Freedom of speech within science

Not so much research but a rant. As you may have heard the chief advisor to the government about drugs policy Prof Nutt was told to resign recently after he gave a lecture stating his views on cannabis classification in the UK and how the government (former Home Secretary Jackie Smith) did not listen to their advice on the matter and increased its classification and punishment for taking it. I do not care about the drug’s classification to be honest. I don’t take it and never have but the current Home Secretary (Alan Johnson, tipped to be future Labour leader, among a couple of others) has had a disagreement with the scientist and dismissed him after he gave a lecture stating his opinion. The reason given was he had crossed the boundary between science and politics. What? A scientist who is an unpaid advisor states his opinion based on evidence then states the government didn’t listen to them when deciding policy is crossing the line?! It sounds like he was starting a debate, using his freedom of speech to discuss topics. The government has decided no, if you don’t agree with us and you are helping us then sod off. Whether you agree with Prof Nutt or not I don’t think it matters. What matters is whether advisors are allowed to criticise the government’s policy when they are not listened to. They believe not. Alan Johnson said: ‘he was not dismissed because of the work of the council but because of his failure to recognise that... his role is to advise rather than criticise’. My PM Gordon Brown said: ‘Prof Nutt had repeatedly undermined Labour's drug message.’ I think it is ridiculous to suggest a scientist cannot talk about such things in public and suggests to me the government is being hypersensitive over these matters. Perhaps because they cannot handle anymore bad press and people going against them. After all the chances of them winning the next election are slim to none.

So far Lord Drayson (a businessman) who determines science policy in the government’s Business department and coordinates with the research councils was strongly against this when he first heard it. Lord Winston FRS, a respected scientist in the public eye and Labour peer, is against the dismissal and Prof John Beddington FRS (who recently recovered an honorary degree from York University in the Summer 2009 graduation ceremony) has spoken the most sense on the matter (http://news.bbc.co.uk/1/hi/sci/tech/8340318.stm). He is the chief advisor to the government on all things sciencey. He points out most scientific committees do not have a problem with the government. This is reassuring but and the send of an email the Home Secretary (with no background what so ever in science) has dismissed an advisor and caused two to quit in sympathy because they don’t think they can work with a government that a) doesn’t listen to them and b) forces out the head of the committee for no good reason. I think this is unacceptable from the government hence I have written to the Home Secretary and feel I should email my local MP as well. Hopefully my next post will contain more science and less ranting. It’s being planned, just need to get some pretty photos.

Ig Nobels 2009

For a few years now that I have been following the Ig Nobels as they are awarded. To continue the tradition, here is a post with the list of this year's winners:

VETERINARY MEDICINE PRIZE
Catherine Douglas and Peter Rowlinson of Newcastle University, Newcastle-Upon-Tyne, UK, for showing that cows who have names give more milk than cows that are nameless.
"Exploring Stock Managers' Perceptions of the Human-Animal Relationship on Dairy Farms and an Association with Milk Production," Catherine Bertenshaw [Douglas] and Peter Rowlinson, Anthrozoos, vol. 22, no. 1, March 2009, pp. 59-69.

PEACE PRIZE
Stephan Bolliger, Steffen Ross, Lars Oesterhelweg, Michael Thali and Beat Kneubuehl of the University of Bern, Switzerland, for determining — by experiment — whether it is better to be smashed over the head with a full bottle of beer or with an empty bottle.
"Are Full or Empty Beer Bottles Sturdier and Does Their Fracture-Threshold Suffice to Break the Human Skull?" Stephan A. Bolliger, Steffen Ross, Lars Oesterhelweg, Michael J. Thali and Beat P. Kneubuehl, Journal of Forensic and Legal Medicine, vol. 16, no. 3, April 2009, pp. 138-42.

ECONOMICS PRIZE
The directors, executives, and auditors of four Icelandic banks — Kaupthing Bank, Landsbanki, Glitnir Bank, and Central Bank of Iceland — for demonstrating that tiny banks can be rapidly transformed into huge banks, and vice versa — and for demonstrating that similar things can be done to an entire national economy.

CHEMISTRY PRIZE
Javier Morales, Miguel Apátiga, and Victor M. Castaño of Universidad Nacional Autónoma de México, for creating diamonds from liquid — specifically from tequila.
"Growth of Diamond Films from Tequila," Javier Morales, Miguel Apatiga and Victor M. Castano, 2008, arXiv:0806.1485.

MEDICINE PRIZE
Donald L. Unger, of Thousand Oaks, California, USA, for investigating a possible cause of arthritis of the fingers, by diligently cracking the knuckles of his left hand — but never cracking the knuckles of his right hand — every day for more than sixty (60) years.
"Does Knuckle Cracking Lead to Arthritis of the Fingers?", Donald L. Unger, Arthritis and Rheumatism, vol. 41, no. 5, 1998, pp. 949-50.

PHYSICS PRIZE
Katherine K. Whitcome of the University of Cincinnati, USA, Daniel E. Lieberman of Harvard University, USA, and Liza J. Shapiro of the University of Texas, USA, for analytically determining why pregnant women don't tip over.
"Fetal Load and the Evolution of Lumbar Lordosis in Bipedal Hominins," Katherine K. Whitcome, Liza J. Shapiro & Daniel E. Lieberman, Nature, vol. 450, 1075-1078 (December 13, 2007). DOI:10.1038/nature06342.

LITERATURE PRIZE
Ireland's police service (An Garda Siochana), for writing and presenting more than fifty traffic tickets to the most frequent driving offender in the country — Prawo Jazdy — whose name in Polish means "Driving License".

PUBLIC HEALTH PRIZE
Elena N. Bodnar, Raphael C. Lee, and Sandra Marijan of Chicago, Illinois, USA, for inventing a brassiere that, in an emergency, can be quickly converted into a pair of protective face masks, one for the brassiere wearer and one to be given to some needy bystander.
U.S. patent # 7255627, granted August 14, 2007 for a “Garment Device Convertible to One or More Facemasks.”

MATHEMATICS PRIZE
Gideon Gono, governor of Zimbabwe’s Reserve Bank, for giving people a simple, everyday way to cope with a wide range of numbers — from very small to very big — by having his bank print bank notes with denominations ranging from one cent ($.01) to one hundred trillion dollars ($100,000,000,000,000).
Zimbabwe's Casino Economy — Extraordinary Measures for Extraordinary Challenges, Gideon Gono, ZPH Publishers, Harare, 2008, ISBN 978-079-743-679-4.

