It's been a while since I last blogged about peer-reviewed science.  In a recent Departmental Journal Club, I discussed a paper outlining the use of transgenic RNAi in Drosophila.  In this paper, the authors utilise the power of Drosophila transgenics to use RNAi mediated gene knockdown to identify components of an important developmental signalling pathway.

In contrast to other systems, such as mammalian cell culture systems, in which RNAi mediated knockdown of gene expression is mediated by the introduction of short double-stranded RNA molecules, in Drosophila researcher use longer double stranded RNA molecules.  There are two method of using RNAi to investigate gene function in Drosophila.

Here's the cover picture from the February 2009 issue of Genetics, which just arrived in my office.I think this counts as art!  The caption is Adult heads of Drosophila melanogaster mass-fractionated for RNA extraction. Or, to paraphrase Edmund Spenser, "Dead skulls and bones of flies, whose life has gone astray." For further details see Telonis-Scott et al. (pp. 421-434).  The full citation is: Telonis-Scott et al (2009) Sex-Specific Splicing in Drosophila: Widespread Occurrence, Tissue Specificity and Evolutionary Conservation. Genetics, Vol. 181, 421-434, doi:10.1534/genetics.108.096743

The latest in the ongoing saga of our fly shipment from the USA is that our packet of flies finally made it to the lab.  They've been in transit for exactly three weeks*, and of course kept in in known conditions.  I'm very grateful to various people at Animal Health, who were able to make an exception to the regulations.

Nonetheless, I think the application of tight control of over the international transport of live insects such as these is a bit over the top - the legislation that I've looked through seems principally aimed at commercially important farm stock and other animals important to the human food chain.   I understand there's a general unhappiness in the UK Drosophila research community, especially since the international postal union recently relaxed its regulations regarding the transport of live Drosophila through regular mail.  I'm not particularly optimistic that we can make a change to the enforcement of the new regulations, but it's most definitely worth a try.

I have been conducting research using the fruit fly Drosophila melanogaster since I started my PhD in 1982.  In that time I have imported countless consignments of fly strains through the post and by courier (such as Federal Express).  On only  one occasion can I recall having difficulty getting them through customs - a box of female-sterile mutants from France got stuck, and was in a frightful state when they arrived (this was during my PhD).

The general practise is for the sender to affix one of those green customs tags, asserting that the contents are a gift, of no commercial value (typically $1 may be quoted), that they are live insects, but not an agruicultural pest or a vector of disease.  So for over 26 years as a practising Drosophila geneticist, I've had but one case where this has presented a problem.

It's all got rather Kafka-esque as I try to resolve the ongoing Drosophila importation crisis!  It transpires that the people who have decided that importation of Drosophila should be covered by legislation aimed quite properly at preventing the import of diseased farm animals are a subsidiary of Defra (the Department for Environment, Food and Rural Affairs) called Animal Health.

Now clearly these people have a vitally important job, particularly in light of recent outbreaks of bluetongue, foot and mouth and the potential threat of avian influenza (to which we can add the problems currently afflicting honey bees).  But nowhere on their website do I see indication of why they feel they need to hold up my harmless flies, which are not an agricultural pest, transmit no disease, are not harmful, and in any case would be unable to survive outdoors anyway.  To add to that list of characteristics, these are weak strains carrying recessive lethal mutations.  This is how they describe themselves and their responsibilities:

Of the many questions in evolutionary biology, the genetic basis of reproductive isolation between species and subspecies is a pretty hot topic. Drosophila pseudoobscura is a new world Drosophila species that has been used in evolutionary biology studies for many years.  This paper looks at the genetic basis of the hybrid sterility and segregation distortion seen in crosses between two subspecies, D. pseudoobscura pseudoobscura (referred to as "USA") and D. pseudoobscura bogotana (referred to as "Bogota"). It's a nice illustration of the impact of the 12 Drosophila genome sequences now available - D. pseudoobscura was the second Drosophila species to have its genome sequenced.

There is only partial reproductive isolation between these subspecies - male progeny from Bogota females crossed with USA males are virtually sterile (though when aged, they apparently yield offspring, though with a distorted sex ratio indicative of segregation distortion).  The female siblings are fertile, as are the offspring of a cross performed between UAS females crossed with Bogota males.  The two subspecies therefore obey Haldane's Rule.

The latest publication from our project investigating a Drosophila homologue of WRN exonuclease is now online.  

Ivan Boubriak, Penelope A. Mason, David J. Clancy, Joel Dockray, Robert D. C. Saunders, Lynne S. Cox (2008). DmWRNexo is a 3′–5′ exonuclease: phenotypic and biochemical characterization of mutants of the Drosophila orthologue of human WRN exonuclease Biogerontology DOI: 10.1007/s10522-008-9181-3

This interesting paper investigates whether there is a relationship between polyandry and selfish genetic elements, in the fruit fly Drosophila pseudoobscura. 

