Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:BIOD00001 (DNase I)
 8,324 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The transcriptional start sites of the endogenous human thrombomodulin (TM) gene and transiently expressed TM promoter/CAT gene constructs were defined by nuclease S1 mapping which showed two closely spaced sites at +1 and +6, respectively. Transient expression and in vitro transcription assays of 5' and internal deletion mutants of the TM promoter/CAT gene constructs reveal that the region from -72 to -29 exhibits a positive acting domain which is essential for transcriptional activity, whereas the region from -373 to -225 possesses two positive acting subdomains, -343 to -277 and -245 to -225, which together augment transcriptional activity by about 40%. Electrophoretic mobility shift assays with a duplex oligonucleotide corresponding to -72 to -29 and DNase I footprinting experiments show two specific interaction products which individually or cooperatively protect the DNA sequence from about -60 to -30. These components are essential for TM gene transcription since affinity fractionation of nuclear extracts with a duplex oligonucleotide corresponding to -72 to -29 depletes the above interaction products and specifically inhibits in vitro transcription activity of the promoter, whereas addition of the eluted components specifically restores in vitro transcription activity of the promoter. Electrophoretic mobility shift assays with duplex oligonucleotides corresponding to -294 to -215, as well as -373 to -295 and DNase I footprinting experiments show two specific interaction products which individually bind to the two subdomains but not -72 to -29 and protect the coding and noncoding strands from -245 to -225, and the noncoding strand from -337 to -314, respectively. Transient expression studies reveal that the TM promoter construct starting at -51 and including the TATA box is responsive to TNF only in cell lines exhibiting sensitivity of the endogenous receptor gene to cytokine, whereas other promoter constructs possessing a TATA box sequence are insensitive to TNF in all cell types. Based upon the above data, the regulatory events involved in TNF-dependent transcriptional regulation of the TM gene can be defined with the experimental tools and conceptual framework developed by the present investigation.
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PMID:Transcriptional regulation of the thrombomodulin gene. 133 Oct 78

The 5'-flanking DNA of the mouse RII beta subunit of the cAMP-dependent protein kinase gene was characterized by transient transfection of RII beta-CAT constructs into mouse neuroblastoma cells (NB2a) and Chinese hamster ovary (CHO) cells and by gel mobility shift and footprinting assays. The minimal promoter of the RII beta gene was composed of two adjacent functional elements. A 3'-element which supported enhanced CAT activity was located between base pairs (bp) -267/-168 from the translation initiation start site. CAT plasmids containing these RII beta sequences showed 12- and 16-fold increased CAT activity in the NB2a and CHO cells, respectively, compared to the basic CAT vector. Plasmids containing 20 additional bp 5' to the -267/-168 fragment showed 2-fold more CAT activity than the shorter fragment in NB2a cells, while CAT activity in CHO cells was nearly the same for both constructs. CAT plasmids containing only this 20-bp fragment showed 9- and 13-fold increased CAT activity in NB2a and CHO cells, respectively. The core promoter of the RII beta gene lacked classical TATA and CAT sequences, but contained 3 copies of the Sp1 core consensus sequence. Gel mobility shift assays using 32P-labeled 5'-flanking DNA containing bp -291/-49 and nuclear extracts from NB2a and CHO cells displayed several retarded bands in the gels suggesting complex formation with nuclear DNA-binding factors. Unlabeled DNA containing bp -291/-49 blocked the appearance of all retarded bands. Competition using an oligonucleotide corresponding to the Sp1 DNA-binding site effectively blocked the appearance of the two more slowly migrating bands but did not affect the major rapidly migrating bands. DNase I footprinting analysis using purified Sp1 protein confirmed that Sp1 could bind to the Sp1 sites. Methylation interference and mutational analysis showed that one of the faster migrating bands was the result of factor binding to the DNA sequence adjacent to the Sp1 sites. Additional tissue-specific nuclear-binding factor sequences were detected upstream of the core promoter. Our data suggest that the core promoter of the RII beta gene can initiate transcription from the DNA around the Sp1 sites but that there are tissue-specific nuclear factor-binding sites located distal to the Sp1 sites.
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PMID:Characterization of a minimal promoter element required for transcription of the mouse type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase. 133 64

Expression of the immediate early 1 and 2 gene (IE-1/2) of human cytomegalovirus, an important pathogen in immunosuppressed patients, is controlled by a strong enhancer/promoter. To define the promoter domain within this large cis-active region of about 550 nucleotides, DNA-protein interactions were studied. DNase I footprinting experiments using procaryotically expressed transcription factor Sp1 revealed an extensive interaction of this transcription factor with both consensus and aberrant recognition elements within the IE-1/2 promoter region. Protection of these Sp1 binding sites could also be observed when nuclear extracts prepared from HeLa cells and permissive human fibroblast cells were used. After in vitro mutagenesis of Sp1 targets and transient expression of mutagenized CAT-expression plasmids, however, no significant reduction in CAT activities was found. By analyzing a series of 5' deletion mutants of the IE-1/2 promoter region, a strong cis-acting element was localized between nucleotides -94 and -78, upstream of sites that interact with Sp1. Gel retardation experiments demonstrated binding of recombinant transcription factor CREB to this motif which reveals it as an aberrant CREB recognition sequence. Thus, this study identifies several previously unknown binding sites for transcription factors Sp1 and CREB within the proximal promoter region of the IE-1/2 gene, which differ markedly in their relevance for constitutive promoter function.
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PMID:Analysis of proteins binding to the proximal promoter region of the human cytomegalovirus IE-1/2 enhancer/promoter reveals both consensus and aberrant recognition sequences for transcription factors Sp1 and CREB. 138 62

