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R Renkawitz

Publications and source records attributed to R Renkawitz.

68 records · Page 4Linked to original sources
PubMed1987

Glucocorticoid induction of the rat tryptophan oxygenase gene is mediated by two widely separated glucocorticoid-responsive elements.

Transcription of the gene coding for tryptophan oxygenase (TO) in rat liver is induced 10-fold by glucocorticoids. To identify DNA elements mediating the glucocorticoid-regulated expression of the TO gene we transfected mouse L cells with a fusion gene consisting of 1.95 kb TO 5'-flanking sequences linked to the coding sequence of the gene for chloramphenicol acetyltransferase (CAT). CAT assay and RNA mapping experiments demonstrate that both transient and stable expression of the TO-CAT fusion gene are inducible by dexamethasone. Analysis of transcripts from 5'-deletion mutants identifies two glucocorticoid-responsive elements (GRE), located 450 bp and 1.2 kb upstream of the cap site. The purified rat glucocorticoid receptor binds to the sequence of each GRE as evidenced from footprinting experiments. Interestingly the protected sequence of the proximal footprint is by itself not sufficient for sequence induction, but requires sequences located immediately upstream.

Adrenalectomy↗
PubMed1985

rRNA processing: removal of only nineteen bases at the gap between 28S alpha and 28S beta rRNAs in Sciara coprophila.

We have determined the sequence of the rDNA region between the 28S alpha and 28S beta rRNA coding segments (termed a "gap") in the insect Sciara coprophila, and have used S1 nuclease mapping and cDNA primer extension to define the 5' and 3' boundaries of the gap. Only 19 bases found in rDNA at the gap region are absent from mature 28S rRNA. Eukaryotic rRNAs contain stretches of nucleotides ("expansion segments") which are absent in E. coli rRNA. The gap region in Sciara is located within expansion segment V. Therefore, the excision of 19 bases in the Sciara gap suggests that a large portion of expansion segment V plays no function in mature ribosomes. Specific sequences conserved in Sciara and Drosophila are considered as candidates for recognition signals for the excision of the gap transcript.

Animals↗
PubMed1984

Tissue-specific DNaseI hypersensitive sites in the 5'-flanking sequences of the tryptophan oxygenase and the tyrosine aminotransferase genes.

The genes for tryptophan oxygenase (TO) and tyrosine aminotransferase (TAT) are expressed in a tissue- and development-specific manner and are regulated by glucocorticoids (TO and TAT) and glucagon or its intracellular mediator cAMP (TAT) in rat liver. We have analyzed the chromatin structure of these genes in the vicinity of the 5' ends with regard to DNaseI hypersensitivity and have found DNaseI hypersensitive sites upstream of each of the promoters. Mapping of this region reveals three closely spaced cleavage sites near the TO promoter and a doublet of sites near the TAT promoter. In both genes additional cleavage sites are found further upstream. All hypersensitive sites of both genes are absent in kidney nuclei and therefore appear to be specific for the tissue expressing the genes. A correlation of expression and modified chromatin structure was also observed in a hepatoma cell line expressing TAT but not TO: hypersensitive sites are present in TAT but not in TO chromatin. Upon glucocorticoid induction an additional hypersensitive site is detected approximately 2 kb upstream of the TAT promoter in liver and hepatoma cells.

Animals↗
PubMed1984

Steroid controlled expression of the chicken lysozyme and the rat tryptophan oxygenase gene after transfer into eukaryotic cells.

To study the mechanism of steroid induced transcriptional control we have introduced recombinants of the chicken lysozyme gene and the rat tryptophan oxygenase (TO) gene into heterologous and homologous cells. To monitor the activity of the TO-promoter, 1.9 kb of the TO 5'-flanking sequences were fused with sequences coding for the bacterial enzyme chloramphenicol acetyltransferase (CAT). Upon transfer into mouse L-cells the transient expression of the TO-CAT recombinant was found to be inducible by dexamethasone. Transient expression of chicken lysozyme gene recombinants after introduction into various cell types could only be detected in chicken oviduct cells, or in conjunction with SV-40 enhancer sequences in human cells. The recombinant gene used in oviduct cells was a fusion between the lysozyme promoter, including 1.4 kb of upstream sequences, and the coding region of the gene for SV 40 T-antigen (plys-T). The expression in oviduct cells was stimulated by dexamethasone or progesterone, whereas SV 40 enhanced expression of lysozyme sequences in human cells could not be regulated by steroids. Using several deletion mutants, a region between -220 bp and -140 bp upstream of the cap site was found to be essential for both regulation by glucocorticoids as well as by progesterone.

