Back to thinking about how to explain alternative splicing in an easy, graphical or pictorial way.
Here’s an attempt at sketching plant cells under a microscope. Grid like arrangement of cells, with chloroplasts (photosynthesis organelles) as greenish circles, and the cell nucleus as dark circles/blobs.
Not entirely sure where this is going…maybe a cartoon. Still hope to include Pandas somewhere along the line…
Are RNA thermosensors more common than we thought? Interesting article in the Journal of Experimental Biology speculating whether RNA thermometers (RNATs), well-studied in Prokaryotes, are prevalent in the other Kingdoms of Life.
Changes in the conformation of RNATs (see their Figure 1, above) typically involve melting of short regions of the mRNA, for example hairpin structures, in response to elevated temperature (a ‘zipper’ mechanism) or a shift between alternative conformations of the mRNA that involve larger regions of the molecule (a ‘switch’ mechanism).
The RNAT contains the Shine–Dalgarno (S–D) sequence (AGGAGG) that, when fully exposed, can bind to the small (30S) ribosomal subunit and allow translation to commence. The start codon (AUG) is often located eight nucleotides downstream from the S–D sequence. Thus melting of the ‘thermometer’ allows the S–D sequence and start codon to interact with the 30S subunit, promoting translation of the mRNA.
Interesting read – I wasn’t familiar with the concept of ‘marginal stability’ – the idea that for RNA secondary and tertiary structures, thermosensor regions must have the right stability – or ‘balancing act’ – to allow temperature-driven changes in shape to take place when (and only when) a signalling function is required.
I particularly liked the section on ‘Differential translation of allelic mRNAs: another way to modulate the proteome?‘ – the concept that natural variants (allozymes) with different thermal optima can provide a species with an opportunity to establish populations with adaptively different thermal optima in regions of its biogeographic range where temperatures differ. Thus a cold-optimised allozyme might be more common in populations living in colder regions of a species’ range, whereas the warm-optimised allozyme would be dominant in warmer regions, and therefore crucially that slight changes in base composition likely alter the thermally sensitive mRNA structures that govern translational ability in a way that ensures differential translation of distinct allelic messages.
The author, George Somero, make an interesting point that we might assume that “changes in temperature often are regarded as having negative influences on macromolecular stability” adding “However, there is also a ‘good’ side to this thermal perturbation: the alteration in conformation of the macromolecule that is caused by a change in temperature can function as a thermosensing mechanism and lead to downstream changes that are adaptive to the cell.”
Exciting times lie ahead for RNA structure and temperature sensing….
Trying to re-learn the language of GATEWAY cloning. ‘BP reactions’, ‘LR cloning’, ‘attB sites’, ‘entry clones’, ‘destination vectors’, ‘binary vectors’…I could go on. Thinking of trying a series of ‘Improved Gateway Binary Vectors (ImpGWBs)‘ to make plants express our genes of interest (GOIs) fused to markers. These are described in this paper:
As for the choice of marker, there is a dizzying choice, so as well as trying to get to grips with GATEWAY, there is the question of what best to use as marker. I’ve had disappointing results fusing our GOIs to green fluorescent protein (GFP) in the past, so maybe time to try something else. As you can see, there is no shortage of choice:
Trying to work out how long to make the upstream promoter region for some plasmid constructs. I’ve been using the SnapGene tool to visually stitch together DNA sequences.
How long is the promoter for a particular plant gene? 500, 1000, 2000 bp? or is it defined by particular features in the promoter? I guess for some genes the upstream region barges into other gene loci.
I tried out this web-tool, plantpromoterdb.
Seems quite useful. It displays some features of the promoter that might be considered when deciding the promoter chunk length e.g. it will show the predicted transcriptional start site, and other cis– features.
Seems, though, that most studies take a nominal 1000 to 2000bp upstream of the translational start codon (ATG). Think it’s good to know some of the promoter ‘landscape’ though. Here’s the reference for plantpromoterdb:
This fly paper caught my eye. It examines how Drosophila monitors daily temperature changes via network of circadian clock regulated neurons. It seems the fly continually integrates temperature informations in order to coordinate sleep and activity patterns.
The work shows that nodes within the circadian network are sensitive to brief changes in temperature, and show that particular neurons are inhibited by heating and excited by cooling. It seems also that light and temperature are processed in distinct ways in the clock neutron network.
