Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Summary

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

Abstract

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Introduction

The four core histone proteins H2A, H2B, H3, and H4 play central roles in the compaction, organization, and function of eukaryotic chromosomes. Two sets of each of these histones form the histone octamer, a molecular spool that directs the wrapping of ~147 base pairs of DNA around itself, ultimately resulting in the formation of a nucleosome¹. Nucleosomes are active participants in a variety of chromosome-based processes, such as the regulation of gene transcription and the formation of euchromatin and heterochromatin across chromosomes, and as such have been the focus of intense research over the course of the past several decades. A number of mechanisms have been described by which nucleosomes can be manipulated in ways that can facilitate execution of specific processes – these mechanisms include posttranslational modification of histone residues, ATP-dependent nucleosome remodeling, and ATP-independent nucleosome reorganization and assembly/disassembly^2,3.

The budding yeast Saccharomyces cerevisiae is a particularly powerful model organism for the understanding of histone function in eukaryotes. This can be largely attributed to the high degree of evolutionary conservation of the histone proteins throughout the domain eukarya and the amenability of yeast to a variety of genetic and biochemical experimental approaches⁴. Reverse-genetic approaches in yeast have been widely used to study the effects of specific histone mutations on various aspects of chromatin biology. For these types of experiments it is often preferable to use cells in which the mutant histones are expressed from their native genomic loci, as expression from autonomous plasmids can lead to abnormal intracellular levels of histone proteins (due to varying numbers of plasmids in cells) and concomitant alteration of chromatin environments, which can ultimately confound the interpretation of results.

Here, we describe a PCR-based technique that allows for targeted mutagenesis of histone genes at their native genomic locations that does not require a cloning step and results in the generation of the desired mutation(s) without leftover exogenous DNA sequences in the genome. This technique takes advantage of the efficient homologous recombination system in yeast and has several features in common with other similar techniques developed by other groups - most notably the Delitto Perfetto, site-specific genomic (SSG) mutagenesis, and cloning-free PCR-based allele replacement methods^5,6,7. However, the technique we describe has an aspect that makes it particularly well-suited for mutagenesis of histone genes. In haploid yeast cells, each of the four core histones is encoded by two non-allelic and highly homologous genes: for example, histone H3 is encoded by the HHT1 and HHT2 genes, and the open reading frames (ORFs) of the two genes are over 90% identical in sequence. This high degree of homology can complicate experiments designed to specifically target one of the two histone-encoding genes for mutagenesis. Whereas the aforementioned methods often require the use of at least some sequences within the ORF of the target gene to drive homologous recombination, the technique we describe here makes use of sequences flanking the ORFs of the histone genes (which share much less sequence homology) for the recombination step, thus increasing the likelihood of successful targeting of mutagenesis to the desired locus. Moreover, the homologous regions that drive recombination can be very extensive, further contributing to efficient targeted homologous recombination.

Protocol

NOTE: The experimental strategy for targeted in situ histone gene mutagenesis includes several steps (summarized in Figure 1). These steps include: (1) Replacement of the target histone gene with the URA3 gene, (2) Generation and purification of PCR products corresponding to two partially overlapping fragments of the target histone gene using primers harboring the desired mutation(s), (3) Fusion PCR of the two partially overlapping fragments to obtain full size PCR products for integration, (4) Co-transformation of full size PCR products and backbone plasmid, and selection for marker on plasmid, (5) Screen for 5-FOA-resistant transformants, (6) Purification of 5-FOA-resistant colonies and loss of backbone plasmid, and (7) Molecular analyses to assay for proper integration of the mutant allele.

