RNA synthesis in puff 2-48BC after experimental induction in drosophila hydei

Cell, Vol . 8, 2 9 9 -304, June 1976, Copyright A 1976 by MIT

RNA Synthesis in Puff 2-48BC after Experimental Induction in Drosophila hydei

T . Bisseling, H . D . Berendes, and N . H . Lubsen Department of Genetics University of Nijmegen Nijmegen, The Netherlands

which can be extracted from isolated puffs, to mRNA . We have chosen to study initially the RNA produced by the puff at 2-48BC, since the terminal location of this puff on the second chromosome makes it relatively easy to isolate by micromanipulation .

Summary

Results

Puffs induced by treatment with vitamin B6 at locus 2-48BC in salivary gland cells of Drosophila hydei were isolated by micromanipulation, and the puff RNA was analyzed electrophoretically . The main RNA species migrated with a mobility of 40S . A similar RNA species was found in this puff after its induction by temperature treatment, except that in this case material with a higher mobility was also found, presumably due to the presence of growing chains . No evidence was found for polyadenylation of the RNA contained within the puff . The RNA profile of nucleoplasm of cells from glands either treated with vitamin B6 or subjected to a temperature shock also displayed a prominent peak with a mobility of 40S which was absent in extracts of nucleoplasm from control glands .

Nucleolar RNA To establish the validity of our method (which was slightly modified from Daneholt et al ., 1969) for isolation of the subcellular components and for extraction of the RNA, we first looked at the newly synthesized RNA from microdissected nucleoli . As Figure 1A shows, after a labeling time of 75 min, virtually all newly synthesized RNA moves with a mobility of about 38S (as calculated from the mobility of E . coli rRNA markers), in agreement with other reports on the size of the ribosomal RNA precursor in insects (Ringborg et al ., 1970 ; Meyer and Hennig, 1974 ; Serfling, Maximofsky, and Wobus, 1974) . After a chase period of 105 min, the relative amount of 38S RNA was reduced, and the main peak of radioactive material now moved with a mobility of 28S (Figure 1 B) . The 28S ribosomal RNA in insects is known to be very sensitive to degradation and is likely to be cut in vivo shortly after its final processing (Shine and Dalgarno, 1973) . Since we find, even after melting the RNA by heating, an intact 28S species in the nucleolus, it is improbable that

Introduction The large size of the dipteran salivary gland polytene chromosomes makes them amenable to direct isolation of specific chromosome sites by micromanipulation . This approach has been very successful in elucidating the nature of the transcript in Balbiani ring 2 of Chironomus tentans (for review see Daneholt, 1975), and we have used this method to study the transcript of an experimentally induced puff in Drosophila hydei . As has been shown previously, interference with the cellular respiratory metabolism leads to the activation of at least five genome loci in Drosophila hydei (for review see Berendes, 1975) . An analogous response is seen in Drosophila melanogaster (Ritossa, 1962 ; Ashburner, 1972) . Gene activation is followed by the appearance of a distinct pattern of de novo polypeptide synthesis (Tissieres, Mitchell, and Tracy, 1974 ; Lewis, Helmsing, and Ashburner, 1975) and, at least in diploid cells of Drosophila melanogaster, a new population of mRNA species which hybridize back in situ to the activated gene loci (McKenzie, Henikoff, and Meselson, 1975 ; Spradling, Penman, and Pardue, 1975) . Presumably the puffed areas code for novel mRNAs which in turn are responsible for the altered pattern of protein synthesis . This system then offers the opportunity of studying the pathway from primary transcript,

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Figure 1 . Electropherogram of RNA from Isolated Nucleoli (A) RNA extracted from 100 nucleoli labeled for 75 min . (B) RNA extracted from nucleoli labeled as in (A), but incubation was continued after labeling for 105 min in medium containing an excess of cold RNA precursors . The mobility of E . coli rRNA is indicated .

