Chironomus tentans epithelial cell lines sensitive to ecdysteroids, juvenile hormone, insulin and heat shock

Copyright 0 1982 by Academic Press. Inc. All rlghts of reproduction in any form reserved 0014-4827/82/060309-I 1$02.00/O

Experimental

CHZRONOMUS

TENTANS

Cell Research 139 (1982) 309-319

EPITHELIAL

CELL LINES

SENSITIVE

TO ECDYSTEROIDS, JUVENILE HORMONE, INSULIN AND HEAT SHOCK c. WYSS’ Institutefor Cell Biology, ETH, CH-8093 Ziirich, Switzerland

SUMMARY A technique for the isolation of Chironomus tentans embryo cells and a medium for their in vitro cultivation are described. For survival and proliferation cells require insulin and undefined supplements like fetal calf serum (FCS), yeast extract components, bactopeptone, and/or Drosophila extract. In such cultures epithelial cells proliferate, to form closed, floating vesicles or sheets attached to the dish. Permanent, diploid, epithelial cell lines readily develop. They respond morphologically and by the cessation of proliferation to physiological concentrations of ecdysteroids. The effect of ecdysteroids is prevented or reduced in the simultaneous presence ofjuvenile hormone. Heat-shocked epithelial cell lines stop the synthesis of most proteins and initiate the production of about ten new proteins.

After the successful development of techniques for the isolation of somatic cell hybrids in Drosophila [ 1,2], it was tempting to investigate whether interspecies hybrids of insect cells show genome incompatibilities similar to those seen in hybrids between cells from different vertebrate species (review in [3]). Because of its extremely large chromosomes the Dipteran Chironomus has, like Drosophila, been extensively used in several laboratories for the study of gene regulation. Therefore it was planned to attempt the production of Drosophila xChironomus somatic cell hybrids. However, nothing has so far been published on the behaviour of non-polytene Chironomus cells in vitro, which are the only cells capable of participating in the formation of proliferating hybrids. I report here a simple procedure to isolate

from embryos of Chironomus tentans cells which are viable and free of microbial contamination and can therefore directly be used for cell fusion experiments. Furthermore such cells put into a culture medium originally devised for Drosophila cells are capable of extensive spontaneous differentiation, and in the case of epithelial cells of long-term proliferation. These readily develop into permanent cell lines, the first insect epithelial cell lines reported. Since such cell lines proved to respond to ecdysteroids, juvenile hormone and insulin, and to changes in temperature, this material will certainly be useful for the investigation of hormone and heat shock responses. * Present address: Department of Oral Microbiology and General Immunology, Dental Institute, University Zurich, CH-8028 Zurich, Switzerland. Exp Cell Res 139 (1982~

310

c. wyss MATERIALS

AND METHODS

Animals tentans were raised under standard laboratory conditions as described by Mahr et al. [4]. Egg masses were provided by Dr R. M&hr; they were collected every morning and stored at 10°C with aeration until use.

Chironomus

Culture medium The synthetic culture medium 20 used in this study is an earlier version of medium ZW described elsewhere [5] for culturing normal Drosophila cells. As shown in Results, the simpler medium ZW is at least as effective for the epithelial cell lines as 20. 20 differs from ZW in the following: L-histidine, 155mg/l; glutathione, reduced, nil; biotin, 0.1 mg/I; calcium pantothenate, 10 mg/l; choline chloride, 20 mg/l; folic acid, 0.1 mg/l; m-inositol, 20 mg/l; niacinamide, 0.1 mg/l; nicotinic acid, 10 r&l; pyridoxal-HCI, 30 mg/l; pyridoxine, 0.1 mg/l; thiamineHC1, 10 mg/l; vitamin B12, 0.1 mg/l; andin place of cholesterol and linoleic acid, 1 ml/l of lipids stock solution. Lipids stock solution is obtained by dissolving in 1000 ml of water the following: 10 mg L-a-lecithin (distearoyl), 10 mg L-o-lecithin (dimyristoyl), 5 mg L-o-lecithin (dipalmityl), 160 mg sodium-cu-glycerophosphate, 130 mg calcium phosphoryl choline, 70 mg phosphorylethanolamine, 2 mg cholesterol, 50 mg oleic acid and 50 mg linoleic acid; after extensive stirring at room temperature undissolved material is removed by 0.2 pm pore size filtration; therefore the actual concentration will be lower for some of these components. Unless otherwise stated this basal medium 20 was sunulemented for cell cultures with: 0.1 mg/l bovine ins& (Sigma), 2% (v/v) heat-inactivated fetal calf serum (FCS) (Gibco-Biocuh, Paisley, Scotland), a high molecular weight fraction of (Difco) yeast extract at a concentration adequate for the clonal proliferation of the Drosophi/a Kc cell line [6], 0.5% (v/v) of a Drosophila extract (see below), as well as phenol red (10 mg/l) and penicillin-streptomycin (Gibco-Biocuh).

