New & Noteworthy
July 28, 2015
Understanding lists and knowing how to work with them is crucial to getting the most out of YeastMine. This set of short videos explains everything you need to know.
YeastMine allows you to save objects in lists. Typically, these objects are genes, but you can also make lists of other objects such as Gene Ontology terms or PubMed IDs. One way to create a list in YeastMine is to run a query and save the results in a list. Another way is to type in or upload your own list.
Whenever you create a list in YeastMine, you’re immediately presented with information about the genes in the list, such as Gene Ontology and pathway enrichment, interactions, orthologs, and more. This can help you decide what kind of further analysis you’d like to do.
And what if you create a list but then realize that you forgot to include a gene? No worries. It’s easy to edit your saved lists.
Once you have a list of genes, you can feed it into any template query whose name begins with “Gene” to get results for all of the genes in the list. This powerful feature lets you run successive queries to narrow down your results. For example, you could make a list of all the proteins in a given size range, then query that list to see which ones are located in the nucleus, and finally ask how many of these nuclear proteins have human homologs.
And finally, once you’ve created and saved lists you can do a lot of different things with them: combine them, find their intersection, find genes that are not shared between two lists, or find genes that are in one list but not another. This provides a powerful way to combine or compare results from different YeastMine queries.
As always, please contact us if you have any questions about YeastMine. We’re happy to help!
July 22, 2015
Tails let animals sense and interact with their environment at a distance from their bodies. It turns out that some proteins use their “tails” in a similar way. Except they take it one step further.
In addition to sensing the environment, they can also use their tails as sort of fishing poles to catch proteins they need to interact with. And they do this with great specificity…it is like having that perfect lure that always catches one type of fish.
A great example of this is the septin family of proteins which includes many members that are “tailed” proteins. Septins are highly conserved proteins that typically have a globular GTP-binding domain adjacent to an elongated C-terminal extension.
Septins form structures that act as the boundaries between different cellular parts. In budding yeast cells, they form the septum that separates the mother and bud, and recruit the cytokinesis machinery that allows the daughter cell to separate from the mother cell. In larger animals, they can be found in places such as the dendritic spines of neurons, sperm flagella, or cilia. And in humans, septin mutations have been linked to cancer and neurological diseases.
Until now, the details of how septins recruit other proteins to boundary sites have been elusive. But in two new papers in GENETICS, Finnigan and coworkers in the Thorner lab at Berkeley dove into this question and gained real insight into the lures these proteins use.
In their first paper they reported an extremely comprehensive genetic analysis to dissect the functions of two of the least characterized septins, Shs1 and Cdc11. In the second paper they used both genetic and physical methods to show how these septins recruit myosin to the septum to form the contractile ring that pinches off the bud from the mother.
The bottom line: their C-terminal tails are extremely important. They intertwine with other proteins’ tails like love-struck seahorses. And their specificity comes from these same tails—certain tails only coil around other tails.
The S. cerevisiae genome encodes a family of septins that assemble with each other to form octameric rods that consist of four different septins. The rods have both end-to-end and side-to-side interactions with each other, forming a ladder-like superstructure.
The septins Cdc11 and Shs1 are the most closely related members of the septin family, and the most recently evolved. They cap the ends of the septin rods. In otherwise wild-type cells, Cdc11 is essential for life while Shs1 is not.
Because SHS1 can be deleted without causing a major phenotype, the first step in investigating its function was to find genetic conditions under which its function becomes more obvious. The authors created four different genetic backgrounds in which the function of other septins was compromised by different mutations. Cells that had mutations in both SHS1 and in other septin genes had obvious problems, such as elongated buds or the inability to grow at high temperatures.
Now Finnigan and colleagues were set to do a detailed genetic analysis to figure out what different parts of Shs1 do by testing mutant versions in these different backgrounds. We can’t possibly recapitulate all the results here, but we’ll do our best to cover the highlights.
Almost all septins, whether in yeast or mammals, end with a tail: a long stretch called the C-terminal extension (CTE) that contains sequence patterns characteristic of a coiled-coil structure. The researchers found that the coiled coil regions of Shs1 and Cdc11were essential to their functions. (And no, they didn’t create any mutations by writing their names in the coiled coil sequence!)
Finnigan and colleagues tried swapping CTEs between different septins. When Cdc11 carried the Shs1 CTE and vice versa, the cells grew just fine. However, this swappability didn’t extend to other septins that are positioned internally in the septin rods. The CTEs of the end subunits Cdc11 and Shs1 could be exchanged for each other, but these CTEs only worked when they were on the ends of the rods.
Since coiled coils are often involved in interactions between proteins, Finnigan and colleagues wondered whether the essential function of the Cdc11 and Shs1 CTEs might be to recruit other proteins to the bud neck.
To test this, they searched published data to identify proteins that are well-known to be localized to the bud neck at the time in the cell cycle when septins are present. They found 30 such proteins, and overexpressed GFP-tagged versions of each in a strain where both Cdc11 and Shs1 lacked their CTEs.
Of the 30 proteins, only overexpressed Bni5 suppressed the growth defect of this strain. To test directly whether binding to Bni5 is a critical function of Shs1, Finnigan and colleagues fused the two genes to each other, so that Bni5 replaced the CTE of Shs1. This fusion protein could compensate for the lack of both Cdc11 and Shs1 CTEs.
