Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila

Abstract

Regulation of DNA replication is critical, and loss of control can lead to DNA amplification. Naturally occurring, developmentally regulated DNA amplification occurs in the DNA puffs of the late larval salivary gland giant polytene chromosomes in the fungus fly, Sciara coprophila. The steroid hormone ecdysone induces DNA amplification in Sciara, and the amplification origin of DNA puff II/9A contains a putative binding site for the ecdysone receptor (EcR). We report here the isolation, cloning, and characterizing of two ecdysone receptor isoforms in Sciara (ScEcR-A and ScEcR-B) and the heterodimeric partner, ultraspiracle (ScUSP). ScEcR-A is the predominant isoform in larval tissues and ScEcR-B in adult tissues, contrary to the pattern in Drosophila. Moreover, ScEcR-A is produced at amplification but is absent just prior. We discuss these results in relation to the model of ecdysone regulation of DNA amplification.

Amplification and expression of a salivary gland DNA puff gene in the prothoracic gland of Bradysia hygida (Diptera: Sciaridae)

“…Several studies identify the hormone 20E as a regulator of DNA replication and transcription in DNA puff forming sites (Alvarenga et al, 1991;Amabis and Amabis, 1984a,b;Basso et al, 2002;Candido-Silva et al, 2008;Foulk et al, 2006Foulk et al, , 2013Penalva et al, 1997). Specifically in B. hygida, BhC4-1 transcription and amplification are detected 16 and 24 h after 20E injection, respectively, which might suggest that these processes are regulated by different sets of as yet uncharacterized factors that act together with the hormone (Basso et al, 2002).…”

The ecdysone receptor (ScEcR-A) binds DNA puffs at the start of DNA amplification in Sciara coprophila

“…ScEcR-A is isoform A of EcR, and it predominates in the salivary glands of fourth larval instar S. coprophila (Foulk et al 2013). A polyclonal antibody to this isoform was produced against the unique ScEcR-A NH 2 -terminus as described (Foulk et al 2013).…”

“…A polyclonal antibody to this isoform was produced against the unique ScEcR-A NH 2 -terminus as described (Foulk et al 2013). Alexa Fluor 488 dye-conjugated goat anti-rabbit IgG was obtained from Molecular Probes (Invitrogen, Eugene, OR, USA).…”

“…EcREs are also present in the promoters of genes II/9-1 and II/9-2 (DiBartolomeis and Gerbi 1989; Bienz-Tadmor et al 1991). Isoform A of the ecdysone receptor (ScEcR-A) predominates in the salivary glands of fourth instar Sciara larvae (Foulk et al 2006, 2013). At the earliest forming DNA puffs, II/9A and II/2B, DNA amplification initiates just before morphological puffing becomes evident (stage 10×5), and replication continues through the onset of puff formation (stage 12×6) and plateaus when a burst of transcription results in maximal puff expansion (stage 14×7) (Wu et al 1993).…”

Genome Duplication

“…The genes encoding the proteins required for pupation are then amplified many fold. A larval-specific ecdysone receptor binds to a specific DNA sequence adjacent to the origin recognition complex-binding site within a site-specific origin of bidirectional replication (Foulk et al, 2013). Similarly, the follicle cells that envelop each oocyte in Drosophila amplify the four genes that produce the proteins for chorion (eggshell) formation.…”