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

A high molecular weight mitochondrial DNA (mtDNA) replication complex, associated with the mitochondrial membrane, was isolated by sucrose gradient centrifugation from purified wheat embryo mitochondria. This complex comprised the mtDNA as well as enzyme activities involved in the replication and transcription of the organelle genome, such as DNA polymerase, RNA polymerase and topoisomerase type I. The isolated complex is active in mtDNA and mtRNA synthesis in vitro. Electron microscopy and lipid analysis confirmed the membrane origin of this complex. Enzyme activities are resistant to physiological ionic strengths, 0.1-0.2 M KC1, while the membrane-mtDNA association is resistant up to 1 M KC1. DNase treatment of the complex released the DNA polymerase activity while protease treatment solubilized mtDNA, suggesting the direct interaction of mtDNA with membrane protein(s). The use of a novel approach to detect mtDNA fragments specifically retained by the mitochondrial membranes after Sal I digestion of the complex suggests that specific mtDNA sequences anchor mtDNA to mitochondrial membranes.
Plant Mol Biol 1991 Feb
PMID:Isolation from wheat mitochondria of a membrane-associated high molecular weight complex involved in DNA synthesis. 189 1

Cruciforms have been suggested as potential recognition structures at or near origins of DNA replication in eukaryotic cells. Monoclonal antibodies with structural specificity for DNA cruciforms have been produced (Frappier et al. J. Mol. Biol. 193, 751, 1987). The effect of these antibodies, when introduced into permeabilized cells, was to increase overall DNA synthesis and relative copy number of genes (Zannis-Hadjopoulos et al. EMBO J. 7, 1837, 1988); this was interpreted to be a consequence of antibody stabilization of the cruciforms located at or near replication origins resulting in multiple initiations of DNA replication at a single site. Fluorescent labeling of nuclei with anti-cruciform antibodies produces a nonuniform pattern of fluorescence in cells arrested at the G1/S boundary which then changes with progression through S-phase (Ward et al. Exp. Cell Res. 188, 235, 1990). In order to determine the relationship of cruciform distribution in DNA with the nuclear matrix/chromosomal scaffold, we assessed the susceptibility of DNA containing cruciforms to digestion with DNase I. The majority of the cruciforms detectable at G1/S and throughout the nucleus are readily digested by DNase, suggesting that cruciform structures may not be intimately associated with matrix proteins. The fraction that is resistant to DNase I appears associated with nuclear membrane and the nucleolus. No cruciforms could be detected in metaphase chromosomes; cruciforms either are not present or are inaccessible--buried in the scaffold. The absence of cruciforms from metaphase chromosomes would be consistent with the viewpoint that the cruciform in vivo is a transient structure dependent upon and interacting with proteins essential for replication or transcription.
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PMID:DNA cruciforms and the nuclear supporting structure. 190 39

Two transcription factors, rat UBF (rUBF) and rat SL-1 are required for the efficient transcription of the rat promoter in vitro. In vitro studies have established that two broadly defined cis-acting domains, the core promoter element and the upstream promoter element, cooperate to direct correct transcription by RNA polymerase I. The ability of UBF to bind to two linker-scanning mutants of the upstream promoter element, which did not respond to the addition of UBF in in vitro transcription assays, was assessed by DNase footprinting. UBF protected the same region of the promoter in the linker-scanning mutant in BSM 129/124 as it did in the wild-type, but did not yield a typical footprint over the promoter in the linker-scanning mutant BSM 106/101. Previously we reported that promoters with mutant core promoters elements, either the guanine at -16 or -7 substituted by an adenine, were inactive in vitro unless the assays were supplemented with UBF. Those results suggested that the binding of UBF upstream of the core was required for the promotion of transcription. The interactions between the core and upstream promoter elements were assessed by constructing double mutants of the promoter. In two constructs the conserved guanines at either -16 or -7 were altered in a deletion mutant (-86) that did not respond to UBF. In a third construct the guanine at -16 in BSM 129/124 was changed to an adenine. These bidomain mutant constructs did not respond to the addition of UBF in an in vitro transcription assay, confirming that the rescue of the core promoter mutants requires an intact and functional upstream promoter element.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem
PMID:Complementary in vivo and in vitro analyses of the interactions between the cis-acting elements of the rat rDNA promoter. 192 91