Figure 1: Overview of the Strategy for Targeted in situ Mutagenesis of Histone Genes in Budding Yeast. In this example the targeted gene is HHT2, but any other core histone gene can also be mutagenized using this strategy. (A) Haploid yeast cells harbor two histone H3-encoding genes (HHT1 and HHT2) and two histone H4-encoding genes (HHF1 and HHF2) arranged as shown in the figure (the HHT1 and HHF1 genes are located on chromosome II and the HHT2 and HHF2 genes are located on chromosome XIV - in each case, the arrows point in the direction of transcription). In the first step of the procedure, the ORF of the HHT2 gene is replaced with the URA3 gene, giving rise to an hht2Δ::URA3 strain. (B) In part 1, a wild-type copy of the HHT2 gene from a genomic DNA sample is used as template for two PCR reactions to generate the two partially overlapping fragments of the gene. The reverse primer for the first reaction includes one or more mismatched nucleotides (indicated with a red circle) that correspond to the desired mutation(s) to be introduced into the genome. The forward primer for the second reaction has the equivalent mismatch in a reverse complementary configuration (also indicated with a red circle). The two PCR products generated in part 1 (products a and b) are then used as templates for fusion PCR using two primers that anneal to products a and b in the fashion shown in part 2. This results in the generation of full-size PCR products (product c in part 3) harboring the desired mutation(s). (C) The hht2Δ::URA3 strain is then co-transformed with the full-size PCR products and a backbone plasmid (a HIS3-marked plasmid in this example), and cells are selected for the presence of the plasmid (on media lacking histidine in this example). Transformants are then screened for 5-FOA resistance - resistant cells are candidates for having undergone a homologous recombination event leading to integration of the PCR product and excision of the URA3 gene, as shown. Subsequent loss of the backbone plasmid by mitotic cell division leads to the final desired histone mutant strain. We have found that selection of the backbone plasmid followed by screening for 5-FOA resistance results in a much higher frequency of identification of correct integration events compared to direct selection on 5-FOA plates, which mostly identifies cells that have acquired spontaneous URA3 mutations. (This figure has been modified from reference¹⁴). Please click here to view a larger version of this figure.

1. Replacement of the Target Histone Gene with the URA3 Gene

1. Perform standard PCR-mediated one-step gene disruption replacing the ORF of target histone gene with the URA3 gene^8,9.
   NOTE: The use of yeast cells carrying the ura3Δ0 is recommended as this mutation removes the entire endogenous URA3 ORF, thus avoiding integration of the PCR product into the URA3 locus⁸. Alternatively, the K. lactis URA3 gene can be used effectively for the generation of the histone replacement in any ura3 background as it is functional in S. cerevisiae but has only partial sequence homology with the S. cerevisiae URA3 gene. The strain should also be auxotrophic for at least one compound that will allow for selection of the backbone plasmid in the transformation experiment (see step 4 of this protocol). This step is not necessary if a target histone geneΔ::URA3 strain is already available.

2. Generation and Purification of PCR Products Corresponding to Two Partially Overlapping Fragments of the Target Histone Gene using Primers Harboring the Desired Mutation(s)

1. Generate PCR products corresponding to two partially overlapping fragments of the target histone gene.
   1. Prepare two PCR reactions as follows:
      1. To generate PCR products corresponding to the first half of the gene (product a in Figure 1B), set up the following reaction: 1 μl template DNA, 5 μl10 μM forward primer, 5 μl10 μM reverse primer, 0.5 μl (1.25 U) thermostable DNA polymerase, 10 μl 5x DNA polymerase buffer, 5 μl dNTP mixture (2 mM each), and 23.5 μl dH₂O.
         NOTE: The template DNA can be genomic DNA derived from a strain wild-type for the target histone gene isolated using standard procedures¹⁰. To account for variations in DNA concentration and level of impurities in different genomic preparations, it is recommended to optimize the reactions by using either undiluted DNA or different dilutions of the genomic preparations (e.g., 1:10 and 1:100). The forward primer should anneal to a region upstream of the target gene. The reverse primer should anneal within the ORF, be ~40 nucleotides in length, and contain the desired mutation(s) somewhere in the middle of it (see Figure 1B-1 and Representative Results section for examples). The use of a high fidelity DNA polymerase is recommended in order to reduce rates of undesired mutations during the synthesis of the PCR products.
      2. To generate PCR products corresponding to the second half of the gene (product b in Figure 1B), set up a reaction as indicated in 2.1.1.1 but with different primers.
         NOTE: The forward primer should anneal within the ORF, be ~ 40 nucleotides in length, and contain the desired mutation(s) somewhere in the middle of it. Note that the mutation(s) in this primer is the reverse complement of the mutation(s) in the reverse primer in step 2.1.1.1. The reverse primer should anneal to a region downstream of the target gene (see Figure 1B-1 and Representative Results section for examples).
   2. Place the reactions in a thermocycler with the following settings: 94 ˚C 30 sec; 30 cycles of the following settings: 98 ˚C 10 sec, 60 ˚C 5 sec, 72 ˚C 1.5 min; and 72 ˚C 10 min.
      Note: Optimization of PCR parameters may be required for specific primer sets and target histone gene.
2. Run 20 - 50 μl of the material from the PCR reactions on a 0.9% low melting point agarose gel in 89 mM Tris base, 89 mM boric acid, 2.5 mM EDTA (TBE) buffer.
3. Cut agarose gel sections containing the PCR products from gel using a clean scalpel or razor blade and transfer each to a 1.5 ml microcentrifuge tube. Store agarose sections containing PCR products at -20 ˚C until ready to use.

