Name: a suppressor screen for the characterization of genetic links regulating chronological lifespan in saccharomyces cerevisiae
Uploaded: 2020-09-17T13:00:00
Description: Watch this Scientific Journal Video about A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae at JoVE.com

James T. Arnone would like to acknowledge the support of the students in the Recombinant DNA Technologies course in 2017 and 2018 at William Paterson University who were involved in this project from its inception, but whose efforts did not cross the threshold for authorship: Christopher Andino, Juan Botero, Josephine Bozan, Brenda Calalpa, Brenda Cubas, Headtlove Essel Dadzie, Irvin Gamarra, Preciousgift Isibor, Wayne Ko, Nelson Mejia, Hector Mottola, Rabya Naz, Abdullah Odeh, Pearl Paguntalan, Daniel Raza&#8217;e, Gabriella Rector, Aida Shono, and Matthew So. You are great scientists and I miss you all!
The authors would like to acknowledge the invaluable support of Instruction and Research Technology at William Paterson University for their help: Greg Mattison, Peter Cannarozzi, Rob Meyer, Dante Portella, and Henry Heinitsh. The authors would also like to acknowledge the Office of the Provost for ART support, the Office of the Dean and the Center for Research in the College of Science and Health their support of this work, and the Department of Biology for supporting this project.

1. Identify potential genetic interactions for screening
<ol>
	<li>Identify the genetic background(s) for characterization, that results in an abnormally shorted chronological life span (CLS) in Saccharomyces cerevisiae using the Saccharomyces Genome Database (the SGD, https://www.yeastgenome.org29,30), which compiles known phenotypic information for this organism.
	<ol>
		<li>Select the Function tab from the options on the to...

Unravelling the genetics of aging is a difficult challenge, with many opportunities for further study that can potentially yield significant insights into the complex interactions that exist. There are many methods that allow for the rapid generation of loss-of-function mutants for the study of null strains of yeast45,46. This method presents a straightforward approach to identify and clone genes onto the pRS315 vector for overexpression suppressor studies. One a...

