scholarly journals In vitro transcription of heat-shock-specific RNA from chromatin of Drosophila melanogaster cells.

1978 ◽  
Vol 75 (2) ◽  
pp. 759-763 ◽  
Author(s):  
H. Biessmann ◽  
B. Levy ◽  
B. J. McCarthy
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
In Vitro Transcription

Cloning the gene for the heat shock response positiwe regulator (sigma 32 homolog) from Pseudomonas aeruginosa

1995 ◽  
Vol 41 (1) ◽  
pp. 75-87 ◽  
Author(s):  
Zerlina M. Naczynski ◽  
Andrew M. Kropinski ◽  
Chris Mueller
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Heat Shock ◽  
Sigma Factor ◽  
Oligonucleotide Probe ◽  
In Vitro Transcription ◽  
Translation System ◽  
Amino Acid Homology ◽  
E Coli ◽  
Transcriptional Start Sites

A 31 base pair synthetic oligonucleotide based on the genes for the Escherichia coli heat shock sigma factor (rpoH) and the Pseudomonas aeruginosa housekeeping sigma factor (rpoD) was employed in conjunction with the Tanaka et al. (K. Tanaka, T. Shiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040–1042) RpoD box probe to identify the location of the rpoH gene in P. aeruginosa genomic digests. This gene was cloned into plasmid pGEM3Z(f+), sequenced, and found to share 67% nucleotide identity and 77% amino acid homology with the rpoH gene and its product (σ32) of E. coli. The plasmid containing the rpoH gene complemented the function of σ32 in an E. coli rpoH deletion mutant. Furthermore, this plasmid directed the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transcription–translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50 °C) conditions. Two promoter sites were identified having sequence homology to the E. coli σ70 and σ24 consensus sequences.Key words: heat shock, Pseudomonas aeruginosa, sigma factor, transcription, oligonucleotide probe.

scholarly journals Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region.

1987 ◽  
Vol 7 (6) ◽  
pp. 2046-2051 ◽  
Author(s):  
A D Garcia ◽  
A M O'Connell ◽  
S J Sharp
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
Upstream Region ◽  
Nucleotide Position ◽  
Wild Type ◽  
5S Rna ◽  
Cell Extracts

We constructed deletion-substitution and linker-scanning mutations in the 5'-flanking region of the Drosophila melanogaster 5S RNA gene. In vitro transcription of these templates in Drosophila and HeLa cell extracts revealed the presence of an essential control region (-30 region) located between nucleotides -39 and -26 upstream of the transcription initiation site: deletion of sequences upstream of nucleotide position -39 had no detectable effect on the wild-type level of in vitro transcription, whereas mutations extending between positions -39 and 1 resulted in templates with decreased transcriptional levels; specifically, deletion and linker-scanning mutations in the -34 to -26 region (-30 region) resulted in loss of transcription. The -30 region is essential for transcription and therefore forms part of the Drosophila 5S RNA gene transcription promoter. Compared with the activity of the wild-type gene, mutant 5S DNAs exhibited no impairment in the ability to sequester limiting transcription factors in a template exclusion competition assay. While we do not know which transcription factor(s) interacts with the -30 region, the possible involvement of RNA polymerase III at this region is discussed.

Correlation between the activity of a 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-insensitive puff and the synthesis of major heat-shock polypeptide, hsp70, in Chironomus thummi

1988 ◽  
Vol 66 (11) ◽  
pp. 1177-1185 ◽  
Author(s):  
D. Barettino ◽  
G. Morcillo ◽  
J. L. Díez ◽  
M. T. Carretero ◽  
M. J. Carmona
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Specific Inhibitor ◽  
In Vitro Transcription ◽  
The Other ◽  
Low Salt ◽  
Transcription Inhibitor ◽  
Chironomus Thummi

The induction of puff III-A3b, a major heat-shock puff in Chironomus thummi salivary cells, was insensitive to the transcription inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), whereas no transcriptional activity could be detected at the other heat-shock puffs in the presence of this drug. In these conditions, a polypeptide with the same Mr and isoform pattern as those of the major heat-shock polypeptide, hsp70, was synthesized. These results suggest that hsp70 is encoded by locus III-A3b. In addition to DRB insensitivity, incorporation of [3H]UTP on puff III-A3b took place in an in vitro transcription assay under low-salt conditions (100 mM NaCl); no labelling could be detected at the other heat-shock puffs under these conditions. Although DRB has been reported as a specific inhibitor of RNA polymerase II-directed transcription, and although the low-salt conditions were not propitious for the activity of this enzyme, RNA polymerase II was detected on puff III-A3b and on the other heat-shock puffs by immunofluorescence with anti-RNA polymerase II antibodies.