Drosophila

extract

Wild-type Drosophila melanogaster were raised in mass culture under standard conditions [7]. Flies were homogenized and acid-ethanol extracted according to a procedure for the isolation of vertebrate insulin~8]. The lyophilized neutral extract was dissolved in water at a concentration corresponding to 200 mg flies/ml. This was cleared by centrifugation at 20000 g and sterilized by 0.2 ,um pore size filtration (Gelman TCM). The extract was stored at -20°C.

Chironomus

embryo cells

Fifty egg masses, mechanically cleared of attaching debris were washed four times in distilled water and transferred to a SO-ml Flacon centrifuge tube. After removal of water the tube was filled with a saturated, filtered solution of sodium hypochlorite and vot-texed for 30 set so that the egg mass jelly dissolved and the Exp Cd Res 139 (19821

eggs were free in suspension. After a further 2 min in hypochlorite (including 1 min centrifugation at 50 a) the eggs were washed four times in sterile distilled water (settling of eggs at 1 g). They were then exposed to 75% ethanol during 5 min, followed by four washes in water. The surface sterilized eggs were then transferred to a glass-Teflon homosenizer of about 0.2 mm clearance. After one wash with 20 containing 20% FCS thev were disruoted in 3 ml of this medium. The volume was then brought to 20 ml with the same medium and the suspension centrifuged for 1 min at 600 g. The pellet was then resuspended in serum-free 20 by gentle pipetting and any unbroken eggs and most vitelline membranes were allowed to settle. The supernatant was again centrifuged for 1 min at 600 g. The pellet was then suspended in 20 ml of complete culture medium and the suspension (about 2x 106cells/ ml) distributed in 2 ml aliquots to 35 mm Falcon tissue culture dishes. Cultures were incubated at 25°C in humidified air.

Karyotype

analysis

Cultures containing epitheha growing attached to the culture dish were treated during 24 h with 1 +g/ml colcemid (Fhrka). After washin in PBS (phosphatebuffered saline: 110 mM NaC f , 10 mM NeHPG,, pH 6.6) cells were swollen during 5 min in PBS diluted 1: 5 with water, fixed in methanol/acetic acid 1: 3 and stained with Giemsa.

[35S]methionine-labelled

proteins

For biosynthetic labelling of proteins well developed vesicles were transferred to a complete culture medium in which ZO was replaced by a minimal Drosophila medium ZR, where most amino acids, including methionine. are nresent at onlv 0.1 mM concentration [9]. After 24-h experiments*were started by addition of hormones (5sfold concentrated in PBS) or PBS control, changes in temperature (immersion hr water bath) and/or addition of labelled methionine to 400 pCi/ml (NEN 1108.5 Ci mmol-I, in water). After 1 h labelling cultures were diluted 20-fold with cold 20, the cells were pelleted and frozen at -70°C. Samples were dissolved in 9.5 M urea, 0.5% Triton X-100 and 5% 2-mercaptoethanol, separated by sodiumdodecylsulphate/lO% polyacrylamide gel electrophoresis, stained with Serva Blue and processed forfluorography according to published methods [ 10, 111.

