We describe techniques and experimental applications for the expression of exogenous protein in Drosophila cell lines, emphasizing the ways in which the Drosophila and baculovirus systems differ in their applications.

During metamorphosis, the reorganization of the nervous system of Drosophila melanogaster proceeds in part through remodeling of larval neurons. In this study, we used in-vitro imaging techniques and immunocytochemistry to track the remodeling of the thoracic ventral neurosecretory cells. Axons of these neurons prune their larval arbors early in metamorphosis and a larger, more extensive adult arbor is established via branch outgrowth. Expression of EcR dominant negative constructs and an EcR inverted repeat construct resulted in pruning defects of larval axon arbors and a lack of filopodia during pruning, but showed variable effects on outgrowth depending on the construct expressed. Cells expressing either UAS-EcR-B1(W650A) or UAS-EcR-A(W650A) lacked filopodia during the outgrowth period and formed a poorly branched, larval-like arbor in the adult. Cells expressing UAS-EcR-B1(F645A), UAS-EcR-B2(W650A) or UAS-IR-EcR (core) showed moderate filopodial activity and normal, albeit reduced, adult-like branching during outgrowth. These results are consistent with the role of activation versus derepression via EcR for successive phases of neuronal remodeling and suggest that functional ecdysone receptor is necessary for some, but not all, remodeling events

Drosophila NURF is an ISWI-containing ATP-dependent chromatin remodeling complex that regulates transcription by catalyzing nucleosome sliding. To determine in vivo gene targets of NURF, we performed whole genome expression analysis on mutants lacking the NURF-specific subunit NURF301. Strikingly, a large set of ecdysone-responsive targets is included among several hundred NURF-regulated genes. Null Nurf301 mutants do not undergo larval to pupal metamorphosis, and also enhance dominant-negative mutations in ecdysone receptor. Moreover, purified NURF binds EcR in an ecdysone-dependent manner, suggesting it is a direct effector of nuclear receptor activity. The conservation of NURF in mammals has broad implications for steroid signaling

A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome

Steroid hormones fulfil important functions in animal development. In Drosophila, ecdysone triggers moulting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homologue Ultraspiracle. Both partners are required for binding to ligand or DNA. Like most DNA-binding transcription factors, nuclear receptors activate or repress gene expression by recruiting co-regulators, some of which function as chromatin-modifying complexes. For example, p160 class coactivators associate with histone acetyltransferases and arginine histone methyltransferases. The Trithorax-related gene of Drosophila encodes the SET domain protein TRR. Here we report that TRR is a histone methyltransferases capable of trimethylating lysine 4 of histone H3 (H3-K4). trr acts upstream of hedgehog (hh) in progression of the morphogenetic furrow, and is required for retinal differentiation. Mutations in trr interact in eye development with EcR, and EcR and TRR can be co-immunoprecipitated on ecdysone treatment. TRR, EcR and trimethylated H3-K4 are detected at the ecdysone-inducible promoters of hh and BR-C in cultured cells, and H3-K4 trimethylation at these promoters is decreased in embryos lacking a functional copy of trr. We propose that TRR functions as a coactivator of EcR by altering the chromatin structure at ecdysone-responsive promoters

The ecdysone receptor is a heterodimer of the two nuclear receptors EcR and ultraspiracle (USP). We have identified the regions of Drosophila EcR and USP responsible for transcriptional activation of a semisynthetic Eip71CD promoter in Kc cells. The isoform-specific A/B domains of EcR-B1 and B2, but not those of EcR-A or USP, exhibit strong activation activity [activation function 1 (AF1)], both in isolation and in the context of the intact receptor. AF1 activity in isoform B1 derives from dispersed elements; the B2-specific AF1 consists of a 17-residue amphipathic helix. AF2 function was studied using a two-hybrid assay in Kc cells, based on the observation that potent hormone-dependent activation by the EcR/USP ligand-binding domain heterodimer requires the participation of both partners. Mutagenesis reveals that AF2 function depends on EcR helix 12, but not on the cognate USP region. EcR helix 12 mutants (F645A and W650A) exhibit a dominant negative phenotype. Thus, in the setting tested, the ecdysone receptor can activate transcription using the AF1 regions of EcR-B1 or -B2 and the AF2 region of EcR. USP acts as an allosteric effector for EcR, but does not contribute any intrinsic function