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Puff RNA It has been shown previously that a puff at the locus at 2-48BC can be induced by a variety of treatments, such as a rise in temperature from 25 to 37°C or addition of a high concentration of vitamin B6 to the incubation medium (Leenders et al., 1973). Especially during the incubation with vitamin B6, this puff becomes very large and is therefore easily identified during the manipulation of the chromosomes. Figure 2 shows the process of isolating this puff: first the chromosomes are separated with two glass needles; the puff is then isolated by cutting the second chromosome behind it; and the isolated puffs are collected on a piece of glass. Clearly the pieces of chromosome isolated in this manner contain not only the puffed area but also the rest of the tip of the second chromosome (248& and 2-48&) and possibly a few bands on the proximal side of the puff. However, autoradiographic analysis of glands pulse-labeled during vitamin B6 treatment has shown that in this region of the

Figure

2. Isolation

of Puff 2-48BC

by Micromanipulation

(A) A piece of a fixed salivary gland. (6) The spreading of the chromosomes (C) Cutting of the second chromosome (D) Isolated puffs.

with glass needles. behind the puff.

chromosomes, aside from the puff area, only region 478 contains some label (Leenders et al., 1973). Thus virtually all of the label found in these isolated pieces of chromosome should originate from the 2-48BC region. A typical electrophoretic analysis of this label is shown in Figure 3. Two symmetrical peaks are seen: one of a size corresponding to 40.5 and one with a mobility of about,l6S. The amount of the latter peak varies with the experiment: occasionally it is not present at all, while in other experiments it is of about the same size as the peak at 40s. Occasionally, a small peak is also observed in the 4s region. This peak, however, is not sensitive to RNAase treatments, while the other two peaks are, and may represent contamination with precursors of the mucopolysaccharides. A similar 4s peak has been observed in Rhynchosiara (F. J. S. Lara, personal communication). Furthermore, a variable amount of RNAase-sensitive radioactivity is always found on top of the gel. This may represent a residual aggregation of the RNA molecules. The symmetrical shape of the two peaks and the absence of low molecular weight RNAase-sensitive material make it improbable that the 16s species is caused



Puff Induction in Drosophila hydei 301

by degradation of the 40S species during the dissection and extraction procedure . The symmetry of the 40S peak found here contrasts sharply with the electrophoretic pattern found by Daneholt and his co-workers (for review see Daneholt, 1975) for RNA extracted from Balbiani ring 2 in Chironomus tentans : they observed a continuous spectrum of material up to a size of 75S . They argued convincingly that the material smaller than 75S represented growing RNA chains . If, however, the rate of transport of the finished product out of the puff area is considerably slower than the rate of synthesis, then the growing chains would constitute only a minor proportion of the total label in the puff area and a continuous spectrum of material would not be observed . Cytological data suggest that such an accumulation indeed occurs in puff 2-48BC . Autoradiographic analysis has shown that incorporated label is chased very slowly from

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the puff during vitamin B6 treatment (J . Derksen, personal communication) . Nevertheless, in electronmicrographs of sectioned salivary gland nuclei after induction of the puff at 2-48BC, the distinctive complex RNP particle produced by this puff can be seen in the nucleoplasm (Derksen, Berendes, and Willart, 1973) . Some release of material from this puff area must therefore occur . The nucleoplasm from labeled glands (again obtained by microdissection) was also analyzed electrophoretically . A distinct peak of 40S material was found in the nucleoplasm of vitamin B6-treated glands (Figure 4B), while nucleoplasm from untreated glands showed a heterogeneous electrophoretic profile (Figure 5) . The relative amounts of 40S and 16S material in the nucleoplasm after vitamin B6 treatment are similar to the amounts found in puffs isolated from the same cells, which also argues against a precursorproduct relation between these two molecules . It cannot be excluded that some of the 40S material derives from other puffs present . We can exclude, however, the idea that the 40S material found in isolated puffs is due to contaminating nucleoplasm, since extracts from other pieces of (inactive) chromosomal material, isolated from the same glands, contained no radioactivity except for some small molecular weight material (see Figure 4A) . Puffs analogous to these induced in Drosophila hydei can be induced in Drosophila melanogaster by a temperature shock (Ritossa, 1962 ; Ashburner, 1972), although Drosophila melanogaster does not seem to contain a locus analogous to 2-48BC and is not sensitive to vitamin B6 treatments (M . Ashburner,

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Figure 3 . Electropherogram of RNA Extracted from Isolated Puffs after Vitamin B6 Induction The extract was obtained from 75 puffs . For the RNAase treatment (dotted line), half of the sample was made 400 1 g/ml in pancreatic RNAase and incubated for 2 hr at 37°C before electrophoresis .

