In panels E and F,poly(A) + RNAs were prepared from control and hormone-treated cells and translated in the reticulocyte lysate. ...... Plasmid pBR322 (Bolivar et al., 1977) was cut with PstI. ... Alwine,J.C., Kemp,D.J. and Stark,G.R. (1977) Proc.
The EMBO Joumal vol.3 no.1 pp.235-243, 1984
cDNA clones for the ecdysone-inducible polypeptide (EIP) mRNAs of Drosophila Kc cells
C.Savakis1, M.M.D.Koehler and P.Cherbas* Department of Cellular and Developmental Biology, Harvard University, Cambridge, MA 02138, USA 'Present address: Department of Genetics, University of Cambridge, Cambridge, UK *To whom reprint requests should be sent Communicated by F.C.Kafatos
The ecdysone-inducible polypeptides (EIPs) 28, 29 and 40 were identified previously as polypeptides whose synthesis is stimulated early in the ecdysone response of Drosophila Kc cells. We have now shown, using two-dimensional gels, that each of these EIPs consists of three species differing in pI, and all stimulated by ecdysone. Translations and hybridarrested translations indicated that the poly(A) + EIP mRNAs increase 10-fold in abundance during the first 4 h of ecdysone treatment. By a differential screen of a cDNA library we have identified cDNA clones corresponding to all three EIPs. Two kinds of clones were isolated: one hybridizes to the EIP 40 mRNA(s); the second hybridizes to the mRNA(s) encoding all the EEPs 28 and 29. The EIP 28/29 and EIP 40 loci detected by these clones are each present at single sites on the polytene chromosomes and each is at or in the vicinity of an ecdysone-regulated puff. Key words: ecdysone/Drosophila/cDNA clones/Kc cells/ steroid -
Introduction Steroid hormones act, at least in part, by regulating gene expression. There is good reason to believe that for those genes which comprise the 'early' or primary hormone response, this regulation occurs by means of a direct interaction between steroid-receptor complexes and DNA in the vicinity of the regulated transcription unit (Yamamoto and Alberts, 1976; Higgins and Gehring, 1978). Thus, in the example of mouse mammary tumor virus (MMTV), a region of 5'-flanking DNA is both necessary and sufficient for hormonal regulation and binds glucocorticoid-receptor complexes (Lee et al., 1981; Payvar et al., 1981; Huang et al., 1981; Ucker et al., 1981; Fasel et al., 1982; Geisse et al., 1982; Govindan et al., 1982; Buetti and Diggelmann, 1983; Hynes et al., 1983; Chandler et al., 1983; Ponta et al., 1983; Parker, 1983; Scheidereit et al., 1983; Majors and Varmus, 1983). One very important step towards understanding both the mechanism and the tissue specificity of steroid responses will, therefore, be to identify those cellular genes which participate in the primary hormonal responses. We have been studying the early effects of ecdysone (see Materials and methods) in the Drosophila Kc cell line, and have sought in particular to identify genes which are regulated as part of the early hormone response in these cells. The Kc line is a permanent cell line which originated from primary cultures of disaggregated whole embryos of D. melanogaster (Echalier and Ohanessian, 1970). Kc cells are © IRL Press Limited, Oxford, England.
ecdysone-sensitive in that hormone-treated cells cease to proliferate and they differentiate both morphologically and enzymatically (Courgeon, 1972a, 1972b; Wyss, 1976; BestBelpomme and Courgeon, 1977, 1978; Cherbas et al., 1977, 1980a, 1980b; Berger et al., 1978; Rosset, 1978; Cherbas and Cherbas, 1981). We have previously reported that most pulselabeled polypeptides resolved by SDS-polyacrylamide gel electrophoresis were unaffected during 1-2 days hormone treatment. However, we were able to identify three ecdysoneinducible polypeptides (EIPs) whose relative synthesis increases markedly within 30 min of hormone addition and reaches an elevated plateau within 4 h (Savakis et al., 1980). These were designated the EIPs 28, 29 and 40 in accordance with their apparent mol. wts. (in kd). EIP induction is a specific effect of ecdysones acting at physiologically reasonable concentrations. Because it occurs rapidly and displays the broad dependence on hormone concentration characteristic of early puffs (Ashburner, 1973), EIP induction may be a primary event in the ecdysone response of Kc cells. We now report studies demonstrating that the increased synthesis of the EIPs in ecdysone-treated cells is accompanied by increases in the abundances of their poly(A) + mRNAs. We have employed a differential colony hybridization screen to identify cDNA clones corresponding to the EIP mRNAs. Results EIP induction analyzed by two-dimensional gels The EIPs 28, 29 and 40 have been defined by their sizes (mobilities in SDS gels) and by the rapid increase in their synthesis during ecdysone treatment. This contrasted with an otherwise essentially invariant pattern of protein synthesis, at least as observed following electrophoresis in one dimension. We have analyzed more thoroughly the effects of ecdysone on protein syntheis in Kc cells using two-dimensional separations. The complete results will be presented elsewhere; however the pertinent ones are summarized below and illustrated in Figure 1. Cells were treated for 4 h with 10-6 M 20-hydroxyecdysone (20-HE) or with the solvent (ethanol) alone. [These are standardized treatments and below we refer to cells thus treated as E-cells and C-cells, respectively.] The cells were then labeled in parallel (20 min) with [3H]leucine. The proteins were extracted, separated by isoelectric focusing and electrophoresis, and detected by autofluorography as described in Materials and methods. In Figure 1, panel A represents control cells and panel B hormone-treated cells. Comparison of these two panels confirms our earlier observation that the relative synthetic rates for most polypeptides are unaffected by hormone treatment. This.generalization applies over the entire range of sizes and charges sampled and not simply to the region shown in Figure 1. In contrast, inducible species of -28, 29 and 40 kd are readily identifiable. Each is composed of three forms differing in pl. For example, the three inducible spots of -28 kd 235
C.Savakis, M.M.D.Koehler and P.Cherbas F1
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Fig. 1. Portions of two-dimensional gels including the EIPs. In panels A - D cells were treated for 4 h with ecdysone or with ethanol alone, then labeled for 20 min with [3H]leucine: A, control cells; B, ecdysone-treated cells. Following the pulse-labeling a portion of each culture was chased 2 h in fresh nonradioactive medium which in the case of the ecdysone-treated cells also contained the hormone: C, control-chase; D, ecdysone-chase. Panel G is a guide to the spots of interest. In panels E and F, poly(A) + RNAs were prepared from control and hormone-treated cells and translated in the reticulocyte lysate. The translation products are shown in E for control cell RNA and F for ecdysone-treated cell RNA. Panel H is a guide to these spots. The autofluorograms have been truncated to show only the relevant regions. The acidic end of the isofocusing dimension is to the right. For details of hormone treatments, labeling and translations, see text. Open arrows (N) indicate the pair of actin spots.
co-migrate with EIP 28 in the SDS dimension (data not shown) and differ inter se by unit charge (A.Bieber, personal communication). We designate these the EIPs 281, 2811 and 28111, with form I being the most acidic. Likewise, EIP 29 is resolved into three forms and is similarly named. EIP 40 also appears as a cluster of three spots. Induction of EIP 4011 is often, as in Figure 1, marginal; however evidence to be presented later indicates that EIP 4011 is properly included in the set of EIP 40 spots. The two-dimensional separations have revealed only one new EIP, a polypeptide of -35 kd which is designated EIP 35 (Figure 1). Since, as we show below, the EIPs 28 are a set of related polypeptides, as are the EIPs 29 and EIPs 40, it is of interest to discover the relationship among the forms differing in pl. One approach is illustrated in Figure 1, panels C and D. These represent control and hormone-treated cells labeled as in A and B, but chased 2 h prior to extraction. The effectiveness of the chase is indicated by the very efficient conversion of the more basic actin III to actin II (Berger and Cox, 1979). With regard to the ensemble of EIP spots, the chase decreases the label present in the EIPs 28111 and 29111. These species are transient; whether the chased label accumulates in the more acidic EIPs 28 and 29 cannot be determined from
236
these experiments.
Cell-free translations From the total cellular RNAs of C and E cells we isolated the corresponding poly(A) + fractions. These C-A + and E-A + RNAs were translated in mRNA-dependent rabbit reticulocyte translation system, and the [3H]leucine-labeled products were analyzed by one- and two-dimensional electrophoresis and autofluorography. Typical results are illustrated in panels E and F of Figure 1. The following observations are pertinent. (i) The translation products include the EIPs 281, II and III, 29I and II, and 401, II and III, all of which can be identified by their characteristic mobilities in the two dimensions. The positions of these spots correspond precisely to those of their in vivo-labeled counterparts. The hierarchies of intensities, namely 2811 > 291I > 281 > 28III and 40111 > 401 > 4011, correspond to those observed following labeling in vivo and a chase. (ii) Each of the eight identifiable EIPs is synthesized at a significantly higher rate from the E-A + than from the C-A + template, i.e., their translatable mRNAs are inducible. (iii) No labeled translation product appears in the expected
cDNA dones for E3IP mRNAs of Kc cells
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Fig. 2. Hybrid-arrested translations. Poly(A) + RNAs from control cells (C) and hormone-treated cells (E) were reverse-transcribed to yield single-stranded C-and E-cDNAs. The RNAs and cDNAs were mixed in all four combinations, hybridized to two different Cots, and translated as described in the text. The figure shows an autofluorogram of a 10%7o SDS-polyacrylamide gel; the various channels represent a single exposure of that autofluorogram. Letters at the top indicate the source of the RNA and the source of the cDNA; the relevant Cot is indicated above each lane. The positions of the EIPs 28, 29 and 40 are indicated at the right and their induction is shown in the leftmost two, unhybridized lanes.
