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Table of Contents
Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
Problem Set 2
VI B. TRANSFORMATION
Transformation is defined as the transfer of genetic information from a donor to a recipient using naked DNA. The recipient takes DNA up from the media, and there is no requirement for cell to cell contact.
While naturally transformable bacterial strains exist, these are sufficiently rare that induced transformation is more important to the geneticist. Induced transformation is the act of tricking a normally non-transformable bacteria into taking up DNA from the media. It involves two hurdles: first, the purification of DNA which can be either plasmid or chromosome, linear or circular; secondly, the generation of "competent" cells, which are cells which can accept DNA. In the case of E. coli, the following considerations apply: (i) you need multivalent cations and some time of incubation at low temperature; (ii) only a fraction of the cells are competent (<10%); (iii) there are two phases, DNA uptake and establishment, and the latter seems limiting; (iv) there appear to be approximately 100 "channels" per cell for DNA uptake.
Alternatively, Gram-positive bacteria can be incubated with degradative enzymes to remove the peptidoglycan layer and thus form protoplasts. When these latter cell forms are incubated with DNA and polyethylene glycol, one obtains cell fusion and concomitant DNA uptake. In both of these examples, if the DNA is linear, it tends to be very sensitive to nucleases so that transformation is most efficient when it involves the use of covalently closed circular DNA. Alternatively, nuclease-deficient cells (RecBC- strains) can be used to improve transformation.
Finally, the technique of electroporation is coming into more use with bacteria. It essentially involves fusing cells with pulses of electric current and can be used for both cell fusion and incorporation of DNA. When optimized for a particular organism, electroporation seems to be good for at least an order of magnitude increase in the frequency of transformants when compared to optimized transformation methods.
There are also cases of natural transformation where bacterial species naturally take up DNA from the environment: Bacillus, Streptococcus pneumoniae, Haemophilus, and Neisseria. With these bacteria, the competence to accept DNA is natural, but often is dependent on the cells being in a particular growth stage. The DNA binding in these bacteria is by particular receptors and, in the case of some of the gram negative cells, particular DNA sequences are required. In Gram-positive bacteria, the process is exceedingly complex and the DNA is cut into fragments and converted to single strands which are protected by DNA binding proteins. Integration into the chromosome is apparently by single-strand invasion of the DNA duplex followed by ligation. In this latter case, plasmids are poorly transformed since they lack homology with the chromosome. This can be solved either using a recipient with a homologous plasmid or using plasmid multimers so that the overlapping single strand fragments can regenerate a plasmid monomer.
In general, transformation allows the transfer of reasonably small, apparently random, pieces of chromosomal or plasmid DNA from a donor to a recipient. As with any gene transfer system, the transferred DNA is selectable only if stably inherited in the recipient cell. This is done either by introducing genes that themselves can replicate (which requires both the ability to circularize and to be associated with a replicon) or can integrate into or recombine with a replicon in the recipient cell. Compared with conjugation, transformation is rather slow and work-intensive, not only because of the requirement for isolation of DNA, but also because competent cells need to be produced. However, it is often the only method available, especially if the DNA in question is a result of in vitroconstructions. Transformation also tends to become extremely inefficient as the size of the DNA increases. One of the solutions has been to package large pieces of DNA into phage heads in vitroand then have the constructed phage inject the DNA into the desired cells.
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