Puffs and PCR: the in vivo dynamics of early gene expression during ecdysone responses in Drosophila

Development ◽  
1993 ◽  
Vol 118 (2) ◽  
pp. 613-627 ◽  
Author(s):  
F. Huet ◽  
C. Ruiz ◽  
G. Richards
Keyword(s):  
Salivary Gland ◽  
Gene Networks ◽  
Larval Instar ◽  
Polytene Chromosomes ◽  
Early Gene ◽  
Rt Pcr ◽  
Response Characteristics ◽  
Larval Salivary Gland

The steroid hormone ecdysone orchestrates insect development by regulating gene networks. In Drosophila the most detailed description of ecdysone action is the sequential activation of early and late puffs in the polytene chromosomes of the late larval salivary gland. A number of these early puffs (2B5, 74EF and 75B) contain complex transcription units (Broad-Complex, E74 and E75 respectively) encoding families of regulatory proteins which are expressed in most if not all tissues. In vitro, transcripts of the different isoforms of these early genes as well as the ecdysone receptor (EcR) present varying dose response characteristics (Karim and Thummel, 1992, EMBO J. 11, 4083–4093). We have developed an in vivo approach using a reverse transcription-polymerase chain reaction assay (RT-PCR) so as to visualise these transcripts in the RNA extracted from a single salivary gland. Using one salivary gland lobe for developmental puff staging and the sister lobe for RT-PCR, we have obtained precise developmental profiles for these transcripts and have extended our study to other tissues and stages where puffing studies were not possible. In the salivary gland we have characterised three distinct ecdysone responses. For the mid and late third larval instar responses our results confirm and extend the conclusions of the in vitro studies concerning the temporal expression of the early gene isoforms. The relatively brief prepupal response contains elements in common with each of the larval responses and all three can be explained by the profiles of the respective ecdysone peaks. Interestingly EcR transcripts respond differently during each response. The analysis of different tissues of the same animal reveals subtle differences in the timing of the ecdysone response and isoform expression and suggests that this may reflect tissue differences in the ecdysone profiles. As these molecules have homologues in vertebrates, our analysis may have general implications for the organisation of hormonal responses in vivo.

Isolation and characterization of fifteen ecdysone-inducible Drosophila genes reveal unexpected complexities in ecdysone regulation

1993 ◽  
Vol 13 (11) ◽  
pp. 7101-7111
Author(s):  
P Hurban ◽  
C S Thummel
Keyword(s):  
Salivary Gland ◽  
Steroid Hormone ◽  
Polytene Chromosomes ◽  
Feedback Regulation ◽  
Primary Response ◽  
Cdna Libraries ◽  
Isolation And Characterization ◽  
Larval Salivary Gland ◽  
Subtracted Cdna Libraries

Our insights into the regulatory mechanisms by which the steroid hormone ecdysone triggers Drosophila melanogaster metamorphosis have largely depended on puffs in the larval salivary gland polytene chromosomes as a means of identifying genes of interest. Here, we describe an approach that provides access to ecdysone-inducible genes that are expressed in most larval and imaginal tissues, regardless of their ability to form puffs in the polytene chromosomes. Several hundred cDNAs were picked at random from subtracted cDNA libraries and subjected to a rapid and sensitive screen for their ability to detect mRNAs induced by ecdysone in the presence of cycloheximide. Of the 15 genes identified in this manner, 2 correspond to early puffs in the salivary gland polytene chromosomes, at 63F and 75B, confirming that this screen functions at the desired level of sensitivity and is capable of identifying novel primary-response genes. Three of the genes, Eig45-1, Eig58, and Eig87, are expressed coordinately with the salivary gland early genes; one of them, Eig58, maps to the 58BC puff that is active when the 74EF and 75B early puffs are at their maximal size. Another gene identified in this screen, Eig17-1, encodes a novel cytochrome P-450. On the basis of its sequence identity and temporal profile of expression, this gene may play a role in steroid hormone metabolism and thus could provide a mechanism for feedback regulation of ecdysone production. Although all 15 genes have patterns of transcription that are consistent with ecdysone regulation in vivo, 5 genes do not appear to be induced by the late larval ecdysone pulse. This indicates that ecdysone induction in larval organs cultured with cycloheximide is not always indicative of a primary response to the hormone.

