Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.
Mol Cell Biol 1991 Feb
PMID:The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells. 199 Feb 64

The physiological significance of in vitro leucine zipper interactions was studied by the use of two strategies which detect specific protein-protein interactions in mammalian cells. Fusion genes were constructed which produce chimeric proteins containing leucine zipper domains from several proteins fused either to the DNA-binding domain of the Saccharomyces cerevisiae GAL4 protein or to the transcriptional activation domain of the herpes simplex virus VP16 protein. Previous studies in mammalian cells have demonstrated that a single chimeric polypeptide containing these two domains will activate transcription of a reporter gene present downstream of the GAL4 DNA-binding site. Similarly, if the GAL4 DNA-binding domain of a chimeric protein could be complexed through leucine zipper interactions with the VP16 activation domain of another chimeric protein, then transcriptional activation of the reporter gene would be detected. Using this strategy for detecting leucine zipper interactions, we observed homo-oligomerization between leucine zipper domains of the yeast protein GCN4 and hetero-oligomerization between leucine zipper regions from the mammalian transcriptional regulating proteins c-Jun and c-Fos. In contrast, homo-oligomerization of the leucine zipper domain from c-Myc was not detectable in cells. The inability of the c-Myc leucine zipper to homo-oligomerize strongly in cells was confirmed independently. The second strategy to detect leucine zipper interactions takes advantage of the observation that the addition of nuclear localization sequences to a cytoplasmic protein will allow the cytoplasmic protein to be transported to and retained in the nucleus. Chimeric genes encoding proteins with sequences from a cytoplasmic protein fused either to the GCN4 or c-Myc leucine zipper domains were constructed. Experiments with the c-Myc chimeric protein failed to demonstrate transport of the cytoplasmic marker protein to the nucleus in cells expressing the wild-type c-Myc protein. In contrast, the cytoplasmic marker was translocated into the nucleus when the GCN4 leucine zippers were present on both the cytoplasmic marker and a nuclear protein, presumably as a result of leucine zipper interaction. These results suggest that c-Myc function requires hetero-oligomerization to an as yet undefined factor.
Mol Cell Biol 1991 Feb
PMID:Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization. 199 Feb 93

The 5' flanking region of the mouse N-ras gene was investigated to determine the elements governing transcriptional activity of the gene. The promoter did not contain typical TATA or CCAAT boxes, and according to primer extension and RNase protection analyses, transcription started at several sites. These assays also confirmed the short nucleotide distance interposed between the N-ras transcription unit and the previously described upstream unr gene. Chromatin studies performed by digestion of nuclei with DNase I revealed the presence of four hypersensitive sites: a, b, c, and d. Deletion mutagenesis of the 5' flanking region revealed sequences responsible for both promotion and inhibition of transcription. These sequences resided within 230 bp upstream of the transcription initiation site. Hypersensitive site b colocalized with the 76-bp segment with promoter activity. The negative regulatory element at position -180 colocalized with hypersensitive site a, was active on the N-ras promoter in stable as well as transient assays, and down-regulated the heterologous herpes simplex virus thymidine kinase promoter. Footprint analysis and in vivo transfection-competition experiments indicated that a trans-acting factor is responsible for the negative effect on transcription. The interaction between the cis-acting negative regulatory element and the promoter region may play a role in the tissue- and developmental-stage-specific patterns of expression of the N-ras gene.
Mol Cell Biol 1991 Mar
PMID:Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element. 199 95

A synthetic, 23-bp ecdysterone regulatory element (EcRE), derived from the upstream region of the Drosophila melanogaster hsp27 gene, was inserted adjacent to the herpes simplex virus thymidine kinase promoter fused to a bacterial gene for chloramphenicol acetyltransferase (CAT). Hybrid constructs were transfected into Drosophila S3 cells and assayed for ecdysterone-inducible CAT expression. In the absence of ecdysterone a tandem pair of EcREs repressed the high constitutive level of CAT activity found after transfection with the parent reporter plasmid alone. After hormone addition very high levels of CAT activity were observed. Insertion of the EcRE pair 3' of the CAT gene also led to high levels of ecdysterone-induced CAT expression, but the repression of high constitutive levels of CAT activity failed to occur. The EcRE-CAT construct was cotransfected with plasmids containing tandem 10-mers or 40-mers of the EcRE but lacking a reporter gene. These additional EcREs led to a reduced level of ecdysterone-induced CAT activity and to an elevation of basal CAT activity in the absence of hormone. The data suggest that the receptor binds to the EcRE in the absence of hormone, blocking basal transcription from a constitutive promoter. In the presence of ecdysterone, receptor-hormone binding to the EcRE leads to greatly enhanced transcription.
Mol Cell Biol 1991 Apr
PMID:Ecdysterone regulatory elements function as both transcriptional activators and repressors. 200 85