RESULTS AND DISCUSSION Isolation

of cells

The procedure described in Methods was adopted after several preliminary experiments. In all experiments the egg surface sterilization procedure appeared to be effective in that no contaminants were found in samples incubated in antibiotic-free me-

Chironomus

epithelial

cells

3 11

dium. Also in the few samples of epithelial put into a new dish. After 1 week in culture cell lines looked at ultrastructurally no bac- all dishes contained numerous foci of epiteria were detected. thelial growth, either attached to the plastic When Chironomus embryos were dis- or more frequently as small, floating vesirupted strictly according to our routine pro- cles. Their origin could not be related to cedure for Drosophila embryos [5] in me- one of the cell types previously seen. In dium 20, large numbers of cells were liber- addition to the new appearance of epithelia ated. However, they rapidly disintegrated many of the round cells found earlier had in suspension and could not be recovered developed into giant, opaque fat body cells. by centrifugation. Only when eggs were During the next 10 days some of these, broken in 20 supplemented with 20% FCS mostly in aggregates, developed the typical could intact cells be isolated. Since these green colour seen in fat body in vivo. Apart cells afterwards remain stable in unsupple- from a few heavily melanizing cells, probmented 20 it may be assumed that during ably hemocytes, no other obvious changes the disruption of eggs toxic material (from were recorded. After about 4 weeks, with the yolk?) is released which can be neu- 1: 1 media change weekly, most cells began tralized by serum and after the centrifugato degenerate, with the exception of epition remains in the supemate. The cell sus- thelial cells which continued to proliferate. pension thus obtained consists mainly of single cells, but some aggregates not easily Long-term culture of epithelia dissociated by pipetting are also present. The epithelial vesicles developed in the For all experiments reported here these ag- primary and secondary cultures could be gregates were not removed by nylon mesh transferred to fresh medium where they filtration in order to avoid cell loss. continued to grow, yielding macroscopic vesicles and/or sheets attached to the dish Primary cultures (fig. la). When vesicles were allowed to Cells from embryos less than 24 h old were grow undisturbed they reached a size of isolated and plated at high density (about about 2 mm diameter until they began to 4x106 cells/35 mm dish/2 ml) in complete collapse and rimple. Typically in such medium. Within 1 h most cells were at- folded epithelia then the initially mostly flat tached to the dish and many had started and transparent cells began to increase in to extend processes. After 24 h an extensive size and turn into more or less spherical, opaque cells often containing crystalline network of nerve cells and of contracting multinuclear muscle cells had formed sur- (birefringent) inclusions. These cells did not rounding many cells not readily classified, continue proliferation. Further epithelial such as spindle-shaped cells, tibroblast-like outgrowth was, however, observed from and macrophage-like cells and many large, those parts of the collapsed vesicle which had apparently remained free of contact round cells. At this time remaining vitelline membranes floating in the medium were re- with other parts of the epithelium. Although moved using a Pasteur pipette under a ster- difficult to control, this intriguing phenomeomicroscope. Then 1 ml fresh medium was enon, suggesting a contact-induced shift added. At 72 h of culture 2 ml fresh medium from a proliferative program to some terwas added per dish. The contents were minal differentiation, merits further invesgently pipetted and 2 ml was withdrawn and tigation. Attempts to reproducibly dissoE.rp Cell Res 139 (1982)

312

C. Wyss

ciate epithelia in order to facilitate subculturing and quantitative growth studies were largely unsuccessful in that cells either completely disintegrated or then remained in epithelial association. For dissociation the following enzymes were tested at several concentrations and incubation times: trypsin, subtilisin, dispase, protease K and protease type VIII. None of these gave any improvement over pure mechanical dissociation using a 5 ml Falcon pipette. Also no improvement in either dissociation or survival was observed when the mechanical treatment was done in PBS, 20, complete culture medium (the routine procedure) or in 20 medium with 20% FCS. Always a large proportion of all cells disintegrated and the others remained in epithelial pieces of varying size. These could either close up to form new small vesicles or attach to the dish and grow as sheets. Occasionally single cells were obtained but in no case could these be observed to enter proliferation. This suggests that the origin of epithelial cells may have been in the aggregates present in the primary cultures. Alternatively they could originate in vitro from a less sensitive precursor. Since epithelia appeared in large numbers in every culture set-up, their growth potential does not appear to be the result of extensive adaptive changes. This notion is supported by observations on the karyotype and on the growth rate of epithelial cells. During 18 months of continuous proliferation no striking change in growth rate suggesting continuous selection of growth rate variants was detected. Due to the technically imposed uncontrolled subculturing this cannot be quantitated, however. Routinely cultures were diluted after mechanical dissociation 1 : 50 every 4 weeks. In view of the extensive destruction of cells during dissociation the effective dilution ~5.v~ Cdl Res 139 11982)