3. Fusion PCR of the Two Partially Overlapping Fragments to Obtain Full Size PCR Products for Integration

1. Prepare template for PCR reactions
   1. Melt agarose gel sections from step 2.3 by placing the microcentrifuge tubes in a heat block set at 65 ˚C for 5 min (or until fully melted). Vortex tubes every 1 - 2 min to facilitate the melting process.
   2. Transfer a set amount of melted agarose from each sample (e.g., 50 μl each, for a total of 100 μl) into a single microcentrifuge tube and mix by vortexing. Use this as the template in the fusion PCR reactions. Place the tube at -20 ˚C until ready to use.
2. Amplify a large quantity of full size PCR product (product c in Figure 1B)
   1. Set up six PCR reactions, each with the following components: 2 μl template DNA, 10 μl 10 μM forward primer, 10 μl 10 μM reverse primer, 1 μl (2.5 U) thermostable DNA polymerase, 20 μl 5x DNA polymerase buffer, 10 μl dNTP mixture (2 mM each), and 47 μl dH₂O.
      NOTE: The number of reactions can be altered depending on the PCR efficiency. The template DNA (see 3.1.2) should be heated to 65 ˚C until melted, mixed by vortexing, and added last to the PCR reaction mix. Once added, mix gently but thoroughly by pipetting the solution up and down several times. To account for variations in DNA concentration in the different samples, it is recommended to first optimize the reactions by using either undiluted template or different dilutions of the template (e.g., 1:10 and 1:100). The two primers used should anneal to the two partially overlapping fragments of the target gene as illustrated in Figure 1B-2 and be designed such that the final PCR products will have at least 40 base pairs on either side homologous to the regions flanking the URA3 ORF that will drive the homologous recombination step (see Representative Results section for examples). The use of a high fidelity DNA polymerase is recommended in order to reduce rates of undesired mutations during the synthesis of the PCR products.
   2. Place the tubes in a thermocycler with the following settings: 94 ˚C 30 sec; 30 cycles of the following settings: 98 ˚C 10 sec, 50 ˚C 15 sec, 72 ˚C 1.5 min; and 72 ˚C 10 min.
      NOTE: Optimization of PCR parameters may be required for specific primer sets and target histone gene.

