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

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

The early proteins of simian virus 40 (SV40) large T and small t antigen (T/t antigen) can each cause the transcriptional activation of a variety of cellular and viral promoters. We showed previously that simian cellular DNA-binding factors (the Band A factors) bind to sequences within the SV40 late promoter which are important for transcriptional activation in the presence of the SV40 early proteins. Band A factors isolated from simian cells which produce T/t antigen (COS cells or SV40-infected CV-1 cells) have altered binding properties in comparison with the factors from normal simian cells (CV-1). This suggests that the transcriptional activation mediated by T/t antigen may be due to either modification of existing factors or induction of new members of a family of factors. We have purified the Band A factors from both COS and CV-1 cells and have determined the binding site by methylation interference and DNase protection footprinting. The COS cell factors have altered chromatographic properties on ion-exchange columns and have higher-molecular-weight forms than the CV-1 cell factors. Major forms of the CV-1 factors migrate between 20 and 24 kilodaltons, while the COS factors migrate between 20 and 28 kilodaltons. The binding sites for the factors from CV-1 and COS cells are similar, covering a rather broad region within the 72-base-pair repeat comprising the AP-1 site and the two-octamer binding protein (OBP100/Oct 1) sites, OBP I and OBP II. Specific binding competition analyses indicate that the two general regions within the binding site (the AP-1-OBP II site and the OBP I site) each retain partial binding ability; however, the factors bind best when the two regions are adjacent in a relatively specific spatial arrangement. The binding site for the Band A factors corresponds very well to sequences necessary for the activation of the late promoter as defined by deletion and base substitution mutagenesis studies (J. M. Keller and J. C. Alwine, Mol. Cell. Biol. 5:1859-1869, 1985; E. May, F. Omilli, M. Emoult-Lange, M. Zenke, and P. Chambon, Nucleic Acids Res. 15:2445-2461, 1987). These data, in combination with the data showing that the Band A factors are modified or induced in the presence of T/t antigen, strongly suggest that T/t antigen mediates its transcriptional activation function, at least in part, through the Band A factors.
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PMID:Activity of simian DNA-binding factors is altered in the presence of simian virus 40 (SV40) early proteins: characterization of factors binding to elements involved in activation of the SV40 late promoter. 215 10

The processes responsible for the multidrug-resistant (Mdr) phenotype in Adriamycin (doxorubicin)-resistant HL-60 leukemia cells (HL-60/AR) are not defined. Since enhanced transcription of resistance-related proteins is associated with Mdr cells, we sought to determine whether changes in the expression of specific transcription factors were a feature characteristic of the Mdr process. Nuclear extracts were prepared from wild-type and resistant cells and compared for their ability to bind DNA consensus sequences for the transcription factors Sp1 and NF kappa B contained in the 5' long terminal repeat region of human immunodeficiency virus type 1. Southwestern (DNA-protein) blots showed a family of DNA-binding proteins of 105 kilodaltons (kDa) that were present only in HL-60/AR cells. Competitive gel shift assays indicated that these factors were related to transcription factor Sp1, and immunoblotting with an Sp1 antibody identified this factor as Sp1. DNase footprinting of the promoter region in the human immunodeficiency virus type 1 5' long terminal repeat showed that protection occurred at two Sp1 sites as well as two NF kappa B sites and the trans-acting region with nuclear extracts only from resistant cells. Preliminary evidence also suggests that phosphorylation may play a negative regulatory role in the activity of Sp1, since calf intestine alkaline phosphatase stimulated the DNA-binding activity of Sp1 in vitro. These results indicate that HL-60/AR cells contain an abundance of DNA-binding proteins, particularly Sp1, which probably interact with other cis-acting regulatory proteins in a cooperative manner.
Mol Cell Biol 1990 Oct
PMID:Increased expression and DNA-binding activity of transcription factor Sp1 in doxorubicin-resistant HL-60 leukemia cells. 220 18

The identities of two cloned, arabinose-inducible promoters were tested by hybridizing promoter DNA fragments with restriction digests of chromosomal DNA containing Mudlac phage inserted in either araFGH or in araE transport operons. One promoter, thought to be araE, is within 10(3) base-pairs of a Mudlac insertion in the araE gene. The second promoter was not found within several thousand base-pairs of either of the known transport genes. This promoter is now named araPJ (araJ). The DNA sequence of the fragment containing the araFGH promoter was determined. The start site of transcription in vivo was located to within +/- 1 base-pair (bp) by S1 nuclease mapping. DNase 1 footprinting revealed that, in comparison with the araBAD and araE promoters, the locations of the AraC and cyclic AMP receptor protein (CRP) binding sites are reversed with CRP lying between AraC and RNA polymerase. The central location of the CRP binding site may explain why the araFGH promoter is more catabolite sensitive than the other ara promoters. AraC and CRP were both required for maximal transcription in vitro, although a low level of transcription was detected with CRP alone. S1 nuclease mapping of mRNA-DNA hybrids from the araJ promoter located the transcription start point to within #/- 3 bp, and demonstrates that the promoter is dependent upon AraC protein and CRP in vivo. DNase footprinting showed that the location of the AraC protein binding site on araJ is adjacent to the RNA polymerase site, as seen at the araBAD and araE promoters. Two CRP sites were observed; one is upstream from the AraC site and one is downstream from the transcription start site.
J Mol Biol 1990 Oct 20
PMID:Characterization of the Escherichia coli araFGH and araJ promoters. 223 17

In designing new DNA recognizing and cleaving reagents, we introduce herein a bisacridine derivative (referred to as bisacridine) in which two acridine heterocycles are connected by a penta(ethylene glycol) bridging chain. This compound offers two possible functions: 1, stabilization of DNA bisacridine intercalator complex by metal ion. The penta(ethylene glycol) chain stabilizes metal ions binding to the phosphate site of DNA, where the penta(ethylene glycol) chain constitutes a part of a pseudomacrocyclic ligand for metal binding; and 2, enhancement of metal-assisted hydrolytic cleavage of DNA by means of a metal concentration effect by the pseudomacrocyclic ethereal chain. The binding isotherms of bisacridine with DNA in the presence of metal ions showed that the binding was mainly governed by the cation exchange reaction on the anionic DNA polymer chain, i.e., the exchange between metal ions and the cationic bisacridine. The bisacridine showed an increase DNA binding ability compared to quinacrine, the monoacridine counterpart, and caused an enhancement of DNA cleavage in the presence of Cu2+ ions. Additional experiments which included DNase 1 footprinting in the presence of bisacridine and the DNA cleavage by Cu2+/bisacridine using a 32P end-labelled DNA fragment, suggested that the Cu2(+)-assisted DNA cleavage sites in the presence of bisacridine were in reasonable overlap with the DNA binding sites of bisacridine.
J Mol Recognit 1990 Aug
PMID:Cleavage of double helical DNA by Cu2+ ion in the presence of bisintercalator containing penta(ethylene glycol) connector chain. 227 32

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.
Mol Endocrinol 1990 Apr
PMID:The uteroglobin promoter contains a noncanonical estrogen responsive element. 228 Jul 77

The expression of the gene for the C3 polypeptide is confined to the ventral prostate of the male rat and is regulated by androgens. To study the mechanism of this tissue-specific and hormone-dependent regulation we have used DNase-I footprinting and band-shift assays to locate binding sites for nuclear proteins isolated from different tissues. In this paper we present evidence that there are tissue-specific differences in the nuclear proteins that are able to bind to a CCAAT motif and a nuclear factor-I consensus that are present in the promoter of the rat C3 gene. Using competition assays and heat denaturation we show that the CAAT box/enhancer binding protein itself may be one of the transcription factors involved.
Mol Endocrinol 1990 Aug
PMID:Tissue-specific differences in the binding of nuclear proteins to a CCAAT motif in the promoter of the androgen-regulated C3 gene. 229 26

The DNA-binding, annealing and recombinational activities of purified RecA-DNA complexes stabilized by ATP gamma S (a slowly hydrolysable analog of ATP) are described. Electrophoretic analysis, DNase protection experiments and observations by electron microscopy suggest that saturated RecA complexes formed with single- or double-stranded DNA are able to accommodate an additional single strand of DNA with a stoichiometry of about one nucleotide of added single-stranded DNA per nucleotide or base-pair, respectively, of DNA resident in the complex. This strand uptake is independent of complementarity or homology between the added and resident DNA molecules. In the complex, the incoming and resident single-stranded DNA molecules are in close proximity as the two strands can anneal in case of their complementarity. Stable RecA complexes formed with single-stranded DNA bind double-stranded DNA efficiently when the added DNA is homologous to the complexed strand and then initiate a strand exchange reaction between the partner DNA molecules. Electron microscopy of the RecA-single-stranded DNA complexes associated with homologous double-stranded DNA suggests that a portion of duplex DNA is taken into the complex and placed in register with the resident single strand. Our experiments indicate that both DNA binding sites within RecA helical filaments can be occupied by either single- or double-stranded DNA. Presumably, the same first DNA binding site is used by RecA during its polymerization on single- or double-stranded DNA and the second DNA binding site becomes available for subsequent interaction of the protein-saturated complexes with naked DNA. The way by which additional DNA is taken into RecA-DNA complexes shows co-operative character and this helps to explain how topological problems are avoided during RecA-mediated homologous recombination.
J Mol Biol 1990 Mar 05
PMID:Characterization of the DNA binding activity of stable RecA-DNA complexes. Interaction between the two DNA binding sites within RecA helical filaments. 231 1


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