was higher. In any case the total multiplication achieved by these cells is sufficient to consider them permanent cell lines. The karyotype of the epithelial cell lines at least numerically is normal diploid [12] (fig. 2). Interestingly the small fourth chromosomes are mostly found in very close association. Fig. 2a shows a metaphase where the two fourth chromosomes are exceptionally well separated. In most other metaphases (fig. 2b) the pair is hardly resolved. In a few metaphases (fig. 2c) the homologues of all four chromosomes are found in close association. The high frequency at which growing epithelia appear in cultum clearly indicates that the population of eplmelial cells thus obtained will be heterogeneous. As already mentioned, cloning of such cells has not been possible. To some extent the expected heterogeneity can be reduced, however, by growing cultures from single small vesicles. Still, in such cultures heterogeneity is also observed. Apart from the possible polyclonality of the starting vesicle this will be largely due to the different behaviour of cells depending on theirposition in the epithelium as already noted for collapsing vesicles. Typically in spherical vesicles morphological heterogeneity of cells is limited (fig. lc, d); occasionally cells are seen protruding from the monolayer into the lumen, or a cap of very widely spread cells is found. However, in non-spherical vesicles (fig. 3), which also lack a free edge as

Fig. I. Morphology of epithelia. (a) Spherical vesicles

and large attached sheet: darkfield. (b-h) Phase contrast. Relatively homogeneous cell populations in (b) sheet and (c, d) spherical vesicles;(e) loosely arranged but oriented cells at edge of sheet bulging (top) off the dish due to central growth; v) cells extending spikelike protrusions across the sheet: (a) canal-like’ structures within a sheet; (h) rolled .;p edge of sheet, later leading to tube and vesicle formation. Bar, (a) 0.2 mm: (&-II) 20fim.

Chironomus epithelial cells

21-821807

3 13

EXQ Cell Res 139 (1982)

Fig. 2. Karyotype of epithelial cells. (a) Metaphase with eight distinctly resolved chromosomes; (b) meta-

phase (most frequent) with the small fourth chromo-

discontinuity, cell shape and size can vary greatly, with elongate, flat cells at constrictions, flat, polygonal cells on extended surfaces, and small, spherical cells at fine tips. In epithelia attached to the culture dish the edge is an obvious source of heterogeneity. The edge can be formed tightly by relatively small cells (fig. 1b, g) or more loosely by large cells which often seem to hold some tension (fig. le). This may be created by active growth zones (with very small cells) in the centre of the epithelium causing the sheet to bulge off the culture dish surface (fig. 1e). Quite often the edge is seen to roll back (fig. lh) thereby forming a tube from which by continued proliferation closed vesicles are again formed. In fig. lfa less frequent situation is shown where some cells extend compact spike-like protrusions across other relatively large, flat cells. Rather exceptional is the formation of canal-like structures within an epithelium (fig. lg) reminiscent of developing tracheae. The frequency of the various morphological appearances varies between different lines. Some lines, e.g., show almost exclusively spherical vesicles, whereas in one line extensive tubiform vesicles are routinely formed. Also the readiness with which epithelia grow attached to the culture Exp Cdl Res 139 (1982)

somes closely associated; (c) metaphase with homologues of all chromosomes in close association. Bar, 5 pm.