Transcription of the RP2 gene in the mouse kidney is induced by androgens. This induction is species specific within the genus Mus. For example, the gene responds to androgens in Mus domesticus, but is refractory to hormone in the distantly related species M. caroli. In the present report we have characterized DNA-binding factors that recognize the 5' flanking region of the RP2 gene. One factor (termed RPBF-1) binds a DNA fragment spanning the region between -157 and -311 relative to the transcriptional start site. RPBF-1 is present in kidney nuclear extracts from both control and androgen-treated M. domesticus as well as from control M. caroli; however, in the latter species a distinct factor (termed RPBF-2) is induced by androgens and replaces RPBF-1. The androgen-dependent replacement of RPBF-1 by RPBF-2 is specific to the kidney of M. caroli. DNase-1 footprinting analyses indicate that the two factors recognize distinct, yet overlapping, regions of the RP2 promoter: RPBF-1 binds the region between -247 and -269, while RPBF-2 binds the region between -265 and -290. The RPBF-2-binding site contains a sequence that is homologous to that recognized by nuclear factor-1 (NF-1), suggesting that RPBF-2 is a NF-1-like factor. This is supported by competition experiments with synthetic oligonucleotides corresponding to the NF-1-binding site within the adenovirus origin of replication. Thus, androgens can modulate, in a species- and tissue-specific manner, DNA-binding factors that recognize promoter regions of genes.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1991 Apr
PMID:Androgen modulation of DNA-binding factors in the mouse kidney. 192 89

Pulmonary infiltrating lymphocytes (PIL) isolated directly from human lung were examined for their surface immune phenotype by monoclonal antibody staining and cytofluorimetry. In order to purify PIL, resected lungs were enzymatically digested with collagenase and DNase and subjected to density centrifugation and nylon-wool column separation. In some cases, CD4+ lymphocytes were further purified with alpha CD8 and complement. The majority of pulmonary lymphocytes were CD2+ (87 +/- 1%) and CD3+ (73 +/- 4%). Virtually all of the CD3+ PIL were Ti alpha beta+. Greater than 90% of both CD4+ or CD8+ PIL were CD45RO+ and CD45RA-, consistent with prior antigen sensitization in vivo. A subset of CD4+ PIL (34 +/- 4%) expressed Leu8, the human congener of the murine MEL-14 lymphocyte homing receptor, whereas most homologous CD4+ peripheral blood lymphocytes were Leu8+ (75 +/- 8; P less than 0.01). HLA-DR surface antigens were expressed by 45 +/- 5% of CD4+ PIL versus 9 +/- 1% of CD4+ peripheral blood lymphocytes (P less than 0.001). There was no significant difference in the percentage of low-affinity interleukin-2 (IL-2) receptor-positive CD4+ lymphocytes in lung and blood (9 +/- 3% versus 13 +/- 2%). Analysis of the DNA synthetic cell cycle showed that approximately 5% of blood CD4+ lymphocytes and approximately 25% of CD4+ PIL were in S/G2/M. Compared to homologous blood T cells, purified PIL displayed enhanced proliferative responses to IL-2 and diminished responses to the lectin phytohemagglutinin. Lectin-stimulated PIL showed greater secretion of interferon-gamma and IL-2 than did blood lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1991 Nov
PMID:Most human pulmonary infiltrating lymphocytes display the surface immune phenotype and functional responses of sensitized T cells. 193 Oct 75

In vitro studies have demonstrated that the estrogen receptor (ER) can bind to the rat PRL estrogen response element (ERE) located 1700 basepairs upstream of the transcriptional start site. However, the mechanism by which the receptor-DNA complex influences the activity of RNA polymerase located in the promoter region is not understood. To begin investigating this process, we developed cell lines derived from GH3 cells that contain steroid-responsive bovine papillomavirus minichromosomes. Within these minichromosomes is a hybrid gene composed of the 5' flanking region of the PRL gene, driving the expression of the Tn5 gene. The episomal PRL DNA sequences responded to 17 beta-estradiol (E2) by increasing the rate of Tn5 gene transcription. Nucleosome mapping experiments using micrococcal nuclease demonstrated that nucleosome-like structures were assembled on the minichromosome in an ordered array separated by 150-200 basepairs of DNA. Novel S1 nuclease as well as DNase-I-hypersensitive sites in the chromatin of the promoter and distal regulatory regions of the episomal PRL gene were detected by indirect end-labeling studies. The nuclease hypersensitive sites in the distal region containing the ERE were modified after treatment of the cells with either E2 or the antiestrogen 4-hydroxytamoxifen. However, only E2 treatment of cells resulted in an increase in the nuclease hypersensitivity of the promoter region and induced gene expression, while antiestrogen treatment had no effect on either parameter. This suggests that complex interactions between factors located at the distal and proximal regulatory regions ultimately determine the transcriptional response of the PRL gene to E2.
Mol Endocrinol 1990 Aug
PMID:An interaction between the 5' flanking distal and proximal regulatory domains of the rat prolactin gene is required for transcriptional activation by estrogens. 196 74

