The development of eye color in Drosophila is known to involve special diffusible substances [1,2]. A genetically vermilion (v) eye will develop wild-type eye color if it is supplied with v+ substance by transplantation or by injection of body fluid of wild type flies. Similarly a genetically cinnabar (cn) eye will develop the color characteristic of wild type if it is supplied with cn+ substance. The present paper summarizes preliminary experiments made to learn something of the nature of the two substances just mentioned. 



During the course of our studies, Ephrussi and Harnly [3] have shown that pupal fluid can be freed of living cells by freezing in liquid air without destroying the v+ and cn+ substances. Khouvine, Ephrussi and Harnly [4] have shown further that these substances can be extracted from Calliphora pupae with 95 per cent alcohol-ether mixtures and with 95 per cent alcohol but not with pure ether. They conclude that these substances are not proteins or enzymes, a conclusion confirmed by our work

Beadle, G. W.

Thimann, Kenneth V.

English

Caltech Authors - Main

Diffusible Substances Concerned
Development of Eye Colors in Drosophila: Extraction of the
Kenneth V. Thimann, and G. W. Beadle 
doi:10.1073/pnas.23.3.143 
 1937;23;143-146 PNAS
 This information is current as of December 2006.
 www.pnas.org#otherarticles
articles: 
This article has been cited by other
 E-mail Alerts
. click hereor
box at the top right corner of the article
articles cite this article - sign up in the 
Receive free email alerts when new
 Rights & Permissions
 www.pnas.org/misc/rightperm.shtml
tables) or in entirety, see: 
To reproduce this article in part (figures,
 Reprints
 www.pnas.org/misc/reprints.shtml
To order reprints, see: 
 Notes:
VOL. 23, 1937 PHYSIOLOGY: THIMANN AND BEADLE
DEVE)LOPMENT OF EYE COLORS IN DROSOPHILA: EXTRA C-
TION OF THE DIFFUSIBLE SUBSTANCES CONCERNED
By KENNETH V. THIMANN AND G. W. BEADLE
BIOLOGICAL LABORATORIES, HARVARD UNIVERSITY
Communicated January 25, 1937
Introduction.-The development of eye color in Drosophila is known
to involve special diffusible substances." 2 A genetically vermilion (v) eye
will develop wild-type eye color if it is supplied with v+ substance by trans-
plantation or by injection of body fluid of wild type flies. Similarly a
genetically cinnabar (cn) eye will develop the color characteristic of wild
type if it is supplied with cn+ substance. The present paper summarizes
preliminary experiments made to learn something of the nature of the
two substances just mentioned.
During the course of our studies, Ephrussi and Harnly3 have shown that
pupal fluid can be freed of living cells by freezing in liquid air without
destroying the v+ and cn+ substances. Khouvine, Ephrussi and Harnly4
have shown further that these substances can be extracted from Calliphora
pupae with 95 per cent alcohol-ether mixtures and with 95 per cent
alcohol but not with pure ether. They conclude that these substances are
not proteins or enzymes, a conclusion confirmed by our work.
Method of Testing for the Substances.-Liquid extracts are tested for v+
substance by injections of approximately 0.4 cubic mm. into the body
cavities of apricot vermilion (wa v) or vermilion brown (v; bw) larvae shortly
prior to puparium formation. In a similar way, cn+ substance is tested for
by injections into cinnabar brown (cn bw) larvae. The test flies normally
have practically colorless eyes, and the presence of an active substance
is indicated by a darkening of the eye color. The maximum modification
results in an eye color similar to apricot or brown, depending on the consti-
tution of the test fly.
It was found desirable to measure responses in a quantitative way. The
test flies were separated into groups of increasing intensities of eye color
and these groups were assigned values from 0 to 5. The value 0 represents
no response and 5 a response such that the color is indistinguishable from
brown control flies. The mean of the values obtained in this way, using
from 10 to 20 injected flies, thus gives a figure which expresses in a rough
quantitative way the intensity of the reaction. It is known from Mal-
pighian tube-transplants5 that the response varies with the number of tubes
implanted and therefore presumably with the quantity of substance
available in the test animal. It is not assumed that the difference in con-
