The 'evolution' of a PhD project—from jumping genes to biochemical pathways

Traffic, Jan, 2007 by Henry Chung

We wanted to find out if Cyp6g1 is produced in the similar tissues in Hikone-R and Celera, to see if resistance to DDT was due to production in a novel tissue type or due to increased production in similar tissues. We performed in situ hybridisation on the larvae of Hikone-R and Celera. What we observed was quite intriguing: in Celera, Cyp6g1 was expressed in the midgut, the Malpighian tubules and the fat body, the tissues 'traditionally' associated with the detoxification of xenobiotics. In Hikone-R, however, while it was expressed in the same tissues the level of expression was much higher, as revealed by darker blue staining. At this stage, what could be concluded was that Cyp6g1 is expressed in the same tissues in both insecticide-susceptible strains and resistant strains of the fruitfly, albeit at higher levels in the latter.

Next, we wanted to see if the regulatory sequences of the gene contained all the elements needed for the expression of Cyp6g1. The regulatory region of a gene contains DNA elements, telling it where to express, when to express and how much to express (Figure 1). Remember that Accord was inserted into the regulatory sequence of the Cyp6g1 in resistant fruitflies. This could affect some of the elements of the Cyp6g1 regulatory sequence. The DNA sequence of the regulatory sequence in Celera is almost identical to the regulatory sequence in Hikone-R, except that the latter contains the Accord insertion. We cloned the regulatory sequence of Cyp6g1 from both Celera and Hikone-R and put them in front of a fluorescence reporter gene called 'green fluorescence protein' (GFP)--isolated from the jellyfish Aequorea victoria, which fluoresces green when exposed to blue light--and injected these into fruitflies. In this way, we could determine what the regulatory sequence does to a gene just by looking at where GFP is produced (i.e. which tissue is fluorescing green). The experiment showed that the pattern of expression shown by GFP is exactly the same as was seen by in situ hybridisation. This confirmed that the regulatory sequences in both strains of flies were wholly responsible for the expression of Cyp6g1 and the only difference between these strains was Accord.

A MOBILE BILLBOARD

Regulatory elements are better known as enhancers. Professor David Arnosti from Michigan State University, a leading researcher on enhancers, describes some of these enhancers as 'billboards' due to the mechanisms of their activity. (12) You can almost imagine a cartoon depiction of an enhancer carrying a billboard saying 'EXPRESS ME HERE'. We wanted to know if Accord itself contains enhancers that direct the expression of Cyp6g1 in resistant fruitflies, so we cloned just the 491 base pair Accord and stuck it in front of GFP and injected this into fruitflies. To our surprise, we saw the same tissues--the midgut, Malpighian tubules and fat body--light up in bright green fluorescence. This showed that Accord, a jumping gene, contains tissue specific enhancers that drive the expression of Cyp6g1 in those three tissues, increasing the amount of Cyp6g1 protein produced, thus leading to insecticide resistance (Figure 2). So Accord can be described as a 'mobile billboard', moving around and switching on the expression of genes near the proximity of its insertion site. After many other experiments and controls, we published our research in the journal Genetics. (13) Our paper was well received and was featured in New Scientist magazine. (14)