BIOLOGY PRIZE:
Fumiaki Taguchi, Song Guofu, and Zhang Guanglei of Kitasato University Graduate School of Medical Sciences in Sagamihara, Japan, for demonstrating that kitchen refuse can be reduced more than 90% in mass by using bacteria extracted from the feces of giant pandas.
"Microbial Treatment of Kitchen Refuse With Enzyme-Producing Thermophilic Bacteria From Giant Panda Feces," Fumiaki Taguchia, Song Guofua, and Zhang Guanglei, Seibutsu-kogaku Kaishi, vol. 79, no 12, 2001, pp. 463-9. [and abstracted in Journal of Bioscience and Bioengineering, vol. 92, no. 6, 2001, p. 602.]

Looking for some science on swine flu

As a medical association in Spain has recently communicated to the press, more than anything there seems to be an epidemic of fear going on. As I am currently living in Portugal, I do not know what the situation is in other countries, but I can tell you how the situation is here. Every news bulletin starts with more news about the flu, even though no one has died here yet. It then finishes with a previously recorded warning on how one should wash hands frequently and keep 1m away from other people while talking to them, quite an ordeal in a country where the common greeting is a couple of kisses in the cheek. In addition, we have ambulances going around with the paramedics dressed as in the photo, right out of films like ‘Outbreak’. Now, most people seem to assume that because I have just graduated with a Biology degree that I should know everything there is to know about the current flu. Besides the generic question ‘So, what do you think about this flu thing?’, I have had some more specific questions, from ‘I heard that it is impossible for this combination of genes to happen in nature, surely this must have been created by evil scientists with corporative interests?’ to ‘This virus is called H1N1, the 1918 pandemic virus was also H1N1, are we all going to die of Spanish flu?’. I try to explain people that at uni we are taught how to get informed about things, rather than becoming specialist on every single biological problems; and that frankly I am so fed up of hearing about this flu that I couldn’t be bothered to look it up. However, I think the hysteria might be affecting me, probably because I’ve just read the book Blindness (if you have read it/watched the film you will know why). So I decided to interrupt my holidays-away-from-science to read a bit about this topic, and thought I would tell you some of my findings.

My first port of call was the website of the World Health Organisation, usually a reliable source for information. I was pleasantly surprised by the lack of over-dramatic information. There is of course a whole section dedicated to this flu, right in the home page, but the answers given in the FAQs seem quite sensible. It tells us that 2009 A(H1N1) is spread as the normal flu, and that the current worries as based on the fact that being a virus which never circulated in humans before, there is no, or very little, immunity. In addition, as stated in their website, the virus is spreading fast in young people (10-45), from a majority of mild cases to some serious illnesses, the majority of which in patients with underlying conditions. The recommendations of the WHO are to take pain killers and drink loads of fluids if you have the flu. And only contact the medical services if you have serious symptoms like shortness of breath, or if the fever lasts for longer than 3 days. Quite unlike what recently happened in Portugal. When a man who had travelled to Britain was diagnosed with this flu, he saw his house invaded by doctors who quickly took him away to a hospital and confined the whole family to their house. Without telling them what to do or when they could leave.

Anyway, I then thought it would be a good idea to read some information from journals, a bit more scientific and hopefully less dramatic source of news. However, as you know, I am currently unable to access most papers, as I am in between universities and am yet to be provided with a username in my new institution. So what follows is based on a couple of papers I could access, and is by no means comprehensive.

As an introduction, some information from the Virology handouts we were given last October. Influenza viruses are RNA viruses that have a segmented genome (8 segments/genome). The glycoprotein spikes, haemagglutinin (HA) and neuraminidase (NA) are important in the entrance of the virus in the host cells. There are 15 known HA and 9 NA serotypes, and their combination provides the name that we see for the viruses such as H1N1. Birds and pigs are important reservoirs for genetic and antigenically diversity and reassortment in these viruses. In particular pigs, as their cells can be infected by both avian and human influenza viruses, making them nice ‘mixing pots’.

In July this year Garten et al., published a paper in Science in which they characterised the 2009 A(H1N1) virus both antigenically and genetically. They start the paper by giving a small introduction on the influenza pandemics of the last century, and how we got from those to the current 2009 A(H1N1). Then it goes on to characterise the actual viruses. The closest ancestral gene for each of the eight segments seems to have a swine origin, although having originally come from avians and sometimes humans in different occasions. This is quite interesting, as swine influenza viruses had not, until now, caused much disease or been incredibly good at human-human transmission. As the authors point out, this virus probably had been circulating for a while in pigs, unnoticed due to lack of monitoring.

Analysis of the virus genome showed that none of the molecular signatures of increased transmissibility or virulence of A(H1N1) viruses can be found in this strain, and that functionally important receptor binding sites on HA do not differ from classic swine influenza viruses. No markers were also found that indicate adaptation to the human host or features of previous pandemic virus. The main important difference seems to be a genetic marker for resistance to adamantine antivirals, but the virus still seems to be sensitive to the other type of antivirals, the neuraminidase inhibitors. So, this study concludes, we must be worried about this virus firstly because we don’t really know what makes it good at replicating in humans, and secondly because it has a genetic composition not seen before, so we don’t really know what to expect.

I then went on to read a couple of papers regarding studies done in mammal models such as ferrets (apparently classic models for influenza studies) and mice.

The first study was published in Science also in July, by Maines et al. This post is becoming quite long already, so I’ll just summarise their results (based on 3 independently isolated 2009 A(H1N1) viruses as compared with a seasonal influenza strain). They basically inoculated 106 p.f.u in ferrets and monitored different indicators such as body weight, viral titres, direct and indirect transmissibility, etc. The main conclusion from the study was that 2009 A(H1N1) caused higher morbidity (weight loss, etc, depending on which virus isolate), showed, unlike seasonal flu, high viral titres in the lower respiratory tract or the intestine, but that it was less efficient at indirect transmission (putting healthy ferrets in cages near to inoculated animals).


The second animal study I looked at was published online by Nature, in what they call a ‘near final version’. They also used virus isolated from infected humans, though not all of which were the same as in the Science paper. They studied the effect of these viruses in mice, macaques, ferrets and pigs, though not all of the virus isolates seem to have been used in all of the experiments. They also looked at more indicators, such as pro-inflammatory cytokines, lung pathology, etc. In ferrets the results were similar to those of the Science paper, except that this time indirect transmission was successful. In mice and macaques, the 2009 A(H1N1) virus seems to be worse than the seasonal virus (at least one of the isolates that they seem to mostly use throughout, CA04) although according with which criteria varies with the model. The inoculated pigs were asymptomatic, explaining why the virus had not been noticed before in this animal.

