Immunocharacterization of the DNA puff BhC4-1 protein of Bradysia hygida (Diptera: Sciaridae)

The DNA puff BhC4-1 gene is amplified and highly expressed in the salivary gland of Bradysia hygida late larvae. Using affinity-purified polyclonal antibodies we have identified the product of the BhC4-1 gene as a 43 kDa polypeptide which is present in extracts of salivary glands from late fourth instar larvae and in the corresponding gland secretion, but not in glands from earlier stages. We also demonstrate that this protein is produced mainly in the S1 and S3 regions of the salivary gland, where BhC4-1 amplification levels are more pronounced and larger amounts of mRNA are produced. By immunoelectron microscopy the BhC4-1 protein was detected in secretory granules of the S1 and S3 regions, and localized in fibrous structures present in the saliva.     

Puffing activities and binding of ecdysteroid to polytene chromosomes of Drosophila melanogaster

Salivary glands of third instar Drosophila melanogaster larvae were incubated in vitro in the presence of 5 x 10(-6) M 20-hydroxy-ecdysone. Steroid hormone was localized on the polytene chromosomes of the salivary gland by a combination of photoaffinity-labeling and indirect immunofluorescence microscopy. Steroid hormone binding to chromosomal loci and their puffing activity was correlated for the larval/prepupal puffing cycle characterized by puff stages 1-10. In general, there was a good correlation between the sequential and temporal puffing activity induced by 20-hydroxy-ecdysone and the binding of ecdysteroid hormone to these puffs. Ecdysteroid hormone was detected at intermolt, and at early and late puffs with two notable exceptions. Ecdysteroid was not detected at the two well-studied puffs at 23E and at 25AC, the former being an early puff, which is activated in the presence of 20-hydroxy-ecdysone, and the latter being an intermolt puff, which regresses more rapidly in the presence of hormone. Ecdysteroid hormone was present at puffs as long as the respective puff was active. Also, it apparently accumulated at late puff sites after induction. Since ecdysteroid binding to chromosomal loci is temporal as well as sequential during the larval/prepupal puffing cycle, additional factors besides steroid hormone are necessary for sequentially regulating puffing and concomitant gene activity during development from larvae to prepupae.     

Molecular analysis of the ecdysterone-inducible 2B5 "early'' puff in Drosophila melanogaster.

The 2B5 region of the X-chromosome in Drosophila melanogaster plays a developmentally important role in the ecdysterone-triggered response of the late third instar salivary gland. Using a combination of transposon-tagging and chromosomal walking techniques, we have isolated 231 kb of contiguous genomic DNA sequences corresponding to this region. We have more precisely aligned this DNA to the 2B1,2 to 2B5-6 interval of the cytogenetic map by locating the position of three well-characterized chromosomal breakpoints by in situ hybridization and genomic DNA blotting experiments. Labeled cDNA, synthesized from poly(A)+ RNA isolated from hormone-induced salivary gland and imaginal disc tissues and hybridized to the cloned DNA, demonstrated that the ecdysterone-inducible sequences mapped to DNA segments corresponding to the 2B3,4 to 2B5-6 interval. Although some of these sequences were inducible in only one tissue type, many were found to be inducible in both salivary glands and imaginal discs. RNA blotting experiments have detected a major 4.5-kb RNA which is hormone inducible in the larval salivary gland and whose quantitative induction is not inhibited by cycloheximide. Thus, the 4.5-kb RNA represents at least one product from the ecdysterone-responsive 2B5 "early' puff.     

The secretory proteins of the larval salivary gland of Drosophila melanogaster

The gene for a major salivary gland secretion protein (Sgs-1) in Drosophila melanogaster has been mapped to chromosome 2 between dp (13.0) and cl (16.5). In the late third instar larva, a puff forms in this region. This puff (25 B) regresses as the ecdysteroid concentration increases prior to puparium formation. Quantitative analysis of the secretory protein 1, showed that, when present in extra dose, region 25 B results in a significant elevation in its relative amount. This suggests that the structural gene for this protein is localized in this region and that its synthesis is directly correlated to the activity of the 25 B puff.     

Ecdysterone induced protein synthesis in salivary glands and in fat body of Drosophila melanogaster larvae

Salivary glands of 3rd instar larvae of Drosophila melanogaster were labeled with ³H-leucine in the presence and absence of ecdysterone. Twentysix ecdysterone inducible proteins were detected. Their induction was correlated with puff stage. Synthesis of fifteen proteins commenced during early puff stage (PS2); synthesis of seven others at late puff stages (PS8–10). Synthesis of four proteins was induced between puff stage 3/4 and 7/8. Thus, the hormonal induction of protein synthesis generally reflected the appearance of early and of late puffs as described by Ashburner (1972). Eleven ecdysterone inducible proteins were detected in larval fat body in vitro. Comparison of the fat body to the salivary gland proteins revealed that one of the ecdysterone induced fat body proteins was identical in molecular weight and charge to one of the proteins induced by ecdysterone in salivary glands.     

Cadherin-17 is required to maintain pronephric duct integrity during zebrafish development

The characterization of DNA puff BhC4-1 expression was extended and its response to 20-hydroxyecdysone investigated in Bradysia hygida and in transgenic Drosophila carrying the BhC4-1 gene. In both organisms the activation of BhC4-1 in salivary glands occurs at the end of the larval stage coinciding with the peak in ecdysone titers which induces metamorphosis. Injections of 20-hydroxyecdysone into mid-fourth instar larvae of B. hygida show that the induction of BhC4-1 expression, as well as amplification and puff C4 expansion, are late events induced by the hormone. This late response of BhC4-1 expression was also observed in transgenic salivary glands cultivated in the presence of 20-hydroxyecdysone. In vitro studies using transgenic Drosophila indicate that both repressor and activator factors regulate the timing of BhC4-1 expression in salivary glands.