Functional characterization of the sciarid BhC4-1 core promoter in transgenic Drosophila

Abstract

BackgroundCore promoters are cis-regulatory modules to which bind the basal transcriptional machinery and which participate in the regulation of transcription initiation. Although core promoters have not been extensively investigated through functional assays in a chromosomal context, the available data suggested that the response of a given core promoter might vary depending on the promoter context. Previous studies suggest that a (-57/+40) fragment constitutes the core promoter of the BhC4-1 gene which is located in DNA puff C4 of the sciarid fly Bradysia hygida. Here we tested this (-57/+40) fragment in distinct regulatory contexts in order to verify if promoter context affects its core promoter activity.ResultsConsistent with the activity of a core promoter, we showed that in the absence of upstream regulatory sequences the (-57/+40) fragment drives low levels of reporter gene mRNA expression throughout development in transgenic Drosophila. By assaying the (-57/+40) fragment in two distinct regulatory contexts, either downstream of the previously characterized Fbp1 enhancer or downstream of the UAS element, we showed that the BhC4-1 core promoter drives regulated transcription in both the germline and in various tissues throughout development. Furthermore, the use of the BhC4-1 core promoter in a UAS construct significantly reduced salivary gland ectopic expression in third instar larvae, which was previously described to occur in the context of the GAL4/UAS system.ConclusionsOur results from functional analysis in transgenic Drosophila show that the BhC4-1 core promoter drives gene expression regardless of the promoter context that was assayed. New insights into the functioning of the GAL4/UAS system in Drosophila were obtained, indicating that the presence of the SV40 sequence in the 3' UTR of a UAS construct does not preclude expression in the germline. Furthermore, our analysis indicated that ectopic salivary gland expression in the GAL4/UAS system does not depend only on sequences present in the GAL4 construct, but can also be affected by the core promoter sequences in the UAS construct. In this context, we propose that the sciarid BhC4-1 core promoter constitutes a valuable core promoter which can be employed in functional assays in insects.

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

“…The BhC4-1 core promoter is located in a 97 bp (À57/+40) fragment and a salivary gland cis-regulatory module that is sufficient to promote BhC4-1-lacZ developmentally regulated expression in the salivary gland is located in a 129 bp (À186/À58) fragment (Garcia et al, 2011;Lecci et al, 2008;Monesi et al, 2003). These functional studies also identified a 67 bp (À253/ À187) cis-regulatory module that promotes BhC4-1-lacZ expression in the ring gland of transgenic animals (Lecci et al, 2008).…”

Chromosomal localization and partial sequencing of the 18S and 28S ribosomal genes from Bradysia hygida (Diptera: Sciaridae)

“…Bradysia impatiens has been reported to be an economically problematic species in Florida (Mead, 1978), Bradysia species are known major insect pests in greenhouses (Meers and Cloyd, 2005), and Bradysia odoriphaga Yang and Zhang is the most serious pest of Chinese chive (Li et al, 2007). Bradysia hygida, first described by Sauaia and Alves (1968), is a sciarid that has been extensively used as a model system to study developmentally regulated gene transcription and amplification (Laicine et al 1984;Monesi et al 1995;Fiorini et al, 2001;Basso Jr et al, 2002;Candido-Silva and Monesi, 2010;Garcia et al, 2011;Passos et al, 2012). In this sciarid, the 5S ribosomal gene is localized in the autosomal A chromosome.…”

Hemocyte-specific Promoter for the Development of Transgenic Silkworm, Bombyx mori

“…One of the most important events in the control of gene expression is the regulation of transcription initiation (Garcia et al, 2011). The major components of the eukaryotic core promoters are composed of several sequence motifs such as the initiator, TATA box, TFIIB recognition element, downstream core promoter element, motif ten element and downstream core element (Smale and Kadonaga 2003;Juven-Gershon et al, 2008;JuvenGershon and Kadonaga 2010;Garcia et al, 2011).…”