Although the estrogen responsiveness and estrogen receptors of Xenopus hepatocytes have been well described, oocytes of this species have not previously been shown to contain estrogen receptors (ER). Recombinant human ER (HER) was expressed in oocytes in a dose dependent fashion as measured by [35S]methionine incorporation into newly synthesized proteins. Chloramphenicol acetyl transferase (CAT) reporter plasmids, driven by a herpes simplex thymidine kinase promotor with or without a 17 base pair estrogen response element (ERE) from the vitellogenin A2 gene, were also injected into oocytes. When injected without the accompanying HER sequences, the construct containing the ERE expressed 10-fold more CAT activity, and this response was saturable as demonstrated by injecting increasing amounts of reporter plasmid. These results suggest either the activity of small amounts of a Xenopus ER (measured here by LH-20 assay), or the presence of some endogenous oocyte protein other than the ER that can interact with this ERE. When HER was co-expressed with ERECAT, CAT expression was suppressed over a wide range of HER concentrations. This unexpected repression may be due to displacement of an estrogen receptor or other endogenous oocyte regulatory protein on the ERE. HER's positive regulatory activity may require transcription factors that are lacking or insufficient in the oocyte. Alternatively the simple 17 base pair ERE may not provide DNA binding sites for such transcription factors.
J Steroid Biochem Mol Biol 1991 Apr
PMID:Human estrogen receptor introduced into the Xenopus oocyte represses expression from an artificial frog estrogen response element. 203 57

Transformed (GH-3) and non-transformed (P3) rat anterior pituitary cells were compared in their ability to direct expression of plasmids containing a variety of eukaryotic transcriptional regulatory elements (TREs). These include the herpes simplex virus thymidine kinase (HSV-TK), Rous sarcoma virus long terminal repeat (RSV-LTR), simian virus 40 early (SV-40E), human cytomegalovirus immediate-early (CMV-IE) and mouse metallothionein 1 (mMT-1) TREs. Chloramphenicol acetyl transferase (CAT) gene expression served as a reporter in this study. Following transient transfection, the cell lines exhibited similar profiles of TRE utilization. In each cell line. CMV-IE was most efficient in directing reporter gene expression, although 2-fold greater activity was observed in GH-3 versus P3 cells. RSV-LTR directed gene expression was lower than that of CMV-IE while both HSV-TK and SV-40E were inactive in each cell line. Also, the mMT-1 promoter was inducible by addition of ZnCl2 to the culture media, though the level required for maximal activation differed between the two cell lines. Transfected GH-3 and P3 cells, therefore, displayed similar TRE utilization profiles yet significant differences were observed in the ability of these cell lines to respond to specific regulatory elements.
Mol Cell Endocrinol 1991 Feb
PMID:A comparison of transcriptional regulatory element activities in transformed and non-transformed rat anterior pituitary cells. 205 Feb 77

