Transformation Procedure

Methods were developed for transferring DNA into fungal cells and to select the transformed cells that have taken up this DNA. In the prototype experiment done in Edward Tatum's laboratory, wild type DNA was introduced into an inositol-requiring mutant

Figure 9.1 Neurospora crassa growing on a sugarcane factory-waste dump. The genes encoding pink-orange color have been used as a visual reporter system in gene-silencing experiments. (Photo: courtesy of P. Maruthi Mohan.) (See color insert following page 140.)

(auxotroph) of Neurospora crassa. The use of an auxotrophic mutant as the recipient allowed the rare, inositol-independent cells (prototroph) to be directly selected on minimal media lacking the nutritional supplement. However, the prototrophic selection method has the disadvantage that the nutritional mutation has to be introduced into the recipient (host) strain by a prior sexual cross. This time-consuming difficulty is overcome by the use of a dominant selectable marker (Hynes, 1996), for example, the mutant p-tubulin gene (BmF), which confers resistance to the fungicide benomyl, or the hygromycin resistance gene (hph) which allows transformants to be selected even in the wild-type background. The DNA-treated conidia are plated on media that contains benomyl or hygromycin on which the untransformed cells cannot grow, allowing the transformed colonies to be selected.

The entry of DNA into cells is facilitated by removal of the cell wall. The cell wall can be removed by treating cells with a commercial mixture of p, 1-3 glucanase and chitinase enzymes (Novozyme) obtained from a soil fungus, Trichoderma viride (see Davis, 2000). The protoplasts (spheroplasts), freed from the constraint of the rigid cell wall, become round and are prone to bursting in a hypertonic environment and, therefore, need to be osmotically stabilized to regenerate a mycelial culture. Because the commercial preparations of the lytic enzymes vary from batch to batch, the protoplasting method yields variable results; it was therefore replaced by the electroporation or the particle bombardment methods for introducing DNA. In the now commonly used electroporation method, conidia are placed in a solution of DNA and subjected to strong electric pulses to facilitate the entry of DNA into the cell, presumably by transiently opening holes in the membrane.

In practice, the DNA to be used for transformation is cut into fragments by DNA-cutting enzymes called restriction enzymes. A fragment is joined into a small circular DNA

molecule called a plasmid (vector) containing a point (restriction site) for joining the foreign DNA, one of which will have the gene of interest. The vector DNA selected is one that contains a selectable marker and regulatory sequences necessary for the expression of the transgene. The ligated, recircularized DNA fragment containing the gene of interest is introduced into the fungal cell (generally conidia) by transformation. Typically, 1 to 20 jag of DNA per 107 cells in 50 jl of a pH 7.5 buffer containing 1M sorbitol or polyethylene glycol 4000 and 50 mM of CaCl2 are incubated before plating the conidia on selective agar media to select the rare, specific colonies of transformed cells. The transformation frequency is about 1 to 20 per microgram of DNA. The specific fragment of DNA from a single chosen transformed colony can be reisolated (cloned) by taking advantage of the fact that it is now tagged with DNA sequences of the transformation vector.

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