Animals↗
PubMed1984

Sequences in the promoter region of the chicken lysozyme gene required for steroid regulation and receptor binding.

We have constructed a series of deletion mutants in the lysozyme promoter region fused to the SV40 T-antigen coding region. Regulated expression was tested after microinjection of the lysozyme deletion mutants into primary cultures of chicken oviduct cells using fluorescent antibodies against T antigen. Deletion of lysozyme gene sequences upstream of position - 164 was accompanied by loss of both progesterone- and glucocorticoid-induced expression. Using the rat liver glucocorticoid receptor for binding studies, two separate binding sites have been identified: a strong binding site that is destroyed by deletion of lysozyme sequences between positions -74 and -39 and a weaker binding site contained between positions -208 and -161 upstream of the lysozyme cap site.

Amino Acid Sequence↗
PubMed1983

Transcriptional regulation of the tryptophan oxygenase gene in rat liver by glucocorticoids.

The enzyme tryptophan oxygenase (EC 1.13.11.11), which is synthesized in rat liver, is induced by glucocorticoids. We have used cloned tryptophan oxygenase genomic and cDNA sequences to study the mechanism of induction. Rat liver poly(A+) RNA was separated on a formaldehyde gel, blotted to nitrocellulose, and hybridized to a nick-translated tryptophan oxygenase cDNA clone to analyze the kinetics of tryptophan oxygenase mRNA accumulation. Transcription in isolated rat liver nuclei was investigated to determine the relative rate of transcription of the tryptophan oxygenase gene. Analysis of the accumulation of albumin mRNA, which is unaffected by glucocorticoids, served as an internal control. We show here that the synthetic glucocorticoid dexamethasone causes a 10-fold increase in the concentration of tryptophan oxygenase mRNA sequences in rat liver, and that this is a consequence of transcriptional activation of the tryptophan oxygenase gene.

Animals↗
PubMed1982

Transient expression of the chicken lysozyme gene after transfer into human cells.

The chicken lysozyme gene was inserted into an SV40-based plasmid vector, and the recombinants were transfected into the human cell lines HeLa and MCF-7. Correct and efficient transient expression directed by the lysozyme promoter was found in both of these cell lines, as determined by S1 nuclease mapping and Northern blot analysis of the RNAs made. SV40 sequences dramatically enhance the expression of the lysozyme gene. This enhancing effect is only acting in cis and is distance/orientation dependent, since clones containing the lysozyme gene in either orientation produce different amounts of correct lysozyme transcripts. The transfected lysozyme gene was not induced by steroid hormone treatment of the cells.

Breast Neoplasms↗
PubMed1982

Expression of a chicken lysozyme recombinant gene is regulated by progesterone and dexamethasone after microinjection into oviduct cells.

We transferred a chicken lysozyme gene recombinant by microinjection into primary cultures of chicken oviduct cells. The recombinant gene is a fusion between the lysozyme promoter, including 1.4 kb of upstream sequences, and the coding region of the gene for SV40 T antigen (plys-T). The expression of plys-T is stimulated by the steroid hormones progesterone and dexamethasone, but not by estradiol. The number of oviduct cells expressing coinjected or separately injected control genes is not increased by steroids. A deletion mutant lacking the lysozyme sequences between -161 and +15 does not express T antigen, indicating that transcription of plys-T starts within the lysozyme promoter region. By screening different cell types we found that microinjected plys-T is expressed in chicken oviduct cells but not in chicken macrophages or fibroblasts or in rat II fibroblasts.

Animals↗
PubMed1979

Ribosomal DNA of fly Sciara coprophila has a very small and homogeneous repeat unit.