So, what are the conclusions I could get, overall, from these three studies? Overall it seems that the 2009 A(H1N1) virus is worse than the seasonal influenza viruses in mammalian models. However, how much worse is probably hard to say. I am not familiar at all with viral studies, so it is hard for me to critically analyse these studies, but it seems to me that in most cases the n numbers were quite small (n=3 in some cases in the Nature study), but this is perhaps not surprising, as they must have been quite quick studies, considering the current need to understand this new virus. And, as it is probably the case in most animal studies in general, I reckon the doses used were probably much higher than what humans would be exposed to in the real world. In addition, there is always the question on how well do these animal models really reflect what happens in humans.

In any case, it seems that so far these studies indicate that 2009 A(H1N1) is, in these models, worse than seasonal flu. Whether it is as bad as some of the pandemics that have been affecting us in the last 100 years it is unknown. The other worry is of course that the virus might evolve into something much more dangerous. Though all the virus isolates obtained so far tend to be quite similar, 5 minor genome variants have already been detected. Restricting the spread of the virus, even if it is not so bad right now, might be a good way of preventing it from acquiring any more deadly features. Whether this data justifies the measures being taken by most government, that is really, as PHD comics puts it so well, beyong the scope of my area of study. I will leave that to those who study the spread of pandemics and know of population health. I think we have had enough speculation for one disease.

Garten, R., Davis, C., Russell, C., Shu, B., Lindstrom, S., Balish, A., Sessions, W., Xu, X., Skepner, E., Deyde, V., Okomo-Adhiambo, M., Gubareva, L., Barnes, J., Smith, C., Emery, S., Hillman, M., Rivailler, P., Smagala, J., de Graaf, M., Burke, D., Fouchier, R., Pappas, C., Alpuche-Aranda, C., Lopez-Gatell, H., Olivera, H., Lopez, I., Myers, C., Faix, D., Blair, P., Yu, C., Keene, K., Dotson, P., Boxrud, D., Sambol, A., Abid, S., St. George, K., Bannerman, T., Moore, A., Stringer, D., Blevins, P., Demmler-Harrison, G., Ginsberg, M., Kriner, P., Waterman, S., Smole, S., Guevara, H., Belongia, E., Clark, P., Beatrice, S., Donis, R., Katz, J., Finelli, L., Bridges, C., Shaw, M., Jernigan, D., Uyeki, T., Smith, D., Klimov, A., & Cox, N. (2009). Antigenic and Genetic Characteristics of Swine-Origin 2009 A(H1N1) Influenza Viruses Circulating in Humans Science, 325 (5937), 197-201 DOI: 10.1126/science.1176225

Maines TR, Jayaraman A, Belser JA, Wadford DA, Pappas C, Zeng H, Gustin KM, Pearce MB, Viswanathan K, Shriver ZH, Raman R, Cox NJ, Sasisekharan R, Katz JM, & Tumpey TM (2009). Transmission and pathogenesis of swine-origin 2009 A(H1N1) influenza viruses in ferrets and mice. Science (New York, N.Y.), 325 (5939), 484-7 PMID: 19574347

Itoh, Y., Shinya, K., Kiso, M., Watanabe, T., Sakoda, Y., Hatta, M., Muramoto, Y., Tamura, D., Sakai-Tagawa, Y., Noda, T., Sakabe, S., Imai, M., Hatta, Y., Watanabe, S., Li, C., Yamada, S., Fujii, K., Murakami, S., Imai, H., Kakugawa, S., Ito, M., Takano, R., Iwatsuki-Horimoto, K., Shimojima, M., Horimoto, T., Goto, H., Takahashi, K., Makino, A., Ishigaki, H., Nakayama, M., Okamatsu, M., Takahashi, K., Warshauer, D., Shult, P., Saito, R., Suzuki, H., Furuta, Y., Yamashita, M., Mitamura, K., Nakano, K., Nakamura, M., Brockman-Schneider, R., Mitamura, H., Yamazaki, M., Sugaya, N., Suresh, M., Ozawa, M., Neumann, G., Gern, J., Kida, H., Ogasawara, K., & Kawaoka, Y. (2009). In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses Nature DOI: 10.1038/nature08260

Investigating the Smallest Bacterial Genome

During the first two terms of my final year at university I took on a dry project based on bacterial genetics. In retrospect this was quite a cheap move since it avoids so many of the problems involved in normal lab work, but in my defence it was the project that interested me most and I really wanted some computing experience before making PhD applications. My work was based on bacterial endosymbionts of insects, which have been of recent interest due to their extremely small genomes. Bacteria in these symbiotic relationships are given a protected, nutrient-rich environment and in return they allow insects to survive on unbalanced diets by synthesising scarce biomolecules.

Primary endosymbiotic bacteria live their entire lives inside insects and are vertically transmitted from generation to generation, a process that leads to coevolution between the bacteria and the insect. One of the results of this coevolution was major changes to the original bacterial genome, which contained many genes that are essential for free-living bacteria but are unnecessary for life within an insect. Consequently, common features of endosymbiotic genomes compared to those of free-living bacteria are severe gene loss, genome compaction and skewing of GC content.

Electron micrograph showing bacteriocytes taken from P. venusta

1 – Bacteriocyte; 2 – C. ruddii; 3 – Unidentified electron-dense mass

My project focused on Carsonella ruddii, the only bacterial endosymbiont of the psyllid, Pachypsylla venusta. It was hailed as the smallest bacterial genome chracterised when it was sequenced in 2006 and still holds that record. Its genome contains only 182 ORFs, less than 3% intergenic DNA and has a GC content of 16.5%. The bacteria appears to be provided with many nutrients by its host and its metabolism has been reduced to a few pathways: ATP synthesis, a section of the pentose phosphate pathway and biosynthesis of certain amino acids.

The early stages of my project involved a reannotation of the C. ruddii genome followed by a sequence-based functional analysis of its metabolic enzymes. Using the enzymes deemed functional in this analysis I built an updated model of the C. ruddii metabolism which could be divided into six pathways involved in amino acid biosynthesis, five of which were incomplete. The only fully intact pathway led to the production of isoleucine and valine. These are both essential amino acids for insects and are severely under-represented in the adult psyllid diet.

Four of the incomplete amino acid pathways were missing only one reaction and the conservation of the rest of each of the pathways suggested that they might still be functional in C. ruddii. The ‘missing’ reactions might occur spontaneously under some conditions or could be catalysed by unidentified enzymes. For three of these four missing reactions I found an example in the literature of a different bacterial endosymbiont which had lost that reaction but had retained the rest of the pathway. This seemed to suggest that the enzymes catalysing these reactions might be expendable and subject to loss during genome reduction in endosymbionts. Based on this and some other evidence from similar situations in endosymbionts I predicted that these pathways are probably functional in C. ruddii and that its main role symbiotic role is to provide the psyllid with essential amino acids.

