J. Mol. Biol.

(1974) 84, 389-398

Protein Synthesis in Salivary Glands of Drosophila melanogaster :

Relation to Chromosome PufFs
ALFRED Trssr&zEst,

HERSCHEL K. MITCHELLAND URSULAM.!IRAcY

Division of Biology
California Institute of Technology
Pasadena,Calif. 91109, U.S.A.
(Received4 September7973)

The salivary glands and other tissues from Drwophib

nwkmoga.eter were dissected
at various times throughout
the prepupal period, as well as after heat shocks and
ecdysterone treatments, and the proteins labelled by incubating the isolated tissues
with [35S]methionine
were separated by electrophoresis
on sodium dodecyl
sulphate-polyaoryhunide
gel. The labelled band patterns from salivary gland, as
seen on the autoradiograph
of the gel, showed striking variations, in a manner
remarkably
similar to variations
in puff patterns during the same prepupal
period. In proteins from Malpighian
tubes, the pattern of bands varied to a lesser
extent and in brain only a few components were modified.
Heat shock brought about the appearance of a number of new bands, while
others were reduced in intensity. This effect was observed with all the tissues
examined, salivary glands, brain and Malpighien
tubes, as well as wing imaginal
discs, tissue lacking polytene chromosomes. The six most heavily labelled bands
induced by heat shock represent about 30%, and one component alone represents
over IS%, of the total label in the sample, as seen in salivary glands, brain and
Malpighian
tubes. The synthesis of RNA at. puff sites was investigated after heat
shock by [3H]uridine
labelling. By correlating the amount of [3H]uridine
in some
puffs with the level of [35S]methionine
in some bands a tentative
relation is
suggested in a few instances.
The effect of ecdysterone treatment was also studied in the salivary glands.
Changes in a number of protein bands were noticed, though they were much less
pronounced than those following heat shock.

1. Introduction
that the puffs of the giant polytene chromosomes
of Diptera
are the sites of rapid RNA synthesis (Beermann,
1952; Pelling, 1964,197O) and thus
of very active genes.Evidence was obtained recently showing that RNA synthesized
in a particular puff was transported to the cytoplasm (Daneholt & Hosick, 1973)
where presumably it functions as messengerRNA and is translated into proteins,
and a direct correlation has been shown between a giant puff from the salivary
glands of Chironomw and a particular polypeptide chain secreted by these glands
It is well established

t On sabbatical leave from Depertement de Biologic Moleculeire,

de 1Ecole de Medecine, Geneve, Switzerlend.
389

Universiti

de Gleneve, 30 Quai

390

A.

TISSI&RES,

H.

K.

MITCHELL

AND

U.

M.

TRACY

(Baud&h
Q Panitz, 1968; Grossbach, 1969). It is thus of particular interest to
study the synthesis of proteins under conditions where it is known that the pattern
of puffs is strikingly
modified as genes are successively turned on and off. This is
the case in Drosophila
during periods of normal development, (Becker, 1959; Ashburner, 1967), and modifications of the puffs can also be induced under the influence
of the hormone ecdysterone (Ashburner, 1972) or by various changesin the environment such as heat shock (Ritossa, 1962) or anaerobiosia (Ashburner, 1970).
In the work described here we have attempted to correlate changes in the pat,terns of prot.ein synthesis with changes in the dist.ribution of puffs, both during
normal development and after inducing
specific modifications
of that distribution.
The period chosen was one of extensive changesin puff pattern (Ashburner, 1967),
from

the latest

larval

stage

to about

13 hours

after

puparium

formation,

during

which time regulation of larval tissue degradation and imaginal tissue synthesis
is most evident. The proteins were labelled by incubating the isolated tissues for
short periods in the appropriate buffer containing [35S]methionine of high specific
activity and the pulse-labelling thus obtained was shown to reflect the rate of protein synthesis that takes place at a given time in a tissue of the whole animal. The
labelled proteins were separated by electrophoresis in sodium dodecyl sulphateacrylamide gels and our findings show that the changes in band pattern seen on
the autoradiographa of such gels are remarkably similar in charact,er to the changes
in patterns of chromosomepuffs.