scholarly journals Functional Analysis of the Heat Shock Regulator HrcA of Chlamydia trachomatis

2002 ◽  
Vol 184 (23) ◽  
pp. 6566-6571 ◽  
Author(s):  
Adam C. Wilson ◽  
Ming Tan
Keyword(s):  
Heat Shock ◽  
Chlamydia Trachomatis ◽  
In Vitro Transcription ◽  
Negative Regulator ◽  
Heat Shock Gene ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Topological State ◽  
Heat Shock Gene Expression

ABSTRACT HrcA is a regulator of bacterial heat shock gene expression that binds to a cis-acting DNA element called CIRCE. It has been proposed that HrcA and CIRCE function as a repressor-operator pair. We have purified recombinant HrcA from the pathogenic bacterium Chlamydia trachomatis and have shown that it is a DNA-binding protein that functions as a negative regulator of transcription. HrcA bound specifically to the CIRCE element in a concentration-dependent manner. HrcA repressed the in vitro transcription of a chlamydial heat shock promoter, and this repression was promoter specific. HrcA-mediated repression appears to be dependent on the topological state of the promoter, as repression on a supercoiled promoter template was greater than that on a linearized template. These results provide direct support for the role of HrcA as a transcriptional repressor in bacteria. This is the first report of the in vitro reconstitution of transcriptional regulation in Chlamydia.

scholarly journals TATA box-dependent protein-DNA interactions are detected on heat shock and histone gene promoters in nuclear extracts derived from Drosophila melanogaster embryos.

1988 ◽  
Vol 8 (8) ◽  
pp. 3204-3214 ◽  
Author(s):  
D S Gilmour ◽  
T J Dietz ◽  
S C Elgin
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Transcriptional Activation ◽  
Tata Box ◽  
Gene Promoters ◽  
Dna Interactions ◽  
Nuclear Extracts ◽  
Protein Dna Interactions ◽  
Exonuclease Digestion

We monitored protein-DNA interactions that occur on the hsp26, hsp70, histone H3, and histone H4 promoters in nuclear extracts derived from frozen Drosophila melanogaster embryos. All four of these promoters were found to be transcribed in vitro at comparable levels by extracts from both heat-shocked and non-heat-shocked embryos. Factors were detected in both types of extracts that block exonuclease digestion from a downstream site at ca. +35 and -20 base pairs from the start of transcription of all four of these promoters. In addition, factors in extracts from heat-shocked embryos blocked exonuclease digestion at sites flanking the heat shock consensus sequences of hsp26 and hsp70. Competition experiments indicated that common factors cause the +35 and -20 barriers on all four promoters in both extracts. The formation of the barriers at +35 and -20 required a TATA box but did not appear to require specific sequences downstream of +7. We suggest that the factors responsible for the +35 and -20 barriers are components whose association with the promoter precedes transcriptional activation.

scholarly journals Insulating DNA directs ubiquitous transcription of the Drosophila melanogaster alpha 1-tubulin gene.

1994 ◽  
Vol 14 (9) ◽  
pp. 6398-6408 ◽  
Author(s):  
K H O'Donnell ◽  
C T Chen ◽  
P C Wensink
Keyword(s):  
Drosophila Melanogaster ◽  
Developmental Stages ◽  
Germ Line ◽  
Core Promoter ◽  
High Sensitivity ◽  
In Vitro Transcription ◽  
Gene Products ◽  
Position Effects ◽  
Germ Line Transformation

We identify DNA regions that are necessary for the ubiquitous expression of the Drosophila melanogaster alpha 1-tubulin (alpha 1t) gene. In vitro transcription showed that two upstream regions, tubulin element 1 (TE1 [29 bp]) and tubulin element 2 (TE2 [68 bp]), and a downstream region activate transcription. Germ line transformation demonstrated that these three regions are sufficient to direct the alpha 1t core promoter to begin transcribing at the stage of cellular blastoderm formation and to continue thereafter at high levels in all tissues and developmental stages. Remarkably, mutation of any one of these regions results in high sensitivity to chromosomal position effects, producing different but reproducible tissue-specific patterns of expression in each transformed line. None of these regions behaves as an enhancer in a conventional germ line transformation test. These observations show that these three regions, two of which bind the GAGA transcription factor, act ubiquitously to insulate from position effects and to activate transcription. The results also provide vectors for ubiquitous expression of gene products and for examining silencer activities.

