Expression of an eclosion hormone gene in insect cells using baculovirus vectors

Eldridge, Russ; Horodyski, Frank M.; Morton, David B.; O’Reilly, David R.; Truman, James W.; Riddiford, Lynn M.; Miller, Lois K.

doi:10.1016/0020-1790(91)90025-a

Cited by 39 publications

(10 citation statements)

References 34 publications

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“…6C). Preincubation of the antiserum with recombinant Manduca EH [45] abolishes the immunostaining in Drosophila (data not shown), supporting the conclusion that the antibody detects the Drosophila homologue of Manduca EH.…”

Section: Expression Of the Eh Genesupporting

confidence: 69%

Isolation, characterization and expression of the eclosion hormone gene of Drosophila melanogaster

Horodyski

Ewer

Riddiford

et al. 1993

European Journal of Biochemistry

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Eclosion hormone (EH) is a neuropeptide that triggers the performance of ecdysis behaviors at the end of a molt. We have isolated the EH gene from Drosophila melanogasfer, and localized the gene to the right arm of chromosome 3 at band position 90B1-2. The 97-amino-acid translation product contains a signal peptide followed by a 73-amino-acid prohormone. The N-terminus of the prohormone has diverged from lepidopteran EH both in its length and amino acid composition, and contains a potential endoproteolytic cleavage site. The deduced sequence of Drosophila EH is 58% identical (36 of 62 amino acids) to that of Manduca EH. The EH gene is expressed as a 0.8-kb transcript in a single pair of brain neurons which extend their processes the entire length of the central nervous system and also to the corpora cardiaca portion of the ring gland. These cells show massive depletion of immunoreactive EH at ecdysis.Over the last 10 years, work in invertebrates in general and insects in particular has led to identification of a large number of neuropeptides, many of which are expressed in a small number of cells within the central nervous system (CNS) [l, 21. These findings raise a number of important questions such as the role of these peptides in the normal physiology and development of the organism and the mechanisms that restrict peptide expression to a select set of cells within the CNS. To answer both questions, Drosophila melunogaster provides a number of distinct advantages. Mutants in a desired gene can be generated with relative ease and P-element transformation technology allows examination of regulatory regions that control peptide expression [3, 41. Consequently, we have turned to Drosophila to further our analysis of the neuropeptide eclosion hormone (EH), that triggers ecdysis of insects [5].Eclosion hormone was first found in moths [6] and later shown to be a 62-amino-acid peptide [7 -91 produced by two pairs of ventromedial neurosecretory cells in the brain [lO-131. In moths it has marked behavioral and developmental actions. It is known to act directly on the CNS to release the stereotyped motor programs that bring about the shedding of the old cuticle at the end of each molt [14]. Circulating peptide also acts on peripheral tissues to cause a diverse set of effects such as an increase in plasticity of wing cuticle [15], discharge of secretion by dermal glands [16], and the programmed degeneration of ecdysial muscles [17]. Yet the full range of EH actions are not known because it is not possible to block selectively either release or action of EH in moths. It appeared feasible to study EH in Drosophila because CNS extracts of both Calliphora [5] and Drosophila (Kimura and Truman, unpublished data) show EH activity in moth bioassays. Moreover, blood from ecdysing fleshflies, Sarcophaga bullata, stimulates premature ecdysis behavior in pharate (before ecdysis) flies [18]. The recent cloning of the EH gene from two species of moths, Munduca sexfa [ll] and Bombyx mori [13], has provided an avenue to search for t...

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Section: Expression Of the Eh Genesupporting

confidence: 69%

Isolation, characterization and expression of the eclosion hormone gene of Drosophila melanogaster

Horodyski

Ewer

Riddiford

et al. 1993

European Journal of Biochemistry

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“…Injections of synthetic EH would aid in the disentangling of direct versus indirect ETH and EH actions. Unfortunately, synthetic Drosophila EH was not available, and synthetic Manduca EH (Eldridge et al, 1991) caused severe behavioral abnormalities and fatal physiological changes when tested on fly larvae (M. L. del Campo and J. Ewer, unpublished observation), so its use was not pursued further.…”

Section: Role Of Eh and Eth In Tracheal Air Fillingmentioning

confidence: 99%

Neuroendocrine Control of Larval Ecdysis Behavior inDrosophila: Complex Regulation by Partially Redundant Neuropeptides

