Category: Research Blog Page 5 of 7

Stringent Analysis of Gene Function and Protein–Protein Interactions Using Fluorescently Tagged Genes

Another invention based on the MiMic gene trap system. From the abstract:

GFP traps have mainly been used to study the endogenous expression patterns of trapped genes or the subcellular localization of their protein products. Here, we show that the GFP tag can also be used to interfere with gene function by RNAi-mediated knockdown of the tagged transcripts. This method, which we refer to as “tag-mediated loss-of-function,” addresses major shortcomings of the classical RNAi approach in which gene-specific sequences are targeted.

The basic idea is that you modify your favorite gene in vivo so it becomes tagged with GFP, and then direct your RNAi targeting constructs against the GFP tag rather than the protein itself. 

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Combinatorial Activation and Repression by Seven Transcription Factors Specify Drosophila Odorant Receptor Expression

This paper (summarizing many years of work) describes the combinatorial code that controls the expression of olfactory receptor proteins in the fly sensory system.  It is unknown at present how much OR expression patterns  differ between species.  But if they do, this code would suggest a clear mechanism for their evolution.

Two papers in Nature Biotechnology describe massively parallel functional dissection of mammalian enhancers:

Massively parallel functional dissection of mammalian enhancers in vivo

Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay

These papers translate the good old idea of systematic structure-function analysis from proteins to regulatory sequences.  The ultimate goal is to characterize the functional significance of every nucleotide position (and, ideally, their interactions) within a cis-regulatory element.  Two make this happen, you need two things: the ability to generate hundreds of thousands of synthetic sequences, and the ability to analyze the output of all these sequences – and you need to accomplish both of these things in an automated, high-throughput way.  That’s exactly what these folks did, in somewhat different ways.  These tricks would not work for complex developmentally regulated enhancers, but it’s a good start.

Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks

I don’t think this will come as a surprise to anyone, but the experiment still had to be done.  It’s nice to know that genes conserved over millions of years are conserved because (gasp) losing them is bad for you.

Seriously though, this is a very nice paper.  As they say:

Almost all eukaryotic genes are conserved, suggesting that they have essential functions. However, only a minority of genes have detectable loss-of-function phenotypes in experimental assays, and multiple theories have been proposed to explain this discrepancy.

They show then that almost genes have significant effects on fitness if you just measure it properly.  Take a look:

The Majority of Animal Genes Are Required for Wild-Type Fitness

RNA Editing Underlies Temperature Adaptation in K⁺ Channels from Polar Octopuses

This is a fun story. I’ll just reproduce their whole abstract here:

To operate in the extreme cold, ion channels from psychrophiles must have evolved structural changes to compensate for their thermal environment. A reasonable assumption would be that the underlying adaptations lie within the encoding genes. Here, we show that delayed rectifier K⁺ channel genes from an Antarctic and a tropical octopus encode channels that differ at only four positions and display very similar behavior when expressed in Xenopus oocytes. However, the transcribed messenger RNAs are extensively edited, creating functional diversity. One editing site, which recodes an isoleucine to a valine in the channel’s pore, greatly accelerates gating kinetics by destabilizing the open state. This site is extensively edited in both Antarctic and Arctic species, but mostly unedited in tropical species. Thus adenosine-to-inosine RNA editing can respond to the physical environment.

Is this weird or what?

Trisha Wittkopp’s lab has just published a very interesting paper where they look at the effects of cis– and trans-regulatory mutations in yeast.  This paper gives a nice picture of the properties of mutations that are potentially available for selection.  Definitely worth reading.

Contrasting Properties of Gene-Specific Regulatory, Coding, and Copy Number Mutations in Saccharomyces cerevisiae: Frequency, Effects, and Dominance

In the last few years, there has been a lot of work (finally!) on how the size of developing organs is controlled.  An animal has to have the correct proportions, so every organ needs to coordinate with every other organs and decide how fast to grow and when to stop growing.  Several pathways have been shown to be required for this coordination.  Now, this paper shows that one of the ways growth is coordinated is through the regulation of tRNA synthesis:

Drosophila RNA polymerase III repressor Maf1 controls body size and developmental timing by modulating tRNA_i^Met synthesis and systemic insulin signaling

How is this relevant to evolution?  Body size and organ proportions (allometry) are among the fastest evolving morphological traits, but we know virtually nothing about how that happens.  Similarly, sexual size dimorphism is very common, but we don’t know how it is accomplished and how in changes in evolution.  Hopefully, better knowledge of the molecular genetics of size control will stimulate more work on the evolution of size and allometry.

Here’s a really cool paper:

DNA Methylation of the Gonadal Aromatase (cyp19a) Promoter Is Involved in Temperature-Dependent Sex Ratio Shifts in the European Sea Bass

They show that temperature-dependent sex determination (or rather, in this case, trans-differentiation) is mediated by the methylation of the aromatase promoter.  An obvious question is whether the environmentally driven methylation of the promoters of genes involved in sexual differentiation could be a general mechanism in species with environmental sex determination.  I am sure lots of people will be asking this soon, at least in vertebrates.

Does anyone known a dipteran with temperature-dependent sex determination?  Can’t think of one right now.