Name: studying age-dependent genomic instability using the s. cerevisiae chronological lifespan model
Uploaded: 2011-09-29T12:00:00
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Description: Watch this Scientific Journal Video about Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model at JoVE.com

We thank S. Jinks Robertson and E. Heidenreich for providing plasmids and yeast strains; P. Pham and M.F. Goodman for help withthe TLS assay. This work was supported, in part, by the American Federation for Aging Research grant and by NIH AG20642, AG025135.

Two lifespan models have being used to study aging in S. cerevisiae: RLS and CLS. The replicative (budding) life span (RLS) is based on the observation that yeast mother cells undergo a finite number of divisions3-6. We will focus on the chronological lifespan (CLS), a model based on the chronological survival of non-dividing yeast in culture or on plates7-11.Wild type yeast grows exponentially for 10-12 hours in synthetic dextrose complete (SDC) medium. When glucose level reduces, yeast switches from fermentation to respiration, a process termed diauxic shift. Cells continue to divide slowly during the post-diauxic phase for approximately 48 hours before entering G0 arrest. The metabolic rate of the cells remains high till day 5-6 (day 0 is the inoculation day). Cell viability over time can be accessed by the percentage of the chronologically aging cells, sampled every two days, which can exit G0 arrest and form colonies on rich YPED plates.
1. Yeast chronological lifespan (CLS) in liquid culture
<ol>
	<li>Inoculate a single colony into 1 ml synthetic complete glucose medium (SDC, Table 1) and incubate overnight with shaking (220 rpm) at 30&deg;C. Three inoculums from independent colonies should be prepared for each strain to provide biological replicas.</li>
	<li>Dilute the overnight culture into fresh SDC medium (usually 10 ml) to OD=0.05-0.1 (&tilde;1:100 dilution) and incubate with shaking (220 rpm) at 30&deg;C. This time point is considered day 0 of the chronological aging. Maintain a 1:5 ratio of culture/flask volume (10 ml culture in 50 ml, or for longer/large experiments 25 ml culture in 125 ml) to ensure proper aeration.</li>
	<li>Starting from day 3, remove 2 aliquots of 10 &mu;l from the flask, dilute 10,000 times in autoclaved water, 10 &mu;l diluted culture (i.e., 104-10) on YEPD (1% yeast extract, 2% bacto peptone, 2% dextrose, 2% agar) plates, and incubate at 30&deg;C for 48-72 hours before colony forming unite (CFU) is counted.</li>
	<li>Day 3 CFU is considered 100% survival. Dilution factors of aging culture in the subsequent days should be adjusted accordingly to ensure &tilde;20-100 colonies in the CFU assay (e.g., 104-10, 104-30, 103-10, etc.). For wild type cells in the DBY746 background, the CLS reaches 1% survival around day 11.</li>
</ol>
Variations of in situ viability assay include:
<ul>
	<li>Calorie restriction (CR) by glucose limitation. Dilution of overnight culture in glucose limited SC medium (such as 0.5 or 0.05% instead of the standard 2% glucose) generally lead to lower population saturation density on day 3, but much extended mean and maximal (10% survival) lifespan12.</li>
	<li>For extreme CR/starvation, wash the day 3 stationary phase culture (usually 10 ml, as in step 3 above) with autoclaved water (resuspend cells in equal volume of water and spin down at 2500 rpm for 5 min, repeat 3 times). Incubationof cells in water mimics the starvation condition that yeast may encounter in the wild. Every two days subsequently, aging culture should be washed with autoclaved water and resuspended in equal volume of water to remove any nutrients released from lysed dead cells. Perform viability assay by sampling the aging culture as described above. This starvation condition will led to almost a doubling of mean CLS in the wild type DBY746 strain12.</li>
</ul>
2. in situ viability assay