dish varies between different lines. How far these differences between lines reflect the different potential of the respective founder cells remains to be investigated. Culture medium Studies on Chironomus cells in vitro have so far been limited to the maintenance of larval or prepupal organs containing highly polytene cells, in particular of salivary glands and fat body. For these experiments mostly the chemically defined, modified Cannon medium [13] has been used, which, however, proved to be unsatisfactory for long-term incubations. More recently, Firling & Kobilka [14] devised a medium specifically modelled to the analysis of Chironomus larval hemolymph. Supplemented with yeastolate and FCS this medium permitted long term experiments with salivary glands in vitro. Work in this laboratory and on that of B. Daneholt (personal communication) showed that the chemically defined medium 20 presented here (cf Methods for limited data on the concentration of some lipophilic compounds) gives excellent results with salivary gland incubations up to several days. This medium has been developed empirically for Drosophila cells, leading to a composition widely differing

Chironomus

Fig. 3. Effect of ecdysterone on epithelia. Non-spherical vesicles were transferred to medium containing 1 M/ml ecdysterone. (we) Vesicle after 0, 15, 40,

epithelial

cells

3 15

64 and 144 h of hormone treatment, respectively; (f-i): vesicle after 0, 3, 6 and 17 days of hormone treatment, respectively. Bar, 0.2 mm.

from the analysis of Drosophila (and Chirocompared with whole salivary glands. nomus) larval hemolymph. Whereas 20 Omission of FCS alone slightly reduced may suffice for long term maintenance, survival, but further development was exeven established Drosophila cell lines re- tremely slow. No obvious adverse effect quire further supplements in order to ac- was seen after the single omission of either tively proliferate, in particular insulin, FCS, Drosophila extract or of yeast extract comyeast extract components and/or extracts of ponents. However, 20 supplemented only adult Drosophila [5, 6, 9, 151. Therefore in with insulin and FCS yielded sparse and exploratory experiments with embryonic slowly developing cultures. The crude Chironomus cells these four supplements to Drosophila extract used here, could be re20 were tested in various combinations and placed by 0.1 mg/l of a ca 80 % pure prepaconcentrations. The conditions as given in ration of CalGF, a highly cationic comMethods were found to give satisfactory re- pound of less than 300 D MW, isolated sults. This was later confirmed with the from adult Drosophila [9]. However, both established epithelial cell cultures. The CalGF and the high MW fraction of yeast single omission of insulin prevented most extract are not easily obtained. Therefore cells from surviving the first day in culture, a more practical replacement for both was indicating a higher sensitivity of these cells sought. Exp Cd Res 139 fl982)

316

C. Wyss

it is added to cultures together with inhibitory amounts of ecdysteroids. The growth inhibition seen with 30 rig/ml ecdysteroid is completely suppressed by JH and even a concentration of 1000 nglml ecdysteroid is not able to completely inhibit vesicle formation in the presence of JH. This interference of JH with ecdysteroid effects is very similar to earlier results with the Drosophila Kc cell line [ 181. In contrast to these Drosophila cells of undefined origin, however, the Chironomus epithelial cells give exactly the same dose response with ecdysone or observations Preliminary ecdysterone. show that these Chironomus cells very rapidly metabolize ecdysone to more polar compounds, among them ecdysterone (A. Dtibendorfer & C. W. unpublished). This Ecdysteroids and juvenile hormone might be an explanation for the unusual efThe new availability of insect epithelial cell fectiveness of ecdysone. If it is a sufficient lines naturally raises many expectations, explanation is not yet clear, since nothing several of them linked to the possibility of is known of the kinetics of hormone mehormone-dependent differentiation. There- tabolism and action. The same reservations fore several experiments were carried out must be made with respect to any explanato determine if these cells are able to re- tion of the combined effects of JH and spond to the two most studied insect hor- ecdysteroids. In a different type of experiment, ecdysmones, ecdysteroids and juvenoids. Freshly dissociated epithelia were dispensed into terone was added to a concentration of media containing different concentrations 1000 rig/ml to cultures containing already of ecdysone or ecdysterone alone or in well developed vesicles. Again a complete combination with juvenile hormone III (JH) inhibition of proliferation was noted. In at a fixed concentration of ca 6x lo-’ M addition, however, in this situation mor(2% of a saturated solution of JH in PBS phological and also biochemical reactions [17]). For both ecdysteroids no effect was of the cells can be followed. One reaction noted on morphology and proliferation with fairly common at least at the edge of epiconcentrations up to 10 nglml. At a concen- thelial sheets on the culture dish (where it is tration of 30 nglml they drastically reduced most easily observed) is the formation of the size and number of developing vesicles multinuclear cells. Most observations were, and at concentrations of 100 rig/ml and however, made on vesicles. As shown in higher completely inhibited vesicle forma- fig. 3 they shrink during the days following tion. JH added alone or in combination with addition of hormone and subsequently the either ecysteroid in concentrations up to 10 cells appear to increase in volume and benglml did not affect cultures visibly. How- come opaque. In this way the vesicles turn ever, a clear effect of JH is seen when into almost compact cell masses. PrelimiTwo solutions were found. One is a different crude extract of adult Drosophila which supports the proliferation of normal Drosophila cells (in hormone-supplemented medium) [5] and probably also contains CalGF. The other possibility is the use of Bactopeptone. In conclusion, the most practical way to culture Chironomus epithelial cell lines at present is medium ZW [5] supplemented with 0.1 mg/l bovine insulin, 1 g/l Bactopeptone (Difco) and 2% (v/v) heat-inactivated FCS. This could certainly serve as a good basis for further work on Chironomus cells in vitro, embryonic cells as well as polytene cells, where the role of insulin could fruitfully be investigated .