2. Materials
(a) St+-ains
For most of the work
quantities
as described
(b)

done
earlier

[%Qnethionine

and Methods
and

culture

here we used the Oregon-R

(Mitchell
& Mitchell,
1964).
labellinq

of proteina

wild-type

in Golatcd

stock

culture

in mass

tissues

The salivary
glands
or other
tissues
were dissected
in medium
A containing
0.03 Msodium
phosphate
buffer
(pH 6*8), 0.04 nf-KCl,
0.011 M-NaCl,
0.003 ~-C&l,
and 0.021 MMgCl,
(Ashburner,
1970) at 23C,
and the intact
tissues,
in general
3 pairs of salivaqr
glands,
were transferred
to a 5-~1 drop of medium
A containing
10 to 40 pCi of [36S]methionins
(spec. act. 50 to 180 Ci per mol) on a piece of Parafllm
about
8 mm X 5 mm
placed
on a microscope
slide. This was then covered
with a small inverted
glass vial containing
a piece of moist
filter paper
to prevent
evaporation.
Incubation
was carried
out
at 23C, usually
for 20 min, and was stopped
by adding,
with
the slide still under
the
microscope,
a drop
of cold
10% trichloroacetic
acid.
The liquid
was removed
and this
wash was repeated
3 times.
The small piece of Parafilm
with
the tissue was transferred
to a 3-ml conical
centrifuge
tube
fllled with
cold
IO?/, trichloroacetic
acid. The tissue,
detached
from
the Parafilm,
sedimented
to the bottom
of the tube. After
20 min in the
cold, the trichloroacetic
acid solution
was pipetted
off, and the tissue was further
washed
for 20 min first in 95%
ethanol,
then in a mixture
of methanol/chloroform
(1: l), and
finally
allowed
to dry at 37C. Twenty
or 30 4 of electrophoresis
sample
buffer
(0.0625
MTris.HCl
(pH 6.8), 1% sodium
dodecyl
sulphate,
1 /b /3-mercaptoethanol,
10% glycerol,
with
about
O.OOlo/o bromophenol
blue)
was added,
the tube wss sealed with
3 layers
of
Parafilm
and heated
in a boiling
water
bath for 10 min. The proteins
were found
to be

efficiently extracted from the tissues by this procedure

for radioactivity
measurement or clectrophoresis.
TO find

out

the

conditionsdescribed,
each

one

containing

time

course
a number
6 salivary

and the samples were then ready

of incorporation
of [35S]methionine
into proteins
under
the
of samples
were prepared
and incubated
from 10 to 80 min,
glands
from larvae
about
6 h before
puparium
formation

PUFFS

AND

PROTEIN

SYNTHESIS

IN

DROSOPHILA

391

and 1.2 x lo6 cts/min of [35S]methionine

in 6 ~1 of medium A. The samples were then
washed (the last trichloroacetic
acid wash being heated for 10 mm at QOC), the proteins
extracted from the tissues in electrophoresis
sample buffer, as above, and radioactivity
determined. The incorporation
of methionine was almost linear for 20 min and by 80 min
it had dropped a little more than twofold (Fig. 1). As in some of the experiments
with
ecdysterone, it was necessary to incubate the tissue in medium A for periods up to 90 min,
the glands were preincubated
in medium A for this length of time at 23C, then samples
were prepared as above. It was found that incorporation
was still fairly active for the
Crst 20 min (Fig. 1).
20,ooc

i
" l0,000
h
5
;
g
B
a

5000

0
Time (mm)

Time course of [35S]methionine incorporationinto

proteins of salivary gl8nds. -O-O-,
Shows the values obtained when the glsnds were incubsted with [36S]methionine in buffer (medium
A) for various lengths of time at 23C. -a-m-,
Represents the values given when the tissue
was incubeted for 90 min at 23C in medium A before being exposed to the label. Experimental
Fro.

conditions

1.

8re described

in Materials

(c)

and Methods.

Gel electrophwesi.3

and

autoradiography

Polyacrylamide
gel electrophoresis
was carried out in a discontinuous
sodium dodecyl
sulphate system (Laemmli, 1970) on slabs A in thick using the modification
of Studier
(1972) of the apparatus described by Reid & Bieleski (1968), with 12.6% acrylamide
containing
O*8o/o bisacrylamide
in the separating gel. The slabs were stained with Cooma&e blue, destained by diffusion, dried according to the method of Fairbanks
et al.
(1965) modified by Maize1 (1970) and autoradiographed
by contact with Kodak no-screen
X-ray films.
necessary to agcertuin the validity
of the method
(i) Does treatment
with pancreatic
ribonuclease
and deoxyribonuclease
modify the
electrophoretic
pattern? To answer this question, salivary glands from larvae, after
[35S]methionine
incorporation,
washing and drying as described above, were incubated
for 15 min at 23C, fhst with 5 pg of pancreatic
ribonuclease/ml
in 0.01 rd-Tris*HCl
(pH 7*4), then with the addition
of 5 ~1 of pancreatic
deoxyribonuclease
I/ml and
1 mna-MgCle. The electrophoretic
pattern of the autoradiograph
was not modified by this
treatment,
which was therefore omitted in the experiments
described below.
(ii) Does [36S]methionine
labelling of isolated tissue in vitro give the same pattern of
electrophoresis
autoradiograph
as does a pulse-labelling
in viva? I.6 &i (O-1 d) of [3bS]rnethionine
was injected into each of several larvae and after 20 min the glands were
(d) Controls

