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.!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. 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 1’Ecole de Medecine, Geneve, Switzerlend.
389

Universiti

de Gleneve, 30 Quai

1970) at 23”C. 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. 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. TISSI&RES. with about O. from the latest larval stage to about 13 hours after puparium formation.03 Msodium phosphate buffer (pH 6*8).ribution of puffs. 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. (Ashburner.terns of prot. TRACY (Baud&h Q Panitz. 0. The proteins were found to be efficiently extracted from the tissues by this procedure for radioactivity measurement or clectrophoresis. After 20 min in the cold. glands from larvae about 6 h before puparium formation .HCl (pH 6. The liquid was removed and this wash was repeated 3 times. 1959. In the work described here we have attempted to correlate changes in the pat. The tissue. 50 to 180 Ci per mol) on a piece of Parafllm about 8 mm X 5 mm placed on a microscope slide.ein synthesis with changes in the dist.0625 MTris. both during normal development and after inducing specific modifications of that distribution. This is the case in Drosophila during periods of normal development. usually for 20 min. 2. 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. sedimented to the bottom of the tube. Incubation was carried out at 23”C. then in a mixture of methanol/chloroform (1: l). MITCHELL AND U. 1967). the trichloroacetic acid solution was pipetted off. Grossbach. were transferred to a 5-~1 drop of medium A containing 10 to 40 pCi of [36S]methionins (spec.er to the changes in patterns of chromosomepuffs. This was then covered with a small inverted glass vial containing a piece of moist filter paper to prevent evaporation. and the intact tissues. act. 1968. (Becker. 1964).390 A.011 M-NaCl. 1962) or anaerobiosia (Ashburner.021 MMgCl. 0. 1970). 10% glycerol. K. and modifications of the puffs can also be induced under the influence of the hormone ecdysterone (Ashburner. a drop of cold 10% trichloroacetic acid. with the slide still under the microscope. the tube wss sealed with 3 layers of Parafilm and heated in a boiling water bath for 10 min. and finally allowed to dry at 37°C. 1 “/b /3-mercaptoethanol.OOlo/o bromophenol blue) was added. 1% sodium dodecyl sulphate. Ashburner. and was stopped by adding. The small piece of Parafilm with the tissue was transferred to a 3-ml conical centrifuge tube fllled with cold IO?/. M.04 nf-KCl.003 ~-C&l. 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. 1969). 1972) or by various changesin the environment such as heat shock (Ritossa. 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.8). and the tissue was further washed for 20 min first in 95% ethanol. trichloroacetic acid. in general 3 pairs of salivaqr glands. and 0. during which time regulation of larval tissue degradation and imaginal tissue synthesis is most evident. TO find out the conditionsdescribed. Twenty or 30 4 of electrophoresis sample buffer (0. H. 0. The period chosen was one of extensive changesin puff pattern (Ashburner. detached from the Parafilm. 1967).

after [35S]methionine incorporation.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 .000 h 5 . 1970) on slabs A in thick using the modification of Studier (1972) of the apparatus described by Reid & Bieleski (1968). The samples were then washed (the last trichloroacetic acid wash being heated for 10 mm at QO’C). dried according to the method of Fairbanks et al. it was necessary to incubate the tissue in medium A for periods up to 90 min. as above. which was therefore omitted in the experiments described below. washing and drying as described above. It was found that incorporation was still fairly active for the Crst 20 min (Fig. then samples were prepared as above. 20. 1). were incubated for 15 min at 23”C. As in some of the experiments with ecdysterone. (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. salivary glands from larvae. The electrophoretic pattern of the autoradiograph was not modified by this treatment. 1). The slabs were stained with Cooma&e blue. The incorporation of methionine was almost linear for 20 min and by 80 min it had dropped a little more than twofold (Fig. conditions 1. necessary to agcertuin the validity of the method (i) Does treatment with pancreatic ribonuclease and deoxyribonuclease modify the electrophoretic pattern? To answer this question.01 rd-Tris*HCl (pH 7*4). (1965) modified by Maize1 (1970) and autoradiographed by contact with Kodak no-screen X-ray films. Gel electrophwesi.2 x lo6 cts/min of [35S]methionine in 6 ~1 of medium A. -O-O-. 8re described in Materials (c) and Methods. with 12. and radioactivity determined. the proteins extracted from the tissues in electrophoresis sample buffer. then with the addition of 5 ~1 of pancreatic deoxyribonuclease I/ml and 1 mna-MgCle. fhst with 5 pg of pancreatic ribonuclease/ml in 0. g B a 5000 0 Time (mm) Time course of [35S]methionine incorporationinto proteins of salivary gl8nds. Shows the values obtained when the glsnds were incubsted with [36S]methionine in buffer (medium A) for various lengths of time at 23°C.PUFFS AND PROTEIN SYNTHESIS IN DROSOPHILA 391 and 1.3 and autoradiography Polyacrylamide gel electrophoresis was carried out in a discontinuous sodium dodecyl sulphate system (Laemmli.ooc i " l0. destained by diffusion. Experimental Fro. -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.6% acrylamide containing O*8o/o bisacrylamide in the separating gel. the glands were preincubated in medium A for this length of time at 23”C.