4. Co-transformation of Full Size PCR Products and Backbone Plasmid, and Selection for Marker on Plasmid

1. Concentration of PCR products
   1. Pool the six PCR reactions (600 μl total) from step 3.2.2 into a single microcentrifuge tube and mix by vortexing.
   2. Split the sample into three 200 μl aliquots in microcentrifuge tubes. Precipitate the DNA in each tube by adding 20 μl of 3M sodium acetate (pH 5.2) and 550 μl of 100% ethanol. Mix the solution thoroughly and place on ice for at least 15 min. Collect DNA by centrifugation at ~14,000 x g for 10 min, rinse the pellet with 200 μl of 70% ethanol, and air dry.
   3. Resuspend each DNA pellet into 25 μl of dH₂O, and pool into a single tube (for a total of 75 μl).
2. Yeast co-transformation
   1. Prepare 10 ml of overnight culture of the strain generated in section 1 in Yeast extract Peptone Dextrose (YPD) liquid medium¹¹.
   2. The following morning, inoculate 400 mL of YPD liquid medium with 8 ml of the saturated overnight culture and incubate by shaking at 30 °C for 4 - 5 h to allow cells to enter logarithmic phase of growth.
   3. Collect the cells by centrifugation at ~3,220 x g for 10 min, discard the liquid medium, and resuspend the cells in 1 volume of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1 M Lithium Acetate solution (TE/LiAc).
   4. Collect the cells by centrifugation at ~3,220 x g for 10 min, and discard the TE/LiAc.
   5. Resuspend the cells in 1 ml TE/LiAc.
   6. Set up the following reaction cocktail in a microcentrifuge tube: 800 μl of cells from step 4.2.5, 40 μl of boiled 10 mg/ml salmon sperm DNA, a total of 12.5 μg of backbone plasmid DNA, and 75 μl of concentrated PCR product from step 4.1.3.
      NOTE: Salmon sperm DNA should be boiled for 5 min and placed on ice for at least 5 min before use in the reaction. Total volume of backbone plasmid DNA added should be kept to a minimum (~80 μl or less). See Representative Results section for an example of a backbone plasmid.
   7. Mix the cocktail tube thoroughly and aliquot evenly into eight microcentrifuge tubes (Tubes 1 - 8).
   8. Set up the following two control transformation reaction tubes:
      1. Tube 9 (no PCR product control): 100 μl of cells from step 4.2.5, 5 μl of boiled 10 mg/ml salmon sperm DNA (boiled for 5 min; see step 4.2.6 Note), a total of 1.56 μg of backbone plasmid DNA and no PCR product added.
      2. Tube 10 (no DNA control): 100 μl of cells from step 4.2.5, 5 μL of boiled 10 mg/mL salmon sperm DNA (see step 4.2.6 Note), no backbone plasmid DNA added, and no PCR product added.
      3. Mix both tubes gently but thoroughly by pipetting up and down several times.
   9. Incubate the ten tubes at 30 ˚C for 30 min.
   10. To each tube, add 1.2 ml of 40% polyethylene glycol (PEG 3350) in TE/LiAc. Mix thoroughly using a P-1000 pipet until the solution is homogeneous.
   11. Incubate the ten tubes at 30 °C for 30 min. Gently mix the solution by pipetting up and down and then incubate tubes at 42 °C for 15 min.
   12. Collect the cells by spinning the tubes in a microcentrifuge at ~14,000 x g for 30 sec. Discard the liquid and resuspend the cells in 1 ml of sterile dH₂O.
   13. Collect the cells by spinning the tubes in a microcentrifuge at ~14,000 x g for 30 sec. Discard the liquid and resuspend the cells in 500 μl of sterile dH₂O.
   14. Pool tubes 1 - 8 together (total volume of 4 ml) and mix thoroughly by pipetting up and down.
   15. Plate 200 μl of the above mixture on each of twenty complete minimal dropout medium plates¹¹ (plates 1-20) for selection of the backbone plasmid.
   16. Plate 200 μl of the mixture from Tube 9 and 200 μl of mixture from Tube 10 each on its own selection plate (plates 21 and 22, respectively).
   17. Incubate the 22 plates at 30 °C for 3 - 5 days to select for plasmid transformants.
   18. Inspect transformation plates after 3 - 5 days of incubation. Approximately 5,000 colonies should be visible on plates 1-21 (see Representative Results for an example) and no colonies should be present on plate 22.

5. Screen for 5-FOA-resistant Transformants

1. Transfer cells from plates 1 - 20 (and transformation plate 21 as a control) to 5-fluoroorotic acid (5-FOA) plates¹¹ by replica-plating¹² in order to screen for loss of the URA3 gene as a result of integration of the PCR products at the desired location.
   1. Remove the plate lid and press the plate containing colonies on a sterile velvet. Transfer the cells from the velvet to a 5-FOA plate by pressing the plate on the velvet. Incubate plates at 30 ˚C for 2 days.
2. Following the 2-day incubation, carefully inspect the 5-FOA plates for growth.
   NOTE: A candidate integration event will be represented by a small asymmetric "squashed" colony on a 5-FOA plate - conversely, small papillae growing on 5-FOA plates are likely representative of spontaneous URA3 mutations that arose during the growth of colonies on the transformation plates, and are thus unlikely to represent the desired integration event (see Figure 3 in the Representative Results section for further elaboration on this point and for some examples).

