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.     

The effect of ecdysterone on nonhistone proteins in salivary gland chromosomes of Sciara coprophila.

Synthesis and transport of proteins to the cell nucleus during puff induction was studied in S. coprophila. Changes in grain distribution along chromosomes (L-methionine [35S] incorporation into protein) were correlated with puffs induced by ecdysterone in vitro; A pattern of specific labelling at the sites of incipient puffs was noted within 2 h after the addition of the hormone, i.e. grains on the chromosomes were in clusters, characteristic for this time point and not seen in the controls (where only non-specific labeling was noted 0-4 h). Characteristic chromosomal puffs appeared between 3-4 h after the addition of ecdysterone. It was concluded that during ecdysterone-induced puff formation in salivary gland chromosomes, proteins which had been previously synthesized were selectively transported from the cytoplasm to specific sites on the chromosomes.     

Photoinduced bonding of endogenous ecdysterone to salivary gland chromosomes of Chironomus tentans

Endogenous ecdysterone has been bonded to chromosomal loci by irradiation of Ch. tentans salivary glands. The hormone has been localized on the polytene chromosomes by indirect immunofluorescence microscopy. Hormone binding to chromosomes is stage-specific. Seven chromosomal loci could be identified which specifically bound hormone in larval salivary glands, and 21 chromosomal loci which specifically bound hormone in prepupal salivary glands. All puffs that have been described by Clever (1961) as being inducible by ecdysterone have been found to contain irreversibly bound ecdysterone in prepupal salivary gland chromosomes. A small number of puff sites in larval salivary gland chromosomes exhibited varying amounts of bound ecdysterone, (as judged by fluorescence intensity) most notably 117B and Balbiani rings 1 and 3 on chromosome IV. In addition to stage specific binding sites, there were many others showing equal binding of the hormone in both, larval and prepupal, stages of development. — Fluorescence intensities (reflecting the amount of bonded hormone) at puff sites along the tip section of the prepupal salivary gland chromosome arm IR have been computed indicating that differences between fluorescence intensities of different puffs can be expressed as multiples of a basic fluorescence intensity. Thus, the amount of fluorescence intensity (bonded hormone) in the various puffs may be quantized. — The data indicate that in Ch. tentans salivary glands ecdysterone acts, at the chromosomal level. The development of larvae into prepupae generates more puff sites and more hormone binding. This is discussed in the light of current models of hormone-receptor function.     

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.     

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.     

Cytogenetic analysis of the 2B3-4-2B11 Region of the X chromosome of Drosophila melanogaster

Larvae homozygous or hemizygous for the l(l) t435 mutation located within the early ecdysteroid puff 2B5, or carrying a deletion of the 2B5 band, die at the end of the third larval instar. In the salivary gland chromosomes of these larvae only intermoult puffs are detected. If these salivary glands are incubated in vitro with 20-OH ecdysone for 6 h the intermoult puff 68 C remains large, some early puffs (74EF and 75B) are induced to 30–40% of their normal size, other early (63F) and all late puffs (62E, 78D, 82F and 63E) are not induced at all. Puff 2B5 reaches its normal size but does not regress after 6h incubation with 20-OH ecdysone, as it does in normal stocks. The data obtained in this study show the existence of a locus (or loci) in the band (puff) 2B5 which is necessary for the normal response of the salivary gland chromosomes to the hormone 20-OH ecdysone.     

Puffing in the regions of the esterase and ecdysterone genes in Drosophila lummei strains differing in beta-esterase expression

Correlation between the activity of esterase and ecdysterone puffs was observed by comparison of puff expression in the areas of alpha- and beta-esterase genes localization and exogenous ecdysterone-sensitive areas (2-G-1-2-G-5). 2-G-5 puff expression and beta-esterase activation correlated with decrease of ecdysterone puffs activity. Constant puffing in 2-G-1 and 2-G-4 matched with constant activity of alpha-specific isoenzymes. The F1 larvae from cross between lines with normal and null-allele beta Est2 gene carried heteromorph puff and its expression depended upon cross direction.     

cDNA clones for the ecdysone-inducible polypeptide (EIP) mRNAs of Drosophila Kc cells

