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.

Differential control of gene activity by isoforms A, B1 and B2 of the Drosophila ecdysone receptor.

The steroid hormone ecdysone initiates molting and metamorphosis in Drosophila via a heterodimeric receptor consisting of EcR that binds hormone, and USP, a homolog of the vertebrate RXR receptor. EcR exists in three isoforms EcRA, EcRB1 and EcRB2 that are thought to direct specific physiological responses to ecdysone. These three isoforms differ only in their N-terminal A/B domain that implies that sequences responsible for the differential physiological effects lie within the A/B domains of the EcR isoforms. In the present study, we set out to determine the capability of the three isoforms and their A/B domains to control gene transcription. When full-length EcR plasmids were cotransfected into mammalian cells with a USP expressing and a cognate reporter plasmid, the three EcR isoforms showed striking differences in their ability to control gene transcription, both in the presence and in the absence of hormone. Furthermore, the A/B domains of EcRB1 and of EcRB2 when fused to the GAL4 DNA binding domain are sufficient to activate transcription of a reporter gene, in yeast as well as in mammalian cells. In contrast, a fusion construct containing the A/B domain of EcRA represses basal transcription of the reporter gene. All these findings emphasize the importance of the A/B domains of the three EcR isoforms for differentially controlling gene transcription. Furthermore, they provide evidence for the existence of an autonomous ligand-independent activation function (AF1) in the A/B domains of EcRB1 and EcRB2 and of an inhibitory function (IF) in the A/B domain of EcRA.

Developmental effects of a chimeric ultraspiracle gene derived from Drosophila and Chironomus.

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.

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.

Immunohistochemical localization of ecdysteroid receptor and ultraspiracle in the epithelial cell line from Chironomus tentans (Insecta, Diptera).

Ecdysteroid receptor (EcR) and its heterodimerization partner, ultraspiracle (USP), were demonstrated in the epithelial cell line from Chironomus tentans by immunohistochemistry. In untreated cells both proteins are present in nuclei as well as in granular compartments of the cytosol. At 1 day after addition of 1-microM 20-OH-ecdysone (20E) total immunofluorescence had increased in the nuclei, whereas the cytoplasmic staining had disappeared. At the 2nd and 3rd days all cells within a vesicle appear identical according to morphological criteria, but the EcR and USP immunoreactivity becomes restricted into patches of neighbouring cells. The hormonally induced changes in the pattern of localization of functional ecdysteroid receptor, the heterodimer of EcR and USP, are discussed in relation to similar effects of 20E on acetylcholinesterase and muscarinic acetylcholine receptor distribution in this cell line.

High level transactivation by the ecdysone receptor complex at the core recognition motif.

Ecdysteroid signaling in insects is mediated by the ecdysone receptor complex that is composed of a heterodimer of the ecdysone receptor and Ultraspiracle. The DNA binding specificity plays a critical role of defining the repertoire of target genes that respond to the hormone. We report here the determination of the preferred core recognition motif by a binding site selection procedure. The consensus sequence consists of a perfect palindrome of the heptameric half-site sequence GAGGTCA that is separated by a single A/T base pair. No binding polarity of the ecdysone receptor/Ultraspiracle heterodimer to the core recognition motif was observed. This core motif mediated the highest level of ligand-induced transactivation when compared to a series of synthetic ecdysone response elements and to the natural element of the Drosophila hsp27 gene. This is the first report of a palindromic sequence identified as the highest affinity DNA binding site for a heterodimeric nuclear hormone receptor complex. We further present evidence that the ligand of the ecdysone receptor preferentially drives Ultraspiracle from a homodimer into a heterodimer. This mechanism might contribute additionally to a tight control of target gene expression.

Torsional state of DNA in a transcriptionally hyperactive Balbiani ring of polytene chromosomes.

The torsional tension of unconstrained double-helical DNA was determined in transcriptionally hyperactive Balbiani ring 2 (BR2) and in inactive polytene chromosome bands of Chironomus tentans. The method used is based on the dual ability of small intercalating ligands to (a) sense, by differential binding, twists that deviate from that of regular B-form DNA and (b) create positive torsional tension in closed double-stranded DNA, thereby compensating for any negative torsional tension that existed before intercalation. Isolated nuclei of salivary glands were stained with the intercalating fluorescent dye ethidium bromide (EtBr) at various concentrations, and the temporal fluorescence intensity changes (deltaI/I per min) occurring in BR2 and in inactive bands were monitored under a confocal laser scanning microscope during the process of DNA nicking by laser irradiation or DNAase I. From the EtBr concentration at which deltaI/I per min was neither positive nor negative after nicking (i.e. at the equivalence point), the relative twist difference (RTD) was calculated. In bands, it was found to be very small, suggesting that their unconstrained DNA is under low torsional stress. In contrast, the RTD of DNA in highly expanded areas of BR2 was estimated to be negative and of a significant magnitude in absolute terms. This indicates that transcriptionally hyperactive DNA is under considerable negative torsional tension.

