Two-step regulation of ecdysone-inducible late puffs in salivary glands of Drosophila melanogaster

Our previous study showed that some ecdysone-inducible late puffs could also be induced by a mild detergent (digitonin) in Drosophila salivary glands. However, they could only be induced at the stage immediately prior to when developmentally programmed puffing occurred, suggesting that these late puff loci were under two-step regulation. Using an in vitro culture of salivary glands, we have examined whether ecdysone or the protein products of early puff genes participate in either of the two steps of late puff regulation. This study has revealed that (i) the acquisition of digitonin-responsiveness (the first step) could be induced in vitro by incubating salivary glands with ecdysone; (ii) the first step could also be induced by protein synthesis inhibition even in the absence of ecdysone; (iii) the second step required both ecdysone and protein synthesis unless treated with digitonin; and (iv) the first step, rather than the second step, determines the timing of normal puff formation in the loci. These results suggest that, during normal development, ecdysone controls both steps by activating two types of early genes; the first type, whose function can be mimicked by cycloheximide, renders the loci responsive to digitonin and the second type, whose function can be mimicked by digitonin, activates the loci to form puffs.     

The prepupal salivary glands of Drosophila melanogaster

Evidence is presented in support of the concept that the larval salivary gland of Drosophila melanogaster continues to function as an important secretory organ throughout prepupal stages and after pupation. Just after puparium formation, and at other later periods, the glands appear to be in the process of disintegration, but each time they recover until after pupation. Nuclear blebbing occurs through the time of survival of the glands, but is shown not to involve transport of RNA out of the nucleus. Transport in and out of the nucleus is clearly rapid and in a steady state as compared to the massive and intermittent export of cytoplasmic substance into the lumen of the gland.This work was supported by grants from the National Science Foundation (GB-23343, PCH-02044).     

Larval saliva in Drosophila melanogaster: production, composition, and relationship to chromosome puffs.

Drosophila melanogaster salivary glands produce a mucoprotein-containing saliva in the third larval instar. At the time of prepupa formation, the protein component of the saliva is more than 30% of the total gland protein. Electrophoresis of reduced and alkylated saliva proteins in acrylamide gels yields four saliva-specific fractions. Two protein fractions contain strongly linked sugar. The molecular weights of the proteins were ascertained in SDS-acrylamide gels. Molecular weights for two sugar-free fractions were found to be 12 × 10³ and 23 × 10³ and, for one fraction containing little sugar, it probably lies below 100 × 10³. The variability of saliva proteins in 67 wild types of D. melanogaster were investigated. With the help of transplantation experiments, it was shown that the salivary glands synthesize saliva autonomously. Saliva proteins could be electrophoretically demonstrated earliest in the salivary glands of 86- to 88-hr-old larvae. After saliva is discharged from the gland lumen at the beginning of prepupa formation, the glands produce another type of saliva during the entire prepupal stage and also secrete it into the gland lumen. The chromosome puffs in section 3C of the X chromosome and in section 68C in the third chromosome show a behavior that is positively correlated with larval saliva synthesis.     

Changes of enzyme activity in larval salivary glands following the induction of respiration dependent puffs in giant chromosomes of Drosophila melanogaster.

Changes in the activity of 3 enzymes--lactate dehydrogenase (LDH), soluble malate dehydrogenase (sMDH), and pyruvate kinase (PK)--in homogenates of larval salivary glands of Drosophila melanogaster were studied before and after the induction of heat sensitive puffs with trinactin. The activities of 2 enzymes (LDH and sMDH) were enhanced after puff induction, an effect that was abolished in the presence of cycloheximide. Pyruvate kinase activity did not change after puff induction. The results indicate a relation between the increase of LDH and sMDH activity due to de novo synthesis and the induction of heat sensitive puffs.     

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.     

Protein synthesis in salivary glands of Drosophila hydei after experimental gene induction.

Several treatments, namely incubation at 37 degrees C, in the presence of arsenite, 2,4-dinitrophenol or vitamin B-6, or release from anaerobiosis induce the same set of puffs in the polythene chromosomes of salivary glands of Drosophila hydei. Analysis of changes in protein-synthetic patterns (as determined by radioautography of sodium dodecyl sulphate-gel electrophoretograms of extracts from [35S]methionine-labelled salivary glands) showed that concomitant with puff induction by these various treatments the same six strongly labelled polypeptide bands appeared. The amount of radioactive label in these peptides accounted for 25% of the total incorporation of [35S]methionine, except during incubation at 37 degrees C when it accounted for about 50%. The rate of synthesis of these peptides was maximal 1 h after the start of the puff-inducing treatment. The rate of decay of the rate of synthesis showed first-order kinetics both after removal of the puff-inducing stimulus or in the presence of actinomycin, with a half-life of approx. 4h.     