Polyandry - where females have multiple mating partners - is widespread in animals, but despite its frequency, little is known of the costs and benefits of this reproductive strategy (though this paper cites evidence that the costs of multiple mating appear to outweigh the benefits.  It is likely that the benefits lie in that polyandry gives the female a greater degree of control over paternity, via sperm competition.  There is also a possibility that selfish genetic elements may promote polyandry by correlating male fitness with sperm competition.

For much of my professional career as a Drosophila geneticist I've worked with polytene chromosomes, and it's always interesting to see papers with interesting tidbits of information about their structure and function. Polytene chromosomes are those rather strange structures formed from high levels of chromosome endoreduplication (see this blog article for a detailed description). Polytene chromosomes are widespread in flies, and in Drosophila are mostly studied in the larval salivary glands where they are easy to work with: Calvin Bridges used salivary gland polytene chromosomes to construct his polytene chromosome map. In this paper, Tom Hartl and colleagues show Condensin complexes (which have a function in chromosome condensation and anaphase chromosome segregation; and in vitro can induce and trap DNA supercoiling) can cause polytene chromosome disassembly and antagonise transvection.  Their data link processes of chromosome condensation and DNA supecoiling with higher order interphase nuclear structure that impacts on gene expression.

Unlike salivary gland polytene chromosomes, those of ovarian nurse cells break down during the development of the nurse cells, at about mid-oogenesis.  In this paper, two mutant alleles of a predicted component of the condensin II complex, Cap-H2 are studied.  In flies mutant for Cap-H2, the nurse cell polytene chromosomes don't disassemble.

A scary reminder of the 1958 movie The Fly, here's writer-illustrator Zina Saunders' (no relation) picture of the ghastly Palin. Check out her website for a some acute political art, and click on this thumbnail for the bigger image.

I think that's me in the lab coat.

 Wolbachia pipientis is a rather peculiar bacterium.  It's an intracellular organism, and is found in a wide variety of tax, including nematodes, crustacea, and arachnids.  About 20% of insect species are thought to have Wolbachia.  Wolbachia has evolved a number of mechanisms to ensure transmission, which is generally maternal.  There are a number of consequences of infection with these intracellular bacteria observed in different species - including reproductive isolation between infected and uninfected strains of the mosquito Culex pipiens.  I also recall seeing a old paper (though I've long-since lost the citation) which claimes a growth advantage of infected Drosophila larvae in crowded culture, though I think this must have predated the identification of Wolbachia.

So some laboratory Drosophila strains are infected with Wolbachia, while some are not.  This can lead to artefacts in many experiments.  For example, in my own lab, we had some perplexing results concerning the effects of particular transgene on viability of some combinations - the involvement of Wolbachia became clear firstly when we noticed the effects depended on which which strain was paternal and which maternal in the cross, and finally the observation that  this effect vanished after treating the transgenic stocks with antibiotic.  These kind of artefacts are particularly important to consider when working with complex characteristics such as lifespan (as we do in my lab).

 Alfred Henry Sturtevant is another of my all time heroes of Drosophila genetics (see also Calvin Bridges).

 As an undergraduate he generated the first genetic map of any organism.  He realised the linear order and relative position of genes on a chromosome would be reflected in the frequency of genetic recombination between them. Using a set of six X-linked mutants (actually two were alleles, so there were only five markers), he assembled a 5 locus map of the X chromosome. [Sturtevant (1913) J. Exp. Biol. 14; 43-59; pdf]
• Sturtevant's discovery of inversion is important because it explains the why/how of certain genetic defects and discovery of inversion allows for its presence to be tested for

We've had another paper on the WRN exonuclease homologue accepted for publication.  In it we demonstrate that DmWNexo is a 3'-5' exonuclease, and describe a new EMS allele. Here's the abstract:

The premature human ageing Werner's syndrome is caused by loss or mutation of the WRN helicase/exonuclease. We have recently identified the orthologue of the WRN exonuclease in flies, DmWRNexo, encoded by the CG7670 locus, and showed very high levels of mitotic recombination in a hypomorphic PiggyBac insertional mutant. Here, we report a novel allele of CG7670 , with a point mutation resulting in the change of the conserved aspartate (229) to valine. Flies bearing this mutation show levels of mitotic recombination 20-fold higher than wild type. Molecular modelling suggests that D229 lies towards the outside of the molecule distant from the nuclease active site. We have produced recombinant protein of the D229V mutant, and assayed its nuclease activity in vitro, and compared activity with that of wild type DmWRNexo and a D162A E164A double active site mutant we have created. We show for the first time that DmWRNexo has 3′ -5′ exonuclease activity and that mutation within the presumptive active site disrupts exonuclease activity. Furthermore, we show that the D229V mutant has very limited exonuclease activity in vitro. Using Drosophila , we can therefore analyse WRN exonuclease from enzyme activity in vitro through to fly phenotype, and show that loss of exonuclease activity contributes to genome instability. 

