More news from the lab and this time its me ranting about the behaviour of my cells.
When investigating the function of a particular gene one tactic geneticists have availible to them is to break the gene and see what happens. One way of achieving this is to ‘knock it out,’ to entirely remove the gene from the genome, or otherwise inactivate it. This allows you to study the phenotype (Characteristics) of the resultant cells to try and work out what the gene was probably doing. However in some cases the gene is very important indeed, and the resultant phenotype is merely ‘dead’ which makes it very difficult to actually do anything with the cells, and impossible to get enough to actually characterise them. Dead isn’t a very usefull phenotype, we want to know exactly what type of dead they are.
One way of overcoming this hurdle is through what is known as a conditional mutant. As the name suggests, this is a mutated version of a gene which only shows its abberent behaviour under certain ‘restrictive’ conditions. One of the most common forms of conditional mutant is the temperature sensitive mutant, which only shows its ‘dead’ phenotype at higher temperatures. Thus most work is conducted at a lower temperature and then the cells are shifted to a higher temperature for testing.
The only problem is that even at the cooler temperature temperature-sensitive mutants are rarely wild-type (normal) and are usually at a slight disadvantage, showing slower growth than normal cells. Now as any student of Darwin will realise we have a bit of a problem. Genomes mutate with a low background frequency which is nethertheless high enough to cause problems, as any mutations that help rescue the slight disadvantages will be selected for, either by outcompetition in liquid culture, or by selection bias (which is basically me selecting the bigest colonies on my plates). Unfortunately sometimes these new mutations also help to make the cells grow better at the higher, normally restrictive temperature, and better than dead happens to be alive.
Now this is very annoying to me, as I want my cells to be dead at the restrictive temperature. For one it identifies where my mutant gene is, and for another is important for other experiements in which I try and make them not dead through other means (largely by making other mutations, or introducing other genes). But seing as I do most my growth at the colder permissive temperature there is no way of telling if my cells have reverted untill I shift them to the higher temperature, at which point they are dead and of no use to me. Fortunately it is possible to replica plate cells, in which a plat of cells is transfered onto velvet, and then ‘copied’ onto two seperate plates, one of which is kept at the restrictive temperature and the other the permissive. This allows me to make sure I choose cells which grow on one plate but not the other.
Unfortunately I am finding that selection is something which must be maintained almost constantly, and even then revertants still seem to slip through the cracks. It seems sometimes you only realise that something has gone wrong when things grow when they shouldn’t have done.
[tags]genetics, pombe, science, phd, mutants[/tags]
Posts Tagged ‘science’
More news from the lab and this time its me ranting about the behaviour of my cells.
Oh dear. I like Wikipedia, its a lot of fun and usually reasonably accurate, but I just stumbled across an article which was almost entirely false. I can understand a few misconceptions sneaking in, but what possesses someone to write about a topic on which they clearly have no idea; especially when its something that specialist knowledge would seem a fairly obvious requirement.
The article in question was discussing the ubiquitin like protein Nedd8, something on which I’m currently doing a PhD. Ubiquitin is well characterised and has several roles, including targeting proteins to the proteasome, the ‘dustbin’ of the cell. Nedd8 is similar to Ubiquitin in structure, and the way it is used to tag other proteins. However it does not target proteins to the proteasome, and instead performs other functions. Yet the wikipedia article suggested that Nedd8 performed exactly the same role as ubiquitin, and accompanied this with ill formed sentences which gave little in the way of information and instead hinted at other severe misconceptions.
Neddylation is a protein degrading process analogous to ubiquitinylation in higher organisms (eukaryotes) in which NEDD8 is a key player. Unlike lysosomal degradation of proteins, a tagging occurs by attaching a small protein chain to mark for an enzyme complex (proteasome) by which they are then broken down. In fact, the term “neddylation” means a special form of ubiquitinylation initiated by an uncommon subset of proteins (see below), of which the initiators as well as the substrates are unique although the same pattern as in ubiquitinylation is followed.
