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J. Mol. Biol. (1974) 84, 389-398

Protein Synthesis in Salivary Glands of Drosophila melanogaster :

Relation to Chromosome PufFs

ALFRED Trssr&zEst, HERSCHEL K. MITCHELL AND URSULAM. I’RAcY

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. pu ff 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

It is well established 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

t On sabbatical leave from Depertement de Biologic Moleculeire, Universiti de Gleneve, 30 Quai

de 1’Ecole de Medecine, Geneve, Switzerlend.

389

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390 A. TISS I&RE S, H. K. MITCHELL AND U. M. TRAC Y

(Baud&h Q Panitz, 1968; Grossbach, 1969).

It

is thus of particular interest to

study the synthesis of proteins under conditions where i t 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; Ash-

burner, 1967), and modifications of the puffs can also be induced under the influence

of the hormone ecdysterone (Ashburner, 1972) or by various changes n the environ-

ment such as heat shock (Ritossa, 1962) or anaerobiosia (Ashburner, 1970).

In the work described here we have attempted to correlate changes n 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 changes n 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 pro-

tein 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 sulphate-

acrylamide 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 and Methods

(a) St+-ains and culture

For most of the work done here we used the Oregon-R wild-type stock culture in mas s

quantities as described earlier (Mitchell & Mitchell, 1964).

(b) [ Qnethionine labellinq of proteina in Golatcd

tissues

The salivary glands or other tiss ues were dissec ted in medium A containing 0.03

M-

sodium phosphate buffer (pH 6*8), 0.04 nf-KCl, 0.011 M-NaCl, 0.003 ~-C&l, and 0.021

M-

MgCl, (Ashburner, 1970) at 23”C, and the intact tissue s, 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 microsco pe slide. This was then covered with a sma ll inverted glass vial con-

taining a piece of moist filter paper to prevent evaporation. Incubation was carried out

at 23”C, usually for 20 min, and was stopped by adding, with the slide still under the

microscop e, 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 w as transferred

to a 3-ml conic al 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 37°C. Twenty or 30 4 of electrophoresis samp le buffer (0.0625

M-

Tris.HC l (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

eff icient ly extracted from the tissues by this procedure and the samples were then ready

for radioactiv ity measurement or clectrophoresis.

TO find out the time course of incorporation of [35S]methionine into proteins under the

conditions escribed, a number of sam ples were prepared and incubated from 10 to 80 min,

each one containing 6 salivary glands from larvae about 6 h before puparium formation

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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 QO’C), 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 23”C, then samples

were prepared as above. It was found that incorporation was still fai rly active for the

Crst 20

min (Fig. 1).

20,ooc

i

l0,000

h

5

;

g

B

a

5000

0

Time (mm)

Fro.

1.

Time course of [35S]methionine incorporationinto proteins o f salivary gl8nds. -O-O-,

Shows the values obtained when the glsnds were incubsted with [36S]methionine in buffer (medium

A) for various lengths o f time at 23°C. -a-m-, Represents the values given when the tissue

was incubeted for 90 min at 23°C in medium A before being exposed to the label.

Experimental

condition s 8re described in Materials and Methods.

(c) 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 Coo-

ma&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.

(d) Controls

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 23”C, 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

26

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392

A. TISS IfiRES, H. K. MITCHELL AND U. M. TRAC Y

dissec ted and prepared for electrophoresis. Autoradiographs thus obtained did not differ

from those where isolated glands from anima ls of the same age were incubated in vitro

with the label.

(e) [3H]uridine labelling

of puffs after

heat shock

The salivary glands of heat-shocked and control larvae were incubated for 10 min at

23’C 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 slide s dipped in ethanol. After

drying, the slide s were dipped in liquid emulsion (Ilford L-4), exposed for 7 days and

developed for rephotography.

