Name: targeted in situ mutagenesis of histone genes in budding yeast
Uploaded: 2017-01-26T15:00:00
Description: Watch this Scientific Journal Video about Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast at JoVE.com

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.

NOTA: La strategia sperimentale per mirata in situ mutagenesi gene istone comprende diversi passaggi (riassunti in Figura 1). Questi passaggi includono: (1) Sostituzione del gene bersaglio istone con il gene URA3, (2) Generazione e purificazione dei prodotti di PCR corrispondenti a due frammenti parzialmente sovrapposti del gene istone target usando primers della mutazione desiderata (s), (3 ) fusione PCR dei due frammenti parzialmente sovrapposti per ottenere pieni prodotti dimensione...

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L&#39;elevata omologia di sequenza tra due geni non alleliche che codificano ciascuno dei quattro proteine essenziali istoniche delle cellule S. cerevisiae aploidi possa rappresentare sfida per investigatori vogliano indirizzare specificamente uno dei due geni per mutagenesi. In precedenza descritto lievito metodologie di mutagenesi, compreso il Delitto Perfetto, genomica mutagenesi sito-specifica (SSG), e PCR-based metodi pezzi allele-free clonazione 5, <sup class="x...

I quattro proteine ​​di base dell&#39;istone H2A, H2B, H3, H4 e giocano un ruolo centrale nella compattazione, l&#39;organizzazione e la funzione dei cromosomi eucariotiche. Due insiemi di ciascuna di tali istoni formano la ottamero istoni, un rocchetto molecolare che dirige l&#39;avvolgimento ~ 147 paia di basi del DNA su se stessa, che si traduce nella formazione di un nucleosoma 1. I nucleosomi sono partecipanti attivi in ​​una varietà di processi cromosomiche-based, come ad esempio la regolazione della trascrizione genica e la formazione di eucromatina e heterochromatin tra cromosomi, e come tali sono stati al centro di un&#39;intensa ricerca nel corso degli ultimi decenni. Un certo numero di meccanismi sono stati descritti da cui nucleosomi può essere manipolato in modi che possono facilitare l&#39;esecuzione di processi specifici - Questi meccanismi comprendono modificazione post-traslazionale di residui degli istoni, rimodellamento nucleosomi ATP-dipendente, e la riorganizzazione nucleosomi ATP-indipendentee montaggio / smontaggio 2, 3. Il lievito Saccharomyces cerevisiae erba è particolarmente potente organismo modello per la comprensione della funzione istone negli eucarioti. Questo può essere in gran parte attribuito all&#39;elevato grado di conservazione evolutiva delle proteine istone tutto il eucarioti dominio e la riconducibilità di lievito ad una varietà di sperimentale genetico e biochimico approcci 4. approcci Reverse-genetici nel lievito sono stati ampiamente utilizzati per studiare gli effetti di specifiche mutazioni istoni su vari aspetti della biologia della cromatina. Per questi tipi di esperimenti è spesso preferibile utilizzare cellule in cui gli istoni mutanti sono espressi dal loro loci genomici nativa, come espressione da plasmidi autonome può portare a anormali livelli intracellulari di proteine ​​istoni (a causa di un numero variabile di plasmidi in cellule) e alterazione concomitante di cromatina environments, che alla fine possono confondere l&#39;interpretazione dei risultati. Qui, si descrive una tecnica PCR-based che consente di mutagenesi mirata di geni istone alle loro posizioni nativi genomiche che non richiede una fase di clonazione e risultati nella generazione della mutazione desiderata (s) senza rimanenti sequenze di DNA esogene nel genoma. Questa tecnica sfrutta l&#39;efficiente sistema di ricombinazione omologa nel lievito e ha diverse caratteristiche in comune con altre tecniche simili sviluppati da altri gruppi - in particolare il Delitto Perfetto, genomica mutagenesi sito-specifica (SSG), e la clonazione senza PCR allele-based metodi sostitutivi 5, 6, 7. Tuttavia, la tecnica descriviamo ha un aspetto che lo rende particolarmente adatto per la mutagenesi dei geni istoni. In cellule di lievito aploidi, ciascuno dei quattro istoni nucleo è codificata da due non-ageni llelic e altamente omologhi: per esempio, l&#39;istone H3 vengono codificati dai geni HHT1 e HHT2, e le fasi di lettura aperte (ORF) dei due geni sono oltre il 90% identica in sequenza. Questo elevato grado di omologia può complicare esperimenti progettati per indirizzare specificamente uno dei due geni istone-codifica per la mutagenesi. Considerando che i suddetti metodi richiedono spesso l&#39;uso di almeno alcune sequenze all&#39;interno della ORF del gene bersaglio di guidare ricombinazione omologa, la tecnica descriviamo qui fa uso di sequenze fiancheggianti le ORF dei geni istoni (che condividono molto meno omologia di sequenza) per la fase di ricombinazione, aumentando così la probabilità di successo di targeting mutagenesi al locus desiderata. Inoltre, le regioni omologhe che guidano la ricombinazione possono essere molto ampia, contribuendo ulteriormente alla efficiente ricombinazione omologa mirato.

