4. Yeast Molecular Techniques
- Prof. Dr. Horst Feldmann1,2
Published Online: 26 SEP 2012
Copyright © 2012 Wiley-VCH Verlag GmbH & Co. KGaA
Yeast: Molecular and Cell Biology, Second Edition
How to Cite
Feldmann, H. (ed) (2012) Yeast Molecular Techniques, in Yeast: Molecular and Cell Biology, Second Edition, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. doi: 10.1002/9783527659180.ch4
Adolf Butenandt Institute, Molecular Biology, Ludwig-Maximilians-Universität M¨nchen, Schillerstr. 44, 80336 M¨nchen, Germany
Ludwig-Thoma-Strasse 22B, 85232 Bergkirchen, Germany
- Published Online: 26 SEP 2012
- Published Print: 22 AUG 2012
Print ISBN: 9783527332526
Online ISBN: 9783527659180
- yeastmolecular techniques;
- genetic engineering;
- reverse genetics;
- more genetic tools;
- yeast genome analyses
• Initially, some conventional methodology is presented (i.e., how yeast cells can be grown and opened in order to isolate particular subcellular constituents). In the present laboratory routine, these methods might no longer be applied. A description of genetic techniques (e.g., genetic mapping, genetic crosses, or tetrad analysis) has been excluded a priori. Preparation of recombinant DNA and transformation of yeast cells are still practiced in many laboratories. As this volume does not aim at describing the protocols for the various procedures, we just explain the principles and refer the reader to the relevant literature. Useful information on equipment, growth media, appropriate yeast vectors, and strains can frequently be found in the brochures or catalogs of the relevant companies.
• The successful transformation of yeast cells by hybrid plasmids in 1978 marked a milestone in molecular biology. Depending on their molecular shaping, these plasmids would autonomously replicate in yeast (as single or multicopy entities) or integrate as single copies into defined loci within the yeast genome. A plasmid endowed with a segment of centromeric DNA and transformed in yeast cells is stabilized; during mitotic cell division this plasmid will be normally replicated once and the two copies segregated according to the rules known for the yeast chromosomes. Shuttle vectors, capable of propagating both in yeast and in bacterial cells, allowed reciprocal transfer of genetic material from one host to the other. Selection of transformed cells was facilitated by inclusion of appropriate genetic markers into the plasmid sequences. Replica plating of yeast cells grown on solid agar or colony hybridization was as easy as for bacterial cells. Expression plasmids carrying yeast-specific promoter (and terminator) sequences could be used to express foreign genes in the yeast system and even to design them for export. Remarkably, this approach also proved that in a multitude of cases human genes were capable of functionally complementing their homologous counterparts in appropriate yeast mutants.
• A suitable extension of yeast transformation by plasmids was offered by the finding that appropriate cosmids of considerable length (up to 40 kb) could serve as shuttle vectors as well. This technique was later applied for the construction of ordered yeast genomic libraries, which turned out to be much more advantageous in the sequencing project than plasmid or phage libraries. A similar line was followed in the construction of YACs. Thus, human DNA fragments up to 1 Mb could be accommodated and propagated. The big hope of using this tool in mapping the human genome, however, finally turned into a disappointment because the YACs suffered rearrangements in the yeast due to its propensity of frequent recombination via short homology regions. This trait has been employed in “one-step gene replacement” of yeast genes as only 20 bp at each border were sufficient to effect disruption and subsequent substitution of a genomic sequence by another.
• A most successful technique, developed in 1989 and still applied, is the yeast two-hybrid system for the detection of protein–protein interactions. The yeast three-hybrid system, developed some 10 years later, was designed as an assay for RNA–protein interactions.
• Finally, we briefly introduce some aspects of techniques to detect and quantitate protein–protein interactions.