Biomedical subjects

M Beato

Publications and source records attributed to M Beato.

At least 109 records · Page 6Linked to original sources

PubMed1990

Structural features of a regulatory nucleosome.

DNA sequences from the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR) position nucleosomes both in vivo and in vitro. Here, were present chromatin reconstitution experiments showing that MMTV-LTR sequences from -236 to +204 accommodate two histone octamers in positions compatible with the in vivo data. This positioning is not influenced by the length of the DNA fragment and occurs in linear as well as in closed circular DNA molecules. MMTV-LTR DNA sequences show an intrinsic bendability that closely resembles its wrapping around the histone octamer. We propose that bendability is responsible for the observed rotational nucleosome positioning. Translational nucleosome positioning seems also to be determined by the DNA sequence. These data, along with the results from reconstitution experiments with insertion mutants, support a modular model of nucleosome phasing on MMTV-LTR, where the actual positioning of the histone octamer results from the additive effect of multiple features of the DNA sequence.

Base Sequence↗

PubMed1990

DNA rotational positioning in a regulatory nucleosome is determined by base sequence. An algorithm to model the preferred superhelix.

MMTV-LTR sequences -190/-45 position a histone octamer both in vivo and in vitro. Experimental evidence suggested that nucleosome rotational positioning is determined by the DNA sequence itself. We developed an algorithm that is able to predict the most favorable path of a given DNA sequence over a histone octamer, based on rotational preferences of different dinucleotides. Our analysis shows that these preferences are sufficient for explaining the observed rotational positioning of the MMTV-LTR nucleosome, at one base pair accuracy level. Computer-generated 3-D models of the experimentally calculated and predicted MMTV-LTR nucleosome show that the predicted orientation is fully compatible with the currently available data in terms of accessibility of relevant sequences to regulatory proteins.

Algorithms↗

PubMed1990

Tissue-specific expression, hormonal regulation and 5'-flanking gene region of the rat Clara cell 10 kDa protein: comparison to rabbit uteroglobin.

The amino acid sequence of rat Clara Cell 10 kDa secretory protein (CC10) shows 55% identity to rabbit uteroglobin. In order to define the relationship between rat CC10 and rabbit uteroglobin in detail, the tissue-specific expression and hormonal regulation of rat CC10 mRNA was analyzed. We report that like rabbit uteroglobin, rat CC10 mRNA is expressed in lung and esophagus, as well as in uteri of estrogen- and progesterone-treated females. Expression of CC10 mRNA in lung is regulated by glucocorticoids. The similarity in expression pattern of rat CC10 mRNA and rabbit uteroglobin mRNA is reflected by a striking similarity in the 5'-flanking regions of the two genes. Despite this overall similarity, two regions of 0.3 kb and 2.1 kb are absent in the rat CC10 upstream gene region. The larger region includes a cluster of hormone receptor binding sites, believed to be responsible for differential regulation of rabbit uteroglobin by glucocorticoids and progesterone. Thus, while the sequence identities in the coding and 5'-flanking regions point towards a common ancestor for the uteroglobin and CC10 gene, later events (deletions/insertions) might have caused species-specific differences in their regulation.

Animals↗

PubMed1990

Progesterone receptor stimulates transcription of mouse mammary tumour virus in a cell-free system.

In higher eukaryotes, steroid receptors are general modulators of gene activity which bind to DNA hormone response elements (HREs) in the vicinity of regulated promoters. Analysis of mutant and chimaeric receptor proteins in gene transfer experiments has identified the domains responsible for hormone binding, interaction with the HREs and transactivation, but the transactivating function of hormone receptors has proved difficult to reproduce in cell-free assays. Here we describe a crude in vitro system in which transcription from the mouse mammary tumour virus (MMTV) promoter is increased up to 10-fold by native progesterone receptor from rabbit uterus. The stimulatory effect must depend on receptor binding to the MMTV-HRE as it is abolished by deletion or mutation of the HRE and by HRE-oligonucleotide competition. The nuclear factor-I binding site immediately downstream of the HRE, however, does not appear to be essential to progesterone receptor-mediated in vitro stimulation of MMTV transcription. The transactivation activity of the progesterone receptor depends on binding of a functional ligand and so this assay should be useful for dissection of the mechanism of transcription activation by steroid hormones.

