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The Pv11, an insect cell line established from the midge Polypedilum vanderplanki , is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.

Pesticides occur in the environment as mixtures, yet the joint toxicity of pesticide mixtures remains largely under-explored and is usually overlooked in ecological risk assessment. In the current study, joint toxicity of a neonicotinoid insecticide (imidacloprid, IMI) and a strobilurin fungicide (azoxystrobin, AZO) was investigated with Chironomus dilutus over a wide range of concentrations and at different effect levels (organism, cell, and gene levels). The two pesticides, both individually and in combination, were found to induce oxidative stress and cause lethality in C. dilutus. Median lethal concentrations for IMI and AZO were 3.98 ± 1.17 and 52.9 ± 1.1 μg/L, respectively. Mixtures of the two pesticides presented synergetic effects at environmentally relevant concentrations whilst antagonistic effects at high concentrations, showing concentration-dependent joint toxicity. Investigation on the expressions of 12 genes (cyt b, coi, cox1, cyp4, cyp12m1, cyp9au1, cyp6fv1, cyp315, gst, Zn/Cu-sod, Mn-sod, and cat) revealed that the two pesticides impaired mitochondrial respiration, detoxification, and antioxidant system of C. dilutus, and the joint effects of the two pesticides were likely due to an interplay between their respective influences on these physiological processes. Collectively, the synergistic effects of the two pesticides at environmentally relevant concentrations highlight the importance to incorporate combined toxicity studies into ecological risk assessment of pesticides., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Pesticides are extensively used for the control of agricultural pests and disease vectors, but they also affect non-target organisms. Cypermethrin (CYP) is a synthetic pyrethroid used worldwide. Otherwise, bioinsecticides like Bacillus thuringiensis var. israelensis (Bti) have received great attention as an environmentally benign and desirable alternative. In order to evaluate the toxicity of those pesticides, Chironomus calligraphus was selected due to its high sensitivity to some toxicants. Third and fourth instars larvae were exposed to serial dilutions of CYP and Bti to determine LC 50 values. In order to evaluate the potentially histopathological alterations as biomarkers, after 96-h of exposure, live larvae were fixed for histological analysis of the mid region of digestive tract. The 96-h LC 50 values were 0.52 and 1.506μg/L for CYP and Bti, respectively. Midgut histological structure of the control group showed a single layer of cubical cells with microvilli in their apical surface and a big central nucleus. The midgut epithelium of larvae exposed to a low concentration of CYP (0.037μg/L) showed secretion activity and vacuolization while at high concentration (0.3μg/L) cells showed a greater disorganization and a more developed fat body. On the other hand, Bti caused progressive histological damage in this tissue. Chironomus calligraphus is sensitive to Bti and CYP toxicity like other Chironomus species. The histopathological alterations could be a valuable tool to assess toxicity mechanism of different pesticides., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Larvae of the African midge Polypedilum vanderplanki show extreme desiccation tolerance, known as anhydrobiosis. Recently, the cultured cell line Pv11 was derived from this species; Pv11 cells can be preserved in the dry state for over 6 months and retain their proliferation potential. Here, we attempted to expand the use of Pv11 cells as a model to investigate the mechanisms underlying anhydrobiosis in P. vanderplanki. A newly developed vector comprising a constitutive promoter for the PvGapdh gene allowed the expression of exogenous proteins in Pv11 cells. Using this vector, a stable Pv11 cell line expressing green fluorescence protein (GFP) was established and retained desiccation tolerance. Gene silencing with GFP-specific siRNAs significantly suppressed GFP expression to approximately 7.5-34.6% of that in the non-siRNA-transfected GFP stable line. Establishment of these functional assays will enable Pv11 cells to be utilized as an effective tool to investigate the molecular mechanisms underlying anhydrobiosis.

