Rbf/E2F1 control growth and endoreplication via steroid-independent Ecdysone Receptor signalling in Drosophila prostate-like secondary cells.

In prostate cancer, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 typically occurs at a late-stage of tumour progression. It appears to regulate a switch to an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that upon mating, binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG), which share some similarities with prostate epithelial cells, switch their growth regulation from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control. This physiological change induces genome endoreplication and allows SCs to rapidly replenish their secretory compartments, even when ecdysone levels are low because the male has not previously been exposed to females. Here, we test whether the Drosophila Rb homologue, Rbf, and E2F1 regulate this switch. Surprisingly, we find that excess Rbf activity reversibly suppresses binucleation in adult SCs. We also demonstrate that Rbf, E2F1 and the cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key regulators of mating-dependent SC endoreplication, as well as SC growth in both virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and endoreplication-associated growth in SCs, mirroring changes seen in CRPC. Furthermore, Bone Morphogenetic Protein (BMP) signalling, mediated by the BMP ligand Decapentaplegic (Dpp), intersects with CycD/Rbf/E2F1 signalling to drive endoreplication in these fly cells. Overall, our work reveals a signalling switch, which permits rapid growth of SCs and increased secretion after mating, independently of previous exposure to females. The changes observed share mechanistic parallels with the pathological switch to hormone-independent AR signalling seen in CRPC, suggesting that the latter may reflect the dysregulation of a currently unidentified physiological process.

Epithelial cell-turnover ensures robust coordination of tissue growth in Drosophila ribosomal protein mutants.

Highly reproducible tissue development is achieved by robust, time-dependent coordination of cell proliferation and cell death. To study the mechanisms underlying robust tissue growth, we analyzed the developmental process of wing imaginal discs in Drosophila Minute mutants, a series of heterozygous mutants for a ribosomal protein gene. Minute animals show significant developmental delay during the larval period but develop into essentially normal flies, suggesting there exists a mechanism ensuring robust tissue growth during abnormally prolonged developmental time. Surprisingly, we found that both cell death and compensatory cell proliferation were dramatically increased in developing wing pouches of Minute animals. Blocking the cell-turnover by inhibiting cell death resulted in morphological defects, indicating the essential role of cell-turnover in Minute wing morphogenesis. Our analyses showed that Minute wing discs elevate Wg expression and JNK-mediated Dilp8 expression that causes developmental delay, both of which are necessary for the induction of cell-turnover. Furthermore, forced increase in Wg expression together with developmental delay caused by ecdysone depletion induced cell-turnover in the wing pouches of non-Minute animals. Our findings suggest a novel paradigm for robust coordination of tissue growth by cell-turnover, which is induced when developmental time axis is distorted.

Wing patterning in faster developing Drosophila is associated with high ecdysone titer and wingless expression.

'Developmental robustness' is the ability of biological systems to maintain a stable phenotype despite genetic, environmental or physiological perturbations. In holometabolous insects, accurate patterning and development is guaranteed by alignment of final gene expression patterns in tissues at specific developmental stage such as molting and pupariation, irrespective of individual rate of development. In the present study, we used faster developing Drosophila melanogaster populations that show reduction of ~22% in egg to adult development time. Flies from the faster developing population exhibit phenotype constancy, although significantly small in size. The reduction in development time in faster developing flies is possibly due to coordination between higher ecdysteroid release and higher expression of developmental genes. The two together might be ensuring appropriate pattern formation and early exit at each development stage in the populations selected for faster pre-adult development compared to their ancestral controls. We report that apart from plasticity in the rate of pattern progression, alteration in the level of gene expression may be responsible for pattern integrity even under reduced development time.

Developmental regulation of regenerative potential in Drosophila by ecdysone through a bistable loop of ZBTB transcription factors.

