Ecdysteroid-Responsive Genes in aDrosophilaCell Line (original) (raw)
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Ecdysteroid-inducible polypeptides in a Drosophila cell line
Cell, 1980
In the Drosophila melanogaster cell line Kc-H, ecdysteroid hormone treatment causes increased relative synthesis of three ecdysteroid-inducible polypeptides (EIPs), named according to their molecular weights (in kilodaltons) EIP 40, EIP 29 and EIP 28. Increased synthesis of the ElPs is detectable within 45 min (EIP 28) or 75 min (EIPs 40 and 29), is maximal at 4-8 hr and continues for almost 2 days. During this period no other major changes in protein synthesis are discernible using one-dimensional gels. At maximum, EIP 28 synthesis is elevated at least 10 fold above its basal level, and ElPs 40 and 29 somewhat less. EIP induction is ecdysteroidspecific and is detectable in the presence of lo-' M 20-hydroxyecdysone. It does not occur in hormone-resistant cells. Apparently identical polypeptides are inducible in another ecdysteroid-responsive cell line, Schneider's line 3. Because EIP synthesis is an early and substantial response to ecdysteroids, this is a promising system for the study of steroid hormone action.
Isolation and chromosomal localization of ecdysterone-responsive genes in a Drosophila cell line
Chromosoma, 1984
Cultured Kc 0% cells of Drosophila melanogaster are responsive to ecdysterone treatment. A library of lambda phages carrying segments of Drosophila genomic DNA was differential screened using poly(A)--RNAs from control and ecdysterone-treated cells. Nine independent recombinant phages that hybridized more intensely with poly(A)+RNA from treated cells and six that hybridized most strongly with poly(A) + RNA from untreated cells were selected. Genomic localization of these "inducible" and "repressible" sequences was determined by hybridization in situ. These results suggest that expression of several unique genes is increased by the hormone. The six "repressible" sequences each contained DNA that hybridized to multiple chromosomal sites and appeared to be mobile elements, suggesting that the steroid hormone might be acting on the transposable elements. These probes will be useful for the study of positive and negative steroid regulation within the same cell.
Insect Biochemistry and Molecular Biology, 1993
We have cloned a cDNA sequence coding for a Chironomus tentans steroid hormone receptor homologue which exhibits extensive amino acid sequence eo-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 (7. tentans protein into D. melanogaster Schneider's line 2 cells leads to transcriptional interference with endogenous dEcR on an ecdysteroid-regulated promoter.
Insect Biochemistry and Molecular Biology, 2004
Inducible expression systems have proven to be of major interest when analysing the function of specific genes or when expressing cytotoxic proteins. In an effort to develop inducible switches allowing for flexible fine-tuning of gene expression levels in insect cells, we have compared the induction capacities of two Drosophila minimal promoters when linked to four consecutive ecdysone response elements. These minimal promoters, either containing a TATA-box or a downstream promoter element, drove the expression of a luciferase reporter gene. Potent induction capacities were observed with the insect moulting hormone, 20-hydroxyecdysone, and with ponasterone A, a plant ecdysteroid. The developed inducible switches further expand the repertoire of molecular tools for functional expression of proteins of interest in insect cells. In addition, the combination of an ecdysone switch with promoters that possess different structural elements can provide novel insights into ecdysteroid-induced transcription in an insect cell line. #
Ecdysteroid receptors in Drosophila melanogaster adult females
Molecular and Cellular Endocrinology, 1989
Ecdysteroid receptors were identified and partially characterized from total cell extracts of whole animals and dissected tissues from Drosophila melunogaster adult females. Binding studies indicated the presence of two ecdysteroid binding components having high affinity and specificity consistent with receptors previously identified in embryos and larvae. The highest affinity binding component in 3-to 4-day females had a dissociation constant of 9.2 x lo-lo M and a maximal binding concentration of approximately 90 pmol/g protein, with a lower affinity component having a dissociation constant of 2.94 X 10e9 M. Receptors at similar concentrations were also observed in abdominal walls containing adult fat body, with relatively lower receptor levels observed in ovaries. These results indicate that the observed ecdysteroid hormone concentrations in adult females can account for a physiological stimulator-y effect on yolk protein synthesis in adult fat body.