<table border="0" cellpadding="0" cellspacing="0" width="1091">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">1 kb DNA Ladder (DNA standards)</td>
			<td style="width:177px;">New England BioLabs</td>
			<td style="width:128px;">N3232L</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose&#160;</td>
			<td>Sigma</td>
			<td>A5093-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric Acid</td>
			<td>Sigma</td>
			<td>B0394-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dNTP mix (10 mM each)</td>
			<td>ThermoFisher Scientific</td>
			<td>R0192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA solution (0.5M, pH 8.0)</td>
			<td>AmericanBio</td>
			<td>AB00502-01000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol (200 Proof)</td>
			<td>Fisher Scientific</td>
			<td>16-100-824</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td>Sigma</td>
			<td>E4884-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lithium acetate dihydrate</td>
			<td>Sigma</td>
			<td>L6883-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MyCycler Thermal Cycler</td>
			<td>BioRad</td>
			<td>170-9703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(ethylene glycol) (PEG)</td>
			<td>Sigma</td>
			<td>P3640-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PrimeSTAR HS DNA Polymerase (high fidelity DNA polymerase)&#160; and 5X buffer</td>
			<td>Fisher Scientific</td>
			<td>50-443-960</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Salmon sperm DNA solution</td>
			<td>ThermoFisher Scientific</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma 7-9 (Tris base, powder form)</td>
			<td>Sigma</td>
			<td>T1378-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium acetate trihydrate</td>
			<td>Sigma</td>
			<td>236500-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Supra Sieve GPG Agarose (low metling temperature agarose)</td>
			<td>AmericanBio</td>
			<td>AB00985-00100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taq Polymerase and 10X Buffer</td>
			<td>New England BioLabs</td>
			<td>M0273X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Toothpicks</td>
			<td>Fisher Scientific</td>
			<td>S67859</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl (1M, pH 8.0)</td>
			<td>AmericanBio</td>
			<td>AB14043-01000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">a-D(+)-Glucose</td>
			<td>Fisher Scientific</td>
			<td>AC170080025</td>
			<td>for yeast media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Fisher Scientific</td>
			<td>DF0140-01-0</td>
			<td>for yeast media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>Fisher Scientific</td>
			<td>DF0118-07-2</td>
			<td>for YPD medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Fisher Scientific</td>
			<td>DF0127-17-9</td>
			<td>for YPD medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-aminobenzoic acid</td>
			<td>Sigma</td>
			<td>A9878-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenine</td>
			<td>Sigma</td>
			<td>A8626-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine hydrochloride</td>
			<td>Sigma</td>
			<td>G2879-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Alanine</td>
			<td>Sigma</td>
			<td>A7627-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Arginine monohydrochloride</td>
			<td>Sigma</td>
			<td>A5131-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Asparagine monohydrate</td>
			<td>Sigma</td>
			<td>A8381-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Aspartic acid sodium salt monohydrate</td>
			<td>Sigma</td>
			<td>A6683-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Cysteine hydrochloride monohydrate</td>
			<td>Sigma</td>
			<td>C7880-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamic acid hydrochloride</td>
			<td>Sigma</td>
			<td>G2128-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine</td>
			<td>Sigma</td>
			<td>G3126-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Histidine monohydrochloride monohydrate</td>
			<td>Sigma</td>
			<td>H8125-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">L-Isoleucine</td>
			<td>Sigma</td>
			<td>I2752-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Leucine</td>
			<td>Sigma</td>
			<td>L8000-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Lysine monohydrochloride</td>
			<td>Sigma</td>
			<td>L5626-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Methionine</td>
			<td>Sigma</td>
			<td>M9625-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Phenylalanine</td>
			<td>Sigma</td>
			<td>P2126-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Proline</td>
			<td>Sigma</td>
			<td>P0380-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Serine</td>
			<td>Sigma</td>
			<td>S4500-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">L-Threonine</td>
			<td>Sigma</td>
			<td>T8625-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tryptophan</td>
			<td>Sigma</td>
			<td>T0254-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tyrosine</td>
			<td>Sigma</td>
			<td>T3754-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Valine</td>
			<td>Sigma</td>
			<td>V0500-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">myo-Inositol</td>
			<td>Sigma</td>
			<td>I5125-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uracil</td>
			<td>Sigma</td>
			<td>U0750-100G</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Sulfate</td>
			<td>Fisher Scientific</td>
			<td>A702-500</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Yeast Nitrogen Base</td>
			<td>Fisher Scientific</td>
			<td>DF0919-07-3</td>
			<td>for complete minimal dropout medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Fluoroorotic acid (5-FOA)</td>
			<td>AmericanBio</td>
			<td>AB04067-00005</td>
			<td>for&#160; 5-FOA medium</td>
		</tr>
	</tbody>
</table>

targeted in situ mutagenesis of histone genes in budding yeast

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.

Descriviamo la generazione di un allele hht2 esprimere un istone H3 mutante proteina ospitare una sostituzione nella posizione 53 da un arginina ad acido glutammico (H3-R53E mutante) come un esempio rappresentativo della mirata strategia situ mutagenesi. Abbiamo generato un ceppo in cui l&#39;intera ORF di HHT2 è sostituito dal gene URA3 (vedi punto 1 del protocollo). Questo ceppo,...

Watch this Scientific Journal Video about Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast at JoVE.com

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

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

Mirata<em&gt; In Situ</em&gt; mutagenesi dell&#39;istone geni in germogliamento lievito

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

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Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

DNA Structure and Function

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