In many organisms, the regenerative capacity of tissues progressively decreases as development progresses. However, the developmental mechanisms that restrict regenerative potential remain unclear. In Drosophila, wing imaginal discs become unable to regenerate upon damage during the third larval stage (L3). Here, we show that production of ecdysone after larvae reach their critical weight (CW) terminates the window of regenerative potential by acting on a bistable loop composed of two antagonistic Broad-complex/Tramtrack/Bric-à-brac Zinc-finger (ZBTB) genes: chinmo and broad (br). Around mid L3, ecdysone signaling silences chinmo and activates br to switch wing epithelial progenitors from a default self-renewing to a differentiation-prone state. Before mid L3, Chinmo promotes a strong regenerative response upon tissue damage. After mid L3, Br installs a nonpermissive state that represses regeneration. Transient down-regulation of ecdysone signaling or Br in late L3 larvae enhances chinmo expression in damaged cells that regain the capacity to regenerate. This work unveils a mechanism that ties the self-renewing and regenerative potential of epithelial progenitors to developmental progression.

Dual Roles of Glutathione in Ecdysone Biosynthesis and Antioxidant Function During Larval Development in Drosophila.

Ecdysteroids, including the biologically active hormone 20-hydroxyecdysone (20E), play essential roles in controlling many developmental and physiological events in insects. Ecdysteroid biosynthesis is achieved by a series of specialized enzymes encoded by the Halloween genes. Recently, a new class of Halloween gene, noppera-bo (nobo), encoding a glutathione S-transferase (GST) in dipteran and lepidopteran species, has been identified and characterized. GSTs are well known to conjugate substrates with the reduced form of glutathione (GSH), a bioactive tripeptide composed of glutamate, cysteine, and glycine. We hypothesized that GSH itself is required for ecdysteroid biosynthesis. However, the role of GSH in steroid hormone biosynthesis has not been examined in any organisms. Here, we report phenotypic analysis of a complete loss-of-function mutant in the γ-glutamylcysteine synthetase catalytic subunit (Gclc) gene in the fruit fly Drosophila melanogasterGclc encodes the evolutionarily conserved catalytic component of the enzyme that conjugates glutamate and cysteine in the GSH biosynthesis pathway. Complete Gclc loss-of-function leads to drastic GSH deficiency in the larval body fluid. Gclc mutant animals show a larval-arrest phenotype. Ecdysteroid titer in Gclc mutant larvae decreases, and the larval-arrest phenotype is rescued by oral administration of 20E or cholesterol. Moreover, Gclc mutant animals exhibit abnormal lipid deposition in the prothoracic gland, a steroidogenic organ during larval development. All of these phenotypes are reminiscent to nobo loss-of-function animals. On the other hand, Gclc mutant larvae also exhibit a significant reduction in antioxidant capacity. Consistent with this phenotype, Gclc mutant larvae are more sensitive to oxidative stress response as compared to wild-type. Nevertheless, the ecdysteroid biosynthesis defect in Gclc mutant animals is not associated with loss of antioxidant function. Our data raise the unexpected hypothesis that a primary role of GSH in early D. melanogaster larval development is ecdysteroid biosynthesis, independent from the antioxidant role of GSH.

Endocrine-related genes are altered by antibacterial agent triclosan in Chironomus riparius aquatic larvae.

Triclosan (TCS) is an antibacterial agent widely used in personal care and consumer products and commonly detected in aquatic ecosystems. In the present study, the effects of TCS on endocrine-related genes of Chironomus riparius aquatic larvae, a reference organism in aquatic toxicology, were evaluated. Twenty-four-hour in vivo exposures at 10µg/L, 100µg/L, and 1000µg/L TCS revealed that this xenobiotic was able to alter the transcriptional activity of ecdysone receptor gene (EcR), the ultraspiracle gene (usp), the estrogen-related receptor gene (ERR), and the E74 early ecdysone-inducible gene, as measured by real-time RT-PCR. Moreover, the hsp70 gene, a heat shock protein gene, was upregulated after exposure to TCS. The results of the present work provide the first evidence of the potential disruptive effects of TCS in endocrine-related genes suggesting a mode of action that mimics ecdysteroid hormones in insects.