The fourth of these incomplete pathways was the most interesting because I was unable to locate another endosymbiont which was missing the same reaction. The reaction was catalysed by the product of a gene, AS, which was present on the C. ruddii genome but which I had labelled as a pseudogene during functional analysis. Although it’s difficult to conclusively say that an enzyme is inactive solely by sequence analysis, multiple alignments showed that this copy of AS was extensively degraded and was missing both of its key catalytic residues as well as its substrate binding residues. However, later in the project when I was scanning an EST set taken from the insect host of C. ruddii I located another copy of AS which also had bacterial origin but which was not present on the C. ruddii genome. Sequence analysis showed that this version of AS seemed to be active and could potentially fill the gap in the pathway.

Where did this copy of AS originate from? It aligned well with the version of AS from P. aeruginosa and appeared to have a bacterial origin but was not found on the C. ruddii genome or the psyllid mitochondrial genome, both of which have been sequenced. Several lines of evidence ruled out the presence of a second bacterial endosymbiont in this symbiosis and since no plasmids had been reported during DNA sequencing of C. ruddii the source of this sequence appeared to be the nuclear genome of P. venusta itself. The presence of this bacterial sequence in the eukaryotic genome suggests that LGT may have taken place between a bacterial genome and the insect nuclear genome. This would be one explanation for the fact that C. ruddii has only 182 ORFs, which is significantly lower than the predicted minimal bacterial genome. However, it is also possible that C. ruddii uses mitochondrial proteins to survive and so LGT is not the only explanation for the low ORF count.

This was my favourite line of investigation during my project but the symbiosis between C. ruddii and P. venusta had many more interesting features that I read about over the year. One of the questions I got in my viva was whether C. ruddii should be labelled as a bacterium or an organelle. I think this question is only really important when considering a minimal bacterial genome and if C. ruddii does turn out to be importing essential proteins from elsewhere then I think that the label organelle is definitely more appropriate. However, the definition of an organelle doesn't seem to be well-established and so whether or not C. ruddii really does have the smallest bacterial genome is a matter of opinion.


Nakabachi A, Yamashita A, Toh H, Ishikawa H, Dunbar HE, Moran NA, & Hattori M (2006). The 160-kilobase genome of the bacterial endosymbiont Carsonella. Science (New York, N.Y.), 314 (5797) PMID: 17038615

Gil, R., Silva, F., Pereto, J., & Moya, A. (2004). Determination of the Core of a Minimal Bacterial Gene Set Microbiology and Molecular Biology Reviews, 68 (3), 518-537 DOI: 10.1128/MMBR.68.3.518-537.2004

Glass, J. (2006). Essential genes of a minimal bacterium Proceedings of the National Academy of Sciences, 103 (2), 425-430 DOI: 10.1073/pnas.0510013103

Thao, M., Moran, N., Abbot, P., Brennan, E., Burckhardt, D., & Baumann, P. (2000). Cospeciation of Psyllids and Their Primary Prokaryotic Endosymbionts Applied and Environmental Microbiology, 66 (7), 2898-2905 DOI: 10.1128/AEM.66.7.2898-2905.2000

Non-B DNA structures and disease

People seem to have started contributing actively to the blog again so I thought it was my turn to try again. And this is an achievement, as facing any science, in particular anything written by me about it, would have been impossible a month ago.

I decided to keep things simple for this one and just give a short introduction on the theme of non-B DNA structures and how they influence disease, the topic of my open essay.

When we think about DNA, we think about its B-DNA structure, the one that Watson and Crick described in that famous Nature paper more than 50 years ago, i.e. a right-handed double helix, 20Å diameter, in which the base pairs are almost perpendicular to the axis of the helix, 10.5bps per turn. Although most DNA will be in this structure, it can also form other structures, the so called non-B DNA structures. Some of these are shown in the figure. These alternative structures have a few common characteristics: firstly, whether or not a structure is formed, and which specific structure, seems to be sequence dependent, as it often involves formation of new pairings of bases. Secondly, most of these structures are in higher energetic state than normal B-DNA, for example, because they require the separation and reformation of hydrogen bonds. Overall, therefore, DNA with a favourable sequence tends to remain in its B-DNA form, requiring events such as DNA replication, transcription or protein binding to be transiently converted to these unusual structures. Sequences with propensity to form unusual structures are quite common in the human genome, and it seems that in some cases they are necessary for the normal functioning of the cell. However, they have also been implicated in disease, and this is what I’ll be focusing till the end of this post.

There is a considerable list of diseases thought to involve, as part of their pathology, the formation of non-B DNA structures, but the mechanisms by which this is though to happen can vary. For example, certain sequences/structures have been thought to cause genomic instability, e.g. by causing chromosomal rearrangements due to the propensity of these structures to promote double strand breaks. Another interesting form of genomic instability associated with these unusual structures is repeat expansion, implicated in motor diseases such as Huntington’s disease. Non-B DNA structures, namely Z-DNA, have also been associated with viral infections.

Now, there isn’t really enough space here to talk about everything, so I’m going to give one examples of a situation in which a non-B DNA structure is though to be involved in disease. Friedreich ataxia (FRDA) is a disease caused by the expansion of GAA•TTC tracts in intron 1 of a gene encoding the protein frataxin, essential for mitochondrial function. In this disease, repeat expansion is associated with loss of protein expression. One of the current models by which frataxin production is thought to be reduced in expanded GAA•TTC repeats is based on their ability to form triplexes (an alternative model suggests that epigenetic changes may also be important). Duplex opening within the repeated region, due to the passage of RNA polymerase, is thought to allow one of the separated single strands to form Hoogsteen hydrogen bonds with the purine strand of a B-DNA duplex within the same repeated sequence. This leads to the formation of a 3-stranded helix. Its formation on the non-template GAA strand in frataxin probably interferes with RNA polymerase progression. The free template strand is then thought to base pair with the newly synthesised RNA transcript, forming stable RNA/DNA dimers and preventing further transcription.

Now, this is only a model, for a specific type of structure within the context of a specific disease. And although different sequences have been shown to form these unusual structures, and these structures to be somehow associated with specific diseases, to neatly demonstrate how exactly one influences the other seems to be quite hard. This is particularly difficult because different groups often use different model organisms or protocols, leading to sometimes contradictory results. Overall, although I liked writing about this topic, since I had never considered how the structure of DNA could have an impact on disease, I got the feeling that a lot of work still needs to be done before convincing evidence is given for how exactly these structures impact on our wellbeing, and what sort of therapeutics can be developed from this knowledge.