Animals↗

PubMed1990

Nucleosome positioning modulates accessibility of regulatory proteins to the mouse mammary tumor virus promoter.

Minichromosomes containing the MMTV hormone responsive element (HRE) exhibit precisely positioned nucleosomes. Chromatin reconstitution of short HRE DNA fragments also results in precise positioning of nucleosomes as revealed by footprinting, which suggests that information for nucleosome phasing is contained within this short sequence. While hormone receptors bind naked DNA and reconstituted nucleosomes with similar affinities (3- to 5-fold difference), NFI, a transcription factor essential for efficient utilization of the MMTV promoter, binds naked DNA very tightly but does not bind the nucleosomally organized promoter. Hormone receptor binding to the MMTV nucleosome does not dissociate the nucleosome but leads to greater accessibility of the promoter-proximal end to exonuclease III. Precise positioning of one nucleosome over the MMTV promoter could repress transcription by preventing NFI binding in the absence of hormone, while still allowing interaction of activated hormone receptor with HRE.

Animals↗

PubMed1990

Nuclear factor I acts as a transcription factor on the MMTV promoter but competes with steroid hormone receptors for DNA binding.

Several steroid hormones induce transcription of the mouse mammary tumor virus (MMTV) promoter, through an interaction of their respective receptors with the hormone responsive elements (HREs) in the long terminal repeat (LTR) region. The molecular mechanism underlying transcriptional activation is not known, but binding of nuclear factor I (NFI) to a site adjacent to the HRE appears to be required for efficient transcription of the MMTV promoter. In JEG-3 choriocarcinoma cells the MMTV promoter is transcribed inefficiently, even after transfection of the receptor cDNA and treatment with glucocorticoids or progestins. These cells contain low levels of NFI as cotransfection of NFI cDNA enhances MMTV transcription and this effect is inhibited by mutation of the NFI binding site. In DNA binding experiments with purified NFI from pig liver, the glucocorticoid and progesterone receptors do not co-operate but rather compete with NFI for binding to their respective sites on the LTR. Similar results are obtained with a functional recombinant NFI synthesized in vitro. Competition for DNA binding is probably due to steric hindrance as the DNase I footprints of the hormone receptors and NFI do overlap. These results suggest that, though NFI acts as a transcription factor on the MMTV promoter, transcriptional activation does not take place through a direct facilitation of DNA binding of NFI by steroid hormone receptors.

Animals↗

PubMed1990

Mutual transrepression of Fos and the glucocorticoid receptor: involvement of a functional domain in Fos which is absent in FosB.

In this study, we show that Fos protein can repress transactivation by the glucocorticoid receptor (GR). In addition, we demonstrate that GR is capable of inhibiting, in a hormone-dependent fashion, Fos-mediated transactivation of AP-1 dependent transcription. Moreover, repression of the serum response element by Fos is abolished by the GR in the presence of hormone. Transrepression of glucocorticoid mediated induction involves a region of Fos, located between amino acids 40 and 111, to which no function has been previously assigned, and which is poorly conserved among Fos, FosB and Fra-1. In agreement with this finding, FosB is not capable of transrepressing GR activation of transcription, representing the first functional difference between Fos and FosB. We have mapped the domain of the GR which is required for repression of AP-1 dependent transcription, to the region of central DNA binding domain. Our results suggest that Fos and the GR may form transcriptionally inactive complexes and point to a regulatory interrelationship between different signal transduction pathways.

Amino Acid Sequence↗

PubMed1990

A comparison of mouse and rabbit embryos for the production of transgenic animals by pronuclear microinjection.