position for EIP 29111. Instead we routinely observe an inducible species of about the expected magnitude and mol. wt. but with a pl intermediate between 281 and 2911. This spot is labeled EIP 29a in Figure IF. (iv) Two additional minor inducible spots, EIPs 29b and 29c, are observed which exhibit pls intermediate between those of the EIP 40 and EIP 28/29 clusters. They are never observed following labeling in vivo. (v) The collection of 11 EIP and putative EIP species just enumerated exhausts the list of inducible translation products we have observed in these experiments. EIP 35 does not appear as an identifiable translation product, nor do we observe any additional inducible species which may represent a precursor of EIP 35. Collectively the EIPs account for a significant fraction of the total synthesis from E-A + templates. In fact the EIPs 2811 and 2911 are each more intensely labeled than any other polypeptide we detect. Overall the array of EIPs 28 and 29 1of the total incorporation. probably represent 51/o In summary, for most of the EIPs detected following labeling in vivo it is possible to identify counterparts among the translation products of poly(A) + RNAs. Each of the EIPs, but no other quantitatively significant translation product, is synthesized more rapidly from E-A + than from C-A + templates. Similar comparisons have been performed using a
variety of template RNA concentrations; although the ratio of EIP 40 to EIP 28 and 29 synthesis is sensitive to template concentration, for each of the EIPs E-A+ RNA is a better template than C-A + RNA at every concentration tested (data not shown). We also note that translations of total cellular RNAs yield similar results. These results suggested that EIP induction is accompanied by, and presumably at least partly caused by, increases in the levels of translatable EIP mRNAs. A priori this could reflect increased translatability of pre-existing mRNAs or increased mRNA abundance. We have employed a hybridizationarrested translation procedure (Paterson and Bishop, 1977; Hastie and Held, 1978) to distinguish between these possibilities. Using E-A + and C-A + RNAs as templates for avian myeloblastosis virus (AMV) reverse transcriptase, we prepared the corresponding cDNA mixtures. The resulting E-cDNA and C-cDNA were hybridized with equivalent weights of the homologous and heterologous poly(A) + RNAs, the hybridizations being carried out to Cot values of 0.05 and 0.5 (mol nucleotide/s). Aliquots of the hybridization reactions were then translated and the products analyzed by one-dimensional electrophoresis and autofluorography. The results shown in Figure 2 consist of the translation products from four pairs of reactions - the four cDNA:RNA combinations, each at two Cots. 237
C.Savakis, M.M.D.Koehler and P.Cherbas
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Fig. 3. Translations of RNAs selected by hybridization to cloned cDNAs: one-dimensional analysis. Channels labeled 'E' represent the translation products of poly(A) + RNA from hormone-treated cells. Channels labeled 'c' and 'e' represent the translation products of control poly(A)+ RNAs (c) and hormone-treated poly(A)+ RNAs (e) selected by hybridization to cDNA-containing plasmids. The numbers at the top identify the plasmids used, pKc441, pKcl7l, pKc252 and pKcl9l. pKcl71 is an example of a plasmid which yielded no detectable product. The product in the pKc441 lanes co-migrates with EIP 40; those in the pKc252 and pKcl91 lanes, with the EIPs 28 and 29.
For present purposes it is sufficient to evaluate this experiment qualitatively. Specific inhibitory effects due to hybridization should be more pronounced at the greater Cot; this contrasts with generalized inhibition by DNA which should be equivalent in all eight lanes. Because the hybridization reactions are (ideally) second order in the concentration of any hybridizing species, the fraction of any given mRNA remaining unhybridized should vary inversely with its initial concentration. That is, more abundant mRNAs should be disproportionately inactivated (Hastie and Held, 1978). If the EIP mRNAs are equally abundant in the C-A+ and E-A+ RNAs, then E-cDNA and C-cDNA should be equivalent in their abilities to arrest EIP synthesis. Figure 2 shows that: (i) translations of E-A + RNA but not of C-A + RNA yield intense bands corresponding to the EIPs; (ii) EIP synthesis is selectively arrested by hybridization with E-cDNA but not with C-cDNA; (iii) no other translation product is differentially inhibited by the two cDNA preparations; (iv) the EIPs are among the products most strongly inhibited by hybridization to E-cDNA. We interpret these results to mean that the EIP mRNAs are more abundant following induction, that they are among the most abundant mRNAs present in the E-A + population, and that no other abundant mRNA species is induced to a comparable extent.
238
191 Fig. 4. Translations of RNAs selected by hybridizations to cloned cDNAs: two-dimensional analysis. The top panel ('Total') shows a portion of an autofluorogram representing a two-dimensional separation of the translation products of poly(A) + RNA from hormone-treated cells. The same RNA was hybridized to pKc441, pKc252 and pKcl9l, and the selected species translated with the results shown. Only the EIP regions are shown; no spots were detectable elsewhere on the autofluorograms.