Demonstration of an ecdysteroid receptor in a cultured cell line of Drosophila melanogaster
Proceedings of the National Academy of Sciences, 1978
ABSTRACr Incubation of the high-speed supernatant from the KC cell line of Drosophila melanogaster with [3lJponasterone A results in significant binding of the ligand as determined by gel filtration and dextran-coated charcoal binding assays. In vivo exposure of the KC line to [3Hjponasterone A for 30 min results in a marked binding of the ligand by a KCI-soluble nuclear extract. With both the cytosol and nuclear preparations the binding has specificity and a low dissociation constant (3 X 10-9 M). In addition the labeling of the nuclear preparation exhibits a saturation at approximately 7 X 10-10 M which probably reflects the molar concentration of cytoplasmic receptors.
Specific transcriptional responses to juvenile hormone and ecdysone in Drosophila
Insect Biochemistry and Molecular Biology, 2007
Previous studies have shown that ecdysone (E), and its immediate downstream product 20-hydroxyecdysone (20E), can have different biological functions in insects, suggesting that E acts as a distinct hormone. Here, we use Drosophila larval organ culture in combination with microarray technology to identify genes that are transcriptionally regulated by E, but which show little or no response to 20E. These genes are coordinately expressed for a brief temporal interval at the onset of metamorphosis, suggesting that E acts together with 20E to direct puparium formation. We also show that E74B, pepck, and CG14949 can be induced by juvenile hormone III (JH III) in organ culture, and that CG14949 can be induced by JH independently of protein synthesis. In contrast, E74A and E75A show no response to JH in this system. These studies demonstrate that larval organ culture can be used to identify Drosophila genes that are regulated by hormones other than 20E, and provide a basis for studying crosstalk between multiple hormone signaling pathways. r
Transcriptional Regulators of Ecdysteroid Biosynthetic Enzymes and Their Roles in Insect Development
Frontiers in Physiology, 2022
Steroid hormones are responsible for coordinating many aspects of biological processes in most multicellular organisms, including insects. Ecdysteroid, the principal insect steroid hormone, is biosynthesized from dietary cholesterol or plant sterols. In the last 20 years, a number of ecdysteroidogenic enzymes, including Noppera-bo, Neverland, Shroud, Spook/Spookier, Cyp6t3, Phantom, Disembodied, Shadow, and Shade, have been identified and characterized in molecular genetic studies using the fruit fly Drosophila melanogaster. These enzymes are encoded by genes collectively called the Halloween genes. The transcriptional regulatory network, governed by multiple regulators of transcription, chromatin remodeling, and endoreplication, has been shown to be essential for the spatiotemporal expression control of Halloween genes in D. melanogaster. In this review, we summarize the latest information on transcriptional regulators that are crucial for controlling the expression of ecdysteroid ...
cDNA clones for the ecdysone-inducible polypeptide (EIP) mRNAs of Drosophila Kc cells
The EMBO journal, 1984
The ecdysone-inducible polypeptides (EIPs) 28, 29 and 40 were identified previously as polypeptides whose synthesis is stimulated early in the ecdysone response of Drosophila Kc cells. We have now shown, using two-dimensional gels, that each of these EIPs consists of three species differing in pI, and all stimulated by ecdysone. Translations and hybrid-arrested translations indicated that the poly(A) EIP mRNAs increase 10-fold in abundance during the first 4 h of ecdysone treatment. By a differential screen of a cDNA library we have identified cDNA clones corresponding to all three EIPs. Two kinds of clones were isolated: one hybridizes to the EIP 40 mRNA(s); the second hybridizes to the mRNA(s) encoding all the EIPs 28 and 29. The EIP 28/29 and EIP 40 loci detected by these clones are each present at single sites on the polytene chromosomes and each is at or in the vicinity of an ecdysone-regulated puff.