Butyl benzyl phthalate (BBP) is a ubiquitous contaminant whose presence in the environment is expected for decades, since it has been extensively used worldwide as a plasticizer in the polyvinyl chloride (PVC) industry and the manufacturing of many other products. In the present study, the interaction of BBP with the ribosome biogenesis pathway and the general transcriptional profile of Chironomus riparius aquatic larvae were investigated by means of changes in the rDNA activity (through the study of the internal transcribed spacer 2, ITS2) and variations in the expression profile of ribosomal protein genes (rpL4, rpL11, and rpL13) after acute 24-h and 48-h exposures to a wide range of BBP doses. Furthermore, cytogenetic assays were conducted to evaluate the transcriptional activity of polytene chromosomes from salivary gland cells, with special attention to the nucleolus and the Balbiani rings (BRs) of chromosome IV. BBP caused a dose and time-dependent toxicity in most of the selected biomarkers, with a general depletion in the gene expression levels and the activity of BR2 after 48-h treatments. At the same time, decondensation and activation of some centromeres took place, while the activity of nucleolus remained unaltered. Withdrawal of the xenobiotic allowed the larvae to reach control levels in the case of rpL4 and rpL13 genes, which were previously slightly downregulated in 24-h tests. These data provide the first evidence on the interaction of BBP with the ribosome synthesis pathways, which results in a significant impairment of the functional activity of ribosomal protein genes. Thus, the depletion of ribosomes would be a long-term effect of BBP-induced cellular damage. These findings may have important implications for understanding the adverse biological effects of BBP in C. riparius, since they provide new sensitive biomarkers of BBP exposure and highlight the suitability of this organism for ecotoxicological risk assessment, especially in aquatic ecosystems., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The karyotype of Camptochironomus pallidivittatus Edwards, 1929 (Diptera, Chironomidae) from five populations of the Lower Volga region and Central Caucasus (the northern macroslope) has been studied. In populations of S. pallidivittatus from the Central Caucasus, 11 banding sequences (BS) were found; one sequence, pal B10, was new to the species. In the Saratov population, 11 BS were also found, three of which were new for the species-pal A3, pal B11, and pal B12. The banding sequences detected for the first time have not yet been found in other parts of the habitat of this species and may be endemic to these regions. In the studied populations ofS. pallidivittatus, banding sequences were found that were nonstandard but fixed in the karyotype. This is indicative of some degree of chromosomal divergence. These banding sequences include pal A2.2 in arm A and pal B10.10 in arm B in the Central Caucasus region, as well as pal B2.2 and pal G2.2 in the Lower Volga region. Arms A, B, D, and G in the Central Caucasian populations and A, B, and D in the Saratov oblast were polymorphic. The composition of heterozygous sequences between populations from different regions coincided only in arm D (pal D 1.2). In arms A and B, the set of heterozygous BS was different: pal A1.2 and pal B1.10 sequences were found in the Central Caucasian populations, and pal A1.3 and B11.12 were found in Saratov oblast. The number of genotypic combinations of S. pallidivittatus was higher in the Central Caucasus region, whereas the number of zygotic combinations was higher in the Saratov population. The percentage of heterozygous larvae in the Central Caucasian populations varied from 20 to 80, whereas all individuals in the Saratov population had heterozygous inversions. Zygotic combinations of larvae in all the studied populations were different.

Cytogenetic analysis was performed on samples of Chironomus plumosus L. (Diptera, Chironomidae) taken from waterbodies of various types in Bryansk region (Russia) and Gomel region (Belarus). Karyotypes of specimens taken from stream pools of the Volga were used as reference samples. The populations of Bryansk and Gomel regions (except for a population of Lake Strativa in Starodubskii district, Bryansk region) exhibit broad structural variation, including somatic mosaicism for morphotypes of the salivary gland chromosome set, decondensation of telomeric sites, and the presence of small structural changes, as opposed to populations of Saratov region. As compared with Saratov and Bryansk regions, the Balbiani ring in the B-arm of chromosome I is repressed in populations of Gomel region. It is concluded that the chromosome set of Ch. plumosus in a range of waterbodies of Bryansk and Gomel regions is unstable.

This article discusses three reviews on the theme of nuclear organization.