Red, GAA repeat strand; blue, GTT complementary strand; orange, RNA transcript; yellow, RNA polymerase. 1, Intramolecular triplex; 2, Stalled RNA polymerase; 3, DNA/RNA hybrid

Hebert, M. (2008). Targeting the gene in Friedreich ataxia Biochimie, 90 (8), 1131-1139 DOI: 10.1016/j.biochi.2007.12.005

Bacolla, A. (2004). Non-B DNA Conformations, Genomic Rearrangements, and Human Disease Journal of Biological Chemistry, 279 (46), 47411-47414 DOI: 10.1074/jbc.R400028200

WELLS, R. (2007). Non-B DNA conformations, mutagenesis and disease Trends in Biochemical Sciences, 32 (6), 271-278 DOI: 10.1016/j.tibs.2007.04.003

Wang, G., & Vasquez, K. (2009). Models for chromosomal replication-independent non-B DNA structure-induced genetic instability Molecular Carcinogenesis, 48 (4), 286-298 DOI: 10.1002/mc.20508

Learning how to tell time with cyanobacteria

Most living things can innately tell the time. If you take a person and put them into continuous darkness for months by themselves they will follow a sleep-wake cycle of about 24-25 hours (as well as going a little mad). Circadian rhythms allow life to predict the raising and setting of the sun to allow us to change our gene expression metabolism and behaviour with the changing of the day. Predicting changes in temperatures and light levels are very important. Circadian clocks have three important features. First of all they are free-running at about 24 hours (in constant conditions like light or darkness they will keep going at ~24 hours). Secondly they are temperature compasatory meaning that changes in temperature do not greatly affect the clock. Finally they must be able to be reset by environmental inputs. The changes of the seasons mean days get longer or shorter so to accurately predict the raising of the sun your clock must be reset. Plus for those who fly great distances we must reset according to the new environment and sadly suffer from jet lag while we are slowly adjusting (Johnson et al., 2008).

These clocks have evolved independently multiple times. In animals, plants, some fungi and recently discovered in cyanobacteria (Johnson et al., 2008). It was once thought because prokaryotes divide so raplidly that a 24 hour rhythm would be useless to them but that is not the case (Johnson et al., 1996). A great deal has been learnt about rhythms in bacteria but there are still many unanswered questions. The fact circadian clocks have evolved multiple times shows it offers a great advantage to those that posses one but was no present in the last common ancestor of life. Changing behaviour to only occur at a specific time of day is a great advantage to organisms, plants to photosynthesize and animals to look for food at the right times (Bell-Pedersen et al., 2005).

Cyanobacteria also rely on the sun for energy so predicting when the sun will rise allows them to start synthesising photosystem proteins before the sun raises while not producing them all night or waiting for the sun to raise. It has been shown when you make a mixed culture of bacteria with a mutant clock (no rhythmic changes in gene expression) and wild-type then wild-type will soon outcompete the mutants. Those that have a different length period (eg 16 hours rather than 24 hours) will always lose to wild-type in 24 hour day conditions but wild-type will lose in conditions that match the internal clock of these mutants (Yan et al., 1998). Interestingly in cyanobacteria the whole genome shows rhythmic expression! (Liu et al., 1995) This is in contrast to most organisms that control 15%-35% of their genome in a circadian dependant manner (McDonald and Rosbach, 2001; Correa et al., 2003; Michael and McClung, 2003). This is controlled by the supercoiled state of the whole genome! (Smith and Williams, 2006) I eagerly await the paper that explains how this achieved.

What is rather fascinating about this system is the oscillator. In most organisms gene expression is negatively regulated by its product in a way it cycles its levels over 24 hours and controls output (Bell-Pedersen et al., 2005). In cyanobacteria, this is not the case. Rhythmic expression of clock proteins occur but they do not repress there own synthesis. Also an in vitro oscillator can be setup. In the test tube the clock protein KiaC will cycle through different phosphorylation states if with KaiA, KaiB and ATP. In the organism this forms the post-translational oscillator (Nakajima et al., 2005). This is temperature compensatory and this is built into the ATPase activity of KaiC (Terauchi et al., 2007). It has been shown even when locked in one phosphorylation state you get a weak rhythm so a transcriptional-translational feedback loop as in eukaryotes is needed for a robust system (Kitayama et al., 2008) posing the question of whether this occurs in eukaryotes.

And to finish off I will add one final oddity about the circadian rhythms of cyanobacteria. Instead of using a photoreceptor to sense light inputs like other organisms they read the state of the metabolism. When light is high photosynthesis is occurring and the redox state of the cell changes and is seen as an input to reset the clock (Ivleva et al., 2005).

References

*Of importance
**Truly significant

Bell-Pedersen, D., Cassone, V.M., Earnest, D.J., Golden, S.S., Hardin, P.E., Thomas, T.L. and Zoran, M.J. (2005) Circadian rhythms from multiple oscillators: Lessons from diverse organisms. Nature Reviews Genetics, 6, 544-556.

Correa, A., Lewis, A.Z., Greene, A.V., March, I.J., Gomer, R.H. and Bell-Pedersen, D. (2003) Multiple oscillators regulate circadian gene expression in Neurospora. Proceedings of the National Academy of Sciences of the United States of America, 100, 13597-13602.

Ivleva, N.B., Bramlett, M.R., Lindahl, P.A. and Golden, S.S. (2005) LdpA: a component of the circadian clock senses redox state of the cell. Embo Journal, 24, 1202-1210.

Johnson, C.H., Golden, S.S., Ishiura, M. and Kondo, T. (1996) Circadian clocks in prokaryotes. Molecular Microbiology, 21, 5-11.

Johnson, C.H., Mori, T. and Xu, Y. (2008) A Cyanobacterial Circadian Clockwork. Current Biology, 18, R816-R825.

**Kitayama, Y., Nishiwaki, T., Terauchi, K. and Kondo, T. (2008) Dual KaiC-based oscillations constitute the circadian system of cyanobacteria. Genes & Development, 22, 1513-1521.

Liu, Y., Tsinoremas, N.F., Johnson, C.H., Lebedeva, N.V., Golden, S.S., Ishiura, M. and Kondo, T. (1995) Circadian Orchestration of Gene-Expression in Cyanobacteria. Genes & Development, 9, 1469-1478.