The three Drosophila EcR isoforms differ only at their N termini; thus, they share the conserved ligand-binding domain transcriptional activation function (AF2) and only differ in the unconserved A/B region, which contains a second, isoform-specific, activation function (AF1). We have developed a dominant-negative mutant EcR (EcR-DN), expressed it in flies with the GAL4/UAS system, and used it to block ecdysone signaling in eight tissues or groups of tissues. Localized EcR-DN arrests ecdysone-dependent development in the target cells and often--because of a molting checkpoint--arrests development globally. Simultaneously expressing individual wild-type EcR isoforms in the same target tissues suppresses the EcR-DN phenotype and identifies the rescuing isoform as sufficient to support the development of the target. Every isoform, and even an N-terminal truncated EcR that lacks any AF1, supports development in the fat body, eye discs, salivary glands, EH-secreting neurosecretory cells and in the dpp expression domain, implying that AF1 is dispensable in these tissues. By contrast, only EcR-A is able to support development in the margins of the wing discs, and only EcR-B2 can do so in the larval epidermis and the border cells of the developing egg chamber. In light of our results, the simplest explanations for the widespread spatial and temporal variations in EcR isoform titers appear untenable

Drosophila cells in culture can be transformed by introducing exogenous DNA carrying a selectable marker. Here we report on the fate of plasmids that contain an extended fragment of Drosophila DNA in addition to the selectable marker. A small minority of the resulting transformants appear to arise from homologous recombination at the chromosomal target. However, the majority of the insertions are the products of illegitimate events in the vicinity of the target DNA, and they often cause mutations in the targeted region. The efficiency of this process, its homology dependence, and the clustering of the products define a novel transformation pathway that we call "parahomologous targeting."

The Drosophila ecdysone receptor (DmEcR) is a member of the nuclear receptor superfamily; it functions as an obligate heterodimer with another nuclear receptor, DmUSP. EcR homologs have now been cloned from several other insects. We report here that one such homolog, BmEcR from the commercial silkmoth, Bombyx mori, is a functional ecdysone receptor. Upon dimerization with BmCF1, the silkmoth homology of DmUSP, BmEcR binds the radiolabeled steroid ligand 125I-iodoponasterone A with Kd = 1.1 nM, indistinguishable from that exhibited by DmEcR/DmUSP. BmEcR/BmCF1 forms a specific complex with an ecdysone response element (EcRE) derived from the heat shock protein 27 (hsp27) gene promoter of Drosophila; and, as with DmEcR/DmUSP, formation of this complex is stimulated by the presence of 20-hydroxyecdysone. Finally, BmEcR can substitute for DmEcR in an EcR-deficient Drosophila tissue culture line, stimulating trans-activation of an ecdysone-inducible reporter gene construct. Thus, BmEcR and BmCF1 are the functional counterparts of DmEcR and DmUSP, respectively and, despite considerable sequence divergence between the Drosophila and Bombyx proteins, the counterparts are--at least qualitatively--functionally equivalent

We report that cells of a Drosophila embryonic cell line (Kc167 cells) can be readily and stably transformed by transposition of P elements from exogenous DNA. Cells are transfected with plasmids carrying methotrexate- or alpha-amanitin-resistance markers expressed from constitutive promoters and co-transfected with a gene encoding a somatically active transposase. Transient expression of the transposase leads to efficient production of transformed, resistant cells. We describe conditions under which most resistant clones are healthy and harbor a small number (1-50) of transposons and few (< or = 5%) retain plasmid sequences derived from illegitimate recombination. Using conditions like these it should prove possible to construct enhancer trap and/or gene libraries using Drosophila cells