Peptidase activities of extracts of salivary glands of Drosophila melanogaster

1. Peptide-splitting enzymes have been studied in buffered glycerine extracts of larval salivary glands of three stocks of Drosophila melanogaster. 2. The ultraviolet absorption spectrum of the glycerine extracts indicates the presence of a considerable amount of nucleic acid. 3. Alanylglycine (AG), leucylglycine (LG), leucylglycylglycine (LGG), glycylglycine (GG), and diglycylglycine (GGG) are split by the gland extracts in descending order of activity. 4. Of the various metals added, manganese was the only one found to give clear cut activation and that only with LGG as substrate. Cysteine inhibited the splitting of both AG and LG. 5. Comparison of the data with those published indicates the presence in the extracts in descending order of activity (at pH 7.6, 40 degrees C.) of at least four enzymes: an AG-dipeptidase, an LG-dipeptidase, a leucineaminopeptidase, and possibly an aminopolypeptidase. 6. Optimum conditions for the measurement of the enzyme splitting AG were determined. The pH activity and kinetic data are typical for an AG-dipeptidase. 7. An enzyme (probably cathepsin II) splitting benzoyl-l-arginineamide (pH 5.0) with cysteine activation was observed to occur with very low activity in gland extracts.     

Three-dimensional organization of telomeres in the Drosophila melanogaster salivary glands nuclei

The 3D-FISH was employed to investigate the telomere topology in polytene nuclei of salivary glands of Drosophila melanogaster. The majorities of telomeres in polytene nuclei of salivary glands in Drosophila strain y(2-717) are localized in the nuclear central area and have no contacts with nuclear membrane. In females of this strain, ectopic contacts between telomeres occur at 25 % higher frequency than in males. HeT-A DNA in y(2-717alk3-2) strain, which is a derivative of y(2-717) carrying an inversion between 1D and 13C bands, is found in region 13 of X chromosome. The frequency of ectopic contacts of telomeres in y(2-717alk3-2) males is 10 % higher than that in y(2-717) strain. The number of ectopic contacts can be significantly different in independent experiments, possibly indicating the role of random factors in the contact formation.     

Messenger-like RNA synthesis and DNA chromosomal puffs in the salivary glands of Rhynchosciara americana

RNA synthesis was studied mainly in the proximal sections of Rhynchosciara salivary glands in late fourth instar at two typical periods of development. These are characterized either by the absence or presence of the so-called “DNA puffs” in the salivary gland chromosomes. It was found that simultaneously with the appearance of the DNA puffs there is a great increase in the synthesis of all RNA species. The greatest increase was found to take place in the rate of synthesis of messenger-like RNA. Four main classes of messenger-like RNA were detected, having mobilities corresponding to 33, 23, 16, and 14 S RNA. There is a correlation between the abundance of the 16 S messenger-like RNA and the degree of opening of the B-2 DNA puff. This species might therefore be transcribed from this puff.     

Experimental puffs in salivary gland chromosomes of Drosophila hydei

Summary In Drosophila hydei abnormal puffing activities could be induced by temperature shocks and treatments with 1.2% and 1.4% KCl solutions. After temperature shocks in vivo and in vitro, a number of puffs showed a similar change in activity.Other puffs were found to show a change in activity only after a distinct treatment.Some of the puffs, specific for temperature shocks, showed a change in activity only at a distinct stage of development.In discussing the results, particular attention is paid to puffs observed in common after all treatments.Dedicated to Prof. H. Bauer on the occasion of his sixtieth birthday.     

Vacuole dynamics in the salivary glands of Drosophila melanogaster during prepupal development

A central function of the Drosophila salivary glands (SGs), historically known for their polytene chromosomes, is to produce and then release during pupariation the secretory glue used to affix a newly formed puparium to a substrate. This essential event in the life history of Drosophila is regulated by the steroid hormone ecdysone in the late‐larval period. Ecdysone triggers a cascade of sequential gene activation that leads to glue secretion and initiates the developmentally‐regulated programmed cell death (PCD) of the larval salivary glands, which culminates 16 h after puparium formation (APF). We demonstrate here that, even after the larval salivary glands have completed what is perceived to be one of their major biological functions – glue secretion during pupariation – they remain dynamic and physiologically active up until the execution phase of PCD. We have used specific metabolic inhibitors and genetic tools, including mutations or transgenes for shi, Rab5, Rab11, vha55, vha68‐2, vha36‐1, syx1A, syx4, and Vps35 to characterize the dramatic series of cellular changes occurring in the SG cells between pupariation and 7–8 h APF. Early in the prepupal period, they are remarkably active in endocytosis, forming acidic vacuoles. Midway through the prepupal period, there is abundant late endosomal trafficking and vacuole growth, which is followed later by vacuole neutralization and disappearance via membrane consolidation. This work provides new insights into the function of Drosophila SGs during the early‐ to mid‐prepupal period.     