centrations of active substance measured by any unit difference in the
arbitrary scale is the same. Indeed the results suggest that a considerably
143
PHYSIOLOG Y: THIMANN AND BEADLE PRoc. N. A. S.
greater increase in concentration is represented by the difference between
4 and 5 than by that between 0 and 1.
Preliminary Experiments.-Wild-type pupae of D. melanogaster, 24 to
60 hours after puparium formation, were crushed and centrifuged into
three layers, an upper one of foam or oil, a second of clear yellowish liquid
and a bottom layer of sludge made up largely of cells and pupa cases. The
aqueous layer, injected into Wa v larvae, produced a very faint or doubtful
intensification in the color of the eyes; the other layers were completely
negative. On the assumption that the substances were being inactivated
by oxidation, pupae were crushed under sesame oil. Fluids obtained in this
way were negative in all tests. The oil layers were likewise consistently
negative even when the volume of oil was kept small to avoid dilution. A
chloroform extract of the pupae, taken up in sesame oil, was also negative.
It appeared that the substances were not lipoid-soluble, and therefore at-
tention was re-directed to the preparation of water extracts. It was soon
found that if extracting and centrifuging were carried out in an atmosphere
of nitrogen, a clear watery layer could be obtained which gave strong posi-
tive tests for both v+ and cn+ substances.
Extractions in Nitrogen.-The procedure finally evolved was to use a
wide tube, drawn out at the base into a thin-walled capillary. At the point
where the bore narrowed a slight constriction was made by thickening the
glass, and on this was rested a pad of glass wool. The pupae were put
into the wide tube, and a piece of narrow glass tubing, sealed at one end,
but with a small hole blown in the side of the seal, was fitted into the wide
tube by means of rubber sleeves. Nitrogen was passed in through this
tube for some minutes, the capillary then sealed off and the pupae thor-
oughly crushed in the atmosphere of nitrogen. The crusher was finally
disconnected from the nitrogen supply and quickly sealed off, and the
whole tube was then centrifuged at about 2500 r. p. m. for 15 minutes.
The glass wool acts as a coarse filter, retaining pupa cases, and a clear
yellowish aqueous layer collects in the capillary.
The fluid so obtained-about 0.1 to 0.2 cc. from 300 pupae-gave posi-
tive results of a strength about 2.5 in v; bw and from 1.5 to 2.0 in cn bw.
The fluid prepared in this way showed a strong precipitate of coagulable
protein on heating in boiling water. Heating the vessel containing pupae
in boiling water for 2-3 minutes, before crushing and extraction, however,
did not affect the results. Subsequent heating of the fluid extracted from
heated pupae produced no further precipitation. The substances are
therefore extremely heat-stable, and hence are not to be classed with
enzymes or true proteins. Further, if the juice, extracted in nitrogen from
boiled pupae, was exposed to air for 24 hours and then injected, the tests
showed no diminution in strength; (fresh juice, 4 ffies v; blw, mean 2.5;
24 hours old juice, 5 flies, mean 2.5). Thus, when freed from enzymes in
144
PHYSIOLOG Y: THIMANN AND BEADLE
this way, the v+ substance is also stable to oxidation. It was also found
that active extracts could be obtained from pupae which had been dried at
1000 in the oven.
On evaporating theextractto drynessinvacuo, andextractingwithacetone,
practically no solids were obtained in the acetone; both tests of this extract
taken up in water were negative (see table 1). The residue, on freeing from
insoluble matter and taking up in water, was still highly active. The
active substance is therefore insoluble in absolute acetone.
TABLE 1
STRENGTH OF RBACTION
PREPARATION IN v; bw IN cn bw
Original juice (2000 pupae in 0.5 cc.) 2.5 1.75
Evaporate; extract with acetone;
test the extract 0 0
Take up residue in 0.2 cc. water and
filter from bulky precipitate 2.8 2.2
Treat with 0.02 cc. 5% H202 0.6 0.5
Add charcoal and boil 0.9 0.1
Oxidation of the Substances.-Extracts made in air from pupae first
boiled in water or dried at 100°C. are active. The susceptibility to oxida-
tion by air evidently is marked only in the presence of the enzymes of the
pupa. Since Dakin6 has shown that hydrogen peroxide closely imitates