To investigate the regulatory processes involved in the expression of the D2 dopamine receptor gene, a rat genomic clone was isolated using a 21-mer oligonucleotide probe made of exon 1 sequences. A 1.3-kb region including all of exon 1, its 5'-flanking region, and part of intron 1 was sequenced. S1 nuclease analysis indicated three consecutive nucleotides as the main transcription start sites; several weaker sites were also noted between 321 and 363 nucleotides upstream from the 3' end of exon 1. The promoter region lacks TATA and CAAT boxes and is rich in G+C content with several putative Sp1 binding sites. Transient expression assays using chimeric constructs of D2 promoter deletion mutants-chloramphenicol acetyl-transferase gene in the neuroblastoma cell line NB41A3 which expresses D2 binding sites indicated strong transcription enhancing activity between nucleotides -75 and -30 and silencing activity between nucleotides -217 and -76. DNase I footprinting studies using nuclear extract from NB41A3 suggested Sp1 binding to its consensus sequence at nucleotide -48 but inhibition of Sp1 binding at nucleotide -86 by the extract. The D2 promoter could not induce transcription of the heterologous CAT gene in C6 glioma, embryonal NIH 3T3, or hepatic Hep G2 cells. It is concluded that the rat D2 gene shares with the human D1A dopamine receptor gene several features typical of "housekeeping" genes but they are both tissue-specific, regulated genes. Unlike the D1A gene, however, the D2 gene has a strong preference for transcription initiation to three consecutive nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Analysis of the promoter region of the rat D2 dopamine receptor gene. 139 Jun 23

The plasma cholesteryl ester transfer protein (CETP), primarily synthesized in the liver of several species, is expressed at very low levels in a number of transformed human liver cell lines. The human CETP gene promoter contains a sequence that closely resembles the binding site for the transcription factor CCAAT/enhancer-binding protein (C/EBP). This site is capable of binding C/EBP, as shown by electrophoretic mobility shift and DNase I footprint analyses. Transient expression of the bacterial chloramphenicol acetyltransferase gene under the control of the human CETP gene promotor gave low activities in the human hepatoma cell line, HepG2. However, in the presence of C/EBP, CAT activity was markedly elevated indicating that CETP gene promoter activity was enhanced. In primary cultures of isolated hepatocytes, CETP mRNA was lost rapidly and in parallel with the C/EBP mRNA. C/EBP may play an important role in the proper maintenance of CETP gene promoter activity, and its low levels in proliferating or cultured cells may account for the low level of the CETP gene expression in immortalized human liver cell lines or cultured hepatocytes.
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PMID:The CCAAT/enhancer-binding protein trans-activates the human cholesteryl ester transfer protein gene promoter. 142 86

The X protein of hepatitis B virus (HBV) is a transactivator to homologous and heterologous viral and cellular transcriptional regulatory elements. One sequence-specific binding protein, whose binding site located from nt 1102 to nt 1117 of HBV DNA, was identified by mobility shift assay and DNase I foot-printing analysis. A CAT assay experiment demonstrated this 16-bp binding site to have a promoter activity in the X gene transcription. The 58-bp DNA fragment (nt 1085 to nt 1142), which contains the above binding site, could be enhanced by the HBV enhancer. Mobility shift assay using the mutated 58-bp DNA fragments as probes, showed that the mutation, which damaged the palindrome structure between nt 1105 and nt 1112, resulted in loss of the binding activity. This mutation also remarkably reduced the promoter activity. The binding site differed from the target sequences of known transcriptional factors. This factor was thus concluded to be a binding protein to the X gene promoter (X-PBP) of HBV. A homology search demonstrated the binding site to be highly homologous to the promoter elements of human laminin receptor (2H5epitope) and lipoprotein receptor-related protein (LRP) genes.
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PMID:Identification of a binding protein to the X gene promoter region of hepatitis B virus. 144 11

The tyrosinase gene is specifically expressed in melanocytes. Understanding the molecular basis of tissue-specific expression of the tyrosinase gene will greatly explain the mechanisms controlling pigmentation. We report a nucleotide sequence, TGATGTATTC, located -236 base pairs upstream of the transcription start site, that enhances tyrosinase gene expression in mouse melanoma cells. The sequence is referred to as the tyrosinase element-1 (TE-1). TE-1 was protected from DNase I cleavage by pigment cell nuclear extracts but was not protected by non-pigment cell nuclear extract. Partial purification of TE-1 binding protein (TEBP-1) was performed from the B16 mouse melanoma cell nuclear extract using biotin-cellulose affinity chromatography. The affinity-purified fraction exhibited binding to the DNA fragment containing TE-1, and to a synthetic oligomer representing TE-1. UV-cross-linking indicated that the size of TEBP-1 is approximately 49 kD. TE-1 also directed enhanced CAT activity in the B16 melanoma cells but not in non-pigment cells. These data indicate that TE-1 may be an enhancer element that is responsible for pigment cell specific expression of the tyrosinase gene.
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PMID:A cis-acting element involved in mouse tyrosinase gene expression and partial purification of its binding protein. 149 37