Aging is the time-dependent loss of integrity in myriad biological processes that ultimately increases the probability of organismal death. Aging is nearly inevitable for all species. On a cellular level there are several well characterized hallmarks that are associated with aging, including: genomic instability, epigenetic alterations, loss-of-proteostasis, mitochondrial dysfunction, deregulated nutrient sensing, cellular senescence, and telomere attrition1,2. In single celled organisms, such as yeasts, this leads to a reduction in replicative potential and chronological life span3,4. These cellular changes manifest in more complex organisms, like humans, as pathologies that include cancers, heart failure, neurodegeneration, diabetes, and osteoporosis5,6,7. Despite the many complexities that characterize the process of aging, there is conservation of these molecular hallmarks underlying this process across widely divergent organisms8,9,10. Identification of alterations to these pathways during aging led to the realization that they can be manipulated via lifestyle changes &#8211; dietary restriction is shown to substantially extend lifespan in many organisms11. These pathways converge and intersect with each other and many other pathways, in complex ways. Elucidation and characterization of these interactions offers potential for therapeutic interventions to prolong lifespan and healthspan12,13,14.
The conservation of the molecular underpinnings of aging allows for functional dissection of genetic interactions underlying the process through the use of simpler model organisms &#8211; including in the budding yeast, Saccharomyces cerevisiae15,16. There are two established types of aging modeled by budding yeast: chronological aging (the chronological lifespan, CLS) and replicative aging (the replicative lifespan, RLS)17. Chronological aging measures the amount of time that a cell can survive in a non-dividing state. This is analogous to the aging that is seen in cells that spend the majority of their life in G0, such as neurons4. Alternatively, replicative lifespan is the number of times that a cell can divide before exhaustion and is a model for mitotically active cell types (e.g., the number of daughter cells that a cell can have)18.
The overall goal of this method is to present a protocol that allows for the functional dissection of the genetics of aging using S. cerevisiae. While there have been many excellent studies performed by many researchers that have led to our current understanding, there remain many opportunities available for budding researchers to contribute to the aging field from early in their academic career. We present a clear methodology that will allow researchers to further advance the field of aging. This protocol is designed to be accessible for all researchers regardless of the stage in their academic career by providing the tools necessary to formulate and test their own novel hypotheses. The advantage&#160;of our approach is that this is a cost effective method readily accessible to all researchers regardless of institution &#8211; and does not require expensive, specialized equipment necessary for some protocols19. There are several different ways to design this type of screen, the approach outlined in this work is particularly amenable to screening null mutants of non-essential genes that exhibit a severe reduction in the chronological lifespan compared to an isogenic wild-type strain of yeast.
As our proof of principle, we clone SIR2, a lysine deacetylase reported as exhibiting both an extended and a shortened CLS when overexpressed. SIR2 overexpression was recently found to increase CLS in winemaking yeasts; however, several groups have reported no link between SIR2 and CLS extension, leaving its role under characterized20,21,22. Due to these conflicting reports in the literature, we selected this gene to add independent research to help clarify the role of SIR2 in chronological aging, if any. Additionally, increasing the copy number of a SIR2 homologue extends lifespan in a nematode worm model system23.
Autophagy is an intracellular degradation system to deliver cytosolic products, such as proteins and organelles, to the lysosome24. Autophagy is intimately linked to longevity through its role in degrading damaged proteins and organelles to maintain cellular homeostasis25. Induction of autophagy depends on orchestrating the expression of many genes, and the deletion of the ATG1 gene results in an abnormally short CLS in budding yeast26. ATG1 codes for a protein serine/threonine kinase that is required for vesicle formation in autophagy and the cytoplasm-to-vacuole (the fungal lysosomal equivalent) pathway27,28. Here, we present our method for an increased copy number screen, testing the effect of increased SIR2 copy on the CLS in a wild type and an atg1-null background. This method is particularly amenable to junior researchers and research groups at primarily undergraduate institutions, many of which serve communities underrepresented in the sciences and have limited resources.

<table>
	<tbody>
		<tr>
			<td>Fungal/Bacterial DNA kit</td>
			<td>Zymo Research</td>
			<td>D6005</td>
			<td></td>
		</tr>
		<tr>
			<td>HindIIIHF enzyme</td>
			<td>New England Biolabs</td>
			<td>R3104S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid miniprep kit</td>
			<td>Qiagen</td>
			<td>12123</td>
			<td></td>
		</tr>
		<tr>
			<td>SacII enzyme</td>
			<td>New England Biolabs</td>
			<td>R0157S</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon sperm DNA</td>
			<td>Thermofisher</td>
			<td>AM9680</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
	</tbody>
</table>

a suppressor screen for the characterization of genetic links regulating chronological lifespan in saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors contribute to alterations in the normal aging process. These factors intersect in complex ways, as evidenced by the wealth of documented links identified and conserved in many organisms. Most of these studies focus on loss-of-function, null mutants that allow for rapid screening of many genes simultaneously. There is much less work that focuses on characterizing the role that overexpression of a gene in this process. In the present work, we present a straightforward methodology to identify and clone genes in the budding yeast, Saccharomyces cerevisiae, for study in suppression of the short-lived chronological lifespan phenotype seen in many genetic backgrounds. This protocol is designed to be accessible to researchers from a wide variety of backgrounds and at various academic stages. The SIR2 gene, which codes for a histone deacetylase, was selected for cloning in the pRS315 vector, as there have been conflicting reports on its effect on the chronological lifespan. SIR2 also plays a role in autophagy, which results when disrupted via the deletion of several genes, including the transcription factor ATG1. As a proof of principle, we clone the SIR2 gene to perform a suppressor screen on the shortened lifespan phenotype characteristic of the autophagy deficient atg1&#916; mutant and compare it to an otherwise isogenic, wild type genetic background.