Expression of EcR and USP in Escherichia coli: purification and functional studies.

The functional ecdysteroid receptor complex consists of a nuclear receptor heterodimer of ecdysteroid receptor (EcR) and ultraspiracle (USP). EcR and USP of both Chironomus tentans and Drosophila melanogaster were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST). Cell lysis and protein solubilization with the anionic detergent sarkosyl yielded preparations of EcR and USP with properties similar to those of the endogenous receptors in various respects. The heterodimer of the expressed proteins specifically bound the labeled ecdysteroid (Ec) [3H]ponasterone A. Furthermore, it preferentially recognized the palindromic ecdysone response element (EcRE) PALI. Interestingly, binding to the PAL1 element was also observed for EcR homodimers. USP homodimers, in turn, preferentially bound to the direct repeat element DR1. When incubated with native polytene chromosomes of Chironomus, EcR/USP specifically accumulated at the early Ec-inducible puff site IV-2B.

Antibodies against the D-domain of a Chironomus ecdysone receptor protein react with DNA puff sites in Trichosia pubescens.

An antiserum (called AScE/D) against the semiconserved D-domain of a Chironomus tentans ecdysone receptor protein (cEcR) gave indirect immunofluorescence signals at DNA puff sites in Trichosia pubescens. The signals varied in maximum intensity at different DNA puff sites. Control experiments using the secondary rhodamine-labeled anti-rabbit IgG alone, preimmune serum, affinity purified AScE/D (called pABcE/D) and AScE/D preabsorbed with expressing bacterial extract or highly purified bacterially expressed cEcR indicated that the signals obtained at these chromosomal sites were likely to be due to specific interaction between an endogenous sciarid EcR and antibodies against cEcR. This conclusion was supported by observation of signals at certain Ec-inducible primary RNA puff sites. AScE/D signals began to appear at DNA puff sites during L3, the stage when amplification initiates, but at most sites their mean intensity was low and not statistically significant. Sites with AScE/D signals of significant mean intensity at this stage already showed evidence of transcription. The number and strength of transcription signals increased during L4. Comparison of the developmental course of signals for AScE/D, DNA synthesis, RNA presence/synthesis, and puff size for several DNA puffs during late larval- prepupal development showed a closer relationship of AScE/D signals with the initiation of RNA synthesis than with the initiation of DNA synthesis. Therefore, although we cannot absolutely eliminate a direct involvement of EcR in the amplification process at some sites, this investigation gives stronger support for its direct involvement in transcription. Since AScE/D signals are observed at DNA puff sites from the time the latter begin amplification/transcription through their regression, it appears that Ec and EcR are necessary as a sustained stimulus at these regions.

The 5' ends of Hantaan virus (Bunyaviridae) RNAs suggest a prime-and-realign mechanism for the initiation of RNA synthesis.

We examined the 5' ends of Hantaan virus (HTN) genomes and mRNAs to gain insight into the manner in which these chains were initiated. Like those of all members of the family Bunyaviridae described so far, the HTN mRNAs contained 5' terminal extensions that were heterogeneous in both length and sequence, presumably because HTN also "cap snatches" host mRNAs to initiate the viral mRNAs. Unexpectedly, however, almost all of the mRNAs contained a G residue at position -1, and a large fraction also lacked precisely one of the three UAG repeats at the termini. The genomes, on the other hand, commenced with a U residue at position +1, but only 5' monophosphates were found here, indicating that these chains may not have initiated with UTP at this position. Taken together, these unusual findings suggest a prime-and-realign mechanism of chain initiation in which mRNAs are initiated with a G-terminated host cell primer and genomes with GTP, not at the 3' end of the genome template but internally (opposite the template C at position +3), and after extension by one or a few nucleotides, the nascent chain realigns backwards by virtue of the terminal sequence repeats, before processive elongation takes place. For genome initiation, an endonuclease, perhaps that involved in cap snatching, is postulated to remove the 5' terminal extension of the genome, leaving the 5' pU at position +1.

Immunological studies on the developmental and chromosomal distribution of ecdysteroid receptor protein in Chironomus tentans.