Table 1. Properties of the plasmids pKc252 and pKc441 Plasmid Size of cDNA insert
Transcript sizes
Translation products selected
Chromosomal site
pKc252 860 nucleotides 1050 N (major) EIPs 281,11,111, 71C3.4-Dl.2 1300 N (minor) 2911,111, 2300 N (minor) 29a,b,c pKc441 450 nucleotides 1500 N (major) ElPs 401,11,111 55B-E 1650 N (minor) 1800 N (minor)
Preparation, identification, and characterization of cloned cDNAs We have obtained cloned EIP cDNAs by a straightforward approach based on the preceding observations. We prepared a cDNA library using oligo(dT) as primer and E-A + RNA as template. This library was screened with probes representing
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Fig. 5. Northern blots probed for the EIP transcripts. Poly(A)+ RNAs of control (C) and hormone-treated (E) cells were fractionated by electrophoresis on a methylmercury-agarose gel. The RNAs were either unreacted ('control') or deadenylated by incubation with RNase H in the presence of oligo(dT) ('RNase H'). 1 gg RNA was present in each lane. Following electrophoresis, the RNA was transferred to DBM-paper and hybridized to nick-translated pKc252 (left panel, '252'). After autoradiography, the paper was rehybridized with nick-translated pKc441 without prior washing (right panel, '252 + 441'). Size markers included in the gel are not shown (see text).
the C-A + and E-A + RNA populations and we identified colonies hybridizing preferentially to the E-A + probe. Details of the library construction are given in Materials and methods. We employed standard procedures, using AMV reverse transcriptase and S 1 nuclease to copy the E-A + RNAs into double-stranded (ds) cDNAs of 200- 400 nucleotides modal length. These were cloned at the PstI site of pBR322 using the dC:dG tailing method of Roychoudhury et al. (1976). Transformed bacteria were plated on selective medium, then replica-plated onto nitrocellulose filters for colony hybridization in situ. The probes were single-stranded 32P-labeled cDNAs synthesized using C-A + and E-A + RNAs as templates and using oligo(dT) as primer. In the initial stage of the screen we examined 30 plates (- 6000 colonies), each probe being hybridized to duplicate filters. Approximately 50Oto of the colonies yielded detectable hybridization signals and 48 colonies exhibited reproducibly stronger signals with the E-A + probe. These candidates were recovered and re-screened by the same method. In the second stage, 22 colonies showed the expected differential hybridization. We prepared plasmid from each and, using a modified version of the hybridizationselection-translation procedure of Ricciardi et al. (1979), tested each plasmid for its ability to select an mRNA of interest. For 19 of the plasmids the selection-translation procedure yielded no detectable translation products. Three plasmids yielded interesting results (Figure 3). When the mRNA(s) selected by pKc252 are translated the products co-migrate with the EIPs 28 and 29. Judging from the yields of the translation products these mRNA(s) are more abundant in E-A + than in C-A+ RNA as anticipated. -
Figure 4 shows the two-dimensional gels which confirm the identification of the products. Evidently pKc252 selects mRNA(s) encoding the entire array of EIPs 28 and 29 normally observed as translation products. As Figures 3 and 4 show, pKc 191 selected the same mRNA(s) but less efficiently. Finally, pKc441 selected mRNA(s) encoding the EIPs 40. These mRNA(s), like those for the EIPs 28 and 29, are more abundant in E-A + RNA as judged by the selections/translations in Figure 3. Table I lists some of the characteristics of the plasmids pKc252 and pKc441. pKc252 contains a cDNA insert of 860 nucleotides with a single internal PstI site. Excision of the insert with PstI yields a short insert fragment of -340 nucleotides and a long insert fragment of 520 nucleotides. The plasmid hybridized in situ to a single site on the salivary gland polytene chromosomes at 71C3-D2. pKc441 contains a PstI-excisable insert of 450 nucleotides and it hybridizes in situ to a single site in the region 55 B-E. Not surprisingly, both pKc252 and pKc441 hybridize to transcripts which are ecdysone-inducible in Kc cells and which ae relatively abundant in the induced cells. Figure 5 shows an RNA blotting experiment which illustrates these points. The magnitude of the induction at the RNA level is similar to the effect on EIP synthesis, namely an 10-fold increase in EIP 28 synthesis after 4 h and a somewhat smaller increase in EIP 40 synthesis (Savakis et al., 1980; A.Bieber and J.Rebers, personal communication). The sizes of the transcripts detected in Figure 5 have been estimated by comparison with DNA restriction fragment standards (not shown). pKc252 hybridizes to a diffuse band of 1050 nucleotides: minor hybridizing species of 1300 239 -
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C.Savakis, M.M.D.Koehler and P.Cherbas
