New & Noteworthy
November 26, 2013
Transcriptional regulation data are now available on new “Regulation” tab pages for virtually every yeast gene. We are collaborating with the YEASTRACT database to display regulation annotations curated both by SGD and by YEASTRACT on these new pages. Regulation annotations are each derived from a published reference, and include a transcriptional regulator, a target gene, the experimental method used to determine the regulatory relationship, and additional data such as the strain background or experimental conditions. The relationships between regulators and the target gene are also depicted in an interactive Network Visualization diagram. The Regulation tab for DNA-binding transcription factors (TFs) includes these items and additionally contains a Regulation Summary paragraph summarizing the regulatory role of that TF, a table listing its protein domains and motifs, DNA binding site information, a table of its regulatory target genes, and an enrichment of the GO Process terms to which its target genes are annotated (view an example). In the coming months we will be adding this extra information to the Regulation pages of other classes of TFs, such as those that act by binding other TFs.
We have also completely redesigned the web display of the Interactions and Literature tab pages, which now include graphical display of data, sortable tables, interactive visualizations, and more navigation options. These pages provide seamless access to other tools at SGD such as GO tools and YeastMine. Please feel free to explore all of these new features from your favorite Locus Summary page and send us your feedback.
November 25, 2013
Stanford offers an innovative class, targeted at sophomore undergraduates, where students use yeast to determine how a mutation in the p53 gene affects the activity of the resulting p53 protein. What makes this class even cooler is that the p53 mutants come from actual human tumors—the undergraduates are figuring out what actual cancer mutations are doing! And the class uses what we think is the most important organism in the world, S. cerevisiae.
To learn more about the course, we decided to interview Jamie Imam, one of the instructors. After reading the interview, you will almost certainly be as excited about this class as we were and it may even get you to wishing that you could teach the class at your institution. With a little help, you can.
The creators of the course, Tim Stearns and Martha Cyert, really want as many people as possible to use this class to teach undergraduates about what real science is and how fun and exciting it can be. To that end, they are happy to help you replicate the course wherever you are. If you are interested, please contact Tim and/or Martha. You’ll be happy you did. Their contact information can be found at the Stearns lab and Cyert lab websites.
Here now is the interview with Jamie. What a great way to get undergraduates excited about the scientific process.
Can you describe the class?
Sure. Bio44X is designed to be similar to an authentic research experience or as close to one as you can replicate in the classroom. During the quarter, students study mutant versions of a gene called p53, a tumor suppressor that is frequently mutated in cancer. Each partner pair in a classroom gets one p53 mutant that has been identified in a human tumor to study in our yeast system. Throughout the course of the 10 weeks, the students study the transactivation ability of their mutant compared to the wild-type version, and then work to figure out what exactly is wrong with the mutant (Can it bind DNA?, Does it localize to the nucleus properly?, etc.). Multiple sections of this course are taught during the Fall and Winter quarters, so several pairs end up studying the same mutant. We bring these students together to discuss and combine their data throughout the quarter, so there is a lot of collaboration involved. I think the students really enjoy having one topic to study in depth over the quarter rather than short individual modules, and the fact that we are studying a gene so important in cancer makes it easier to get them to care about the work they are doing.
Tell me a little bit about how this class was started.
Previously, Bio44X at Stanford was the more traditional “cookbook” type lab course. Every 2 weeks, the topic would change and students would work through set protocols that had a known correct answer. In 2010, Professors Martha Cyert and Tim Stearns set out to design and pilot a research-based course on a medically relevant topic (the tumor suppressor p53) in response to some national calls for biology lab course reform. Two years and many changes later, the new research-based lab course replaced the previous version and is now taken by all of the students that need an introductory lab course in Biology.
What kinds of experiments do the students get to do in the class?
Students get exposed to a variety of lab techniques that can be used beyond our classroom. We start with sterile technique and pipetting during the very first week (some students have never pipetted before!). During the first class, the students also spot out some yeast strains so they can start collecting data on the transactivation ability of their p53 mutant right away. Once they have some basic information about the function of their mutant, the students then extract protein from their yeast strains. Throughout the rest of the quarter, students use this protein to conduct a kinetic assay, Western blot, and assess DNA binding ability of their mutant p53. They also get some exposure to fluorescence microscopy when they use a GFP-tagged version of their mutant to determine whether it can localize properly to the nucleus. But the most important thing of all is that students learn how to analyze the data and think critically about it. Not only do they “crunch the numbers” but they must use that information to draw some actual conclusions about what is wrong with their mutant by the end of the quarter.
How hard is it to set up and run the class?