The metabolism of chromosomal ribonucleic acid in Drosophila salivary glands and its relation to synthesis of desoxyribonucleic acid

Incorporation of adenine-8-C(14) into chromosomal nucleic acids of Drosophila salivary glands has been observed with the autoradiographic technique. RNA-C(14) and DNA-C(14) were detected as the fractions extractable by ribonuclease digestion and resistant to ribonuclease, respectively. Extractions with desoxyribonuclease and acids were also used to identify the nucleic acids. Time-course curves were determined from grain counts. RNA-C(14) concentration reached a maximum in 2 hours, and decreased after the 4th hour. DNA-C(14) concentration reached its maximum within 8 hours, and showed no decreases during a 48-hour experiment. In the latter part of the period of observation, morphological differentiation of the gland occurred, the decrease in RNA-C(14) became very rapid, and a large increase in DNA-C(14) was observed. Marked decrease in RNA-C(14) and increase in DNA-C(14) were detectable in a few hours when isotope was administered shortly before visible differentiation of the gland. Measurements of nuclear size indicated no significant decreases in RNA-C(14) amount prior to the period of differentiation. During this later period, a large decrease in RNA-C(14) amount occurs suddenly, and the same amount of C(14) is added simultaneously to the DNA fraction, as expected if RNA-C(14) is utilized in the synthesis of DNA.     

Ultrastructural histochemical localization of acid phosphatase in salivary glands of Drosophila melanogaster.

The ultrastructural histochemical localization of acid phosphatase in salivary glands of third instar larvae of Drosophila melanogaster has been studied. Using Gomori's lead phosphate method for acid phosphatase detection, the optimal incubation time in the reaction medium was determined to be 30 min. When glands having wild-type acid phosphatase activity are incubated for this time, deposition of the final reaction product is observed in essentially every lysosome and artifactual staining is minimal.     

Development of DNA puffs and patterns of polypeptide synthesis in the salivary glands of Bradysia hygida

In the three salivary gland regions of Bradysia hygida (Diptera, Sciaridae) the patterns of polypeptide synthesis, as revealed by electrophoresis and fluorography, are very stable during the fourth larval instar until about 30 h before the pupal molt. At this age the patterns of polypeptide synthesis start to undergo marked changes. The striking correlations between these changes and the development of two distinct groups of DNA puffs support the proposal that DNA puffs are causally related to the synthesis of specific proteins in the salivary glands.     

20-OH-ecdysone swells nuclear volume by alkalinization in salivary glands of Drosophila melanogaster

Ecdysteroids play an important role in the larval moulting process of insects. Ecdysone-induced stimulation causes specific puffs in polytene chromosomes of salivary gland cells resulting in nuclear swelling. During this process, changes of intracellular ion composition are thought to act as an early regulatory mechanism of gene activation. By use of video-imaging analysis and electrophysiological techniques, we examined ecdysone-induced nuclear swelling in Drosophila salivary glands in situ and its dependence on pH and calcium. Isolated glands of the third larval stage were superfused with a solution mimicking the haemolymph. Addition of 5×10^–6 mol/l 20-OH-ecdysone led, after a lag period of 50 min, to a sustained Ca²⁺-dependent increase of nuclear volume by 23.0±2.3%. Amiloride, a blocker of plasma membrane Na⁺/H⁺ exchange, prevented 20-OH-ecdysone-induced nuclear swelling. Decreasing pH in the superfusate from 7.15 to 6.8 led to nuclear shrinkage by 16.9±3.9%. Measurments of pH in salivary gland cells with ion-sensitive microelectrodes disclosed an alkalinization of 0.23±0.05 pH units after stimulation with 20-OH-ecdysone. We postulate that 20-OH-ecdysone activates the amilorde-sensitive plasma membrane Na⁺/H⁺ exchanger. This leads to intracellular alkalinization and concomitant decondensation of the nuclear chromatin visible as nuclear swelling. Thus, cell alkalinization could be a potentially important stimulatory mechanism in mediating ecdysteroid-induced activation of the cell nucleus.     

Acid phosphatase activity in the larval salivary glands of developing Drosophila melanogaster.

Both the biochemical profile and the optical and fine structural localization of acid phosphatase activity in the larval salivary glands of developing Drosophila melanogaster is described. Biochemically, acid phosphatase shows peak activity in the glands of feeding larvae, followed by a marked decline. Directly preceding the onset of cell histolysis however, enzyme activity increases 1.5 fold and is maintained at this level. Histochemically, acid phosphatase activity initially appears as discrete point or lysosomal sources. As development proceeds, an intense and diffuse form of enzyme is seen, accompanying an extremely vacuolated cytoplasm. Ultrastructurally, the enzyme is located in lysosomes, Golgi elements, multivesicular bodies and both within, and on the extracisternal surface of the rough endoplasmic reticulum. This extracisternal or cytosolic form appears directly preceding cell lysis and eventually shows a comprehensive cellular distribution. Large numbers of acid phosphatase positive haemocytes are attached to the basal glandular surface at all developmental stages. In morphologically intact gland cells, discrete extracisternal enzyme activity appears associated with local areas of degradation.     

The synthesis of compound bands in Drosophila melanogaster salivary gland chromosomes

Small chromosome aberrations were utilized to construct compound bands in the 3C region of Drosophila melanogaster salivary gland chromosomes. The results imply that one should expect to find multiple functions associated with single bands, especially heavy bands. It is suggested that the natural occurrence of compound bands needs to be recognized and that exceptions to the one gene: one band correspondence are expected to occur.Journal Paper No. J-9459 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 1985