Safety
EMS is a mutagen; and a carcinogen.; It should therefore be handled in a hood
with gloves. EMS solutions can be deactivated in a solution of 4g NaOH and 0.5 ml
thioglycolic acid in 100 ml. We store contaminated dry waste in sealed plastic bags
under a hood, which are periodically removed by University safety officers.
Screen design
Normally one treats males and then crosses them to untreated female virgins. In a
simple screen these females will be marked with the mutation of interest and the F1
progeny can be screened for new alleles. There are many more sophisticated types of
screen. In order to distinguish the chromosome bearing the new allele from the maternal
chromosome one of these may be marked with another (at best closely linked) mutation,
ideally a recessive also present on the appropriate balancer. It may be desirable to
screen only the F1 sons in order to avoid recombination between the marker and new
allele. It is best to screen for some other RK1 marker while screening for the gene of
interest, (probably the best are eye colour mutations such as white;, cinnabar ;or scarlet);
to give you something to find and as a running positive control for the effectiveness of
the treatment.
Males
It is best to use more males than you would normally as their virility can be
affected by the treatment. Avoid the marker yellow,; which reduces virility and increases
mortality during treatment. Ideally treat 3 day old males. Those that are too young will
not yet be fertile, those that are too old may suffer from Portnoy's complaint.
Dosage
Dosage is controlled by EMS concentration and can be monitored by scoring for
X-linked lethals. This is best done by crossing some of the treated males en masse to
virgins bearing an X-chromosome balancer (such as FM6;). The resulting F1 daughters are
allowed to mate freely to their brothers, and after several days are placed in tubes
individually and allowed to lay for a few days, and are then discarded. The F2 sons
must inherit the X-chromosome from their F1 mothers which are now a single isolates
(called *) from the original mutagenised grand-fathers. The F2 males can thus be either
FM6/Y or */Y. In some lines * can carry a newly induced lethal mutation and thus only
FM6/Y sons will appear. Thus the % of lines with no Bar+ F2 sons is the % newly
induced X-linked lethals;. Typically 25 mM EMS will yield about 30% X-linked lethals,
which equates to roughly one hit per autosome on average, and 1 hit in 2000 to 5000 for
most loci. The effective dosage of EMS may vary with different lab conditions or stocks,
so a pilot run to test several EMS concentrations may be worthwhile.
Mosaicism
Orthodoxy holds that EMS mutated post-meiotic sperm undergo DNA repair in
the embryo. This may well occur after the first cleavage division so it is quite common
for the F1 progeny to be somatic and sometimes germ-line mosaics for a new allele. Thus
often apparently mutant F1 individuals can yield a mixture of F2 progeny, or no F2
individuals bearing the new allele. This is tiresome but unavoidable, and the rate of such
"false positives;" will depend in part on the distance on the zygotic fate map between
the organ which is the focus of your mutation (e.g. the eye for white mutations) and the
germ line. This rate of "false positives" can easily be 50%.
Clusters;
If progeny are recovered from cells that were pre-meiotic during EMS treatment,
several identically mutant sperm could result from their subsequent mitotic division.
Thus the same allele could be recovered multiply. As spermatogenesis requires 5 days at
25°C, so long as the parents are discarded on the fourth day after treatment this should
not occur. It may be that the mutagenesis rate is not constant in these different daily
cohorts of sperm.
References
These topics are well discussed in Tom Grigliatti's chapter 2 Mutagenesis in
David Roberts' book Drosophila a practical approach (IRL press, Oxford, Washington DC
1986), and in Michael Ashburner's Drosophila, A Laboratory Handbook (Cold Spring
Harbor Press, Cold Spring Harbor New York, 1989).
Treatment :
1) Make solution I (100mM Tris Ph7.5 and 10% sucrose). Autoclave and store at room
temperature.
2) On the day of use make deactivating solution (4g NaOH + 0.5 ml thioglycolic acid
in 100 ml). Put this in the hood along with plenty of paper towels etc. and a plastic
bag for dry waste such as gloves and yellow tips.
3) In the hood, on a diaper, dilute solution I 1+9 and add EMS. 26.5 µl EMS in 10 ml yields a 25mM EMS solution.
4) Make up glass tubes with wadded kimwipes or filter papers in the bottom, and cotton bungs on top. Make two such for every 100 males to be treated.
5) Transfer the males to empty bunged vials and allow to dehydrate for 30 minutes.
6) In the hood, on a diaper, pour the EMS solution from tube to tube so as to thoroughly soak the paper in half of your prepared tubes and dispose of the residue by dilution in deactivating solution. Do not allow the cotton bung to become wet (this will prevent the tube from ventilating when the males are in it and they will die).
7) In the hood, on a diaper, transfer the dehydrated males to the EMS tubes, and leave them there over night. Leave a sign up on the hood to notify others of the presence of EMS.
8) In the morning transfer the males on to the dry tubes to allow them to wipe their feet and hopefully shit-out most of the EMS.
9) After 30 minutes transfer the males to freshly yeasted ordinary fly food tubes and properly dispose of all of the dry waste. The males may now be removed from the hood. Allow them a couple of hours to recover, then cross them.
10) Sub on the crosses every day for four days and discard the adults on the fifth.