heat-killed pathogenic strain and the live nonpathogenic strain, however, did cause the mice to die. Live pathogenic strains of Pneumococcus were recovered from the mice that died of the mixed infection.
Griffith showed that a particular factor in the virulent S strain could transform the nonvirulent R strain of pneumococcus into a lethal form, even when the S strain had been killed after exposure to high temperatures. We now know that DNA had escaped from the dead S cells and that the living R cells had then picked up the DNA within the body of a living mouse (Purves et al., 1998).
HOW DOES IT WORK?
Not all bacteria take up free-floating DNA in the environment. The genera that generally exhibit transformation include: Bacillus, Streptococcus, Azotobacter, Haemophilus, Neisseria, and Thermus. The recipient cells must be competent (able to transform). Competence is a phenotype conferred by one or more proteins. It has been shown that competence occurs late in the exponential phase of bacterial growth. The duration of competence varies from a few minutes in Streptococcus to hours in Bacillus (Krawiec, 2002).
Dubnau and Provvedi (2000) described the proteins involved in the steps of B. subtilis transformation as shown in table 1 below.
Table 1. B. subtilis competence proteins
|I||ComEA||Bitopic integral membrane protein, with C-terminus outside||DNA receptor and presentation of DNA to transport machinery|
|II (PSTC)||ComC||Polytopic integral membrane protein (inferred from sequence)||Processing protease|
Peripheral membrane protein on inner face of membrane
|Access of DNA across wall|
|II (PSTC)||ComGB, ComGF||Integral membrane proteins (inferred from sequence)||
Access of DNA across wall
|II (PSTC)||ComGC, ComGD, ComGE, ComGG||Integral membrane proteins, translocated to wall when processed by ComC||Access of DNA across wall|
|III||ComEC||Polytopic integral membrane protein (inferred from sequence)||DNA transport: (channel?)|
|IV||ComFA||Integral membrane protein, exposed on inner face of membrane||DNA translocator|
In essence, double stranded DNA binds to the surface of a competent cell and is cleaved into fragments of about 15 kb. The double stranded DNA, which is still external to the cell, is then separated into single stranded DNA. One fragment of the single stranded DNA is degraded, while the other is transported across the membrane and into the cell (Dubnau et al., 2000). Upon entry into the cell, a single strand of the foreign DNA is incorporated into the recipient cell via recombination (Krawiec, 2002).
Fig. 2. Illustration of bacterial transformation. DNA from dead cells gets cut into fragments and exits the cell. The free floating DNA can then be picked up by competent cells. The exogenous DNA is incorporated into the host cell's chromosome via recombination.
IN THE LAB
Transformation is used in the lab in order to assess linkage. Extraction of
the donor DNA causes inevitable breakage. The relative map distance is determined
according to the percentage of two genes being on the same transforming segment
(double transformation). For example, two genes that are close to each other
on the chromosome will have a high occurrence of double transformation and a
low occurrence of being carried on separate transforming segments (Griffiths
et al., 2000).
In performing linkage experiments, the major problem of inducing transformation must be over come. The exterior surface of the plasma membrane is negatively charged due to the phospholipid head groups. This results in repulsion of the negatively charged DNA. The charge repulsion can be temporarily fixed by neutralizing the DNA with calcium salts. The plasma membrane is permeable to DNA when the charge has been neutralized. This technique is used on both prokaryotic and eukaryotic cells being transformed. In plants and fungi, however, the cell wall must first be removed with enzymes, which results in protoplasts (Griffiths et al., 2000).
FOR MORE ON TRANSFORMATION.....
Dubnau, D., Provvedi, R. (2000). Internalizing DNA. Research in Microbiology, 151: 475-480.
Griffiths, A.J.F., Gelbart, W.M., Miller, J.H., Lewontin, R.C. (2000). Modern Genetic Analysis. New York: W.H. Freeman and Company.
Krawiec, S.S. (2002). Prokaryotic Genetics. Lehigh University. 12 Feb 2003. www.lehigh.edu/~sk08/courses/Bios324/Module9.htm.
Purves, W.K., Orians, G.H., Heller, H.C., Sadava, D. (1998). Life. Sunderland, Massachusetts: Sinauer Associates, Inc.
Unknown. Recombinant Protein Production in Eukaryotic Cells. 12 Feb 2003. http://classweb.gmu/archriste/385-Ch07appt/385-Ch07a-PwrPt.
Please send any questions or comments to: Monica
Molecular Biology at Davidson College
Back to my molecular biology home page