(

)

Chemicals

[36S]methionine and [3H]uridine were obtained from New England Nuclear, Boston,

Mass. and ecdystero ne from Schwarz-Mann, Orangeburg, 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 n 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

seen n Plate I(b) and Figure 2 and only one of these, moving at 20 mm, is present

at all the stages, hough it changes n 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 migra-

tion 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 changes n the band

pattern (2 or 3 bands only) during the prepupal period than do samples rom 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.

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PUFFS AND PROTEIN SYNT HESIS IN DROSOPH1LA

393

FIG.

2. Densitometer traaings of the autorediogreph of Plate I, showing changes in l&be lling

patterns of proteins from the salivary glands during the prepupal period. The numbers indicate

the time 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 37.5°C water bath for 20 minutes (Ashburner, 19’70).

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 23°C

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 23°C and otherwise treated in

the same way at the s&me time. The extracted proteins were electrophoresed on

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392 -4. TISS I&RES , I-1. I(. MITCHELL ;\SD LJ. JI. TRAC Y

60

a 0

L

4o0

I I

I

I

I

I I

20 40 60

Miaration (mm)

FIG 3. Heat-shock effect on densitometer tracing of autoradiograph for the -2-h salivary gland

samp les. The tracing of the control wtw subtracted from that of the heat shock.

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 a ll 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 addit ion 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 heat-

treated salivary gland sample.

The heat shock brought about the labelling of new bands at the same position

in all three tissues at al l 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 lim ited 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 forma-

tion, one treated for 20 minutes at 37.5”C, the other mainta ined at 25°C 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 imag inal

disc samples after heat shock, the appearance of the characteristic bands (results

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PUFFS AND PROTEIN SYNTHES IS IN DROSOPHILA

395

TABLE 1

Amount of label in some protein bands induced by heat shock

Distance of

band migrmtion

on gel

(mm)

salivary

glands

-2h

Salivary

glands

+9h

B&n

-2h

Melpighian

tubes

+9h

9.5 19.0 16.0

15.0 17.0

12

2.2

2.4 3.6

2.3

26 1.4

2.2

2.7 0.7

38

1.7 1-7 3-6 2.5

44

1.7 I-7 3.5 2.5

46 4.5 2.7

6.2

6.7

V&e s are given aa percentage of toti lebel in each sample. They were obtained by measuring

the surface of peaks on densitometer tmcinga from the autoradiograph 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 37.5”C for 20 minutes and the other left at 25°C.

At the end of this period, the flies were again etherized and their brains dissected

and incubated at 23°C 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 repre-

sents 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 labell ing 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 labe l 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.

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396 A. TISS IKRES , H. K. MITCHELL AND U. M. TRAC Y

TABLE 2

Effects of ecdysterone n some rotein bands

Distanoe of

band migration

on gel

(-4

Tim e of exposure to ecdysterone

60 min 90 min

27 +O.Q

31 +0.46 +:.74

39

- 0.43 -0*72

42

+0.16 + 062

60 + 0.43 +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 23°C 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 cases he changesobserved were less han 1y0 of the total label incorporated

and thus far less han those resulting from heat shocks, as seen n Table 1.

4. Discussion

In the work described here, the proteins were labelled in vitro by incubating the

isolated tissue with [36SJmethionine or 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 changes n 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 suggest hat 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 37G’C 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%,

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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 n 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,

suggests hat 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 chromosome V 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 com-

pelling. In the case of the heat shock, we have extended the observations of Ash-

burner (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 atten-

tion to chromosome3 in the pictures shown, but the ninth puff at 33B (Ashburner,

1970) s also abelled 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 responsible or 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 compo-

nents 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 pro-

ducts but they can provide useful guidelines for further experiments.

The system used here, in which drastic changes n 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, ,he system would seemwell suited to the study of the puff proteins (Clever,

8/18/2019 1-s2.0-0022283674904471-main


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 com-

ponents 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 segments nvolved at each puff locus could be done, as was described by Lam-

bert (1972) for Balbini rings in

Chironomus.

This work was supported in part by grants from the National Science Foundation

(GB-23343) and the Swiss National Fund for Scientific Research (3811.72).

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