Interested to see the use of a fluorescent protein tool called CaMPARI that photo-converts from green to red in proportion to Ca2+ levels – could this be used in plant work? Would require both light (photo-activation) and temperature manipulation…
The kinetics of temperature response was monitored by measuring intracellular Ca2+ concentrations using a calcium sensor called GCaMP6m and showed that particular neurons showed increases in intracellular calcium during cooling and decreases during heating.
The authors state that their findings reveal that the circadian network transduces brief and transient temperature changes and prolonged increases in temperature in distinct ways.
Thermoreceptors are found in structures in the antennae, called the aristae. Each arista contains both cold-sensitive and heat-sensitive cells. From their figure (below), they found that the responses to cooling and heating were attenuated when the aristae was removed.
This work is interesting to use since we are trying to understand how plants respond to everyday changes in temperature – both short-term (daily fluctuations) and long term (seasonal) changes in temperature.
Always amuses me the infographic from Matt Might called ‘The illustrated guide to a Ph.D,’ (click here for the full infographic page). Very useful for new Ph.D. candidates, but it I guess it’s just as valid for anyone working at the coal-face of scientific research. We are all just trying to make small dents at the boundary of scientific knowledge – ‘suppose that makes us all eternal stu-dents!
Science is a pretty earnest endeavour, sometimes it’s healthy to poke fun at it. The Independent Standard is a serious sounding web-site, but it’s effectively along the lines of the ‘Daily Mash‘ or ‘The Poke‘ for the Sciences.
I would recommend visiting this site if you want to cut-free from the Science wheel for a while. They are all spoof stories but sometimes the parody is so deep that it actually makes you think about the process of Science.
Some good examples are:
Human Tears Are The Best Thing For Precipitating DNA…..worth thinking about the next time a DNA/RNA prep comes around
An ‘Eppendorf For Life’ Scheme Launched…we have shopping bags for life…what about Eppendorfs for life..
and Leeds Scientist Refuses To Do Southern Blot….reporting regional variations in attitudes to molecular biology techniques
As a follow-up to the earlier ‘Counting Cobras‘ post, Hugh pointed out this recent opinion article from The EMBO Journal written by Alain Prochiantz.
The author recommends playing the science ‘game’ – for example by trying to publish in the so-called high ranking journals even if they do not necessarily reflect novelty and importance, because this is important for obtaining grant funding. Interestingly the idea of developing side projects appealed to me – the concept of planting seeds that, although would probably not get published in stellar journals or even accepted by peers short-term (see “You should stop science”, “you are making a fool of yourself”), but nonetheless with time might grow and branch out into research with high impact.
Other notable comments were the idea that “…there never really is a golden age [in science research]” and ” …it was not better yesterday but [..] it will be better tomorrow, provided that we never forget to defend the “Value of science”.
and finally…”Science remains a game, a game that must be taken very seriously, but nevertheless enjoyed”.
I suppose it’s trying to keep that balance – to play the game enjoyably!?
I’m liking the logo on my ice bucket today….what fun typefont is that?!
‘Polishing’ my RNA today – thats when we remove contaminating genomic DNA that might have co-purified during the RNA isolation stage. I’m using TURBO DNase from Invitrogen (…or is it ThermoFisher?)
In this case RNA:good :-), DNA:bad 😦
We don’t want DNA since it might interfere with measuring gene expression levels later on.
One thing that I find increasingly challenging is actually getting into the lab. A combination of major rebuilding a the University of Glasgow and Glasgow City Council imposing a ‘ring of no-free-parking steel’ around the West End of the city results in a unseemly work-day scramble to get the few remaining reasonably cost-effective parking spaces near the University.
Thus I often find myself zig-zagging across the city to eventually end up back across the Clyde river to do park-and-ride with the Glasgow Underground (aka the Clockwork Orange) at either the Shields Road or Kelvinbridge stations to get back over to the West End.
I suppose I shouldn’t complain too much, however. At least I get to see this great mural of Glasgow’s West End by Alastair Gray, author of Lanark. The mural adorns the entrance/exit foyer of Hillhead underground station – the station nearest the University of Glasgow main campus.
The Bower Building – where the Glasgow collaborators are based – can be spotted on the mural map ! Here it is – located right next to the modern Woolfson Medical School Building: