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Biomedical subjects

O Pongs

Publications and source records attributed to O Pongs.

At least 145 records · Page 8Linked to original sources
PubMed1985

Characterization of glucocorticoid receptor in HeLa-S3 cells.

Glucocorticoid receptor of the human cell line HeLa-S3 has been characterized and has been compared to rat and to mouse glucocorticoid receptors. If HeLa cells were lysed in the absence of glucocorticoid, glucocorticoid receptor was isolated in a nonactivated form, which did not bind to DNA-cellulose. If HeLa cells were preincubated with glucocorticoid, glucocorticoid receptor was isolated in an activated, DNA-binding form. HeLa cell glucocorticoid receptor bound [3H]triamcinolone acetonide with a dissociation constant (KD = 1.3 nM at 0 degrees C) that was similar to those of mouse and rat glucocorticoid receptors. Similarly, the relative binding affinities for steroid hormones decreased in the order of triamcinolone acetonide greater than dexamethasone greater than promegestone greater than methyltrienolone greater than aldosterone greater than or equal to moxestrol. Nonactivated and activated receptors were characterized by high-resolution anion-exchange chromatography (FPLC), DNA-cellulose chromatography, and sucrose gradient centrifugation. Human, mouse, and rat nonactivated glucocorticoid receptors had very similar ionic and sedimentation properties. Activated glucocorticoid receptors were eluted at similar salt concentrations from DNA-cellulose columns but at different salt concentrations from the FPLC column. A monoclonal mouse anti-rat liver glucocorticoid receptor antibody [Westphal, H.M., Mugele, K., Beato, M., & Gehring, U. (1984) EMBO J. 3, 1493-1498] did not cross-react with HeLa cell glucocorticoid receptor. Glucocorticoid receptors of HeLa, HTC, and S49.1 cells were affinity labeled with [3H]dexamethasone and with [3H]dexamethasone 21-mesylate. The molecular weights of [3H]dexamethasone 21-mesylate labeled glucocorticoid receptors (MT 96 000 +/- 1000) were undistinguishable by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1985

In vitro transcription with extracts of nuclei of Drosophila embryos.

An in vitro transcription system has been developed from 0.3M NaCl extracts of nuclei of Drosophila embryos. Optimal transcription in the Drosophila embryo extract (DEX) was at 5mM MgCl2, 70mM KCl, 25 degrees C and with promoter concentrations of 0,75-1.0 pmol/assay. In vitro transcription from the Adenovirus-2 major late and the Drosophila histone gene promoters was studied in particular. S1-nuclease protection experiments showed that in vitro transcription from these promoters was accurate. In vitro transcription from the Adenovirus-2 major late promoter was less efficient than from histone gene H3 and H4 promoters in DEX. Vicecersa, in vitro transcription from Adenovirus-2 major late promoter was more efficient in HeLa whole cell extracts. The efficiencies of transcription from histone gene promoters decreased in DEX in the order H4 greater than or equal to H3 greater than H2a. Transcription from H2b and H1 promoters was not detected in DEX. The transcription from the Adenovirus-2 major late promoter was completely inhibited by histone H3 and H4 promoters. Preincubation of DEX with the adenoviral template, however, did not inhibit transcription from histone H3 and H4 promoters. The transcription start sites of histone genes H3 and H4 are separated by 160 base pairs. The H3 and H4 transcription start sites were subcloned separately. Now, a competition of transcription from the H3/H4 promoters with the Adenovirus-2 major late promoter was observed. The competition studies suggest that preincubation of DEX with the adenoviral template inhibited transcription from the H3 promoter more strongly than from the H4 promoter.

Adenoviridae↗
PubMed1984

Cloning of a gene localized and expressed at the ecdysteroid regulated puff 74EF in salivary glands of Drosophila larvae.