As there are conflicting reports on the role of SIR2 during aging, we chose this gene for study as a potential suppressor of the atg1&#916; mutant&#8217;s shorten CLS phenotype26. The role of SIR2 is somewhat controversial, with conflicting reports on its role in extending CLS, however it has been clearly linked to increased CLS in at least one yeast background, with a role in both autophagy and mitophagy22,31<su...

Watch this Scientific Journal Video about A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae at JoVE.com

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors ...

Aging is characterized by the time-dependent deterioration of multiple cellular processes that ultimately increases the probability of organismal death. Within a population, this process is variable and a number of genetic and environmental factors can affect it. One of the best studied and understood variables is the role of diet on lifespan.Dietary restriction is an intervention that ultimately delays the onset of aging and aging-associated changes. This increases an organism's lifespan and health span, or the length of time when an organism has a normal, healthy life. Our current understanding has led to the identification of a distinct set of cellular changes that have been termed the hallmarks of aging, such as the accumulation of mutations, dysregulation of telomeres, a loss of protein homeostasis, respiration problems, and reactive oxygen species, among others.Now much of this understanding comes from studies that use many different model organisms. There've been many genetic screens that have identified genes that both shorten and extend lifespan. And these include studies in the budding yeast Saccharomyces cerevisiae, which is the focus of this methodology, as well as studies in more complex multicellular organisms, like C.elegans and the murine system.Of these though, the budding yeast is particularly amenable to aging studies due to its short natural lifespan and a number of genetic approaches that can unravel the complex genetic relationships, making it an excellent system for functional dissection of the genetics of aging. Yeast can be utilized to study several different aspects of aging. And we will be focusing on chronological lifespan, which is the duration that a cell can survive.The objective of this work is to present a straightforward genetic approach to perform an over-expression suppressor screen. We will utilize a genetic background that results in an abnormally shortened chronological lifespan. And we'll utilize a targeted approach to identify genes that can rescue or suppress the shortened lifespan phenotype, attempting to identify genes that restore a normal lifespan.The first genetic background is an autophagy-deficient strain of yeast. Autophagy, which loosely translates as self-eating, is an intracellular degradation system to deliver cytosolic products, such as proteins and organelles, to the lysosome. This helps maintain cellular homeostasis by eliminating damaged proteins, as well as those that have outlived their life.We will be working with a mutant strain that has a deletion of the ATG1 gene. ATG1 codes for a protein serine/threonine-protein kinase that is required for vesicle formation and autophagy in the cytoplasm-to-vacuole targeting pathway. The ATG1 null mutant has a phenotype of having an abnormally short chronological lifespan.In this aging lab, we will design and test for genetic factors that can rescue, or suppress, the abnormally shortened lifespan characteristic of the ATG1 null mutant. However, this can be expanded to other short-lived genetic backgrounds as well. The first step in this process is to identify a gene that is linked to extending lifespan.We're going to focus specifically on the genes that extend the chronological lifespan when they are over-expressed. To do this, we are going to use the publicly-available data that is accessible from the yeast genome database. We're going to go up here.Search by phenotype. And about midway down, we will see we can select the chronological lifespan. To look for genes that increase the lifespan, what you can see is the wealth of data available that has genes linked to this particular phenotype.Today, we will be studying SIR2, a histone lysine deacetylase that is linked to extended chronological lifespan when it is over-expressed. The first thing we will do is access the DNA sequence for the coding region, as well as the regulatory regions that flank either side of this gene. We will take 400 base pairs upstream and downstream to be sure that we encompass all the regulatory regions.We will use this DNA sequence to design PCR primers that will allow us to clone this gene into our plasmid vector. We will use PCR to amplify our gene and to clone it into the pRS315 vector. In order to do this, we will design primers that contain restriction digestion