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Figure 4 . Comparison of the Electrophoretic Profile of Labeled RNA from Isolated Puffs and Nucleoplasm during Vitamin B6 Treatment Puffs (A) and nucleoplasm (B) were obtained from the same glands . 75 puffs were used, while nucleoplasm was obtained from 50 cells . The profile of the label contained in 50 pieces of inactive chromosomes (about half a chromosome long) is shown by the dotted line in (A) . Note the virtual absence of 16S material in this particular puff extract as compared to that shown in Figure 3 .



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quoted by Derksen, 1975) . It has been shown that during temperature treatment of diploid cells of Drosophila melanogaster, distinctive new RNA species appeared in the cytoplasm . Most of these RNA species contained poly(A) (McKenzie et al ., 1975 ; Spradling et al ., 1975) . Furthermore, since in mammalian cells some of the hnRNA contains poly(A), part of which may serve as a precursor for the poly(A) containing mRNA (for review see Lewin, 1975), it was of interest to see whether or not the puff RNA contained poly(A), Extracts from isolated puffs were therefore passed over a poly(U)-Sepharose column, and the flow-through and the bound material were analyzed by gel electrophoresis . All of the labeled material was present in the flow-through fraction and had a mobility close to 28S (Figure 6) . This higher mobility is presumably due to RNAase action during the chromatographic procedure . We thus conclude that the RNA present in the puff does not

contain poly(A), and if poly(A) is added to this RNA, it must occur after transport of the material out of the puff area . As mentioned above, the puff at 2-48BC can also be induced by a temperature shock . Under these conditions, the puff is smaller than during vitamin B6 induction and, furthermore, does not remain but regresses after about 2 hr (Berendes, 1968) . The salivary glands, however, are as active in uridine and methionine incorporation as untreated glands, while treatment with vitamin B6 severely inhibits both . On the other hand, this induction procedure is less specific for 2-48BC and many more chromosome loci are active (although not puffed), as indicated by autoradiographic analysis (Berendes, 1968) . Thus RNA synthesized in bands close to 2-48BC may occasionally contaminate the isolated puffs . However, judging from the relative grain den-

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Figure 5 . Electropherogram of a Nucleoplasmic Extract from Uninduced Cells Nucleoplasm was obtained from 25 cells labeled for 75 min at 25°C as described in Experimental Procedures . Note that the incorporation of radioactive precursors is much higher than during vitamin B6 . It is improbable, however, that a "40S" peak is present but hidden by the high incorporation at other chromosome loci in this profile, since the uptake of radioactive precursors is severely inhibited during incubation with vitamin B6 (N .H .L ., unpublished observations) .

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Figure 6 . Poly(U)-Sepharose Chromatography of Extracts from Isolated Puffs after Vitamin B6 Induction The extract from 70 puffs was passed over a 0 .5 ml poly(U)Sepharose column as described by Lindberg and Persson (1972) . (Under these conditions, labeled poly(A), obtained from Miles, was retained completely) . Both the unbound (solid line) and the bound (dotted line) fractions were analyzed electrophoretically .



Puff Induction in Drosophila hydei 303

sities over the puffed area and adjacent bands in autoradiograms, such contamination would constitute only a minor proportion of the total labeled material extracted from the isolated puffs, especially since the technique used precludes the isolation of small puffs . To compare the RNA present in the puff area under these conditions, RNA was extracted from temperature-induced puffs and analyzed by gel electrophoresis . A more complex pattern was obtained than after vitamin B6 treatment : a peak with a mobility of 40S is still present, but heterogeneous material with a higher electrophoretic mobility was also seen (Figure 7A) . This heterogeneous material might be produced by processing of the 40S species, or it might represent growing RNA chains . The first possibility is improbable since 40S material is also found in the nucleoplasm (Figure 7B), and preliminary results show no conversion of 40S material to smaller species during a chase . If the heterogeneous material represents growing RNA chains, then, by analogy to the situation in BR2, the 40S species must represent the primary transcript, since a continuous spectrum of material with a lower mobility is not observed unless processing starts during transcription . As in the vitamin B6-induced puffs, any modification of RNA chains within the puff area does not include addition of poly(A), since none of the radioactive material in extracts of isolated puffs was retained by poly(U) filters (determined by the method of Sheldon, Jurale, and Kates, 1972) . Discussion Although both during vitamin B6 and temperature treatment the main species of RNA found in the puff area has a mobility of 40S, there is a qualitative difradioactivity (cts /min . )