scholarly journals SOURCES OF LARVAL SALIVARY GLAND SECRETION IN THE DIPTERAN CHIRONOMUS TENTANS

1969 ◽  
Vol 40 (1) ◽  
pp. 61-78 ◽  
Author(s):  
D. Doyle ◽  
H. Laufer
Keyword(s):  
Salivary Gland ◽  
De Novo ◽  
Salivary Secretion ◽  
Disc Electrophoresis ◽  
Protein Fractions ◽  
Chironomus Tentans ◽  
Salivary Gland Secretion ◽  
Larval Salivary Gland

The soluble proteins in the hemolymph, the salivary gland, and the salivary secretion of fourth instar Chironomus tentans were examined by disc electrophoresis in acrylamide gels. Of the 11 protein fractions detected in buffered saline extracts of the gland, 10 are present also in the hemolymph. Amino acid isotope incorporation experiments indicate that the protein fractions shared by the salivary gland and the hemolymph are not synthesized in the gland but are synthesized in other larval tissues. Immunochemical studies show that most of these proteins eventually are secreted from the gland. The salivary gland in vivo and in vitro is active in de novo protein synthesis. The protein synthesized tends to form large molecular weight aggregates. As demonstrated by radioautography, at least 80% of this protein is secreted from the 30 large cells forming most of the gland. The proteins synthesized in the salivary gland cannot be detected in the hemolymph. The results of this investigation are consistent with a mechanism of secretion formation involving both de novo synthesis of some secretion proteins and the selective uptake, transport, and secretion of hemal proteins by the salivary gland.

The Drosophila E74 gene is required for the proper stage- and tissue-specific transcription of ecdysone-regulated genes at the onset of metamorphosis

Development ◽  
1995 ◽  
Vol 121 (5) ◽  
pp. 1411-1421 ◽  
Author(s):  
J.C. Fletcher ◽  
C.S. Thummel
Keyword(s):  
Salivary Gland ◽  
Critical Role ◽  
Polytene Chromosomes ◽  
Early Gene ◽  
Secondary Response ◽  
Phenotypic Characterization ◽  
Large Set ◽  
Late Genes ◽  
Larval Salivary Gland ◽  
Maximal Induction

The steroid hormone ecdysone directly induces a small set of early genes, visible as puffs in the larval salivary gland polytene chromosomes, as it signals the onset of Drosophila metamorphorsis. The products of these genes appear to function as regulators that both repress their own expression and induce a large set of secondary-response late genes. We have identified recessive loss-of-function mutations in the early gene E74, a member of the ets protooncogene family that encodes two related DNA-binding proteins, E74A and E74B. These mutations cause defects in pupariation and pupation, and result in lethality during metamorphosis. Here we extend our phenotypic characterization of the E74A and E74B mutant alleles to the molecular level by examining their effects on the transcription of over 30 ecdysone-regulated genes. We show that the transcription of most ecdysone primary-response genes during late larval and prepupal development is unaffected by the E74 mutations. Rather, we find that E74 is necessary for the appropriate regulation of many ecdysone secondary-response genes. E74B is required for the maximal induction of glue genes in mid third instar larval salivary glands, while E74A is required in early prepupae for the proper timing and maximal induction of a subset of late genes. E74 activity is also necessary for the correct regulation of genes expressed predominantly in the fat body, epidermis or imaginal discs. These observations confirm that E74 plays a critical role in regulating transcription during the early stages of Drosophila metamorphosis. In addition, the widespread effects of the E74 mutations on transcription indicate that E74 functions in regulatory hierarchies not only in the larval salivary gland, but throughout the entire organism.

scholarly journals Isolation and characterization of fifteen ecdysone-inducible Drosophila genes reveal unexpected complexities in ecdysone regulation.

