Our modern concepts of molecular genetics began with the idea that was once commonly referred to as the "Central Dogma": DNA makes RNA makes protein. In this way, the genotypic information carried in DNA is transferred by way of RNA intermediates to proteins, which usually are the basis of phenotype. It is now commonplace to introduce DNA into cells in order to construct new strains. The process of introducing DNA into cells to change their phenotype is referred to as genetic transformation.
Genetic transformation is a phenomenon which was first discovered with bacteria in the 1930's and 1940's and was instrumental in demonstrating that DNA was the genetic material. In early experiments, it was shown that extracts from heat-killed Pneumococcus bacteria could cause disease in mice if injected together with a live non-virulent strain (which alone was unable to induce pneumonia in the mice). Somehow, the non-virulent strain had become "transformed" into a virulent strain. We know now that the "transformation" was due to the genes from the virulent bacteria becoming incorporated into the genome of the nonvirulent strain, causing a permanent change in its properties. Nowadays, transformation of workhorse Esherichia coli bacteria is commonplace in molecular manipulation of genes.
The modern definition of transformation would be something like "the permanent (or nearly permanent) acquisition of a novel phenotypic trait as a result of the uptake of exogenous DNA molecules carrying the gene(s) for that trait". As such, transformation is not limited to bacteria. In fact, it is not only possible but a frequently used methodology to transform yeast or mammalian or plant cells by treating them in certain ways and then adding exogenous DNA. The techniques that are routinely used in research labs and industry can be used in the classroom as well. To make the process easy, it is important that the particular gene which we introduce into the cells causes an easily observed change in the colony, such as a color change. It is also necessary to have gene whose phenotype can be selected on an appropriate growth medium. The red adenine mutants satisfy both of these requirements.
Transformation is more frequent when special DNA molecules called plasmids are used to carry the genetic information to be introduced into the cell. Plasmids are independent DNA molecules, like small versions of chromosomes, which can carry genetic information and cause themselves to be propagated. Some plasmids exist naturally and are like viruses in that they exist to propagate themselves inside cells. These natural plasmids have been exploited and engineered so that they have useful properties for biotechnology and research.
The yeast plasmids which we use in the transformation experiment contain genes derived from yeast chromosomes which allow them to be replicated and transmitted during cell division. They also contain two genes whose products are involved in amino acid and nucleotide biosynthesis, LEU2 and ADE1. These genes code for enzymes necessary to produce the amino acid leucine, and the nucleotide precursor adenine, respectively. We introduce these plasmids into a yeast strain (HA1L) which contains a chromosomal mutation blocking leucine biosynthesis (Leu-) and one blocking adenine biosynthesis (Ade-). This strain is therefore unable to grow on minimal medium lacking leucine or adenine. Moreover, the adenine mutation causes the cells to accumulate a red pigment and so Ade- colonies growing on medium containing adenine will appear pink or red, rather than the normal creamy white color. If this strain takes up a plasmid that carries "good" (wild type) copy of the leucine gene and one of the adenine gene, its phenotype (and genotype) will be different. The strain containing the plasmid will be able to grow in the absence of leucine (Leu+) and adenine (Ade+) and will form cream-colored, rather than pink, colonies. Thus, we will have "transformed" the yeast from the Leu- Ade- red phenotype to a Leu+ Ade+ cream-colored phenotype.
Yeast geneticists have engineered a number of plasmids that are useful for transforming yeast. While they differ in may details, many of them share the following characteristics:
1. They are circular molecles containing DNA from yeast and from E. coli. 2. They contain some yeast DNA sequence that allows them to replicate within the cell. This may be either a sequence found on yeast chromosomes called ARS (for autonomous replication sequence), or part of the replication origin of the naturally occuring yeast plasmid, known as the 2-micron circle. 3. They contain an analogous origin of replication from the E. coli genome. 4. They contain one or more selectable genes from yeast. 5. They contain one or more selectable genes from E. coli.
Clearly, these plasmids can replicate in either yeast or E. coli. Therefore, they are called shuttle vectors. The E. coli part is useful because it is much easier to grow and isolate DNA from bacteria than from yeast. We have engineered two such vectors for classroom experiments. One, YCpADE1, uses a yeast chromosal ARS, and also contains a yeast centromere sequence. Its anatomy is illustrated in Figure 1. The other, YEpADE1, uses the replication origin of the 2-micron plasmid. Its anatomy is illustrated in Figure 2.
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Last updated Wednesday, 04-Dec-2002 20:16:56 UTC