Eukaryotic gene expression is the result of the integrated action of multimolecular machineries. These machineries associate with gene transcripts, often already nascent precursor messenger RNAs (pre-mRNAs). They rebuild the transcript and convey properties allowing the processed transcript, the mRNA, to be exported to the cytoplasm, quality controlled, stored, translated, and degraded. To understand these integrated processes, one must understand the temporal and spatial aspects of the fate of the gene transcripts in relation to interacting molecular machineries. Improved methodology is necessary to study gene expression in vivo for endogenous genes. A complementary approach is to study biological systems that provide exceptional experimental possibilities. We describe such a system, the Balbiani ring (BR) genes in polytene cells in the dipteran Chironomus tentans. The BR genes, along with their pre-mRNA-protein complexes (pre-mRNPs) and mRNA-protein complexes (mRNPs), allow the visualization of intact cell nuclei and enable analyses of where and when different molecular machineries associate with and act on the BR pre-mRNAs and mRNAs.

Purpose: Chironomus ramosus is one of the recently reported radiotolerant insects. Salivary gland cells of fourth instar larvae respond to ionizing radiations with increases in the levels of antioxidant enzymes and chaperone proteins. Here we made an attempt to study the state of nuclear DNA after exposure of larvae to a lethal dose for 20% of the population (LD(20)) of gamma radiation (2200 Gy, at a dose rate 5.5 Gy/min)., Materials and Methods: Genomic DNA preparations were subjected to competitive ELISA (Enzyme linked immunosorbent assay) for detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and dynamic light scattering (DLS) to monitor any radiation-induced damage. Single salivary gland cells were subjected to alkaline single cell gel electrophoresis (ASCGE), comet assay and pulsed field gel electrophoresis (PFGE) to check for DNA double-strand breaks., Results: Results from all four experimental procedures confirmed damage of nucleobases and fragmentation of nuclear DNA immediately after radiation. Some 48 h after radiation exposure, modified 8-oxodG residues returned to basal level, homodispersity of genomic DNA reappeared, the length of comet tail regressed significantly (ASCGE) and PFGE pattern matched with that of high molecular weight unirradiated DNA., Conclusion: Chironomus ramosus larvae showed control of DNA damage as observed over 48 h in post irradiation recovery which could be attributed to their ability to tolerate gamma radiation stress.

Set and frequencies of inversion banding sequences in polytene chromosomes in the Palearctic natural populations of a Holarctic chironomid Glyptotendipes barbipes (2n = 8) (the Northwest of Russia, Irkutsk region, Kirghizia, Armenia) have been investigated. The majority of larvae were heterozygotes on inversions. Distinctions in a set and frequencies of inversions between populations form different regions have been ascertained. We have established that G. barbipes karyotype in the populations studied includes inversion banding sequences that are endemic for Palearctic (p'barG2), and also the general sequences met both in Palearctic, and in Nearctic (h'barB3, h'barC3, h'baE2, h'barF2).

Scanning electron microscopy (SEM) has made remarkable progress and has become an essential tool for observing biological materials at microscopic level. However, various complex procedures have precluded observation of living organisms to date. Here, a new method is presented by which living organisms can be observed by field emission (FE)-SEM. Using this method, active movements of living animals were observed in vacuo (10(-5)-10(-7) Pa) by protecting them with a coating of thin polymer membrane, a NanoSuit, and it was found that the surface fine structure of living organisms is very different from that of traditionally fixed samples. After observation of mosquito larvae in the high vacuum of the FE-SEM, it was possible to rear them subsequently in normal culture conditions. This method will be useful for numerous applications, particularly for electron microscopic observations in the life sciences., (© The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

