Larval Salivary Glands Research Articles

A comparison of the multiple Drosophila heat shock proteins in cell lines and larval salivary glands by two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis of Drosophila proteins from heat-shocked salivary glands resolves the major heat shock proteins into over 14 different molecular weight species. Most of these proteins are in one of the six molecular weight classes previously reported for heat shock proteins: 83,000, 70,000, 68,000, 34,000, 26,000 to 28,000 and 21,000 to 23,000 M r. A few minor proteins, which range in molecular weight from 44,000 to 66,000, are also observed. The 70,000 M r heat shock proteins are resolved into seven slightly different molecular weight groups; within each group, there are several isoelectric point variants. The 83,000, 34,000, and 68,000 M r proteins are single molecular weight species, although there are three isoelectric point variants of the 68,000 M r protein. The 26,000 to 28,000 M r proteins separate into eight major components in two different molecular weight groups plus a run of basic proteins that probably are a result of post-translational modifications of the major proteins. The 21,000 to 23,000 M r proteins consist of three components in two different molecular weight groups. Separation of heat shock proteins from two Drosophila cell lines shows a similar pattern of multiple components, although some of the minor variants visible in salivary glands are not synthesized in the cell lines. In vitro translation of mRNA from heat-shocked salivary glands in a reticulocyte lysate results in a pattern of multiple heat shock proteins nearly identical to that observed in vivo.

Tissue-specific and substrate-specific detection of aldehyde and pyridoxal oxidase in larval and imaginal tissues of Drosophila melanogaster.

The substrate specificities of aldehyde and pyridoxal oxidases in Drosophila melanogaster have been determined with a variety of aliphatic and aromatic aldehydes. This analysis has led to the discovery that 2,4,5-trimethoxybenzaldehyde is a specific substrate for pyridoxal oxidase, as based on the histochemical distribution of oxidase activity, the absence of enzymatic activity in the lpo strains, and the dosage dependence on the number of 1po+ genes present. The tissue-specific localization of aldehyde oxidase (AO) and pyridoxal oxidase (PO) in the larval and adult structures showed that AO was present in all the major internal organs of the larvae and adults, including brain, imaginal discs, Malpighian tubules, digestive system, and reproductive structures. Pyridoxal oxidase is present in many of the same structures which possess AO, but is missing from the cardia, crop, imaginal discs, ovarian follicle cells, paragonia, pericardial cells, and wreath cells. The only structure which possesses PO but lacks AO is the larval salivary gland. These histochemical differences in AO and PO distribution were also confirmed by enzymatic analysis of the activities present in homogenates of ovaries, paragonia, and salivary glands. The general pattern of enzyme expression appears to be established during embryogenesis and maintained throughout the life of the individual.

Genetic analysis of the larval secretion gene Sgs-4 and its regulatory chromosome sites in Drosophila melanogaster.

Larval salivary gland secretion from seven wild-type stocks of Drosophila melanogaster was electrophoretically analyzed. Considerable variability occurs in the X-chromosomally coded secretion protein 4, both qualitatively, as expressed by differences in electrophoretic mobilities, and quantitatively as seen by its relative amount in the secretion. Drosophila stocks with "normal" amounts of protein 4 show approximately 80-90% dosage compensation in the males, whereas in two stocks with lower amounts of protein 4 there is no indication of dosage compensation. Genetic analysis showed that the properties of secretion protein 4 and the level of expression of the Sgs-4 gene are controlled by the X-chromosome. Recombination experiments indicate that the stock-specific characteristics of protein 4 are properties of the structural gene Sgs-4 itself or of a chromosome region immediately adjacent to Sgs-4. One recombinant (R+79), manifesting an intermediate level of dosage compensation, indicates that a chromosome segment closely distal to Sg-4 is responsible for the regulation of the gene and for dosage compensation in particular. Accordingly, Sgs-4 must be transcribed from distal to proximal. Its position on the genetic map is 3.6. Two stocks, Hikone-R and Kochi-R, which were originally described as 0-mutants produce very low amounts of a specific secretion protein, 4 h, as revealed by a transvection effect and also by fluorography of overloaded gels.