The puffing cycle of salivary gland chromosomes of Drosophila larvae, which initiates the developmental path to pupariation, is induced by ecdysteroid hormone. Its action leads to prominent puffs at loci 2B5, 74EF and 75B. Fragments of the 74EF puff of the D. melanogaster 3L chromosome were microdissected from salivary gland squashes. EcoRI-digested DNA of these fragments was cloned into lambda phage. Clones were screened with puff stage-specific cDNA probes. Thirteen out of 650 clones hybridized preferentially with puff stage 4-specific cDNA. The prominent early puffs at 74EF and 75B are most active between puff stage 4 and 6. Therefore, one of the 13 lambda phages was chosen for further analysis. It was used to isolate 24 kb of Drosophila DNA from genomic libraries. The DNA hybridized in situ to locus 74F. The 74F DNA coded for a transcript, which was made in salivary glands, but not in fat body of third instar larvae. It accumulated in K(C) cells in response to ecdysteroid treatment. The polyadenylated transcript size was 2.7 kb as judged by Nothern blot analysis. The transcription start site of the 74F gene has been mapped. Sequences upstream of the transcription site contain several sequence elements common to other eucaryotic genes, including potential Z-DNA forming sequences. Also, there is sequence homology to upstream sequences, which have been involved in the regulation of transcription of the salivary gland glue protein 4 gene.

Journal Article↗
PubMed1983

Puffing activities and binding of ecdysteroid to polytene chromosomes of Drosophila melanogaster.

Salivary glands of third instar Drosophila melanogaster larvae were incubated in vitro in the presence of 5 x 10(-6) M 20-hydroxy-ecdysone. Steroid hormone was localized on the polytene chromosomes of the salivary gland by a combination of photoaffinity-labeling and indirect immunofluorescence microscopy. Steroid hormone binding to chromosomal loci and their puffing activity was correlated for the larval/prepupal puffing cycle characterized by puff stages 1-10. In general, there was a good correlation between the sequential and temporal puffing activity induced by 20-hydroxy-ecdysone and the binding of ecdysteroid hormone to these puffs. Ecdysteroid hormone was detected at intermolt, and at early and late puffs with two notable exceptions. Ecdysteroid was not detected at the two well-studied puffs at 23E and at 25AC, the former being an early puff, which is activated in the presence of 20-hydroxy-ecdysone, and the latter being an intermolt puff, which regresses more rapidly in the presence of hormone. Ecdysteroid hormone was present at puffs as long as the respective puff was active. Also, it apparently accumulated at late puff sites after induction. Since ecdysteroid binding to chromosomal loci is temporal as well as sequential during the larval/prepupal puffing cycle, additional factors besides steroid hormone are necessary for sequentially regulating puffing and concomitant gene activity during development from larvae to prepupae.

Animals↗
PubMed1982

Ecdysterone induced protein synthesis in salivary glands and in fat body of Drosophila melanogaster larvae.

Salivary glands of 3rd instar larvae of Drosophila melanogaster were labeled with 3H-leucine in the presence and absence of ecdysterone. Twenty-six ecdysterone inducible proteins were detected. Their induction was correlated with puff stage. Synthesis of fifteen proteins commenced during early puff stage (PS2); synthesis of seven others at late puff stages (PS8-10). Synthesis of four proteins was induced between puff stage 3/4 and 7/8. Thus, the hormonal induction of protein synthesis generally reflected the appearance of early and of late puffs as described by Ashburner (1972). Eleven ecdysterone inducible proteins were detected in larval fat body in vitro. Comparison of the fat body to the salivary gland proteins revealed that one of the ecdysterone induced fat body proteins was identical in molecular weight and charge to one of the proteins induced by ecdysterone in salivary glands.

Adipose Tissue↗
PubMed1982

Identification and characterization of the ecdysterone receptor in Drosophila melanogaster by photoaffinity labeling.

Salivary glands of third-instar larvae of Drosophila melanogaster as well as Drosophila K(c) tissue culture cells have been irradiated in the presence of ecdysterone. Irradiation covalently links ecdysterone to a single cellular protein, which is similar, if not identical, in salivary glands and in K(c) cells. This protein has a molecular weight of 130,000 and it has the characteristics of a typical hormone-receptor molecule in terms of hormone-binding properties, translocation into the nucleus, and sedimentation characteristics. The yield of the photoinduced bonding of ecdysterone to receptor protein is around 15%. Ponasterone A competed with ecdysterone for the bonding. Also, ponasterone A itself reacted upon photoactivation with the beta-ecdysterone receptor protein in Drosophila tissue culture cells. We have previously shown that ecdysterone can be bonded upon irradiation to specific hormone-controlled puffs of polytene chromosomes of D. melanogaster third-instar larvae [Gronemeyer, H. & Pongs, O. (1980) Proc. Natl. Acad. Sci. USA 77, 2108-2112]. Because we have now identified the molecular target of the ecdysterone photoreaction, these data show that a hormone-receptor complex translocates to the nucleus and directly binds to the genes, which are under hormonal control. A quantitative assay of hormone-receptor complex in K(c) cells before and after hormone stimulation showed that ecdysterone does not regulate the synthesis and the available amount of its receptor. It was also observed that the translocated hormone-receptor complex resides in the nucleus as long as the hormone is present in the tissue culture medium.

Journal Article↗
PubMed1981

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.

Animals↗
PubMed1980

Translation initiation factor 2 alters transcriptional selectivity of Escherichia coli ribonucleic acid polymerase.

Preparations of translation initiation factor IF-2 strongly stimulate the production of rRNA by Escherichia coli RNA polymerase but have little effect on the synthesis of other RNA species. The factor alters the sedimentation characteristics of RNA polymerase holoenzyme. We suggest a mechanism by which IF-2 alters the pattern of transcriptional selectivity of RNA polymerase.

DNA-Directed RNA Polymerases↗
PubMed1980

Formylmethionyl-tRNA alters RNA polymerase specificity.

Escherichia coli fMet-tRNAfMet alters the pattern of promoter selection of E. coli RNA polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6), affecting RNA synthesis from the rRNA, suIII+tRNA, and lac promoters in different ways. The in vitro synthesis of the stable RNA species is selectively decreased, whereas that of lac RNA from both the wild-type and mutant UV5 promoters is selectively increased at high ionic strength. The functional effect of fMet-tRNAfMet resembles that of the nucleotide guanosine 3'-diphosphate 5'-diphosphate (ppGpp). This nucleotide competes with the binding of fMet-tRnafMet to RNA polymerase.

Binding, Competitive↗
PubMed1980

Localization of ecdysterone on polytene chromosomes of Drosophila melanogaster.

Ecdysterone has been crosslinked in situ to polytene chromosomes of salivary glands of Drosophila melanogaster by photoactivation. The crosslinked hormone has been localized on the chromosomes by indirect immunofluorescence microscopy. At different developmental stages the hormone was detected at different chromosomal loci. These chromosomal sites correspond to ecdysterone-inducible puff sites. Thus, the hormone binds directly to chromosomal loci, whose transcription depends on the presence of the hormone. Ecdysterone was also crosslinked to one puff site that regresses during larval development. This indicates that (i) hormone binding to polytene chromosomes activates transcription at specific loci, and (ii) hormone-dependent regression of intermolt puff 68C is mediated by direct binding of ecdysterone. Ecdysterone was not detected on puff sites that are independent of hormone action or in chromosomal interbands. After heat shock, no ecdysterone became crosslinked to polytene chromosomes.

Animals↗
PubMed1978

Identification of cysteine-10 of protein S18 as part of the mRNA-binding site of Escherichia coli ribosomes by affinity-labeling studies with a chemically reactive A-U-G analog.

The reaction of a bromoacetamidophenyl derivative of the initiation codon A-U-G (A-U-G) with tight couples of Escherichia coli ribosomes leads to an exclusive crosslinking of label to protein S18. This crosslinking inhibits A-U-G-directed fMet-tRNAfMet binding into the puromycin-sensitive site of ribosomes and stimulates elongation-factor-dependent binding of Met-tRNAmMet. It is, therefore, concluded that protein S18 is located at or near the aminoacyl-tRNA binding site of E. coli ribosomes. Peptide as well as amino acid analysis shows that the reaction between A-U-G and ribosomes took place at cysteine-10 of protein S18. A-U-G could not be crosslinked to ribosomal proteins of the temperature-sensitive E. coli strain 258ts, where arginine-11 of protein S18 is replaced by a cysteine residue.

Affinity Labels↗