In Drosophila melanogaster, three temporally distinct ecdysone-responsive puff sets, the so-called intermoult, early and late puffs, have been described on the salivary gland polytene chromosomes. We have analyzed in detail a DNA segment of the 3C polytene region, from which the originates one of the most prominent intermoult puffs, with the aim of identifying ecdysone response elements (EcREs). Here we report that two putative EcREs of identical sequence are located at this puff site. Interestingly, these elements display a novel structural feature, being composed of directly repeated half-sites. Our results show that the EcR/USP heterodimer known to constitute the ecdysone functional receptor complex is able to bind to and transactivate through target elements composed of directly repeated half-sites. In addition, we show that these elements are also able to bind efficiently USP alone, suggesting that USP and EcR/USP could compete for their binding to DNA

The Eip28/29 gene of Drosophila is an example of a tissue- and stage-specific ecdysone-responsive gene. Its diverse patterns of expression during the third larval instar and a synopsis of those patterns in terms of expression groups have been reported previously. Here we have studied the expression (in transgenic flies) of reporter genes controlled by Eip28/29-derived flanking DNA. During the middle and late third instar, most tissues exhibit normal expression patterns when controlled by one of two classes of regulatory sequences. Class A sequences include only 657 Np of 5' flanking DNA from Eip28/29. Class B sequences include an extended 3' flanking region and a minimal (< or = 93 Np) 5' flanking region. The class B sequences include all those elements known to be important for ecdysone induction in cultured cells. They are sufficient to direct the normal premetamorphic induction of Eip28/29 in the lymph glands, hemocytes, proventriculus, and Malpighian tubules. This is consistent with our suggestion that Kc cells are derived from embryonic hematopoietic cells. It is remarkable that the epidermis requires only class A sequences. These are sufficient to up-regulate expression at mid-instar and to down-regulate expression at metamorphosis. It follows that the epidermis uses EcREs distinct from those that function in Kc cells. It is possible that the Upstream EcRE, which is nearly silent in Kc cells, is active in the epidermis

Although the biological activity of the insect moulting hormone ecdysone, is manifested through a hormonally regulated transcriptional cascade associated with chromosomal puffing, a direct association of the receptor with the puff has yet to be established. The cloned ecdysone receptor (EcR) is by itself incapable of high-affinity DNA binding or transcriptional activation. Rather, these activities are dependent on heterodimer formation with Ultraspiracle (USP) the insect homologue of vertebrate retinoid X receptor. Here we report that native EcR and USP are co-localized on ecdysone-responsive loci of polytene chromosomes. Moreover, we show that natural ecdysones selectively promote physical association between EcR and USP, and conversely, that high-affinity hormone binding requires both EcR and USP. Replacement of USP with retinoid X receptor produces heterodimers with distinct pharmacological and functional properties. These results redefine the ecdysone receptor as a dynamic complex whose activity may be altered by combinatorial interactions among subunits and ligand

Approximately 25% of arthropod RNA polymerase II-transcribed promoters contain one or more copies of the sequence TCAGT beginning within the interval (-10, +10). The clear statistical overrepresentation of this sequence and, to a lesser extent, of its cognates ACAGT, GCAGT, and TCATT, implies that they may be significant promoter elements. Their collective sequence similarity to vertebrate initiators (Inrs) of the TdT class suggests that the vertebrate and arthropod elements are homologous. Prior work in vertebrate systems has emphasized the role of the Inr in promoters lacking TATA boxes, where it can serve as an alternate staging site for polymerase II initiation. However, it is clear that the Inr sequence is by no means restricted to TATA-deficient promoters. Functional tests using the TATA-containing Drosophila gene Eip28/29 support the idea that the Inr is a facultative promoter element, required for efficient transcription under some conditions. For example, the Inr protects basal expression of Eip28/29 from the silencing effect of ecdysone response elements. In addition, the Inr is required for the function of an enhancer of basal activity in Eip28/29. We conclude that Inrs are promoter elements found sporadically throughout the higher eukaryotes, that the requirement for an Inr depends upon the array of other promoter elements which may be present in a given gene, and that Inrs may permit enhancers to discriminate among promoters

Those of us who study ecdysone action share at least two important long-range goals: (i) to understand the developmental specificity of steroid action in full molecular detail, by integrating ecdysone action with our rapidly expanding knowledge of the molecular biology of insect development, and (ii) to better understand the nature of the steroid response and its evolution by taking advantage of the unparalleled opportunities for both genetic and comparative study afforded by the diversity of the "ecdysone world". However, until recently, the molecular fundamentals of the ecdysone system were unknown and our efforts have, of necessity, been devoted to their elucidation. Now that the situation has changed: we have a small but varied catalog of ecdysone-responsive genes for study and it is clear that some of these are tissue- and stage-specific in their expression. The ecdysone receptor (EcR), like other steroid receptors a member of the nuclear receptor family, is now accessible to molecular study, and we have a preliminary understanding of the DNA sequences (EcREs) that bind receptor and specify a gene as ecdysone-responsive. With these tools in hand and with the opportunity to turn to larger questions, it is a propitious moment to consider the nature of those questions and how ecdysone can contribute to the answers

The Drosophila genes Eip28/29 and Eip40 are expressed in Kc cells and are rapidly induced by the steroid hormone ecdysone. The molecular basis for Eip28/29's regulation in those cells has been studied in some detail. To determine how this regulation relates to normal development, we have examined the expression of both genes throughout Drosophila development, with special attention to Eip28/29 and the final larval instar. Eip28/29 expression is complex; there are tissues in which it is never expressed, others in which it is continuously expressed at a low level and tissues in which its expression is regulated without obvious relationship to endocrine events. However high-level Eip28/29 expression always correlates with the presence of ecdysone and there is good evidence that Eip28/29 is directly regulated by the hormone in some tissues and at some stages. Most striking are the induction of Eip28/29 transcripts in numerous tissues at the last larval molt, their induction in the epidermis at the time of the 'late 3rd transition', their extinction in the same tissue by the premetamorphic ecdysone peak, and their induction by that peak in the lymph gland, hemocytes and proventriculus. These contrasting regulatory behaviors provide a well-defined model for studying the developmental specificity of steroid responses. Eip40 appears to be ecdysone-inducible only in the lymph gland and there only at the premetamorphic peak. The similarities been Eip28/29 and Eip40 regulation in the lymph gland and Kc cells support the idea that Kc cells are derived from a hematopoietic ancestor

The neurogenic loci Notch and Delta, which both encode EGF-homologous transmembrane proteins, appear to function together in mediating cell-cell communication and have been shown to interact at the cell surface in vitro. To examine the role of the EGF repeats in this interaction, we performed an extensive deletion mutagenesis of the extracellular domain of Notch. We find that of the 36 EGF repeats of Notch, only two, 11 and 12, are both necessary and sufficient to mediate interactions with Delta. Furthermore, this Delta binding ability is conserved in the corresponding two repeats from the Xenopus Notch homolog. We report a novel molecular interaction between Notch and Serrate, another EGF-homologous transmembrane protein containing a region of striking similarity to Delta, and show that the same two EGF repeats of Notch also constitute a Serrate binding domain. These results suggest that Notch may act as a multifunctional receptor whose 36 EGF repeats form a tandem array of discrete ligand-binding units, each of which may potentially interact with several different proteins during development

The steroid hormone ecdysone triggers coordinate changes in Drosophila tissue development that result in metamorphosis. To advance our understanding of the genetic regulatory hierarchies controlling this tissue response, we have isolated and characterized a gene, EcR, for a new steroid receptor homolog and have shown that it encodes an ecdysone receptor. First, EcR protein binds active ecdysteroids and is antigenically indistinguishable from the ecdysone-binding protein previously observed in extracts of Drosophila cell lines and tissues. Second, EcR protein binds DNA with high specificity at ecdysone response elements. Third, ecdysone-responsive cultured cells express EcR, whereas ecdysone-resistant cells derived from them are deficient in EcR. Expression of EcR in such resistant cells by transfection restores their ability to respond to the hormone. As expected, EcR is nuclear and found in all ecdysone target tissues examined. Furthermore, the EcR gene is expressed at each developmental stage marked by a pulse of ecdysone

