Name: studying ribonucleotide incorporation: strand-specific detection of ribonucleotides in the yeast genome and measuring ribonucleotide-induced mutagenesis
Uploaded: 2018-07-26T15:00:00
Description: Watch this Scientific Journal Video about Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis at Jo

We thank all current and former Kunkel Lab members for their work and discussions related to protocol and reused data presented here. This work was supported by Project Z01 ES065070 to T.A.K. from the Division of Intramural Research of the National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS).

1. Alkaline Hydrolysis and Strand-specific Southern Blot (Figure 1)
<ol>
	<li>Cell growth and genomic DNA isolation
	<ol>
		<li>Isolate yeast genomic DNA using a yeast DNA purification kit following the manufacturer&#8217;s instructions. Use the cultures that are in the exponential phase of growth (between an optical density of 0.5 and 1). Resuspend the final DNA pellet in 35 mL of 1x TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA)).</li>
		<li>Add 1 mL of 5 ...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topoisomerase+1-mediated+removal+of+ribonucleotides+from+nascent+leading-strand+DNA.">Topoisomerase 1-mediated removal of ribonucleotides from nascent leading-strand DNA.</a> Molecular Cell. 49 (5), 1010-1015 (2013).</li><li>Clausen, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+replication+enzymology+in+vivo+by+genome-wide+mapping+of+ribonucleotide+incorporation.">Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation.</a> Nature Structural &amp; Molecular Biology. 22 (3), 185-191 (2015).</li><li>Foster, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+determining+spontaneous+mutation+rates.">Methods for determining spontaneous mutation rates.</a> Methods Enzymology. 409, 195-213 (2006).</li><li>Williams, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+processing+of+ribonucleotides+in+DNA+by+topoisomerase+1+is+leading-strand+specific.">Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific.</a> Nature Structural &amp; Molecular Biology. 22 (4), 291-297 (2015).</li><li>Pavlov, Y. I., Shcherbakova, P. V., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+consequences+of+putative+active+site+mutations+in+yeast+DNA+polymerases+alpha,+epsilon,+delta,+and+zeta.">In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta.</a> Genetics. 159 (1), 47-64 (2001).</li><li>Nick McElhinny, S. A., Kissling, G. E., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+correction+of+lagging-strand+replication+errors+made+by+DNA+polymerases+{alpha}+and+{delta}.">Differential correction of lagging-strand replication errors made by DNA polymerases {alpha} and {delta}.</a> Proceedings of the National Acaddemy of Sciences U S A. 107 (49), 21070-21075 (2010).</li><li>Clark, A. B., Lujan, S. A., Kissling, G. E., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mismatch+repair-independent+tandem+repeat+sequence+instability+resulting+from+ribonucleotide+incorporation+by+DNA+polymerase+epsilon.">Mismatch repair-independent tandem repeat sequence instability resulting from ribonucleotide incorporation by DNA polymerase epsilon.</a> DNA Repair (Amst). 10 (5), 476-482 (2011).</li><li>Lujan, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mismatch+repair+balances+leading+and+lagging+strand+DNA+replication+fidelity.">Mismatch repair balances leading and lagging strand DNA replication fidelity.</a> PLoS Genetics. 8 (10), e1003016 (2012).</li><li>Reijns, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+removal+of+ribonucleotides+from+DNA+is+essential+for+mammalian+genome+integrity+and+development.">Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development.</a> Cell. 149 (5), 1008-1022 (2012).</li><li>Lujan, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+polymerase+fidelity+and+mismatch+repair+bias+genome+variation+and+composition.">Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition.</a> Genome Research. 24 (11), 1751-1764 (2014).</li></ol>

Here, we describe the protocols for two sets of experiments that are frequently used to semi-quantitatively analyze ribonucleotides incorporated during DNA replication and the mutagenic effects of unrepaired ribonucleotides. Although these approaches involve the model eukaryote S. cerevisiae, these techniques can be easily adapted to other microbes and even higher eukaryotes.
Probing for unrepaired ribonucleotides in DNA using alkaline-agarose electrophoresis coupled with Southern blo...

