Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Enhancer activities have been observed in DNA fragments up to 1.36 kb long located on the 3'-side of the cluster of the three alpha-type globin-encoding genes in duck [Kretsovali et al., C.R. Acad. Sci. Paris 307 (1988) 563-568] and chicken [Knezetic and Felsenfeld, Mol. Cell. Biol. 9 (1989) 893-901]. We report here the identification of a chicken silencer element placed upstream from the three GATA-1 sites which constitute the core enhancer element in both species. This silencer element can autonomously reduce the activity of promoters for thymidine kinase and alpha D globin. Band shifts and DNase I footprinting experiments using nuclear extracts from thermosensitive avian erythroblastosis virus-transformed chicken erythroblasts led to the delineation of three sites for DNA-binding proteins within the silencer element.
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PMID:Silencer and enhancer elements located at the 3'-side of the chicken and duck alpha-globin-encoding gene domains. 810 Jul 90

The murine alpha B-crystallin gene (a member of the small heat shock protein family) is expressed constitutively at high levels in the lens and at lower levels in many other tissues, including skeletal muscle. We have previously used the herpes simplex virus thymidine kinase promoter fused to the human growth hormone gene to identify an alpha B-crystallin enhancer at positions -427 to -259 that has high activity in muscle and low activity in lens cell lines. In the study reported here, we performed DNase I footprinting, transfection, mutagenesis, and electrophoretic mobility shift experiments using the murine C2C12 muscle and alpha TN4-1 lens cell lines and the rabbit N/N1003A lens cell line to identify sequences responsible for activity of this enhancer. Enhancer activity in both the muscle and lens cells was dependent on novel elements called alpha BE-1 (-407 to -397), alpha BE-2 (-360 to -327), and alpha BE-3 (-317 to -306). These elements were also weakly occupied by nuclear proteins in L929 cells, which appear to express the alpha B-crystallin gene at a very low level (detectable only by the polymerase chain reaction). A fourth element containing a consensus muscle regulatory factor-binding site called MRF (-300 to -288) was occupied and used only by the C2C12 muscle cells. Cotransfection in NIH 3T3 cells and antibody-gel shift experiments using C2C12 nuclear extracts indicated that MyoD, myogen, or a similar member of this family can activate the alpha B-crystallin enhancer by interaction with the MRF site. Taken together, we conclude that the alpha BE-1, alpha BE-2, and alpha BE-3 elements are shared by both lens and muscle cells, but the MRF element is used only in muscle cells, providing the first example of a muscle-specific control element in a crystallin gene.
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PMID:The murine alpha B-crystallin/small heat shock protein enhancer: identification of alpha BE-1, alpha BE-2, alpha BE-3, and MRF control elements. 841 3

Regulation of galanin gene expression in the anterior pituitary (AP) is positively influenced by estrogen in rodents and undetermined in humans. The objective of this study was to investigate the mechanism behind estrogen induction of galanin by identifying any putative estrogen receptor (ER) binding sequences within the human galanin promoter that may function as estrogen response elements (ERE). Two regions, gERE1 and gERE2, were identified in the galanin 5'-flanking sequence with similarity to the full 13-base ERE consensus previously defined in the vitellogenin gene (vERE). Both sequences were tested in mobility shift assays for the ability to bind nuclear proteins isolated from rat AP tissue or MtTW-10 pituitary tumors. Only the distal sequence at -527 (gERE1) yielded an ERE-specific DNA/protein complex distinguished by mobility and cross-competition with vERE. The gel mobility pattern of the DNA/protein complex was comparable between the pituitary tissue and tumor extracts. However, DNA/protein affinity estimations demonstrated a greater affinity of pituitary proteins for gERE1 over the vERE sequence. Evidence that the human ER (hER) does recognize the gERE1 sequence in the human galanin gene was provided by electrophoretic mobility shift assays (EMSAs) with Sf9 extracts enriched in recombinant hER. In addition, antibodies specific for the hER recognized the gERE1/protein complex in supershift experiments. Enhancer activity by gERE1 was detected in transient transfections of the rat GH3 pituitary cell line, resulting in a 4-fold induction of expression driven by the heterologous thymidine kinase promoter in the presence of estrogen. Evidence for ER regulation of the gERE1 enhancer was demonstrated by: 1) inhibition of enhancement using the specific ER antagonist ICI 164,384; and 2) enhancement in HeLa cells that was dependent upon coexpression with hER. Enhancement by gERE1 was half the magnitude as that from the vERE element and may reflect a difference in affinity or composition of the ER complex between the two sequences. These data demonstrate the presence of a functional ERE sequence within the human galanin gene that could potentially function as a regulatory element for estrogen action in the AP.
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PMID:An estrogen receptor binding site within the human galanin gene. 934 90