McDonald, M.J. and Rosbach, M. (2001) Microarray analysis and organization of circadian gene expression Drosophila. Cell, 107, 567-578.

Michael, T.P. and McClung, C.R. (2003) Enhancer trapping reveals widespread circadian clock transcriptional control in Arabidopsis. Plant Physiology, 132, 629-639.

*Nakajima, M., Imai, K., Ito, H., Nishiwaki, T., Murayama, Y., Iwasaki, H., Oyarna, T. and Kondo, T. (2005) Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science, 308, 414-415.

Smith, R.M. and Williams, S.B. (2006) Circadian rhythms in gene transcription imparted by chromosome compaction in the cyanobacterium Synechococcus elongatus. Proceedings of the National Academy of Sciences of the United States of America, 103, 8564-8569.

Terauchi, K., Kitayama, Y., Nishiwaki, T., Miwa, K., Murayama, Y., Oyama, T. and Kondo, T. (2007) ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria. Proceedings of the National Academy of Sciences of the United States of America, 104, 16377-16381.

Yan, O.Y., Andersson, C.R., Kondo, T., Golden, S.S. and Johnson, C.H. (1998) Resonating circadian clocks enhance fitness in cyanobacteria. Proceedings of the National Academy of Sciences of the United States of America, 95, 8660-8664.

The Life and Death of Elysia Chlorotica

Since the end of university I’ve had some trouble letting go of science as a constant occupation. Fortunately I was given the chance to post here about an old journal club presentation rather than go cold turkey, so here is a short piece on one of the most interesting organisms I’ve read about.

The picture on the left is of a sea slug of the genus Elysia which has evolved to blend in with its surroundings by looking almost exactly like a small leaf. The green colour that makes this camouflage so convincing is produced when the slug feeds on algae and steals their chloroplasts, subsequently storing them throughout its body. The use of chloroplast pigments to produce a leaf colour is common among sea slugs but the subject of this post is exceptional among the rest of its family. While in the majority of sea slugs the chloroplasts stop working within days or weeks of incorporation, those taken up by Elysia chlorotica can remain stable and active within the slug for at least ten months. As a result the slug is a rare example of a photosynthetic animal and it can survive without food for a long time, as long as it has light and carbon dioxide.

E. chlorotica has coevolved with a specific species of algae named Vaucheria litorea, which is a source of food and chloroplasts for the slug. The metamorphosis of E. chlorotica larvae into their slug form takes place only if the larvae are attached to filaments of V. litorea and in the absence of this algal species the larvae will die. Immediately after metamorphosis the slugs feed on the algae to which they are attached and take up their chloroplasts, incorporating them into their branched digestive tract.

As mentioned previously, most species of sea slug lose their chloroplast functions soon after obtaining them from algae. This is expected to be because the majority of genes required for chloroplast survival and function are encoded on the algal nucleus. Since animals do not have genes involved in chloroplast maintenance the question of how E. chlorotica manages to sustain its stolen plastids was investigated by several labs. In a recent study, the chloroplast genome of V. litorea was sequenced to determine whether it had greater genetic autonomy than previously sequenced chloroplasts, however, the chloroplast genome was found to be largely normal and was missing many genes essential for photosynthesis.

One of these missing genes, psbO, encodes a component of photosystem II which is vulnerable to damage during photosynthesis and needs to be regularly resynthesised for chloroplast function. Rumpho et al. were able to amplify the whole of this gene from sea slug DNA using primers based on seqeuence databases. The amplified sequence was shown to correspond exactly to the version of psbO from V. litorea, indicating that lateral gene transfer had taken place between the algae and the slug. The same sequence could be amplified from sea slug eggs which had not yet encountered algae, confirming that the sequence had entered the E. chlorotica germline. The authors behind this study speculate that all of the other genes needed for chloroplast survival and function have also been transferred to the slug germline, explaining the unique ability of E. chlorotica to maintain its chloroplasts for its entire life. So it seems that the sea slugs have stolen both DNA and organelles from algae, which is quite an achievement.

Another interesting property of E. chlorotica is that its generations are separated from each other. Whether slugs are collected in the lab or monitored in their natural environment, the entire adult population undergoes a synchronous death every year after they have laid their eggs. Pierce et al. observed that viral particles could be seen in the cytoplasm and nuclei of slugs just before their mass death and suggested that they may be responsible for the mortalities. The morphology of the viruses and the presence of reverse transcriptase activity suggest that they are retroviruses and, since the particles were found even under controlled laboratory conditions, it is likely that the virus is encoded on the genome of the sea slug. The group who made this observation suggested that the viruses may respond to annual environmental changes and kill the sea slugs in the spring, though this hypothesis has not been tested so far.

Unfortunately, the last paper I found on research into the viruses within the sea slugs was in 1999 and so research into this interesting aspect of the organism may not be ongoing. However I'm hoping to find some more about it in the future.

Rumpho, M. (2000). Solar-Powered Sea Slugs. Mollusc/Algal Chloroplast Symbiosis PLANT PHYSIOLOGY, 123 (1), 29-38 DOI: 10.1104/pp.123.1.29

Pierce, S., Maugel, T., Rumpho, M., Hanten, J., & Mondy, W. (1999). Annual Viral Expression in a Sea Slug Population: Life Cycle Control and Symbiotic Chloroplast Maintenance Biological Bulletin, 197 (1) DOI: 10.2307/1542990

Pierce, S., Massey, S., Hanten, J., & Curtis, N. (2003). Horizontal Transfer of Functional Nuclear Genes between Multicellular Organisms Biological Bulletin, 204 (3) DOI: 10.2307/1543594

Rumpho, M., Worful, J., Lee, J., Kannan, K., Tyler, M., Bhattacharya, D., Moustafa, A., & Manhart, J. (2008). From the Cover: Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica Proceedings of the National Academy of Sciences, 105 (46), 17867-17871 DOI: 10.1073/pnas.0804968105

Flu flu go away, come again another day (when I don't have exams)

Right I should be revising for those finals but with my arm hurting I am using it as an excuse not to write practice essays today and typing doesn't put as much strain on it (I think).