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Figure 7 . Electropherograms of Puff and Nucleoplasmic Extracts after a Temperature Shock (A) Extract from 75 puffs . (B) Nucleoplasm was isolated from 50 cells as described in Experimental Procedure, except that glands were labeled for 70 min .

ference between the RNA profiles obtained . We have argued that this difference is due to a high ratio of growing chains to finished molecules in the puffs induced by a temperature shock . This is also to be expected from the differences in the size of the puff . Clearly, once the size of a puff has reached a steady state, the rate of RNA synthesis and the rate of transport of material from the puff area must be the same . The much larger size of the puff during vitamin B6 induction, as compared to temperature-induced puffs, thus means that the ratio of growing chains to finished chains is much lower in vitamin B6-induced puffs . It must be noted that labeling times were long enough to label completely all the material present in the puffs : labeling was for 2 .5 hr during vitamin B6 treatment and for 60 min at 37°C, while the times required to chase label from the puff area are at least 90 min (J . Derksen, personal communication) and 20 min (Berendes, 1968), respectively (both chase times were determined in the presence of actinomycin D and the inductive stimulus) . We have not found any evidence for processing of the RNA chains within the puff area : neither a conversion of the 40S species to a smaller RNA was seen nor was the RNA polyadenylated . Studies of RNA processing in the nucleoplasm are hampered by the fact that, especially at 37°C, many other chromosome loci are active . A 2-48BC origin for an RNA species therefore cannot be assigned unambiguously . The presence of a large peak of 40S material in the nucleoplasm during vitamin 66 treatment does suggest that possible processing products do not accumulate . It is possible that the lowering of the ATP level of the cells during puff induction (Leenders et al ., 1974) causes an increase in the stability of nuclear RNA, as has been shown to occur in hepatoma cells (Avram and Hersko, 1975) . It must be pointed out further that despiralization of the DNP fibers of one locus during puff induction also leads to despiralization of DNP fibers in neighboring loci . In particular, the puff area at 2-48BC includes five small bands, that is, 2-48B 4 through 2-48C2 . Neither by autoradiography nor in electronmicrographs can the transcriptional activity be assigned unambiguously to any of these bands . Thus the possibility cannot be excluded that more than one band in this region is transcriptionally active after puff induction . Indeed, some of the autoradiographic data obtained with vitamin B6-induced puffs have been interpreted to show that more than one band is active (see Berendes et al ., 1973) . Since the activation of other bands could vary with the induction procedure used, this could explain the variable appearance of the 16S species during vitamin B6 induction .

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Experimental Procedures

Edstrom, J .-E . (1964) . In Methods of Cell Physiology, D . Prescott, ed . (New York : Academic Press), p. 417 .

Isolation and Labeling of Salivary Glands Salivary glands were isolated by hand from late third instar larvae of a laboratory stock of Drosophila hydei . For puff induction with vitamin 86, 5-7 pairs of glands were incubated for 2 .5 hr at 25°C in 25 µl of Poels' (1972) medium containing 2 .5 x 10-2 M vitamin B6, 50 µCi 3 H-uridine (40-44 Ci/mM), 25 µCi 3H-adenosine (20-27 C/mM), 25 µCi 3H-cytidine (25-29 Ci/mM), and 5 µCi 3H-guanosine (5-6 Ci/mM) . For puff induction by temperature shock, glands were incubated at 37°C for 55 min in Poels' medium containing twice as much labeled precursors as during vitamin B6 treatment .

Leenders, H . J ., Derksen, J ., Maas, P . M . J . M ., and Berendes, H . D . (1973) . Chromosoma 41, 447 .

