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Structure and function of the 5'-flanking sequence of the human cytosolic selenium-dependent glutathione peroxidase gene (hgpx1).
J Biol Chem 1992; 267(9):5949-58JB

Abstract

Human selenium-dependent glutathione peroxidase (hGPx1) (EC 1.11.1.9) is thought to be involved in many critical cellular functions as a result of its role in glutathione-mediated reduction of toxic peroxides, and it is implicated as a mechanism of resistance against oxygen free radicals. Previous studies have demonstrated that the gene encoding hGPx1 (hgpx1) is more highly expressed in multidrug-resistant AdrR MCF-7 human breast cancer cells than in the parental WT MCF-7 cell line. In order to further study the transcriptional regulation of hgpx1, we have cloned the genomic hgpx1 gene and determined its nucleotide sequence. The 2550-base pair (bp) 5'-flanking sequence of hgpx1 contained the terminal 511 bp of the 3' end of a previously reported rhoH12 cDNA (Yeramian, P., Chardin, P., Madaule, P., and Tavitian, A. (1987) Nucleic Acids Res. 15, 1989), a ras-related oncogene. Further downstream from rhoH12, but before the start of transcription of hgpx1, RNase protection analysis revealed a transcribed sequence of at least 270 bp which we have called mid. RNA transcripts homologous to both rhoH12 (1.8 and 1.5 kilobase pairs (kb)) and mid (1.8 kb) are also more highly expressed in AdrR MCF-7 cells than in WT MCF-7 cells. We screened an AdrR MCF-7 cDNA library with the mid sequence and isolated a partial cDNA clone which contains both mid and rhoH12 sequences and is colinear with the genomic sequence which extends from 10 bp 3' to the rhoH12 stop codon to 810 bp 5' to the start of transcription of hgpx1. The start of transcription of hgpx1 in AdrR MCF-7 cells was determined by primer extension analysis. The promoter and 2 kb of the 5'-flanking sequence of hgpx1 was fused to the bacterial chloramphenicol acetyltransferase gene (hGPx1-CAT1). Analysis of deletion constructs of hGPx1-CAT1 revealed three possible cis-acting regulatory regions. The transcriptional regulation of hgpx1 was examined using the hGPx1-CAT hybrid genes and nuclear run-on studies. We found no evidence that increased mRNA transcript formation could account for different levels of hgpx1 RNA either in different breast cancer cell lines or in response to selenium.

Authors+Show Affiliations

Medicine Branch, National Cancer Institute, Bethesda, Maryland 20892.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

1556108

Citation

Moscow, J A., et al. "Structure and Function of the 5'-flanking Sequence of the Human Cytosolic Selenium-dependent Glutathione Peroxidase Gene (hgpx1)." The Journal of Biological Chemistry, vol. 267, no. 9, 1992, pp. 5949-58.
Moscow JA, Morrow CS, He R, et al. Structure and function of the 5'-flanking sequence of the human cytosolic selenium-dependent glutathione peroxidase gene (hgpx1). J Biol Chem. 1992;267(9):5949-58.
Moscow, J. A., Morrow, C. S., He, R., Mullenbach, G. T., & Cowan, K. H. (1992). Structure and function of the 5'-flanking sequence of the human cytosolic selenium-dependent glutathione peroxidase gene (hgpx1). The Journal of Biological Chemistry, 267(9), pp. 5949-58.
Moscow JA, et al. Structure and Function of the 5'-flanking Sequence of the Human Cytosolic Selenium-dependent Glutathione Peroxidase Gene (hgpx1). J Biol Chem. 1992 Mar 25;267(9):5949-58. PubMed PMID: 1556108.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Structure and function of the 5'-flanking sequence of the human cytosolic selenium-dependent glutathione peroxidase gene (hgpx1). AU - Moscow,J A, AU - Morrow,C S, AU - He,R, AU - Mullenbach,G T, AU - Cowan,K H, PY - 1992/3/25/pubmed PY - 1992/3/25/medline PY - 1992/3/25/entrez SP - 5949 EP - 58 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 267 IS - 9 N2 - Human selenium-dependent glutathione peroxidase (hGPx1) (EC 1.11.1.9) is thought to be involved in many critical cellular functions as a result of its role in glutathione-mediated reduction of toxic peroxides, and it is implicated as a mechanism of resistance against oxygen free radicals. Previous studies have demonstrated that the gene encoding hGPx1 (hgpx1) is more highly expressed in multidrug-resistant AdrR MCF-7 human breast cancer cells than in the parental WT MCF-7 cell line. In order to further study the transcriptional regulation of hgpx1, we have cloned the genomic hgpx1 gene and determined its nucleotide sequence. The 2550-base pair (bp) 5'-flanking sequence of hgpx1 contained the terminal 511 bp of the 3' end of a previously reported rhoH12 cDNA (Yeramian, P., Chardin, P., Madaule, P., and Tavitian, A. (1987) Nucleic Acids Res. 15, 1989), a ras-related oncogene. Further downstream from rhoH12, but before the start of transcription of hgpx1, RNase protection analysis revealed a transcribed sequence of at least 270 bp which we have called mid. RNA transcripts homologous to both rhoH12 (1.8 and 1.5 kilobase pairs (kb)) and mid (1.8 kb) are also more highly expressed in AdrR MCF-7 cells than in WT MCF-7 cells. We screened an AdrR MCF-7 cDNA library with the mid sequence and isolated a partial cDNA clone which contains both mid and rhoH12 sequences and is colinear with the genomic sequence which extends from 10 bp 3' to the rhoH12 stop codon to 810 bp 5' to the start of transcription of hgpx1. The start of transcription of hgpx1 in AdrR MCF-7 cells was determined by primer extension analysis. The promoter and 2 kb of the 5'-flanking sequence of hgpx1 was fused to the bacterial chloramphenicol acetyltransferase gene (hGPx1-CAT1). Analysis of deletion constructs of hGPx1-CAT1 revealed three possible cis-acting regulatory regions. The transcriptional regulation of hgpx1 was examined using the hGPx1-CAT hybrid genes and nuclear run-on studies. We found no evidence that increased mRNA transcript formation could account for different levels of hgpx1 RNA either in different breast cancer cell lines or in response to selenium. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/1556108/Structure_and_function_of_the_5'_flanking_sequence_of_the_human_cytosolic_selenium_dependent_glutathione_peroxidase_gene__hgpx1__ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=1556108 DB - PRIME DP - Unbound Medicine ER -