We have developed a plasmid-based system for isolating gene rearrangements in mammalian cells by selection for reversion of a promoterless drug resistance gene. pNH4 contains the selectable marker gene neo under the control of the herpes simplex virus, thymidine kinase (tk) promoter and, upstream and in the opposite orientation, a dormant promoterless hygromycin B resistance gene (hph) that can be expressed following rearrangement events. An NIH 3T3 cell line stably transfected with pNH4 that has a spontaneous frequency of generation of Hphr colonies of approximately 10(-8) was isolated. Treatment of this line with ethyl methanesulfonate raised the frequency of Hphr colony formation approximately 100-fold. Approximately 60% (21 of 35) of ethyl methanesulfonate-induced Hphr clones showed rearrangements detectable by Southern blot analysis within a 40-kb region surrounding the integrated construct, including a nonhomologous recombination event and, possibly, a large insertion. Additionally, three Hphr clones showed evidence of gene amplification. Northern (RNA) blot analysis of hph mRNA suggests that the rearrangements may provide a function that allows the tk promoter to initiate transcription off the opposite strand, thus yielding hph transcripts. Cell lines harboring pNH4, or modifications of it, may be valuable for studying recombination mechanisms responsible for the various types of genetic rearrangements found in cancer cells.
Mol Cell Biol 1991 Aug
PMID:A novel, plasmid-based system for studying gene rearrangements in mammalian cells. 207 98

The collagen alpha 1(I) promoter, which is efficiently transcribed in NIH 3T3 fibroblasts, contains four binding sites for trans-acting factors, as demonstrated by DNase I protection assays (D. A. Brenner, R. A. Rippe, and L. Veloz, Nucleic Acids Res. 17:6055-6064, 1989). This study characterizes the DNA-binding proteins that interact with the two proximal footprinted regions, both of which contain a reverse CCAAT box and a G + C-rich 12-bp direct repeat. Analysis by DNase I protection assays, mobility shift assays, competition with specific oligonucleotides, binding with recombinant proteins, and reactions with specific antisera showed that the transcriptional factors nuclear factor I (NF-I) and Sp1 bind to these two footprinted regions. Because of overlapping binding sites, NF-I binding and Sp1 binding appear to be mutually exclusive. Overexpression of NF-I in cotransfection experiments with the alpha 1(I) promoter in NIH 3T3 fibroblasts increased alpha 1(I) expression, while Sp1 overexpression reduced this effect, as well as basal promoter activity. The herpes simplex virus thymidine kinase promoter, which contains independent NF-I- and Sp1-binding sites, was stimulated by both factors. Therefore, expression of the collagen alpha 1(I) gene may depend on the relative activities of NF-I and Sp1.
Mol Cell Biol 1991 Aug
PMID:Transcription factors nuclear factor I and Sp1 interact with the murine collagen alpha 1 (I) promoter. 207 9

The mouse gene Krox-24 is transiently activated during cell cycle reentry. It encodes a protein with three zinc fingers similar to those of the transcription factor Sp1. Here we present a biochemical characterization of the gene products. Krox-24 mRNA is translated into two proteins of 82 and 88 kilodaltons, designated p82Krox-24 and p88Krox-24, respectively. p82Krox-24 is initiated at the first AUG codon of the open reading frame, whereas synthesis of p88Krox-24 starts at a non-AUG codon located upstream. Both proteins were synthesized in HeLa cells infected with recombinant vaccinia viruses expressing Krox-24 cDNAs. Under these conditions, they were found phosphorylated on serine residues and glycosylated. The availability of the proteins made possible the determination of the DNA recognition sequence. In vitro, Krox-24 bound specifically to the sequence 5'-GCG(C/G)GGGCG-3'. This sequence is similar but not identical to the Sp1 target sequence. Insertion of an oligomer for the binding site in cis, close to the herpes simplex virus thymidine kinase promoter, rendered this promoter responsive to Krox-24. Krox-24 is therefore a sequence-specific transcriptional activator. Krox-24-binding sites were found upstream of several serum-inducible genes, raising the possibility that Krox-24 is involved in the regulation of these genes.
Mol Cell Biol 1990 Jul
PMID:The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator. 211 74

A wild-type strain of herpes simplex virus type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) and a tk- mutant strain (HSV-1:PTK3B) were used to study the role of the viral tk in the repair of UV-irradiated HSV-1 in human cells. UV survival of HSV-1:PTK3B was substantially reduced compared with that of HSV-1:KOS when infecting normal human cells. In contrast, the UV survival of HSV-1:PTK3B was similar to that of HSV-1:KOS when infecting excision repair-deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV-irradiated HSV-1 in human cells depends, in part at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair.
Environ Mol Mutagen 1990
PMID:Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet-irradiated herpes simplex virus in human cells. 215 40


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>