many types of biological oxidation, an attempt was made to reproduce
the action of the enzymes of the pupa by this reagent. To an active
extract, H202 was added up to a final concentration of 0.2 per cent. The
solution was then gently boiled. This treatment greatly weakened the
responses to both tests (table 1). No marked difference in the susceptibility
of the two substances was indicated, although numerous experiments were
carried out under varying conditions with H202, especially with concentra-
tions '/lo to 1/20 of the above. It appears that the activities of both cn+
and v+ substances are more or less completely destroyed by peroxide.
Comparison of v+ and cn+ Substances.-In all tests of the aqueous ex-
tracts of pupae, the response in the test for cn+ substance was consistently
weaker than that for v+ substance, by from 0.5 to 1 point. On the other
hand, Beadle5 finds that implantation of Malpighian tubes gives results
which are very nearly equal in the two tests. The above experiments give
no evidence that cn+ substance is any more easily destroyed by oxida-
tion than the v+ substance. It follows either that the cn+ substance is less
easily extractable than the v+ substance, or that it takes more of the cn+ sub-
stance to produce an equivalent effect. Strong positive results (cn 3.0) ob-
tained with hot Ringer extract of Malpighian tubes from only 15 larvae,
would suggest that the bulk of the substances are present in these organs,
and hence the differences are ascribable to the differences in the ease with
which the two substances are extracted. This conclusion is supported by an
VOL. 23, 1937 145
PHYSIOLOGY: G. W. BEADLE
experiment with cold Ringer extract of wild type Malpighian tubes; this ex-
tract, in v; bw, gave a color-value of 2.0, but in cn bw only about 0.8.
Finally, proof that the two tests cannot be due to the same substance,
previously adduced by transplantation experiments, was given by extract-
ing cinnabar pupae, which should contain only the v+ substance. About
200 pupae were heated to 1000, crushed and centrifuged in nitrogen as
described above, and the clear juice injected; it gave a v+ test of about 2.5,
but the cn test was completely negative.
Summary.-l. The substances capable of changing vermilion and
cinnabar eye color toward wild type may be obtained in cell-free extracts
from wild-type pupae, or the former alone from cinnabar pupae.
2. These substances are heat-stable and water-soluble, and apparently
insoluble in acetone or sesame oil. They are, therefore, presumably
neither proteins nor enzymes.
3. The cn+ substance is less readily extracted than is v+ substance.
4. Both substances are rapidly destroyed by oxidizing enzymes in the
pupa juice, or, in the absence of enzymes, by dilute solutions of H202.
5. A simple method is described for roughly determining the concen-
tration of the substances.
' Ephrussi, B., and Beadle, G. W., Bull. Biol. Fr. Belg., 71, 54-74 (1937).
2 Beadle, G. W., and Ephrussi, B., Genetics, 23, 76-86 (1937).
3 Ephrussi, B., and Harmly, M. H., C. R. Acad. Sci., Paris, 203, 1028 (1936).
4 Khouvine, Y., Ephrussi, B., and Harnly, M. H., C. R. Acad. Sci., Paris, 203,1542
(1936).
6 Beadle, G. W., these PROCEEDINGS, 23, 146-152 (1937).
6 Dakin, H. D., Oxidations and Reductions in the Animal Body, New York (1922).
DEVELOPMENTOFEYE COLORSINDROSOPHILA: FATBODIES
AND MALPIGHIAN TUBES AS SOURCES OF DIFFUSIBLE
SUBSTANCES
By G. W. BEADLE
BIOLOGIcAL LABORATORIEs, HARVARD UNIVERSITY
Communicated January 25, 1937
From the work of Ephrussi and Beadle (see Ephrussi and Beadle' and
Beadle and Ephrussi2 for summaries) it is known that three diffusible
substances are involved in the production of wild type eye color in Dro-
sophila melanogaster. This paper is concerned with the normal sources
of two of these, v+ and cn+ substances. Under certain conditions of genetic
constitution these two substances may be produced by eye tissue itself.1 2
Sturtevant's studies of mosaics of D. simulans indicated that v+ substance
146 PROC. N. A. S.


Development of Eye Colors in Drosophila: Extraction of the Diffusible Substances Concerned

http://authors.library.caltech.edu/10379/1/THIpnas37.pdf

Development of Eye Colors in Drosophila: Extraction of the Diffusible Substances Concerned

Abstract

Similar works

Full text

Available Versions

Caltech Authors - Main

Caltech Authors - Main