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.     

Induction of ecdysterone-stimulated chromosomal puffs in permeabilized Drosophila salivary glands: A new method for assaying the gene-regulating activity of cytoplasm

A simple assay system for gene regulation using chromosomal puffing as an index of gene activity was established. Salivary glands of Drosophila melanogaster treated with a mild detergent, digitonin, were permeable to high molecular substances, including β-galactosidase (MW 465,000). The permeabilized salivary glands retained the ability to form puffs at the ecdysterone-stimulated loci (74EF and 75B) in response to the hormone. Incubation of the permeabilized salivary glands at puff stage 1 (PS1) for 2 hr in a medium containing both ecdysterone and a homogenate of intact salivary glands at puff stage 8–9 (PS8–9) induced a puff at 78C, where puffing occurs only at puff stages 6–11 in vivo. The puff at 78C was not induced when the permeabilized PS1 glands were incubated with the combination of ecdysterone and a homogenate of the PS1 salivary glands. Likewise, the 78C puff was not induced in intact PS1 salivary glands by a 2-hr incubation with ecdysterone and PS8–9 gland homogenate. These results indicate that a factor(s) required for 78C puff formation is present in PS8–9 but not in PS1 salivary glands and that factor(s) can permeate digitonin-treated salivary glands but not intact glands. The effectiveness of the permeabilized salivary glands as an assay system for gene-regulating factors is discussed.     

Drosophila: the genetics of two major larval proteins.

A series of irradiation-induced deficiencies covering 62 polytene chromosome bands in chromosome arm 3L of Drosophila melanogaster includes the loci of two abundant developmentally regulated larval proteins. The structural gene for larval serum protein 2 (LSP 2) lies at 68E3 or 4, and that for salivary glue secretion protein 3 between 68A8 and 68C11, coincident with a major intermoult puff active in the salivary gland at the time of glue synthesis. The structural genes for esterase 6 and four visible recessive loci lie within the same region.     

Specific repression of a puff in the salivary gland chromosomes of Drosophila virilis after injection of glucosamine

Five hours after the injection of glucosamine into the hemolymph of Drosophila virilis larvae, puff 55E in the salivary gland chromosomes is significantly reduced in its activity. This observation in connection with the circumstances under which the activity of puff 55E decreases during normal development led to the proposition that its activity is involved in mucoprotein synthesis in the salivary glands during the third larval instar. Factors that may regulate the activity of puff 55E are discussed.     

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.     

Puff induction in permeabilized Drosophila salivary glands in a chemically defined medium

We showed previously that digitonin-permeabilized salivary glands form prominent puffs in response to ecdysterone only when the incubation medium is supplemented with a homogenate of intact glands. To develop a chemically defined medium that supports puff formation in permeabilized salivary glands, we examined the requirement of ribonucleoside triphosphates (NTPs), precursors of RNA synthesis, for puff formation in permeabilized salivary glands. We found that prominent ecdysone puffs were induced in permeabilized salivary glands when the concentration of each NTP in the medium was higher than 0.5 mM. The puff size was significantly reduced if the volume of the medium were more than 2.0 microliter per gland. This suggests the existence of a factor(s), in addition to NTPs, which is required for puff formation and is diffusible from permeabilized glands.     

Digitonin Activates Different Sets of Puff Loci Depending on Developmental Stages in Drosophila melanogaster Salivary Glands

We showed previously that treatment of Drosophila melanogaster salivary glands with a mild detergent, digitonin, induces heat shock puffs and many developmentally regulated puffs. To find if the mechanism underlying the puff induction by digitonin is related to the temporal control of gene expression in salivary glands, we examined effects of digitonin on salivary glands at various puff stages from late third instar larva to white prepupa. The results indicate that (a) all the heat shock puffs are induced by digitonin irrespective of the developmental stage of the treated glands, (b) intermolt and early puff loci are always irresponsive to digitonin, and (c) late puff loci respond to digitonin to form puffs only before the stage of their developmentally programmed puffing. Based on the stage at which the locus becomes digitonin responsive, the digitonin-responsive late puff loci were divided into two groups: group A loci, responsive to digitonin continuously from PS1 until programmed puffing begins, and group B loci, responsive to digitonin only in a short period of time immediately before the programmed puffing. The results suggest that a digitonin-sensitive suppression mechanism(s) is involved in the temporal control of gene expression in Drosophila salivary glands.     

Ecdysone-regulated chromosome puffing in the salivary glands of the Mediterranean fruit fly, Ceratitis capitata.

The effect of ecdysone on the puffing activity of the polytene chromosomes of Ceratitis capitata has been studied in organ cultures of late-larval salivary glands. Culture of glands from 120-h-old larvae (puff stage 1) in the presence of ecdysone resulted in the initiation of the late-larval puffing cycle that is normally observed in 145-h-old larvae (puff stage 4). During a 7-h period in the presence of ecdysone, the puffing patterns of most loci resembled the in vivo patterns observed in the period between puff stages 4 and 10, indicating that the first puffing cycle can be initiated by the hormone and proceed almost to completion, in vitro. Culture of salivary glands in the presence of ecdysone and a protein-synthesis inhibitor, as well as ecdysone withdrawal and readdition experiments, indicated that most of the ecdysone-regulated puffs could be categorized into three classes: (i) the puffs that were suppressed immediately by ecdysone, even in the absence of protein synthesis; (ii) the puffs that were induced directly by ecdysone; and (iii) the puffs that were induced indirectly by ecdysone, that is, they were induced after a lag period of a few hours and required protein synthesis for their induction.     

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.