A Kunitz type protease inhibitor related protein is synthesized in Drosophila prepupal salivary glands and released into the moulting fluid during pupation

From the Drosophila virilis late puff region 31C, we microcloned two neighbouring genes, Kil-1 and Kil-2, that encode putative Kunitz serine protease inhibitor like proteins. The Kil-1 gene is expressed exclusively in prepupal salivary glands. Using a size mutant of the KIL-1 protein and MALDI-TOF analysis, we demonstrate that during pupation this protein is released from the prepupal salivary glands into the pupation fluid covering the surface of the pupa. 3-D-structure predictions are consistent with the known crystal structure of the human Kunitz type protease inhibitor 2KNT. This is the first experimental proof for the extracorporal presence of a distinct Drosophila prepupal salivary gland protein. Possible functions of KIL-1 in the context of the control of proteolytic activities in the pupation fluid are discussed.     

Biochemical and ontogenetic aspects of glycoprotein synthesis in Drosophila virilis salivary glands

After SDS-polyacrylamide gel electrophoresis two glycosylated glue proteins are found in the salivary glands of Drosophila virilis late third instar larvae. Synthesis of larval glue protein 1 occurs in three successive steps: at first a precursor protein with a molecular weight of about 138,000 daltons is formed. This is modified by two subsequent steps of glycosylation, the first one involving hexosamine, the second one hexoses. Studies with tunicamycin and β-hydroxynorvaline suggest that glycosylation occurs at threonine residues. Larval glue protein 2 has a molecular weight of approximately 15,000 daltons and is weakly glycosylated. The synthesis of glue proteins is stage specific. It starts at about 120 hr after oviposition and attains its maximal rate about 20 hr later. At this time the larvae leave the food. Between ecdysone release and puparium formation (146–151 hr) larval glue protein synthesis is terminated. Throughout the prepupal stage a different set of glycoproteins is synthesized. Thus, the larval-prepupal transition is accompanied by the reprogramming of glycoprotein synthesis in salivary glands. The secretion products formed during the two developmental stages seem to possess different biological functions.     

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.     

Balbiani ring 3 in Chironomus tentans encodes a 185-kDa secretory protein which is synthesized throughout the fourth larval instar

We have continued to map and identify genes encoding a family of secretory proteins. These proteins are synthesized in larval salivary glands of the midge, Chironomus tentans, and assemble in vivo into insoluble silk-like threads. The genes for several secretory proteins exist in Balbiani rings (BRs) on salivary-gland polytene chromosomes. A randomly primed cDNA clone, designated pCt185, hybridized in situ to BR3 and was shown on Northern blots to originate from a salivary gland-specific 6-kb poly(A) + RNA. The partial cDNA sequence contained 483 nucleotides including one open reading frame (ORF) encoding 160 amino acids (aa). A striking feature of the ORF was the periodic distribution of cysteine residues (Cys-X-Cys-X-Cys-X6-Cys) which occurred approximately every 22 aa. A cDNA-encoded 18-aa sequence was selected for chemical peptide synthesis. When affinity-purified antipeptide antibodies were incubated with a Western blot containing salivary-gland proteins they reacted specifically with a 185-kDa secretory protein (sp185). Developmental studies showed that sp185 and its mRNA were present in salivary glands throughout the fourth larval instar. Thus sp185 and a family of 1000-kDa secretory proteins are encoded by a class of genes that are expressed throughout the fourth instar. This contrasts with the developmentally regulated expression of the sp140 and sp195 genes whose expression is maximal during the prepupal stages of larval development.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster,: IV. The mid prepupal period

In the mid prepupal period of development of Drosophila melanogaster two major changes in gene activity occur in the salivary glands: (a) The mid prepupal puffs (e.g., 63E, 75CD) are induced and (b) the late prepupal puffs (e.g., 62E, 74EF, 75B, and 93F) acquire the competence to respond to ecdysone. These events can be studied in vitro. Both require that the ecdysone titre be very low (<5 × 10^?9, M) and both events depend upon protein synthesis.     