MITCHELL dissected and prepared for electrophoresis. H. and a few changes in some weaker bands. When the proteins were stained with Coomassie blue (Plates I(a) and II(a)) the patterns were relatively constant throughout the post-larval stagesexamined. which disappeared at the time of puparium formation. the stained patterns from brain and Ralpighian tubes remained constant.5 h. act. On the other hand. obtained from New England Nuclear. 3. 2) show extreme variations during that period of prepupal development. Boston. 9 mm. moving at 20 mm.392 A. M. over 52 bands with different migration distances could be detected. exposed for 7 days and developed for rephotography. The minor bands undergo the same type of variation. 3. This observation was reproduced in several separate experiments. K. In contrast. Of the remaining 16 bands. The stained patterns of gel electrophoresis in the three tissues at three different stages of development are shown in PI&e II(e). . with the exception of one rather strong band at 43 mm. 13 h. After drying. Results (a) Protein patterns during prepupal development Sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns of salivary gland proteins are shown in Plate I. and ecdysterone [3H]uridine were from Schwarz-Mann. about 22 rather strong bands or peaks can be seenin Plate I(b) and Figure 2 and only one of these. the changes are more pronounced. the preparation dipped in liquid nitrogen. 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 23’C with 50 PCi of [3H]uridine (spec. stained and squashed (Ashburner. some persist through many stages and others through only a few. N. 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 glands were rapidly washed in medium A. Orangeburg.7 Ci per mmol) in 3 ~1 of medium A.though it changesin intensity. the slides were dipped in liquid emulsion (Ilford L-4). 47 and 55 mm). the coverslips removed and the slides dipped in ethanol. 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. the autoradiograph of the samegel (Plate I(b)) and the densitometer tracings therefrom (Fig.Y. Chromosome figures were photographed. In Malpighian tubes. however. While a few changes took place in the salivary gland samples during the same period (see Plate I(a)). 1967). five components are seen clearly at only one stage (2 h. affecting about a dozen bands (Plate II(b)). On careful examination of the autoradiograph. fixed in 45% acetic acid for 30 min. is present at all the stages. Excluding the larval sample (-3 h). (e) [3H]uridine Autoradiographs animals of the labelling AND same U. TISSIfiRES. ( f) Chemicals [36S]methionine and Mass. from those where isolated glands from with the label. 24 mm and 49 mm. 9.

The weights. ~wl~~togrcster during the prcpupal $ formation.5 gel 3. The numhrrs given below each sample the distance of migration from t. nw shown T chymutryp&opvn 81 2( (a) represent mm.5 7.5 9 11 .3 25.700 60. The scales on the. 13 .5 Ch) ?5 olectrophoresis of labelled the time in hours before position of the mol. Stained patterns (a) and autoradiograph 2 glands of 1).000 MoLwt I -3 (b) of a sodium dodocyl sulphate-polyacrylamide period. after puparium usrd.5 Time 5.he origin in brtwwn the 2 photographs xvith their molecular 5-5 Time(h) PLATE T. sides indicate and myoglobin. wt 2 Cb) proteins (-) and markers 9 11 from salivaq.-3 0. 0.5 2 3. catalaw.

