New & Noteworthy

Adding Introns to Synthetic Biology’s Toolbox

July 3, 2014

As any good handyman knows, the more tools you have in your tool chest, the better the chance that you can find what you need to solve a problem.  The same goes for synthetic biologists.  The more parts they can mix and match, the more likely they are to engineer the exact level of gene expression they need.

Synthetic biologists have added introns to their tool chest. Image from Wikimedia Commons

In the last few years synthetic biologists have amassed a wide variety of transcription and translation elements that can be combined in different ways to exquisitely tune the level of expression of their gene of interest.  And now, in a new study out in PLOS Genetics, Yofe and coworkers have added introns to the list of parts available for our favorite yeast Saccharomyces cerevisiae.

Yeast isn’t loaded with introns, but it does have a reasonable number that can be co-opted for synthetic biology.  The authors inserted 240 of these introns individually into the same position near the 5’ end of the yellow fluorescent protein (YFP) gene and monitored the level of fluorescence of each individual strain over a 24 hour period.  They chose the 5’ end of the gene because yeast has a bias for introns being located there.

The authors found that these reporters spanned a 100-fold range of gene expression, that every intron caused a decrease in the level of gene expression, and that even though many of these introns respond to environmental stimuli in their natural context, their effect on gene expression here was immune to the environmental changes the authors tested.  Taken together, these results suggest that introns could be used in yeast systems for dampening over-exuberant gene expression in ways that are independent of growth conditions.  If all of this holds up, introns will prove to be very useful tools indeed.

Yofe and coworkers next wanted to use this library to figure out some of the rules for why some introns cause lowered activity compared to others.  The simplest possibility, that longer introns cause a larger decrease in gene expression, turned out not to be true.  There was no correlation between the size of the intron and its effect on the level of fluorescence. 

Next they scanned the sequences of their constructs to look for elements that might increase or decrease splicing efficiency.  These splicing regulatory elements (SREs) are better understood in larger eukaryotes, but there is evidence that they are important in yeast as well.  The authors identified a number of intron splicing enhancers (ISEs) and intron splicing silencers (ISSs) that were highly enriched near the splice sites. 

To confirm that these sequences did in fact affect splicing efficiency (and hence gene expression), they showed that mutating the enhancer motif TTTATGCT to the silencer motif TTTGTGTA in two reporters resulted in a 22% and a 13% decrease in gene expression.  This proof of principle experiment suggests that future synthetic biologists may be able to further tweak the expression of their genes by manipulating these SREs.

In a final set of experiments the authors used the library to identify rules that can be used to predict how inserting various introns into different positions will affect a gene’s activity.  They found that the most important features were the presence of SREs and the RNA structures at the intron-exon junction.  Synthetic biologists should be able to use these rules to intelligently design their reporter systems.

These experiments are the first step towards adding introns to the ever growing set of tools available to synthetic biologists for modulating gene expression.  We are getting closer to figuring out how genes are controlled and being able to use that knowledge to our advantage.  Or to put it another way, we have taken another baby step towards being able to control a gene as well as a yeast cell does.

Polygamous DNA Replication

June 26, 2014

Regulation ensures that most replication origins fire only once during a cell cycle, just as social mores ensure that most people have only one spouse at a time. But the exceptions can be interesting. Portrait of a Young Married Couple by Jacob Jordaens, image from Wikimedia Commons

Once someone is married, there are lots of things that keep them from starting a second marriage at the same time.  Laws, fear of losing the first spouse, social mores and so on all create a situation where the vast majority of people have only a single spouse at any one time. 

As each of these inhibitions is lifted, people will be more or less inclined towards polygamy, depending on who they are and the culture they live in.  For example, if having multiple spouses becomes acceptable socially, then some people might dive right in while others might hold off.

It turns out that origins of replication are similar.  There are many layers of control that keep an origin from firing more than once during any cell cycle.  But just like people and polygamy, when a few inhibitory layers are removed, some origins are more likely to fire more than once in a cell cycle than are others.

In a new study out in PLOS Genetics, Richardson and Li have identified a DNA sequence that makes nearby origins of replication fire more than once during a cell cycle when certain regulatory mechanisms have been disabled.  The authors hypothesize that these reinitiation promoters (RIPs) may be important for promoting genetic diversity by causing genomic duplication of specific regions under certain circumstances. 

This lab had previously shown that the origin ARS317 reinitiates more frequently when global regulation is removed from some key players in initiation: Cdc6p, the Mcm2-7 complex, and the origin recognition complex (ORC).  They disabled the regulation of all three of these by mutating each to prevent their recognition by the master regulator cyclin-dependent kinase (CDK, whose catalytic subunit is Cdc28p). In this study, they identified a second origin, ARS1238, that also reinitiated more often under these conditions.  The authors next set out to identify why these origins reinitiated under these conditions.

The first thing they found was that chromosomal context didn’t matter a whole lot.  Both origins reinitiated at around the same rate when they were in their natural context or when moved to other chromosomes.  The ability to reinitiate must be contained in the sequence of the DNA that was moved.

They next showed through deletion and linker scanning analysis that the two origins both required an AT-rich, ~60 base pair sequence to reinitiate.  This sequence needed to be within around 35-75 base pairs of the origin to promote reinitiation. Not any old stretch of AT-rich DNA would do; a specific DNA sequence was necessary, suggesting that this DNA is not required for reinitiation just because it is more easily unwound. 

These authors have shed light on a key process in the life of a cell—the firing of an origin of replication once and only once during any cell cycle.  It is critical for a cell that origins do not routinely reinitiate to prevent widespread genomic duplications that left unchecked would be very dangerous to the cell.

Richardson and Li have shown that not all origins are created equally, in that some are more likely to reinitiate under certain conditions than are others. If similar regions in mammalian cells turn out to be hotspots for genetic changes in cancers, then scientists may be able to target them to prevent the cancer’s genetic progression.  We may be able to reintroduce laws to keep polygamy at bay.

Shared Domains and Phosphorylation Sites on Protein Pages

June 24, 2014

We have redesigned the Protein page to include a new tabular display of protein domains. This table provides the identifier for each domain and illustrates the respective locations of the domains within the protein. In addition to this new table, the domains are displayed in an interactive network diagram that presents the proteins that share these domains with your protein of interest (see figure below, left).

Another new feature on the Protein page is the display of phosphorylation sites within the protein’s sequence (as curated by BioGRID). This feature is available for both the reference strain S288C and other commonly used S. cerevisae strains, using the pull-down to select the desired strain view (see figure below, right) .

Left: Proteins (gray circles) that share domains (colored squares) with Fas1p (yellow circle). Right: an example of some of the phosphorylation sites in Swe1p (red residues).

Proteins that share domains with Fas1p

Swe1p protein sequence and phophorylation sites highlighted in red.

YGM Early Registration Deadline is Approaching!

June 23, 2014

There are only a few days left to register for the Yeast Genetics Meeting at the early registration rate. After midnight on Thursday, June 26, the fees will increase by $75. Conference housing is filling up fast too. This is a meeting you don’t want to miss, so don’t delay!

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