The post-embryonic changes in Melipona quadrifasciata anthidioides Lep. (Hym. Apoidea). II. Development of the salivary glands system

The paper deals with the development of the salivary gland system in Melipona quadrifasciata anthidioides, which begins in the prepupal stage. The silk glands degenerate by autolysis at the end of the larval stage. Degeneration is characterized by cytoplasmic vacuolization and pycnosis of the nuclei of the secretory cells. The glandular secretory portion of degenerated silk glands separates from the excretory ducts. The salivary glands develop from the duct of the larval silk glands. The thoracic salivary glands develop from the ducts of the secretory tubules and the head salivary glands from the terminal excretory duct. The mandibular glands appear in the prepupa as invaginations of mandibular segments, and their differentiation to attain the adult configuration occurs during pupation. The hypopharyngeal glands have their origin from evaginations of the ventral anterior portion of the pharynx. A long tubule first appears with walls formed by more than one cellular layer. Then some cells separate from the lumen of the duct, staying attached to it by a cuticular channel in part intracellular. The initial duct constitutes the axial duct, in which the channel of the secretory cells opens. During the development of salivary and mandibular glands, they recapitulate primitive stages of the phylogeny of the bees. During the development of salivary glands system, mitosis accounts for only part of the growth. Most of the growth occurs by increase in size of cells rather than by cell division. In brown-eyed and pigmented pupae six days before emergence, the salivary gland system is completely developed, although not yet functioning.     

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.     

Ecdysterone responsive functions in the mutantl(1)su(f) ts67g ofDrosophila melanogaster

Summary Transferring the temperature sensitive mutantl(1)su(f) ^ts67g from 25° C to 30° C before or early in the third larval instar blocks the increase in the ecdysterone titer that normally occurs at the end of the larval period. Feeding exogenous ecdysterone to these hormone-deficient larvae results in the formation of pseudopupae. The mutant was used to study ecdysterone-inducible functions in late larval salivary glands by preparing three animal samples with different hormone titers: the titer was low in one sample because of an earlier temperature shift, high in a second sample because the larvae were subsequently transferred to ecdysterone-supplemented food, and also high in a third sample that was kept at 25°C, providing a control for normal development. The effect of the different hormone conditions was studied by³⁵S-methionine labeling of the salivary gland proteins during the larval to prepupal transition and the prepupal period. The results indicate that synthesis of several of the proteins normally appearing during the transition and prepupal period is induced by exogenous ecdysterone.     

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).     

Histone synthesis in Rhynchosciara salivary glands. I. Site and timing of synthesis.

The site of histone synthesis was studied in polytene cells of the salivary glands of the Rhynchosciara americana (Diptera). It was found that, as is the case in non-polytene systems, these proteins are synthesized in the cytoplasm in a class of light polysomes which contain 3-4 ribosomes. This class of polyribosomes is most active at about 5 days before pupation when the nuclei are most active in DNA synthesis and the chromosomes of the gland show many open 'DNA puffs'.     

家蚕蛹-成虫变态期中肠和涎腺超微结构变化与功能

为进一步明确家蚕Bombyx mori变态期间消化系统的生理功能以及溶茧酶的来源器官,通过透射电镜观察和酶活性检测,对家蚕蛹 成虫变态期中肠和涎腺的超微结构、水解酶活力以及中肠内容物的变化进行了观察和分析。结果表明: 蛹第7日到羽化前1日家蚕中肠细胞和刚羽化成虫的涎腺细胞中,均可观察到大量的分泌泡、分泌颗粒、微绒毛等分泌细胞的结构特征以及活跃的分泌现象。潜成虫的中肠和涎腺中都存在活性较高的水解酶活力,其中每毫克中肠组织中蛋白酶活力、酯酶活力和纤溶酶活力分别为726 U、751 U和263 U,每毫克涎腺组织中上述3种酶的活力分别为603 U、523 U和147 U,说明中肠和涎腺可能都具有分泌溶茧酶的功能。家蚕蛹期中肠内容物的主要成分是蛋白质、脂质和糖,三者占内含物总量的95%以上,其中蛋白质含量占78.8%～80.2%。中肠内容物的重量在刚化蛹时为20.1～21.9 mg/头,化蛹后7日内无明显变化,化蛹第9日内容物重量减少63.01%～66.17%,到成虫羽化时已所剩无几,可能是因内容物被消化吸收所致。据此推测,在家蚕变态期中肠还具有贮存和释放营养物质的功能,而溶茧酶的另一个功能可能是分解消化中肠内容物。     

Protein mapping of the salivary complex from a hematophagous leech

The salivary complex of leeches contains many components able to modulate physiological mechanisms, such as coagulation and fibrinolysis, and it is composed by the salivary glands and proboscis, encompassing two different proteomes. The bidimensional electrophoretic pattern of the salivary complex from the Haementeria depressa leech revealed a total of 352 spots, 103 in common with the muscular tissue and 249 exclusive from the salivary complex as detected by silver staining; these spots showed isoelectric points from 3.5 to 9.5 and covered an apparent molecular weight range from 10 to 105 kDa. The following isoforms of proteins were identified by mass spectrometry analysis: antiplatelet protein, myohemerythrin and carbonic anhydrase. Since the leeches were not fed for about 2-3 months to stimulate the secretion of proteins that facilitates the blood metabolism, these most abundant proteins in the salivary complex excised from leeches, are expected to play a role during feeding and might have some anti-hemostatic properties. Furthermore, by zymography, a gelatinolytic and a fibrinolytic protein were identified.     

