The utilization during mitotic cell division of loci controlling meiotic recombination and disjunction in Drosophila melanogaster.- Bruce S. Baker, Adelaide T. C. Carpenter, and P. Ripoll
- Genetics 1978 90: 531-578. [Abstract] [PDF]
As a postdoc, I worked for several years on the cell cycle in Drosophila. At that time, the field was just beginning to take off, not least because of the efforts of my then group leader. Nowadays, I study the ageing process, still using Drosophila, and including modelling the function of WRN, the gene responsible for the progeroid condition Werner's syndrome (WS). WRN encodes a RecQ DNA helicase (unwinds the DNA double helix), but unusually has a second activity, a DNA exonuclease (removes nucleotide bases from the end of a DNA strand). We are currently studing the Drosophila homologue of the WRN exonuclease (which we have named DmWRNexo, and which is encoded by the CG7670 locus).
When trying to demonstrate that a mutant of CG76700 indeed displayed characteristic comparable with defects seen in cell lines derived from WS patients, I recalled reading this paper at the beginning of my postdoc position.
The paper itself is lengthy, coming in at 47 pages. I guess the reason I like this paper, and regard is as having "classic" status, is down to the clarity of exposition. The authors state clearly why one might expect mutations that affect meosis to also affect mitosis, clearly lay out the basis for the assay they choose to apply, and explain clearly what the take-home message is. In the days before most, if any, of the genes being studied had been molecularly cloned, this needed to be a classical genetics work.
The assay for mitotic effects is summarised in the figure below. Essentially, mitotic defects leading to chromosome fragmentation, chromosome breaks, recombination or aneuploidy are revealed by using recessive cuticular markers such as yellow (y), forked (f) and multiple wing hairs (mwh), indicated schematically by a and b in the diagram. Note in particular that some of the events lead to aneuploid cells, while some continue as euploid cells: the cells generated by these events continue to proliferate, leading to patches (or clones) of cells. Aneuploid cells will proliferate less rapidly than euploid cells.
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As an example, the picture below shows some mwh clones (each cell normally bears a single hair - mutant cells bear a tuft of hairs) in the wing blade. Smaller clones (in terms of number of mutant cells in the clone) reflect more recent events in the cell lineage forming the wing blade than do the larger clones.
Above: multiple wing hair clones in wing blades (from my paper on DmWRNexo)
The important point for me is the analysis of clone frequencies. The graphs below illustrate the analysis. The plot the log of clone frequency against clone size (expressed as the number of cell divisions). The expectation for normally growing euploid cells is that 1 cell clones will be twice as numerous as 2 cell clones, four times as numerous as 4 cell clones, etc. The expected distribution (on the log plot) would therefor be a straight line of specific gradient - indicated by the bold line in the graph below. distributions of steeper gradient indicate slower growing clones, for example due to aneuploidy.
Above: sample data from the paper, showing clone frequencies resulting from euploidy and aneuploidy.
Notice how steep the data in the top left panel are: these are for mei-41 mutant flies. The molecular nature of the defect in these flies was unknown in 1978: now we know that mei-41 encodes the Drosophila homologue of ATR, a component of the system that identifies double strand breaks: chromosome fragmentation (leading to aneuploidy) is frequent in these mutants.
In cases such as our CG7670 mutant (that gives rise to the wing blade clones shown above), we see no evidence of aneuploidy, and conclude that homologous exchange is the principal cause of wing blade clones.
This "classic paper" is a fine read, established commonalities of genetic control of meiosis and mitosis, and furthermore set up a technique that I for one have found invaluable 30 years after publication.