Cytogenetic and genetic mapping of a salivary gland secretion protein in Drosophila melanogaster.

The larval salivary gland secretion of Drosophila melanogaster is separated, by acid-urea polyacrylamide electrophoresis, into several different protein components. The genes specifying three of these had hitherto been located both genetically and cytogenetically, and shown to map close to sites that form prominent puffs in larval salivary gland chromosomes. A fourth component of the secretion (SGS-6), present in some, but not all, stocks of D. melanogaster, is shown to be under the control of a gene that maps to 3-42.0 and to bands 71 C1,2-71 F3,5. In those stocks that possess SGS-6 a puff is active in 71 C3-4 in Puff Stage 1, but not in older larvae. We conclude that this puff is the manifestation of a transcriptionally active Sgs-6 gene.

DNA replication in polytene chromosomes: similarity of termination patterns in somatic and germ-line derived polytene chromosomes of Anopheles stephensi Liston (Diptera: Culicidae).

DNA replication is initiated in the ovarian follicles of the anautogenous Culicid Anopheles stephensi in response to a blood meal. The chronology of nurse cell polytene chromosome3H-thymidine labelling patterns has been defined and the terminal labelling patterns of the nurse cell and larval salivary gland polytene X-chromosomes have been compared and found to be essentially similar.

Cell-specific synthesis and glycosylation of secretory proteins in larval salivary glands of Chironomus thummi.

Synthesis and glycosylation of larval salivary gland secretory proteins of Chironomus thummi were analyzed with respect to cell specific differences in the Balbiani ring (BR) pattern and glycoprotein composition of secretion formerly detected by histochemical staining procedures. In the secretion of a special cell type in salivary glands, which is characterized by the appearance of an additional BR, and additional polypeptide with a relative molecular weight (Mr) of 160 kD was found differing in its antigenic properties and tryptic fingerprint pattern from main cell secretion proteins. This so-called ssp-160 component is preferentially synthesized and glycosylated in the special cells. In the same cells, both the synthesis and glycosylation of all other major secretory proteins was found to be diminished or even repressed. In contrast to the conspicuous cell-specific differences at the level of protein synthesis. RNA analyses show the prominent synthesis of 75S RNA in both main and special cells and gave no clear indication of the synthesis of a smaller RNA fraction as expected from the size of ssp-160 component.--These and further data on synthesis and properties of secretory proteins as well as expression of BR DNA are discussed with regard to the assumption that at least some of the eight major secretory polypeptides are coded for by BR DNA. The BR gene(s) might have originated by manifold duplications and modifications of short repetitive prototype DNA sequences, which are coordinatively expressed.

Homologous banding patterns in the polytene chromosomes from the larval salivary glands and ovarian nurse cells of Anopheles stephensi Liston (Culicidae).

A comparison of the banding patterns of two homologous polytene chromosome arms from the larval salivary gland and ovarian nurse cell complement of Anopheles stephensi is presented. The homologous chromosomes from the somatic larval salivary glands and germ-line derived ovarian nurse cells have essentially the same band-interband organisation. An analysis of the 3H-uridine labelling patterns of a small chromosome segment from the two tissues indicates that germ-line polytene chromosomes are not radically different from somatic polytene chromosomes in their patterns of gene expression.

Location of the 18/28S ribosomal RNA genes in two Hawaiian Drosophila species by monoclonal immunological identification of RNA.DNA hybrids in situ.

Using both heterologous rabbit antisera and mouse monoclonal antibody to RNA.DNA hybrids, we have mapped the in situ hybridization locus of the 18/28S ribosomal RNA fraction to a single large band on polytene autosome 3 in Drosophila heteroneura and Drosophila silvestris. This portion of the chromosome is not physically connected with the nucleolus at the end of larval salivary gland development. In mature larvae, little or no hybridization with the material in the nucleolus can be detected. In younger larvae, hybridization of the ribosomal RNA probe to the nucleolus itself can be observed. The chromosome 3 locus is the only band in the polytene genome that shows variation in size and intensity of staining between populations and species. The interband chromosome regions that are immediately distal or proximal to the 18/28S rRNA locus have been involved in a disproportionately large number of natural inversion breaks observed in the euchromatic portion of the polytene chromosome. In 104 species of Hawaiian Drosophila in which chromosome 3 polytene sequences have been determined, 15 breaks occur in these two regions. On a random basis, only one such break is expected. We propose that this locus may be flanked by substantial heterochromatic blocks which are not represented in the salivary gland chromosome.