Micromanipulation After incubation, glands were fixed in alcohol :acetic acid (3 :1 v/v) for 15 min at 4°C, rinsed twice for 15 min with 70% alcohol, and stored in alcohol :glycerol (1 :1) for at least 90 min at-20°C . Cellular components were isolated from the fixed glands essentially according to Edstrom (1964) with a Fonbrune micromanipulator . The fragments were collected on a small piece of glass (1 .5 mm2) . Extraction and Electrophoresis of RNA The small piece of glass containing the cellular fragments was rinsed with chloroform to remove the paraffin oil . It was then transferred to a small tube containing 25 id of extraction medium [0 .02M Tris-HCI (pH 7 .4), 2 .5% SDS, 0 .1 mg/ml polyvinyl sulfate, and 1 mg/ml E . coli rRNA] . The sample was immediately heated at 90°C for 3 min and applied to the gel . Electrophoresis was performed on 2 .4% acrylamide gels according to Bishop, Claybrook, and Spiegelman (1967) . After the electrophoretic run, gels were sliced, and the gel slices were dissolved in 200 µl hydrogen peroxide for 6 hr at 60°C and counted in 10 ml of toluene:triton scintillation fluid . Materials 3H-uridine and 3H-adenosine were obtained from Amersham Radiochemical Centre ; 3 H-guanosine and 3 H-cytosine were from New England Nuclear . E . coli rRNA was supplied by BDH biochemicals, and poly(U)-Sepharose was obtained from Pharmacia . All other chemicals used were reagent grade . Acknowledgments The authors would like to thank Dr . J . Derksen for his helpful comments during the course of this work and preparation of the manuscript . Received December 8, 1975 ; revised February 19, 1976 References Ashburner, M . (1972) . In Results and Problems in Cell Differentiation, 4, W. Beermann, ed . (Berlin : Springer-Verlag), p . 100 . Avram, M ., and Hersko, A . (1975) . Biophys . Biochem . Res . Commun . 65, 1303 . Berendes, H . D . (1968) . Chromosoma 24, 418 . Berendes, H . D . (1975) . FEBS Letters, in press . Berendes, H . D ., Alonso, C., Helmsing, P . J., Leenders, H . J ., and Derksen, J . (1973) . Cold Spring Harbor Symp . Quant . Biol . 38, 645. Bishop, D . H . L ., Claybrook, J . R ., and Spiegelman, S . (1967) . J . Mol . Biol . 26, 373 . Daneholt, B . (1975) . Cell 4, 1 . Daneholt, B ., Edstrom, J .-E ., Egyhazi, G ., Lambert, B ., and Ringborg, U . (1969) . Chromosoma 28, 418 . Derksen, J . (1975) Cell Differentiation 4, 1 . Derksen, J ., Berendes, H . D ., and Willart, E . (1973) . J . Cell Biol . 59, 661 .

Leenders, H . J ., Kemp, A ., Koninkx, J . F . J . G ., and Rosing, J . (1974) . Exp . Cell Res . 86, 25 . Lewin, B . (1975) . Cell 4, 11, 77 . Lewis, M ., Helmsing, P . J ., and Ashburner, M . (1975) Proc . Nat . Acad . Sci . USA 72, 3604 . Lindberg, U ., and Persson, T . (1972) . Eur . J . Biochem . 31, 246 . McKenzie, S . L ., Henikoff, S ., and Meselson, M . (1975) . Proc . Nat . Acad . Sci . USA 72, 1117 . Meyer, G . F ., and Hennig, W . (1974) . Chromosoma 46, 121 . Foals, C . L . M . (1972) . Cell Differentiation 1, 63 . Ringborg, U ., Daneholt, B ., Edstrom, J .-E ., Egyhazi, E ., and Lambert, B . (1970) . J . Mol . Biol . 51, 327 . Ritossa, F . (1962) . Experientia 18, 571 . Serfling, E ., Maximovsky, L. F ., and Wobus, U . (1974) . Eur. J . Biochem . 45, 277 . Sheldon, R ., Jurale, C ., and Kates, J . (1972) . Proc . Nat . Acad . Sci. USA 69, 417 . Shine, J ., and Dalgarno, L . (1973) . J . Mol . Biol . 75, 57 . Spradling, A., Penman, S ., and Pardue, M . L . (1975) . Cell 4, 395 . Tissieres, A ., Mitchell, H . K ., and Tracy, U . (1974) . J . Mol . Biol . 84, 389 .