Transcriptome analysis to identify genes for peptides and proteins involved in immunity and reproduction from male accessory glands and ejaculatory duct of Bactrocera dorsalis.

In the male reproductive system of insects, the male accessory glands and ejaculatory duct (MAG/ED) are important organs and their primary function is to enhance the fertility of spermatozoa. Proteins secreted by the MAG/ED are also known to induce post-mating changes and immunity responses in the female insect. To understand the gene expression profile in the MAG/ED of the oriental fruit fly Bactrocera dorsalis (Hendel), that is an important pest in fruits, we performed an Illumina-based deep sequencing of mRNA. This yielded 54,577,630 clean reads corresponding to 4.91Gb total nucleotides that were assembled and clustered to 30,669 unigenes (average 645bp). Among them, 20,419 unigenes were functionally annotated to known proteins/peptides in Gene Orthology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes pathway databases. Typically, many genes were involved in immunity and these included microbial recognition proteins and antimicrobial peptides. Subsequently, the inducible expression of these immunity-related genes was confirmed by qRT-PCR analysis when insects were challenged with immunity-inducible factors, suggesting their function in guaranteeing fertilization success. Besides, we identified some important reproductive genes such as juvenile hormone- and ecdysteroid-related genes in this de novo assembly. In conclusion, this transcriptomic sequencing of B. dorsalis MAG/ED provides insights to facilitate further functional research of reproduction, immunity and molecular evolution of reproductive proteins in this important agricultural pest.

Extensive transcriptional response associated with seasonal plasticity of butterfly wing patterns.

In the eastern United States, the buckeye butterfly, Junonia coenia, shows seasonal wing colour plasticity where adults emerging in the spring are tan, while those emerging in the autumn are dark red. This variation can be artificially induced in laboratory colonies, thus making J. coenia a useful model system to examine the mechanistic basis of plasticity. To better understand the developmental basis of seasonal plasticity, we used RNA-seq to quantify transcription profiles associated with development of alternative seasonal wing morphs. Depending on the developmental stage, between 547 and 1420 transfrags were significantly differentially expressed between morphs. These extensive differences in gene expression stand in contrast to the much smaller numbers of differentially expressed transcripts identified in previous studies of genetic wing pattern variation in other species and suggest that environmentally induced phenotypic shifts arise from very broad systemic processes. Analyses of candidate endocrine and pigmentation transcripts revealed notable genes upregulated in the red morph, including several ecdysone-associated genes, and cinnabar, an ommochrome pigmentation gene implicated in colour pattern variation in other butterflies. We also found multiple melanin-related transcripts strongly upregulated in the red morph, including tan and yellow-family genes, leading us to speculate that dark red pigmentation in autumn J. coenia may involve nonommochrome pigments. While we identified several endocrine and pigmentation genes as obvious candidates for seasonal colour morph differentiation, we speculate that the majority of observed expression differences were due to thermal stress response. The buckeye transcriptome provides a basis for further developmental studies of phenotypic plasticity.

The developmental control of size in insects.

The mechanisms that control the sizes of a body and its many parts remain among the great puzzles in developmental biology. Why do animals grow to a species-specific body size, and how is the relative growth of their body parts controlled to so they grow to the right size, and in the correct proportion with body size, giving an animal its species-characteristic shape? Control of size must involve mechanisms that somehow assess some aspect of size and are upstream of mechanisms that regulate growth. These mechanisms are now beginning to be understood in the insects, in particular in Manduca sexta and Drosophila melanogaster. The control of size requires control of the rate of growth and control of the cessation of growth. Growth is controlled by genetic and environmental factors. Insulin and ecdysone, their receptors, and intracellular signaling pathways are the principal genetic regulators of growth. The secretion of these growth hormones, in turn, is controlled by complex interactions of other endocrine and molecular mechanisms, by environmental factors such as nutrition, and by the physiological mechanisms that sense body size. Although the general mechanisms of growth regulation appear to be widely shared, the mechanisms that regulate final size can be quite diverse.