Replication of DNA in larval salivary glands of Drosophila after in vivo synchronization.

The replication of DNA in the giant chromosomes in different cells of Drosophila larval salivary glands is asynchronous. A method of in vivo synchronization of the nuclei has been successfully devised by a 5'-fluorodeoxyuridine (FdU) block-release-thymidine chase technique, and the patterns of replication sequences have been examined by 3H-thymidine autoradiography. When the larvae of Drosophila melanogaster are fed on FdU for 48 h, and the block is released thereafter, most cells are found in mid-replication phase (termed 3C). When the larvae are subjected to a chase in normal Drosophila medium (or sucrose), a series of cells arrive at 3C phase about every 8 h. When they are chased in sucrose containing thymidine, the number of cells in 3C phase rises to 70%, and then drops rapidly to 1-2% of all labelled cells. The terminal phases (3D, 2D and 1D) reach a peak between 4-8 h. At 12-14 h of chase the 3D-1D peaks decline and a third peak consisting mostly of the initial phases (DD-1C) is found at 14--16 h. The replication of DNA in polytene chromosomes of Drosophila thus seems to proceed in a regular sequence of DD-3C-1D.

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.

Satellite DNA of Anopheles stephensi Liston (Diptera: Culicidae). Chromosomal location and under-replication in polytene nuclei.

Four satellite DNAs in the Anopheles stephensi genome have been defined on the basis of their banding properties in Hoechst 33258-CsCl density gradients. Two of these satellites, satellites I and II, are visible on neutral CsCl density gradients as a light density peak forming approximately 15% of total cellular DNA. Hoechst-CsCl density gradient profiles of DNA extracted from polytene tissues indicates that these satellites are underreplicated in larval salivary gland cells and adult female Malpighian tubules and possibly also in ovarian nurse cells. The chromosomal location of satellite I on mitotic and polytene chromosomes has been determined by in situ hybridisation. Sequences complementary to satellite I are present in approximately equal amounts on a heterochromatic arm of the X and Y chromosomes and are also present, in smaller amounts, at the centromere of chromosome 3. A quantitative analysis of the in situ hybridisation experiments indicates that sequences complementary to satellite I at these two sites differ in their replicative behaviour during polytenisation: heterosomal satellite I sequences are under-replicated relative to chromosome 3 sequences in polytene larval salivary gland and ovarian nurse cell nuclei.

In situ transcription analysis of chromatin template activity of the S-chromosome of Drosophila following high molar NaCl treatment.

The chromatin template activity of the polytene chromosomes in larval salivary glands of Drosophila hydei has been assayed by in situ transcription, on the fixed chromatin using E. coli RNA polymerase holoenzyme and 3H-UTP as the monitoring substrate, both with optimal salt and high salt concentration (1 M NaCl). Results reveal an increase in the net transcription of all chromosomes in the high salt treatment series in comparison with the control. The X-chromosomes of the male larval gland also shows an increase in the labelling following the high salt treatment, but the increase is significantly less than that of the autosomes of the same nucleus. On the basis of these findings it has been suggested that the X-chromosomal hyperactivity of the male, as normally known to exist, might be guided by an inherent modulation of the structure of the X-chromatin.

Giant polytene chromosomes from the ovaries of a Drosophila mutant.

The chromosomes of the ovarian nurse cells of Drosophila melanogaster fall apart during their cycles of endoreduplication. However, chromosomal synapsis occurs in the pseudonurse cells produced in certain mutant females. The resulting polytene chromosomes undergo developmental changes that are strikingly different from those recorded for the giant chromosomes of the larval salivary gland cells.