1993 ◽  
Vol 13 (11) ◽  
pp. 7101-7111 ◽  
Author(s):  
P Hurban ◽  
C S Thummel
Keyword(s):  
Salivary Gland ◽  
Steroid Hormone ◽  
Polytene Chromosomes ◽  
Feedback Regulation ◽  
Primary Response ◽  
Cdna Libraries ◽  
Isolation And Characterization ◽  
Larval Salivary Gland ◽  
Subtracted Cdna Libraries

Our insights into the regulatory mechanisms by which the steroid hormone ecdysone triggers Drosophila melanogaster metamorphosis have largely depended on puffs in the larval salivary gland polytene chromosomes as a means of identifying genes of interest. Here, we describe an approach that provides access to ecdysone-inducible genes that are expressed in most larval and imaginal tissues, regardless of their ability to form puffs in the polytene chromosomes. Several hundred cDNAs were picked at random from subtracted cDNA libraries and subjected to a rapid and sensitive screen for their ability to detect mRNAs induced by ecdysone in the presence of cycloheximide. Of the 15 genes identified in this manner, 2 correspond to early puffs in the salivary gland polytene chromosomes, at 63F and 75B, confirming that this screen functions at the desired level of sensitivity and is capable of identifying novel primary-response genes. Three of the genes, Eig45-1, Eig58, and Eig87, are expressed coordinately with the salivary gland early genes; one of them, Eig58, maps to the 58BC puff that is active when the 74EF and 75B early puffs are at their maximal size. Another gene identified in this screen, Eig17-1, encodes a novel cytochrome P-450. On the basis of its sequence identity and temporal profile of expression, this gene may play a role in steroid hormone metabolism and thus could provide a mechanism for feedback regulation of ecdysone production. Although all 15 genes have patterns of transcription that are consistent with ecdysone regulation in vivo, 5 genes do not appear to be induced by the late larval ecdysone pulse. This indicates that ecdysone induction in larval organs cultured with cycloheximide is not always indicative of a primary response to the hormone.

Comparative Evaluation of In Vitro and In Vivo Assays for the Detection of Avian Infectious Bronchitis Virus as a Contaminant of Live Poultry Vaccines

1998 ◽  
Vol 26 (5) ◽  
pp. 629-634
Author(s):  
Emiliana Falcone ◽  
Edoardo Vignolo ◽  
Livia Di Trani ◽  
Simona Puzelli ◽  
Maria Tollis
Keyword(s):  
Infectious Bronchitis Virus ◽  
Serological Response ◽  
Avian Infectious Bronchitis Virus ◽  
Animal Testing ◽  
Rt Pcr ◽  
Pcr Assay ◽  
In Vivo Test ◽  
Infectious Bronchitis

A reverse transcriptase polymerase chain reaction (RT-PCR) assay specific for identifying avian infectious bronchitis virus (IBV) in poultry vaccines, and the serological response to IBV induced by the inoculation of chicks with a Newcastle disease vaccine spiked with the Massachusetts strain of IBV, were compared for their ability to detect IBV as a contaminant of avian vaccines. The sensitivity of the IBV-RT-PCR assay provided results which were at least equivalent to the biological effect produced by the inoculation of chicks, allowing this assay to be considered a valid alternative to animal testing in the quality control of avian immunologicals. This procedure can easily be adapted to detect a number of contaminants for which the in vivo test still represents the only available method of detection.

Genetic Analysis of the Drosophila 63F Early Puff: Characterization of Mutations in E63-1 and maggie, a Putative Tom22

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 229-244
Author(s):  
Martina Vaskova ◽  
A M Bentley ◽  
Samantha Marshall ◽  
Pamela Reid ◽  
Carl S Thummel ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Genetic Analysis ◽  
Polytene Chromosomes ◽  
Reading Frame ◽  
Protein Levels ◽  
Ef Hand ◽  
Larval Salivary Gland ◽  
Complementation Groups ◽  
Inducible Genes ◽  
Early Puff