and 2300 nucleotides, also diffuse and also inducible, appear on overexposed autoradiograms. pKc441 hybridizes to a band of 1500 nucleotides and also to minor species of 1650 and 1800 nucleotides. Because the transcripts detected by pKc252 must include the mRNAs for the EIPs 28 and 29 and because those mRNAs might be distinct but differ only slightly in length, we reasoned that the diffuse bands might include multiple RNAs masked by heterogeneity in the lengths of the poly(A) tails. Therefore we removed poly(A) by hybridizing the C-A + and E-A + RNAs to oligo(dT) and subsequently digesting with RNase H (Vournakis et al., 1975). To eliminate the possibility that some RNA species were degraded internally we translated the deadenylated RNAs and separated the products on two-dimensional gels. The resulting autofluorographic patterns included 200-300 discrete spots which were indistinguishable in relative intensity and approximately so in absolute intensity from the products of the undigested RNAs. In particular, all the EIP species were represented at their usual relative intensities (data not shown). Figure 5 shows that although deadenylation sharpens the bands (with the new modal size approximately equal to the original lower limit) there is no obvious indication of heterogeneity either for the transcripts detected by pKc252 or those detected by pKc441. After deadenylation the major species detected by pKc252 is 920 nucleotides long and the minor species which are probably precursors are -1100 and 2200 nucleotides. In the case of pKc441 the deadenylated sizes are 1400 nucleotides for the major species and 1550 and 1730 nucleotides for the minor species. -
-
-
-
-
Discussion Our previous observations concerning protein synthesis in ecdysone-treated Kc cells suggested that the domain of regulated polypeptides may be quite small. The striking and early induction of the EIPs 28, 29 and 40 occurred against a background in which other major synthetic products were essentially invariant. This observation has now been confirmed at the higher resolution afforded by two-dimensional separations. We can identify only 10 polypeptides whose synthesis changes detectably and reproducibly in response to ecdysone. Nine of these are the various EIPs 28, 29 and 40. The tenth is the previously undetected EIP 35. Thus, among the several hundred polypeptides sampled in our experiments, early ecdysone-induced changes are quite rare. They are no more frequent among the many barely detectable species revealed by the two-dimensional gels than among the rapidly synthesized species assayed by the one-dimensional separations. This suggests that the domain of early ecdysone effects may be quite small though it does not exclude the possibility of more frequent and/or important responses among minor species undetected in these experiments. That such changes are exceptional rather than common among major species has of course been important for the success of our cloning strategy. In the unrelated but ecdysone-responsive Schneider 3 cell line we observe induction of the EIPs plus induction of hsp23 as reported by Ireland and Berger (1982). However hsp23 induction does not occur in Kc cells. The in vivo product we have called EIP 35 cannot be identified among the translation products of C-A + and E-A + RNAs. Possibly it is encoded by an mRNA which does not fractionate as poly(A) +; however, translations of total RNA 240
also fail to yield a spot of the expected size and pl (M.M.D. Koehler, unpublished observations). Alternatively, EIP 35 may be synthesized as a primary product having quite different properties. So far we have identified among translation products no inducible species which might be a candidate for this hypothetical precursor. In contrast, the majority of the EIP 28, 29 and 40 species known from in vivo labeling experiments are readily identifiable as products of cell-free translation using poly(A)+ templates. Indeed the EIPs 28 and 29 appear to be somewhat more prominent among the poly(A)+ translation products than they do in vivo or in translations of total RNA. We suspect that this reflects a genuine if minor enrichment for these mRNAs which carry unusually long poly(A) tails. The EIPs 40 synthesized in the reticulocyte lysate appear to be identical in relative intensity, in pl, and in mobility to the species detected in vivo. The same is true for the EIPs 28 except that EIP 28111 is underrepresented among the translation products as it is following a 2 h chase in vivo. For the EIPs 29 the relationship between the in vivo pattern and the translation products is more problematic. Translation products corresponding in intensity and position to the EIPs 29I and II are readily apparent. However the minor species EIP 29111 is missing and is replaced by a more acidic spot of comparable intensity, EIP 29a. In addition, the translations yield the minor new spots 29b and 29c. Preliminary experiments suggest that EIP 28111 and 29111 may be the primary translation products which normally give rise to the more acidic species by co-translational or post-translation acetylation (A.Bieber and M.M.D.Koehler, unpublished observations). This is in accord with the pulse-chase results shown in Figure 1. However, we cannot yet explain the aberrant forms 29a, b and c though we suspect that they are processing artifacts. The critical observation is that the levels of the EIP mRNAs are elevated in the poly(A) + RNA fraction prepared from 4 h hormone-treated cells. This was shown initially by experiments like that shown in Figure 1, panels E and F. Translations of complex RNA populations are complicated by the effects which the particular conditions and mixtures of templates may exert on the translational efficiency for any one template (Stewart et al., 1973; Palmiter, 1974; McKeehan, 1974). However, in the comparison of C-A + and E-A + RNAs, the overall composition of the population remained constant and the results were independent of the amount of input RNA. E-A + RNA is a substantially better template for EIP synthesis than C-A + RNA. The hybridization-arrested translations (Figure 2) measure the levels of hybridizable mRNA irrespective of its translatability. The crucial result is that E-cDNA is much more effective than C-cDNA in arresting the translation of the EIPs. This implies that species homologous to the EIP mRNAs are substantially more abundant in the E-A + than the C-A + population. While the experiment must be considered only semi-quantitative in the absence of kinetic standards, the substantial and specific inhibition by hybridization with E-cDNA to Cot = 0.5 implies that the EIP transcripts are relatively abundant (Hastie and Held, 1978). This is consistent with the implications of the translation experiments assuming the EIP mRNAs to have average translational efficiencies. On the basis of the in vivo protein synthetic patterns, of the translations, and of the hybridization-arrested translations, we estimated that the EIP 28 mRNA represents 1I1% of the E-A + RNA, and 10-fold less or 0O. I o of the -
