Bradysia hygida (Diptera, Sciaridae) presents two eukaryotic Elongation Factor 1A gene homologues: partial characterization of the eukaryotic Elongation Factor 1A-F1 gene

Abstract

Elongation factor 1A is a highly conserved protein that participates in translation. We report the occurrence of two genes homologous to the eukaryotic Elongation Factor 1A in Bradysia hygida and describe the partial cloning and characterization of the B. hygida eukaryotic Elongation Factor 1A-F1 (BheEF1A-F1) gene. The pattern of BheEF1A-F1 expression in the salivary gland at the end of the fourth larval instar was investigated using real-time PCR. The results showed that BheEF1A-F1 expression levels are relatively constant at the time when rapid changes in protein synthesis occur in this tissue. In situ hybridization experiments coupled to Southern blot analyses showed that the BheEF1A-F1 gene is located at position 3d of the A chromosome and a second gene homologous to eEF1A is located at position 6a of the X chromosome. Southern blot analyses showed that both the BheEF1A-F1 gene and the second gene homologous to eEF1A constitute non-amplified genes. The present results contribute to the molecular characterization of a sciarid eEF1A gene.

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

“…Total RNA was isolated from salivary glands (10 pairs), prothoracic glands (25 pairs) and integument (10 integuments) of larvae at age E1 (larval age when there is no BhC4-1 expression in the salivary gland) and at age E7 + 4 h (larval age when BhC4-1 is maximally expressed in the salivary gland) (Basso et al, 2002). The dissected tissues were washed twice in 70% ethanol and total RNA was extracted as previously described (Candido-Silva and Monesi, 2010). Total RNA concentrations were estimated by absorbance measurements at 260 nm, assuming that 40 lg/mL RNA has an absorbance of 1.0.…”

“…BhC4-1 transcript levels were normalized to Bh28S transcript levels, which do not vary during the larval stage (Monesi et al, 2001). Previous characterization of the BhEF1A-F1 gene has shown that it is not amplified during the larval stage (Candido-Silva and Monesi, 2010), and this gene was chosen for normalizing the BhC4-1 amplification levels in the genomic DNA samples. The presented data were obtained from at least two independent biological samples.…”

“…To determine whether the BhC4-1 gene is also amplified in prothoracic glands, qPCR assays were performed using genomic DNA extracted from embryos, prothoracic and salivary glands of larvae at ages E1 (before gene amplification) and at age E7 + 16 h (after gene amplification), and adult flies. BhC4-1 amplification levels were normalized by the BhEF1A-F1 gene amplification levels, which is a gene that is not amplified in B. hygida (Candido-Silva and Monesi, 2010). As a calibrator, we employed the normalized BhC4-1 amplification levels in salivary glands at age E1, the larval age prior to the onset of DNA amplification .…”

“…Genomic DNA extracted from B. hygida embryos (E), salivary glands (SG), prothoracic glands (PG) of larvae at age E1 (E1) and at age E7 + 16 h (E7+) and from adult flies (F) were employed as templates in qPCR reactions. The BhC4-1 amplification levels were normalized in relation to the BhEF1A-F1 gene, employed as a reference gene (Candido-Silva and Monesi, 2010). The amplification levels were estimated relative to the amplification levels in salivary glands at age E1, which was employed as a calibrator.…”

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.…”