In the liquid culture, a small fraction of the surviving cells may re-entered the cell cycle and grow utilizing remaining nutrients or those released form lysed dead cells in the medium, a phenotype termed regrowth/gasping13. We developed a viability-on-a-plate system that utilizes the auxotrophy of the DBY746 strain (trp1)to circumvent the regrowth/gasping problem and also allow the testing of the effect of constant exposure or deprivation of various external nutrients or stimuli on yeast CLS11. This in situ viability assay also mimics the replicative lifespan model such that cells are constantly exposed to abundant nutrients for the duration of the lifespan analysis.
<ol>
	<li>Inoculate a single colony into 1 ml synthetic complete glucose medium (SDC) and incubate overnight with shaking (220 rpm) at 30&deg;C. At least three inoculums from independent colonies should be prepared for each strain to provide biological replicas.</li>
	<li>Let the culture to grow to &tilde;108 cells/ml (OD600=&tilde;10), dilute the culturewith autoclaved water to 100-200 cell/10 &mu;l (usually 104-fold dilution) and 1,000 cells/10 &mu;l (103-fold dilution).</li>
	<li>Plate two aliquots of 10-30 &mu;l of diluted culture onto one tryptophan dropout SDC plate. For each strain, prepare a set of 8 to 20 plates and labeled according to plating density(e.g., 104-fold diluted, plate 10 &mu;l or 104-10, 104-30, 103-10, 103-30, etc.), which ensure 10-100 colonies can be counted in anticipation of decreased viability during aging.</li>
	<li>Incubate the plate set at 30&deg;C for the duration of the lifespan analysis.</li>
	<li>On the day of plating and every 2 days subsequently, remove one plate and drop-wise add 0.5 ml Tryptophan (2 mg/ml) to allow viable cells to grow. Incubate the plate at 30&deg;C for additional 48-72 hours. Count the CFU for viability on the day of tryptophan addition. The CFU on the day of plating is considered 100% survival.</li>
</ol>
Variations of in situ viability assay include:
<ul>
	<li>Age cells on agar plates (extreme starvation condition). Add 1 ml 2x YPED instead of tryptophan on subsequent days to allow growth and CFU count14.</li>
	<li>Age cells on plates with tryptophan dropout synthetic complete medium with various carbon sources, e.g. various concentrations of glucose (calorie restriction by glucose limitation), various carbon sources such as ethanol, glycerol, acetic acid14. Add 1 ml glucose/tryptophan solution (20% glucose and 1mg/ml tryptophan) to allow growth and CFU count.</li>
	<li>Other nutrient manipulation, such as nitrogen.</li>
</ul>
3. DNA damage and mutation frequency during chronological aging
Canavanine resistance (Canr) and can1 sequencing 
Spontaneous mutation frequency can be evaluated by measuring the frequency of canavanine resistance (Canr) in chronologically aging cultures. Mutations in the CAN1 (YEL063) gene, which encodes the plasma arginine permease, render cells resistant to the arginine analogue L-canavanine.Canr colonies collected at different time points can also be saved for extraction of genomic DNA and subsequent sequencing of the CAN1 gene, which can provide mutation spectrum data (with Mutation Surveyor, SoftGenetics).
Base substitutions (Trp+ reversions)
Strains with trp1-289 contain an amber mutation (C403T) in the TRP1 coding sequence. Measurement of frequency of trp1-289 to Trp+ reversion15 allows the estimation of base substitution rate during yeast chronological aging.
Frame-shift mutations
The Lys- strain EH150 (MATa, lys2&Delta;BglII, trp1-&Delta;, his3-&Delta;200, ura3-52, ade2-1o) harbors a lys2&Delta;BglII mutation that was constructed by inserting 4 nucleotides to create a BglII restriction enzyme site in the LYS2 gene. The resulting +4 shift in the open reading frame results in auxotrophy for lysine that can be reversed by small insertion/deletion mutations16-17.
Gross chromosomal rearrangements (GCRs)
To detect gross chromosomal rearrangements (GCRs), we generated a mutant strain, in which HXT13 (YEL069), encoding a highly redundant hexose transporter, was disrupted by a URA3 cassette18. HXT13 is located 7.5 kb telomeric to CAN1 on chromosome V. Mutations in both CAN1 and URA3 genes render cells resistance to L-canavanine and 5-fluoroorotic acid (5FOA), respectively. Considering the low frequency of point mutations that occur in both genes, analysis of the Canr 5FOAr frequency provides an estimation of GCRs that result in the loss of both genes.