-3 <a) 3 S 10 -3 Time Ch) 3 B 10 -3 M 3 10 I I <b) CHC -2 4 S HCH 10 Time(h) CH -2 B CHC H M C H I .

.

a 6 .

Berendes. The salivary glands. Here we have selected heat shock as B suitable treatment which could show a correlation between puff p&&x-n and specific polypeptides. Densitometer traaings of the autorediogreph of Plate I. Ashburner. patterns the time AND PROTEIN SYNTHESIS IN DROSOPH1LA 393 2. 1070). brain end Malpighian tubes. Besides salivary glands. 19’70). two other tissues. four brains and Malpighi~n tubes from six animals. brain and Malpighian tubes were dissected at 23°C and incubated with [35S]methionine as described in Materials and Methods using six salivary glands. Control animals were maintained at 23°C and otherwise treated in the same way at the s&me time.PUFFS FIG. for each sample. 1962. To this end the animals were placed in a closed vessel which was immersed in a 37. showing changes in l&belling of proteins from the salivary glands during the prepupal period. The numbers indicate in hours before (. were used. as unlike salivary glands they represent the major tissues which survive larvctl histolysis. The extracted proteins were electrophoresed on . The origin is at the right. respectively.) end after pup8rium formation. 1968.5°C water bath for 20 minutes (Ashburner. (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.

\SD LJ. 38. In all the samples. were dissected from each of two sets of larvae four hours before puparium formation. salivary gland gel and the resulting autoradiograph is shown in Plate II(b). the other maintained at 25°C and serving as a control. Moreover. At all stages examined in salivary glands. the appearance of the characteristic bands (results . Drastic changes in patterns of labelled proteins did occur as a result of the heat shock. The tracing FIG I 40 effect on densitometer tracing of the control wtw subtracted I I 60 (mm) of autoradiograph for the -2-h from that of the heat shock. The autoradiograph pattern thus obtained showed.5 mm. the amount of label in each new band was roughly proportional in the three tissues. ten wing imaginal discs. TRACY 60 a 0 L 4o0 I I 20 I I Miaration 3. shown in Plate II. JI. also appeared. tissues known to lack polytene chromosomes. 44 and 45. In addition new clear bands at 12. as seen in Table 1. The heat shock brought about the labelling of new bands at the same position in all three tissues at all the stages examined. Heat-shock samples. I(. and others more difficult to ascertain at 6. and the component at 9.5 mm was immediately obvious. one treated for 20 minutes at 37. To investigate whether the effect of heat shock was limited to cells having polytene chromosomes. in the imaginal disc samples after heat shock. These results are summarized for the -2-h salivary gland stage in Figure 3.5”C. 26. at 18. MITCHELL . six of the new bands account for about one-third. then treated and analysed by electrophoresis as described in Materials and Methods.5 and 16 mm.66 and 69 mm.392 -4. I-1. thus removing common peaks present equally in control and heattreated salivary gland sample.5 mm itself accounts for one-sixth of the total incorporation. Here the densitometer tracing of the autoradiograph. of the control was subtracted from that of the heat shock. brain and Malpighian tubes the appearance of a very heavily labelled component at 9. Some bands present in the controls were found reduced in intensity after heat shock. Each of the two sets of ten discs was incubated with [35S]methionine for 30 minutes. 33. TISSI&RES.

0 2.6 2. As shown in Plate III(b) and (c). They surface of peaks on densitometer tmcinga from the autoradiograph 17.3 0.5 6. separated into two lots.2 1.2 V&es are given aa percentage of toti lebel in each sample.5 2. it seemed likely that this would also be seen in the amount of RNA synthesized at puff sites. It should be noted that the label at position 88EF (Plate III(c) and (d)) is a background puff not induced by heat shock.0 3.2 1-7 I-7 2.7 Melpighian tubes +9h 15. not shown) as seen in salivary glands. In view of this extraordinary rate of protein synthesis exhibited in one single band.4 1.7 1.5 6. 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). the flies were again etherized and their brains dissected and incubated at 23°C with [35S]methionine for 30 minutes. one-day-old flies were etherized.7 3-6 3. . Non-heated controls gave a number of such background puffs as expected but none was labelled to a greater extent than this one. 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.7 were obtained by measuring shown on Plate II. brain and Malpighian tubes samples from heat shocked animals.5”C for 20 minutes and the other left at 25°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).5 12 26 38 44 46 the salivary glands Salivary glands B&n -2h +9h -2h 19. 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).e II(b)) is coded for by a. one being placed at 37. In order to answer this question. To investigate this question.PUFFS AND PROTEIN SYNTHESIS TABLE IN DROSOPHILA 395 1 Amount of label in some protein bands induced by heat shock Distance of band migrmtion on gel (mm) 9.7 2. Thus it may be that the protein band which appears after heat shock at 9.4 2.7 4.5 mm on the autoradiograph (Pls.0 2. At the end of this period. gene in the 87B region of chromosome 3R.t. (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. 1967) and autoradiography.5 16. Furthermore the amount of label at position 87B was very much larger than at sny other locus. We also asked whether the effect of heat shock could be seen in tissues of the adult fly.0 2.