During eukaryotic nuclear DNA replication, ribonucleotides are incorporated into the genome by all three major DNA replicases, DNA polymerases (Pols) &#945;, &#949;, and &#948;1,2. RNase H2-dependent ribonucleotide excision repair (RER3) removes the majority of these embedded ribonucleotides.
A ribonucleotide in DNA is susceptible to hydrolysis, as the 2&#39; hydroxyl group of the sugar moiety can attack the adjacent phosphodiester bond, releasing one end with a 2&#39;-3&#39; cyclic phosphate and the other with a 5&#39;-OH4. Alkaline conditions can greatly accelerate this reaction. Thus, the hydrolysis of embedded ribonucleotides during incubation in a basic solution causes fragmentation of genomic DNA, which can be visualized by alkaline-agarose electrophoresis5. This DNA can be transferred to a membrane and probed by Southern blot analysis using strand-specific probes that allow the identification of alkali-sensitive sites caused by ribonucleotides incorporated into the nascent leading- or lagging-strand DNA, respectively.
In yeast cells lacking RNase H2 activity, removal of ribonucleotides can be initiated when topoisomerase I (Top1) nicks the DNA at the embedded ribonucleotide6,7. However, when Top1 cleaves on the 3&#39; side of the ribonucleotide, this generates 5&#39;-OH and 2&#39;-3&#39; cyclic phosphate DNA ends that are refractory to religation. Failure to repair, or aberrant processing of these &#39;dirty ends&#39; can lead to genomic instability. In addition, if the incision occurs within a repeat DNA sequence, the repair process can lead to deletion mutations. This is particularly problematic for tandem repeats, where short deletions (of between two and five base pairs) are commonly observed in RNase H2-deficient cells. The Top1-dependent deleterious effects in the absence of yeast RNase H2 activity are exacerbated in a DNA polymerase&#160;&#949; mutant (pol2-M644G) promiscuous for ribonucleotide incorporation during nascent leading strand synthesis.
Processing of ribonucleotides in DNA leads to spontaneous mutations and this mutagenesis can be detected by using appropriate reporter genes and selecting for the accompanying phenotypic change. A fluctuation test or Luria and Delbr&#252;ck experiment is one of the most commonly used methods to measure spontaneous mutation rates using selectable reporter genes8,9. In yeast, the URA3 and CAN1 genes can be used as reporters in a forward mutation assay, which allows for the detection of all mutation types that result in the loss of gene function. The spontaneous mutation rate is estimated as the median of that observed for multiple parallel cultures started from single colonies without mutations in the target reporter gene. A yeast RNase H2-deficient strain such as rnh201&#916; has a moderately elevated overall spontaneous mutation rate that is largely caused by an elevated incidence of 2 - 5 bp deletions in tandem repeat sequences. Thus, to fully characterize the mutagenic effects of ribonucleotides in the genome, specific mutation rates need to be determined. In this case, the URA3 or CAN1 reporter genes can be amplified and sequenced to determine the types and locations of the mutations, and specific mutation rates can be calculated. Compiling mutations identified in multiple independent URA3 or CAN1 mutants can then be used to generate a mutation spectrum.