To confirm that the important function of the CTEs is to hold Bni5 in the right place, they came up with an alternative test using a “nanobody”, which is a very small, very high-affinity single-chain antibody. They replaced the CTE of either Cdc11 or Shs1 with a nanobody that recognized GFP, and expressed a GFP-Bni5 fusion in these strains. In both cases, tethering Bni5 to the septin via the nanobody obviated the need for the CTE.
Finally, the authors asked why it is important for Bni5 to be located on the septin rods. Previous work had suggested that Bni5 recruits Myo1 (myosin), an important component of the contractile ring at the bud neck. They used the same nanobody constructs to test this, simply expressing GFP-Myo1 in the strains where the nanobody replaced the CTEs of Cdc11 or Shs1. Sure enough, tethering Myo1 to the terminal septins eliminated the need for Bni5.
So we now know that tails are absolutely essential for the functions of the alternative terminal septins Shs1 and Cdc11. These fishing poles let them hold on to the Bni5 “bait,” which in turn catches Myo1 to provide the muscle for cytokinesis to occur. Since septins are so highly conserved, it’s probable that these results will be directly applicable to higher organisms: there are mammalian septins that also occupy the end positions of septin rods, analogous to Cdc11 and Shs1. And that’s no fish story!
Not only septins use their tails to fish.
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
July 13, 2015
The eminent Drosophila geneticist Michael Ashburner famously said: “Biologists would rather share their toothbrush than share a gene name.” It’s true that assigning names to genes is often a sticky subject.
In the Saccharomyces cerevisiae community we’re very lucky to have well-defined guidelines for genetic nomenclature, an established system for reserving gene names, and criteria for making them “standard,” or official, names. This system was agreed upon by yeast researchers nearly two decades ago and has served the community well.
Take a look at this video to get an overview of how the gene naming system works. And as always, please contact us with any questions or suggestions.
July 8, 2015
Gifts can be hard to buy for some people. They have everything they need and not many outside interests. What to do?
You could name a star after them or get them some knick knack they don’t need. Or you could design a personalized protein that has their name in it, solve the structure and present them with the picture.
This is what Deiss and coworkers did to celebrate the 50th birthday of their colleague Andrei N. Lupas, a key figure in studying coiled-coil proteins. They created a personalized protein based on Gcn4 from Saccharomyces cerevisiae. And of course Gcn4 is a coiled-coil protein!
Coiled-coil proteins are the perfect clay for biosculpting a personalized protein. They follow a relatively simple set of rules which makes it easy to predict how they will fold. There isn’t much of the “protein folding problem” with these user-friendly proteins.
Basically these proteins consist of repeated 7 amino acid motifs that each form an alpha helix. They have hydrophobic residues down one face of the helix so that they will tend to oligomerize with each other to keep the hydrophobic residues away from the water. These helices spontaneously coil up like a rope (hence their name).
The 7 amino acids of a repeat are usually represented as a-b-c-d-e-f-g and are arranged in the pattern hxxhcxc, with h being hydrophobic residues, c being charged residues and x being most any other amino acid. So a and d must be hydrophobic, and e and g charged. That’s pretty much it!
Deiss and coworkers used the name Andrei N. Lupas to create a personalized coiled coil. They replaced 12 amino acids in Gcn4 with the amino acids represented by the letters in his name. Well, they were able to do that for most of the letters.
First off, they had to Roman things up a bit and turn the U into a V (there is no amino acid with the single amino acid code U). So here is the amino acid sequence they used and how they lined it up with the 7-amino acid repeats:
In this arrangement, the hydrophobic residues are asparagine, isoleucine, and valine, and the charged residues are aspartic acid, glutamic acid, proline, and serine. Obviously the last two are not optimal, especially the proline. Proline has an especially rigid conformation and is known to wreak havoc with alpha helices.
When the authors analyzed the protein, they found that as predicted, the proline disrupted the part of the alpha helix with which it was associated. But not enough to completely destroy the coiled coil structure. X-ray diffraction showed that this protein was still able to trimerize properly. They had created a distorted but functional personalized protein. What other kind would anyone want!
And it isn’t as if proline is completely absent from the heptad repeats of coiled-coil proteins. A quick search by the authors found two viral fusion proteins, 1ZTM and 3RRT, that could form a trimer even though they too had prolines. In both of these proteins the proline is in the f position.
They also found 4 dimers with a proline in a heptad repeat. In these cases the proline is at b or c. So no known natural coiled-coil proteins have a proline at the e position. Talk about personalized!
How cool is all of this, and who wouldn’t want a protein of their very own? Unfortunately, not everyone can easily have one.
For example, President Barack Obama would have real trouble since there are no amino acids designated with a B or an O and there is no obvious way to transform these letters into ones that are present in the single letter code. Jeb Bush is out too, but maybe we can do something with Hillary Clinton. Let’s see if we can line up the amino acids of her first name to create a personalized Gcn4 just for her.
“HILLARY” isn’t too bad by itself. All the letters are amino acids (yay) and a and d are hydrophobic (isoleucine and alanine). Aspartic acid works very well for e and while probably not perfect, histidine isn’t too bad for g. The tyrosine at position f is not ideal either but is way better than a proline. This thing might replace one heptad repeat in Gcn4 without causing too many problems.
So what about your name? Can you turn yours into a heptad repeat to create your own personalized Gcn4?
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics