Markus Lezzi

Cloning of a Chironomus tentans cDNA encoding a protein (cEcRH) homologous to the Drosophila melanogaster ecdysteroid receptor (dEcR).

We have cloned a cDNA sequence coding for a Chironomus tentans steroid hormone receptor homologue which exhibits extensive amino acid sequence co-linearity with the ecdysteroid receptor of Drosophila melanogaster (dEcR; cell 67, 59-77). The DNA-binding domain has 95% and the hormone-binding domain 75% amino acid sequence identity with the cloned dEcR. The gene for this C. tentans protein is located on chromosome II, region 17C, as determined by in situ hybridization to polytene chromosomes of salivary glands. On Northern blots cDNA probes of the cloned gene hybridize to polyadenylated RNA of ca 4.2 kb. The expression of the cloned gene seems to be developmentally regulated and correlates to changes in ecdysteroid titer. Transfection of this C. tentans protein into D. melanogaster Schneiders line 2 cells leads to transcriptional interference with endogenous dEcR on an ecdysteroid-regulated promoter.

Ecdysteroid Receptors and their Applications in Agriculture and Medicine

Publisher Summary This chapter summarizes the present knowledge on the ecdysteroid receptor (EcR) structure and function with special emphasis on those aspects that are relevant for gene switch applications, and describes gene switch technology with a focus on EcR‐based gene switches. The EcR is a member of the nuclear receptor superfamily and exhibits the typical modular structure composed of the N‐terminal A/B domain, the DNA‐binding C domain, the hinge (D) region, the ligand‐binding E domain, and the C-terminal F domain. The ligand‐binding domain is multifunctional and includes ligand‐dependent dimerization and transactivation functions, while ligand‐independent transactivation and dimerization functions are found in the terminal domains and in the region spanning the DNA binding domain and the N‐terminal region of the hinge, respectively. The EcR heterodimerizes with other members of the nuclear receptor superfamily noticeably with the ultraspiracle protein (USP), which is an orthologue of the vertebrate retinoic acid X receptor (RXR). The progress in made in EcR research has shown that the ligand‐binding pocket of EcR is highly flexible and adaptable. This property of EcR permits the development of receptor systems for practical application in medicine and agriculture. The application of EcR‐based gene switch technology in cellular systems toward functional genomics, drug and ligand discovery, and therapeutic protein production has already commenced grow rapidly in near future. The application of EcR‐based gene switches in plants has a great potential and benefits greatly from the fact that several potential ligands represent registered insecticides and have a track record of environmental safety.

Functional characterization of two Ultraspiracle forms (CtUSP-1 and CtUSP-2) from Chironomus tentans

Two forms, CtUSP-1 and CtUSP-2, of the Chironomus tentans homolog of Ultraspiracle (new nomenclature: Chironomus NR2B4) were described and verified as components of the functional ecdysteroid receptor. The two forms differed from each other in the most N-terminal regions of the A/B domain and were tested for several properties. Both forms showed the ability to heterodimerize with CtEcR and interact with a variety of direct repeat and palindromic EcREs, and both conferred specific ligand binding when heterodimerized with EcR. CtUSP-2 showed a twofold higher ponasterone-binding potential than CtUSP-1. Both USP forms demonstrated the ability to activate ecdysteroid-inducible transcription in HeLa cells and the variations in the A/B domain of these forms were not associated with detectable differences in transcriptional activation. Thus, the two forms function similarly. Among species for which USP forms have been reported, Chironomus is the most closely related one evolutionarily to Drosophila. Despite this proximity, a variety of structural differences were noted in both the A/B and E domains of USP between the two species. The Chironomus USP forms lack many of the amino acid residues associated with the ligand-dependent AF2 transactivation function found in all other RXRs and USPs reported so far.

The activity of Balbiani rings 1 and 2 in salivary glands of Chironomus tentans larvae under different modes of development and after pilocarpine treatment

The activity of the Balbiani rings 1 and 2 (BR1 and BR2) in the salivary gland was followed during development of fourth instar larvae of Chironomus tentans under different modes of development, with or without a previous pilocarpine treatment. The activity was determined in parallel by two different methods, by incorporation of [3H]uridine into BR-RNA (75 S) and by morphometry of BR1 and of BR2. In glands of untreated larvae BR2 does not change dramatically except for a depression of activity during oligopause (resting phase). BR1 is completely inactive during this phase but exhibits a pronounced activity maximum in the middle of the prepupal period, in subitaneously developing (i.e., uninterrupted) as well as in postoligopause cultures. After pilocarpine treatment the activity of BR2 (rather than of BR1) is generally increased. The extent of this stimulation, however, is strongly development dependent. A most striking activity difference is observed in postoligopause between animals of stage 5 and of stage 6. The relationship between BR2 activity and degree of emptying of the salivary gland lumen was investigated. A model is proposed in which BR2 activity is conceived as being regulated by two parameters: by the degree of filling of the gland lumen and by the stage and mode of development of the larva.

Comparative toxicity and ecdysone receptor affinity of non-steroidal ecdysone agonists and 20-hydroxyecdysone in Chironomus tentans

Ecdysone agonists belonging to the bisacylhydrazine class of compounds are a new generation of insecticidal compounds that cause premature lethal molts in susceptible intoxicated insects. While two of the bisacylhydrazines (coded as RH-5992 and RH-2485) are predominantly toxic to lepidopteran pests, RH-5849, which has not been commercialized, has a broader spectrum of toxicity. We have carried out toxicity bioassays with last (4th) instar Chironomus tentans L. larvae, radioligand binding assays using bacterial fusion proteins of C. tentans ecdysone receptor and ultraspiracle (CtEcR, CtUSP), and C. tentans imaginal disc development assays to compare the relative potencies of the three bisacylhydrazine compounds as well as of 20-hydroxyecdysone (20E). In all three assays, the potency of the three bisacylhydrazines was in the order RH-2485>RH-5992>RH-5849. While in toxicity assays 20E was ineffective, most likely due to rapid metabolism, it was more potent than RH-5849 but less so than RH-5992 and RH-2485 in imaginal disc assays. In summary, we compared the potencies of the ecdysone agonists for C. tentans at three levels: whole organism, imaginal discs and the receptor level, and our results indicate that the increased toxicity of the non-steroidal ecdysone agonists for C. tentans has a high correlation to the affinity of these compounds for CtEcR/CtUSP bacterially expressed proteins. Our results, though, do not exclude reasons of metabolic stability of the compounds in C. tentans, which we have not investigated in this report.

Non-genomic ecdysone effects and the invertebrate nuclear steroid hormone receptor EcR--new role for an "old" receptor?

The ecdysteroids (Ec), invertebrate steroid hormones, elicit genomic but also non-genomic effects. By analogy to vertebrates, non-genomic responses towards Ec may be mediated not only by distinct membrane-integrated but also by membrane-associated receptors like the classical nuclear ecdysteroid receptor (EcR) of arthropods. This is supported by a comparison of physiological properties between invertebrate and vertebrate steroid hormone systems and recent findings on the subcellular localization of EcR. The measured or predicted high degree of conformational flexibility of both Ec and the ligand binding domain (LBD) of EcR give rise to a conformational compatibility model: the compatibility between conformations of the cognate receptors ligand binding domain and structures or conformations of the ligand would determine their interaction and eventually the initiation of genomic versus non-genomic pathways. This model could also explain why specific non-genomic effects are generally not observed with non-steroidal agonists of the bisacylhydrazine group.

The giant secretory proteins of Chironomus tentans salivary glands: the organization of their primary structure, their amino acid and carbohydrate composition

It is generally accepted that the giant secretory proteins of the larval salivary glands of Chironomus tentans are encoded by genes located in Balbiani rings (1 and 2). The major part of the DNA of these genes consists of tandem repeats of 200–240 base pair (bp). It was investigated whether this repetitive sequence arrangement is also expressed at the level of the gene products, the giant secretory proteins (SPs). These proteins were partially digested by an endogenous protease or by papain. It was found that SP Ia and Ib (nomenclature: Rydlander and Edström 1980) contain a repetitive primary structure over much of their length. The basic repeat unit is 18,000 daltons. Amino acid analysis of SP I (a+b) revealed a biased distribution which is characteristic for structural proteins. Studies of the carbohydrate composition of SP I (a+b) revealed an unexpectedly high total sugar content of 17.4% (w/w). It was composed of N-acetylglucosamine, glucose, mannose, and galactose in a ratio of 3.4∶6.3∶3.2∶1.0.

Developmental Effects of a Chimeric ultraspiracle Gene Derived From Drosophila and Chironomus

Summary: The ultraspiracle (usp) gene encodes a nuclear receptor that forms a heterodimer with the ecdysone receptor (EcR) to mediate transcriptional responses to the insect steroid hormone, 20‐hydroxyecdysone (20HE). The responses ultimately elicit changes associated with molting and metamorphosis. Although Ultraspiracle (USP) is required at several developmental times, it is unclear whether USP plays stage‐specific roles in Drosophila. A chimeric transgene (d/cusp), produced by replacing the ligand‐binding domain (LBD) of Drosophila USP with the equivalent domain from another Diptera, Chironomus tentans, was tested for its ability to rescue Drosophila usp mutants from early larval lethality. A single copy of the d/cusp was sufficient to rescue transformants from several lines through larval development but they died suddenly during the late third instar. Additional doses of d/cusp were required to allow survival through the adult stage, but they did not restore a normal prepupal contraction. Thus, the arrest at the onset of metamorphosis apparently is caused by the impaired ability of the chimeric USP to mediate a stage‐specific function associated with the LBD. genesis 28:125–133, 2000.

Presence of an ecdysteroid-specific binding protein (“receptor”) in epithelial tissue culture cells of Chironomus tentans

Abstract In an epithelial cell line of Chironomus tentans , the presence of a high-affinity binding component for moulting hormones is demonstrated using whole cells or cell extracts. The K d -value for ponasterone A binding to whole cells is 0.17 × 10 −9 M, and to cell extracts it is 0.65 × 10 −9 M or 0.17 × 10 −9 M as determined by Scatchard or kinetic analyses, respectively. Competition assays revealed the relative affinities of this component to the three ecdysteroids ponasterone A, 20-hydroxyecdysone and ecdysone to be 1, 0.013 and 0.0029 Hydrocortisone, a steroid hormone from vertebrates, does not compete with ponasterone A for binding. Binding of ponasterone A is reduced by increasing salt concentrations. The apparent half-life of the binding component at 4°C is 41 h, and it is increased to 98 h when cell extracts are stored in the presence of ligand. The binding component is sensitive to proteinase and to agents blocking sulfhydryl groups.

Hormonal control of gene activity in polytene chromosomes

Abstract The most recent published work on the roles of ions and hormones on gene activation in Chironomus polytene chromosomes is discussed along with a series of as-yet-unpublished experiments. Studies utilizing ecdysone and juvenile hormone in vivo reveal the existence of specific chromosomal regions that are sensitive to one or the other hormone. Similar effects can be elicited by changing the ionic milieu of the chromosomes including isolated chromosomes. The hormones do not appear to induce specific puffing patterns in isolated chromosomes; however, alternate possibilities are suggested for this lack of effect. Studies are reported on the normal sequence of puffing at the hormone-sensitive regions of the polytene chromosomes of both the salivary glands and the Malpighian tubules during development. The advantage of the latter structures is that, unlike the salivary glands, they remain intact during pupal life. Correlations are also drawn between the activity of an ecdysone-sensitive region and the appearance of an electrophoretically identifiable protein in the secretion of the salivary glands. These new data are consistent with our belief that insect hormones act indirectly to elicit gene activity by first causing alterations in the ionic constitution of the chromosomal milieu.