Målrettet<em> I Situ</em> Mutagenese af histon-gener i knopskydende gær
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
De fire centrale histonproteiner H2A, H2B, H3 og H4 spille centrale roller i komprimeringen, organisation og funktion af eukaryote kromosomer. To sæt af hver af disse histoner danne histon octamer, en molekylær spole, der dirigerer indpakning af ~ 147 basepar af DNA rundt om sig selv, i sidste ende resulterer i dannelsen af en nukleosom 1. Nukleosomer er aktive deltagere i en bred vifte af kromosom-baserede processer, såsom regulering af gen transskription og dannelsen af euchromatin og heterochromatin tværs kromosomer, og som sådan har været genstand for en intens forskning i løbet af de sidste mange årtier. En række mekanismer er blevet beskrevet, hvorved nukleosomer kan manipuleres på måder, der kan lette udførelsen af særlige processer – disse mekanismer indbefatter posttranslationelle modifikation af histon-rester, ATP-afhængig nukleosom remodeling, og ATP-uafhængige nukleosom reorganiseringog montering / demontering 2, 3.
Den spirende gær Saccharomyces cerevisiae er en særlig kraftig model organisme for forståelsen af histon funktion i eukaryoter. Dette kan i vid udstrækning tilskrives den høje grad af evolutionære konservering af histonproteinerne hele domænet Eukarya og modtagelighed af gær til en række genetiske og biokemiske eksperimentelle tilgange 4. Reverse-genetiske metoder i gær er ofte blevet brugt til at undersøge effekten af specifikke histon mutationer på forskellige aspekter af kromatin biologi. For disse typer af eksperimenter er det ofte at foretrække at anvende celler, hvori de mutante histoner udtrykkes fra deres native genomiske loci, som ekspression fra autonome plasmider kan føre til unormale intracellulære niveauer af histon-proteiner (på grund af varierende antal plasmider i celler) og samtidig ændring af kromatin environments, hvilket i sidste ende kan forvirre fortolkning af resultaterne.
Her beskriver vi en PCR-baseret teknik, som muliggør målrettet mutagenese af histon-gener ved deres native genomiske placeringer, der ikke kræver et kloningstrin og resulterer i dannelsen af den ønskede mutation (er) uden efterladenskaber exogene DNA-sekvenser i genomet. Denne teknik drager fordel af effektive homolog rekombination-system i gær og har flere træk til fælles med andre lignende teknikker udviklet af andre grupper – især den Delitto Perfetto, stedsspecifik genomisk (SSG) mutagenese og kloning-fri PCR-baseret allel udskiftning metoder 5, 6, 7. Men den teknik beskriver vi har et aspekt, der gør det særligt velegnet til mutagenese af histon-gener. I haploide gærceller, er hver af de fire kernehistoner indkodet af to ikke-allelic og meget homologe gener: for eksempel histon H3 kodet af HHT1 og HHT2 gener, og de åbne læserammer (ORF'er) af de to gener er over 90% identisk i sekvens. Denne høje grad af homologi kan komplicere eksperimenter designet til specifikt at målrette en af de to histon-kodende gener for mutagenese. Hvorimod de førnævnte fremgangsmåder kræver ofte anvendelse af mindst nogle sekvenser i ORF af målgenet til at køre homolog rekombination, teknikken beskriver vi her gør brug af sekvenser, der flankerer ORF'erne af histon-gener (som deler meget mindre sekvenshomologi) for rekombinationen trin, hvilket øger sandsynligheden for vellykket målretning af mutagenese til den ønskede locus. Desuden kan de homologe områder, der driver rekombination være meget omfattende, hvilket yderligere bidrager til effektiv målrettet homolog rekombination.
Protocol
Representative Results
Discussion
Det høje niveau af sekvenshomologi mellem de to ikke-allele gener, der koder for hver af de fire centrale histonproteiner i haploide S. cerevisiae-celler kan repræsentere en udfordring for forskere, der ønsker at specifikt mod et af de to gener for mutagenese. Tidligere beskrevne gær mutagenese-metoder, herunder Delitto Perfetto, stedspecifik genomisk (SSG) mutagenese, og kloningsvektorer-free PCR-baserede allel udskiftning metoder 5, 6,</su…
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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