Unexpected role of the steroid-deficiency protein ecdysoneless in pre-mRNA splicing.

The steroid hormone ecdysone coordinates insect growth and development, directing the major postembryonic transition of forms, metamorphosis. The steroid-deficient ecdysoneless1 (ecd1) strain of Drosophila melanogaster has long served to assess the impact of ecdysone on gene regulation, morphogenesis, or reproduction. However, ecd also exerts cell-autonomous effects independently of the hormone, and mammalian Ecd homologs have been implicated in cell cycle regulation and cancer. Why the Drosophila ecd1 mutants lack ecdysone has not been resolved. Here, we show that in Drosophila cells, Ecd directly interacts with core components of the U5 snRNP spliceosomal complex, including the conserved Prp8 protein. In accord with a function in pre-mRNA splicing, Ecd and Prp8 are cell-autonomously required for survival of proliferating cells within the larval imaginal discs. In the steroidogenic prothoracic gland, loss of Ecd or Prp8 prevents splicing of a large intron from CYP307A2/spookier (spok) pre-mRNA, thus eliminating this essential ecdysone-biosynthetic enzyme and blocking the entry to metamorphosis. Human Ecd (hEcd) can substitute for its missing fly ortholog. When expressed in the Ecd-deficient prothoracic gland, hEcd re-establishes spok pre-mRNA splicing and protein expression, restoring ecdysone synthesis and normal development. Our work identifies Ecd as a novel pre-mRNA splicing factor whose function has been conserved in its human counterpart. Whether the role of mammalian Ecd in cancer involves pre-mRNA splicing remains to be discovered.

Molecular cloning and transcription expression of 3-dehydroecdysone 3α-reductase (3de 3α-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L.

Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, and reproduction etc. The decreased ecdysteroid in titre results from enhanced ecdysteroid inactivation reactions including the formation of 3-epiecdyson under ecdysone oxidase and 3-dehydroecdysone 3α-reductase (3DE 3α-reductase). In this paper, we cloned and characterized 3-dehydroecdysone 3α-reductase (3DE 3α-reductase) in different tissues and developing stage of the silkworm, Bombyx mori L. The B. mori 3DE 3α-reductase cDNA contains an ORF 783 bp and the deduced protein sequence containing 260 amino acid residues. Analysis showed the deduced 3DE 3α-reductase belongs to SDR family, which has the NAD(P)-binding domain. Using the Escherichia coli, a high level expression of a fusion polypeptide band of approx. 33 kDa was observed. High transcription of 3DE 3α-reductase was mainly presented in the midgut and hemolymph in the third day of fifth instar larvae in silkworm. The expression of 3DE 3α-reductase at different stages of larval showed that the activity in the early instar was high, and then reduced in late instar. This is parallel to the changes of molting hormone titer in larval. 3DE 3α-reductase is key enzyme in inactivation path of ecdysteroid. The data elucidate the regulation of 3DE 3α-reductase in ecdyteroid titer of its targeting organs and the relationship between the enzyme and metamorphosis.

Accessory gland as a site for prothoracicotropic hormone controlled ecdysone synthesis in adult male insects.