Procedures for the production of transgenic animals have low overall efficiency. To evaluate factors responsible for low efficiency, zygotes of two species, varying intensities of microscope light, different qualities of injection pipettes, and six different genes were tested for their influence on the efficiency of pronuclear gene injection for the production of transgenic rabbits and mice. Rabbit zygotes were less sensitive to mechanical manipulation during injection than mouse zygotes. Exposing zygotes to a microscope light intensity of 5550 lx significantly reduced their cleavage rate, while a lower intensity (2280 lx) did not. Using pipettes with a filament for pronuclear gene injection of mouse zygotes resulted in a higher cleavage rate of zygotes after injection than when pipettes were used without filament (30.3 vs 20.6%). Implantation rates varied between 2.9% (HB72CAT) and 23.1% (ts 58-2) depending on the gene used. No transgenic animals were obtained after injection of uteroglobin-CAT-hybrid genes (B2B3UGCAT, HB72CAT), while all other genes used (UG 11.8, UGTAg, RSV lacZ, ts 58-2) resulted in transgenic embryos, fetuses, and newborn animals.

Journal Article↗

PubMed1990

Contacts between steroid hormone receptors and thymines in DNA: an interference method.

Understanding the mechanisms by which regulatory proteins recognize genetic information stored in DNA relies on the availability of methods to analyze their interaction with individual nucleotides and their reactive groups. Here we describe the use of KMnO4 to analyze the contacts between steroid hormone receptors and thymines within a hormone responsive element. Although several pyrimidine residues are highly conserved among different receptor binding sites their participation in sequence recognition has not been directly studied. Using an interference procedure based on selective modification of the thymine ring by KMnO4, we detect intimate contacts between the glucocorticoid or progesterone receptors and three thymine residues within the promoter distal receptor binding site of the mouse mammary tumor virus (-190/-160). A comparison of binding data obtained with oligonucleotides containing desoxyuridine, bromodeoxyuridine, cytosine, or 5'-methylcytosine instead of thymines demonstrates that the methyl group of those three thymines contributes to the free energy of binding. This simple method could be of general utility for the study of sequence-specific protein-DNA interactions.

Animals↗

PubMed1990

Efficient binding of glucocorticoid receptor to its responsive element requires a dimer and DNA flanking sequences.

A combination of the gel retardation assay and interference by hydroxyl radical modification (missing nucleoside technique) was used to analyze the interaction of the glucocorticoid receptor (GR) with various glucocorticoid responsive elements (GRE). Short oligonucleotides containing the 15-bp GRE and 1 to 3 flanking base pairs on each side, are bound with very low affinity. The same GREs, when positioned in the center of a large DNA fragment (40-50 bp), show high affinity for the receptor. However, when the GRE is positioned at the border of a 54-bp fragment, the affinity of the GR for the GRE decreases markedly. The DNA binding affinity increases linearly with each added flanking base pair and optimal binding is observed with 8-10 flanking bp. Thus, the nonconserved DNA sequences flanking the GRE contribute significantly to the free energy of receptor binding to DNA. Using larger DNA fragments (greater than 100 bp) and a smaller form of the receptor (40 kD), two retarded complexes are found that correspond to monomeric and homodimeric receptor DNA complexes. The DNA-binding domain of the GR (20 kD), expressed in bacteria, binds to the GRE as a monomer as well as a dimer and can form heterodimers with the native 94-kD GR. Insertion or deletion of one single base pair between the two halves of the GRE reduces the affinity for the homodimeric form of the native GR, and inhibits the function of the GRE in gene transfer experiments, suggesting that a dimer of the GR is the functional entity that binds to the GRE.

Animals↗

PubMed1990

Binding of the glucocorticoid receptor induces a topological change in plasmids containing the hormone-responsive element of mouse mammary tumor virus.

