Brief Background on Yeast Plasmids
Yeast plasmids have a lot of features like those found in the plasmids that you
worked with in previous classes and that you will read about in your textbook. For
example, yeast plasmids contain a selectable marker. However, yeast selectable markers
are typically based on auxotrophies (or nutrient requirements) rather than antibiotic
resistance. So, if you transform a ura3-52 mutant (that cannot grow on media lacking
uracil) with a plasmid containing URA3 as the selectable marker, only cells that take up
the plasmid will be able to grow on SDC-ura plates. To maintain plasmids that do not
integrate into an endogenous yeast chromosome, transformed strains should always be
grown in/on selective media.
The three major classes of plasmids commonly used in yeast are described below
and include high-copy, low-copy, and integrating.
The most common type of high-copy yeast plasmids are the "two micron" (2μ)
vectors. The 2μ DNA fragment that gives these vectors their name is derived from the
endogenous 2μ plasmid carried in nearly all laboratory yeast strains. This 2μ plasmid
encodes proteins that allow cells to maintain 20-50 copies of any plasmid carrying the 2μ
origin of replication. Because 2μ plasmids are maintained at such high copy numbers,
they provide a convenient way to monitor the effects of overproduction of a particular
gene product.
When plasmids need to be maintained at a low copy number, CEN-based vectors
provide an attractive alternative. Because they carry a centromere sequence and a normal
yeast origin of replication (as opposed to a 2μ ori), these vectors are recognized and
replicated like small chromosomes, and strains typically carry only one copy of this type
of plasmid.
As the name implies, integrating plasmids typically insert themselves, or
integrate, into the host cell's genome. This integration results from a recombination event
between any yeast sequences carried on both the plasmid, and the host’s endogenous
DNA. One feature that facilitates the integration of this type of plasmid is its lack of a
yeast origin of replication, also known as an autonomous replication sequence, or ARS.
Plasmid Complementation
In the lab, complementation analysis can be achieved not only through mating but
also by transforming cells with a plasmid carrying a "good" allele of a mutant gene.
Today you will transform your HePCr mutant strain with a plasmid to test the
hypothesis that your mutant carries a hypomorphic allele of LEM3 (if you need to review
what “hypomorphic” means, you might try looking it up in your textbook).
Previous studies have shown that loss-of-function mutations in LEM3 make yeast
cells resistant to ether lipid drugs like HePC, but not other drugs (Hanson et al. 2003).
The construct we will use to test
for complementation is a CENbased plasmid carrying a
fragment of genomic DNA that
encodes LEM3. You will also
transform your mutant with the
empty vector (pGFP-N-FUS) as a
control (Note that this empty
vector was designed by
Niedenthal et al. (1996)).
Diagrams representing both the
control vector and the LEM3containing plasmid are shown to
the right. After following the
transformation protocol found
below, you will plate the
transformed cells onto SDC-ura
to select those cells that have
taken up the plasmid.
Transformation of Yeast
with pGFP-N-FUS::LEM3
Lithium acetate transformation protcol
adapted from Gietz and Woods (2002).
The day before your lab period,
you must come in to lab and
start a culture of your mutant
strain. The culture should be
grown in YPD and placed in the
shaking water bath at 30oC
1. During your lab period, briefly
vortex your culture, then pipette 1ml
of it into each of two microfuge
tubes.
2. Pellet the cells by spinning in a
microfuge for one minute at 13,000rpm.
3. Use a pipetman to remove the media from each tube without disturbing the cell pellets.
4. Resuspend the each pellet separately in 500μl of sterile water by gently pipetting the solution
up and down.
5. Pellet the cells by spinning in a microfuge for one minute at 13,000rpm.
6. Use a pipetman to remove the water without disturbing the cell pellets.
7. Resuspend each pellet in 200μl of 100mM lithium acetate.
8. Pellet the cells by spinning in a microfuge for one minute at 13,000rpm.
9. Use a pipetman to remove the lithium acetate solution without disturbing the cell pellets.
10. Repeat steps 7, 8, and 9.
11. Add the following ingredients on top of each cell pellet in the order listed:
240 μL PEG3350 (50% w/v)
36μL 1.0 M LiAc
25μL carrier DNA (2mg/mL)
To one of the tubes, add 50μL plasmid DNA (pGFP-N-FUS::LEM3) and label this tube with
your initials and the letter “L” for LEM3.
To the other tube, add 50μL of the empty vector (pGFP-N-FUS) and label this tube with
your initials and the letter “C” for control.
12. Vortex the tubes vigorously until the cell pellets have been completely resuspended (about 1
minute).
13. Incubate both at 30oC for 30 minutes.
14. Heat shock both samples in a water bath at 42oC for 20-25 minutes.
15. Pellet cells by spinning in a microfuge for one minute at 13,000 rpm.
16. Carefully remove the supernatant from each sample.
17. Resuspend each pellet in 1ml of sterile water by gently pipetting up and down.
18. Pellet cells by spinning in a microfuge for one minute at 13,000 rpm..
19. Remove approximately 800μL of water from each tube, resuspend the pellets in the remaining
liquid and plate each sample on its own SDC-ura plate. Both plates should be incubated at 30oC.
In three days, return to lab and you should find colonies of transformed yeast on your
SDC-ura plates. Streak two of your LEM3 transformants and two of your empty vector
transformants onto a single SDC-ura plate containing HePC. As an additional control on the
same SDC-ura + HePC plate, you should also streak a Δlem3 (Δ refers to the fact that the gene
has been deleted) strain carrying a LEM3 plasmid and a Δlem3 strain carrying the empty vector
pGFP-N-FUS. When complete your plate should contain six streaks total. Incubate this plates at
30oC and in two to three days, return to lab to record your results with the BioDoc-It system.
References
Gietz, R.D. and R.A. Woods. (2002) Transformation of yeast by the Liac/SS carrier DNA/PEG method.
Meth.Enzymol. 350: 87-96.
Hanson, P.K., Malone,L., Birchmore, J.L. and J. W. Nichols (2003) Lem3p is essential for the uptake and
potency of alkylphosphocholine drugs, edelfosine and miltefosine. J. Biol. Chem. 278: 36041-50.
Niedenthal, R.K., Riles, L., Johnston, M., and J.H. Hegemann (1996) Green fluorescent protein as a marker
for gene expression and subcellular localization in budding yeast. Yeast. 12: 773-86.