Tissue distribution of isocitrate dehydrogenase in Drosophila melanogaster

Distribution of the soluble form of isocitrate dehydrogenase (ICDH) in imaginal discs, some larval and some adult Drosophila melanogaster structures was determined. ICDH distribution in imaginal discs provides an opportunity to study in morphologically similar cells the degree of biochemical differentiation, the ontogeny of ICDH pattern formation, tissue determination at the disc level and in some instances the position of developmental compartments. Differential activity for ICDH was observed in the eye-antennal disc, dorsal prothoracic disc, all leg discs, wing and haltere discs, genital disc, larval gut, plus the adult ovaries and the male genital structures. Uniform activity was found in the labial disc, larval salivary gland cells, nests of abdominal histoblasts and in larval fat bodies. No detectable ICDH activity was observed in the imaginal cells of the clypeus-labrum, the imaginal cells of the foregut, the imaginal ring of the hindgut, or in clusters of imaginal cells of ICDH negative regions of the midgut. It is concluded that ICDH, like aldehyde oxidase, is non-random in its tissue distribution and does provide an additional internal histochemical marker for developmental studies.

Molecular limits on the size of a genetic locus in Drosophila melanogaster.

This report places outer limits on the size of the DNA region required for expression of a Drosophila gene. This region, termed the unit of expression, includes not only the structural gene but also any cis-acting sequences that modulate its activity. The locus we have chosen, Sgs-4, codes for one of the glue proteins secreted by larval Drosophila salivary glands. Cytological deficiencies have been identified that eliminate sequences on one side or the other of Sgs-4 without affecting its expression. The ends of these deficiencies have been localized accurately with respect to restriction endonuclease sites in and near the locus. These endpoints limit the Sgs-4 structural gene and essential flanking sequences to a 16- to 19-kilobase region of the X chromosome. The results also show that there is no DNA sequence rearrangement in the Sgs-4 region during development of either the polytene larval salivary glands or adult flies.

Changes of the membrane potential during formation of heat shock puffs induced by ion carriers in Drosophila salivary glands

Effects of the cation carriers trinactin, 2,4-dinitrophenol (DNP), and the calcium ionophore A23187 on induction of heat shock puffs and membrane potential of Drosophila larval salivary glands have been studied. The membrane potential is found to be a function of extracellular potassium concentration and it is not increased by the K +-ionophore trinactin. Furthermore, trinactin depolarizes the cell membrane to 75% of the initial value during 1 h with a rate faster in a 10 mM than 30 mM containing medium. Puff induction by trinactin becomes more rapid and more intense with increasing K + concentration in the medium. The proton carrier DNP produces a concentration dependent depolarization of the cell membrane and this is enhanced by decreasing pH of the medium. In contrast puff induction by DNP is largely depressed at low medium pH. A23187 alone has no effect on the membrane potential, nor does it induce heat shock puffs. However, trinactin- and DNP-induced puffing activity is depressed and proton carrier-induced membrane depolarization is diminished in the presence of A23187. We suggest that changes of intracellular membrane permeabilities, especially of the mitochondrial membrane by ion carriers, result in disturbance of respiratory functions which is reflected on the membrane potential and responsible for the induction of heat shock puffs.

Differential histone phosphorylation during Drosophila development.

During the ontogeny of Drosophila larval salivary gland cells, nuclear DNA is endoreduplicated and organized into polytene chromosomes. All DNA sequences are not reduplicated to the same extent. Satellite DNA’s, which are concentrated in the centromeric heterochromatin of diploid cells, are coalesced in the chromocenter of polytene cells (Gall et al. 1971) and selectively underreplicated (Dickson et al. 1971; Gall et al. 1971). In D. virilis, for instance, three satellites comprise about 45% of the diploid DNA (Gall et al. 1971; Blumenfeld and Forrest 1972) but less than 1% of polytene salivary gland cell DNA (Gall et al. 1971). Underreplication presumably involves specific interactions between chromosomal proteins and satellite DNA’s, or nucleotide sequences lying near satellites. Proteins that can be correlated with satellites migth be involved with these interactions. With this in mind, consider that the underreplication of satellite DNA is correlated with the decreased phosphorylation of Hl histones (Blumenfeld et al. 1978).