The ecdysone-inducible polypeptides (EIPs) 28, 29 and 40 were identified previously as polypeptides whose synthesis is stimulated early in the ecdysone response of Drosophila Kc cells. We have now shown, using two-dimensional gels, that each of these EIPs consists of three species differing in pI, and all stimulated by ecdysone. Translations and hybrid-arrested translations indicated that the poly(A)⁺ EIP mRNAs increase ˜10-fold in abundance during the first 4 h of ecdysone treatment. By a differential screen of a cDNA library we have identified cDNA clones corresponding to all three EIPs. Two kinds of clones were isolated: one hybridizes to the EIP 40 mRNA(s); the second hybridizes to the mRNA(s) encoding all the EIPs 28 and 29. The EIP 28/29 and EIP 40 loci detected by these clones are each present at single sites on the polytene chromosomes and each is at or in the vicinity of an ecdysone-regulated puff.     

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.     

Ecdysteroid-inducible polypeptides in a Drosophila cell line

In the Drosophila melanogaster cell line Kc-H, ecdysteroid hormone treatment causes increased relative synthesis of three ecdysteroid-inducible polypeptides (EIPs), named according to their molecular weights (in kilodaltons) EIP 40, EIP 29 and EIP 28. Increased synthesis of the EIPs is detectable within 45 min (EIP 28) or 75 min (EIPs 40 and 29), is maximal at 4-8 hr and continues for almost 2 days. During this period no other major changes in protein synthesis are discernible using one-dimensional gels. At maximum, EIP 28 synthesis is elevated at least 10 fold above its basal level, and EIPs 40 and 29 somewhat less. EIP induction is ecdysteroid-specific and is detectable in the presence of 10(-8) M 20-hydroxyecdysone. It does not occur in hormone-resistant cells. Apparently identical polypeptides are inducible in another ecdysteroid-responsive cell line, Schneider's line 3. Because EIP synthesis is an early and substantial response to ecdysteroids, this is a promising system for the study of steroid hormone action.     

Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs

The salivary glands and other tissues from Drosophila melanogaster were dissected at various times throughout the prepupal period, as well as after heat shocks and ecdysterone treatments, and the proteins labelled by incubating the isolated tissues with [³⁵S]methionine were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel. The labelled band patterns from salivary gland, as seen on the autoradiograph of the gel, showed striking variations, in a manner remarkably similar to variations in puff patterns during the same prepupal period. In proteins from Malpighian tubes, the pattern of bands varied to a lesser extent and in brain only a few components were modified.Heat shock brought about the appearance of a number of new bands, while others were reduced in intensity. This effect was observed with all the tissues examined, salivary glands, brain and Malpighian tubes, as well as wing imaginal discs, tissue lacking polytene chromosomes. The six most heavily labelled bands induced by heat shock represent about 30%, and one component alone represents over 15%, of the total label in the sample, as seen in salivary glands, brain and Malpighian tubes. The synthesis of RNA at puff sites was investigated after heat shock by [³H]uridine labelling. By correlating the amount of [³H]uridine in some puffs with the level of [³⁵S]methionine in some bands a tentative relation is suggested in a few instances.The effect of ecdysterone treatment was also studied in the salivary glands. Changes in a number of protein bands were noticed, though they were much less pronounced than those following heat shock.     

Similarities in chromosomal puffing induced by temperature shocks and dinitrophenol in Drosophila

A comparison between chromosomal puffs induced by temperature shocks and dinitrophenol in regions 87 A and 87 B of salivary gland chromosome 3 R of Drosophila melanogaster is presented. The size of both puffs can be regulated by either the temperature of a heat-shock or by the concentration of dinitrophenol. The two agents are additive with respect to their effects upon puff size, supporting the idea that both result in a change in ATP synthesis within the cell. A possible relationship between these agents and puff formation and RNA metabolism is discussed.This investigation was supported by research grant No. 119-70 from the Cancer Association of Greater New Orleans.     