Homologous and homeologous recombination
To monitor the level of homologous (100%) and homeologous recombination (91%) during chronological aging, we generated mutants in which linearized plasmids (HIS3::intron-IR-URA3) carrying either 100% homologous inverted repeats (IRs) (pSR406) or 91% homeologous IRs (pSR407) at the HIS3 locus19. Recombination between the IRs allows the expression of functional His3 protein.
<ol>
	<li>In parallel to normal viability assay (as described above), remove an appropriate amount of cells from the aging culture,
	<ol>
		<li>for Canr mutation, start with &tilde;2x107 cells (200 &mu;l of day 3 culture);</li>
		<li>for Trp+ reversion, start with &tilde;108 cells (500-1000 &mu;l of day 3 culture);</li>
		<li>for Lys+ frame-shift mutation, start with &tilde;108 cells (500-1000 &mu;l of day 3 culture);</li>
		<li>for GCRs, start with 2-3x108 cells (2-3 ml of day 3 culture);</li>
		<li>for homologous and homeologous recombination, start with &tilde;5x107 cells (200-500 &mu;l of day 3 culture);</li>
	</ol>
	adjust the plating amount according to the decrease in total viability and the increase in mutation frequency during chronological aging (Table 2). In case of strains with hypermutator phenotype (such as those with deficiencies in DNA repair), reduce the sampling amount accordingly.</li>
	<li>Pellet the cells with bench-top centrifuge machine (6000 rpm for 5 min).</li>
	<li>Resuspend cells in 1 ml autoclaved water, and pellet the cells again.</li>
	<li>Resuspend cell pellet in 100 &mu;l of water. Plate cells,
	<ol>
		<li>for Canr mutation, on arginine-dropout synthetic complete medium plates (SDC-Arg, supplemented with 60 &mu;g/ml L-canavanine sulfate);</li>
		<li>for Trp+ reversion, on tryptophan-dropout plates (SDC-Trp);</li>
		<li>for Lys+ frame-shift mutation, on lysine-dropout plates (SDC-Lys);</li>
		<li>for GCRs, on arginine-dropout plates (SDC-Arg)supplemented with 5FOA (1 mg/ml) and L-canavanine sulfate (60 &mu;g/ml);</li>
		<li>for homologous and homeologous recombination, on histidine-dropout plates supplemented with galactose (2%).</li>
	</ol>
	</li>
	<li>Count CFUs after 3-4 days' incubation at 30&deg;C. The mutation frequency is normalized to the number of viable cells.</li>
</ol>
Complementary to the in situ viability assay, age-dependent Trp+ reversion, Lys+ frame-shift mutation, recombination, or Canr can also be studied in cells aged on plate.
<ol>
	<li>Plate cells (&tilde;108 cells/plate) on to Trp-, Lys-, His-dropout, or L-canavanine synthetic complete medium plates (as described above).</li>
	<li>Every two days (or at the time point of interest), score the newly emerging colonies.</li>
	<li>Mutation frequency is estimated by normalizing newly emerging colonies to the total number of viable cells of the specific day.</li>
</ol>
4. Translesion synthesis (TLS)
The age-dependent mutation frequency increase may involve increased macromolecule damage, diminished cellular protection/repair, and increased erroneous DNA repair (such as Pol&zeta;-dependent translesion synthesis, TLS) during aging. For example, the long-lived sch9&Delta; mutants exhibit elevated expression of SOD2, and decreased expression of the error-prone DNA repair enzyme Rev120. Here we describe an assay combining damaged DNA template and whole nuclear extract to evaluate the TLS in vitro.
<ol>
	<li>Prepare nuclear extract as described by Wang et al.21; quantify the protein concentration by BCA assay (Pierce).</li>
	<li>Add 0, 5, 10 or 20 &mu;g of nuclear extracts to 50 &mu;l (final volume) reactions containing TLS Buffer (20 mM HEPES, 7 mM MgCl2, 1 mM DTT, 25 mM NaCl, pH 7.8), 200 &mu;MdNTPs and 100 nM 5'-32P-labelled 12-mer primer (5'-CGA TGG TAC GGA-3') annealed to a 48-mer template containing DNA damage of interest (5'-TCG ATA CTG GTA CTA ATG ATT AAC GAC TXA AGC ACG TCC GTA CCA TCG-3', in which X is the damaged site, e.g., abasic site, 8-oxo-G, etc.).</li>