During the austral summer, larvae of the terrestrial midge Belgica antarctica (Diptera: Chironomidae) experience highly variable and often unpredictable thermal conditions. In addition to remaining freeze tolerant year-round, larvae are capable of swiftly increasing their cold tolerance through the rapid cold-hardening (RCH) response. The present study compared the induction of RCH in frozen versus supercooled larvae. At the same induction temperature, RCH occurred more rapidly and conferred a greater level of cryoprotection in frozen versus supercooled larvae. Furthermore, RCH in frozen larvae could be induced at temperatures as low as -12°C, which is the lowest temperature reported to induce RCH. Remarkably, as little as 15 min at -5°C significantly enhanced larval cold tolerance. Not only is protection from RCH acquired swiftly, but it is also quickly lost after thawing for 2 h at 2°C. Because the primary difference between frozen and supercooled larvae is cellular dehydration caused by freeze concentration of body fluids, we also compared the effects of acclimation in dehydrated versus frozen larvae. Because slow dehydration without chilling significantly increased larval survival to a subsequent cold exposure, we hypothesize that cellular dehydration caused by freeze concentration promotes the rapid acquisition of cold tolerance in frozen larvae.

Real-time observation of single molecules or biological nanoparticles with high spatial resolution in living cells provides detailed insights into the dynamics of cellular processes. The salivary gland cells of Chironomus tentans are a well-established model system to study the processing of RNA and the formation and fate of messenger ribonucleoprotein particles (mRNPs). For a long time, challenging imaging conditions limited the access to this system for in vivo fluorescence microscopy. Recent technical and methodical advantages now allow observing even single molecules in these cells. We describe here the experimental approach and the optical techniques required to analyze intranuclear trafficking and export of single native mRNPs across the nuclear envelope.

The translocation of single mRNPs (mRNA-protein complexes) from the nucleus to the cytoplasm through the nuclear pore complex (NPC) is an important basic cellular process. Originally, in order to visualize this process, single mRNP export was examined using electron microscopy (EM) in fixed Chironomus tentans specimens. These studies described the nucleocytoplasmic translocation of huge mRNPs (~30 kb) transcribed from the Balbiani-ring genes. However, knowledge of the in vivo mRNP kinetics in cell compartments remained poor up until recently. The current use of unique fluorescent protein tags, which are able to bind to mRNA transcripts, has allowed the detection and measurements of single mRNP kinetics in living cells. This has demonstrated that mRNP movement is affected by the size of the transcript and the splicing process. It was found that mRNP rates of translocation are slower in the nucleus compared to the cytoplasm and that the cell nucleus contains interchromatin tracks in which mRNPs diffuse. In order to track single mRNP movement in living cells, it is important to be able to identify single mRNP molecules transcribed from a certain gene, at the single-cell level. Single-molecule analysis of gene expression requires advanced imaging systems and analytical software in order to detect and follow the movement of single mRNPs. In this chapter we describe the methods required for the detection and tracking of single mRNP movement in living mammalian cells.

Using Clark oxygen electrode, dependence of mitochondrial functions on Ca(2+)-release channels activity of Chironomus plumosus L. larvae salivary glands suspension was investigated. Cells were ATP-permeabilized in order to enable penetration of exogenous oxidative substrates. Activation of plasmalemmal P2X-receptors (as well as P2Y-receptors) per se does not modify the endogenous respiration of salivary gland suspension. That is, Ca(2+)-influx from extracellular medium does not influence functional activity of mitochondria, although they are located along the basal part of the plasma membrane. Activation of RyRs intensifies endogenous respiration and pyruvate-malate-stimulated respiration, but not succinate-stimulated respiration. Neither activation of IP3Rs (via P2Y-receptors activation), nor their inhibition alters endogenous respiration. Nevertheless, IP3Rs inhibition by 2-APB intensifies succinate-stimulated respiration. All abovementioned facts testify that Ca2+, released from stores via channels, alters functional activity of mitochondria, and undoubtedly confirm the existence of endoplasmic-mitochondrial Ca(2+)-functional unit in Ch. plumosus larvae salivary glands secretory cells. In steady state of endoplasmic-mitochondrial Ca(2+)-functional unit the spontaneous activity of IP3Rs is observed; released through IP3Rs, Ca2+ is accumulated in mitochondria via uniporter and modulates oxidative processes. Activation of RyRs induces the transition of endoplasmic-mitochondrial Ca(2+)-functional unit to the active state, which is required to intensify cell respiration and oxidative phosphorylation. As expected, the transition of endoplasmic-mitochondrial Ca(2+)-functional unit to inactivated state (i. e. inhibition of Ca(2+)-release channels at excessive [Ca2+]i) limits the duration of signal transduction, has protective nature and prevents apoptosis.