So Swine flu/Mexican flu/H1N1 flu has brought Mexico to a halt and scared the rest of the world into a panic. Big mouth Vice-president Biden of the USA has done it again saying he wouldn't trust travelling etc. fair point but you are trying to calm the masses as a leader. From what is humble virologist wannabe can see is this new hybrid strain made from genes of avian, swine and human flu is doing is spreading very quickly among people. For some reason it has killed lots in Mexico (allegedly) but only causing mild cases elsewhere. Today however the researcher at Mill Hill have suggested this strain is not very deadly. This paints a rather paradoxical picture. We see many deaths in Mexico from a fast spreading flu. Yes the world should be worried and has done the right thing by preparing for the worse. But we have only seen mild cases in most people outside Mexico and its genome suggests it is only mild. It doesnt have the make-up that causes cytokine storm that kills young people (like Spanish flu did). That is one thing not to worry about (right now). Plus it only infects cells near the top of the respiratory track so doesn't cause infections deep in the lungs, making it easy to spread but not as deadly as some.

So two things need to be thought about 1) why has it killed so many people in Mexico? and 2) what shall we call it? I am not going to tackle the first but I have some thought. The second I think should be Mexican flu. Swine flu is hurting the pig farming industry (some countries have banned pork imports!) while H1N1 is just rubbish. Loads of flu strains are called H1N1. Some seasonal flu is H1N1 and Spanish flu of 1919 was H1N1!!! I personally think the H and N naming system is out dated and inaccurate. It is not the H and N proteins that make a virus what is is per se but the many other changes with them.

Closing thought though, Flu has a high mutation rate with its RNA genome (no where near HIV but still more than what we would like) so changes may occur soon that could be game changers.

http://news.bbc.co.uk/1/hi/health/8028371.stm

When a bit more fat is not that bad

Nothing is as simple as it looks in Biology. We can’t even rely on gene or protein sequences to tell us what is going to happen. At the chromosomal level, the new field of epigenetics is showing how the nucleotide sequence does not define everything. At protein level we have to consider post-translational modifications. My final year project, now at the final write up stages, concentrated on an aspect of Trypanosoma brucei molecular biology which I very pompously described as a potential drug target and the cure for African sleeping disease. This bit was probably ‘paper talk’ though. In any case, the project focused on protein palmitoylation.

Palmitoylation is part of a group of several lipid modifications that can occur in proteins. Examples include N-myristoylation, prenylation and GPI-anchors. N-myristoylation is the reason why I thought it was not pushing it to much to say that we can kill parasites by targeting these modifications. N-myristoylation corresponds to the co-translational addition of myristate to an N-terminus glycine, and is catalyzed by N-myristoyltransferases (NMT). It so happens that NMTs were shown to be essential for parasite viability, and several antifungal NMT inhibitors apparently work on reducing NMT activity in T.brucei as well(1).

But back to palmitoylation, the post-translational addition of palmitic acid to a cysteine residue (reviewed in 2 and 3). It can occur in any place in the protein, and so far no consensus palmitoylation motif has been identified. Often it occurs in an N-terminus cysteine, just next to a myristoylation site. These proteins are said to be dually acylated. Palmitoylation can also happen in close proximity to prenylation sites. Palmitoylation can perform many functions in the cell, the most obvious being protein tethering to membranes and cellular localization. It has also been shown to interfere with protein-protein interactions and even with protein degradation. For example, the yeast SNARE Tgl1, when is not palmitoylated, can interact with the ubiquitin ligase Tul1(4).

Those paying attention (or still reading) will think that it is a bit strange that palmitoylation can regulate protein degradation like that. In fact, most of these lipid modifications, N-myristoylation for example, last for the life time of the protein. Well, palmitoylation is characterized by a unique feature: it is reversible. This means that it can determine processes that other lipidations cannot. Dynamic trafficking of proteins of proteins, for example. This is the case of Ras protein, which through an deacylation/reacylation cycle is able to exchange between the plasma membrane and the Golgi apparatus (5). Reversibility can also be important for signalling. Regulator of G-protein signalling 2 (RGS2) was shown to increase or decrease its GTPase-activating ability depending on which of its cysteine residues are palmitoylated(6).

The process of palmitoylation in itself is catalyzed by the enzymes palmitoyl-acyltransferases (PATs). These were pretty hard to identify, apparently. Not only they were difficult to purify, but the reaction itself can occur spontaneously with biological significance(7), which made many sceptic on whether these enzymes existed at all. However, they were identified, first in yeast, now in mammals and in my dear T.brucei.

And what was supposed to be a short post if already long enough. I finish by adding that my project was about finding proteins that were palmitoylated and that would change their localization in the absence of this modification, in an attempt to identify substrates for RNAi PAT studies in T.brucei. I must say that the project was not particularly successful, but I developed the important skill of writing long discussions based on few results, which I bet will come handy in the future.

I now challenge Mel to continue in this post-translational modification topic and tell us about his project on glycosylation.


References:

1. Price, H. (2002). Myristoyl-CoA:Protein N-Myristoyltransferase, an Essential Enzyme and Potential Drug Target in Kinetoplastid Parasites Journal of Biological Chemistry, 278 (9), 7206-7214 DOI: 10.1074/jbc.M211391200

2. Nadolski MJ, Linder ME. Protein lipidation. FEBS J 2007;274:5202-10.

3. Greaves J, Chamberlain LH. Palmitoylation-dependent protein sorting. J Cell Biol 2007;176:249-54.

4. Valdez-Taubas J, Pelham H. Swf1-dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation. EMBO J 2005;24:2524-32.

5. Rocks O, Peyker A, Kahms M, et al. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 2005;307:1746-52.

6. Ni J, Qu L, Yang H, et al. Palmitoylation and its effect on the GTPase-activating activity and conformation of RGS2. Int J Biochem Cell Biol 2006;38:2209-18.

7. Kummel D, Heinemann U, Veit M. Unique self-palmitoylation activity of the transport protein particle component Bet3: a mechanism required for protein stability. Proc Natl Acad Sci U S A 2006;103:12701-6.

Faith of a different sort

So having finals upon us should be no excuse to stop the advancement of scientific and philosophical thought on here. So when I wandered into town today to do a favour for my dad (30 min walk for me, 3 hour drive for him) I saw a stall with a sign talking about truth. I still have no idea what a truth is so I thought I would have a look at what truths they were talking about. It was a stall full of Jehovah’s witnesses and I was very nice and polite before you say anything! I always am to there face. Being nasty and telling them, ‘you are probably was wrong as you can be’ doesn’t help win them over to your cause! Richard Dawkins QED. But over our chat I mentioned I supported evolution and was an atheist. But they suggested trusting some of the gaps in evolution had an explanation needed faith. This is an interesting one. It doesn’t but it requires something. I know very little physics and geology. If someone was to question the age of the earth in front of me I would have to turn around I say ‘I don’t know how they worked it out and don’t understand the sciences behind it’ so is it faith in these people I have in other scientists? Probably. But is it bad or wrong for me. I think it is a different sort of faith. First of all it is not blind absolute trust. I know I can easily be wrong about many things (it doesn’t happen often enough though) and I know other can be.