A region located remotely upstream of the human pituitary GH (GH-N) gene and required for efficient GH-N gene expression in the pituitary of transgenic mice was cloned as a 1.6-kb Bg/II (1.6G) fragment. The 1.6G fragment in the forward or reverse orientation increased -496GH-N promoter activity significantly in pituitary GC and GH3 cells after gene transfer. The 1.6G fragment was also able to stimulate activity from a minimal thymidine kinase (TK) promoter which, unlike -496GH-N, lacked any Pit-1/GHF-1 element. Enhancer activity was localized by deletion analysis to a 203-bp region in the 3'-end of the 1.6G fragment and was characterized by the presence of a diffuse 136-bp nuclease-protected site, observed with pituitary (GC) but not nonpituitary (HeLa) cell nuclear protein. A major low-mobility complex was observed by electrophoretic mobility shift assay (EMSA) with GC cell nuclear protein, and the pattern was distinct from that seen with a HeLa cell extract. The nuclease-protected region contains three A/T-rich Pit-1/ GHF-1-like elements, and their disruption, in the context of the 203-bp region fused to the TK promoter, reduced enhancer activity significantly in pituitary cells in culture. A mutation in this region was also shown to decrease enhancer activity in transgenic mice and correlated with a decrease in the 203-bp enhancer region complex observed by EMSA. The participation of Pit-1/GHF-1 in this complex is indicated by competition studies with Pit-1/GHF-1 elements and antibodies, and direct binding of Pit-1/GHF-1 to the A/T-rich sequences was shown by EMSA using recombinant protein. These studies link the A/T-rich sequences to the distal enhancer activity associated with the GH locus control region in vitro and in vivo.
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PMID:A role for A/T-rich sequences and Pit-1/GHF-1 in a distal enhancer located in the human growth hormone locus control region with preferential pituitary activity in culture and transgenic mice. 1044 1

Myeloperoxidase (MPO) is a granule protein, transiently expressed during the promyelocyte stage of myeloid differentiation. It is transcribed in a stage and lineage specific manner. Studies of MPO gene regulation can help to elucidate the mechanism of normal and abnormal myeloid differentiation. Our preliminary data indicated the lack of basal promoter activity in the region immediately 5' to the MPO cDNA. Here, we report the results of the detailed molecular studies of the human MPO promoter region. To locate potential promoter elements active in HL60 cells, we made promoter deletion constructs ranging in size from 200 bp to 4.5 kb of the 5' region of the hMPO gene, cloned into the chloramphenicol acetyl transferase (CAT) reporter vector. Following electroporation of the promoter constructs into HL60 cells, CAT enzyme production was found only in the construct containing approximately the 1 kb region upstream of the reported MPO cDNA. A separate set of constructs was made to look for putative MPO enhancer elements. Several fragments upstream of the MPO promoter showed prominent transactivation of the TK promoter, indicating a possible enhancer. Tissue specificity of MPO promoter fragments was determined in myeloid cells arrested either before induction of MPO expression (KG1), during MPO expression (HL60), or after it had ceased (U937), as well as in non-MPO expressing non-myeloid cells. The construct containing an approximate 1000 bp fragment of the 5' region of MPO was found to direct CAT expression only in HL60 cells. The 3'-truncations of this promoter region resulted in loss of tissue-specificity, while the promoter activity remained largely unchanged. A negative regulatory element was found upstream of the MPO promoter which repressed heterologous promoters in all the tested cell lines. Enhancer elements showed no tissue- or stage-specificity that were characteristic for native MPO gene. Sequence analysis of the putative MPO promoter region showed a number of potential transcription factor binding sites. Of special interest is the region containing the purine-rich site that can bind proteins from the ets-family of transcription factors and a duplicate GATA-like site. When inserted upstream of a reporter containing the minimal Herpes simplex viral thymidine kinase (HSV-TK) promoter (into pBL2CAT plasmid) this site strongly activated the TK promoter in transfected myeloid cells. Further studies showed that oligonucleotides derived from the MPO promoter region bind multiple proteins in a band-shift assay. Taken together, our experiments located the regulatory elements important for human MPO gene expression in HL60 promyelocytes.
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PMID:Molecular analysis of the human myeloperoxidase promoter region. 1063 85

Activation of the human growth hormone gene (hGH-N) is linked to a locus control region (LCR) containing four (I-III, V) hypersensitive sites (HS). Pit-1 binding to HS I/II is required for efficient pituitary expression. However, inclusion of HS III and V, located about 28 and 32 kb upstream of the hGH-N gene, respectively, is also required for consistent hGH-N expression levels in vivo. HS V is referred to as a boundary for the hGH LCR, but no specific enhancer blocking or barrier function is reported. We examined a 547 bp fragment containing HS V sequences (nucleotides -32,718/-32,172 relative to hGH-N) for enhancer-blocking activity using a well-established transient gene transfer system and assessed these sequences for CCCTC binding factor (CTCF), which is linked to enhancer-blocking activity. The 547 bp HS V fragment decreased enhancer activity with a reverse-orientation preference when inserted between HS III enhancer sequences and a minimal thymidine kinase promoter (TKp). These sequences are associated with CTCF in human pituitary and nonpituitary chromatin. Enhancer-blocking activity with an orientation preference was further localized to a 45 bp sub-fragment, with evidence of CTCF and upstream binding factor 1 (USF1) binding; USF1 is linked more closely with barrier function. The presence of yin and yang 1 (Yy1) that cooperates with CTCF in the regulation of X-chromosome inactivation was also seen. A decrease in CTCF and Yy1 RNA levels was associated with a significant reduction in enhancer-blocking activity. Assessment of CpG-dinucleotides in the TKp indicates that the presence of HS V sequences are associated with an increased incidence of CpG-dinucleotide methylation of the GC box region. These data support association of CTCF and enhancer-blocking activity with HS V that is consistent with a role as a (LCR) boundary element and also implicates Yy1 in this process.
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PMID:Enhancer-blocking activity is associated with hypersensitive site V sequences in the human growth hormone locus control region. 2171 Nov 61