It takes a lot of organization because we have around 200 or more students that take this class every year! Fortunately, we have a great team to help organize the setup of the labs so that the instructors can focus on the teaching. Nicole Bradon manages a small staff that sets up the classrooms and prepares all of the reagents for the lab each week. Dr. Daria Hekmat-Scafe, who is one of the instructors, constructs many of the yeast strains that we give to the students. The team of lecturers (Dr. Shyamala Malladi, Dr. Daria Hekmat-Scafe and I) all work together on lectures and other course materials so everyone gets a similar experience. All together, it takes a lot of behind-the-scenes work, but then the students really get to focus on the experiments and their results.
Do you enjoy teaching the class? What is your favorite part? Your least favorite part?
I love teaching this class! It is so fun to go through this research experience with so many students and they all bring their unique perspectives to the course (we get engineers, psych majors, bio majors, econ majors and others). Also, each section has only 20 students so you really get the chance to get to know them over the course of the 10 weeks. Sometimes the experiments don’t work as planned (like real science) but overall it ends up being a great learning experience.
What do you hope the students will learn and get out of the class? And are they learning/getting it?
We hope that students learn to think critically and what it really means to “think like a scientist”. Too often, science is boiled down to a series of facts that students are expected to memorize and that isn’t what science really is! Science is all about finding exciting questions and constructing experiments that try and answer those questions. The beauty of a research-based lab course is that students can also feel more in charge of their own learning. We have performed assessments of the class and have found that over the course of the quarter, students develop a more sophisticated understanding of what it means to “think like a scientist” and a large portion are more interested in becoming involved in scientific research. I think this is great, as I feel that undergraduate research helped me understand science so much more deeply than many of the courses I had taken.
How would someone at another University go about replicating this course? Are there resources available to help them get started and/or keep it running?
Our group is willing to share our course materials and knowledge with others that are interested in replicating this at other institutions. Anyone who is interested should feel free to contact us! Also, there is a paper in preparation that will describe some of the key aspects of the course as well as more details about what we have learned from the assessments of the course over the past few years.
There you have it…a great class that uses the awesomeness of yeast to teach undergraduates how to think like scientists. Again, if you’re interested in learning more, please contact Tim Stearns and/or Martha Cyert at Stanford.
November 18, 2013
Imagine you have the instructions for building a car but you don’t know what any of the specific parts do. In other words, you can build a working car but you don’t understand how it works.
One way to figure out how the car works would be to remove a part and see what happens. You would then know what role that part played in getting a car to run.
So if you remove the steering wheel, you’d see that the thing runs into a wall. That part must be for steering. When you take out the radiator, the car overheats so that part must be for cooling the engine. And so on.
Sounds like a silly way to figure out how the car works, but this is essentially one of the key ways we try to figure out how a cell works. Instead of parts, we knock out genes and see what happens. A new study by Teng and coworkers is making us rethink this approach.
See, one of the big differences between a machine and a cell is that the cell can react and adapt to the loss of one of its parts. And in fact, it not only can but it almost certainly will.
Each cell has gone through millions of years of evolution to adapt perfectly to its situation. If you tweak that, the cell is going to adapt through mutation of other genes. It is as if we remove the radiator from the car and it evolves an air cooling system like the one in old Volkswagen Bugs.
Teng and coworkers decided to investigate whether or not knocking out a gene causes an organism to adapt in a consistent way. In other words, does removing a gene cause a selection pressure for the same subset of mutations that allows the organism to deal with the loss of the gene. The yeast knockout (YKO) collection, which contains S. cerevisiae strains that individually have complete deletions of each nonessential gene, gave them the perfect opportunity to ask this question.
There have long been anecdotal reports of the YKO strains containing additional, secondary mutations, but the authors first needed to assess this systematically. They came up with an assay that could detect whether secondary mutations were occurring, and if so, whether separate isolates of any given YKO strain would adapt to the loss of that gene in a similar way. The assay they developed had two steps.
The first step was to fish out individual substrains from a culture of yeast that started from a single cell in which a single gene had been knocked out. This was simply done by plating the culture and picking six different, individual colonies. Each colony would have started from a single cell in the original culture.
The second step involved coming up with a way to distinguish differently adapted substrains. The first approach was to see how well each substrain responds to increasing temperatures. To do this, they looked for differences in growth at gradually increasing temperatures using a thermocycler.
They randomly selected 250 YKO strains and found that 105 of them had at least one substrain that reproducibly responded differently from the other substrains in the assay. In contrast, when they looked at 26 isolates of several different wild type strains, including the background strain for the YKO collection, there were no differences between them. This tells us that the variation they saw in the knockout substrains was due to the presence of the original knockout.
So this tells us that strains can pretty quickly develop mutations but it doesn’t tell us that they are necessarily adapting to the knocked out gene. To see if parallel evolution was indeed taking place, the authors chose to look at forty strains in which the same gene was independently knocked out. They found that 26 of these strains that had at least one substrain with the same phenotype, and fifteen of those had mutations that were in the same complementation group. So these 15 strains had evolved in similar ways to adapt to the loss of the same gene.
