142 H. K. MITCHELL AND J. R. SIMMONS 
Fraction 2 (lipid-soluble) is the most obviously different and since studies on it have 
been described elsewhere’ it will not be considered further here. That Fractions 1 and 3 
are also distinct and have little if any overlap has been demonstrated in two ways. 
First, addition of [C] valine to a homogenate before separation led to a recovery of 
more than 99.8% of the radioactivity in Fraction 1. Second, as described below, on 
column chromatography Fractions 1 and 3 contain few if any substances in common. 
Thus, the use of the preliminary fractionation simplifies further separation and iden- 
tification of the many components indicated by the data shown in Fig. 2. 
The extraction methods described above have been applied to a study of similarities 
and differences among larvae of wild type Drosophila and two lethal mutants, lethal 
tvanslucida (l-tr) and lethal meander (l-me). Both of the mutants have been shown by 
CHEN AND HaAporn!™,1!! to have abnormalities in protein synthesis. Third instar 
larvae were used (when the mutant phenotypes are expressed). Fractionations were 
carried out using Dowex-50-4X but use was made of a linear gradient and a triethyl- 
amine buffer. The buffer can be removed sufficiently for chromatography without 
heating. Details of the system are given with the data in Fig. 5 along with the summary 
of results. In the figure, elution patterns for Fraction I are given on the left where all 
the common amino acids are eluted between Fraction 48 and 103 (positions of some 
are shown as an index). Nevertheless a large amount of material including some 
peptides appears earlier as shown previously (Fig. 2). In contrast, elution patterns 
of Fraction 3 (shown at the right in Fig. 5) showed negligible quantities of ninhydrin- 
reacting material prior to tube No. 85. Furthermore, the material obtained after 
Fraction 85 gave a large increase in amino acids (10—30-fold) on hydrolysis. Un- 
fortunately, in these experiments, the column separations were completed before the 
analyses and the columns were not continued long enough for complete recovery of 
the material in Fraction 3. The conclusion is very clear, however, that the extraction 
method provides a clean separation of materials into Fractions 1 and 3 and thus 
permits more satisfactory subsequent separations. 
With respect to similarities and differences among the mutants and wild type 
there are a number of considerable interest. Considering Fraction 1 first, the mutant 
l-ty shows a large accumulation of materials all the way from tube No. 5-48 and a 
striking deficiency in tubes No. 75 and 76. Mutant /-me shows accumulations at 
tubes No. 36, 37 and 78 anda general increase in peptides that appear after tube No. 60. 
These differences at least (and probably more) appear highly significant since the 
NH, 
| 
C,H—CH—COOH 
| 
“a 
WO 
Tig. 6. Tyrosine-O-phosphate. 
References p. 146 