Antisera were raised against different domains of a putative ecdysteroid receptor (cEcRH) of Chironomus tentans. All the antisera reacted with a 68,000 dalton protein exhibiting DNA binding properties. Additionally, we were able to demonstrate that the antisera immunoprecipitate protein which binds a radioactively labeled ecdysteroid (Ec), i.e., [3H]ponasterone A, with high specificity. These properties indicate that the antisera recognize specifically an endogenous ecdysteroid receptor protein (cEcR) in C. tentans cells and thus are suitable for the following quantitative and qualitative immunological and immunohistochemical investigations. The cellular level of cEcR varies during development, and it is particularly low in oligopausing larvae. In polytene chromosomes of prepupal salivary glands, cEcR is located at approximately 50 transcriptionally active loci. These loci include both early ecdysteroid (Ec)-inducible puff sites, such as the locus containing the gene coding for the homolog of the E75 protein in Drosophila melanogaster, as well as late Ec-inducible puff-sites. The latter group comprises a locus of a gene specifying the homolog of the D. melanogaster ultraspiracle protein. However, loci of genes coding for salivary gland secretory proteins (e.g., Balbiani ring forming chromosome regions) do not specifically react with the antisera. Thus, the developmental regulation of these genes is not directly controlled by Ec. Polytene chromosomes of oligopausing larvae show hardly any loci that contain cEcR. The few detected correspond, with few exceptions, to the most potent cEcR binding sites found in prepupae.

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 Schneider's line 2 cells leads to transcriptional interference with endogenous dEcR on an ecdysteroid-regulated promoter.

Structural and developmental analysis of a gene cloned from the early ecdysterone-inducible puff site, I-18C, in Chironomus tentans.

A gene (I-18C) cloned from the early ecdysterone (EDS)-inducible puff site, I-18C, in Chironomus tentans salivary glands, codes by differential processing for at least five transcripts which can be grouped into spliced and unspliced transcripts. The spliced group comprises several RNAs of approx. 4.6 kb, and the unspliced group comprises the 1.8-kb and 6.5-kb RNAs. All these transcripts have a similar or the same transcription start point. The steady-state level of the spliced 4.6-kb RNAs reveals some parallels with EDS concentrations in animals during the various developmental stages analyzed, while the levels of the unspliced 1.8-kb and 6.5-kb RNAs do not correlate with the EDS concentrations.

Identification of a developmentally regulated gene for a 140-kDa secretory protein in salivary glands of Chironomus tentans larvae.

Secretory proteins are synthesized in salivary glands of the insect, Chironomus tentans, and assemble in vivo into silk-like threads which aquatic larvae use to construct tubes for filter feeding and pupation. Thus far, all known secretory protein genes contain repetitious protein-coding sequences and are located in cytological structures known as Balbiani rings, giant puffs found on polytene secretory cell chromosomes. In this paper we describe the identification of another secretory protein gene which is comprised of repeated sequences; however, this gene is not located in a Balbiani ring. Two partial cDNA clones from a 3.6-kilobase pair poly(A)+ RNA were sequenced and found to contain two open reading frames for protein synthesis. Antibodies were raised against synthetic oligopeptides whose sequences were derived from these two open reading frames. An immunoaffinity-purified antibody for one of these peptides bound specifically to a 140-kDa secretory protein (sp140). The cDNA sequences contain tandem repeats of 42 base pairs which encode a repeat of 14 amino acids with a composition and oligopeptide sequence similar to other secretory proteins. The C. tentans genome contains about 70 copies of this 42-base pair repeat organized as a contiguous block of 3 kilobase pairs or less. The sp140 gene was mapped by in situ hybridization to polytene chromosome band I-17-B. Developmental studies of protein accumulation, steady-state levels of mRNA, and relative transcription rate suggested that the sp140 gene is developmentally regulated so that maximal expression is achieved during the prepupal stages of the fourth larval instar. Based upon these results we proposed that sp140 gene belongs to a prepupal class of secretory protein genes. While the sp140 gene shares structural and expression characteristics with other secretory protein genes, its unique chromosomal location shows that this multigene family is not restricted to Balbiani rings.

Developmental changes in the responsiveness to ecdysterone of chromosome region I-18C of Chironomus tentans.