though varying in intensity. Discussion In the work described here. K. ecdysterone treatment of isolated salivary glands in puff stage 1 yielded obvious changes in [35S]methionine incorporation in five bands. beside a few weaker ones. labelled by injecting [35S]methionine into the animals. These data suggestthat all the protein synthesis occurring at high rates in prepupal salivary glands results directly from chromosomepuffs.43 +0.46 . The salivary glands from 90-h larvae (puff stage 1) were inoubated in medium A at 23°C with and without lo-* M-eodystarone. 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. (d) Effects of ecdysterone As shown in Table 2. the proteins were labelled in vitro by incubating the isolated tissue with [36SJmethioninefor a short time. one only.16 + 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. After 60 and 90 min each semple of 6 glands was incubated for 20 min in [9]methionine and prepared for electrophoresis and autoradiography. 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. TRACY 2 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. In both casesthe changesobserved were lessthan 1y0 of the total label incorporated and thus far lessthan those resulting from heat shocks. The six principal bands account for about 30%. During the prepupal period. in one single electrophoretic band. four showing an increaseand one a decrease. followed by dissection and incorporation of [36S]methionine after one and three hours. in which the animals were exposed to 37G’C for 20 minutes. TISSIKRES. 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.396 A. 4. . and to the disappearance of others. Under the conditions used.Similar results were obtained by injection of ecdysterone into larvae. can be seenat all stagesand five are visible at one stage only. as seenin Table 1.43 +:.Q +0. The heat shock. Of the great many bands which can be seen during the 16-hour period examined.74 -0*72 + 062 +0. or of a group of individual proteins as the case may be. to give a concentration of 10m4M in the hemolymph. M. led to the rapid appearance of six rather strong bands. MITCHELL TABLE AND U. H.0.

Daneholt & Hosick (1973) have shown that a 75 S RNA transcribed in the Balbini ring 2 region of chromosomeIV in Chirowmm tentans. 1966). These could correspond to puff loci induced early (Ashburner. For inst. Considering the amount of label at 87A and 67B. as shown in salivary glands.he system would seemwell suited to the study of the puff proteins (Clever. So far our evidence for the direct relation of a given puff to a polypeptide band is circumstantial.ance. Thus it may well be that temperature variations have profound effects on development and select. 70A. short-lived. but was again weaker by 90 minutes. However. as they are the next most heavily labelled protein bands (Table 1). consistently showed several times greater label than any of the others. and which mimic the pattern of puffs on chromosomes. we have extended the observations of Ashburner (1970) that the treatment induces new puffs at the loci 63BC. 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. 87A. seemssuitable for the study of several questions related to control mechanisms at the DNA level. in which drastic changes in the protein l&belling patterns were observed. it is possible that these loci are responsiblefor the protein bands at 46 mm and 38 mm. and that they are. tissue known to lack polytene chromosomes. increased to 60 minutes. In the case of the heat shock. suggeststhat the messengerRNAs synthesized at puff sites are rapidly transported to the cytoplasm and translated. while the loss could correspond to locus 25AC. and it is reasonable to postulate that this component is coded for by messengerRNA produced at locus 87B. In this set. . Further work will be necessary to find out whether such stable RNAs are also present in the system used here. one locus. This behavior is similar to that described by Ashburner (1972) for locus 23E. but the ninth puff at 33B (Ashburner. The system used here. 64F. 87B. In the in vitro experiments. and to messengerRNAs and their translation. 3) appeared in amounts several times greater than any of the others. 67B. 74EF and 75B. the information obtained by observing gains and losses in polypeptide products after heat shock and ecdysterone treatment is quite compelling. The rapid appearance or disappearanceof bands. and found in high amounts in the cytoplasm. seenduring the prepupal period and following the heat shock. 93D and 95D by the demonstration of uridine incorporation in isolated glands at each of these positions (Plate III).ion due to differential modulation of gene activities. stage 1 salivary glands treated in vivo and in vitro with ecdysterone show new polypeptide components at 27.t. Obviously these approachescannot give unequivocal associations between chromosome loci and specific polypeptide products but they can provide useful guidelines for further experiments. We have confined our attention to chromosome3 in the pictures shown. on the average.5 mm (Plate II and Fig. At the same time and at other developmental stagesalso. 87B. the polypeptide component at 27 mm appeared by 30 minutes. In imaginal discs. is remarkably stable. brain and Malpighian tubes (Table 1). one protein band at 9. 1970) is also labelled after heat shock. 42 and 51 mm and the loss of an existing component at 39 mm. Similarly.PUFFS AND PROTEIN SYNTHESIS IN DROSOPHILA 397 and one of them alone for over 15% of the total protein synthesis in the sample. 1972) such as 23E. the heat shock induced the appearance of the same characteristic bands and this effect was also seenon brains of adult flies. 1955. Mitchell. 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. respectively.

