"Parahomologous" gene targeting in Drosophila cells: an efficient, homology-dependent pathway of illegitimate recombination near a target site.

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."

Genetic transformation of Drosophila cells in culture by P element-mediated transposition.

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.

Bombyx EcR (BmEcR) and Bombyx USP (BmCF1) combine to form a functional ecdysone receptor.

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.

The moulting hormone ecdysone is able to recognize target elements composed of direct repeats.

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.

Tissue-specific regulation by ecdysone: distinct patterns of Eip28/29 expression are controlled by different ecdysone response elements.

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.

Functional ecdysone receptor is the product of EcR and Ultraspiracle genes.

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.

The IVth Karlson Lecture: ecdysone-responsive genes.

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 arthropod initiator: the capsite consensus plays an important role in transcription.

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.

Tissue-specific ecdysone responses: regulation of the Drosophila genes Eip28/29 and Eip40 during larval development.

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.

Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor.

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 Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily.

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.

Ecdysteroid receptors in the central nervous system of Manduca sexta: their changes in distribution and quantity during larval-pupal development.

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.

Identification of ecdysone response elements by analysis of the Drosophila Eip28/29 gene.

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.

Ionic coupling and mitotic synchrony of siblings in a Drosophila cell line.

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.

Diverse expression of overlapping genes: the Drosophila Eip28/29 gene and its upstream neighbors.

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.

Effects of juvenile hormone on the ecdysone response of Drosophila Kc cells.

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.

26-[125I]iodoponasterone A is a potent ecdysone and a sensitive radioligand for ecdysone receptors.

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.