<table>
	<tbody>
		<tr>
			<td>YPD media</td>
			<td></td>
			<td></td>
			<td>20 g dextrose, 20 g peptone, 10g yeast extract, in deionized H2O up to 1 L, add 20 g Bacto agar for solid media, autoclave.</td>
		</tr>
		<tr>
			<td>COM plates</td>
			<td></td>
			<td></td>
			<td>1.3 g SC dropout mix, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar deionized H2O up to 1 L. Adjust pH to 5.8. Autoclave and 30 &#8211; 35 ml per plate.</td>
		</tr>
		<tr>
			<td>CAN plates</td>
			<td></td>
			<td></td>
			<td>1.3 g SC-Ura dropout powder, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar, 25 mg uracil and H2O up to 1 L. Autoclave for 15 mins at 121 &#176;C and cool down to 56 &#176;C. Add 6 mL of filter-sterilized 1% canavanine sulfate solution.</td>
		</tr>
		<tr>
			<td>5-FOA plates</td>
			<td></td>
			<td></td>
			<td>1.3 g SC-Ura dropout powder, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar, 25 mg uracil and H2O up to 800 mL. Autoclave for 15 mins at 121 &#176;C and cool down to 60 &#176;C. Add 200 mL of filter-sterilized 0.5% 5-FOA solution.</td>
		</tr>
		<tr>
			<td>L-Canavanine sulfate</td>
			<td>US Biological</td>
			<td>C1050</td>
			<td></td>
		</tr>
		<tr>
			<td>5-FOA</td>
			<td>US Biological</td>
			<td>F5050</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass culture tube</td>
			<td></td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr>
			<td>Culture rotator in 30 &#176;C incubator</td>
			<td></td>
			<td></td>
			<td>Shaker incubator can be used instead</td>
		</tr>
		<tr>
			<td>96 well round bottom plates</td>
			<td></td>
			<td></td>
			<td>Sterile, any brand</td>
		</tr>
		<tr>
			<td>3 mm glass beads</td>
			<td>Fisher Scientific</td>
			<td>11-312A</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>12-channel pipettes</td>
			<td></td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr>
			<td>Ex Taq DNA Polymerase</td>
			<td>TaKaRa</td>
			<td>RR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Epicentre MasterPure Yeast DNA Purification Kit</td>
			<td>Epicenter</td>
			<td>MPY80200</td>
			<td></td>
		</tr>
		<tr>
			<td>3 M sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S7899</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 Fluorometer</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td>Newer model available</td>
		</tr>
		<tr>
			<td>dsDNA BR Assay kit</td>
			<td>Invitrogen</td>
			<td>Q32850</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma-Aldrich</td>
			<td>221473</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Dot Scientific Inc.</td>
			<td>DSF10400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromocresol green</td>
			<td>Eastman</td>
			<td>6356</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene cyanol FF</td>
			<td>International Technologies Inc.</td>
			<td>72120</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl (pH 8.0)</td>
			<td>Teknova</td>
			<td>T5080</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Gold Nucleic Acid Gel Stain</td>
			<td>Invitrogen</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>UV transilluminator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Nylon membrane Hybond-N+</td>
			<td>GE Healthcare</td>
			<td>RPN303B</td>
			<td></td>
		</tr>
		<tr>
			<td>3 MM CHR Chromotography paper</td>
			<td>Whatman</td>
			<td>3030-392</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Caledon</td>
			<td>7560-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Stratalinker 1800</td>
			<td>Stratagene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>G-25 spin column</td>
			<td>GE Healthcare</td>
			<td>27-5325-01</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Sodium phosphate buffer (pH 7.2)</td>
			<td>Sigma-Aldrich</td>
			<td>NaH2PO4 (S9638); 
			Na2HPO4 (S9390)</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>L4522</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma-Aldrich</td>
			<td>47671</td>
			<td></td>
		</tr>
		<tr>
			<td>Geiger counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

studying ribonucleotide incorporation: strand-specific detection of ribonucleotides in the yeast genome and measuring ribonucleotide-induced mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Treatment of genomic DNA with alkali followed by alkaline gel electrophoresis allows for semi-quantitative detection of the DNA fragmentation due to the abundance of stably incorporated ribonucleotides. Figure 2 shows the gel images of yeast genomic DNA treated with or without KOH5. The M644L variant of Pol2, the catalytic subunit of Pol&#949;, has reduced the ability to incorporate ribonucleotides while the M644G mutant incorporates m...

Watch this Scientific Journal Video about Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis at Jo