cDNA dones for E1P mRNAs of Kc ceOls
C-A+ RNA. Our success in identifying EIP cDNA clones by a plusminus screen confirms this. The differential screen was in fact relatively inefficient. For example, we recovered two EIP 28/29 cDNA clones in a screen of 6000 colonies. When the same library was re-screened using the insert of pKc252 as 1 %o of the colonies as pKc252probe, we identified homologous; that these represent identical or closely-related mRNA sequences has been confirmed by sequencing (C.Savakis and L.Cherbas, unpublished observations). Similarly, only one EIP 40 cDNA clone was identified by the differential screen, but re-screening with the EIP 40 insert yielded -0.3070 of the colonies as positives (J.Rebers, personal communication). The inefficiency is reflected in false negative results and is attributable to the poor signals and resulting variability arising from the use of complex probes. False positive results represented by the 19 plasmids whose DNAs selected no translatable mRNA are more difficult to explain but probably arise from the same problem of signal variability. That these are false positives has been confirmed for many of these plasmids by Northern analysis (C.Savakis, unpublished observations). Despite these difficulties the differential screen has permitted us to isolate cDNA clones corresponding to all of the EIPs 28, 29 and 40. It is entirely consistent with the earlier analysis that we recovered no cDNA clones corresponding to other polypeptides. The hybridization-selected translations (Figures 3 and 4) permitted unambiguous assignment of the cDNAs to EIP mRNAs. These experiments provide the first evidence that the EIPs 28 are inter-related, as are the EIPs 29 and the EIPs 40. It was more surprising that the EIPs 28 and 29 are homologous to one another. The plasmids pKc252 and pKcl91 each select mRNAs which can be translated to yield all eight of the EIP 28 and 29 species which regularly appear in translations. We have been unable to discriminate among these species by increasing the stringency of the hybridizations (C.Savakis, unpublished observations). Thus there may be a family of EIP 28 and 29 genes which are highly conserved for part of their sequence and which individually encode the various EIPs 28 and 29. Alternatively, a single EIP 28/29 gene might give rise to a variety of different mRNAs or there might be a single mRNA and a single primary translation product which undergoes novel post-translational modifications. Finally, Kc cells might be heterozygous at the EIP 28/29 locus and some of the resulting species might be allelic. Our initial attempt to clarify this situation is illustrated in Figure 5. If a set of EIP 28/29 mRNAs was obscured by their polydisperse poly(A) tails, this set might be revealed after enzymatic deadenylation. The deadenylation was successful and yielded relatively sharp bands shorter by 100-120 nucleotides than the original pKc252 and pKc441-hybridizing species. However, the deadenylated bands remained unimodal. If there is more than one EIP 28/29 transcript these do not differ in size by more than -50 nucleotides. We will present evidence elsewhere to show that there is, in fact, a unique EIP 28/29 gene which when homozygous yields two distinct EIP 28/29 mRNAs which differ in length by 12 nucleotides (L.Cherbas and P.Cherbas; R.A.Schulz, L.Cherbas and P.Cherbas, in preparation). Figure 5 also illustrates the increased abundances of the EIP 28/29 and EIP 40 transcripts by the end of 4 h of hormone treatment. This result confirms the earlier inferences based on translations and hybridization-arrested translations. -
-
Both pKc252 and pKc441 hybridize to single sites on the polytene chromosomes. Both sites 71CD and 55B-D are interesting. The hybridization at 71CD is at or very near the location of a puff which increases in size during the first hour of ecdysone treatment of salivary glands (Ashburner, 1972a; Velissariou and Ashbumer, 1981). Similarly pKc441 hybridizes near the center of a puff which develops within 4 h of ecdysone addition (Ashbumer, 1972b). Thus both loci identified by our screen for early-induced Kc cell RNAs may arise from the sites of ecdysone-induced puffs in the salivary gland. Whether this is the case will require study of the transcripts induced in the salivary gland. If it is true, this correlation would suggest that a few loci may participate in the early ecdysone response in diverse tissues (Holden and Ashburner, 1978; Richards, 1982). Our goal is to identify and study genes whose activities change as part of the early or primary response to ecdysone. We suppose that the mechanism of this regulation, its tissue specificity, and the nature of the early gene products themselves will all have significant implications for our understanding of eukaryotic regulatory pathways and development. The isolation of cDNA clones for the EIPs 28, 29 and 40 provides us with the tools for this analysis.
