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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Junctional sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were pretreated with 0.1-500 microM ryanodine under equilibrium conditions optimal for receptor binding, followed by the removal of bound alkaloid by several washes in Ca(2+)- and ryanodine-free buffer. Pretreatment with > 100 nM ryanodine results in a concentration-dependent decrease in the Bmax of the high affinity sites and a complete loss of measurable low affinity binding sites that persist for > 48 hr. Quantitative analysis of residual ryanodine using three different methods demonstrates that the inhibition is not the result of residual ryanodine bound to its receptor. Ca2+ transport measurements made with antipyrylazo III show that actively loaded ryanodine-pretreated SR exhibits a persistent insensitivity to ryanodine- and daunomycin-induced Ca2+ release that is not seen with washed control vesicles. Lipid bilayer membranes fused with SR vesicles exhibit rapidly fluctuating single-channel events with a conductance of 468 pS in asymmetric CsCl solutions. Ryanodine (10 microM) produces a unidirectional transition to a slowly fluctuating half-conductance state that is not reversed by perfusion of the bilayer with Ca(2+)-free buffer and subsequent addition of dithiothreitol. However, dithiothreitol added in the ryanodine pretreatment medium offers marked protection against ryanodine-induced loss of binding sites and allows complete restoration of native gating behavior of single channels in bilayer lipid membrane. Using three different experimental approaches, the data demonstrate that the alkaloid at micromolar concentration persistently alters SR Ca2+ release channel function, perhaps by uncoupling four negatively cooperative binding sites. The oxidation of critical receptor thiols is implicated in the process.
Mol Pharmacol 1992 Dec
PMID:Ryanodine induces persistent inactivation of the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum. 148 Jan 32

Thyroid hormone receptors (TRs) bind as dimers to specific DNA response elements. We have used a genetic approach to identify amino acid sequences required for dimerization of the TR beta isoform. Bacteria expressing a chimeric repressor composed of the DNA binding domain of the bacteriophage lambda cl repressor fused to the TR beta ligand binding domain are immune to lambda infection as a consequence of homodimerization activity provided by the receptor sequences. The phenotypes of deletions and point mutations of the TR beta sequences map dimerization activity to a subregion of the ligand binding domain that is highly conserved among all members of the nuclear hormone receptor superfamily. These results confirm and extend previous findings indicating that this subregion plays an important role in the dimerization of TR beta and other superfamily members.
Mol Endocrinol 1992 Nov
PMID:Thyroid hormone receptor dimerization function maps to a conserved subregion of the ligand binding domain. 148 Jan 76

The regulation of human corticotropin-releasing hormone (hCRH) gene promoter activity by inducers of cAMP was investigated by transient transfection with a construct containing the hCRH gene promoter fused to the chloramphenicol acetyltransferase gene. Expression of hCRH-chloramphenicol acetyltransferase was strongly enhanced by forskolin in the neuroblastoma SK-N-MC and choriocarcinoma JAR cell lines. Overexpression of the catalytic subunit of protein kinase A dispensed the need for forskolin, and cotransfection of cAMP-responsive element-binding protein cDNAs enhanced forskolin-dependent expression of the hCRH promoter. Progressive 5'-end deletions of the hCRH promoter delineated a cAMP- responsive region between -226 and -164 base pairs. This fragment contained the sequence TGACGTCA at -221 base pairs, consistent with the consensus motif for a CRE. A homologous oligonucleotide responded to cAMP when cloned in either orientation in front of the thymidine kinase promoter. However, the level of constitutive and inductive cAMP expression was dependent on the cell line and on intrinsic properties of the promoter. Mutation of the wild type CRH-CRE sequence into an AP-1 site (TGAGTCA) completely abolished stimulation by cAMP. In contrast, coexpression of the catalytic subunit of protein kinase A dispensed the need for stimulation with forskolin, which showed that the CRH-CRE oligonucleotide served as a functional equivalent of the native CRE element.
Mol Endocrinol 1992 Nov
PMID:Identification and characterization of a 3',5'-cyclic adenosine monophosphate-responsive element in the human corticotropin-releasing hormone gene promoter. 148 Jan 79

A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active HlyD-LacZ fusion proteins were only generated when lacZ was fused to hlyD within the first 180 bp (60 amino acids). HlyD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD). Active insertions of phoA into the middle region of hlyD were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA+ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA+ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Gen Genet 1992 Jul
PMID:A topological model for the haemolysin translocator protein HlyD. 149 79

Previous work in our laboratory has shown that the 5' nontranscribed promoter region of the gene for ribosomal protein (rp) S16A-1 of Saccharomyces cerevisiae, when fused to a lacZ gene, is necessary and sufficient to cause an increase in expression of the heterologous lacZ gene fusion product after cells have been shifted from a glycerol to glucose carbon source. This increase in expression is characteristic of that observed with the native rp gene. We have sought to define more precisely those areas of the promoter that may be involved in the differential expression/regulation of RPS16A-1 when host cells are subjected to a variety of nutritional environments. It has already been demonstrated by others that the promoter regions of most rp genes contain at least one consensus element, designated UASrpg, which is necessary for the transcriptional activation and maintenance of expression of the gene during steady-state growth in rich media. Our main experimental approach has been to create a series of 5' end deletions in the promoter region of RPS16A-1. The individual truncated promoter fragments were then ligated to a lacZ fusion reporter construct. By assaying the cells for production of beta-galactosidase and determining the abundance of lacZ mRNA, we have been able to determined the extent of fusion product expression. We assayed cells under three physiological conditions: steady-state growth in glucose, steady-state growth in glycerol and during sporulation. We report four main findings of our work.
Mol Gen Genet 1992 Jul
PMID:The role of promoter elements of a ribosomal protein gene in Saccharomyces cerevisiae under various physiological conditions. 149 81