Ecdysteroids act initially by binding to nuclear and possibly also extranuclear receptors. The presence and expression of these receptors in the insect brain was investigated in the present study as a means of defining these neurons involved in ecdysteroid-regulated processes at different developmental stages. Early in the fifth larval stadium of Manduca sexta, when endogenous ecdysteroid levels are low, receptors for ecdysteroids in cerebral neurons are either absent or present at low levels. Receptors can be reliably detected only on day 0 and are not found again until day 3.5, at the beginning of the commitment peak in the ecdysteroid titer, when they occur in a small stage-specific population of cells. At this time, ecdysteroid receptors are found mainly in nuclei but are also observed at low levels in cytoplasm. By day 4.8, ecdysteroid receptors are exclusively nuclear, and the number of target cells has increased dramatically in several brain regions, including those with known neurosecretory cell groups. This population and organization of ecdysteroid target cells is constant up to day 6, after which time the number of target neurons declines. By day 7.8, only 10% of the number of labelled neurons seen on days 4.8-6.8 remain in peripheral areas. In the pupal brains, ecdysteroid receptors reappear in a new population of neurons. The results indicate changes in the genomic regulation of a varying neuron population by ecdysteroids during fifth stadium development

We have identified ecdysone-response elements (EcREs) by studying regulation of the steroid-responsive Drosophila Eip28/29 gene. First, functional assays of deletion mutants identified large sequence regions required for the response; then a blotting method using the specifically labeled steroid receptor as probe identified receptor-binding regions. Three short receptor-binding regions near Eip28/29 have been identified: Prox and Dist [521 and 2295 nucleotides, respectively, downstream of the poly(A) site] are probably required for the Eip28/29 response in cell lines; Upstream (-440) is unnecessary for that response. We have also demonstrated that an EcRE-containing region from hsp27 contains a receptor-binding site. Each of these four receptor-binding regions functions as an EcRE when placed upstream of an ecdysone nonresponsive promoter and each contains an imperfect palindrome, suggesting the consensus 5'-RG(GT)TCANTGA(CA)CY-3'. Furthermore, a synthetic 15-bp fragment containing an imperfect palindrome similar to the consensus is a fully functional EcRE. The presence of any of the EcREs leads, in the absence of hormone, to depressed gene expression. When hormone is added, it relieves this repression and causes additional activation. The similarity of the EcRE sequence to response elements for estrogen, thyroid hormone, and retinoic acid receptors suggests that the steroid receptors and their signal transduction mechanisms have been strongly and broadly conserved

Following mitosis in many cell lines, siblings remain adjoined in dyads until further cell division. We report here a series of experiments designed to ascertain the nature of this apposition in the embryonic Kc cell line of Drosophila melanogaster. We have found that (1) cell division in siblings is highly synchronized when compared to that in nonsiblings: (2) siblings in dyads are dye coupled with respect to Lucifer Yellow, but intercellular diffusion of larger molecules (FITC-dextran at 6 and 24 kDa) is retarded: (3) siblings are electrically coupled by an ungated low-resistance intercellular connection which is resistant to treatment with octanol and CO2, both known to close gap junction channels: and (4) members of a dyad are joined by a cytoplasmic bridge. Structures resembling septate junctions are also found between siblings and between cells in aggregates. The evidence accumulated here suggests that cytokinesis in Kc dyads is incomplete, resulting in an intercellular pathway that may provide for the passage of a molecular or electrical signal that regulates subsequent mitosis

The Eip28/29 gene of Drosophila is known to be regulated by the steroid hormone ecdysone in Kc cells and other cell lines. An investigation of Eip28/29 gene expression in intact animals has led to the discovery of two new genes on its 5' flank. Together these three genes generate at least seven distinct transcripts with diverse patterns of developmental expression. gonadal (gdl) is transcribed on the same strand as Eip28/29 and is expressed in two modes. gdlM transcripts, observed exclusively in the testes, are 1200 and 1500 N long, differing by their polyadenylation site but probably otherwise identical; gdlF transcripts are 900 and 1200 N long and share their terminal exons with the two gdlM transcripts. In adults they are exclusively ovarian, but they are also present in early embryos and, at a lower abundance, in Kc cells. In each mode, the longer transcript results from use of a polyadenylation site within the 5' exon of Eip28/29. The shared region includes 3' noncoding sequences of gdl transcripts and 5' flanking DNA and 5' noncoding sequences of Eip28/29. gdl expression in Kc cells is, however, unaffected by ecdysone. z600 is more distal to Eip28/29 but still contained at least in part within the 2 kb upstream of that gene. Its 600 N transcript is expressed predominantly during the first few hours of embryogenesis. Finally, the Eip28/29 transcripts are present at low levels during most developmental stages