Exp Cell Res 139 (1982)

Chironomus

epithelial

cells

3 17

Fig.

4. Protein pattern of epithelial cells after ecdysterone treatment or heat shock, analysed by sodiumdodecylsulfate/lO% polyacrylamide gel electrophoresis. (Left) Total protein pattern as revealed by Serva Blue stain; (right) protein synthesis pattern as revealed by fluorography (10 days exposure) of [35S]methioninelabelled proteins. I, control cells; 2-4, cells exposed during 4 h to ecdysterone (1000, 100 and 10 ngfml,

respectively), with labelling during the last 60 min of treatment; 5-10, cells incubated at 3PC for 4, 3, 21, 2, 14 and 1 h, respectively, with labelling during the last 60 min of treatment; 11, MW standards: 220 kD fibronectin, 94 kD phosphorylase b, 57 kD pyruvate kinase, 40 kD creatine kinase and 25 kD a-chymotrypsinogen.

nary ultrastructural observations (C. Reinhardt, M. Miller & C. W. unpublished) indicate that after ecdysterone treatment the intercellular coupling, which is already very pronounced in control epithelia, increases markedly. Also, very impressive arrays of microtubules are formed and occasionally extracellular material is detectable at the apical microvilli. Obviously these initial encouraging observations should be followed up by more refined experiments, keeping in mind the experience from other in vitro systems where the importance of both culture conditions and exact hormone treatment schedule have recently been reemphasized [e.g. 193.

In order to detect early changes in protein synthesis by vesicles exposed to ecdysterone, cultures in ZR-complete medium were treated with ecdysterone (IO, 100 and 1000 rig/ml) for 3 h before the addition of [35S]methionine. After 1 h labelling samples were extracted and analysed on sodiumdodecylsulfate/ 10% polyacrylamide gels. As shown in fig. 4 within the resolution of this method no difference in protein synthesis could be detected. Heat shock

In recent years the heat shock response originally described in Drosophila [review in 201 has also been studied in Chironomus Exp Cell Rcs 139 f 1982)

