Reversal of Ethionine Inhibition by Methionine — Hohl and Hamamoto 
535 
Fig. 1 . Growth of Dictyostelium discoideum on 
Escherichia coli in the presence of various concentra- 
tions of ethionine. The ordinate indicates cells/ml at 
the end of the growth period ( 46 hours ) . The inocu- 
lum consisted of 1.0 X 10 5 myxamoebae/ml. 
cell adherence or "stickiness” is not suppressed 
by ethionine. This result corroborates the ex- 
periments to be reported next, where it was 
found that even high concentrations of ethionine 
do not completely inhibit streaming and a type 
of massing of cells reminiscent of aggregation. 
In order to test the effect of ethionine on the 
developmental stages per se, the myxamoebae 
were grown in the absence of ethionine first 
and then washed populations were subjected 
to ethionine as described under materials and 
methods. Figure 2 presents the effects of various 
concentrations of ethionine on the development 
ETHIONINE 
DIFFERENTIATION 
INTO 
TYPICAL 
TYPE 
CONCENTRATION (M) 
SPORES 
SHEATH 
STALK 
FORMS 
NO. 
0 
+ 
+ 
CELLS 
+ 
iil^ 
1 
7.5 xIO' 4 
+ 
+ 
+ 
M 
2 
1.5 xIO' 3 
- 
+ 
+ 
ULAt. 
3 
3.0xl0' 3 
m 
+ 
(IKAA 
4 
6.0xl0' 3 
- 
- 
- 
5 
1.5x10-2 
- 
- 
6 
Fig. 2. Influence of increasing concentrations of 
ethionine on the development of Dictyostelium dis- 
coideum. The results represent the final stages of de- 
velopment regularly reached after 24 to 30 hours. 
of D. discoideum. Ethionine did not appreciably 
reduce the viability of the myxamoebae during 
the time of the experiment. Hence, the effects 
observed cannot be attributed to a partial killing 
of the cell population. A concentration of 
1.5 X 10 -2 M completely inhibits morpho- 
genesis, although some streaming may occur 
resulting in the formation of vaguely defined 
clumps that usually disappear within 24 hours. 
At 6.0 X 10 -3 M large, flat mounds of myx- 
amoebae are formed out of which tiny papillae 
may protrude, rudimentary signs of induction 
of polarity and fruiting body formation. The 
large number of these small papillae per mound 
indicates further that the critical mass, i.e., the 
mass of cells capable of integrated behavior 
(Hohl and Raper, 1964), has been drastically 
reduced. At a concentration of 3.0 X 10~ 3 M 
the myxamoebae collect into large aggregates 
that split up to form many finger-like pro- 
trusions. These protrusions are made up typi- 
cally of a heavy mass of cells at the base, a short, 
thick stalk-like structure oftentimes carrying 
at its apical end a lobose mass of cells some- 
what resembling a sorus. Up to this stage all 
the structures are made up of roundish cells 
without signs of spore differentiation or produc- 
tion of a cellulose sheath. Some large vacuolated 
cells, however, are present and we inter- 
pret them as representing stalk cells. At a con- 
centration of 1.5 X 10 _3 M stalks are formed 
and are ensheathed in a smooth cellulose 
envelope. The outside of the sheath is often 
covered with masses of undifferentiated cells, 
thus making the structures as a whole appear 
distorted. No spores are found in these fruiting 
bodies, but clumps of undifferentiated cells 
may occur at the tip of the stalk. Because of 
the still heavy base and the stalk without sorus 
the whole structure at this level often resembles 
a bowling pin. At a concentration of 7.5 X 
10 -4 M the fruiting bodies, apart from 
occasional distorted forms, are normal in ap- 
pearance though reduced in size. No apparent 
effect can be observed at lower levels of 
ethionine. For convenience the different levels 
of inhibition have been numbered from 1 to 
6, as indicated in Figure 2. In general, spore 
differentiation is most sensitive to ethionine, 
followed by sheath production, stalk cell dif- 
ferentiation, and lastly morphogenetic move- 