A strong positive element within the proximal promoter region of the human beta-myosin heavy chain (beta-MHC) gene that is required for high level expression in primary cultures of fetal rat heart cells was localized by transient assays and DNase I footprinting to positions- 277/-298. Using gel shift studies, this sequence was found to bind specifically at high affinity (Kd approximately 4 x 10(-9) M) to a transcriptional factor (beta F1) found in nuclear extracts from rabbit heart. Dimethyl sulfate interference studies suggested that beta F1 may bind as a dimer to two hexameric imperfect direct repeats containing the consensus sequence 5'-(C/G)-T-G-(T/A)-G-G-3'. Gel shift analyses suggested that beta F1 is related to the M-CAT factor, which is known to control muscle-specific expression of the cardiac troponin T gene. A clustered mutation of the region between the putative binding half-sites and within the "M-CAT"-like domain abolished beta-MHC promoter activity. The sequence of the positive element also contains binding motifs for several transcriptional factors that regulate viral and cellular genes, including AP4, AP5, TEF-1, and MyoD-like proteins. When multiple copies of the beta-MHC element were inserted downstream from the transcriptional initiation site of the thymidine kinase gene, it did not act as a classical enhancer, showing some dependence upon orientation.
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PMID:Characterization of a strong positive cis-acting element of the human beta-myosin heavy chain gene in fetal rat heart cells. 157 22

The latency associated transcript (LAT) gene is the only viral genomic region that is abundantly transcribed during herpes simplex virus type 1 (HSV-1) neuronal latency. As such, it may play an important role in HSV-1 latency and/or reactivation. The regulation of the LAT gene is complex and appears to include a combination of positive and negative functional elements in and near the LAT promoter. In this study, transient CAT assays were used to map the minimal promoter necessary for constitutive activity in neuronal and nonneuronal cells to between nucleotide positions -161 and -2 (relative to the start of LAT transcription). The region from -283 to -161 was able to slightly increase promoter activity of the minimal promoter and appeared to have a larger effect in neuronal derived cells. Gel-shift experiments using nuclear extracts from neuronal and nonneuronal derived cells detected a major factor that bound specifically to the -161 to -2 probe. We designated this factor LAT promoter binding factor (LPBF). Two additional minor factors also bound specifically to the minimal promoter. DNase I footprint analysis and gel-shift competition experiments demonstrated that LPBF bound to a region that includes the palindromic sequence CCACGTGG located at nucleotides -72 to -65. Deletion of this palindrome resulted in a loss of binding of LPBF from the minimal promoter region and an 8- to 30-fold reduction in promoter activity in both neuronal and nonneuronal cells. Thus, LPBF appears to play a major role in LAT promoter regulation.
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PMID:Identification of a major regulatory sequence in the latency associated transcript (LAT) promoter of herpes simplex virus type 1 (HSV-1). 185 Sep 7

Mouse lactoferrin is expressed in a variety of tissues under different types of control. To understand how molecular mechanisms govern the mode of lactoferrin expression, we isolated and characterized the 5'-flanking region of the lactoferrin gene. Several clones containing lactoferrin gene fragments were isolated from a mouse (129/J) genomic library including clone lambda J14, which contains a 7.5-kilobase pair 5'-flanking sequence. Sequence analysis of the region flanking the transcription initiation site revealed the following: a TATA-like sequence, two CAAT boxes, three GC boxes including one within the first intron, an AP2 site, seven PU boxes, an AC-rich region, a B1 sequence, and an estrogen-responsive element consensus sequence over-lapping with a chicken ovalbumin upstream promoter-binding element. Footprinting analysis demonstrated that several regions, including the putative estrogen-responsive element region, in the 5'-flanking sequence were protected from DNase I digestion. Promoter fragments were cloned into a chloramphenicol acetyltransferase receptor plasmid to study functional activity. The mouse lactoferrin gene promoter was active in human endometrium carcinoma RL 95-2 cells and in rat glioma C6 cells. Multiple upstream elements modulated the basal transcriptional promoter activity. The transcription level directed by this minimal promoter was controlled by both positive (between -1739 and -922) and negative (between -2644 and -1739, and between -589 and -291) regulatory sequences. A tissue-specific regulatory sequence was critical for the establishment of lactoferrin expression in human endometrium carcinoma cells, but not in rat glioma cells located between -1739 and -922. Reporter plasmid 0.6 mL14-CAT, containing the estrogen-responsive element sequence, was estrogen-responsive in the presence of estrogen receptor in human endometrium carcinoma RL 95-2 cells.
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PMID:Characterization of estrogen-responsive mouse lactoferrin promoter. 193 12


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