We all need to have this kind of faith to get through life. We cannot know everything so we need to trust others or ‘the system’. Imagine a friend offers you a life in there car but you have never seen them drive before in your life. You don’t know if they are good or bad or will get you killed or not. But you get in anyway. Perhaps because you are so lazy you don’t care if you could die as long as it gets you there faster like me. Or because you have faith that having passed a driving test and having belief in there abilities means you can trust them. It is like knowing peer reviewing produces mostly good knowledge and the author is confident in the results even if you don’t understand the experiments or the maths used to analyse it. This evidence based faith is not a bad thing but needs use to take the results with a large pinch of results. It is not perfect. Sometimes someone who you think would be a good driver turns out not to be while taking a ‘fact’ at face value because you trust the scientist you get it from could mean you are wrong but sometimes we need to take these ‘leaps of faith’. Just know that is what you are doing when you do.

Agree or not?

Drosophila: Virgins, balancers and jump-outs

A wise man once said that the sooner you find your model organism, the better. Ignoring the fact that the wise man I am quoting is actually me, I think I generally agree with him. This year I am working on an exciting project which extensively uses the model organism Drosophila melanogaster, geneticists’ favourite little invertebrate. In the 15 short weeks I have been working with flies, I have learned a great deal from these wonderful creatures and experienced emotions from adoration to amusement to frustrated rage in my dealings with them.

Briefly, this is your average everyday “fruit fly” (even though a more appropriate name would be “vinegar fly”) with the complete package of a head, a thorax with 2 wings and 6 legs, an abdomen, and everything else that makes flies flies. I will spare you the tediously detailed anatomy and physiology lecture and will instead jump straight to the cool stuff that interests us geneticists and you readers of genetics blogs.

One of the most interesting things about Drosophila is in fact the people that use them in research. These people are commonly known as “fly pushers”, since they do in fact spend a lot of their time pushing flies around under a microscope using an old half-mangled paintbrush. One thing that you will inevitably hear about once you start hanging out with fly pushers is how interested they are in virgin females. Flies, that is. You see, since experiments in fly genetics involve crossing male flies from one line to female flies of another in order to obtain progeny of the required genotype, one must ensure that the females have not already been fertilised by males before picked out and crossed to a different line. Collecting virgins involves looking for flies that have a dark spot in their abdomen called the meconium, which is visible for 2-3 hours from eclosion (i.e. emergence of the adult from the pupa). Females will not be receptive to males for up to 8 hours from eclosion so any fly that has a meconium can be safely considered a virgin. The most efficient way to collect virgins is to empty a vial of eclosing flies in the morning, selecting females with visible meconium, and returning in the evening. Any females in that vial will be virgins even if the meconium isn’t visible, since less than 8 hours will have passed since your last collection. Much like the world we live in these days, virgins can be rather hard to find, so if you ever hear a scientist worrying about the fact that they need as many virgins as possible, you now have an idea of what they are talking about.




Now let’s delve into some actual genetics. One of the multitude of reasons Drosophila are an excellent test tube for genetic experimentation is something called a “balancer chromosome”. A line of flies carrying a recessive lethal mutation can be maintained by tracking the mutation using dominant markers, or by “balancing” it over a different lethal mutation on its homologous chromosome. This way each chromosome “rescues” the mutation the other is carrying. However, if recombination takes place between these chromosomes, it is likely that you will then have one chromosome carrying both mutations over a wild type chromosome, which will quickly take over the stock and the mutations will be lost. You could always outcross males every generation (there is no recombination in male Drosophila) but that would be very laborious. In order to get around this problem, fly geneticists initially discovered and subsequently intentionally generated balancer chromosomes. These are full chromosomes which contain multiple inversions (essentially all the genes are still there and intact, but their order is jumbled up) in order to suppress recombination with its homologous chromosome and make any gametes with recombined chromosomes non-viable due to aneuploidy. They also carry some dominant visible markers such as Cy (curly wings), Tb (tubby), Sb (stubble) and others, and some recessive lethal markers, meaning that they can never go homozygous.

So, to illustrate this, if you have a recessive lethal mutation on chromosome 2 (let’s now call that chromosome “Yorf”), in order to establish a line that will self-perpetuate and not need constant attention, all you need to do is cross it to a line that carries a balancer second chromosome, for example the chromosome CyO. You will now have a line whose flies will always have the same genotype, i.e. Yorf/CyO. As the Yorf/CyO flies interbreed, any Yorf/Yorf progeny will not survive because the mutation will be lethal, and any CyO/CyO flies will also not survive because of the recessive lethal marker that CyO carries. There will also never be any recombination between the Yorf and CyO chromosomes because of the multiple inversions in CyO that prevent homologous pairing. Wonderful, isn’t it?

Another great thing about Drosophila is insertional mutagenesis. P-elements are transposons, short stretches of DNA which, in the presence of transposase and absence of inhibitor, are excised from the genome and reinserted randomly into another site. Some P-elements are rendered inactive by having the transposase gene deleted. This temporarily immobilises the transposon, until that fly is crossed to a fly carrying an active transposase gene. In the progeny, the P-element will jump out, and if it inserts into the coding sequence of a gene, it can disrupt its function. A mutagenesis screen can then be carried out and the insertion locus can be mapped using standard complementation or cytological mapping. There is however an application of P-element mutagenesis that has accelerated Drosophila genetics in recent years.

The targeted gene knock-out system developed in mice is an excellent way to study the role of single genes, but the long generation time and ethical concerns make the mouse a rather cumbersome model organism. An analogous targeted gene disruption tool has been developed in Drosophila, taking advantage of a property of P-element transposition. When a P-element is transposed, its excision from the genome can sometimes be imprecise, taking some of its flanking DNA along with it, thus causing a deletion in that gene. Since an extensive library of mapped P-element inserts already exists (in over half the fly genome), a line carrying a P-element insert in a particular gene can be obtained and the P-element “jumped out” by crossing to a transposase line. In the cases where the jump-out has been imprecise, the result will effectively be a targeted gene knock-out.

This is, of course, only scratching the surface of the range of genetic tools that Drosophila offers. The best way to understand fly genetics is to try some crosses on paper yourself. Some quite amazing things can be achieved within a few weeks’ time simply by performing a series of crosses and studying the progeny. The waiting time is still a little too long for impatient people like myself, but careful time planning can ensure that you always have some flies to play with and are never stuck staring at a pupa, waiting for the damn fly to emerge.