Studying giant nuclei of Chironomus plumosus in situ (Makarov, Chentsov, 2010), we concluded that polythene chromosome structure appears after 2 M NaCl and DNase treatment in presence of 2 mM CuCl2. Cu2+ -ions may stabilize bonds between specific non-histone components, arranged into non-histone matrix of polythene chromosome. Here, we investigated the non-histone matrix of pig embryo mitotic chromosomes in situ, using 2 mM CuCl2-stabilization method. In 2 mM CuCl2-stabilized cells the residual chromosome body (non-histone matrix) could be visualized in every stage of mitosis. Mitotic chromosome non-histone matrix had the same reaction on preliminary hypotonic treatment as normal chromosome: different decondensation of non-histone material was observed. Topoisomerase IIalpha and SMC 1 had uniform localization inside chromosomal body and did not form any axial structures.

Dry-preservation of nucleated cells from multicellular animals represents a significant challenge in life science. As anhydrobionts can tolerate a desiccated state, their cells and organs are expected to show high desiccation tolerance in vitro. In the present study, we established cell lines derived from embryonic tissues of an anhydrobiotic chironomid, Polypedilum vanderplanki, designated as Pv11 and Pv210. Salinity stress induced the expression of a set of anhydrobiosis-related genes in both Pv11 and Pv210 cells, suggesting that at least a part of cells can autonomously control the physiological changes for the entry into anhydrobiosis. When desiccated with medium supplemented with 300 mM trehalose or sucrose and stored for 4 weeks in dry air (approximately 5% relative humidity), a small percentage of the cells was found to be viable upon rehydration, although surviving cells seemed not to be able to multiply. We also attempted dry-preservation of organs isolated from P. vanderplanki larvae, and found that a proportion of cells in some organs, including fat body, testis, nerve and dorsal vessel, tolerated in vitro desiccation., (Copyright 2009 Elsevier Inc. All rights reserved.)

Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins.

Morphometric analysis of changes in nucleolar organizer (NO), Balbiani rings (BR)--BR(B), BR(1G), BR(2G) and chromosome I arm B puff activities, and in chromosome compactness of Chironomus plumosus (Diptera) polytene chromosomes was carried out in acute period under separate and combined influence of atropine and pilocarpine. Supression effect of cholinotropic preparations mixture was revealed. Suppression of NO activity with atropine concentration increase in the mixture served as criterion of toxicity.

In many insects, the rapid cold-hardening (RCH) response significantly enhances cold tolerance in minutes to hours. Larvae of the Antarctic midge, Belgica antarctica, exhibit a novel form of RCH, by which they increase their freezing tolerance. In this study, we examined whether cold-sensing and RCH in B. antarctica occur in vitro and whether calcium is required to generate RCH. As demonstrated previously, 1 h at -5 degrees C significantly increased organismal freezing tolerance at both -15 degrees C and -20 degrees C. Likewise, RCH enhanced cell survival of fat body, Malpighian tubules, and midgut tissue of larvae frozen at -20 degrees C. Furthermore, isolated tissues retained the capacity for RCH in vitro, as demonstrated with both a dye exclusion assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based viability assay, thus indicating that cold-sensing and RCH in B. antarctica occur at the cellular level. Interestingly, there was no difference in survival between tissues that were supercooled at -5 degrees C and those frozen at -5 degrees C, suggesting that temperature mediates the RCH response independent of the freezing of body fluids. Finally, we demonstrated that calcium is required for RCH to occur. Removing calcium from the incubating solution slightly decreased cell survival after RCH treatments, while blocking calcium with the intracellular chelator BAPTA-AM significantly reduced survival in the RCH treatments. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) also significantly reduced cell survival in the RCH treatments, thus supporting a role for calcium in RCH. This is the first report implicating calcium as an important second messenger in the RCH response.