Insect steroid hormones (ecdysteroids) are important for female reproduction in many insect species and are required for the initiation and coordination of vital developmental processes. Ecdysteroids are also important for adult male physiology and behavior, but their exact function and site of synthesis remains unclear, although previous studies suggest that the reproductive system may be their source. We have examined expression profiles of the ecdysteroidogenic Halloween genes, during development and in adults of the flour beetle Tribolium castaneum. Genes required for the biosynthesis of ecdysone (E), the precursor of the molting hormone 20-hydroxyecdysone (20E), are expressed in the tubular accessory glands (TAGs) of adult males. In contrast, expression of the gene encoding the enzyme mediating 20E synthesis was detected in the ovaries of females. Further, Spookiest (Spot), an enzyme presumably required for endowing tissues with competence to produce ecdysteroids, is male specific and predominantly expressed in the TAGs. We also show that prothoracicotropic hormone (PTTH), a regulator of E synthesis during larval development, regulates ecdysteroid levels in the adult stage in Drosophila melanogaster and the gene for its receptor Torso seems to be expressed specifically in the accessory glands of males. The composite results suggest strongly that the accessory glands of adult male insects are the main source of E, but not 20E. The finding of a possible male-specific source of E raises the possibility that E and 20E have sex-specific roles analogous to the vertebrate sex steroids, where males produce primarily testosterone, the precursor of estradiol. Furthermore this study provides the first evidence that PTTH regulates ecdysteroid synthesis in the adult stage and could explain the original finding that some adult insects are a rich source of PTTH.

The steroid hormone ecdysone functions with intrinsic chromatin remodeling factors to control female germline stem cells in Drosophila.

Steroid hormones are known systemic regulators of multiple normal and cancerous tissues; however, whether or how they impact the fate and function of adult stem cells is unclear. In the Drosophila ovary, insulin signals modulate the proliferation and self-renewal of germline stem cells (GSCs), yet despite evidence that additional systemic factors control GSC activity, these have remained largely unknown. Here, we report that ecdysone, a steroid hormone structurally related to mammalian sex steroids, directly regulates adult GSC proliferation and self-renewal independently of insulin signaling. Ecdysone controls GSCs through a functional interaction with the chromatin remodeling factors ISWI, an intrinsic epigenetic factor required for GSC fate and activity, and Nurf301, the largest subunit of the ISWI-containing NURF chromatin remodeling complex. Our findings support a link between systemic steroid hormones and the intrinsic chromatin remodeling machinery as a potential mechanism to promote broad transcriptional programs required for adult stem cell self-renewal.

Bacterial, but not baculoviral infections stimulate Hemolin expression in noctuid moths.

Lepidopteran larvae are regularly infected by baculoviruses during feeding on infected plants. The differences in sensitivity to these infections can be substantial, even among closely related species. For example, the noctuids Cotton bollworm (Helicoverpa zea) and Tobacco budworm (Heliothis virescens), have a 1000-fold difference in sensitivity to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection. Recent data were interpreted to indicate that the lepidopteran immunoglobulin protein, Hemolin, is synthesized upon viral injection and therefore to participate in anti-viral responses. To investigate whether Hemolin synthesis is affected by a natural viral infection, specific transcription in fat bodies and hemocytes of H. zea and H. virescens larvae was monitored following per os infection with the baculovirus HzSNPV (H. zea single nucleopolyhedrovirus). Both moths showed the same expression pattern as seen in uninfected animals and coincided with ecdysone responses, previously known to induce Hemolin expression. In contrast, injection of lyophilized Micrococcus luteus resulted in increased Hemolin expression supporting a role for Hemolin as an immuno-responsive protein in these species. The combined data are consistent with the suggestion that while Hemolin seems to participate in the response to virus infection in the superfamily Bombycoidea, this is not true in the Noctuoidea.

A phosphoproteomics approach to elucidate neuropeptide signal transduction controlling insect metamorphosis.

In insects, the neuropeptide prothoracicotropic hormone (PTTH) stimulates production of ecdysone (E) in the prothoracic glands (PGs). E is the precursor of the principal steroid hormone, 20-hydroxyecdysone (20E), that is responsible for eliciting molting and metamorphosis. In this study, we used quantitative phosphoproteomics to investigate signal transduction events initiated by PTTH. We identified Spook (CYP307A1), a suspected rate-limiting enzyme for E biosynthesis, and components of the mitogen-activated protein kinase (MAPK) pathway, as major phosphorylation targets of PTTH signaling. Further, proteins not previously linked to PTTH and ecdysone biosynthesis were identified as targets of PTTH signaling. These include proteins involved in signal transduction, endosomal trafficking, constituents of the cytoskeleton and regulators of transcription and translation. Our screen shows that PTTH likely stimulates E production by activation of Spook, an integral enzyme in the E biosynthetic pathway. This directly connects PTTH signaling to the pathway that produces E. A new mechanism for regulation of E biosynthesis in insects is proposed.