Drosophila Kc cells are ecdysone-responsive: hormone treatment leads rapidly to increased synthesis of several ecdysone-inducible polypeptides (EIPs) and to commitment to eventual proliferative arrest. Later, the treated cells undergo morphological transformation, cease to proliferate, and develop new enzymatic activities, notably, acetylcholinesterase (AChE) activity. These responses have proven useful as models for studying ecdysone action. Here we report the sensitivity of Kc cells to another important insect developmental regulator--juvenile hormone (JH). We find that JH inhibits some, but not all, aspects of the ecdysone response. When Kc cells are treated with ecdysone in the presence of either natural JHs or synthetic analogues, the morphological and proliferative responses are inhibited and AChE induction is blocked. Most striking is that JHs protect the cells from the rapid proliferative commitment induced by ecdysone alone. The JH effects exhibit reasonable dose-response curves with half-maximal responses occurring at very low JH concentrations. Nonetheless, even at high JH concentrations the inhibitory effects are incomplete. It is interesting that EIP induction appears to be refractory to JH. It seems clear that JH is not simply a generalized inhibitor of ecdysone-induced responses

The effects of ecdysone, the steroid molting hormone of arthropods, are of considerable interest both to insect physiologists and to those studying steroid-regulated gene expression. Yet progress in understanding ecdysone receptors has been inhibited by the lack of a suitable highly radioactive hormone analog with high affinity for the receptor. Here we report that the synthetic ecdysteroid 26-iodoponasterone A is one of the most active ecdysones known, inducing half-maximal morphological transformation in Drosophila Kc167 cells when present at 0.5 nM. 26-[125I]Iodoponasterone A can be prepared at a specific activity of 2175 Ci/mmol (1 Ci = 37 GBq) by reaction of the precursor 26-mesylinokosterone with carrier-free Na125I. The radiolabeled material binds to Kc167 cell ecdysone receptors specifically and with affinity (Kd ca. 3.8 X 10(-10) M). Thus, 26-[125I]iodoponasterone A appears to be a superior radioligand for ecdysone receptors on grounds both of affinity and of specific activity. Its ready availability should greatly facilitate studies of these receptors

The Drosophila Eip28/29 gene encodes two primary translation products, ecdysone-inducible polypeptide (EIP) 28III and EIP 29III. When cells of the Kc cell line are treated with the steroid hormone ecdysone, the number of Eip28/29 transcripts and the synthesis of the various forms of EIP 28 and 29 increase rapidly. We have reported the sequence of the Eip28/29 gene and of its major transcript. Here we describe a minor or short-form transcript that is about 25% of the total Eip28/29 gene transcripts in both untreated and hormone-treated cells. This transcript is formed by the use of an alternative splice donor sequence 12 nucleotides upstream from the major donor site at the end of the second exon. Evidently the relative abundance of the two products is not hormonally regulated. The short form translation product should lack only an internal dibasic tetrapeptide. The long and short forms probably represent distinct mRNAs for EIP 28III and EIP 29III, respectively

Crystals of insecticyanin, a blue protein from the haemolymph of the tobacco hornworm Manduca sexta L. have been obtained. They are hexagonal, space group P6(1)22 (or P6(5)22), with unit cell dimensions a = b = 79.1 A, c = 312.1 A. The crystals diffract to 2.5 A resolution, and they are suitable for X-ray diffraction analysis

