8 Transformation
8.1 Yeast Vector and DNA Fragments
In general, transformation is the introduction into cells of exogenously added DNA and the subsequent inheritance and expression of that DNA. The most important advances in the molecular characterization and controlled modification of yeast genes have relied on the use of shuttle vectors which can be used to transform both yeast and E. coli.
The following three main methods are currently used to transform yeast: (i) those using spheroplasts; or (ii) cells treated with lithium salts; and (iii) the use of electroporation.
Spheroplasts for transformations are prepared by the action of hydrolytic enzymes to remove portions of the cell wall in the presence of osmotic stabilizers, typically 1 M sorbitol. Cell-wall digestion is carried out either with a snail-gut extract, usually denoted Glusulase, or with Zymolyase, an enzyme from Arthrobacter luteus. DNA is added to the spheroplasts, and the mixtures is co-precipitated with a solution of polyethylene glycol (PEG) and Ca2+. Subsequently, the cells are resuspended in a solution of sorbitol, mixed with molten agar and then layered on the surface of a selective plate containing sorbitol. Although this protocol is particularly tedious, and efficiency of transformation can vary by over four orders of magnitude with different strains, very high frequencies of transformation, over 104 transformants/
mg DNA, can be obtained with certain strains.Most investigators use cells treated with lithium salts for transformation. After treating the cells with lithium acetate, which apparently permeabilizes the cell wall, DNA is added and the cells are co-precipitated with PEG. The cells are exposed to a brief heat shock, washed free of PEG and lithium acetate, and subsequently spread on plates containing ordinary selective medium. Increased frequencies of transformation are obtained by using specially-prepared single-stranded carrier DNA and certain organic solvents.
A commonly-used method for transforming a wide range of different species of cells is based on the induced permeability to DNA by exposure to electrical fields. The interaction of an external electric field with the lipid dipoles of a pore configuration is believed to induce and stabilize the permeation sites, resulting in cross membrane transport. Freshly-grown yeast cultures are washed, suspended in an osmotic protectant, such as sorbitol, DNA is added, and the cell suspension is pulsed in an electroporation device. Subsequently, the cells are spread on the surface of plates containing selective media. The efficiency of transformation by electroporation can be increased over 100-fold by using PEG, single-stranded carrier DNA and cells that are in late log-phase of growth. Although electroporation procedures are simple, the specialized equipment and the required cuvettes are costly.
8.2 Synthetic Oligonucleotides
A convenient procedure has been described for producing specific alterations of chromosomal genes by transforming yeast directly with synthetic oligonucleotides. This procedure is easily carried out by transforming a defective mutant and selecting for at least partially functional revertants. Transformation of yeast directly with synthetic oligonucleotides is thus ideally suited for producing a large number of specific alterations that change a completely nonfunctional allele to at least a partially functional form. The oligonucleotide should contain a sequence that would correct the defect and produce the desired additional alterations at nearly sites. The method is apparently applicable to all mutant alleles whose functional forms can be selected. Although it is a general procedure, so far it has been extensively used only with mutations of CYC1, that encodes iso-1-cytochrome c, and CYT1 that encodes cytochrome
c1. The transformation is carried out by the usual lithium acetate procedure, using approximately 50 mg of oligonucleotides that are approximately 40 nucleotides long.8.3 Mitochondrial Transformation
Standard methods for transformation of nuclear genes are ineffective for mitochondrial DNA genes. However, DNA can be delivered to the mitochondrial matrix by high-velocity bombardment of yeast cells with tungsten microprojectiles carrying mitochondrial DNA. Several high-velocity microprojectile bombardment devices are commercially available, and these are powered by gunpowder charge or compressed gas.
This method was used to demonstrated that
ro strains can be converted to stable "synthetic r-" strains by transformation with bacterial plasmids carrying mitochondrial genes (see Table 6.2). Similar to natural r- mitochondrial DNA, the synthetic r- mitochondrial DNA can recombine with r+ mitochondrial DNA, thus providing means to replace r+ wild-type genes with mutations generated in vitro.Synthetic
r- strains are isolated by bombarding a lawn of ro cells on the surface of a petri plate with YEp or YCp plasmids carrying both a selectable marker, such as URA3, and the mitochondrial gene of interest. The nuclear and mitochondrial genes may either be on separate or the same plasmid. Ura+ colonies, for example, are then screen for the presence of the mitochondrial gene by crossing the colonies to an appropriate mit- tester strain and scoring the diploids for Nfs+ (see Table 3). The efficiency of mitochondrial transformation varies from experiment to experiment, and can be from 2 x 10-3 to less than 10-4 mitochondrial transformants per nuclear transformant.