22. U. In the case where a high rate of synthesis of specific protein or RNA components is observed. C~omoaoma. B. Biophys. 136-187. 5. (1964). H. Ashburner. Chromoeoma. Exptl Cell Res. 31.S. 5. 12. (1970). J. E. Science. A. . Studier.. K. Nature (London). J. 367-376. R. M.. (1966). Maizel. Chromoeoma. Dros. Biochem. Ashburner. Boyd. Laemmli. E. M. Insect Phyeiol. H. 17.398 A. 49. 755-765. Lethuifaktoren. Beermann. J. Anal. 35. M. C. (1966).. K. 71-122. 38. 393-399. S. F. (1972). Becker. Nat. Method8 in Vkology. Mitchell. H.!. Fairbanks. (1966). (1968). C. H. Inf. J. 671-573. Biochem. Lambert. Elgin. 72. Ritossa. & Bieleski. Hadorn. (1964). Foundation REFERENCES Ashburner. H. 1968) and/or of the non-histone chromosomal proteins (Elgin et al. W. in the press. Chromoeoma. 135-137. L. & Mitchell. J. (1970). 179-246. 139-198. W. (1973). S. C. Wray. 39. 24. L. 309-322. Quant. R. Cold &wing Harbor Symp. (1970)..72). D. Berendes. Clever. & Wu. 21. (1952). Biol. Berendes. U. U. (1969). 227. (1968). K. 654-678. Chromosoma. 28. 70. B. With purified messengerRNAs. Proc. TISSIERES. Baud&h. Levinthal. 25&281. (1968). 1~. 38. R. Rea. & Panitz. (1959). 20. 521-531. R. Grossbach. as was described by Lambert (1972) for Balbini rings in Chironomus. (1970). Chromoeoma. Georg Thieme Verlag. (1966). Chromosoma. 418-437. 15. (1972). M. 1973). Biol. B. & Hosick. M. 470-476... 65-75. H.A. C. V. Chromoeoma. the gene products should not be difficult to isolate and purify. & Reeder. 398-428. Hood. W. Ezperientia. Chromosoma. 680-685. C. 10. (1962). as in some of the stages of the development and particularly after heat shock. Sci. Quant. F. 356-370. (1967). Cold Spring Harbor Symp. Mitchell. Reid. 18. Mol. Serv. This work was supported in part by grants from the National Science (GB-23343) and the Swiss National Fund for Scientific Research (3811. Daneholt. W. H. Rio. Pelling. Stuttgart. 374-381. 442-446. Commun. Pelling. G. MITCHELL AND U. (1973). (1972). TRACY 1966. analysis of the arrangement of information in the DNA segmentsinvolved at each puff locus could be done. Acud. 176. U. F.