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

A procedure for the isolation and purification of a specific hybrid between rat 28S and 18S ribosomal RNA's and nucleolar DNA is described. The method employed includes the following steps: 1) isolation of the nucleolar DNA, 2) hybridization of [14C]rRNA with the nucleolar DNA, and 3) isolation and purification of the rRNA-DNA hybrid complex by chromatography on hydroxylapatite and centrifugation in a CsCl density gradient. In the isolated hybrid complex the RNA:DNA ratio is close to 1:1, and the degree of enrichment of the DNA by the rRNA cistrons is about 1500 times. The hybrid obtained has a sedimentation constant on the order of 20S, is resistant to the action of pancreatic RNase and RNase T1 and sheep brain DNase, and is characterized by high thermostability. Acording to the physicochemical tests used, the rRNA-DNA hybrid complex is a double-stranded poly-nucleotide with an ordered secondary structure.
Mol Biol (Mosk)
PMID:Isolation of a hybrid between rat ribosomal RNA and DNA. 102 44

The principal regulator of erythropoiesis is the glycoprotein erythropoietin, which interacts with a specific cell surface receptor (EpoR). A study aimed at analyzing EpoR gene regulation has shown that both pluripotent embryonal stem cells and early multipotent hematopoietic cells express EpoR transcripts. Commitment to nonerythroid lineages (e.g., macrophage or lymphocytic) results in the shutdown of EpoR gene expression, whereas commitment to the erythroid lineage is concurrent with or followed by dramatic increases in EpoR transcription. To determine whether gene activity could be correlated with chromatin alterations, DNase-hypersensitive sites (HSS) were mapped. Two major HSS located in the promoter region and within the first intron of the EpoR gene are present in all embryonal stem and hematopoietic cells tested, the intensities of which correlate well with EpoR expression levels. In addition, a third major HSS also located within the first intron of the EpoR gene is uniquely present in erythroid cells that express high levels of EpoR. Transfection assays show that sequences surrounding this major HSS impart erythroid cell-specific enhancer activity to a heterologous promoter and that this activity is at least in part mediated by GATA-1. These data, together with concordant expression levels of GATA-1 and EpoR in both early multipotent hematopoietic and committed erythroid cells, support a regulatory role of the erythroid cell-specific transcription factor GATA-1 in EpoR transcription in these cells. However, the lack of significant levels of GATA-1 expression in embryonal stem cells implies an alternative regulatory mechanism of EpoR transcription in cells not committed to the hematopoietic lineage.
Mol Cell Biol 1992 Apr
PMID:The gene for erythropoietin receptor is expressed in multipotential hematopoietic and embryonal stem cells: evidence for differentiation stage-specific regulation. 131 71

A rat genomic clone containing 4.5 kilobases of 5'-flanking DNA and the first exon of the type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase was isolated, restriction mapped, and sequenced. The proximal 400-basepair promoter region was GC rich, lacked TATA/CAAT box motifs, and initiated transcription at multiple sites. Bandshifting and DNase-I footprinting experiments using this region of the RII beta promoter detected several related specific DNA-protein complexes formed using crude and fractionated nuclear extracts from rat ovary, brain, adrenal gland, and liver. All binding in these experiments mapped to a domain within the same region found to confer cAMP inducibility to a chloramphenicol acetyltransferase (CAT) reporter gene when transfected into primary cultures of rat granulosa cells. Although GC boxes (putative SP1-binding sites) and activator protein-2 (AP-2) elements were present in this functional region, and although expression vectors containing AP-2 sites conferred high levels of cAMP regulation of the CAT gene in cultured ovarian cells, neither the GC boxes nor the AP-2 sites were protected by footprint analyses or required for band shift activity of nuclear extract protein. These known regulatory elements, therefore, may be involved in functional activity of the RII beta promoter, but additional cis-acting DNA and trans-acting factors (yet to be characterized) also appear to interact with the functional promoter of the RII beta gene and regulate the hormone-specific expression of the A-kinase subunit in ovarian and neuronal cells.
Mol Endocrinol 1992 Apr
PMID:Identification and characterization of the GC-rich and cyclic adenosine 3',5'-monophosphate (cAMP)-inducible promoter of the type II beta cAMP-dependent protein kinase regulatory subunit gene. 131 46