An amino acid polymorphism in the couch potato gene forms the basis for climatic adaptation in Drosophila melanogaster

Most organisms are faced with dealing with seasonal variations in environmental conditions.  As winter approaches, physiological changes need to be implemented: deciduous trees drop their leaves, mammals may hibernate, and so forth.  In the case of many insects, the strategy is to move into a diapause state.  This may be in any of the life stages - pupal diapause, larval diapause etc, and in the case of Drosophila melanogaster reproductive diapause, in which ovarian activity is shut down in response to a combination of short day length and low temperature.  My interests in diapause are two-fold in origin: firstly, diapause appears to have an impact on lifespan in Drosophila, and secondly, my father identified the existence of reproductive diapause in Drosophila back in 1989.  

The utilization during mitotic cell division of loci controlling meiotic recombination and disjunction in Drosophila melanogaster.

Bruce S. Baker, Adelaide T. C. Carpenter, and P. Ripoll 

Genetics 1978 90: 531-578. [Abstract] [PDF]

As a postdoc, I worked for several years on the cell cycle in Drosophila.  At that time, the field was just beginning to take off, not least because of the efforts of my then group leader.  Nowadays, I study the ageing process, still using Drosophila, and including modelling the function of WRN, the gene responsible for the progeroid condition Werner's syndrome (WS). WRN encodes a RecQ DNA helicase (unwinds the DNA double helix), but unusually has a second activity, a DNA exonuclease (removes nucleotide bases from the end of a DNA strand). We are currently studing the Drosophila homologue of the WRN exonuclease (which we have named DmWRNexo, and which is encoded by the CG7670 locus).

When trying to demonstrate that a mutant of CG76700 indeed displayed characteristic comparable with defects seen in cell lines derived from WS patients, I recalled reading this paper at the beginning of my postdoc position.

The header images are all related to Drosophila:

Above: These are the giant polytene chromosomes found in a variety of tissues in Drosophila - these are from the salivary gland cells of the third instar larva.  Calvin Bridges (see picture of the fly lab below) devised maps based on the banding patterns of these chromosomes - maps still in use today.

Here's an cool page showing academic pedigrees of Drosophila workers: FlyTree.

 It's interesting to see how few steps it takes to get back as far as T. H. Morgan!  For example, here's where I fit in the grand scheme: Robert in FlyTree.

Drosophila RNAi screen identifies host genes important for influenza virus replication.

Linhui Hao, Akira Sakurai, Tokiko Watanabe, Ericka Sorensen, Chairul A. Nidom, Michael A. Newton, Paul Ahlquist & Yoshihiro Kawaoka 

A. Bhutkar, S. W. Schaeffer, S. M. Russo, M. Xu, T. F. Smith, W. M. Gelbart (2008). Chromosomal Rearrangement Inferred From Comparisons of 12 Drosophila Genomes Genetics, 179 (3), 1657-1680 DOI: 10.1534/genetics.107.086108

Back when I was a carefree postdoc, one of the projects I worked on was the assembly of a molecular physical map of the Drosophila melanogaster genome. Of course, Drosophila researchers had for years been using a physical map, the polytene chromosome map, and indeed we used this as the framework on which we assembled our molecular map using cosmid clones. These papers take the genome sequences of 11 Drosophila species (plus the sequence of Drosophila melanogaster, determined back in 2000), fit them to the polytene chromosome maps, and examine chromosome rearrangments seen in inter-species comparisons.  It seems to me there isn't anything hugely sexy in this work, but there is a huge amount of work that sets the evolutionary relationships between these Drosopholids in context.  It's also an opportunity to expound on chromosomes in Drosophila!

Calvin Bridges is, with Alfred Sturtevant, one of my heroes of Drosophila genetics.  Among his achievements were the demonstration and confirmation of the chromsome theory of inheritance, and the establishment of the polytene chromosome maps (for more about polytene chromosomes, see this article).  Bridges was one of the early members of the Morgan fly lab, and stayed there for his entire (though unfortunately short) career.  Kohler, in his excellent history of Drosophila genetics, characterises Bridges as the "blue collar" member of the lab, the worker who would invest huge energy in the technical development of Drosophila genetics.