Teng and coworkers designed a second assay independent of the original heat sensitivity assay and tested a variety of single knockout strains. They obtained similar results that support the idea that knocking out a gene can lead cells to adapt in similar ways. This is both good and bad news.
The bad news is that it makes interpreting knockout experiments a bit trickier. Are we seeing the effect of knocking out the gene or the effect of the secondary mutations that resulted from the knockout? Are we seeing the loss of the radiator in the car or the reshaping that resulted in air cooling? We may need to revisit some earlier conclusions based on knockout phenotypes.
The good news is that not only does this help us to better understand and interpret the results from yeast and mouse (and any other model organism) knockout experiments, it also gives us an insight into evolution and maybe even into the parallel evolution that happens in cancer cells, where mutations frequently co-occur in specific pairs of genes. And while we may never be able to predict if that knock you hear in your engine really needs that $1000 repair your mechanic says it does, we may one day be able to use results like these to predict which cells containing certain mutated genes will go on to cause cancer and which ones won’t.
November 7, 2013
We all know that it’s important to get enough vitamins in our diet. Scary-sounding conditions like scurvy, rickets, and beriberi can all happen when you don’t get enough of them. And that’s not all.
Fairly recently, scientists discovered that when pregnant women get too little folate, their children are at a higher risk for neural tube defects. This connection is so strong that since 1998, the U.S. and Canada have successfully reduced the number of neural tube defects by adding extra folate to grain products.
While these kinds of effects are easy to see, it’s not always so obvious what is going on at the molecular level. But in a new study in GENETICS, Sadhu and coworkers showed that folate and methionine deficiencies can affect us right down to our DNA. And of course, they figured this out by starting with our little friend S. cerevisiae.
Folate and its related compound methionine are pretty important molecules in cellular metabolism. You need folate to make purine nucleotides, and it is essential for keeping just the right levels of methionine in a cell.
And methionine is, of course, one of the essential amino acid building blocks of proteins. But it is more than that. It’s also the precursor for S-adenosyl-methionine (SAM), which provides the methyl groups for protein methylation.
Protein methylation is a big deal for all sorts of things. But one of its most important jobs is undoubtedly controlling levels of gene expression through methylation of histones.
Since folate or methionine deficiency should affect SAM levels, in principle they could affect histone methylation too. But so far this connection had never been shown directly. Sadhu and colleagues set out to see what happens when you deprive S. cerevisiae of these nutrients.
Unlike humans, yeast can synthesize both folate and methionine. So the first step was to make folate- and methionine-requiring strains by deleting the FOL3 or MET2 genes, respectively. These mutant yeast strains couldn’t grow unless they were fed folate or methionine.
Now it was possible to starve these mutant strains by giving them low levels of the nutrients they needed. Starvation for either folate or methionine caused the methylation of a specific lysine residue (K4) of histone H3 to be reduced. Not only that, but expression of specific genes was lower, consistent with their reduced histone methylation.
To see how general this effect was, the authors performed essentially the same experiments in Schizosaccharomyces pombe, which is about as evolutionarily distant from S. cerevisiae as you can get and still be a yeast. In this beast, methionine deficiency also reduced histone methylation. For unknown reasons, folate deficiency didn’t have a significant effect.
Sadhu and coworkers wondered whether this effect was so general that they could even see it in human cells. Since humans are folate and methionine auxotrophs, this experiment was easier to set up. When they grew human cells with starvation levels of folate or methionine, their histone methylation and gene expression were both reduced. So starvation conditions have an impact right down to the level of gene expression, across a wide range of organisms.
The simple explanation for this effect would be that reduced folate leads to reduced SAM levels, and therefore fewer methyl groups are available to modify histones. But the researchers got a surprise when they measured intracellular SAM levels in S. cerevisiae under the starvation conditions: they were the same as in wild type! This conclusion was so surprising that they tried two different, sophisticated methods, but both gave the same result.
They explain this by postulating a kind of metabolic triage. Basically, the cell maintains a certain level of SAM in the cell but there is a pecking order for who gets to use it. At very low nutrient levels, the cell uses the available folate or methionine for the most essential processes such as purine synthesis or translation, and sacrifices histone methylation. As more nutrients become available, then other less critical functions can use them.
This kind of triage might provide an explanation for the link between folate deficiency and neural tube defects, and also for the effectiveness of antifolates against cancer. And it adds to the growing body of evidence that environmental conditions such as famine can have effects that persist across generations. This is an important reminder that any decisions we make today about feeding the hungry could have consequences that reach far into the future.
November 3, 2013
Wish you were going to Cold Spring Harbor for the Cell Biology of Yeasts meeting this week, November 5-9? SGD will be live tweeting from CSHL, highlighting topics from talks and posters. Keep up with events at the meeting by following @yeastgenome on Twitter or searching #YCB2013 for all tweets!