Dynamic control of cell cycle and growth coupling by ecdysone, EGFR, and PI3K signaling in Drosophila histoblasts.

Regulation of cell proliferation has been extensively studied in cultured cell systems that are characterized by coordinated growth and cell-cycle progression and relatively uniform cell size distribution. During the development of multicellular organisms, however, growth and division can be temporally uncoupled, and the signaling pathways that regulate these growth programs are poorly understood. A good model for analyzing proliferation control in such systems is the morphogenesis of the Drosophila adult abdominal epidermis by histoblasts. These cells undergo a series of temporally regulated transitions during which neither cell size nor division rate is constant. The proliferation of histoblasts during metamorphosis is uniquely amenable to clonal analysis in combination with live imaging. Thereby, we show that abdominal histoblasts, which grow while in G2 arrest during larval stages, enter a proliferative stage in the pupal period that is initiated by ecdysone-dependent string/Cdc25 phosphatase transcription. The proliferating histoblasts have preaccumulated stores of Cyclin E, which trigger an immediate S phase onset after mitosis. These rapid cell cycles lack a G1 phase and result in a progressive reduction of cell size. Eventually, the histoblasts proceed to a stage of slower proliferation that, in contrast to the preceding, depends on epidermal growth factor receptor (EGFR) signaling for progression through the G2/M transition and on insulin receptor/PI3K-mediated signaling for growth. These results uncover the developmentally programmed changes coupling the growth and proliferation of the histoblasts that form the abdominal epidermis of Drosophila. Histoblasts proceed through three distinct stages: growth without division, division without growth, and growth-coupled proliferation. Our identification of the signaling pathways and cell-cycle regulators that control these programs illustrates the power of in vivo time-lapse analyses after clone induction. It sets the stage for the comprehensive understanding of the coordination of cell growth and cell-cycle progression in complex multicellular eukaryotes.

Characterization of the RSL1-dependent conditional expression system in LNCaP prostate cancer cells and development of a single vector format.

BACKGROUND: Conditional expression systems are useful tools for the study of gene function but the use of these systems in prostate cancer cells is limited by the undesired biological effects of the inducing ligands. The RheoSwitch system employs RheoSwitch Ligand 1 (RSL1), a non-steroidal analog of the insect hormone ecdysone, to activate a modified nuclear receptor heterodimer that controls target gene expression via GAL4 response elements. This system has not been tested in prostate cancer cells. METHODS: We established LNCaP human prostate cancer cell lines that constitutively express RheoSwitch transcription factors to quantify RSL1-dependent expression and assess the effects of RSL1 on cell proliferation and endogenous gene expression. Potential RSL1-responsive genes were identified using Affymetrix microarrays and validated by Northern blot hybridization. A single-vector format was developed to establish cell lines that conditionally produce a recombinant protein. RESULTS: Stable cell lines displayed tight and potent (over several orders of magnitude) RSL1-dependent regulation of a transiently transfected luciferase reporter gene. RSL1 did not affect basal or androgen-stimulated cell proliferation and exerted minimal effects on the expression of endogenous genes. Cell lines established using the single-vector system also displayed strictly RSL1-dependent production of the recombinant protein encoded by the stably integrated RSL1-responsive expression cassette. CONCLUSIONS: The RheoSwitch system is well suited for conditional gene expression in prostate cancer cells. The single-vector format should facilitate the production of stable cell lines. This system should be useful for the study of proteins involved in prostate cancer in both cell and animal models of the disease.