Nature of female sterility in mutants at the ecs locus controlling sensitivity to ecdysterone in Drosophila melanogaster]

Using interstrain level ovary transplantations function of ovaries was estimated in females bearing mutations at the ecs locus mapped in the 2B3-5 region of the X-chromosome of Drosophila melanogaster. Dissection of recipient flies and genetic analysis of their progeny demonstrated that the donor ovary in fertile flies was capable of normal functioning in the case of connection of the donor ovary in fertile flies with the recipient oviducts. Therefore, female-sterile mutations at the ecs locus are somatic line specific. SDS-PAGE electrophoresis revealed normal level of the yolk proteins in such females. Thus, the sterility of the ecs mutants may arise from abnormal morphogenesis of their genital disc, due to the loss of normal sensitivity to ecdysterone.     

THE EFFECT OF CHANGES IN THE RESPIRATORY METABOLISM UPON GENOME ACTIVITY : A Correlation between Induced Gene Activity and an Increase in Activity of a Respiratory Enzyme

The in vitro regression of experimentally induced chromosome puffs was investigated in explanted salivary gland chromosomes of Drosophila hydei. It was observed that the regression of the puffs 2-32A, 2-36A, 2-48C, and 4-81B is accelerated if substrates for the respiratory metabolism are supplied to the cells. A similar effect can be produced by addition of KCN or oligomycin to medium in which intact salivary glands are incubated. The acceleration of puff regression by these substances occurs not only if the puff-inducing stimulus is removed but as well under conditions in which the stimulus is maintained. Regression of the puffs 2-32A, 2-36A, and 4-81B is inhibited if cycloheximide is present in the incubation medium. Chloramphenicol has no effect on puff regression. Measurements on nicotinamide adenine dinucleotide-dehydrogenase activity in homogenates of salivary glands revealed an increase in enzyme activity of 41 %. Maximum increase is attained at 30 min after the induced puffs have reached their maximum size. The increase in enzyme activity does not occur if the glands are kept in a medium containing either actinomycin D or cycloheximide. Chloramphenicol does not inhibit the increase in enzyme activity. The possible relationship between puff activity and its control as a result of changes in the respiratory metabolism is discussed.     

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.     

Developmental Studies in Drosophila

The salivary gland chromosomes of 3rd instar Drosophila pseudoobscura larvae were observed for puffing changes after injection of larvae with ecdysterone solution. Chromosomes from the salivary glands of 3rd instar larvae and prepupae were similarly examined after incubation in ecdysterone-containing medium. The larvae, after treatment, showed advancement of the puffing process with the occurrence of a pattern similar to that observed during the pre-spiracle eversion period of normal development. At least 92 puffs showed changes in size. For the prepupae, the puffing changes resembled those occurring normally during the late prepupal period. A group of puffs were selected for detailed study. Among these were four puffs on the XR chromosome which exhibited large increases before spiracle eversion and pupation in normal development. As in normal development, two of these became the most prominent puffs observed within h after hormone treatment. In chromosomes from larval glands, the other two XR chromosome puffs were among the largest puffs to appear later in the sequence. However, in chromosomes from prepupal glands one of these later puffs failed to appear. The significance of this large number of hormone-inducible puffing changes at two different periods in development is discussed.     

Local protein accumulation during gene activation

Relative values for the dry mass in puff-forming regions of Drosophila hydei salivary gland chromosomes were established with a Leitz double beam interference microscope. All measurements were made after RNA digestion.Optical path differences per unit area of the induced puffs 2-48C and 4-81B (temperature induced) and a cytoplasm-free background were recorded. In each of the nuclei used for these measurements, the same procedure was applied to two reference regions in the vicinity of the puff, region 2, 47A-48B and region 4, 81C-82C respectively. For comparison of the dry mass values of a puff region at various time intervals after the onset of puff induction, ratios of the optical path differences of the puff region over that of the reference region were calculated.These ratios were established at 5, 10, 30, 60 and 120 min after the onset of a temperature treatment and compared with the ratio in non-treated animals. From the data it can be concluded that the dry mass in the induced puffs increases gradually up to 30 min. At this time the dry mass ratio for puff 2-48C has reached a value of 150% and that of puff 4-81B, 210%. It is concluded that this increase in dry mass is due almost entirely to a local accumulation of non-histone protein.