sites that incorporate HindIII and SacII restriction sites.As you can see, the plasmid contains both restriction sites, HindIII and SacII, that will facilitate our cloning. You should design your PCR primers to contain about 21 or 22 nucleotides of sequence complimentary to the region that you want to amplify. In addition to the sequence, you will add onto the five prime end of them the appropriate restriction site, either HindIII on the forward primer, or SacII on the reverse primer, shown here in red and purple respectively, and further upstream of that, add several nucleotides that will allow the restriction enzyme to bind and create the restriction digestion site at a higher efficiency.Grow a five milliliter culture of the wild type yeast overnight to post-log phase in enriched media, such as YPAD. Harvest genomic DNA using a commercially available kit that is specific for fungal genomic isolation. It is important that the kit is specific for yeast, as they have a very rigid and tough cell wall to penetrate.Zymolyase treatment is effective at digesting the cell wall and greatly increases the genomic yield. Use a spectrophotometer to determine the quantity and the quality of the DNA. To produce an amplicon that is suitable for cloning, it is necessary to utilize a high fidelity PCR polymerase to avoid mutations during the amplification process.There are many different high fidelity PCR kits that are commercially available. To facilitate the optimization of the reaction, we recommend choosing a kit that provides multiple buffering systems, one that is a standard high fidelity buffer, and one optimized for high GC content in complex amplicons. We are going to add 200 nanograms of our genomic DNA.We're going to add five microliters of our buffer. We're going to add 2 1/2 microliters of our forward and our reverse primers. We're going to add one microliter of our polymerase, two microliters of D NTPs.And lastly, we're going to QS the whole reaction to 50 microliters with sterile distilled water. We'll run the reaction according to the protocol specific for the enzyme we're using, as well as for the primers that we have designed in this experiment. Cleanup and concentrate the PCR reaction.Grow a five milliliter culture of E.coli overnight in selective media. Pellet the culture by centrifugation and discard the supernatant. Resuspend and lyse the cells in the provided chaotropic buffer for five minutes.Neutralize the reaction and mix the two by inversion five or six times. Centrifuge your sample for 10 minutes at maximum speed. Transfer the supernatant and to a silica column and wash with the provided buffer.Discard the flow through and repeat the wash with the other appropriate buffers, with 30 second centrifugations in between, discarding your flow through after each. At the end, centrifuge for two minutes more to remove any residual ethanol and elute your plasmid into 20 microliters elution buffer and determine the quantity and quality by spectrophotometer. Now it is time to set up a restriction digestion of the vector and the insert.Add 625 nanograms of DNA, either the vector or the insert, five microliters of CutSmart buffer, one microliter of SacII, one microliter of HindIII, and QS the reaction with water to bring the final reaction volume to 50 microliters. Incubate the restriction digests at 37 centigrade for three hours, followed by 80 centigrade for 20 minutes to heat and activate the enzymes. At this point, digests can be stored at four degrees prior to proceeding to the next step.Next, we're going to set up a 15 microliter ligation using our digested genomic fragment, as well as the vector that we just completed. We're going to start by adding six microliters of water. We are going to add two microliters of our digested vector.This is our pRS315 plasmid. We're going to add four microliters of our insert, the SIR2 gene. We're going to add two microliters of our ligase buffer, case the 10X buffer.And lastly, we're going to add one microliter of the ligase. We're going to incubate this at 16 degrees overnight, and then heat kill the enzyme at 80 degrees centigrade for 20 minutes. To screen for our successfully created plasmid, we're going to transform it into E.coli.We're going to use 50 microliters of chemically competent cells. We're going to thaw them on ice, and as soon as they thaw, we are going to add our ligation reaction, we're going to add the entire contents of it. So we're going to add 15 microliters of the ligation with the insert, and we're going to add 15 microliters of our no-insert control as well to a separate reaction.Once it's been added, flick the tube a few times in order to mix it. And we're going to incubate this on ice. After completing the 30 minute incubation on ice, we're going to add our samples into a 42 degree heating block where we will incubate and heat shock for 20 seconds, which