Clark¹,

Campo²,

Ewer³

2004

J. Neurosci.

109

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To complete each molting cycle, insects display a stereotyped sequence of behaviors to shed the remains of the old cuticle. These behavioral routines, as well as other related physiological events, are critical for proper development and are under the control of several neuropeptides. Their correct deployment and concatenation depends on the complex actions and interactions among several peptide hormones: ecdysis triggering hormone (ETH), eclosion hormone (EH), and crustacean cardioactive peptide (CCAP). Numerous theories, some in conflict, have been proposed to define the functional hierarchies by which these regulatory factors operate. Here we use wild-type Drosophila and transgenic flies bearing targeted ablations of either EH or CCAP neurons, or ablations of both together, to reevaluate their roles. Consistent with findings in moths, our results suggest that EH and ETH affect the release of each other via a positive feedback, although ETH can also be released in the absence of EH. We show that EH and ETH both contribute to the air filling of the air ducts (trachea) of the next stage but that EH may play a primary role in this process. We present evidence that EH, whose actions have always been placed upstream of CCAP, may also regulate ecdysis independently of CCAP. Finally, we confirm that flies lacking EH neurons do not ecdyse prematurely when injected with ETH peptides. These findings are surprising and not easily explained by currently available hypotheses. We propose that important additional neuropeptides, and additional interactions between known regulators, contribute to the mechanisms underlying insect ecdysis behaviors.

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“…The peptide hormones include prothoracicotropic hormone (PTTH; Kawakami et al, 1990; Kataoka et al, 1991; Ishizaki and Suzuki, 1994; Sauman and Reppert, 1996), pre‐ecdysis and ecdysis‐triggering hormones (PETH and ETH 1996; Zitnan et al, 1996, 1999), eclosion hormone (EH; Marti et al, 1987; Kono et al, 1990; Kataoka et al, 1991), and crustacean cardioactive peptide (CCAP; Stangier et al, 1988). Additionally, antibodies against PTTH, EH, ETH, and CCAP have been developed, and the genes for them have been cloned (Nagasawa et al, 1984; Horodyski et al, 1989; Kawakami et al, 1990; Kono et al, 1990; Eldridge et al, 1991; Zitnan et al, 1999; Loi et al, 2001).…”

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confidence: 99%

Cellular localization of bursicon using antisera against partial peptide sequences of this insect cuticle‐sclerotizing neurohormone

Honegger

Market

Pierce

et al. 2002

J of Comparative Neurology

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Bursicon is the final neurohormone released at the end of the molting cycle. It triggers the sclerotization (tanning) of the insect cuticle. Until now, its existence has been verified only by bioassays. In an attempt to identify this important neurohormone, bursicon was purified from homogenates of 2,850 nerve cords of the cockroach Periplaneta americana by using high performance liquid chromatography technology and two-dimensional gel electrophoresis. Bursicon bioactivity was found in four distinct protein spots at approximately 30 kDa between pH 5.3 and 5.9. The protein of one of these spots at pH 5.7 was subsequently microsequenced, and five partial amino acid sequences were retrieved. Evidence is presented that two of these sequences are derived from bursicon. Antibodies raised against the two sequences labeled bursicon-containing neurons in the central nervous systems of P. americana. One of these antisera labeled bursicon-containing neurons in the crickets Teleogryllus commodus and Gryllus bimaculatus, and the moth Manduca sexta. A cluster of four bilaterally paired neurons in the brain of Drososphila melanogaster was also labeled. In addition, this antiserum detected three spots corresponding to bursicon in Western blots of two-dimensional gels. The 12-amino acid sequence detected by this antiserum, thus, seems to be conserved even among species that are distantly related.

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Expression of an eclosion hormone gene in insect cells using baculovirus vectors

Cited by 39 publications

References 34 publications

Isolation, characterization and expression of the eclosion hormone gene of Drosophila melanogaster

Isolation, characterization and expression of the eclosion hormone gene of Drosophila melanogaster

Neuroendocrine Control of Larval Ecdysis Behavior inDrosophila: Complex Regulation by Partially Redundant Neuropeptides

Cellular localization of bursicon using antisera against partial peptide sequences of this insect cuticle‐sclerotizing neurohormone

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