Abstract The 63F early puff in the larval salivary gland polytene chromosomes contains the divergently transcribed E63-1 and E63-2 ecdysone-inducible genes. E63-1 encodes a member of the EF-hand family of Ca2+-binding proteins, while E63-2 has no apparent open reading frame. To understand the functions of the E63 genes, we have determined the temporal and spatial patterns of E63-1 protein expression, as well as undertaken a genetic analysis of the 63F puff. We show that E63-1 is expressed in many embryonic and larval tissues, but the third-instar larval salivary gland is the only tissue where increases in protein levels correlate with increases in ecdysone titer. Furthermore, the subcellular distribution of E63-1 protein changes dynamically in the salivary glands at the onset of metamorphosis. E63-1 and E63-2 null mutations, however, have no effect on development or fertility. We have characterized 40 kb of the 63F region, defined as the interval between Ubi-p and E63-2, and have identified three lethal complementation groups that correspond to the dSc-2, ida, and mge genes. We show that mge mutations lead to first-instar larval lethality and that Mge protein is similar to the Tom22 mitochondrial import proteins of fungi, suggesting that it has a role in mitochondrial function.

scholarly journals Design of PSMA ligands with modifications at the inhibitor part: an approach to reduce the salivary gland uptake of radiolabeled PSMA inhibitors?

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Veronika Barbara Felber ◽  
Manuel Amando Valentin ◽  
Hans-Jürgen Wester
Keyword(s):  
Salivary Gland ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
In Vivo Studies ◽  
Tumor Xenograft ◽  
Tumor Uptake ◽  
Lncap Cells ◽  
High Affinity

Abstract Aim To investigate whether modifications of prostate-specific membrane antigen (PSMA)-targeted radiolabeled urea-based inhibitors could reduce salivary gland uptake and thus improve tumor-to-salivary gland ratios, several analogs of a high affinity PSMA ligand were synthesized and evaluated in in vitro and in vivo studies. Methods Binding motifs were synthesized ‘on-resin’ or, when not practicable, in solution. Peptide chain elongations were performed according to optimized standard protocols via solid-phase peptide synthesis. In vitro experiments were performed using PSMA+ LNCaP cells. In vivo studies as well as μSPECT/CT scans were conducted with male LNCaP tumor xenograft-bearing CB17-SCID mice. Results PSMA ligands with A) modifications within the central Zn2+-binding unit, B) proinhibitor motifs and C) substituents & bioisosteres of the P1′-γ-carboxylic acid were synthesized and evaluated. Modifications within the central Zn2+-binding unit of PSMA-10 (Glu-urea-Glu) provided three compounds. Thereof, only natLu-carbamate I (natLu-3) exhibited high affinity (IC50 = 7.1 ± 0.7 nM), but low tumor uptake (5.31 ± 0.94% ID/g, 1 h p.i. and 1.20 ± 0.55% ID/g, 24 h p.i.). All proinhibitor motif-based ligands (three in total) exhibited low binding affinities (> 1 μM), no notable internalization and very low tumor uptake (< 0.50% ID/g). In addition, four compounds with P1′-ɣ-carboxylate substituents were developed and evaluated. Thereof, only tetrazole derivative natLu-11 revealed high affinity (IC50 = 16.4 ± 3.8 nM), but also this inhibitor showed low tumor uptake (3.40 ± 0.63% ID/g, 1 h p.i. and 0.68 ± 0.16% ID/g, 24 h p.i.). Salivary gland uptake in mice remained at an equally low level for all compounds (between 0.02 ± 0.00% ID/g and 0.09 ± 0.03% ID/g), wherefore apparent tumor-to-submandibular gland and tumor-to-parotid gland ratios for the modified peptides were distinctly lower (factor 8–45) than for [177Lu]Lu-PSMA-10 at 24 h p.i. Conclusions The investigated compounds could not compete with the in vivo characteristics of the EuE-based PSMA inhibitor [177Lu]Lu-PSMA-10. Although two derivatives (3 and 11) were found to exhibit high affinities towards LNCaP cells, tumor uptake at 24 h p.i. was considerably low, while uptake in salivary glands remained unaffected. Optimization of the established animal model should be envisaged to enable a clear identification of PSMA-targeting radioligands with improved tumor-to-salivary gland ratios in future studies.

scholarly journals Interaction of periodontitis and orthodontic tooth movement—an in vitro and in vivo study