	<li>Incubate the mixture at 30&deg;C for 30 min, terminate the reaction with the addition of 2.5 &mu;l EDTA (20 mM) and 2 &mu;l of proteinase K (10 mg/mL).</li>
	<li>Purify DNA with phenol extraction and ethanol precipitation.</li>
	<li>Resuspend DNA in 50 &mu;l of gel loading buffer (98% formamide, 20 mM EDTA, and dye).</li>
	<li>Resolve the translesion synthesis products on 19% polyacrylamide gels.</li>
	<li>Quantify the TLS using phosphorimaging (Figure 1).</li>
</ol>
5. Representative Results
We typically used the CFU counts on day 3 as 100 percent survival in the standard liquid CLS analysis. The percentage survival in the subsequent days can be fitted to calculate the mean (50% survival) and maximum (10% survival) life spans12. Life span results obtained from the in situ viability assay are generally consistent with those using the liquid CLS assay, but with reduced the mean life span, which is due in part to the fact that cells are constantly exposed to nutrients. Presence of glucose inhibits the activity of cellular protection as showed by the reduced transactivation of stress response transcription factor such as Msn2/4 and Gis112.
Age-dependent mutation frequency varies greatly depending on the strain background, genetic manipulation, culture conditions, and age. Table 2 shows the typical results obtained in the wild-type strain (DBY746).
<table border="1">
	<tbody>
		<tr>
			<th>
				Component</th>
			<th>
				g/L</th>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Nitrogen base (-AS/-AA)</td>
			<td>1.8</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>1.4</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>mg/L</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>80</td>
		</tr>
		<tr>
			<td>L-Arginine</td>
			<td>40</td>
		</tr>
		<tr>
			<td>L-Aspartic acid</td>
			<td>100</td>
		</tr>
		<tr>
			<td>L-Glutamic acid</td>
			<td>100</td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>80</td>
		</tr>
		<tr>
			<td>L-Isoleucine</td>
			<td>60</td>
		</tr>
		<tr>
			<td>L-Leucine</td>
			<td>120</td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>60</td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>80</td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>60</td>
		</tr>
		<tr>
			<td>L-Serine</td>
			<td>400</td>
		</tr>
		<tr>
			<td>L-Threonine</td>
			<td>200</td>
		</tr>
		<tr>
			<td>L-Tryptophan</td>
			<td>80</td>
		</tr>
		<tr>
			<td>L-Tyrosine</td>
			<td>40</td>
		</tr>
		<tr>
			<td>L-Valine</td>
			<td>150</td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>80</td>
		</tr>
	</tbody>
</table>
Table 1. Synthetic complete glucose medium, SDC (adjust to pH 6.0 with NaOH). 4-fold excess of histidine, leucine, tryptophan, and uracil are included to compensate the auxotrophy of the DBY746 strain.
<table border="1">
	<tbody>
		<tr>
			<th>
				 </th>
			<th>
				Day 3 (mean &plusmn; SEM)</th>
			<th>
				Up to (during aging)</th>
		</tr>
		<tr>
			<td>Canr</td>
			<td>1.76 &plusmn; 0.12 x10-6</td>
			<td>6-8 x10-6</td>
		</tr>
		<tr>
			<td>Trp+ reversion</td>
			<td>6.60 &plusmn; 1.70 x10-8</td>
			<td>1.60 x10-6</td>
		</tr>
		<tr>
			<td>Lys+ frame-shift mutation</td>
			<td>3.00 &plusmn; 0.78 x10-8</td>
			<td>0.70 x10-6</td>
		</tr>
		<tr>
			<td>GCRs</td>
			<td>0.62 &plusmn; 0.10 x10-8</td>
			<td>0.30 x10-6</td>
		</tr>
		<tr>
			<td>Homologous recombination</td>
			<td>5.55 &plusmn; 2.74 x10-6</td>
			<td>27.00 x10-6</td>
		</tr>
		<tr>
			<td>Homeologous recombination</td>
			<td>0.12 &plusmn; 0.04 x10-6</td>
			<td>0.48 x10-6</td>
		</tr>
	</tbody>
</table>
Table 2.Typical mutation frequencies of wild type cells (DBY746) during chronological aging.
<img alt="Figure 1" src="/files/ftp_upload/3030/3030fig1.jpg" /> 
Figure 1. Result of translesion synthesis (TLS)20. Nuclear extracts from 3 day-old stationary phase wild-type (DBY746) and sch9&Delta; mutant cells were incubated with undamaged or abasic site-containing DNA templates for 30 min at 30&deg;C. TLS products are indicated by solid (with undamaged template) or dotted (with damaged template) lines. There was no translesion synthesis observed in nuclear extract from sch9&Delta; mutants. Free primers are indicated by the open arrow.

