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.

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.

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.

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.

A modification of Heidenhain's iron-haematoxylin technique, as used to stain smears of Drosophila larval polytene chromosomes and neuroblast cells

1971 ◽  
Vol 49 (1) ◽  
pp. 132-133 ◽  
Author(s):  
Albert E. Moorman
Keyword(s):  
Acetic Acid ◽  
Salivary Gland ◽  
Methyl Alcohol ◽  
Polytene Chromosomes ◽  
Larval Salivary Gland

Acetic-acid-fixed smears of Drosophila larval salivary gland chromosomes and of neuroblast cells from the larval ganglion undergoing mitosis are prepared by a modification of Heidenhain's iron-haematoxylin technique, in which absolute methyl alcohol is the solvent of all reagents used in the staining process.

p75, a polypeptide component of karyoskeletal protein-enriched fractions associated with transcriptionally active loci of Drosophila melanogaster polytene chromosomes

1988 ◽  
Vol 8 (5) ◽  
pp. 1877-1886
Author(s):  
B M Benton ◽  
S Berrios ◽  
P A Fisher
Keyword(s):  
Tissue Culture ◽  
Drosophila Melanogaster ◽  
Transcriptional Regulation ◽  
Salivary Gland ◽  
Indirect Immunofluorescence ◽  
Polytene Chromosomes ◽  
Nuclear Interior ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Larval Salivary Gland

A 75-kilodalton polypeptide has been identified which copurifies with karyoskeletal protein-enriched fractions prepared from Drosophila melanogaster embryos. Results of indirect immunofluorescence experiments suggest that this protein, here designated p75, is primarily associated with puffed regions of larval salivary gland polytene chromosomes. In nonpolytenized Schneider 2 tissue culture cells, p75 appeared to be localized throughout the nuclear interior during interphase. In mitotic cells, p75 was redistributed diffusely. A possible role for karyoskeletal elements in transcriptional regulation is discussed.

Chromocentre polymorphism in polytene chromosomes of Simulium costatum (Diptera: Simuliidae)

Genome ◽  
1989 ◽  
Vol 32 (4) ◽  
pp. 510-515 ◽  
Author(s):  
C. Brockhouse ◽  
J. A. B. Bass ◽  
N. A. Straus
Keyword(s):  
Salivary Gland ◽  
Polytene Chromosome ◽  
Polytene Chromosomes ◽  
Black Fly ◽  
Weinberg Equilibrium ◽  
Larval Salivary Gland ◽  
Hybrid Introgression ◽  
Hardy Weinberg Equilibrium ◽  
Two Populations

The polytene chromosomes of the black fly species Simulium (Nevermannia) costatum are joined at the centromeres in a strongly heterochromatic chromocentre. Examination of the larval salivary gland chromosomes revealed two populations with a unique polymorphism for attachment to the chromocentre involving all centromeres. All three homologous pairs of chromosomes are polymorphic for centromeres that do not join to the chromocentre. Samples from one of these populations were large enough for thorough study. In this population, the attachment polymorphism is in Hardy-Weinberg equilibrium for two of the centromeres and was in the same frequency for 2 successive years of sampling. The polymorphism could be either primary, retained from an ancestral nonchromocentric state, or secondary, evolving independently or introduced via hybrid introgression. The evolution of chromocentres is discussed in the context of species in the Simulium vernum group.Key words: black fly, polytene chromosome, chromocentre, polymorphism, evolution.

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.

scholarly journals p75, a polypeptide component of karyoskeletal protein-enriched fractions associated with transcriptionally active loci of Drosophila melanogaster polytene chromosomes.

1988 ◽  
Vol 8 (5) ◽  
pp. 1877-1886 ◽  
Author(s):  
B M Benton ◽  
S Berrios ◽  
P A Fisher
Keyword(s):  
Tissue Culture ◽  
Drosophila Melanogaster ◽  
Transcriptional Regulation ◽  
Salivary Gland ◽  
Indirect Immunofluorescence ◽  
Polytene Chromosomes ◽  
Nuclear Interior ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Larval Salivary Gland

A 75-kilodalton polypeptide has been identified which copurifies with karyoskeletal protein-enriched fractions prepared from Drosophila melanogaster embryos. Results of indirect immunofluorescence experiments suggest that this protein, here designated p75, is primarily associated with puffed regions of larval salivary gland polytene chromosomes. In nonpolytenized Schneider 2 tissue culture cells, p75 appeared to be localized throughout the nuclear interior during interphase. In mitotic cells, p75 was redistributed diffusely. A possible role for karyoskeletal elements in transcriptional regulation is discussed.

scholarly journals The Drosophila salivary gland chromocenter contains highly polytenized subdomains of mitotic heterochromatin.

Genetics ◽  
1995 ◽  
Vol 139 (2) ◽  
pp. 659-670 ◽  
Author(s):  
P Zhang ◽  
A C Spradling
Keyword(s):  
Salivary Gland ◽  
Dna Sequences ◽  
Polytene Chromosomes ◽  
Centromeric Heterochromatin ◽  
Mitotic Chromosomes ◽  
Satellite Dnas ◽  
Central Mass ◽  
P Elements

Abstract Peri-centromeric regions of Drosophila melanogaster chromosomes appear heterochromatic in mitotic cells and become greatly underrepresented in giant polytene chromosomes, where they aggregate into a central mass called the chromocenter. We used P elements inserted at sites dispersed throughout much of the mitotic heterochromatin to analyze the fate of 31 individual sites during polytenization. Analysis of DNA sequences flanking many of these elements revealed that middle repetitive or unique sequence DNAs frequently are interspersed with satellite DNAs in mitotic heterochromatin. All nine Y chromosome sites tested were underrepresented > 20-fold on Southern blots of polytene DNA and were rarely or never detected by in situ hybridization to salivary gland chromosomes. In contrast, nine tested insertions in autosomal centromeric heterochromatin were represented fully in salivary gland DNA, despite the fact that at least six were located proximal to known blocks of satellite DNA. The inserted sequences formed diverse, site-specific morphologies in the chromocenter of salivary gland chromosomes, suggesting that domains dispersed at multiple sites in the centromeric heterochromatin of mitotic chromosomes contribute to polytene beta-heterochromatin. We suggest that regions containing heterochromatic genes are organized into dispersed chromatin configurations that are important for their function in vivo.

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