Sequence analysis of the human glycoprotein hormone α-subunit gene 5'- flanking DNA and identification of a potential regulatory element as an Alu repetitive sequence

Margaret A. Scofield, Wanfen Xiong, Michael J. Haas, Yongjun Zeng, G. Stanley Cox

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The nucleotide sequence of the human glycoprotein hormone α-subunit (GPHα) gene 5'-flanking DNA was determined from -1637 to +49 relative to the cap site (+1). Comparison of the upstream sequence of the human gene with those of rhesus and mouse demonstrates regions with variable identity. When the 1.7 kb fragment was used to drive the expression of chloramphenicol acetyltransferase (CAT) in transiently transfected HeLa cells, it was found that CAT activity was elevated about 3-fold when the fragment was truncated from -1637 to -846, suggesting the presence of a negative regulatory element in the distal 5'-flanking DNA. This overlaps an Alu repetitive sequence (ARS) located between nucleotides -1330 and -1007. Gel mobility shift and DNase protection analyses identified a protein binding site centered around -1100 in the ARS second monomer. The GPHα upstream ARS was cloned in both orientations in positions upstream and downstream from the bacterial CAT gene under control of the herpes simplex virus thymidine kinase (tk) promoter. DNA-mediated transient transfection of these plasmids revealed a marked inhibition (79-82%) of CAT production by the ARS when it was cloned upstream from the tk promoter and in the same orientation as that found in the GPHα 5'-flanking DNA. Smaller decreases (29-57%) were produced by the ARS cloned upstream from the tk promoter in the reverse orientation. In marked contrast, the Alu repetitive element had little or no effect when cloned in either orientation downstream from the tk-CAT gene. Introduction of a second ARS downstream from the CAT reporter gene in vectors already containing an ARS upstream from the tk promoter significantly reduced the strong negative effect elicited by the upstream repetitive element. When compared to the Blur 8 Alu element, the GPHα upstream ARS differs markedly with respect to its effect on tk-CAT expression in transient assays and as a substrate for DNA binding proteins present in HeLa nuclear extracts. Together, the transient expression results demonstrate that ARS elements can influence expression of nearby class II promoters. The extent of this effect depends on element position and orientation, cell type, the particular ARS (e.g., GPHα or Blur 8), and whether copies were present both upstream and downstream from the transcription unit. (C) 2000 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)302-318
Number of pages17
JournalBiochimica et Biophysica Acta - Gene Structure and Expression
Volume1493
Issue number3
DOIs
StatePublished - Oct 2 2000

Fingerprint

Alu Elements
Sequence Analysis
Glycoproteins
Chloramphenicol O-Acetyltransferase
Genes
Hormones
Thymidine Kinase
DNA
Nucleotides
Deoxyribonucleases
DNA-Binding Proteins
Transcription
Viruses
Simplexvirus
Reporter Genes
HeLa Cells
Protein Binding
Assays
Transfection
Plasmids

Keywords

  • Alu DNA binding protein
  • Alu repetitive DNA
  • Gene expression
  • Glycoprotein hormone

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Genetics

Cite this

Sequence analysis of the human glycoprotein hormone α-subunit gene 5'- flanking DNA and identification of a potential regulatory element as an Alu repetitive sequence. / Scofield, Margaret A.; Xiong, Wanfen; Haas, Michael J.; Zeng, Yongjun; Cox, G. Stanley.

In: Biochimica et Biophysica Acta - Gene Structure and Expression, Vol. 1493, No. 3, 02.10.2000, p. 302-318.

Research output: Contribution to journalArticle

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abstract = "The nucleotide sequence of the human glycoprotein hormone α-subunit (GPHα) gene 5'-flanking DNA was determined from -1637 to +49 relative to the cap site (+1). Comparison of the upstream sequence of the human gene with those of rhesus and mouse demonstrates regions with variable identity. When the 1.7 kb fragment was used to drive the expression of chloramphenicol acetyltransferase (CAT) in transiently transfected HeLa cells, it was found that CAT activity was elevated about 3-fold when the fragment was truncated from -1637 to -846, suggesting the presence of a negative regulatory element in the distal 5'-flanking DNA. This overlaps an Alu repetitive sequence (ARS) located between nucleotides -1330 and -1007. Gel mobility shift and DNase protection analyses identified a protein binding site centered around -1100 in the ARS second monomer. The GPHα upstream ARS was cloned in both orientations in positions upstream and downstream from the bacterial CAT gene under control of the herpes simplex virus thymidine kinase (tk) promoter. DNA-mediated transient transfection of these plasmids revealed a marked inhibition (79-82{\%}) of CAT production by the ARS when it was cloned upstream from the tk promoter and in the same orientation as that found in the GPHα 5'-flanking DNA. Smaller decreases (29-57{\%}) were produced by the ARS cloned upstream from the tk promoter in the reverse orientation. In marked contrast, the Alu repetitive element had little or no effect when cloned in either orientation downstream from the tk-CAT gene. Introduction of a second ARS downstream from the CAT reporter gene in vectors already containing an ARS upstream from the tk promoter significantly reduced the strong negative effect elicited by the upstream repetitive element. When compared to the Blur 8 Alu element, the GPHα upstream ARS differs markedly with respect to its effect on tk-CAT expression in transient assays and as a substrate for DNA binding proteins present in HeLa nuclear extracts. Together, the transient expression results demonstrate that ARS elements can influence expression of nearby class II promoters. The extent of this effect depends on element position and orientation, cell type, the particular ARS (e.g., GPHα or Blur 8), and whether copies were present both upstream and downstream from the transcription unit. (C) 2000 Elsevier Science B.V.",
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