Yeast one hybrid system

The Yeast One-Hybrid-System ( Y1H , in German 'Yeast-One-Hybrid-System') is used to identify new protein-DNA interactions , but is also suitable for confirming and characterizing known or suspected protein-DNA interactions .

principle

To find new interactions ( screening ), a distinction is made between a protein-centered and a DNA-centered approach. In both cases, the activation of a reporter gene that z. B. the baker's yeast Saccharomyces cerevisiae gives an antibiotic resistance that it comes to an interaction.

Finding DNA binding sequences for a protein

The cDNA of the protein to be examined is cloned into a vector which codes for a transcriptional activation domain , usually that of the yeast transcription factor GAL4 . A yeast strain is stably transformed with the DNA amplified in Escherichia coli . The cells express the protein to be examined as a fusion protein with an activation domain. The yeasts are now with a reporter - library transformed into the promoter ever before, a DNA fragment, z. B. Restriction fragments of mouse genomic DNA, has been cloned. If there is an interaction with the fusion protein, the reporter gene is activated and the yeast forms colonies on suitable plates .

Look for proteins that bind to an element of DNA

In the first step, yeast cells are transformed with a reporter gene plasmid, in whose promoter the DNA element to be investigated was cloned. The yeasts are now transformed with a cDNA library of plasmids which code for a transcriptional activation domain . Yeasts that express fusion proteins that bind to the target sequence acquire antibiotic resistance . The search for transcription factors for regulatory sequences from promoters and enhancers is used more frequently . In both cases the plasmids are isolated from the selected yeast colonies and identified. As a rule, the protein-DNA interaction is verified using other methods, such as EMSA .

The Bacterial One-hybrid system is a method derived from the Yeast-One-Hybrid-System for the determination of DNA-protein interactions in bacteria. The Yeast-Two-Hybrid-System is a research method for protein-protein interactions .

Web links

Yeast One-Hybrid System clontech
M. Liao, F. Fang: [Yeast one-hybrid system-one effective method studying DNA-protein interaction]. In: Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae. Volume 22, Number 4, August 2000, pp. 388-391, PMID 12903457 (review).
Yeast 1 and 2 hybridization (PDF; 1.5 MB) p. 17

Individual evidence

↑ JS Reece-Hoyes, AJ Marian Walhout: Yeast one-hybrid assays: a historical and technical perspective. In: Methods (San Diego, Calif.). Volume 57, number 4, August 2012, ISSN 1095-9130 , pp. 441-447, doi: 10.1016 / j.ymeth.2012.07.027 , PMID 22884952 , PMC 3448804 (free full text).
^ M. Sieweke: Detection of transcription factor partners with a yeast one hybrid screen. In: Methods in molecular biology (Clifton, NJ). Volume 130, ISSN 1064-3745 , 2000, pp. 59-77, PMID 10589421 .

[1] JS Reece-Hoyes, AJ Marian Walhout: Yeast one-hybrid assays: a historical and technical perspective. In: Methods (San Diego, Calif.). Volume 57, number 4, August 2012, ISSN 1095-9130 , pp. 441-447, doi: 10.1016 / j.ymeth.2012.07.027 , PMID 22884952 , PMC 3448804 (free full text).

[2] M. Sieweke: Detection of transcription factor partners with a yeast one hybrid screen. In: Methods in molecular biology (Clifton, NJ). Volume 130, ISSN 1064-3745 , 2000, pp. 59-77, PMID 10589421 .