Materials and methods Nomenclature In the nomenclature of Goodwin et al. (1978) ecdysteroid is the generic name for compounds structurally related to ecdysone (a-ecdysone). We employ ecdysone as the generic name for compounds with the appropriate biological activity, in analogy with estrogen or progestin. This usage accords with much of the literature. Cells, hormone treatments and labeling The Kc-H strain of Kc cells has been described (Cherbas et al., 1977) as has the clone A4E6 derived from it (Cherbas et al., 1980a, 1980b; Cherbas and Cherbas, 1981). We used the A4E6 clone exclusively, growing the cells as described (Cherbas et al., 1977) save for the omission of antibiotics, and harvesting them in exponential growth (2-8 x 106 cells/ml). All hormone treatments were with 10-6 M 20-HE, which is saturating for EIP induction (Savakis et al., 1980). 20-HE was purchased from Simes, Milan. The hormone was delivered in ethanol which was present at 0.01 (lo (v/v) in the incubation. Control cells were treated similarly with ethanol alone. For in vivo protein labeling, cells were concentrated and pulse-labeled 20 min with [3H]leucine precisely as described by Savakis et al. (1980). RNA isolation Cells for RNA isolation were grown in spinner flasks, harvested and centrifuged at low speed, washed once with Robb's saline (Robb, 1969), then lysed with -20 volumes of lysis buffer (Holmes and Bonner, 1973). The lysate was extracted twice with phenol:chloroform:iso-amyl alcohol (50:50:1, v/v/v) containing 0.1% (w/v) 8-hydroxyquinoline. The aqueous phase was extracted 4 times with ether and nucleic acids were precipitated twice from 0.2 M sodium acetate, pH 6.0, with two volumes of ethanol. The dried pellet was redissolved in water to A260 = 10 and digested with RNase-free DNase at 50 yg DNase per mg nucleic acid, in 10 mM MgCl2, 10 mM NaCl, 10 mM Tris.HCl, pH 7.4. RNA was purified by phenol extraction and ether washes, concentrated by ethanol precipitation, and dissolved in water at 1-5 mg/ml. Poly(A) + RNA was isolated by two cycles of chromatography on oligo(dT)cellulose as described by Efstratiadis and Kafatos (1976). About 2.50/o of the total RNA behaves as poly(A) + (average of six experiments). All RNA fractions were concentrated by ethanol precipitation, dissolved in water, and stored frozen in liquid N2-
Gel electrophoresis SDS-polyacrylamide gel electrophoresis of radiolabeled proteins was modified from our previous procedure (Savakis et al., 1980) to improve the separation of cell-free translation products. The resolving gel contained 0.5 M Tris buffer, both stacking and resolving gels contained 0.5%'o SDS, and the reservoir buffer was 9.9 mM Tris, 76.8 mM glycine, 0.1 0o SDS. Two-dimensional electrophoresis differed from the procedure of O'Farrell (1975) in the following details of the isofocusing dimension: gels containing 9.5 M urea and 2%7o ampholytes [1.6% pH 5-7 (LKB), 0.2407o pH 3.5-10 (LKB), 0.16%7o pH
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C.Savakis, M.M.D.Koehler and P.Cherbas 6-8 (BioRad)] were poured in 200 1l disposable capillary pipets (ClayAdams), and samples were applied in 20 p1. The gels were run for 14.5 kV-h; the potential was maintained at 800 V during the final hour. All protein gels were processed for autofluorography and exposed using pre-flashed Kodak RP film (Bonner and Laskey, 1974; Laskey and Mills, 1975). RNA was fractionated by electrophoresis on vertical agarose gels (1.5 mm thick, 1.5% agarose, w/v) in the presence of methylmercury hydroxide (10 mM), as described by Bailey and Davidson (1976). Cell-free translation RNA was translated in the mRNA-dependent rabbit reticulocyte lysate system (Pelham and Jackson, 1976), containing [3H]leucine (New England Nuclear, > 110 Ci/mmol before addition to the lysate). For some experiments the mRNA-dependent lysate was purchased from New England Nuclear, and used according to the supplier's protocols. Except where otherwise indicated, poly(A) + RNA was added to the translation mixture at a final concentration of 40 Ag/ml. Hybridization-arrested translation Hybridizations were performed at 60°C in a 10 Al mixture containing 1 /4g poly(A)+ RNA, 1 Mg single-stranded cDNA, 20 mM Hepes pH 7.0, and 0.4 M KCI. Aliquots of 3 pl were removed at 3 min and at 30 min and added directly to a reticulocyte lysate translation mixture. Synthesis of single-stranded cDNA (ss-cDNA) Protocols for the synthesis of cDNA are based on the technique of Efstratiadis and Villa-Komaroff (1979). The reaction mixture contained 0.1 M Tris.HCl pH 8.3 (at 42°C), 0.01 M MgCl2, 0.02 M KCI, 0.03 M 2-mercaptoethanol, 0.625 mM of each dNTP, 0.2 mg/ml poly(A)+ RNA, 0.1 mg/ml oligo(dT)2 _l8 (Collaborative Research), and 4600 units/mI AMV reverse transcriptase (a gift from Dr.J.W.Beard). A small amount of [a-32p]dNTP (final specific activity 0.04 Ci/mmol for each nucleotide) was included. The concentration of KCI, dNTPs, and enzyme given above were determined empirically to give an optimal yield and length of cDNA; we obtained - 0.2 Mkg cDNA per yg poly(A) + RNA, with a modal