目标<em>原位</em>组蛋白突变的基因在芽殖酵母
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
四个核心组蛋白H2A，H2B，H3和H4中的压实，组织，和真核染色体的功能发挥中心作用。两套各这些组蛋白的形成的组蛋白八聚体，即指示本身周围的DNA的〜147碱基对的环绕一个分子卷轴，最终导致核小体1的形成。核小体是在各种基于染色体过程，如基因转录的调节和常染色质的形成和异跨染色体积极参与者，因此已经深入研究在过去几十年的过程中的焦点。已经描述了许多机制由核小体可以在方法，可以方便特定进程的执行被操纵 – 这些机制包括组蛋白残基的翻译后修饰，依赖ATP的核小体重构，和ATP依赖性核重组和装配/拆卸2,3。
在芽殖酵母是组蛋白功能的真核生物的理解一个特别强大的模型生物。这在很大程度上归因于整个域真核生物高度组蛋白的进化保守的和酵母的各种遗传和生化实验的方法顺从4。在酵母反向遗传方法已被广泛用于研究染色质生物学的各个方面的具体组蛋白突变的影响。对于这些类型的实验，常常优选使用，其中所述突变体组蛋白从其天然基因组位点表达的细胞，如从自主质粒表达可导致组蛋白的异常细胞内水平（由于细胞不同质粒的数量），并染色质恩伴随变更vironments，它可以混淆最终结果的解释。
在这里，我们描述了一种基于PCR的技术，其允许在不需要在所需突变的生成而不在基因组中剩余的外源DNA序列的克隆步骤和结果其天然基因组的位置的组蛋白的基因的靶向诱变。这种技术利用了有效的同源重组系统的在酵母和具有几个共同的特征与其它组开发的其他类似的技术-尤其是在Delitto普菲 ，位点特异性基因组（SSG）诱变和克隆-自由基于PCR的等位基因替换方法5，6，7。然而，我们描述的技术存在，使得它特别适合于组蛋白基因的突变的一个方面。在单倍体酵母细胞中，每四个核心组蛋白是由两个非一个编码llelic和高度同源的基因：例如，组蛋白H3由HHT1和HHT2基因编码，而两个基因的开放阅读框（ORFs）在序列90％以上相同。这种高度的同源性可以变得复杂设计成特异性靶向诱变两个组蛋白编码基因中的一个实验。而上述的方法通常要求使用的靶基因的ORF内的至少一些序列来驱动同源重组，我们在这里描述的技术利用侧翼组蛋白基因的开放阅读框（共用少得多的序列同源性）序列的的重组步骤，从而增加至所需的轨迹诱变成功定位的可能性。此外，驱动重组同源区可以是非常广泛的，从而进一步提高效率的有针对性的同源重组。
Protocol
Representative Results
Discussion
序列同源性的高水平的两个非等位基因，对于每个在单倍体酿酒酵母细胞中的四个核心组蛋白的代码可以表示谁希望特异性靶向两个基因诱变之一调查一个挑战之间。先前描述的酵母诱变方法，包括Delitto普菲 ，位点特异性基因组（SSG）诱变和自由克隆基于PCR的等位基因替换方法5，6，7，以及更近的酵母基于CRISPR?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Reine Protacio for helpful comments during the preparation of this manuscript. We express our gratitude to the National Science Foundation (grants nos. 1243680 and 1613754) and the Hendrix College Odyssey Program for funding support.
Materials
| 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 (0.5M, 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 (1M, 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 |
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