Developmental studies on two ecdysone deficient mutants ofDrosophila melanogaster.

This paper describes two ecdysone-deficient, recessive-lethal mutants,lethal(1)giant ring gland (grg) andlethal(1)suppressor of forked mad-ts (mad-ts: Jürgens and Gateff 1979) and compares their ecdysteroid titers with that of the wild-type. Mutant larvae show a much reduced ecdysteroid content, amounting to 1/10 to 1/30 of the wild-type values, but never a true titer peak. They fail to pupate and die after 1-3 weeks. Ecdysteroid feeding elicits different responses in the larvae of the two mutants.mad-ts larvae pupate within 24 h, thus showing that their low ecdysteroid titer is directly connected to their inability to pupate.mad-ts resembles the mutantlethal (3)ecdysone-1 ts (Garen et al. 1977). Thegrg mutant larvae, on the other hand, fail to pupate after 20-hydroxyecdysone feeding as well as injection. The primary defect of thegrg mutant is not entirely clear. Thegrg larval salivary gland cells appear to possess normal ecdysteroid receptors. Furthermore, the low ecdysteroid titer ingrg is not the result of an increased ecdysteroid catabolism. The primary defect in the mutant may lie in the malfunctioning neurosecretory cells which do not show neurosecretion in histological preparations. Further support for this notion comes from electronmicrographs of the enlargedgrg ring glands which, in contrast to the wild-type, do not possess nerve endings.In the wild-type three ecdysteroid peaks were found: one shortly before puparium formation, the second at approximately 12 h and the third at about 30 h after pupation. The ecdysteroid titer peak in late third instar, wild-type larvae is mainly due to the presence of 20-dydroxyecdysone as shown by radioimmunoassays after thin layer chromatography and derivatization followed by gas liquid chromatography and mass spectroscopy. In addition, a number of unidentified polar and apolar metabolites were also present.

The differentiation of pseudonurse cells in the ovaries of fs231 females of Drosophila Melanogaster meigen (diptera : drosophilidae)

Drosophila melanogaster females homozygous for the fs231 mutation are sterile because they fail to produce normal egg chambers. The germaria of females reared at 23°C generate ovarian chambers filled with mitotically active cells that resemble cystocytes. At 18°C chambers containing pseudonurse cells (pnc) also occur. A shift from 23°C to 18°C stimulates differentiation of pnc from germarial cystoblasts. The clusters of pnc generated vary greatly in size, but clusters of 8 and 16 pnc are the most common. The chromosomes of pnc undergo endoreduplication and produce banded, polytene chromosomes in which homologues are paired, but regions of asynapsis are common. The pericentromeric heterochromatin in these chromosomes may be under-replicated to a greater degree than in the chromosomes of larval salivary gland cells, since a chromocenter is not observed in squashed preparations of the polytene chromosomes of pnc.

The salivary gland secretions of a neotropical bumblebee.

The larval salivary gland secretions ofBombus atratus were studied with cytochemical and cytophysical methods. In the young feeding larvae a proteic filamentous secretion depicting striations perpendicular to the long axis of its fibrillary threads and exhibiting a wellordered macromolecular array was found. It appears not to differ from the silk secretion described for the fully grown larvae of an europeanBombus species. However, in the fully grown larvae ofB. atratus changes which have not yet been reported for other bees occur involving the salivary gland secretion. Two secretion types are then distinguishable. One is composed of carboxylated and sulfated acid glycosaminoglycans and glycoprotein(s) (mucous secretion), and the other has the same composition as that of the filamentous secretion of the young larvae, although differing morphologically and in terms of macromolecular alignment (flocculent secretion). The filamentous secretion is assumed to be involved inB. atratus with the spinning of the “silken partitions” which at a relatively early stage separate the larvae reared within a common cell from one another. The mucous and flocculent secretions will participate in the cocoons which will cover the pupating larvae. The filamentous and flocculent secretions appear to contain an α-helical fibroin, glycoprotein(s) and lipoprotein(s), but not collagen-type proteins.