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.

Ultrastructural studies of Chironomus salivary gland cells in different states of Balbiani ring activity.

Chironomus tentans fourth instar larvae were awakened from oligopause (diapause) and treated with pilocarpine to stimulate Balbiani ring transcriptional activity. Salivary glands were fixed, sectioned and examined by conventional and stereo-electron microscopy. Balbiani ring RNP lateral fiber densities were measured in well-formed regions with clear Balbiani ring granules arranged along chromatin transcription axes. Despite an up to ten-fold range in Balbiani ring transcriptional activity [10, 12, 14], we observed no significant difference in lateral fiber densities. These data are discussed in terms of the variety of mechanisms for modulating overall transcriptional activity, including recruitment of parallel transcription units in the polytene chromosome. The lack of other major ultrastructural changes at the nuclear envelope and within the domains of tubular rough endoplasmic reticulum argue strongly against a major dismantling of subcellular structure during the induced oligopause.

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.

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.

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.

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.

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.

Elemental analysis of solid microscopic samples in the flameless atomic absorption cuvette.

Solid microscopic samples are precisely located by means of a newly developed applicator under microscopic control in the center of a flameless graphite tube cuvette. The parts of the applicator that reach into the cuvette are made of quartz which can be cleaned by heating. The values for the K and Na content of samples such applied are highly reproducible. No contamination could be detected. Solid samples yield higher signals than liquid samples. A method of calibration is described which uses lyophilized pieces of egg albumin containing known amounts of K and Na as standards.

A gravimetric system for lyophilized samples in the sub-microgram range.

A new quartz fiber balance is described which is evacuated and electrically shielded. This balance minimizes the problems of moisture uptake by the lyophilized sample, air convection inside the balance and static electricity. Besides it offers a wider useful weighing range and a higher handling comfort. It could be shown that in the regular (i.e. not evacuated) balance the moisture uptake varies considerably with the humidity of the air and with the kind of sample analyzed, and that it might be greater than previously reported.

Control of gene activities in the polytene chromosomes of Chironomus tentans by ecdysone and juvenile hormone.

The injection of ecdysone (molting hormone) into chironomid larvae is known to result in the stimulation of specific gene activities (increased RNA synthesis). The morphological manifestation of this gene activation is the formation of a puff. Cytological and autoradiographic analysis of several regions of the salivary gland chromosomes of Chironomus tentans revealed that ecdysone also stimulates Balbiani ring 1. In contradistinction to the puffs, the Balbiani rings are tissue specific and account for a significant percentage of the cell's non-nucleolar RNA.The application of juvenile hormone results in a decreased activity of Balbiani ring 1, suggesting that the two hormones may act antagonistically. In addition, juvenile hormone induces a puff at chromosome region I-19-A and this puff becomes less active after a short-term treatment with ecdysone. These data demonstrate an effect of juvenile hormone on puffing and are the first to demonstrate activation of a Balbiani ring by ecdysone.

Heat shock phenomena in Chironomus tentans I. In vivo effects of heat, overheat, and quenching on salivary chromosome puffing.

Incubation of 4th instar larvae of Chironomus tentans at elevated temperatures leads in salivary and Malpighian chromosomes to the appearance of 4-5 new puffs. Previously present puffs, particularly Balbiani rings in salivary chromosomes, become drastically reduced. The reactions of region IV-5C and Balbiani ring 1 and 2 in salivary glands are quantitatively analyzed. Statistically significant heat shock effects are observed already after 5 min and reach a maximum between 30 and 60 min. The effective temperature range is small (between 33 to 40 degrees C) with an optimum at 37 degrees C. Above 40 degrees C, i.e., at overheat shock temperatures, heat shock reactions are suppressed. Larvae heat or overheat shocked for 1-7 h or 15-30 min, respectively, survive when returned to normal culturing temperatures. The recovery from heat shock of the puffing pattern occurs in two phases: a fast one (10-20 min) and a slow one (up to 5 h) sometimes separated by a period of backlash. Quenching of overheat shocked larvae does not result in a delayed heat shock reaction.

Isolation of DNA from single microsurgically excised bands of polytene chromosomes of Chironomus.

A method is described for excising by a glass knife single bands of isolated polytene chromosomes of the salivary glands of Chironomus tentans larvae. DNA strands were isolated from cut-out bands and their contour lengths were determined on electron micrographs. The mean contour length of DNA strands isolated from the double band I-8A was about twice that of the single band I-11B, namely 63 versus 34 micrometers. The described method may be applicable for molecular studies on single bands (e.g., by DNA cloning).

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.

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.

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.

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.

Mass isolation ofpolytene nuclei from Chironomus salivary glands.

In this paper we describe a method for the rapid mass isolation of polytene nuclei from Chironomus salivary glands. The procedure for the isolation of glands involves 5 principal steps; (a) freezing Chironomus larvae in liquid propane; (b) breaking open frozen animals in a pre-cooled mortar; (c) thawing the fragments in sucrose medium, free of divalent cations; (d) pressing the suspension of broken animals through a system of regularly spaced capillary constrictions of free organs; and (e) enrichment of glands by differential sedimentation and removal of contaminating material under a dissecting microscope. The nuclear isolation procedure is a large scale modification of a method previously described by Robert, using digitonin as a non-ionic detergent to solubilize cytoplasma and secretion without affecting the nuclear membrane. Nuclei obtained by this method show structural integrity and an unchanged chromosomal banding pattern. Their incorporation of UTP is within the same range as reported by other authors for nuclei by hand dissection.

Correlated changes of Balbiani ring expansion and secretory protein synthesis in larval salivary glands of Chironomus tentans.

Salivary glands of various stages of the last larval instar of Chironomus tentans were quantitatively analyzed with respect to the expansion of their Balbiani rings (B1, B2, B3) by a fast green staining procedure as well as to the rate of synthesis of their secretory proteins (S1, S2, S3) by a scintillation counting procedure of electrophoretic fractions. The extent of expansion of B1, B2 and B3 correlates positively with the rate of synthesis of S3, S2 and S1, respectively. With B1 and S3 these parameters undergo a parallel and developmentally specific change being rather depressed in intermolt, and particularly in diapausing animals.