26

392

A.

TISSIfiRES,

H.

K.

MITCHELL

dissected
and prepared
for electrophoresis.
from
those where
isolated
glands
from
with the label.

(e) [3H]uridine

Autoradiographs
animals
of the

labelling

AND

same

U.

M.

TRACY

thus obtained
did
age were incubated

not
in

differ
vitro

of puffs after heat shock

The salivary
glands
of heat-shocked
and control
larvae
were incubated
for 10 min at
23C with 50 PCi of [3H]uridine
(spec. act. 9.7 Ci per mmol)
in 3 ~1 of medium
A. The glands
were rapidly
washed
in medium
A, fixed
in 45%
acetic
acid for 30 min,
stained and
squashed
(Ashburner,
1967).
Chromosome
figures
were
photographed,
the preparation
dipped
in liquid
nitrogen,
the coverslips
removed
and the slides dipped
in ethanol.
After
drying,
the slides were
dipped
in liquid
emulsion
(Ilford
L-4),
exposed
for 7 days and
developed
for rephotography.

( f) Chemicals
[36S]methionine
and
Mass. and ecdysterone

[3H]uridine
were
from
Schwarz-Mann,

obtained from New England Nuclear,

Orangeburg,

Boston,

N.Y.

3. Results
(a) Protein patterns during prepupal development
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns of salivary
gland proteins are shown in Plate I. When the proteins were stained with Coomassie
blue (Plates I(a) and II(a)) the patterns were relatively constant throughout the
post-larval stagesexamined, with the exception of one rather strong band at 43 mm,
which disappeared at the time of puparium formation, and a few changes in some
weaker bands.
In contrast, the autoradiograph of the samegel (Plate I(b)) and the densitometer
tracings therefrom (Fig. 2) show extreme variations during that period of prepupal
development. This observation was reproduced in several separate experiments.
Excluding the larval sample (-3 h), about 22 rather strong bands or peaks can be
seenin Plate I(b) and Figure 2 and only one of these, moving at 20 mm, is present
at all the stages,though it changesin intensity. On the other hand, five components
are seen clearly at only one stage (2 h, 24 mm and 49 mm; 3.5 h, 9 mm; 13 h, 47
and 55 mm). Of the remaining 16 bands, some persist through many stages and
others through only a few. The minor bands undergo the same type of variation.
On careful examination of the autoradiograph, over 52 bands with different migration distances could be detected.
It should be noted that the amount of label incorporated into the total proteins
of the salivary glands remained roughly constant during the period of prepupal
development.
The autoradiographs of the brain samplesshow many fewer changesin the band
pattern (2 or 3 bands only) during the prepupal period than do samplesfrom the
salivary glands. In Malpighian tubes, however, the changes are more pronounced,
affecting about a dozen bands (Plate II(b)).
The stained patterns of gel electrophoresis in the three tissues at three different
stages of development are shown in PI&e II(e).
While a few changes took place in
the salivary gland samples during the same period (see Plate I(a)), the stained
patterns from brain and Ralpighian tubes remained constant.

-3

0.5

3.5

7.5

11

;3

25,700

60,000

MoLwt

-3

(b) of a sodium
dodocyl
sulphate-polyacrylamide
period.
The
numhrrs
given
below
each
sample
the
distance
of migration
from
t,he origin
in
brtwwn
the 2 photographs
xvith
their
molecular

5-5

Time(h)

PLATE
T. Stained
patterns
(a) and autoradiograph
2 glands
of 1). ~wl~~togrcster
during
the prcpupal
$ formation.
The
scales
on the, sides
indicate
and myoglobin,
nw shown
T chymutryp&opvn

81

2(

(a)

represent
mm.
The
weights.