Pituitary lactotroph cell function and PRL gene expression are highly regulated by the cAMP-protein kinase-A (PKA) pathway. To further our understanding of the molecular mechanisms by which cAMP/PKA regulates rat (r) PRL promoter activity and to determine whether cAMP regulation is cell type specific, we 1) transected intact (-425), internal and 5'-deletion, and site-specific mutants of the rPRL promoter ligated to the firefly luciferase reporter gene into both pituitary and nonpituitary cell lines; and 2) assessed the role of the cAMP-cAMP response element-binding protein (CREB) pathway in GH4 rat pituitary cells. The data show that deleting the rPRL promoter from -425 to -116 did not abolish cAMP regulation, implying that proximal elements, such as the basal transcription element (-112/-80) or the pituitary-specific footprint (FP) I (-67/-45), mediate the cAMP response. However, nucleotide changes within FP I or FP II (-130/-120) did not alter the rPRL promoter response to 1 microM forskolin (FSK), despite the 77% and 26% reductions in basal rPRL promoter activity caused by these mutations, respectively. Furthermore, internal deletion of either the basal transcription element of FP I element also failed to affect cAMP regulation of the rPRL promoter, again despite the 90% and 93% reductions in basal promoter activity by these deletions, respectively. Since these internal deletion constructs otherwise contain rPRL promoter sequences from -425 to +73, including the up-stream pituitary-specific FPs III and IV, the data suggest that any one of these cell-specific elements is capable of imparting cAMP regulation to the proximal rPRL promoter. To directly test the implication that the cAMP response of the rPRL promoter is restricted to the pituitary-specific cell type, we took advantage of a 5'-deletion mutant truncated at position -116 and a FP II site-specific mutant, since constructs containing these rPRL promoters are active in nonpituitary cells. Despite the 6.6- and 18.5-fold stimulations over wild-type rPRL promoter activity in nonpituitary cells, respectively, these mutations remained completely unresponsive to FSK treatment. To document that the cAMP-CREB pathway was functional in GC/GH4 rat pituitary cells, CREB was affinity purified from GC rat pituitary cells, and DNase-I protection studies showed that it does not bind to the proximal rPRL promoter. Also, the human glycoprotein alpha-subunit promoter was induced 10-fold by FSK in GH4 rat pituitary cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Endocrinol 1992 Dec
PMID:Cyclic adenosine 3',5'-monophosphate activation of the rat prolactin promoter is restricted to the pituitary-specific cell type. 133 42

Human CG is composed of two subunits, alpha and beta. In addition to its eutopic synthesis in normal and malignant trophoblasts, the hormone is produced ectopically by a variety of tumor cell lines of nonplacental origin. Regulation of the alpha CG gene in trophoblasts appears to differ from that in nontrophoblasts. To determine whether these differences are reflected in the chromatin structure at the alpha CG locus, DNase I-hypersensitive sites within this domain were mapped in human tumor cell lines that differentially express the gene. Two hypersensitive sites were detected in DNA from cell lines that produce the alpha-subunit. The latter includes trophoblastic (JAr and JEG-3 choriocarcinoma) and nontrophoblastic (HeLa cervical carcinoma and ChaGo bronchogenic carcinoma) tumor cell lines. The most prominent site (HS 1) was located approximately 100 base pairs upstream from the transcription start site. In trophoblasts, accessibility of HS 1 increased substantially upon induction of the gene by cAMP, likely reflecting alterations in DNA-protein interactions at the cAMP response element and/or tissue-specific enhancer. In nontrophoblasts, where alpha-subunit synthesis is enhanced by sodium butyrate but not by cAMP, neither butyrate nor cAMP altered the accessibility of HS 1. The HS 2 is comprised of multiple sites with weak to moderate DNase sensitivity located downstream at +1600 to +4000 in cell lines that produce alpha-subunit. Cell lines that do not express the alpha CG gene possess a distinct hypersensitive site (HS 3) within the first intron at about +600; these include 3A-Sub-E (SV40 transformed placenta), CBT (glioblastoma multiforme), and CaSki (cervical carcinoma). Cleavage by DNase at HS 1 and HS 2 is not evident in nuclei from cell lines that do not produce alpha-subunit. These results suggest that HS 1 and HS 3 are characteristic of active and inactive states of the alpha CG gene, respectively, and that the accessibility of HS 1 generally correlates with the level of expression.
Mol Endocrinol 1992 May
PMID:Deoxyribonuclease-hypersensitive sites in the glycoprotein hormone alpha-subunit gene from trophoblastic and nontrophoblastic human tumor cell lines: correlation with expression and effect of chemical inducers. 137 9