Additional chromosomes present only in the germ line are a specific feature of the Orthocladiinae, a subfamily of the Chironomidae. During the complex chromosome cycle in the orthocladiid Acricotopus lucidus, about half of the germ-line-limited chromosomes (Ks) are eliminated in the first division of the primary germ cells. Following normal gonial mitoses, the reduction in the number of Ks is compensated for, in the last mitosis prior to meiosis, by a monopolar movement of the unseparated Ks, while the somatic chromosomes (Ss) segregate equally. This differential mitosis produces daughter cells with different chromosome constitutions and diverse developmental fates. A preferential segregation of mitochondria occurs to one pole associated with an asymmetric formation of the mitotic spindle. This has been detected in living gonial cells in both sexes by using MitoTracker probes and fluorochrome-labelled paclitaxel (taxol). In males, the resulting unequal partitioning of mitochondria to the daughter cells is equalised by the transport of mitochondria through a permanent cytoplasmic bridge from the aberrant spermatocyte to the primary spermatocyte. This asymmetry in the distribution and in the segregation of cytoplasmic components in differential gonial mitosis in Acricotopus may be involved in the process of cell-fate determination.

The fine structure of spermatozoa from several species of chironomids, of Culicoides sp. (Ceratopogonidae) and of Odagmia pontina (Simulidae) was studied. A synapomorphic feature, consisting of nine kidney-shaped structures forming the centriole adjunct, was found in the chironomid species. All members of Chironomoidea share a mono-layered acrosome and a flagellar axoneme, provided with accessory tubules with 15 protofilaments in their tubular wall. The axoneme has a 9+9+2 pattern, but in an unidentified species of chironomid, a 9+9+0 model was observed where the central complex and the spokes are missing. Sperm motility is, however, maintained in all the examined species. The spermatozoa of this taxon have the tendency to complete maturation during their progression along the deferent ducts. Thus, in the proximal region of these ducts, they often show remnants of the spermatid cytoplasm.

In eukaryotes the ribosomal gene population shows two different states in terms of chromatin structure. One subset is organized as nucleosomes (silent copies) while the other has a non-nucleosomal configuration (active copies). Insect cells are not the exception and this bimodal distribution of ribosomal chromatin also occurs in salivary gland cells, and cells of other larval tissues, of the midge Chironomus thummi. In run-on experiments on salivary glands cells we confirmed that transcribed rRNA genes show a non-nucleosomal configuration. The proportion of rRNA genes adopting an open, non-nucleosomal configuration was found to be tissue-dependent, suggesting that the population of unfolded ribosomal chromatin in C. thummi was established during cell differentiation. We propose that cell differentiation determines the fraction of non-nucleosomal rRNA gene copies and thus defines the range of possible rRNA synthesis rates in a particular cell type. In the salivary gland the fraction of unfolded chromatin was not significantly affected when transcription was repressed. However, transcription activation by pilocarpine led to a moderate increase in this fraction. These findings indicate that, in addition to a possible increase in the number of RNA-polymerases per transcribing rDNA unit, the proportion of transcribed ribosomal genes in differentiated cells can be modulated in response to an exceptional rRNA synthesis requirement.

During germ line-soma differentiation in early syncytial embryonic development of the chironomid Acricotopus lucidus, a complement of supernumerary chromosomes, the so-called germ line limited chromosomes (Ks), is excluded from the future somatic nuclei in the course of elimination mitoses. The Ks lag behind in the equatorial plane, while the somatic chromosomes (Ss) segregate equally. After elimination mitoses, the Ks are only present in the pole cells, the primary germ cells. In the divisions before their elimination, the Ks frequently showed delayed separation of sister chromatids with high-frequency formation of anaphasic bridges and lagging in pole movement as detected in 4',6-diamidino-2-phenylindole (DAPI)-stained squash preparations of early embryos. To determine if all of the Ks are eliminated in one step during a single mitosis, a fluorescence in situ hybridization (FISH) analysis of early embryonic divisions was performed using probes of germ line specific repetitive DNA sequences, which specifically label the Ks in their centromeric regions. In most cases, all of the Ks are lost in one mitosis; however, occasionally one or several of the Ks can escape their elimination by segregating and moving poleward together with the Ss. The escaping Ks will then be eliminated in one of the following mitoses. This clearly indicates that the specific conditions to eliminate Ks are not restricted to only one division. Possible mechanisms of elimination of Ks are discussed.

