It is now possible to detect the most inconspicuous mutants -- even those having only slight aberrations
all genetic research begins with isolation of mutants: genes or organisms that appear different from the normal ones (the wild-types). Keith Williams and colleagues from the School of Biological Sciences in Macquarie University, Sydney, Australia, have found a solution known as fluorescence-activated cell sorting (facs) for identifying mutants that differ just a little from the wild-type.
However, the solution works only when the wild-type possesses the protein marker that the mutant lacks. This new technique helps locate such mutants by labelling the wild-type with an antibody joined to a fluorescent probe (a dna fragment marked with a radioactive chemical which helps locate a gene or dna sequence). With this method, a mutant can still be separated even if present in a very small proportion (Cytometry, Vol 25, 1996).
Most mutants tend to be grossly aberrant in appearance or behaviour, so spotting them is usually not a problem. Quite often, they are so aberrant that they die at an early stage of development, making it difficult to experiment with them.
Going by the theory of evolution by natural selection, what we think of as the wild-type today must have arisen as a mutant sometime in the distant past. However, it is clear that aberrant mutants cannot provide (or have provided) the raw material for evolution; they are obviously too 'sick' to evolve. From this point of view, only those mutants that are only slightly different from the wild-type can be taken into account.
Williams and the team illustrate the power of the fluorescence method by selecting mutants lacking a surface glycoprotein (a protein linked to a carbohydrate) in the amoeba Dictyostelium discoideum. After synthesis, many proteins have sugar residues -- glucose, galactose or some other compound -- stuck on to them. Such 'secondary modifications' are important for a host of activities and are probably also necessary for the proper recognition of one protein by another. Each of these modifications requires specific enzymes to catalyse it and a sequence of modifications correspondingly requires a series of enzymes acting one after the other.
When one of the enzymes is absent because the gene that encodes it is mutated, the sugar residue is not attached where it should be. At times, this causes an obvious defect in the system -- for example, cells may not stick to one another. But at other times, the only defect is an invisible one -- an antibody that recognises the modified protein and binds to the cell surface is no longer able to do so. This is the case with many glycosylation mutants (proteins deficient in sugar modification) of the Dictyostelium discoideum, in which the normal protein is recog-nised by a highly specific (monoclonal) antibody.
The researchers induced the mutants by disrupting genes with the help of an exciting new technique called remi (restriction enzyme-mediated integration). In this technique, one uses a marked molecule of dna in combination with a restriction enzyme (that can cut dna in one of the many recognition sites). This mixture is introduced into the cell. If by chance, the restriction enzyme finds a site that it recognises within the gene of interest, that gene is cut and the foreign dna inserted. The gene gets mutated as a result.
The advantage of remi is that once a mutation is identified, it is very easy to 'fish out' the gene that is responsible for it. All that one needs to do is to fish out the marked molecule of dna that has been inserted. However, it is to be noted that the procedure of generating mutants does not guarantee that one can get any chosen gene mutated. What it does do, on the other hand, is to ensure that if such an event occurs, one can, without much difficulty, close in on the gene itself.
After generating mutations with the remi technique, Williams' group proceeded to screen only those putative mutations that did not have any obvious developmental defects. These constituted about 0.1 per cent of the starting population. Beginning with this subset, they labelled the sample of remaining cells with a fluorescent antibody against a surface glycoprotein. It is to be remembered that they were looking for the very rare cell within this sample that did not have any antibodies sticking to it -- the proverbial needle in the haystack.
At this stage, the researchers made use of the facs procedure. In facs, a laser light of chosen wavelength is shone on a rapidly falling series of drops of cells. If the cell fluoresces, an electric field is switched on to deflect the cell to one side so that it does not fall straight down. If it does not fluoresce, it falls vertically as dictated by the laws of gra-vity. It turned out that facs was sensitive enough to separate one non-fluorescing cell in a background of a hundred thousand that did fluoresce.
Subsequently, it was easy to allow the one aberrant cell to grow in an appropriate medium and verify that it had the subtle defect that had been hypothesised. This study opens up the field for further work on mutants that can be detected by the loss of an antibody-binding capability but are, for all other purposes, quite invisible.
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