In vitro gene fusions were constructed between the polygalacturonase-encoding pehA gene of the Erwinia carotovora subsp. carotovora (Ecc) strain SCC3193 and the bla gene of pBR322. The gene fusions obtained (75-2, 75-5 and 75-6) encoded hybrid proteins with the entire signal peptide and 70, 260 or 327 amino acids (aa) of the mature 376 aa PehA protein, respectively, fused to the mature part of the periplasmic beta-lactamase. All three hybrid proteins remained cell-bound in Ecc. High-level expression of the longer fusions 75-5 and 75-6 in Ecc led to reduced growth and viability of the cells. This phenotype was utilized to select for spontaneous extragenic mutations restoring normal cell growth. Two classes of regulatory mutants were obtained by this selection. First, mutants impaired in the production of several exoenzymes, including polygalacturonase, were found. These were phenotypically similar to the previously characterized Exp- mutants. Secondly, mutants specifically impaired in the production of polygalacturonase (designated PehR-), but producing and secreting wild-type levels of pectate lyase and cellulase, were obtained. The PehR- mutations were shown to affect transcriptional activation of the pehA gene. Furthermore, the PehR- as well as PehA- mutants exhibited a reduced virulence phenotype suggesting that polygalacturonase is a virulence factor in Ecc.
Mol Gen Genet 1992 Jul
PMID:Expression of pehA-bla gene fusions in Erwinia carotovora subsp. carotovora and isolation of regulatory mutants affecting polygalacturonase production. 149 88

The postimplantation development of human and animal triploid embryos is well documented, but there is little informative data on their preimplantation development. An analysis of cell number at appropriate times during this period and thus their cleavage rate would give an indication of the potential triploids have for further development and may explain some problems associated with their postimplantation development. To rule out any effects of technical procedures on cleavage rate, appropriate controls were used. Diandric triploid embryos were produced using standard micromanipulatory techniques, which involved the injection of a male pronucleus into a recipient one-cell-stage embryo. The karyoplast was fused to the cytoplasm by electrofusion, and the resulting tripronucleate diandric triploid embryos were transferred to appropriate pseudopregnant recipients. At specific times after the transfer, the embryos were recovered and cell numbers established. The results were plotted and regression lines drawn. Three controls were used 1) micromanipulated diploid embryos from which the male pronucleus had been removed and immediately reinserted and fused to restore diploidy, 2) diploid embryos that had been briefly incubated in cytochalasin D and colcemid to find out the effects these agents had on development, and 3) diploid embryos that had been isolated and briefly incubated in tissue culture medium. All embryos were subsequently transferred to recipients. After isolation at specific times during the preimplantation period, cell numbers were also established and the results plotted.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1992 Jul
PMID:Cleavage rate of diandric triploid mouse embryos during the preimplantation period. 149 74

Meiosis in Saccharomyces cerevisiae requires the induction of a large number of genes whose mRNAs accumulate at specific times during meiotic development. This study addresses the role of mRNA stability in the regulation of meiosis-specific gene expression. Evidence is provided below demonstrating that the levels of meiotic mRNAs are exquisitely regulated by both transcriptional control and RNA turnover. The data show that (i) early meiotic transcripts are extremely unstable when expressed during either vegetative growth or sporulation, and (ii) transcriptional induction, rather than RNA turnover, is the predominant mechanism responsible for meiosis-specific transcript accumulation. When genes encoding the early meiotic mRNAs are fused to other promoters and expressed during vegetative growth, their mRNA half-lives, of under 3 min, are among the shortest known in S. cerevisiae. Since these mRNAs are only twofold more stable when expressed during sporulation, we conclude that developmental regulation of mRNA turnover can be eliminated as a major contributor to meiosis-specific mRNA accumulation. The rapid degradation of the early mRNAs at all stages of the yeast life cycle, however, suggests that a specific RNA degradation system operates to maintain very low basal levels of these transcripts during vegetative growth and after their transient transcriptional induction in meiosis. Studies to identify specific cis-acting elements required for the rapid degradation of early meiotic transcripts support this idea. A series of deletion derivatives of one early meiosis-specific gene, SPO13, indicate that its mRNA contains determinants, located within the coding region, which contribute to the high instability of this transcript. Translation is another component of the degradation mechanism since frameshift and nonsense mutations within the SPO13 mRNA stabilize the transcript.
Mol Cell Biol 1992 Sep
PMID:Early meiotic transcripts are highly unstable in Saccharomyces cerevisiae. 150 96

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.
Mol Cell Biol 1992 Sep
PMID:Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice. 150 98

Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.
Mol Cell Biol 1992 Sep
PMID:A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo. 150 99


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