Selection of hormone resistant subclones in the continuous presence of the insecticide and ecdysteroid mimick RH 5992 (tefubenozide) resulted preferentially in clones with defects in ecdysteroid receptor function. RH 5992 is already degraded to polar products in wild-type cells; no increase in metabolism of tefubenozide is observed in resistant clones. According to Western blots, ecdysteroid receptor (EcR) and its heterodimerization partner ultraspiracle (USP) are present in all resistant clones. The concentrations are comparable to wild-type cells, but in three clones the extent of phosphorylation of USP is diminished. With regard to hormone binding several types of hormone resistance are distinguished: (1) The same two high-affinity hormone recognition sites are present as in wild-type cells (K(D1)=0.31+/-0.28 nM, K(D2)=6.5+/-2.4 nM) but the number of binding sites is reduced. (2) The binding site with the lower affinity (K(D2)) is missing. (3) The binding site with the higher affinity (K(D1)) is missing. (4) No specific binding is observed. Ponasterone A binding can be rescued by addition of EcR but not by USP. (5) Ligand specificity is altered. RH 5992 can not compete [(3)H]-ponasterone A as efficient as in wild-type cells.

The elimination of chromatin or whole chromosomes from the future somatic nuclei during germ line-soma differentiation in early embryogenesis is a genetic phenomenon found in a wide variety of animal species. Less is known about the origin, structure, and function of the germ line-limited chromosomes. In the chironomid Acricotopus lucidus fluorescence in situ hybridization (FISH) with labeled soma DNA to "Keimbahn" chromosomes (Ks) and soma chromosomes (Ss) of spermatogonial mitoses revealed that each of the nine different K types possesses large S-homologous sections, mostly in the distal parts of both chromosome arms. Painting probes of the three Ss and of each of their chromosome arms were generated by microdissection of polytene salivary gland chromosomes and subsequent amplification by the degenerate oligonucleotide-primed polymerase chain reaction. Multicolor FISH demonstrated that each of the Ks, with the exception of one K type, was painted by only one of the three S probes. Furthermore, in seven Ks, one chromosome arm was painted by the long-arm probe and the other by the short-arm probe of the S concerned. The hybridization pattern strongly suggests that each of these K types is derived from a specific S. One function of the S-homologous K sections is thought to be determination of the regular occurrence of crossover events, with the resulting chiasmata in these sections ensuring correct segregation of the K homologs during meiosis. Reverse chromosome painting on polytene S sets with a probe generated from metaphase Ks corroborates the above results and produces conclusive evidence for the hypothesis that during evolution the Ks have developed from the Ss by endopolyploidization and rearrangements followed by the accumulation of germ line-specific repetitive DNA sequences in the centromeric regions.

Specific premessenger ribonucleoprotein (pre-mRNP) particles, the Balbiani ring (BR) granules in the salivary glands of the dipteran Chironomus tentans, can be visualized in the electron microscope when they assemble on the genes, move through nucleoplasm, and bind to and translocate through the nuclear pores. As shown by BrUTP labeling and immunoelectron microscopy, newly synthesized BR RNP particles, released from the BR genes, appear early in all nucleoplasmic regions of the cell nucleus and they saturate the nucleoplasmic pool of BR particles after 2 h of labelling. It is concluded that within the nucleus the BR particles move randomly. Furthermore, estimates of minimum diffusion coefficients for the BR particles are compatible with the view that the particles diffuse freely in the interchromosomal space, although it is not excluded that the random movement could be slightly retarded. Once the particles get bound to the nuclear pore complexes, they seem committed to translocation through the nuclear pores., (Copyright 1999 Academic Press.)