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

This method can help answer questions such as how many ribonucleotides are incorporated into nascent, leading, and lagging strand DNA in the genome. Though this method can provide quantitative and qualitative information regarding ribonucleaotide density in the yeast genome it can also be applied to other systems such as mammalian genomes. The alkaline gel electrophoresis provides a semi quantitative view of ribonucleotide incorporation in the whole genome and strand specific southern blotting allows for greater sensitivity and quantitation at a specific genomic locus.To begin this procedure, add six microliters of one molar potassium hydroxide to each sample of freshly prepared yeast genomic DNA and incubate at 55 degrees celsius for two hours. After two hours, incubate the samples on ice for five minutes. Then add to each sample four microliters of 6-6 alkaline DNA loading buffer.Cast an alkaline gel comprised of one percent agarose, 50 millimolar sodium hydroxide and one millimolar EDTA pH eight with a comb that allows for 24 microliters of DNA sample to be loaded per lane. Spin down the tubes of samples briefly. Load the entire 24 microliters of each sample into the alkaline agarose gel.Include an appropriate DNA size marker. Electrophorese at 30 volts for 30 minutes. Then lower the voltage to 10 volts and electrophorese for 18 to 20 hours.On the following day, neutralize the gel. Immerse the gel in neutralization buffer one and incubate at room temperature with gentle agitation for 45 minutes. Replace the neutralization buffer blend with fresh buffer.And incubate for another 45 minutes. To stain the DNA, soak the neutralized gel in 200 milliliters of water containing a one to 10000 dilution of sybr gold stain at room temperature with gentle shaking for two hours. Visualize the DNA using a UV transilluminator.The DNA from the alkaline gel will be transferred to a positively charged nylon membrane by capillary action. First soak the alkaline gel at room temperature for 15 minutes in alkaline transfer buffer. Next, wet the nylon membrane briefly with water and soak it in alkaline transfer buffer for five minutes.Set up the transfer platform by inverting three 100 milliliter glass beakers in a glass baking dish that is slightly larger than the size of the agarose gel. Set a glass plate on top of the glass beakers. Drape two large pieces of three MMCHR chromatography paper over the glass plate.Pour alkaline transfer buffer over the chromatography paper and half fill the glass dish. Smooth out the bubbles using a five milliliter glass pipette. Place the agarose gel on top.Again, smoothing out the bubbles. Surround all edges of the gel with parafilm. Pour a small amount of alkaline transfer buffer onto the gel.Place the presoaked nylon membrane on top of the gel and smooth out the bubbles. Put two pieces of chromatography paper cut to the size of the agarose gel. Place them on top of the membrane and smooth out any bubbles.Place a large stack of paper towels on top of the transfer sandwich and carefully place a glass plate on top of the paper towels. Add a weighted object to the top and let the transfer proceed at room temperature overnight. On the following day, carefully take the transfer stack apart.Soak the membrane at room temperature in neutralization buffer two for 15 minutes. Cross link the DNA to the charged nylon membrane using a UV cross linker. Use the auto cross link setting.Prior to starting the hybridization procedure, prepare the radiolabeled probe as described in the text protocol. Roll the wetted membrane into a hybridization tube and add 25 milliliters of freshly prepared hybridization buffer. Incubate a 65 degrees celsius with rotation in a hybridization oven for one to two hours.Carefully pour off the solution and add 25 milliliters of hybridization buffer plus the probe. Incubate at 65 degrees celsius for 16 to 18 hours with rotation. Next, pour off the solution into a radioactive waste container.Add phosphate STS washing solution one to the hybridization tube and rotate at room temperature for five minutes. In this manner, perform five washes of five minutes each with phosphate STS washing solution one. After the fifth wash with phosphate STS washing solution one wash the membrane twice with phosphate STS washing solution two for 15 minutes each time at 65 degrees celsius with rotation.Use tweezers to remove the membrane from the hybridization tube and place the membrane in an opened plastic sleeve protector. Cover and expose to an imaging plate. If necessary, strip and reprobe the blot with a different probe.To strip, cover the membrane with 25 milliliters of a 50 percent formamide 2XSSPE solution and incubate at 65 degrees celsius for two hours. Pour off the solution into a radioactive waste container. Add phosphate STS washing solution two to the hybridization tube, and rotate at 65 degrees celsius for 15 minutes.Check the membrane with a geiger counter to confirm the absence of signal before performing the prehybridization and hybridization as demonstrated earlier. These gel images have yeast genomic DNA treated with or without potassium hydroxide show that the cells lacking RNH2 retain more ribonucleotides in the genome. The M644L variant of polymerase two reduces the ability to incorporate ribonucleotides while the M644G mutant incorporates more ribonucleotides than the wild type polymerase.An alkaline agarose gel can be further probed by strand specific southern blot analysis. In this example, the probes anneal to the nascent leading DNA strand within the URA3 reporter gene that has been inserted close to ARS306 in two opposite orientations. Representative results of southern blotting using the nascent leading strand specific probes show a DNA fragmentation pattern caused by alkali cleavage at ribonucleotides and nascent leading strands DNA in a wild type strain and in strains expressing the variants of polymerases alpha, delta, and epsilon that are promiscuous for ribonucleotide incorporation.This graph shows the quantification of the radioactive signal by fraction of the total in each lane from the southern blot. While attempting the procedure, it is important to remember to always use freshly isolated DNA from lock face cells. After its development, this technique paved the way for researchers in the field of DNA repair to quantify the density and the strand into which ribonucleotides have been incorporated by the replicative DNA polymerases.Following this procedure, other methods like hydrolated end sequencing or HydEn-seq, can be performed in order to answer additional questions like the location of the ribonucleotides in the genome. Don't forget that working with radioactive materials can be extremely hazardous and precautions should always be taken to avoid contamination of the workbench and equipment while performing this procedure.

studying-ribonucleotide-incorporation-strand-specific-detection

Research

JoVE Journal

Biology

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Measuring Crop Motility and Food Passaging in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

Measuring Crop Motility and Food Passaging in Drosophila

The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...