Transient transfection studies have been used to determine the DNA sequences of the glycoprotein hormone alpha-subunit gene that are required for tissue-specific expression. In the initial phase of these studies, a variant mouse alpha gene was identified which contains a fully palindromic cAMP response element (CRE). The corresponding region of a previously cloned and sequenced mouse alpha gene contains a single point mutation that disrupts the symmetrical nature of this element. DNase footprint studies demonstrate that the fully palindromic CRE binds the CRE-binding protein with much higher affinity than the imperfect palindrome. Transfection experiments using both mouse alpha gene variants demonstrate differences in basal and cAMP-induced expression. Studies of the cAMP response of the human alpha gene indicated that this gene contains sequences other than the known CRE that are sufficient to permit a transcriptional response to cAMP in both placental and pituitary cells. Expression of human and mouse alpha-subunit genes has been examined in cells of the gonadotrope, thyrotrope, and trophoblast lineages to identify DNA sequences that mediate selective transcription of the alpha gene in these cells. The results demonstrate that sequences between about -500 and -200 are important for expression in the pituitary, but not the placenta. Clustered point mutations were used to further characterize sequences required for expression in the pituitary. Two regions, one at positions -445 to -438 and one at positions -337 to -330, were required for expression in cells of the gonadotrope lineage. One of these regions, at -337 to -330, is also important for expression in thyrotropes. When linked to a minimal promoter, multiple copies of the -344 to -300 region had transcriptional enhancer activity in gonadotropes and thyrotropes, but not in several other cell types. These results are consistent with a model involving different combinations of regulatory elements that determine cell-specific alpha expression in gonadotropes and thyrotropes.
Mol Endocrinol 1992 Jun
PMID:Analysis of DNA sequences required for pituitary-specific expression of the glycoprotein hormone alpha-subunit gene. 137 72

The nature of a DNA element located in the -100 to -85 region of the rat PRL gene has been characterized. Previous studies demonstrated that this region may contribute to basal and hormonally regulated expression of the PRL gene. As this region contains a sequence with similarity to a consensus cAMP-responsive element (CRE), a possible role for the cAMP response element binding protein (CREB) has been explored. A point mutation which made the PRL CRE-like sequence less like a consensus CRE had little effect on basal or cAMP-stimulated expression of a PRL-luciferase reporter gene. DNase footprint studies demonstrated that the proximal region of the PRL gene does not contain a high affinity CREB binding site. Mobility shift experiments demonstrated that the major GH3 nuclear protein which interacts with the -100 to -85 region of the PRL gene in vitro is not CREB. Transfection of a dominant inhibitor of CREB action had little or no effect on expression of an indicator gene containing the PRL proximal region. Thus, the PRL proximal region does not contain a high affinity CREB binding site, and it is unlikely that CREB plays a major role in expression of the PRL gene. The functional capabilities of the -100 to -85 region of the PRL gene were then tested in a transfection assay. Synthetic multimers of this region were found to be sufficient to permit a transcriptional response to cAMP or TRH in GH3 cells and cAMP in Rat-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1992 Jun
PMID:Characterization of a non-tissue-specific, 3',5'-cyclic adenosine monophosphate-responsive element in the proximal region of the rat prolactin gene. 138 49

