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Focus: Plasmid DNA Isolation from Yeast
A Simple and Reproducible Method to Isolate Plasmid DNA from Yeast after a Two-Hybrid
Screening
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| The Wizard® Plus
SV Minipreps DNA Purification System (Cat.# A1330,
A1340, A1460 and A1470) can be used to quickly isolate plasmid DNA from
yeast after a two-hybrid screening procedure. The plasmid DNA isolated with this system is
suitable for many applications, including PCR amplification. Show Me the Data! |
By Gaël Nicolas, Ph.D., Catherine Fournier, Ph.D., Colette Galand, B.S., and
Marie-Christine Lecomte, Ph.D.
Faculty of Medicine, Xavier Bichat, France
Introduction
The yeast two-hybrid system is a widespread method used to study
protein-protein interactions (1-3). In this system, one protein, the "bait"
molecule, is fused to a DNA-binding domain (e.g., Escherichia coli LexA protein),
and the other partner, the "prey" molecule, is fused to an activation domain
(e.g., yeast GAL4 protein). When these two hybrid proteins interact, a bipartite
transcription factor is reconstituted and can transactivate reporter genes, such as lacZ
(encoding beta-galactosidase) or his3 (encoding imidazole acetol phosphate
transaminase enzyme), which are downstream of DNA-binding sites for the bait protein's
DNA-binding domain. The system is also of great use for detecting and characterizing new
binding partners for a specific protein that is fused to the DNA-binding domain. This is
achieved by screening a library of cDNAs fused to the sequence of the activation domain.
In a typical screening protocol, the plasmid DNA from each yeast clone must be isolated in
order to identify the cDNA.
Here we describe a procedure to isolate high-quality plasmid DNA from
yeast, suitable for PCR amplification and E. coli transformation. This method
couples digestion with lyticase, an enzyme that hydrolyzes poly(beta-1,3-glucose) of the
yeast cell wall, followed by the Wizard® Plus SV Minipreps DNA
Purification System(a) protocol. The total procedure is
simple, reproducible and can be used routinely to isolate plasmid DNA from yeast. Lyticase
digestion was preferred over disruption with glass beads because lyticase digestion is
more reproducible and allows a higher yield of DNA.
Procedure
Collect yeast from a liquid culture (2ml) and resuspend in 50µl of
lysis buffer (50mM Tris-HCl [pH 7.5], 1.2M sorbitol, 10mM EDTA and 10mM
beta-mercaptoethanol added just before use). Alternatively, if the yeast clone has been
spread on a plate in order to form an area of strong growth (typically a 2-3cm2
patch), it can be collected with a sterile applicator or pipette tip. Mix vigorously by
vortexing.
Digest the cell wall by adding 200 units of lyticase (30 units/µl,
Sigma Cat.# L2524) to each tube. Incubate at 37°C overnight without shaking.
Collect yeast by centrifugation for 5 minutes at 4,000rpm. Remove and
discard the supernatant.
Perform plasmid DNA isolation on the yeast pellet with the Wizard®
Plus SV Minipreps DNA Purification System as described in Technical Bulletin #TB225 (see Section III.B, Step 2;
reference 4). (Note: The plasmid DNA will be in the spheroplast pellet at
this point.)
Following isolation from the Saccharomyces cerevisiae strain L40*, the plasmid DNA was used as template in PCR amplification as shown
in Figure 1. Purified plasmid can also be used to transform E. coli (1µl is
typically used to transform electrocompetent HB101 strain; data not shown). To obtain
enough yeast plasmid for restriction digestion analysis, transformation of E. coli
followed by plasmid purification is necessary (5).
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Figure 1. Plasmid was isolated from yeast using the
Wizard® Plus SV Minipreps DNA Purification System. Plasmid
DNA was isolated as described in the text, and 1µl of the purified plasmid was used as
template in PCR amplification using vector-specific primers (lanes 1-15). Due to the
absence of plasmid exclusion in yeast, a single clone may contain several different
plasmids. This can result in the amplification of multiple bands in the same sample.
(Lanes M, DNA size markers.) |
References
Vojtek, A.B., Hollenberg, S.M. and Cooper, J.A. (1993) Mammalian Ras
interacts directly with the serine/threonine kinase Raf. Cell 74,
205.
Chien, C. T. et al. (1991) The two-hybrid system: a method to
identify and clone genes for proteins that interact with a protein of interest. Proc.
Natl. Acad. Sci. USA 88, 9578.
Fields, S. and Song, O. (1989) A novel genetic system to detect
protein-protein interactions. Nature 340, 245.
Wizard® Plus
SV Minipreps DNA Purification System Technical Bulletin #TB225, Promega
Corporation.
Ausubel, F.M. et al. (1992) In: Current Protocols in
Molecular Biology, Vol. 2, Greene Publishing Associates, Inc., and John Wiley
and Sons, NY.
*Genotype of Strain L40: MATa
his3-delta200 trp1-901 leu2-3,112 ade2 lys2-801am
LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lacZ GAL4; see
reference 1 for more information.
(a)U.S. Pat. No. 5,981,235.
Wizard is a trademark of Promega Corporation and is
registered with the U.S. Patent and Trademark Office.
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