318

C. Wyss

salivary glands, where by microdissection the intracellular distribution of the heat shock products could be studied [21]. Obviously for many biochemical investigations, as well as questions of tissue dependence of response, the salivary gland system could advantageously be complemented by more readily available material. Therefore experiments were carried out to determine the suitability of the newly available material for such purposes. In fig. 4 the results of an experiment which also included some ecdysterone-treated cultures are shown. After 24 h in ZR-complete medium cultures (50 ~1) were transferred to a 37°C waterbath and [35S]methionine was added for the final 60 min of heat treatment. Total heat exposure was for periods between 1 and 4 h. As expected for experiments of this timespan the total protein pattern as revealed by Serva Blue staining is not notably changed (fig. 4~). Since the protein content of parallel cultures due to the uncontrolled dissociation, is so variable, it is also shown for the interpretation of the fluorogram (fig. 4b). A comparison of fig. 4a vs b indicates low labelling intensity. If this is due to low synthetic activity in ZR-complete medium or to the low specific activity of label (ca 4 Cilmmol, disregarding intracellular dilution) is not clear. Nevertheless, these cells obviously respond to heat shock (lanes 5-10: 4, 3, 24, 2, lt, and 1 h at 37°C respectively) by reduced or absent synthesis of most proteins made by control cells (lane 1) and by the new synthesis of about ten proteins not made by control cells. Most of these will correspond to the proteins described by Vincent & Tanguay [21], namely proteins of 90, 73, 68, ,28, 25 and 22 kD. Whether the broad band around 36 kD has any relation with the 34 kD heat shock protein found in salivary glands only in the nucleus [21] is an open Exp Cd

Res 139 (1982)

question. At variance with the salivary gland data in the epithelial cells the 25 kD (and not the 68 kD) protein is most prominent and two additional heat shock products of about 17 and 19 kD are detected. In addition to the obviously limited experience with the present material, there are several technical differences which could contribute to differences in results. The most important difference may be that salivary glands after a 10 min heat treatment (in vivo) were incubated at 20°C for labelling [21]. This certainly demonstrates the long-lasting effect of heat shock. In the present experiments heat treatment was for periods up to 4 h and labelling was performed at the elevated temperature. These results therefore also include effects of the increased temperature on the protein synthesis itself. It will be interesting to investigate, furthermore, how far the difference in the cell types analysed is responsible for the different results. I thank Dr R. M;ihr for Chironomus egg masses, Dr A. Lang for help with protein analysis, and professor J. Sang for critical comments on the manuscript. This work was supported by a ETH grant to C.W. and H. Eppenberger.

REFERENCES 1. Wyss, C, Somatic cell genetics 5 (1979) 29. 2. - Exp cell res 125(1980) 121. 3. Ringertz? N A & Savage, R E, Cell hybrids. Academic hess, New York (1976). 4. M&r, R, Meyer, B, Daneholt, B & Eppenberger, H, Dev biol 80 (1980) 409. WY% C, Exp cell res 139 (1982) 297. 2: - Somaticcell genetics5 (1979) 23. I. Mitchell, H L Mitchell, A, Drosophila inf serv 39 (1964) 135. 8. Havrankova, J, Schmechel, D, Roth, J & Brownstein, ?v&Proc nati ___ . acad sci US 75 (1978) 5737. 9. Wyss, C, Insect b&hem (1982). In press. 10. Lihnmli, U K, Nature 227 (1970) 680. 11. Banner, W H & Laskey, R A, Eur j biochem 46 (1974) 83. 12. fieer&um, W, Chromosoma 5 (1952) 139. 13. Ringborg,U, Daneholt, B, Edstriim, J-E, EgyhBzi, E Jt Rydlander, L, J mol biol51 (1970) 679.

Chironomus epithelial cells 14. Firling, C E & Kobilka, K B, J insect physiol 25 (1979) 93. 15. Schneider, I & Blumenthal, A B, The genetics and biology of Drosophila (ed M Ashbumer & T R F Wright) vol. 2a, p. 266. Academic Press, London (1978). 16. Wyss, C, Experientia 37 (1981) 664. 17. Giese, C, Spindler, K D & Emmerich, H, 2 naturforsch 32c (1977) 158. 18. Wyss, C, Experientia 32 (1976) 1272.

Printed

in Sweden

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19. Yund, M A, Invertebrate systems in vitro (ed E Kurstak, K Maramorosch & A Diibendorfer) p. 229. Elsevier, North-Holland, Amsterdam (1980). 20. Ashburner, M & Bonner, J J, Cell 17 (1979) 241. 21. Tanguay, R M & Vincent, M, Can j biochem 59 (1981) 67. Received September 22, 1981 Revised version received January 12, 1982 Accepted January 16, 1982

Exp CellRes 139 (1982)