To identify cis-acting elements involved with the expression of the rat carboxypeptidase-E (CPE) gene, constructs containing various regions of the 5'-flanking region of the CPE gene attached to the luciferase reporter gene were transiently expressed in cell lines derived from pituitary (AtT-20 and GH4C1), liver (SK-HEP-1), and kidney (HEK293 and COS1). Regions of the CPE gene spanning the major transcription initiation site (-12 to 47) are sufficient for low levels of transcription. Activity is enhanced 3- to 15-fold by sequences present between -12 and -395 in all cell lines examined. Sequences between -395 and -3081 influenced transcription activity up to 5-fold in some, but not all, cell lines. There was no correlation between the transcription activities of the various constructs and the level of endogenous CPE mRNA in the cell lines, indicating that the tissue-specific elements responsible for the large variations in endogenous CPE mRNA levels are not present within -3081 to 47. The region between -395 and 45 was examined in greater detail using transient expression assays and DNase-I protection analysis. Transcription activity is enhanced in GH4C1 and HEK293 cells by sequence present between -12 and -84; this region contains a potential GC box, which binds factors present in GH4C1 nuclear extracts. Other regions between -340 and 80 that bind proteins in the GH4C1 nuclear extracts include the major transcription initiation site, which has homology to the initiator sequence; the pituitary-specific transcription initiation sites (-101 and -105); and sequences with homology to NF-1, Pan-1, simian virus-40 enhancer core, and AP-2-binding sites. Taken together, these results suggest that basal expression of the CPE gene from its major transcription initiation site, which does not contain an up-stream TATA box, is primarily under the control of an initiator-like element together with an upstream GC box.
Mol Endocrinol 1992 Dec
PMID:Expression of the rat carboxypeptidase-E gene in neuroendocrine and nonneuroendocrine cell lines. 149 89

Binding of nuclear proteins from wild oat aleurone protoplasts to the promoter regions of two gibberellin-regulated wheat alpha-amylase genes (alpha-Amy1/18 and alpha-Amy2/54) has been studied by gel retardation and DNase 1 footprinting. Gel retardation studies using 300-430 bp fragments of the promoters showed similar binding characteristics with nuclear extracts from both gibberellin A1-treated and untreated protoplasts. DNase 1 footprints localised binding of nuclear proteins from gibberellin A1-treated aleurone protoplasts to regions in both promoters. Similar sequence elements in the promoter regions of both genes were protected from digestion although the location and number of footprints in each promoter region were different. Each footprint contained either a sequence similar to the cAMP and/or phorbol ester response elements, or a hyphenated palindrome sequence. The presence of cAMP and/or phorbol ester response element-like sequences in the footprints suggests that transcription factors of the bZIP type may be involved in the expression of alpha-amylase genes in aleurone cells. Footprints containing hyphenated palindrome sequences, found in the promoter regions of both genes, suggest the possible involvement of other classes of transcription factor. The conserved alpha-amylase promoter sequence TAA-CAGA was also shown to bind nuclear protein in the alpha-Amy2/54 promoter. These observations are discussed in relation to alpha-amylase gene expression in aleurone and to functional data concerning these genes.
Plant Mol Biol 1992 Sep
PMID:Aleurone nuclear proteins bind to similar elements in the promoter regions of two gibberellin-regulated alpha-amylase genes. 151 Nov 35

Outer membrane protein P6 is an important antigen expressed on the surface of all strains of Haemophilus influenzae. The predicted amino acid sequence of P6 contains a region of alpha helices that shares sequence identity with a family of helix-turn-helix DNA-binding proteins. A search for sequence-specific binding sites that resemble an operator region within the gene revealed a sequence with striking homology to the consensus operator sequence for lambda Cro and repressor. To test the hypothesis that P6 binds its own gene, purified P6 on nitrocellulose was probed with plasmid DNA containing the P6 gene. P6 bound the P6 gene in this Southwestern blot assay. To further test the observation, gel shift analysis was performed. Gel shift assays using a P6-specific monoclonal antibody demonstrated that P6 in crude cell extracts binds to the region of the gene containing the putative binding site. Competition with a synthetic oligonucleotide corresponding to the putative binding site inhibited binding of P6 to the P6 gene on nitrocellulose and in the gel shift assay. In addition, this oligonucleotide bound directly to P6 on nitrocellulose. Finally, DNase footprinting confirmed that P6 bound specifically to the same region of the P6 gene. These results indicate that P6 binds to a sequence-specific site within its own gene, suggesting that P6 regulates its own expression. This represents the first example of a Gram-negative outer membrane protein binding to its own gene and has potentially important implications as a mechanism for regulation of expression of outer membrane antigens.
Mol Microbiol 1992 Feb
PMID:Outer membrane protein P6 of Haemophilus influenzae binds to its own gene. 156 Jul 83


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