Patterns of protein synthesis in salivary glands ofDrosophila melanogaster during larval and prepupal development

Summary Patterns of protein synthesis in the salivary glands ofDrosophila melanogaster have been studied throughout late larval and prepupal development by pulse labelling the tissues with³⁵S-methionine. Specific changes to the pattern of proteins synthesized during development are found and the significance of these changes is discussed in view of the known changes in gene (puffing) activity which occur at the same times. We review the problem of salivary gland function in prepupalDrosophila.     

Relationship of developmental stage to initiation of replication in polytene nuclei

The various types of autoradiographic patterns occurring in salivary gland nuclei ofD. melanogaster following a short pulse of tritiated thymidine have been described and the probable order of their sequence within a polytenic replication cycle assigned. On the basis of that assignment, the distribution of those patterns in the nuclei of salivary glands of 160 staged larvae and prepupae has been assessed and the following interpretations made: Throughout the third larval instar and prepupal period there is a continuing decrease in frequency of labeled nuclei. Within the larval period that decrease is reflected in a proportional decrease in label patterns characteristic of both initiation and propagation phases of the replication cycle. At the beginning of the prepupal period there is a rise in the proportion of labeled cells displaying initiation type patterns followed by an abrupt cessation of such patterns. It is proposed that termination of initiation of polytenic replication is amongst the physiologic manifestations of the induction of pupation.     

The timing of drosophila salivary gland apoptosis displays an l(2)gl-dose response

During Drosophila metamorphosis, larval tissues, such as the salivary glands, are histolysed whereas imaginal tissues differentiate into adult structures forming at eclosion a fly-shaped adult. Inactivation of the lethal(2)giant larvae (l(2)gl) gene encoding the cytoskeletal associated p127 protein, causes malignant transformation of brain neuroblasts and imaginal disc cells with developmental arrest at the larval-pupal transition phase. At this stage, p127 is expressed in wild-type salivary glands which become fully histolysed 12 - 13 h after pupariation. By contrast to wild-type, administration of 20-hydroxyecdsone to l(2)gl-deficient salivary glands is unable to induce histolysis, although it releases stored glue granules and gives rise to a nearly normal pupariation chromosome puffing, indicating that p127 is required for salivary gland apoptosis. To unravel the l(2)gl function in this tissue we used transgenic lines expressing reduced ( approximately 0.1) or increased levels of p127 (3.0). Here we show that the timing of salivary gland histolysis displays an l(2)gl-dose response. Reduced p127 expression delays histolysis whereas overexpression accelerates this process without affecting the duration of third larval instar, prepupal and pupal development. Similar l(2)gl-dependence is noticed in the timing of expression of the cell death genes reaper, head involution defective and grim, supporting the idea that p127 plays a critical role in the implementation of ecdysone-triggered apoptosis. These experiments show also that the timing of salivary gland apoptosis can be manipulated without affecting normal development and provide ways to investigate the nature of the components specifically involved in the apoptotic pathway of the salivary glands.     

Stage and segment specificity of the secretory cell of the dermal glands of the tobacco hornworm, Manduca sexta

The pair of epidermally derived Verson's glands on each segment of the tobacco hornworm, Manduca sexta, secretes at ecdysis proteinaceous products which coat the epicuticle. These proteins are produced by a single secretory cell which displays both stage- and segment-specificity during development. Three major 12-kDa polypeptides are synthesized at the larval molts, while higher molecular weight (14-93 kDa) polypeptides are produced at the pupal molt. In the pupa, but not in the larva, there are three segment-specific protein patterns, each involving both qualitative and quantitative differences: (1) thoracic (T) segments 1 and 2; (2) T3 and abdominal (A) segment 1; (3) A2-A8. Larval-specific proteins were found to be synthesized in low amounts throughout the penultimate fourth instar, with enhanced synthesis occurring during the molt, coincident with the molting surge of ecdysteroids. Synthesis of the major pupal products commenced about the time of wandering, with enhanced synthesis occurring throughout prepupal development, coincident with the prepupal surge in ecdysteroids. The onset of synthesis of the major pupal products differed, both within and between segments. Culture of fifth instar Day 2 glands in vitro showed that this synthesis depended on 20-hydroxyecdysone. The differential regulation within and between segments observed in vivo was also seen in vitro.     

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.