6. Purification of 5-FOA-resistant Colonies and Loss of Backbone Plasmid

1. Using sterile toothpicks, pick the candidate colonies from the 5-FOA plates described in step 5.2 and streak for single colonies onto YPD plates. Incubate for 2 - 3 days at 30 °C.
2. Following the incubation, replica-plate each YPD purification plate to a fresh YPD plate, a drop-out plate lacking uracil to check for loss of the URA3 gene, and a second drop-out plate to monitor for the presence or absence of the backbone plasmid. Incubate for 1 - 2 days at 30 °C.
3. Following the incubation, identify a colony from each candidate sample that is growing on the YPD plate but not growing on either drop-out plate (such a colony is expected to have lost the URA3 gene through the recombination event and lost the backbone plasmid during mitotic cell division). Restreak such colonies on fresh YPD plates. These colonies are the integration candidates and will be analyzed further in step 7.

7. Molecular Analyses to Assay for Proper Integration of the Mutant Allele

1. Isolate genomic DNA from the candidate samples using standard procedures¹⁰.
2. Amplify genomic region encompassing the target site.
   1. Set up the following PCR reaction for each sample: 0.5 μl template DNA, 5 μl 10 μM forward primer, 5 μl 10 μM reverse primer, 0.5 μl (2.5 units) Taq DNA polymerase, 5 μl 10x Taq DNA polymerase buffer, 5 μl dNTP mixture (2 mM each), and 29 μl dH₂O.
      NOTE: Template DNA is the genomic DNA derived from the candidate samples. It is recommended to also include two control reactions: one using genomic DNA derived from the original histone geneΔ::URA3 strain as template and another using genomic DNA from a wild-type histone strain as template. To account for variations in DNA concentration and level of impurities in different genomic preparations, it is recommended to optimize the reactions by using either undiluted DNA or different dilutions of the genomic preparations (e.g., 1:10 and 1:100). It is important to make sure that these primers anneal to DNA sequences outside the region encompassed by the putatively integrated PCR product - this way, the size of the PCR products in these reactions can be used as a diagnostic tool for integration of the products at the correct genomic location (see Representative Results for an example).
   2. Place the reactions in a thermocycler with the following settings: 94 °C 3 min; 30 cycles of the following settings: 94 °C 45 sec, 50 °C 45 sec, 72 °C 2 min; and 72 °C 10 min.
      NOTE: Optimization of PCR parameters may be required for specific primer sets and target histone gene.
3. Processing of PCR products
   1. Run 20 μl from each reaction on a 0.8% TBE agarose gel.
   2. Assess the size of the PCR products using DNA standards as a reference to determine if the URA3 gene has been successfully replaced by the putatively mutated histone gene (see Representative Results for an example).
      NOTE: In certain cases, the desired mutation(s) introduced into the histone genes either create or destroy a restriction site. If this is the case, the presence of the desired mutation in the PCR products of the size indicative of correct integration can be assessed by subjecting the products to digestion with the corresponding restriction enzyme followed by gel electrophoresis analysis (see Representative Results for an example).
   3. Subject PCR products of the size indicative of correct integration to DNA sequencing to confirm the presence of the desired mutation(s) and to ensure that no additional mutations have been introduced into the genome.

Results

We describe the generation of an hht2 allele expressing a histone H3 mutant protein harboring a substitution at position 53 from an arginine to a glutamic acid (H3-R53E mutant) as a representative example of the targeted in situ mutagenesis strategy.

We generated a strain in which the entire ORF of HHT2 is replaced by the URA3 gene (see step 1 of the protocol). This strain, yAAD156, also harbo...