Glucocorticoid hormones stimulate transcription of the mouse mammary tumor virus (MMTV) promoter by means of an interaction of the hormone receptor with a set of upstream DNA-binding sites called the hormone-responsive element (HRE). In an attempt to understand the underlying molecular mechanism, we have analyzed the influence of receptor binding on the topological state of plasmids carrying different parts of the HRE. Incubation of negatively supercoiled plasmids containing the intact HRE with the 94-kD purified glucocorticoid receptor, followed by extensive digestion with topoisomerase I, leads to a distribution of topoisomers shifted by two turns toward positively supercoiled circles. If relaxed plasmids are incubated with receptor and treated with topoisomerase I, the average linking number of the resulting distribution of topoisomers is also increased by two with respect to the controls. This effect depends on the presence of an intact HRE and is almost undetectable with the 40-kD form of the receptor. Thus, binding of the hormone receptor to functional DNA regulatory sites located in closed circular plasmids results in a topological change that could be related to the hormonal activation of transcription.

Animals↗

PubMed1990

Hormonal induction of transfected genes depends on DNA topology.

Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors.

Animals↗

PubMed1990

The uteroglobin promoter contains a noncanonical estrogen responsive element.

Uteroglobin is expressed in various tissues of the rabbit under complex hormonal control. In the endometrium the uteroglobin gene is transcribed only in epithelial cells after administration of ovarian hormones. In this paper we demonstrate that within the promoter region of the rabbit uteroglobin gene, there is a functional estrogen-responsive element (ERE) located between -265 and -252. Hybrid constructions containing sequences of the uteroglobin promoter up to -299, linked to the chloramphenicol acetyltransferase gene of E. coli respond to estrogens in gene transfer experiments, whereas a deletion that removes half of the ERE does not. A synthetic oligonucleotide corresponding to the putative ERE is able to confer estrogen inducibility to an otherwise unresponsive promoter. Binding experiments with purified estrogen receptor from calf uterus reveal a DNase-I footprint over the ERE. Within this protected region six guanine residues that have been shown to be contacted by the receptor in other EREs are protected against methylation by dimethylsulfate in the presence of the estrogen receptor. We compare this ERE with the vitellogenin A2 ERE from Xenopus and find that the relative affinity of the uteroglobin ERE is slightly lower than that of the vitellogenin ERE. Thus, this uteroglobin ERE could be involved in physiological regulation of uteroglobin expression in the genital tract.

Animals↗

PubMed1990

Establishment of rat endometrial cell lines by retroviral mediated transfer of immortalizing and transforming oncogenes.

To study hormone responsive genes in differentiated epithelial cells and as a model for endometrial carcinoma, lines were established from primary rat endometrial cells by infection with replication-defective retroviruses carrying oncogenes and the selectable gene neo. The initial step involved immortalization through the large T antigen of SV40 to generate a line we designate RENT4, or with the E1a gene of adenovirus to generate lines referred to as RENE1 and RENE2. Additionally, lines generated by large T antigen of SV40 were superinfected with a replication-defective retrovirus harboring the v-Ha-ras oncogene and selected by the ability to form colonies in soft agar. The latter cell lines appeared fully transformed and were designated RENTR01 and RENTR03. Five established lines were characterized for steroid hormone receptors, alkaline phosphatase activity and their complement of the intermediate filaments vimentin and cytokeratin. With the exception of the RENE1 cells all other lines have normal levels of glucocorticoid receptor, whereas only RENE1, RENE2 and RENT4 were positive for the progesterone receptor. RENTR01, RENTR03 and, to a lesser extent, RENE1 exhibited differential expression of cytokeratins dependent upon whether the cells were grown on a substrata of NIH3T3 cells. When grown on formalin-fixed NIH3T3 cells, RENTR01 and RENTR03 cells appeared to differentiate or rearrange themselves in culture. Individual islands of cells showed a heterogeneous pattern of intermediate filaments with vimentin-positive cells localized to the outer portion of the islands whereas cytokeratin-positive cells are seen on the insides of these structures.

Alkaline Phosphatase↗

PubMed1989