Detection of low-level gene induction using in vitro transcription heat shock genes

1985 ◽  
Vol 2 (6) ◽  
pp. 100-107 ◽  
Author(s):  
Jack D. Love ◽  
Alfred A. Vivino ◽  
Kenneth W. Minton
Keyword(s):  
Heat Shock ◽  
Gene Induction ◽  
In Vitro Transcription ◽  
Heat Shock Genes ◽  
Low Level

Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region

1987 ◽  
Vol 7 (6) ◽  
pp. 2046-2051
Author(s):  
A D Garcia ◽  
A M O'Connell ◽  
S J Sharp
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
Upstream Region ◽  
Nucleotide Position ◽  
Wild Type ◽  
5S Rna ◽  
Cell Extracts

We constructed deletion-substitution and linker-scanning mutations in the 5'-flanking region of the Drosophila melanogaster 5S RNA gene. In vitro transcription of these templates in Drosophila and HeLa cell extracts revealed the presence of an essential control region (-30 region) located between nucleotides -39 and -26 upstream of the transcription initiation site: deletion of sequences upstream of nucleotide position -39 had no detectable effect on the wild-type level of in vitro transcription, whereas mutations extending between positions -39 and 1 resulted in templates with decreased transcriptional levels; specifically, deletion and linker-scanning mutations in the -34 to -26 region (-30 region) resulted in loss of transcription. The -30 region is essential for transcription and therefore forms part of the Drosophila 5S RNA gene transcription promoter. Compared with the activity of the wild-type gene, mutant 5S DNAs exhibited no impairment in the ability to sequester limiting transcription factors in a template exclusion competition assay. While we do not know which transcription factor(s) interacts with the -30 region, the possible involvement of RNA polymerase III at this region is discussed.

scholarly journals Protective Effect of Diploschistes ocellatus Against Heat Shock-Mediated Defects on Function of Reproductive Organs in Drosophila melanogaster

2019 ◽  
Vol 4 (2) ◽  
pp. 51-55
Author(s):  
Mohammad Haddadi ◽  
Javad Payam
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Aqueous Extract ◽  
Therapeutic Potential ◽  
Culture Media ◽  
Reproductive Function ◽  
Control Group ◽  
Reproduction Rate ◽  
Protective Function

Introduction: Repeated heat shock (HS) stresses reduce the reproduction rate of Drosophila flies. Heat shock proteins (HSPs) protect cells against irreversible damages inducing heatinduced.Oxidative stress declines protective function of HSPs. Diploschistes ocellatus lichen aqueous extract possesses a strong antioxidant potential in vitro. Antioxidants can preserve HSPs function. Therefore, the present study for the first time investigated the cytoprotective effects of D. ocellatus aqueous extract against HS-mediated deleterious effects on reproductive function in Drosophila melanogaster. Methods: Three different types of culture media including control, 30% lichen extract, and 60%lichen extract were prepared. Adult D. melanogaster flies were placed on Delcour medium and allowed to lay eggs for 2 hours. Then the eggs were equally distributed between the culture media. After flies completed their life cycle, the adult enclosed flies were exposed to HS. To assess reproductive function, the newly emerged adult flies were transferred to the freshly prepared regular culture medium every three days for 3 times and finally adult offspring born to these flies were enumerated.Results: HS negatively affected the reproduction rate in flies in control group. Quantification of adult enclosed flies born to the D. ocellatus extract treated flies showed that lichen extract could negate the deleterious effects of HS on reproduction function of D. melanogaster in a dose-dependent manner.Conclusion: Diploschistes ocellatus aqueous extract attenuated the harmful effects of HS stress on reproductive function of D. melanogaster. The secondary metabolites present in D. ocellatus can be considered as a bona fide candidate in novel drug development to target reproductive diseases in which oxidative stress is involved. Moreover, it can be concluded that D. melanogaster is an ideal model organism to induce cellular stress in vitro and study therapeutic potential of lichen extracts.

scholarly journals In vitro transcription of Drosophila actin and 70,000-dalton heat shock protein genes.

1983 ◽  
Vol 258 (20) ◽  
pp. 12618-12623
Author(s):  
W C Nierman ◽  
A E Miller ◽  
S L Tobin ◽  
T D Ingolia ◽  
F Sanchez ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
In Vitro Transcription ◽  
Heat Shock Protein Genes
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