Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027819 (neuroblastoma)
 27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human manganese-containing superoxide dismutase (MnSOD) is a nuclear encoded mitochondrial protein that scavenges potentially toxic superoxide radicals by dismuting O2- to O2 plus H2O2. To understand the molecular mechanism governing the transcriptional regulation of the human MnSOD gene, I have isolated and sequenced a genomic clone containing the 5' flanking region of the human MnSOD gene. One major transcription start site was mapped by primer extension to a guanine residue 67 base pairs upstream from the translation start site. Eight putative Sp1 binding elements and one AP1 consensus sequence, but no TATA or CAAT box, were found in the promoter region. Furthermore, a series of chimerical/CAT reporter gene constructs were used to transfect human hepatocellular carcinoma(HepG2) human neuroblastoma and human skin fibroblast cell lines to characterize the promoter and regulatory region of the human MnSOD gene. The results show that human MnSOD gene expression is governed by one promoter and that the basic promoter is located between nucleotides -34 and +38. The results also indicate that both positive and negative elements are involved in the regulation of the cell-type specific expression of the human MnSOD gene. The functional studies indicate that the Sp1 binding sites or G+C rich regions play an important role in regulation of expression of the human MnSOD gene in vivo.
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PMID:Characterization of the 5' flanking region of the human MnSOD gene. 860 39

The predominant animal model in which the pharmacology of cannabinoids is studied is the mouse. Nonetheless, the structure and functional expression of the mouse cannabinoid receptor (CB1) gene have not been reported. We have cloned and expressed the gene for the mouse CB1 receptor and compared its properties with those of native mouse CB1 receptors in brain and N18TG2 neuroblastoma cells. The mouse CB1 gene was isolated from a mouse 129 strain genomic library. Sequence analysis of a 6-kb BamHI fragment of the mouse CB1 genomic clone indicates 95% nucleic acid identity between mouse and rat (99.5% amino acid identity) and 90% nucleic acid identity (97% amino acid identity) between mouse and human. Examination of the 5' untranslated sequence of the mouse CB1 genomic clone revealed a splice junction site approximately 60 bp upstream from the translation start site, indicating the possibility of splice variants of the CB1 receptors. The coding region of the mouse CB1 receptor was stably expressed in 293 cells, and binding by [3H]SR 141716A and [3H]CP-55,940 was determined. The Bmax and Kd values obtained with [3H]SR 141716A (921 +/- 58 fmol/mg and 0.73 +/- 0.13 nM, respectively) were similar to those of native mouse CB1 receptors in brain (Bmax of 1.81 +/- 0.44 pmol/mg, Kd of 0.16 +/- 0.01 nM) and N18TG2 cells (Bmax of 197 +/- 29 fmol/mg, Kd of 0.182 +/- 0.08 nM). The mouse CB1 receptor genomic clone will be a useful tool for studying the function and regulation of the CB1 receptor in mice.
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PMID:Isolation and expression of a mouse CB1 cannabinoid receptor gene. Comparison of binding properties with those of native CB1 receptors in mouse brain and N18TG2 neuroblastoma cells. 903 53

We recently cloned cDNAs encoding three subtypes of human alpha1-adrenergic receptors (alpha1ARs), alpha1a, alpha1b, and alpha1d (Schwinn, D. A., Johnston, G. L., Page, S. O., Mosley, M. J., Wilson, K. H., Worman, N. P., Campbell, S., Fidock, M. D., Furness, L. M., Parry-Smith, D. J., Peter, B., and Bailey, D. S. (1995) J. Pharmacol. Exp. Ther. 272, 134-142) and demonstrated predominance of alpha1aARs in many human tissues (Price, D. T., Lefkowitz, R. J., Caron, M. G., Berkowitz, D., and Schwinn, D. A. (1994) Mol. Pharmacol. 45, 171-175). Several lines of evidence indicate that alpha1aARs are important in clinical diseases such as myocardial hypertrophy and benign prostatic hyperplasia. Therefore, we initiated studies to understand mechanisms underlying regulation of alpha1aAR gene transcription. A genomic clone containing 6.2 kb of 5'-untranslated region of the human alpha1aAR gene was recently isolated. Ribonuclease protection and primer extension assays indicate that alpha1aAR gene transcription occurs at multiple initiation sites with the major site located 696 base pairs upstream of the ATG, where a classic initiator sequence is located. Transfection of luciferase reporter constructs containing varying amounts of 5'-untranslated region into human SK-N-MC neuroblastoma cells indicate that a region extending 125 base pairs upstream from the main transcription initiation site contains full alpha1aAR promoter activity. Furthermore, distinct activator and suppressor elements lie 2-3 and 3-5 kilobase pairs upstream, respectively. Although the alpha1aAR promoter contains neither TATA or CAAT elements, gel shift mobility assays targeting three GC boxes immediately upstream of the main transcription initiation site confirm binding of Sp1. Activity of the alpha1aAR promoter is cell-specific, demonstrating highest activity in cells endogenously expressing alpha1aARs. The human alpha1aAR gene also contains several cis regulatory elements, including several insulin and cAMP response elements. Consistent with these observations, we provide the first evidence that treatment of SK-N-MC cells with insulin and cAMP elevating agents leads to an increase in alpha1aAR expression. In conclusion, these data represent the first characterization of the alpha1aAR gene; our findings should facilitate further studies designed to understand mechanisms regulating alpha1AR subtype-specific expression in healthy and diseased human tissue.
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PMID:Transcriptional regulation of the human alpha1a-adrenergic receptor gene. Characterization Of the 5'-regulatory and promoter region. 935 75

Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and the last 6 of the 104 amino acids to a genomic clone that encoded the remaining amino acid residues [Newton, D. L., et al. (1997) Protein Eng. 10, 463-470]. The resulting protein product expressed in Escherichia coli exhibited little enzymatic or cytotoxic activity due to the unprocessed N-terminal Met amino acid residue. In this study, we demonstrate that modification of the 5'-region of the gene to encode [Met-(-1)]Ser or [Met-(-1)]Tyr instead of the native pyroglutamate results in recombinant onconase derivatives with restored activities. [Met-(-1)]rOnc(E1S) was more active than [Met-(-1)]rOnc(E1Y) in all assays tested. Consistent with the action of native onconase, [Met-(-1)]rOnc(E1S) was a potent inhibitor of protein synthesis in the cell-free rabbit reticulocyte lysate assay, degrading tRNA at concentrations that correlated with inhibition of protein synthesis. An interesting difference between the recombinant onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibitor, PRI (onconase is refractory to PRI inhibition). [Met-(-1)]rOnc(E1S) and [Met-(-1)]rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma cells with IC50's very similar to that of onconase (IC50 3.5, 10, and 10 microg/mL after 1 day and 0.16, 0.35, and 2.5 microg/mL after 5 days for onconase, [Met-(-1)]rOnc(E1S), and [Met-(-1)]rOnc(E1Y), respectively). Similar to that of onconase, cytotoxic activity of the recombinant derivatives was potentiated by monensin, NH4Cl, and retinoic acid. Brefeldin A completely blocked the enhancement of cytotoxicity caused by retinoic acid with all three proteins. Thus, drug-induced alterations of the intracellular trafficking of the recombinant derivatives also resembles that of onconase. Stability studies as assessed in serum-containing medium in the presence or absence of cells at 37 degreesC showed that the recombinant proteins were as stable to temperature and cell culture conditions as the native protein. Therefore, exchanging the Glu amino acid residue at the amino terminus of onconase with an amino acid residue containing a hydroxyl group produces recombinant proteins with ribonuclease and cytotoxic properties similar to native onconase.
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PMID:Single amino acid substitutions at the N-terminus of a recombinant cytotoxic ribonuclease markedly influence biochemical and biological properties. 954 48

Co-2 is one of the R-genes against anthracnose identified in common bean. A RAPD marker, cloned as PvH20, was previously shown to contain 6 imperfect leucine-rich-repeats and to reveal a family of related sequences in the vicinity of the Co-2 locus. Using PvH20 as probe, a genomic clone and 2 partial cDNAs were isolated. DNA sequencing revealed that the 6.1 kb genomic fragment contains sequences encoding both NBS-LRR (ORF1) and kinase-like (ORF2) products. The 2 partial cDNAs (cD7 and cD8) belong to the NBS-LRR subfamily as do most of the resistance genes cloned to date. The LRR domain of ORF1 is interrupted by 2 stop codons suggesting that it corresponds to a non-functional member of the multigene family and ORF2 appears to be a kinase pseudogene. The 3 NBS-LRR polypeptides share a high level of amino acid identity and represent different members of a related family. By genetic mapping ORF1, cD7, and cD8 were found to span a genetic distance of 3 cM: cD8 maps at 2 cM from Co-2 and 3 cM from ORF1, cD7 maps at 1 cM from ORF1 and co-segregates with Co-2, thus cD7 might be a putative candidate for the Co-2 R-gene.
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PMID:Cloning and molecular characterization of three members of the NBS-LRR subfamily located in the vicinity of the Co-2 locus for anthracnose resistance in Phaseolus vulgaris. 1023 59

To study the transcriptional mechanisms by which expression of the dopamine receptor regulating factor (DRRF) gene is regulated, a murine genomic clone was isolated using a DRRF cDNA as probe. A 24 kb genomic fragment which comprises 13 kb upstream of the transcription initiation site was sequenced. The promoter region lacks a TATA box and CAAT box, is rich in G+C content, and has multiple putative binding sites for the transcription factor Sp1. The DRRF gene also has consensus sequences for AP1 and AP2 binding sites. The transcriptional activity of five deletion mutants of a 1.5 kb fragment was analyzed by modulating transcription of the heterologous chloramphenicol acetyltransferase (CAT) gene in the promoterless plasmid pCAT-Basic. All mutants showed significant transcriptional activity in the murine neuroblastoma cell line NB41A3, except the construct stretching from -901 to +17. These transient expression assays suggested the presence of positive regulators between -1153 and -901 and between -118 and -93 while a negative regulator was found in the region between -901 and -118. Comparison among cell types revealed strong transcriptional activity of the DRRF promoter in neuronal NB41A3 cells and moderate activity in hepatic HepG2 and renal OK cells, but none in skeletal muscle C2C12 or glial C6 cells. These findings confirm the tissue-specific activity of the DRRF promoter and suggest that this gene shares structural and functional similarities with the dopamine receptor genes that it regulates.
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PMID:Genomic organization and promoter characterization of the murine dopamine receptor regulating factor (DRRF) gene. 1256 28


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