0.5

gel

3.5

Time

5.5

Ch)

?5

olectrophoresis
of labelled
the time
in hours
before
position
of the
mol.
wt

Cb)

proteins
(-)
and
markers

11

from
salivaq.
after
puparium
usrd,
catalaw.

13

-3

<a)

10

-3

Time

Ch)

10

-3

3
10

<b)

CHC
-2

HCH
10

Time(h)

CH
-2

CHC

a
6

PUFFS

FIG.

patterns
the time

AND

PROTEIN

SYNTHESIS

IN

DROSOPH1LA

393

2. Densitometer
traaings
of the autorediogreph
of Plate I, showing
changes in l&belling
of proteins
from the salivary
glands during
the prepupal
period.
The numbers
indicate
in hours before (- ) end after pup8rium
formation.
The origin is at the right.

(b) Effect of heat shock

Modification of normal pufIing patterns of salivary glands chromosomes brought
about by physical or chemical environmental stresses have been described by several
workers (Ritosse, 1962; Berendes, 1968; Ashburner, 1070). Here we have selected
heat shock as B suitable treatment which could show a correlation between puff
p&&x-n and specific polypeptides. To this end the animals were placed in a closed
vessel which was immersed in a 37.5C water bath for 20 minutes (Ashburner, 1970).
Besides salivary glands, two other tissues, brain end Malpighian tubes, were used,
as unlike salivary glands they represent the major tissues which survive larvctl
histolysis. The salivary glands, brain and Malpighian tubes were dissected at 23C
and incubated with [35S]methionine as described in Materials and Methods using
six salivary glands, four brains and Malpighi~n tubes from six animals, respectively,
for each sample. Control animals were maintained at 23C and otherwise treated in
the same way at the s&me time. The extracted proteins were electrophoresed on

392

-4. TISSI&RES,

I-1.

I(.

MITCHELL

;\SD

LJ. JI.

TRACY

60

L
4o0

I
20

Miaration

3. Heat-shock
samples. The tracing
FIG

40

effect on densitometer
tracing
of the control wtw subtracted

60

(mm)

of autoradiograph
for the -2-h
from that of the heat shock.

salivary

gland

gel and the resulting autoradiograph

is shown in Plate II(b). Drastic changes in
patterns of labelled proteins did occur as a result of the heat shock. At all stages
examined in salivary glands, brain and Malpighian tubes the appearance of a very
heavily labelled component at 9.5 mm was immediately obvious. In addition new
clear bands at 12, 26, 38, 44 and 45.5 mm, and others more difficult to ascertain at
6.5 and 16 mm, also appeared. Some bands present in the controls were found reduced
in intensity after heat shock, at 18, 33,66 and 69 mm. These results are summarized
for the -2-h salivary gland stage in Figure 3. Here the densitometer tracing of the
autoradiograph,
shown in Plate II, of the control was subtracted from that of the
heat shock, thus removing common peaks present equally in control and heattreated salivary gland sample.
The heat shock brought about the labelling of new bands at the same position
in all three tissues at all the stages examined. Moreover, the amount of label in each
new band was roughly proportional in the three tissues, as seen in Table 1. In all
the samples, six of the new bands account for about one-third, and the component
at 9.5 mm itself accounts for one-sixth of the total incorporation.
To investigate whether the effect of heat shock was limited to cells having polytene
chromosomes, ten wing imaginal discs, tissues known to lack polytene chromosomes,
were dissected from each of two sets of larvae four hours before puparium formation, one treated for 20 minutes at 37.5C, the other maintained at 25C and serving
as a control. Each of the two sets of ten discs was incubated with [35S]methionine
for 30 minutes, then treated and analysed by electrophoresis as described in Materials
and Methods. The autoradiograph
pattern thus obtained showed, in the imaginal
disc samples after heat shock, the appearance of the characteristic
bands (results

PUFFS

AND

PROTEIN

SYNTHESIS
TABLE

IN DROSOPHILA

395

Amount of label in some protein bands induced by heat shock

Distance
of
band migrmtion
on gel
(mm)

9.5
12
26
38
44
46

the

salivary
glands

Salivary
glands

B&n

-2h

+9h

-2h

19.0

2.2
1.4
1.7
1.7
4.5

16.0
2.4

2.2
1-7
I-7
2.7

Melpighian
tubes
+9h

15.0
3.6
2.7
3-6
3.5
6.2

V&es
are given aa percentage
of toti
lebel in each sample. They
surface of peaks on densitometer
tmcinga
from the autoradiograph

