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....

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 transfo....

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

List of materials used in this article

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