Molecular characterization of the B-2 DNA puff gene of Rhynchosciara americana

Abstract

We have sequenced a 2.5-kb DNA fragment of the B-2 DNA puff from the sciarid Rhynchosciara americana and have defined its transcription unit. This puff is active during the formation of the communal cocoon, which is important for successful metamorphosis of this species and coincides with the final cycle of polytenization in its salivary glands. The B-2 polypeptide, together with the products of two other previously characterized DNA puffs, seems to be engaged in an interaction that results in a gradual modification and hardening of the cocoon structure. The B-2 messenger is temporally regulated in apparent coordination with the other puff products. The predicted polypeptide has characteristics similar to polypeptides from previously sequenced DNA puff genes, in particular those from the R. americana C-8 gene and the Bradysia hygida C-4 gene. The cloned sequence of the B-2 puff is differentially amplified in the three gland regions examined, achieving its highest amplification level of approximately fourfold (two extra cycles) in the anterior segment of the gland. The C-3 DNA puff sequence was also found to be differentially amplified in the different gland regions. Implications of the widespread presence of DNA amplification as a form of gene regulation in the Sciaridae are discussed.

Developmental control of the DNA replication and transcription programs

“…Moreover, gene expression in these tissues also likely occurs over a longer developmental window, and this could negate a need for amplification. Another example of developmental gene amplification occurs in the sciarid flies, in which genes encoding cocoon proteins are amplified in the larval salivary glands (Glover et al 1982;Wu et al 1993;Santelli et al 2004) In the case of the eggshell protein genes in the Drosophila follicle cells and the cocoon protein genes in the sciarid flies, it may be that when confronted with the developmental demand to express a limited set of genes at extremely high levels over a brief developmental window, increased copy number of a subset of genes above the overall ploidy of the genome has been selected.…”

Functional and bioinformatics analyses reveal conservation of cis‐regulatory elements between sciaridae and drosophilidae

“…These genomic regions constitute sites of developmentally regulated gene amplification and transcription (Basso et al, 2002;Foulk et al, 2006;Gerbi et al, 1993;Lara et al, 1991;Stocker et al, 1996). The formation of DNA puffs is induced by ecdysone and is concomitant with the transcription of the genes which present the highest levels of amplification within the amplicons (Fontes et al, 1992;Gerbi et al, 1993;Glover et al, 1982;Paçó-Larson et al, 1992;Penalva et al, 1997;Santelli et al, 1991Santelli et al, , 2004Wu et al, 1993;Yokosawa et al, 1999).…”

“…The BhC4-1 protein is expressed at the time DNA puff C4 is formed and is found both in secretory granules, in the salivary gland, and in fibrous structures present in the saliva of fourth instar larvae (Monesi et al, 2004), a subcellular localization consistent with the proposed role for DNA puff proteins. It is noteworthy that in spite of the availability of complete genome sequences for different insect species, the only identified proteins that present similarity to the BhC4-1 protein are the products of the C8 and B2 DNA puffs of Rhynchosciara americana (Frydman et al, 1993;Penalva et al, 1997;Santelli et al, 2004), strongly suggesting that the BhC4-1 protein is a member of a protein family unique to sciarids.…”

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

“…Taken together, these data further support the existence of two paralogous copies of the eEF1A gene in the nematoceran Dipteran B. hygida. Sciarid DNA puffs have been previously characterized as genomic regions in which developmentally regulated gene amplification occurs (33)(34)(35)(36)(37)(38). The only DNA puff-forming regions found on chromosomes A and X constitute the A14 and X4 regions, respectively (6).…”