2021 ◽  
Author(s):  
Birgit Rath-Deschner ◽  
Andressa V. B. Nogueira ◽  
Svenja Beisel-Memmert ◽  
Marjan Nokhbehsaim ◽  
Sigrun Eick ◽  
...  
Keyword(s):  
Alveolar Bone ◽  
Tooth Movement ◽  
Mechanical Strain ◽  
Orthodontic Tooth Movement ◽  
Fusobacterium Nucleatum ◽  
In Vivo Study ◽  
Rt Pcr ◽  
Periodontal Inflammation

Abstract Objectives The aim of this in vitro and in vivo study was to investigate the interaction of periodontitis and orthodontic tooth movement on interleukin (IL)-6 and C-X-C motif chemokine 2 (CXCL2). Materials and methods The effect of periodontitis and/or orthodontic tooth movement (OTM) on alveolar bone and gingival IL-6 and CXCL2 expressions was studied in rats by histology and RT-PCR, respectively. The animals were assigned to four groups (control, periodontitis, OTM, and combination of periodontitis and OTM). The IL-6 and CXCL2 levels were also studied in human gingival biopsies from periodontally healthy and periodontitis subjects by RT-PCR and immunohistochemistry. Additionally, the synthesis of IL-6 and CXCL2 in response to the periodontopathogen Fusobacterium nucleatum and/or mechanical strain was studied in periodontal fibroblasts by RT-PCR and ELISA. Results Periodontitis caused an increase in gingival levels of IL-6 and CXCL2 in the animal model. Moreover, orthodontic tooth movement further enhanced the bacteria-induced periodontal destruction and gingival IL-6 gene expression. Elevated IL-6 and CXCL2 gingival levels were also found in human periodontitis. Furthermore, mechanical strain increased the stimulatory effect of F. nucleatum on IL-6 protein in vitro. Conclusions Our study suggests that orthodontic tooth movement can enhance bacteria-induced periodontal inflammation and thus destruction and that IL-6 may play a pivotal role in this process. Clinical relevance Orthodontic tooth movement should only be performed after periodontal therapy. In case of periodontitis relapse, orthodontic therapy should be suspended until the periodontal inflammation has been successfully treated and thus the periodontal disease is controlled again.

Transcriptional activation by herpes simplex virus type 1 VP16 in vitro and its inhibition by oligopeptides

1994 ◽  
Vol 14 (5) ◽  
pp. 3484-3493
Author(s):  
T J Wu ◽  
G Monokian ◽  
D F Mark ◽  
C R Wobbe
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transcriptional Activation ◽  
Deletion Mutant ◽  
Full Length ◽  
Early Gene ◽  
Immediate Early ◽  
Simplex Virus

VP16 is a herpes simplex virus (HSV)-encoded transcriptional activator protein that is essential for efficient viral replication and as such may be a target for novel therapeutic agents directed against viral gene expression. We have reconstituted transcriptional activation by VP16 in an in vitro system that is dependent on DNA sequences from HSV immediate-early gene promoters and on protein-protein interactions between VP16 and Oct-1 that are required for VP16 activation in vivo. Activation increased synergistically with the number of TAATGARAT elements (the cis-acting element for VP16 activation in vivo) upstream of the core promoter, and mutations of this element that reduce Oct-1 or VP16 DNA binding reduced transactivation in vitro. A VP16 insertion mutant unable to interact with Oct-1 was inactive, but, surprisingly, a deletion mutant lacking the activation domain was approximately 65% as active as the full-length protein. The activation domains of Oct-1 were necessary for activation in reactions containing the VP16 deletion mutant, and they contributed significantly to activation by full-length VP16. Addition of a GA-rich element present in many HSV immediate-early gene enhancers synergistically stimulated VP16-activated transcription. Finally, oligopeptides that are derived from a region of VP16 thought to contact a cellular factor known as HCF (host cell factor) and that inhibit efficient VP16 binding to the TAATGARAT element also specifically inhibited VP16-activated, but not basal, transcription. Amino acid substitutions in one of these peptides identified three residues that are absolutely required for inhibition and presumably for interaction of VP16 with HCF.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3694-3704
Author(s):  
C Prives ◽  
Y Murakami ◽  
F G Kern ◽  
W Folk ◽  
C Basilico ◽  
...  
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Early Gene ◽  
Wild Type ◽  
The Core ◽  
Cell Extracts ◽  
Sequence Requirements

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

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