The annoyance of temperature sensitive mutants

Jun 10th, 2007

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]

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