DNA binding and transactivation properties of the Schistosoma mansoni constitutive androstane receptor homologue.

SmCAR (Schistosoma mansoni constitutive androstane receptor) is a schistosome homologue of the CAR/PXR/VDR group of nuclear receptors. The P box sequence in the DNA binding domain (DBD) of SmCAR, which is essential in determining the DNA binding specificity of nuclear receptors, is different from its vertebrate homologues. Previous data demonstrates that SmCAR binds to a hormone response element containing a single half site AGTGCA as a monomer. SmRXR1 and SmRXR2 are two S. mansoni homologues of vertebrate retinoid X receptors (RXRs). RXRs usually heterodimerize with various nuclear receptors. Yeast-two hybrid analyses, in vitro pull-down and co-immunoprecipitation assays demonstrated that SmCAR interacts with SmRXR1 but not SmRXR2. Using chimeras consisting of the DBD of SmCAR and the ligand binding domain (LBD) of mouse (m) CAR, we show that despite a different P box, SmCAR DBD shares DNA binding specificity with mCAR. However, the SmCAR DBD does exhibit some of the DNA binding properties specific to SmCAR. Studies of the chimeras also demonstrated that the SmCAR DBD is able to heterodimerize with the DBD of human RXR, allowing high affinity DNA binding. Based on this study and previous results, we conclude that SmCAR may recognize its cognate hormone response element via two mechanisms: binding to DNA monomerically or heterodimerizing with SmRXR1. We also demonstrate that a transcription activation function-1 (AF-1) is located in the SmCAR A/B domain.

A molecular genetic approach to the biosynthesis of the insect steroid molting hormone.

Insect growth, development, and molting depend upon a critical titer of the principal molting hormone of arthropods, 20-hydroxyecdysone (20E). Although the structure of 20E as a polyhydroxylated steroid was determined more than five decades ago, the exact steps in its biosynthesis have eluded identification. Over the past several years, the use of the fly database and the techniques and paradigms of biochemistry, analytical chemistry, and molecular genetics have allowed the cloning and sequencing of four genes in the Halloween gene family of Drosophila melanogaster, all of them encoding cytochrome P450 (CYP) enzymes, each of which mediates one of the four terminal hydroxylation steps in 20E biosynthesis. Further, the sequence of these hydroxylations has been determined, and developmental alterations in the expression of each of these genes have been quantified during both embryonic and postembryonic life.

Comparative analysis of inducible expression systems in transient transfection studies.

Ectopic protein expression in mammalian cells is a valuable tool to analyze protein functions. Increasingly, inducible promoters are being used for regulated gene expression. Here, we compare expression maxima, induction rates, and "leakiness" of the following promoter systems: (I) two tetracycline-responsive Tet systems (Tet-On, Tet-Off), (II) the glucocorticoid-responsive mouse mammary tumor virus promoter (MMTVprom), (III) the ecdysone-inducible promoter (EcP), and (IV) the T7 promoter/T7 RNA polymerase system (T7P). The systems were analyzed by expressing an enhanced green fluorescent protein (EGFP) luciferase fusion reporter protein in transiently transfected cells. Expression was assessed qualitatively by fluorescence microscopy of the EGFP component and quantitatively by measuring the enzymatic activity of the luciferase component of the fusion protein. Basal expression levels ("leakiness") were ranked Tet-On>Tet-Off>MMTVprom>EcP>T7P. Induction rates were EcP>MMTVprom>T7P>Tet-Off>Tet-On. Expression maxima were ranked. Tet-On>Tet-Off>MMTVprom>EcP>T7P. To increase T7-promoter-mediated expression we inserted an internal ribosomal entry site element into the T7 expression cassette. In presence of T7 RNA polymerase this modified T7 promoter achieved expression levels of 42% of a Rous Sarcoma virus promoter, while keeping basal expression extremely low.