Transcription of the thyroglobulin (TG) gene in rat thyroid FRTL-5 cells is stimulated by two hormones, TSH and insulin-like growth factor-I (IGF-I). The effect of TSH is mimicked by cAMP. Promoter regions of the rat TG gene responsible for hormonal action as well as the nuclear regulatory proteins that interact with these regions were characterized. Minimal promoter that responds to both hormones has been found to be up to -171 basepairs from the transcription initiation site. In DNase-I footprinting analysis, nuclear extracts from cells treated with either of these hormones protected the same two major regions within the minimal promoter. Mutations in these two regions abolished basal, TSH-stimulated, as well as IGF-I-stimulated expression of the fused reporter gene chloramphenicol acetyltransferase. DNA mobility shift assay revealed that cAMP and IGF-I induce binding of similar nuclear proteins to these promoter regions. These results suggest that rat TG gene transcription is regulated by the convergent action of two distinct signaling pathways, possibly involving similar DNA-binding nuclear proteins and regulatory sequences of the TG gene promoter.
Mol Endocrinol 1990 Dec
PMID:Similar nuclear factors mediate stimulation of rat thyroglobulin gene transcription by thyrotropin and insulin-like growth factor-I. 196 92

The beta-subunit gene of TSH is specifically expressed in thyrotrope cells of the anterior pituitary gland. To define the particular TSH beta-subunit gene sequences responsible for tissue-specific expression, TSH beta promoter fragments were assessed for promoter activity by gene transfer into TSH-expressing thyrotropic tumor cells (TtT-97). Previous studies have shown that the murine TSH beta gene promoter was more efficiently used in TtT-97 cells compared to other pituitary-derived cells or nonpituitary fibroblasts and that a 191-basepair DNA sequence of the 5' flanking region between -271 and -80 was sufficient for maximal promoter activity in thyrotropes. Further deletional analysis within this region has localized the area responsible for expression in thyrotropes to a 37-basepair region between -117 and -80 up-stream of the major transcriptional initiation site. DNase-I protection assays demonstrated that this functionally defined 5' flanking area, in addition to the adjacent sequences immediately up-stream and down-stream, interacts with protein factors present in nuclear extracts from TtT-97 tumor cells. When fused to a heterologous promoter, fragments derived from the region between -271 and -80 exhibited cell-specific activity, although this was not conferred solely by the TSH beta promoter fragment from -117 to -80. Heterologous promoter activity was further stimulated when fragments containing the areas from -271 or -201 to -77 were used, suggesting combinatorial cis interactions between these regions of the TSH beta promoter. DNase-I protection studies suggest that there are multiple protein-binding domains in the mouse TSH beta 5' flanking sequence. Only the more proximal domains, which encompass important promoter elements, appear to be required for efficient expression in thyrotropes, whereas other more up-stream sites of protein interaction may be involved in regulatory aspects of TSH beta gene expression.
Mol Endocrinol 1990 Dec
PMID:Protein factors in thyrotropic tumor nuclear extracts bind to a region of the mouse thyrotropin beta-subunit promoter essential for expression in thyrotropes. 208 88

The polymerase chain reaction (PCR) was carried out with the highly conserved E. coli ribosomal RNA gene sequences 1376-1395 and 1521-1540. Using these primers and reaction conditions specified by the manufacturer(s), a 165 bp fragment was synthesized using Taq polymerase from three different sources in the absence of any added template. Restriction enzyme analysis suggests the source of this bacterial DNA is neither E. coli nor Thermus aquaticus. A variety of different methods to eliminate it such as treatment with DNase, restriction enzyme digestion, and CsCl2 density gradient centrifugation were unsuccessful. Since the bacteria in which the Taq polymerase is produced are not the source of the DNA, some step(s) in the purification or reagents added to the enzyme must be involved. Thus it is likely other biological products are similarly contaminated. Although the problem is easily dealt with by running a no-template control and choosing other primers if a problem exists, it is important to recognize the potential for a false-positive result.
Mol Cell Probes 1990 Dec
PMID:Taq polymerase contains bacterial DNA of unknown origin. 208 33

To determine whether enhancer elements in addition to the highly conserved octamer (OCTA)-nucleotide motif are important for lymphoid-specific expression of the immunoglobulin heavy-chain (IgH) gene, we have investigated the effect of mutating the binding site for a putative additional lymphoid-specific transcription factor, designated NF-microB, in the murine IgH enhancer. We demonstrate that the NF-microB-binding site plays a critical role in the IgH enhancer, because mutation of the microB DNA motif decreased transcriptional activity of the IgH enhancer in cells of the B-cell lineage but not in nonlymphoid cells. This effect was comparable to or even stronger than the effect of a mutation in the OCTA site. Moreover, combined mutation of both microB and OCTA sites further reduced enhancer activity in lymphoid cells. Interestingly, alteration of either the microB or E3 site in a 70-base-pair fragment of the IgH enhancer that lacks the binding site for OCTA abolished enhancer activity in lymphoid cells completely. Nevertheless, a multimer of the microB motif alone showed no enhancer activity. DNase footprinting analysis corroborated the functional data showing that a lymphoid-specific protein binds to the microB DNA motif. Our results suggest that the microB element is a new crucial element important for lymphoid-specific expression of the IgH gene but that interaction with another enhancer element is essential for its activity.
Mol Cell Biol 1990 Jun
PMID:Involvement of a second lymphoid-specific enhancer element in the regulation of immunoglobulin heavy-chain gene expression. 211 47


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