In this report we show by hybridization of restriction fragments and by Miller spreads that the unit repeat of the fly Sciara coprophila is only 8.4 kb which is the smallest known for a multicellular eukaryote. The 8.4 kb EcoR1 fragment containing a complete unit of Sciara rDNA was cloned in pBR322, and mapped by the method of Parker (1977) and also by double digestions. The coding regions for 28S, 18S, and 5.8S RNA were localized by the method of Berk and Sharp (1977). From these data we conclude that the nontranscribed spacer, external transcribed spacer, and internal transcribed spacer are all shorter than in other organisms, thereby giving rise to the shorter overall rDNA repeat unit of Sciara. At least 90% of the Sciara rDNA repeats are homogeneous, with a length of 8.4 kb, but a 700 bp ladder of minor bands can also be found in digestions of total genome DNA. This profile of major and minor bands is identical between the X and X' chromosomes, as seen by a comparison of several genotypes. There are only 45 rRNA genes per X chromosome of Sciara (Gerbi and Crouse, 1976). These can easily be counted by low magnification Miller spreads which show that virtually all gene copies are actively being transcribed in the stage of spermatogenesis examined. This is the first demonstration for any reiterated gene family where all copies are shown to be simultaneously active.

Animals↗
PubMed1975

Independent replication of the ribosomal RNA genes in the polytrophic-meroistic ovaries of Calliphora erythrocephala, Drosophila hydei, and Sarcophaga barbata.

By filter saturation hybridizations the ribosomal (r)DNA contents of the ovaries Calliphora erythrocephala, Drosophila hydei, and Sarcophaga barbata have been measured in comparison to the rDNA percentages of their diploid brains. The measurements of the ovarian rDNA have been carried out on ovaries where the nurse cells in the distal egg chamber of the ovarioles had reached their highest ploidy level. The diploid rDNA content of each of the respective species was chosen as a 100% standard and the rDNA amounts of the ovaries were related to this 100% level. The results show that the ovaries of C. erythrocephala contain 135% rDNA whereas the rDNA contents in the ovaries of D. hydei and S. barbata are only 51% and 47%, respectively. Measurements carried out on isolated nuclei of the nurse cells and follicle cells in D. hydei show that both have a reduced rDNA content in comparison to the brains (45% and 70%, respectively). The data are discussed in relation to the problem of an rDNA amplification in the germ cells and an rDNA underreplication in polyploid nuclei.

Animals↗
PubMedDate not supplied

Glucocorticoid and progesterone receptors bind to the same sites in two hormonally regulated promoters.

The glucocorticoid receptor of rat liver recognizes nucleotide sequences near the promoter of mouse mammary tumour virus (MMTV) required for hormonal induction in gene transfer experiments. Similar nucleotide sequences have been found in the human metallothionein gene IIA and in the chicken lysozyme gene, the later induced also by oestrogen, progesterone and androgens. In microinjection experiments, deletion of only 44 base pairs (bp) of the lysozyme promoter (from -208 to -164) results in coordinated loss of progesterone and glucocorticoid-dependent gene expression. We show here that purified glucocorticoid receptor from rat liver and progesterone receptor from rabbit uterus yield similar or overlapping exonuclease III footprints in the promoter regions of MMTV and chicken lysozyme. Thus, the regulatory elements for different steroid hormones may be similar or at least share structural features.

Animals↗
PubMedDate not supplied

Promoter specific sensitivity to inhibition of histone deacetylases: implications for hormonal gene control, cellular differentiation and cancer.

Alterations in histone acetylation status appear to play a central role in the regulation of neoplasia, tumor suppression, cell cycle control, hormone responsiveness and senescence. These alterations of chromatin control gene transcription. The histone acetylation status is regulated by the equilibrium of histone acetyl-transferase activity (HAT) and the histone deacetylase activity (HDAC). Commonly, DNA-transfection assays are used to measure the effect of histone acetylation and deacetylation on gene transcription. Here we have analyzed the response of various viral long terminal repeats and vertebrate promoters to the specific histone deacetylase inhibitor trichostatin A (TSA). We show that the activity of many, but not all, promoters is increased upon TSA treatment. Interestingly, the lysozyme promoter exhibited TSA resistance, while the activity of metallothionine, the human growth hormone, and the thymidine kinase promoters was increased. Furthermore, we found that all tested viral promoters are induced by TSA. Analysis of the transcriptional behaviour of the thyroid hormone receptor (TR), the cellular homologue of the v-erbA oncogene, revealed that TSA reduced the gene silencing function but had no influence on the hormone-induced gene activation function of the receptor. These results on gene specific effects, together with the HDAC structural data (1), may be a basis for the development of HDAC inhibitors as antitumor agents.

Animals↗