Antibodies against the D-domain of a Chironomus ecdysone receptor protein react with DNA puff sites in Trichosia pubescens

Abstract

An antiserum (called AScE/D) against the semiconserved D-domain of a Chironomus tentans ecdysone receptor protein (cEcR) gave indirect immunofluorescence signals at DNA puff sites in Trichosia pubescens. The signals varied in maximum intensity at different DNA puff sites. Control experiments using the secondary rhodamine-labeled anti-rabbit IgG alone, preimmune serum, affinity purified AScE/D (called pABcE/D) and AScE/D preabsorbed with expressing bacterial extract or highly purified bacterially expressed cEcR indicated that the signals obtained at these chromosomal sites were likely to be due to specific interaction between an endogenous sciarid EcR and antibodies against cEcR. This conclusion was supported by observation of signals at certain Ec-inducible primary RNA puff sites. AScE/D signals began to appear at DNA puff sites during L3, the stage when amplification initiates, but at most sites their mean intensity was low and not statistically significant. Sites with AScE/D signals of significant mean intensity at this stage already showed evidence of transcription. The number and strength of transcription signals increased during L4. Comparison of the developmental course of signals for AScE/D, DNA synthesis, RNA presence/synthesis, and puff size for several DNA puffs during late larval- prepupal development showed a closer relationship of AScE/D signals with the initiation of RNA synthesis than with the initiation of DNA synthesis. Therefore, although we cannot absolutely eliminate a direct involvement of EcR in the amplification process at some sites, this investigation gives stronger support for its direct involvement in transcription. Since AScE/D signals are observed at DNA puff sites from the time the latter begin amplification/transcription through their regression, it appears that Ec and EcR are necessary as a sustained stimulus at these regions.

Maintenance of the DNA puff expanded state is independent of active replication and transcription

“…Decondensation of chromatin to form sciarid DNA puffs is induced by ecdysone (Crouse 1968;Stocker and Pavan 1974;Berendes and Lara 1975;Fresquez 1979;Ferreira and Amabis 1980;Amabis and Amabis 1984a, b;Stocker et al 1984;Dessen and Perondini 1985;Alvarenga et al 1991) and correlates with occupancy by the ecdysone receptor at these loci (Stocker et al 1997). In normal development of late Sciara larvae, DNA amplification initiates (stage 10×5) before the DNA puffs are evident, but DNA synthesis continues (stage 12×6) when these puffs begin to form (Wu et al 1993).…”

“…Like puffs of other Diptera, they are also sites of intense RNA synthesis (Gabrusewycz-Garcia and Kleinfeld 1966), but unlike the Drosophila RNA puffs or Chironomus Balbiani rings (Rudkin 1955;Hägele 1970;Rasch 1970b), there are about four rounds of "extra" DNA replication, resulting in DNA amplification at the DNA puff loci with respect to the rest of the genome, as suggested by Feulgen staining (Breuer and Pavan 1955) and [ 3 H]thymidine uptake (Ficq and Pavan 1957) and proven by microspectrophotometry (Rudkin and Corlette 1957;Swift 1962;Crouse and Keyl 1968;Rasch 1970a) and molecular hybridization (Glover et al 1982;Paçó-Larson et al 1992;Wu et al 1993). DNA puff amplification provides for additional DNA templates for the burst of mRNA synthesis that occurs shortly thereafter at the DNA puffs (Santelli et al 1991;Paçó-Larson et al 1992;Wu et al 1993;Stocker et al 1997), which encode polypeptides to be secreted in the saliva to form the pupal case (Phillips and Swift 1965;Winter et al 1977a, b;deToledo and Lara 1978;Ferreira and Amabis 1983;Laicine et al 1984).…”

Developmental regulation of BhSGAMP‐1, a gene encoding an antimicrobial peptide in the salivary glands of Bradysia hygida (Diptera, Sciaridae)

“…The S1 regions of the salivary glands were removed from the larvae at age E7, when the first group of DNA puffs is active, and were fixed/squashed according to Stocker et al (1997). In situ hybridization was performed using digoxigenin-labeled probes, as described in the protocol of the Berkeley Drosophila Genome Project (http://www.fruitfly.org/about/methods/cytogenetics.html).…”

Indirect immune detection of ecdysone receptor (EcR) during the formation of DNA puffs in Bradysia hygida (Diptera, Sciaridae)

“…The primary antibody (antiBhEcR) was recognized using goat anti-rabbit secondary antibody conjugated with alkaline phosphatase, followed by detection with NBT/BCIP. Immunofluorescence confocal microscopy B. hygida salivary gland S1 regions, from larvae of various ages, were dissected in saline and squashed (Stocker et al 1997). Chromosome preparations were treated for 2 h with blocking solution (PBS, 0.1% Tween 20 and 2% BSA), followed by incubation overnight with anti-BhEcR (1:10) and monoclonal anti-RNA polymerase II (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:100) in blocking solution.…”

“…The finding that the two groups of DNA puffs are induced by different levels of 20E makes B. hygida an interesting model to understand the role of this hormone and its receptors on both processes: gene amplification and the transcriptional control of the amplified genes. The only known study, in which EcR was immunologically detected in polytene chromosomes of sciarid (T. pubescens), was carefully made by Stocker et al (1997). In T. pubescens, unlike B. hygida, the processes of gene amplification and DNA puff activity demand a constant high level of 20E .…”