Discussion

The high level of sequence homology between the two non-allelic genes that code for each of the four core histone proteins in haploid S. cerevisiae cells can represent a challenge for investigators who wish to specifically target one of the two genes for mutagenesis. Previously described yeast mutagenesis methodologies, including the Delitto Perfetto, site-specific genomic (SSG) mutagenesis, and cloning-free PCR-based allele replacement methods^5,6

Materials

Name	Company	Catalog Number	Comments
1 kb DNA Ladder (DNA standards)	New England BioLabs	N3232L
Agarose	Sigma	A5093-100G
Boric Acid	Sigma	B0394-500G
dNTP mix (10 mM each)	ThermoFisher Scientific	R0192
EDTA solution (0.5 M, pH 8.0)	AmericanBio	AB00502-01000
Ethanol (200 Proof)	Fisher Scientific	16-100-824
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)	Sigma	E4884-500G
Lithium acetate dihydrate	Sigma	L6883-250G
MyCycler Thermal Cycler	BioRad	170-9703
Poly(ethylene glycol) (PEG)	Sigma	P3640-1KG
PrimeSTAR HS DNA Polymerase (high fidelity DNA polymerase)  and 5x buffer	Fisher Scientific	50-443-960
Salmon sperm DNA solution	ThermoFisher Scientific	15632-011
Sigma 7-9 (Tris base, powder form)	Sigma	T1378-1KG
Sodium acetate trihydrate	Sigma	236500-500G
Supra Sieve GPG Agarose (low metling temperature agarose)	AmericanBio	AB00985-00100
Taq Polymerase and 10x Buffer	New England BioLabs	M0273X
Toothpicks	Fisher Scientific	S67859
Tris-HCl (1 M, pH 8.0)	AmericanBio	AB14043-01000
a-D(+)-Glucose	Fisher Scientific	AC170080025	for yeast media
Agar	Fisher Scientific	DF0140-01-0	for yeast media
Peptone	Fisher Scientific	DF0118-07-2	for YPD medium
Yeast Extract	Fisher Scientific	DF0127-17-9	for YPD medium
4-aminobenzoic acid	Sigma	A9878-100G	for complete minimal dropout medium
Adenine	Sigma	A8626-100G	for complete minimal dropout medium
Glycine hydrochloride	Sigma	G2879-100G	for complete minimal dropout medium
L-Alanine	Sigma	A7627-100G	for complete minimal dropout medium
L-Arginine monohydrochloride	Sigma	A5131-100G	for complete minimal dropout medium
L-Asparagine monohydrate	Sigma	A8381-100G	for complete minimal dropout medium
L-Aspartic acid sodium salt monohydrate	Sigma	A6683-100G	for complete minimal dropout medium
L-Cysteine hydrochloride monohydrate	Sigma	C7880-100G	for complete minimal dropout medium
L-Glutamic acid hydrochloride	Sigma	G2128-100G	for complete minimal dropout medium
L-Glutamine	Sigma	G3126-100G	for complete minimal dropout medium
L-Histidine monohydrochloride monohydrate	Sigma	H8125-100G	for complete minimal dropout medium
L-Isoleucine	Sigma	I2752-100G	for complete minimal dropout medium
L-Leucine	Sigma	L8000-100G	for complete minimal dropout medium
L-Lysine monohydrochloride	Sigma	L5626-100G	for complete minimal dropout medium
L-Methionine	Sigma	M9625-100G	for complete minimal dropout medium
L-Phenylalanine	Sigma	P2126-100G	for complete minimal dropout medium
L-Proline	Sigma	P0380-100G	for complete minimal dropout medium
L-Serine	Sigma	S4500-100G	for complete minimal dropout medium
L-Threonine	Sigma	T8625-100G	for complete minimal dropout medium
L-Tryptophan	Sigma	T0254-100G	for complete minimal dropout medium
L-Tyrosine	Sigma	T3754-100G	for complete minimal dropout medium
L-Valine	Sigma	V0500-100G	for complete minimal dropout medium
myo-Inositol	Sigma	I5125-100G	for complete minimal dropout medium
Uracil	Sigma	U0750-100G	for complete minimal dropout medium
Ammonium Sulfate	Fisher Scientific	A702-500	for complete minimal dropout medium
Yeast Nitrogen Base	Fisher Scientific	DF0919-07-3	for complete minimal dropout medium
5-Fluoroorotic acid (5-FOA)	AmericanBio	AB04067-00005	for  5-FOA medium