So I have already addressed that Neddylation is not ‘a protein degrading process’ but furthermore neither is ubiquitination. The latter itself can result in protein degradation in some circumstances, but it itself is not protein degradation.
‘In which Nedd8 is a Key player.’ This badly formed sentence tells us little, and can serve to mislead. To suggest that Nedd8 is a key player in Neddylation is to suggest that bread is a ‘key player’ in a sandwich. Without the Nedd8 it simply isn’t Neddylation.
‘Unlike lysosomal degradation of proteins, a tagging occurs by attaching a small protein chain to mark for an enzyme complex (proteasome) by which they are then broken down.’ Surprisingly, if you can translate the sentence to English, this isn’t too objectionable; apart from the fact that it doesn’t belong here as Nedd8 doesn’t direct proteins to the proteasome. Furthermore as the sentence as it stands makes no sense, its possible that I’m just being charitable in my interpretation.
‘ In fact, the term “neddylation” means a special form of ubiquitinylation initiated by an uncommon subset of proteins (see below), of which the initiators as well as the substrates are unique although the same pattern as in ubiquitinylation is followed.’ A charitable reading could almost accept this, but its so garbled that such a reading is unlikely to occur to anyone not familiar with the subject. ‘A special form of ubiquitinylation’ is woolly at best. The process of Neddylation is analogous to that of ubiquitination (Or ubiquitylation, ubiquitinylation is a strange fusion of the two forms.*) and Nedd8 itself shows a great degree of homology (Similarity) to ubiquitin. But ultimately Neddylation is a ‘special form of ubiquitination’ in the same way a Renault is a special kind of Ford. It is misleading to say that the substrates are ‘unique’ as there are some targets which appear to be both Neddylated and Ubiquitinated in particular p53, however it would be true to say that their targets and target specificities are not identical. By initiators I will assume that the writer is referring to the enzyme cascade which leads to Neddylation. Again however they are incorrect, while the first two enzymes in the pathway, E1 and E2, are Nedd8 specific, no Nedd8 specific E3 ligases (the third enzyme) have been found, and it is suspected that the ubiquitin E3 ligases show dual specificity.
So ultimately there is only one idea in there which is remotely true, that ubiquitination and Neddylation are analogous processes. On reflection I get the impression that this is the only ‘fact’ the author knew.
I’ve pruned the article down to remove inaccuracies and will expand it later this week.
* Edited to add: Although a quick Google suggests this form isn’t completely unknown.
EDIT: I have closed this entry to comments as it was attracting a lot of spam.
I recently did some work at the Edinburgh Science Festival, which attempts to engage the public with science, particularly young children. While I’d like to say that this work was voluntary, it wasn’t, and this underlies something which I see as a far more serious problem.
The supposed aim of the Science Festival is to make science accessible, and to introduce it to the public, who may otherwise remain uninterested. This goal, I think, is worthwhile, particularly when you consider various reports that have appeared this year. However I can’t help thinking that the festival fails on two accounts:
- It only attracts people who are already interested in science.
- It charges for entry, and for a number of the ‘attractions.’
While the former is difficult to avoid, short of breaking into peoples living-rooms and offering to extract DNA from their bacon sandwich, the latter poses a serious and potentially avoidable problem. Charging has two effects, firstly discouraging people from dropping in casually, but more importantly excluding those from less financially secure backgrounds.
Science should be accessible to everyone, not just those with enough money. Yet an event which is meant to increase the accessibility of science still sees fit to exclude a significant number of people on a financial basis.
Now I’m aware that these things cost money to run, yet there are ways of helping meet costs. For one get people to work voluntarily, many will be happy to if actually given a push. Raise other revenue by increased sponsorship (There was some already) and voluntary donations. Alternatively, if this results in a shortfall the least you could do is offer free trips to schools from under-privileged areas.
I know this isn’t all idealistic pipe-dreams as I have seen it achieved in other organisations and groups, such as CHAOS. Why the Edinburgh Science Festival cannot achieve the same thing, I don’t know.