point we will take them out, and we will add recovery media.After the heat shock, we're going to allow for a recovery period. We are going to take our samples and we will add 450 microliters of SOC media. We will incubate these at 37 degrees centigrade to allow the plasmids to be picked up and to allow the ampicillin resistance gene to begin to be expressed.We will allow these samples to recover at 37 degrees for 45 minutes. After that incubation, we set up a series of dilutions, one to 10, and one to 100, and we're going to plate these on LB/Amp media and allow the cells to grow up overnight. We will plate them using sterile technique.We'll take the appropriate plate, which we have labeled accordingly and contains our selectable marker. We will take 150 microliters of our transformed E.coli. Using a sterile inoculating loop, we will then gently spread the cells throughout and across the entirety of the plate so we get a nice even distribution.Once we have finished plating all our dilutions, we will incubate these plates overnight at 37 degrees centigrade to see what grows. After about a day, we should see colonies growing up that hopefully contain the plasmid we were attempting to create. They should look something like this.Using sterile technique, inoculate potential transformants that grew from your transformation into five milliliters LB plus ampicillin following the previously outlined procedure. Isolate the plasmids from every potential transformant and run a restriction digestion as previously described. Run the reaction products on an agarose gel, comparing the potential transformants to the empty vector.Save any transformants that result in excision of a properly-sized insert for further study. Having successfully created our plasmid, we are going to transform it into the ATG1 null yeast background. We're going to take 15 mils of cells and we're going to pellet them, which I have already done right here.After they've pelleted, remove the YPAD media that they were growing in and wash the cells with distilled water. After that, we're going to take the cells and we're going to resuspend them in 100 millimolar lithium acetate. We're going to resuspend them in 200 microliters of that.Resuspend the cell pellet by gentle pipetting up and down. Split the cells into separate microfuge tubes for each of the transformations that you will perform, using 50 microliters of this mix per transformation. To each of the transformations, you will need to add 240 microliters of 50%PEG.PEG is very viscous, pipette and measure very carefully. Mix by pipetting after the addition of the PEG and after every one of the additional components. Next add 36 microliters of one molar lithium acetate, five microliters of salmon sperm DNA, or other carrier DNA, that has been boiled for five minutes and placed on ice, and finally five microliters of the plasmid for each transformation you will be performing.Vortex each tube thoroughly to mix. Incubate your samples at 30 degrees centigrade for 45 minutes. Heat shock them at 42 centigrade for 10 minutes, and then wash the cells once in sterile water, finally resuspending them in 200 microliters of sterile water.Set up one to 10 and one to 100 dilutions of each of the transformed strains of yeast and plate 150 microliters onto SC minus leu plates to select for the plasmids. Spread the cells uniformly and evenly, and allow the plates to dry before inverting and incubating them at 30 degrees centigrade, allowing them to grow for 48 to 72 hours. Once we have successfully cloned our gene to test, we'll test the effect on the chronological lifespan of the organism.We will grow the yeast, monitoring the number of viable colony forming units that remain as a function of time. Grow the yeast first for 72 hours in SC minus leucine liquid media with shaking at 30 degrees centigrade. Using a hemocytometer, determine the dilution necessary to allow you to plate 500 cells.Using sterile technique, plate the cells onto an SC minus leu plate, allowing it to dry and incubate inverted for three days at 30 degrees centigrade, at which point they will grow up and you can score the colony growth and record that time point. After making the plate, continue to incubate your yeast at 30 degrees centigrade without shaking. Initially, we repeat the plating at weekly intervals, taking more frequent time points if our preliminary data suggests a suppression of the aging phenotype.In order to determine the percent viability, we normalize to the day one time point for analysis. Be sure to plate the same dilution at every interval. Continue with this process until you no longer see any colonies growing up.It's helpful to enter your information into an Excel spreadsheet. This will allow you to quickly determine the viability of each strain at the conclusion of the experiment.

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