17.0
2.3
0.7
2.5
2.5
6.7
were

obtained
by measuring
shown on Plate II.

not shown) as seen in salivary glands, brain and Malpighian tubes samples from heat
shocked animals.
We also asked whether the effect of heat shock could be seen in tissues of the
adult fly. In order to answer this question, one-day-old flies were etherized, separated
into two lots, one being placed at 37.5C for 20 minutes and the other left at 25C.
At the end of this period, the flies were again etherized and their brains dissected
and incubated at 23C with [35S]methionine for 30 minutes. The characteristic
bands observed on prepupel tissues from heat-shocked animals were seen on the
electrophoretic
pattern of the autoradiograph
obtained therefrom
(results not
shown).
(c) RNA

synthesis at the sites of puffs following heat shock

We have seen that the heat shock induces a rapid appearance of new labelled
protein bands on the autoradiograph
from the gel electrophoresis, and that one of
the new bands contains three to ten times as much label as the others and represents 15 to 19% of the total label incorporated in the sample (Plate II(b) and Table 1).
In view of this extraordinary
rate of protein synthesis exhibited in one single band,
it seemed likely that this would also be seen in the amount of RNA synthesized at
puff sites. To investigate this question, salivary glands from 108-h heat-shocked
larvae and controls (puff stage 1 of Ashburner (1967)) were incubated with [3H]uridine in medium A for ten minutes and squashes prepared for staining (Ashburner,
1967) and autoradiography.
As shown in Plate III(b) and (c), the chromosomes
from shocked animals yielded strong labelling at eight sites and the position of these
puffs was identical with the heat-shock-induced
puffs observed by Ashburner (1970).
Furthermore
the amount of label at position 87B was very much larger than at
sny other locus. Thus it may be that the protein band which appears after heat
shock at 9.5 mm on the autoradiograph
(Pls,t.e II(b)) is coded for by a. gene in the
87B region of chromosome 3R. It should be noted that the label at position 88EF
(Plate III(c) and (d)) is a background puff not induced by heat shock. Non-heated
controls gave a number of such background puffs as expected but none was labelled
to a greater extent than this one.

396

A.

TISSIKRES,

H.

K.

MITCHELL

TABLE

AND

U.

M.

TRACY

Effects of ecdysteroneon someprotein bands

Distanoe
of
band migration
on gel
(-4
27
31
39
42
60

Time

of exposure

to ecdysterone

60 min

90 min

+O.Q
+0.46
- 0.43
+0.16
+ 0.43

+:.74
-0*72
+ 062
+0.67

Values were obtained

from densitometer
treoings
of autoradiographs
and are given as change
in percentage
of total inoorporation
between
oontrol
and ecdysterone-treated
glands. The salivary
glands from 90-h larvae
(puff stage 1) were inoubated
in medium
A at 23C with and without
lo-* M-eodystarone.
After
60 and 90 min each semple of 6 glands was incubated
for 20 min in
[9]methionine
and prepared
for electrophoresis
and autoradiography.

(d) Effects of ecdysterone

As shown in Table 2, ecdysterone treatment of isolated salivary glands in puff
stage 1 yielded obvious changes in [35S]methionine incorporation in five bands,
four showing an increaseand one a decrease.Similar results were obtained by injection
of ecdysterone into larvae, to give a concentration of 10m4M in the hemolymph,
followed by dissection and incorporation of [36S]methionine after one and three hours.
In both casesthe changesobserved were lessthan 1y0 of the total label incorporated
and thus far lessthan those resulting from heat shocks, as seenin Table 1.

4. Discussion
In the work described here, the proteins were labelled in vitro by incubating the
isolated tissue with [36SJmethioninefor a short time. The autoradiograph from gel
electrophoresis of the proteins from salivary glands labelled in this way was identical
to that of proteins from the same tissue, labelled by injecting [35S]methionine into
the animals. Under the conditions used, the in vitro labelling represents a pulse and
therefore the intensity of a band on the autoradiograph reflects the rate of synthesis
of an individual protein, or of a group of individual proteins as the case may be,
in one single electrophoretic band.
During the prepupal period, the patterns of labelled polypeptides from salivary
glands seen on the autoradiograph undergo striking changeswith time in a manner
remarkably similar to changesin patterns of chromosomepuffs. Of the great many
bands which can be seen during the 16-hour period examined, one only, though
varying in intensity, can be seenat all stagesand five are visible at one stage only.
These data suggestthat all the protein synthesis occurring at high rates in prepupal
salivary glands results directly from chromosomepuffs.
The heat shock, in which the animals were exposed to 37GC for 20 minutes,
led to the rapid appearance of six rather strong bands, beside a few weaker ones,
and to the disappearance of others. The six principal bands account for about 30%,