The presence of nascent RNA in chromosome region I-18C was revealed by the method of induced hybrid formation (IEH) and by use of anti-RNA:DNA antibodies. IEH signals were quantitated, and used as a measure of transcriptional activity in the region. The expansion of the I-18C region was also determined and used as an indication of local chromatin decondensation. Chironomus tentans larvae may undergo oligopause or they may develop subitaneously. It was found that the course of basal activity in I-18C differs substantially in these two modes of development. Most interestingly they also differ in activation of I-18C in response to in vitro ecdysterone treatment. Changes in hormonal responsiveness of I-18C parallel changes in the preexisting state of I-18C decondensation. Oligopause stage 5 animals, being most refractory in this respect, exhibit the most condensed I-18C chromosome region.

Two transcripts of the same ecdysterone-controlled gene are differentially associated with ribosomes.

The ecdysterone-controlled gene I-18C of Chironomus tentans produces two transcripts by differential splicing: a 1.8-kb RNA, with an open reading frame (ORF) of 417 nucleotides (nt) and a 4.6-kb RNA, with no ORF longer than 270 nt. Centrifugation of cytoplasmic extracts in velocity sedimentation sucrose gradients and CsCl-buoyant-density gradients demonstrates that the 1.8-kb RNA is incorporated into polysomes. The 4.6-kb RNA, however, exhibits characteristics of free ribonucleic acid-protein complexes and monosomes.

Developmentally regulated expression of a Balbiani ring 1 gene for a 180-kD secretory polypeptide in Chironomus tentans salivary glands before larval/pupal ecdysis.

The expression of a Balbiani ring 1 gene that codes for a salivary gland-specific 180-kD secretory polypeptide (sp180) is regulated developmentally. Immunoblots of salivary gland protein incubated with an affinity-purified nonapeptide-reactive antibody demonstrated that the salivary gland content of sp180 increases as much as 10-fold between stages 8 and 10 of the fourth larval instar. Hybridization of RNA dot-blots with an oligonucleotide probe indicated that the observed increase in sp180 was preceded by a parallel 20-fold increase in the steady state level of its mRNA beginning between stages 7 and 8. In vitro nuclear transcription experiments demonstrated that there was a 10-fold acceleration in the rate of sp180 gene transcription between stages 6 and 10. The limited period of expression of the sp180 gene contrasted dramatically with the expression of Balbiani ring genes BR1, BR2 alpha, BR2 beta, and BR6, which code for the sp-I family of fibrous secretory polypeptides. The appearance of sp180 in secretion coincided with microscopically visible changes in the bundling of these fibrous polypeptides. At the same time, we noticed changes in the appearance and consistency of feeding tubes that larvae construct with this secretion. These results lead us to propose that sp180 may modify the structure or utilization of fibrous secretory polypeptides specifically for the assembly of pupation tubes necessary for larval/pupal ecdysis.

Structural transition in inactive Balbiani ring chromatin of Chironomus during micrococcus nuclease digestion.

We have analysed by micrococcus nuclease digestion the chromatin structure of genes in the Balbiani ring (BR) regions of a Chironomus cell line. Gel electrophoresis of the DNA fragments reveals a repeating structure which consists of two repeat sizes, a long repeat seen in the large fragments and a small repeat seen in the small fragments. The two repeats hardly overlap, except in a narrow transition zone which is at a different fragment size in the BR 2.2 and the BR 2.1 gene. The sizes of the large repeats fit the repeat of the underlying DNA sequence. The short repeats are between 170 and 180 bp, and after H1 depletion the short repeat in the BR 2.2 gene is 160 bp. Our most favoured interpretation of these data is that in intact chromatin the nucleosomes in the BR genes are phased with respect to the repeating DNA sequence, whereas micrococcus nuclease digestion leads to loss of a nucleosome-positioning constraint and hence to rearrangement of the nucleosomes. Our results imply a possible artefact of nuclease digestion of chromatin, which has to be taken into account in mapping nucleosome positions.

Low degree of ubiquitination of histone 2A in the dipteran Chironomus tentans.

A polyclonal antibody to ubiquitin has been prepared and shown to react with both ubiquitin and ubiquitinated histone 2A (uH2A). Applying this antibody in Western blotting experiments, we have observed that the salivary glands of Chironomus tentans contain an unusually low amount of uH2A (1% of histone 2A), while the amount of free ubiquitin is as abundant as in other animal cells, e.g. HeLa cells. The same low content of uH2A was also found in diploid epidermal cells of Chironomus origin suggesting that the low amount is not a characteristic of the polytene state of chromatin in salivary gland cells but rather a property of C. tentans as a species. The significance of the low degree of ubiquitination is discussed in relation to the information available on the organization of Chironomus chromatin into unusually large chromomeric entities.