The morphogenetic changes in an epithelial cell line from Chironomus tentans that are evoked by molting hormones and molting hormone agonists are accompanied by transient changes in the concentration of actin and beta-tubulin protein and mRNA. As compared to controls, actin protein and mRNA concentrations increase by about 50%, whereas tubulin reaches maxima of 100% increase. The proportion between globular and filamentous actin remains constant after hormone treatment.

DNA-binding features of EcR and USP were investigated using a 0.4 M NaCl extract of the epithelial cell line of Chironomus tentans by means of electrophoretic mobility shift assays (EMSAs). It is shown that the DNA-binding is enhanced by hormone administration and that in the hormone dependent shift, both EcR and USP, are present. Furthermore, we demonstrate that under these conditions, EcR/USP form a unique complex on inverted repeat elements (PAL1 and hsp27-EcRE), while on direct repeat elements (DR1-5), a second complex with higher mobility is formed. In this second complex, neither EcR nor USP are present. Thus, an additional difference between PAL1 and DR-elements is the competition of other factors for DR-elements, modulating its function as an EcRE. A competition EMSA, using PAL1 as radiolabeled probe, reveals the following order of binding strength: PAL1>DR4/5>DR1>DR2/3/hsp27. Surprisingly, using DR1 as radiolabeled probe, shows a different order of binding strength: DR1>DR2>DR3/4/5/PAL1>hsp27. This indicates that the complexes formed on PAL1 are not identical to the ones formed on DR1 and that both are not easily convertible. Furthermore, the affinity of the EcR/USP complex may be altered under various conditions or by interaction with cofactors. Upon hormone administration, DNA binding of the receptor complex is enhanced, but the difference to hormone-free binding reactions decreases in course of time, indicating an additional hormone independent activation. Arch., (Copyright 1999 Wiley-Liss, Inc.)

Pole cell formation in embryos of the parthenogenetic midge, Smittia sp., can be delayed or inhibited by irradiation of the posterior egg pole with ultraviolet light (uv). This leaves the schedule of nuclear divisions and chromosome eliminations virtually unaffected. However, uv irradiation delays the precocious migration to the posterior pole of one nucleus, which normally becomes included in the first pole cell. This effect is photoreversible, i.e., mitigated by application of blue light after uv. Photoreversibility indicates that a nucleic acid component is involved as an effective target. During normal development of Smittia a number of chromosomes are eliminated during mitosis V, not only from somatic nuclei but also in the germ line. In the latter, this mitosis takes place during the first gonial division in the larva. After uv irradiation, the first pole cell nucleus has undergone supernumerary mitoses before pole cell formation and, as a result, is driven into mitosis V precociously as the pole cell divides. This is frequently associated with chromosome elimination from pole cells, which in turn is correlated with subsequent disappearance of already formed pole cells. Adults derived from embryos without pole cells do not form ovaries. Pole cell formation, pole cell preservation, and ovary development are separately inhibited by uv, and inhibition of each step is photoreversible. The results are discussed in the context of germ cell determination, protection against chromosome elimination, and the role of chromosomes limited to the germ line.

Treatment of Chironomus polytene chromosomes with the ultimate carcinogen benzo[a]pyrene diol epoxide I or in vivo administration of the parent hydrocarbon to larvae indicates that the carcinogen interacts with the genome in a nonrandom manner. Visualization of the carcinogen-DNA binding sites by immunofluorescence reveals that, in vivo, some sites are preferentially modified. The combined effects of DNA sequence, chromatin structure, and gene localization may lead to selective targeting of carcinogens to specific genomic regions. In polytene chromosomes the targeting effect is amplified, thereby making these chromosomes a uniquely suitable system for visualizing and studying site-specific interactions of carcinogens with the genome.

Rapid morphological changes were observed in some cells of hand-isolated salivary glands of Ch. thummi larvae. The nuclear envelope, routinely closely fitting the tightly packaged polytene chromosomes, was seen to lose its contact with the chromosomes and to attain a smooth round shape. Then unfolding of the chromosomes occurred, their banding patterns becoming clearly evident, probably through widening the interband regions; the chromosome length increased by about 20%. We argue that the changes observed were induced during gland isolation by lesions of the cell basal envelope in the sites of the fat body connections to the salivary gland.