Transformation of yeast

Figure 4.7. In vivo recombinational cloning in yeast. Two successive PCR reactions are performed. Each set of primers contains 5 flanking sequences that are eventually used for homologous recombination with vector sequences after transformation of yeast with the 2nd PCR fragment and the linearized vector.

Gietz, R. D., and Woods, R. A. (2002). Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87-96. 

Fig. 6. Ure2p filaments are infectious. (A, B) Negatively stained micrographs of Ure2p filaments (A) before and (B) after sonication, which was found to be necessary for effective transformation of filaments. (C) Color spectrum of [URE3] variants in randomly chosen colonies after transformation of yeast cells with Ure2p filaments. (D) Conversion efficiency to [URE3] status is dependent on Ure2p filament concentration. All panels adapted from Figure 2 of Brachmann et al (2005).

Approximately 1 p% of wild-type or mutant pCLI plasmid DNA is used for transformation of yeast cells. Spheroplasts of Saccharomyces cerevisiae strain GRF 18023,24 are prepared following the protocol of Burgers and Percival25 with some modification. High copy number transformants are directly selected on minimal medium plates lacking leucine and containing 8% glucose (to repress lysozyme expression) and 1 M sorbitol as an osmotic stabilizer. 

Protocol 3 Transformation of Yeast with Lithium Acetate.164... 

Again, this has been carried out both for scFv [45] and Fab [46] libraries. While transformation of yeast cells is somewhat more difficult than that of E. coli, methodologies do exist to achieve this. With antibodies displayed on the surface of yeast, not only a mechanical enrichment (e.g., with magnetic beads) is possible, but also the use of cell sorters together with a fluorescently labeled target. Therefore, thresholds for affinity can be defined and the affinity can be measured directly on... 

By far the most exciting development of this work was the demonstration by Schaller and Bleecker [31] that transformation of yeast with the wild type ETR gene conferred the ability to bind ethylene in a saturable and reversible manner and with an appropriate Kp (2.4xl0 M). These results taken together with those outlined above provide almost conclusive evidence that the ETR gene product is indeed an ethylene receptor. In the same work expression of truncated forms of ETRI in yeast provided further evidence that the N-terminal hydrophobic domain of the protein is the site of ethylene binding. 

Moerschell, R. P., Tsunasawa, S., and Sherman, F. (1988). Transformation of yeast with synthetic oligonucleotides. Proc. Natl. Acad. Set. USA 85, 524-528. 

S., and Davis, R.W. (1979) High-frequency transformation of yeast autonomous replication of hybrid DNA... 

BurgersPM,PercivalKJ(1987)Transformation of yeast spheroplasts without cell fusion. Anal Biochem 163 391-397... 

Becker, D M, Guarente L. High-efficiency transformation of yeast by electroporation. Meth Enzymol, 1991 194 182 - 187. 

Fermentation An anaerobic bioprocess. An enzymatic transformation of organic substrates, especially carbohydrates, generally accompanied by the evolution of gas as a byproduct. Fermentation is used in various industrial processes for the manufacture of products (e.g., alcohols, organic acids, solvents, and cheese) by the addition of yeasts, moulds, and bacteria. 

The term fermentation was obtained from the Latin verb fervere which describes the action of yeast or malt on sugar or fruit extracts and grain. The boiling is due to the production of carbon dioxide bubbles from the aqueous phase under the anaerobic catabolism of carbohydrates in the fermentation media. The art of fermentation is defined as the chemical transformation of organic compounds with the aid of enzymes. The ability of yeast to make alcohol was known to the Babylonians and Sumerians before 6000 bc. The Egyptians discovered the generation of carbon dioxide by brewer s yeast in the preparation... 

Whole cells are grown for a variety of reasons. The cells may perform a desired transformation of the substrate, e.g., wastewater treatment the cells themselves may be the desired produce, e.g., yeast production or the cells may produce a desired product, e.g., penicillin. In the later case, the desired product may be excreted, as for the penicillin example, and recovered in relatively simple fashion. If the desired product is retained within the cell walls, it is necessary to lyse (rupture) the cells and recover the product from a complex mixture of cellular proteins. This approach is often needed for therapeutic proteins that are created by recombinant DNA technology. The resulting separation problem is one of the more challenging aspects of biochemical engineering. However, culture of the cells can be quite difficult experimentally and is even more demanding theoretically. 

P450 systems (Sariaslani 1991), their widespread role in the transformation of xenobiotics (Smith and Davis 1968), and their occurrence and activities in yeasts (Kappeli 1986). The essential features of prokaryotic and eukaryotic cytochrome P450 systems are compared in Figure 3.17. 

A cytochrome P450 has been purified from Saccharomyces cerevisiae that has benzo[a]pyrene hydroxylase activity (King et al. 1984), and metabolizes benzo[fl]pyrene to 3- and 9-hydroxybenzo[fl]pyrene and benzo[fl]pyrene-7,8-dihydrodiol (Wiseman and Woods 1979). The transformation of PAHs by Candida Upolytica produced predominantly monohydroxyl-ated products naphth-l-ol from naphthalene, 4-hydroxybiphenyl from biphenyl and 3- and 9-hydroxybenzo[fl]pyrene from benzo[fl]pyrene (Cerniglia and Crow 1981). The transformation of phenanthrene was demonstrated in a number of yeasts isolated from littoral sediments and of these, Trichosporumpenicillatum was the most active. In contrast, biotransformation of benz[fl]anthracene by Candida krusei and Rhodotorula minuta was much slower (MacGillivray and Shiaris 1993). 

Each of the -6000 PCR products was then co-transformed into yeast along with the recipient vector that had been linearized using a restriction enzyme that digests the plasmid at the desired cloning site. The 70 bp of homologous flanking sequence on each end of the PCR products is sufficient for the yeast homologous recombination system to act upon and insert the PCR product into the vector (Hudson et al., 1997 Ma et al., 1987). 

Schiestl, R.H. and Gietz, R.D. (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Current Genetics, 16 (5-6), 339-346. 

Lin-Cereghino, J., Wong, W.W., Xiong, S. et al. (2005) Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques, 38 (1), 44, 46, 48. 

Nitro PAHs have been shown to exhibit a large variety of biological activities. Included in these are the induction of mutations in bacterial (Table I) and eukaryotic cells (9,17,54-57), the neoplastic transformation of cultured mammalian cells (58-59), and the induction of DNA strand breaks (60), DNA repair (61-62), sister chromatid exchanges (63-64), and chromosomal aberrations (65-66). Nitro PAHs have also been demonstrated to bind cellular DNA in bacteria (67-73) and mammalian cells (74-77), to inhibit preferentially the growth of repair-deficient bacteria (78), to have recombinogenic activity in yeast (66,79-80) and to induce tumors in experimental animals (Table II). 

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