The EIPs 28 and 29 are a family of polypeptides identified originally by their ecdysone inducibility in Drosophila cell lines. At least two family members, 28III and 29III, appear to be primary translation products. Here we describe a unique Eip28/29 gene that must encode both primary products. The Eip28/29 gene is unique because the cloned genomic DNA hybridizes to both EIP 28 and 29 messenger RNAs under stringent conditions, but does not anneal detectably to other genomic sequences even under mild conditions. Furthermore the diverse products of this gene are not alleles because flies homozygous for the chromosomal region (71CD) containing the Eip28/29 gene produce mRNAs that translate to yield all the EIPs 28 and 29. We report here the sequence of a 2855-nucleotide region encompassing the Eip28/29 gene. By comparisons with complementary DNA sequences and by nuclease protection experiments we have derived a complete structure for the Eip28/29 transcription unit. The primary transcript is 2146 nucleotides long and is processed by the removal of three introns to yield the predominant mature transcript in tissue culture cells (979 nucleotides). This transcript probably corresponds to the 28III mRNA. Neither the start of the transcription unit nor the structure of the predominant transcript is affected by the hormone ecdysone. The genomic sequence reveals a series of heptanucleotide and octanucleotide repeats of unknown function that fall at about 50-nucleotide intervals within the first 150 nucleotides upstream from the transcription unit. In addition this sequence, when combined with previously published data, suggests that the consensus cap site sequence in Drosophila may be extended to include 13 nucleotides centered on the heptanucleotide core previously recognized by Snyder et al. (1982)

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 hybrid-arrested 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 EIPs 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

In the Drosophila melanogaster cell line Kc-H, ecdysteroid hormone treatment causes increased relative synthesis of three ecdysteroid-inducible polypeptides (EIPs), named according to their molecular weights (in kilodaltons) EIP 40, EIP 29 and EIP 28. Increased synthesis of the EIPs is detectable within 45 min (EIP 28) or 75 min (EIPs 40 and 29), is maximal at 4-8 hr and continues for almost 2 days. During this period no other major changes in protein synthesis are discernible using one-dimensional gels. At maximum, EIP 28 synthesis is elevated at least 10 fold above its basal level, and EIPs 40 and 29 somewhat less. EIP induction is ecdysteroid-specific and is detectable in the presence of 10(-8) M 20-hydroxyecdysone. It does not occur in hormone-resistant cells. Apparently identical polypeptides are inducible in another ecdysteroid-responsive cell line, Schneider's line 3. Because EIP synthesis is an early and substantial response to ecdysteroids, this is a promising system for the study of steroid hormone action

When cells of the Drosophila Kc-H line are treated with larger than or equal to 10(-8) molar beta-ecdysone, they extend long processes and acquire acetylcholinesterase activity. Thus, this permanent line, derived originally from embryol cultures, may be composed of cells having some neural or glial characteristics

In the absence of added hemin, protein synthesis in a rabbit reticulocyte lysate declines abruptly (shuts off) after about 5 min at 30 degrees. In these studies we have examined the basis for the lag period preceding shut-off. The initiation factor that binds Met-tRNAf, previously shown to be rate-limiting in inhibited, heme-deficient lysates, is found to be used stoichiometrically in the presence of excess inhibitor. We suggest that a principal effect of the inhibitor is to impair the recycling of the Met-tRNAf-binding factor; the lag period is attributable largely to the presence of a pool of excess Met-tRNAf-binding factor, which, once used in initiation, cannot be recycled because of the action of the inhibitor

We have re-examined the several papers which appear to us to represent the principal lines of evidence for what we call the Kroeger hypothesis. To do this we have stated this hypothesis in its simplest, most concrete form, a form that has been repeatedly and forceably enunciated in the literature (Kroeger, 1963a, 1965, 1966, 1967, 1968; Kroeger and Lezzi 1966; Lezzi and Frigg, 1971). The evidence suggests to us that ecdysone's effect on puffing is probably not mediated by the [K+]/[Na+]. While such a model cannot, even now, be excluded, we see little reason to believe in it. We take the general issues raised by Kroeger's ideas very seriously. Nucleoprotein complexes are exquisitely sensitive to changes in salt concentration and ionic selectivity is a well-known property of proteins (and of ion-exchangers in general, see, for example, Diamond and Wright, 1969). Thus it might not be shocking if cells utilized this specificity in some general control over chromsosome structure, perhaps a second-layer of control superimposed upon other transcriptional controls. Therefore it is our feeling that Kroeger's data merits very careful and critical study, the more so because the experiments involved are intrinsically difficult. It is in this vein that we have tried to review Kroeger's data