length of - 400 nucleotides. High specific activity 32P-labeled cDNA for use as probe was synthesized in a similar reaction mixture, except that dNTPs were present at 0.1 mM each, and [C-32P]dATP was included at 8 mCi/ml. The reaction mixture was incubated at 42°C for 2.5 h. RNA was then hydrolyzed by the addition of NaOH to 300 mM and EDTA to 20 mM, and incubation at 37°C for 12 h. Salts and free nucleotides were removed by gel filtration in Sephadex G-100. Synthesis of double-stranded cDNA (ds-cDNA) The second strand of the cDNA was synthesized by reverse transcriptase, using the same reaction conditions as the first strand save that oligo(dT), KCI, and [32P]dNTPs were omitted, and the reaction time was 4 h. After de-salting and concentration, the double-stranded product was dissolved in 0.1 M NaCI, 0.05 M NaOAc pH 4.5, 1 mM ZnCl2 and treated with S1 nuclease (Miles) (10 units/Al, 15 min at 37°C, then 2 h at 18°C) to remove single-stranded ends and the hairpin loop. The DNA was then extracted with phenol and ether, and dialyzed against 0.1 mM CoC12, 14 mM potassium cacodylate, 3 mM Tris pH 6.9, in preparation for the tailing reaction. The yield of ds-cDNA was - 10%, based on input ss-cDNA, and the modal length was - 300 bp. Construction of recombinant DNA plasmids Plasmid pBR322 (Bolivar et al., 1977) was cut with PstI. The linearized vector was tailed with oligo(dG) and the ds-cDNA with oligo(dC), using terminal deoxynucleotidyl transferase (a gift from Dr.V.E.Ratliff) as described by Roychoudhury et al. (1976). Reaction times were chosen to give - 15 residues per 3' end. The tailed DNAs were extracted with phenol and ether and dialyzed against 0.1 M NaCl, 1 mM EDTA, 10 mM Tris.HCI pH 8.0. The tailed vector (5 Ag/ml) and ds-cDNA (0.3 /g/ml) were mixed, heated to 70°C for S min and then incubated at 42°C for 2 h to permit annealing of the tails. Transformation and screening The annealed recombinant plasmids were used to transfect Escherichia coli K12 strain HBIOI (Boyer and Rouland-Dussoix, 1969), using the procedure of Wensink et al. (1974). Tetracycline-resistant bacterial colonies were grown, replica-plated, and fixed to nitrocellulose filters, using the procedures of Hanahan and Meselson (1980) and Grunstein and Hogness (1975). After baking, the filters were shaken gently at 37°C in 2 x SSC (1 x SSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS (w/v), 0.1 mg/ml proteinase K. They were then washed for 2 days at 65°C in four changes of 4 x SSC, 5 x Denhardt's solution (Denhardt, 1966), 0.2% SDS, with 100 Ag/ml denatured, sonicated calf-thymus DNA included in the last wash solution. The filters were hybridized to 32P-labeled ss-cDNA probes (1.7 x 106 c.p.m. per filter) for 48 h in the same solution plus 10 jg/ml poly(A). The hybridization was carried out in heat-sealed plastic bags. After hybridization, the filters were washed in 4 x SSC, 5 x Denhardt's solution, 0.5% SDS (65°C, four 30 min washes)
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and 2 x SSC (room temperature, one 30 min wash). The filters were exposed to Kodak RP film, using an intensifying screen for some exposures. Plasmid purification Clones of transformed bacteria were grown in liquid culture and the plasmids amplified by the addition of chloramphenicol. Cleared lysates were prepared from these cells, and the plasmid DNA was purified by isopycnic sedimentation, as described by Clewel (1969). Hybrid-selected translation RNA was purified by hybridization to cloned DNA immobilized on a filter (Ricciardi et al., 1979). Phenol-extracted cleared lysates (Ploegh et al., 1980) were spotted, hybridized, and eluted as described by Kafatos et al. (1979). The purified RNA was then translated in the reticulocyte lysate system. RNA blotting and hybridization to plasmid probes Diazobenzyloxymethyl-derivatized paper (DBM-paper) was prepared as described by Alwine et al. (1977). RNA was transferred from gels to the DBM-paper, using 0.2 M sodium acetate buffer (pH 4.0). Paper strips containing transferred RNAs were incubated for 4- 12 h in 50% formamide, 5 x SSC, 0.025 M sodium phosphate pH 6.0, 0.207o SDS, 1 mg/ml sonicated denatured calf-thymus DNA, 1% glycine, 5 x Denhardt's solution. Hybridization was carried out at 42°C in the same solution from which glycine was omitted and to which dextran sulfate (10% w/v) was added. Probes were prepared by nick-translation of plasmid DNA (Rigby et al., 1977); hybridizations included 105 c.p.m./ml of 32P-labeled probe, at 1 -5 x 108 c.p.m./,ug. After hybridization, the blot was washed in 4 x SSC, 0.20%o SDS (three 20 min washes at room temperature), and 0.1 x SSC, 0. 1% SDS (two 20 min washes at 45°C).
Acknowledgements We thank M.Ashburner and W.M.Gelbart for interpreting in situ hybridizations for us. We are grateful to Lucy Cherbas for help with cell culture and in situ hybridizations, for scientific counsel, and for advice on the manuscript. This work was supported by grants for P.T.C. from the National Science Foundation and the American Cancer Society.
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