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F., JinksRobertson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+crossovers+between+diverged+sequences+are+regulated+by+mismatch+repair+proteins+in+Saccaromyces+cerevisiae.">Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae.</a> Mol Cell Biol. 16, 1085-1093 (1996).</li><li>Madia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogene+homologue+Sch9+promotes+age-dependent+mutations+by+a+superoxide+and+Rev1/Polzeta-dependent+mechanism.">Oncogene homologue Sch9 promotes age-dependent mutations by a superoxide and Rev1/Polzeta-dependent mechanism.</a> J Cell Biol. 186, 509-523 (2009).</li><li>Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+expression+of+recombinant+genes+and+preparation+of+cell-free+extracts+in+yeast.">Controlled expression of recombinant genes and preparation of cell-free extracts in yeast.</a> Methods Mol Biol. 313, 317-331 (2006).</li><li>Fabrizio, P., Pletcher, S. D., Minois, N., Vaupel, J. W., Longo, V. 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No conflicts of interest declared.

Liquid aging cultures sometime exhibit regrowth/gasping phenotype13, which complicates the CLS analysis. Regrowth typically occurs when more than 90-99% of the population has lost viability. This phenotype is often associated with increased oxidative stress and/or decreased protection in the cell. For example, the frequency of this phenotype more than doubles in cells lacking cytosolic superoxide dismutase and greatly reduces in long-lived mutants (e.g., sch9&Delta; or ras2&Delta;) or mutants overexpressing superoxide dismutases22. In practice, we define regrowth as an increase in viability or stabilization in viability for 3 consecutive samplings in the high mortality phase during chronological aging.
Age-dependent frequencies of different DNA mutations vary greatly depending on the strain background, genetic manipulation, culture conditions, as well as regrowth/gasping and extremely low survival. Pre-experiments should be carried out at time points such as the mean and maximum survival with various plating density for mutation assays to determine mutation frequency range before performingthe full scale life span analysis. The wild type strain should always be included in a life span or mutation frequency study in parallel to any treatment or genetic mutant, such that inter-experiment-variations can be accounted for. Multiple biological replicas should be included in the study; and, both liquid and in situ viability/mutation assays should be carried out to corroborate the results.
Instead of focusing on one specific type of DNA mutation, profiling of age-dependent genomic instability using multiple assays in combination, may shed light on specific DNA damage and DNA damage repair system(s) that contribute to age-dependant genomic instability. For example, a significant increase in gross chromosomal rearrangements (GCRs), compared to those of other DNA mutations, is observed in wild type yeast suggesting an elevationof double strand break and/or an impairment in non-homologous end joining (NHEJ) during yeast chronological aging (Table 2). The can1 mutation spectrum (sequencing of the CAN1 gene) obtained in the wild-type aging yeast suggested an increase in oxidative damage during chronological aging and error-prone DNA repair; whereas, in long-lived sch9&Delta; mutants, less oxidative DNA damage and much reduced error-prone translesion synthesis were observed20.
Methods described here can be further expended to study age-dependant genomic instability. For example, quiescent and non-quiescent cells can be isolated from yeast stationary-phase cultures using the density gradient method described by Allen et al.23. Combined with the mutation assays described here, we have reported that a large portion of age-dependent mutations arises from quiescent cells, rather than the dividing, damaged or apoptotic cells20,24.

studying age-dependent genomic instability using the s. cerevisiae chronological lifespan model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Watch this Scientific Journal Video about Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model at JoVE.com

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

studying-age-dependent-genomic-instability-using-s-cerevisiae

Research

JoVE Journal

Biology

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...