PUFFS

AND

PROTEIN

SYNTHESIS

IN DROSOPHILA

397

and one of them alone for over 15% of the total protein synthesis in the sample,
as shown in salivary glands, brain and Malpighian tubes (Table 1). In imaginal
discs, tissue known to lack polytene chromosomes, the heat shock induced the
appearance of the same characteristic bands and this effect was also seenon brains
of adult flies. The induction by heat of the sameprotein components in all the tissues
examined implies that the susceptibility is locus specific and the same loci may
be susceptible at all stagesof development. The heat shock given is within the range
of possible exposure of the fly in nature and also of treatments known to produce
pheno-copies (Hadorn, 1955; Mitchell, 1966). Thus it may well be that temperature
variations have profound effects on development and select,ion due to differential
modulation of gene activities.
The rapid appearance or disappearanceof bands, seenduring the prepupal period
and following the heat shock, and which mimic the pattern of puffs on chromosomes,
suggeststhat the messengerRNAs synthesized at puff sites are rapidly transported
to the cytoplasm and translated, and that they are, on the average, short-lived.
Daneholt & Hosick (1973) have shown that a 75 S RNA transcribed in the Balbini
ring 2 region of chromosomeIV in Chirowmm tentans, and found in high amounts
in the cytoplasm, is remarkably stable. Further work will be necessary to find out
whether such stable RNAs are also present in the system used here.
So far our evidence for the direct relation of a given puff to a polypeptide band
is circumstantial. However, the information obtained by observing gains and losses
in polypeptide products after heat shock and ecdysterone treatment is quite compelling. In the case of the heat shock, we have extended the observations of Ashburner (1970) that the treatment induces new puffs at the loci 63BC, 64F, 67B,
70A, 87A, 87B, 93D and 95D by the demonstration of uridine incorporation in
isolated glands at each of these positions (Plate III). We have confined our attention to chromosome3 in the pictures shown, but the ninth puff at 33B (Ashburner,
1970) is also labelled after heat shock. In this set, one locus, 87B, consistently showed
several times greater label than any of the others. At the same time and at other
developmental stagesalso, one protein band at 9.5 mm (Plate II and Fig. 3) appeared
in amounts several times greater than any of the others, and it is reasonable to
postulate that this component is coded for by messengerRNA produced at locus
87B. Considering the amount of label at 87A and 67B, it is possible that these loci
are responsiblefor the protein bands at 46 mm and 38 mm, respectively, as they are
the next most heavily labelled protein bands (Table 1). Similarly, stage 1 salivary
glands treated in vivo and in vitro with ecdysterone show new polypeptide components at 27, 42 and 51 mm and the loss of an existing component at 39 mm. These
could correspond to puff loci induced early (Ashburner, 1972) such as 23E, 74EF
and 75B, while the loss could correspond to locus 25AC. In the in vitro experiments,
the polypeptide component at 27 mm appeared by 30 minutes, increased to 60
minutes, but was again weaker by 90 minutes. This behavior is similar to that
described by Ashburner (1972) for locus 23E. Obviously these approachescannot give
unequivocal associations between chromosome loci and specific polypeptide products but they can provide useful guidelines for further experiments.
The system used here, in which drastic changes in the protein l&belling patterns
were observed, seemssuitable for the study of several questions related to control
mechanisms at the DNA level, and to messengerRNAs and their translation. For
inst,ance,t,he system would seemwell suited to the study of the puff proteins (Clever,

398

A. TISSIERES,

H. 1~. MITCHELL

AND

U. M. TRACY

1966; Berendes, 1968) and/or of the non-histone chromosomal proteins (Elgin et al.,
1973). In the case where a high rate of synthesis of specific protein or RNA components is observed, as in some of the stages of the development and particularly
after heat shock, the gene products should not be difficult to isolate and purify.
With purified messengerRNAs, analysis of the arrangement of information in the
DNA segmentsinvolved at each puff locus could be done, as was described by Lambert (1972) for Balbini rings in Chironomus.
This work was supported in part by grants from the National
Science
(GB-23343) and the Swiss National Fund for Scientific Research (3811.72).

Foundation

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