Identification and characterization of the nucleotide sequence of the 5′-flanking region of the human gilz gene. (A) Mapping of the transcription start site of the gilz gene by primer extension. RNA from NKL cells was prepared and submitted to reverse transcription using a 32P-radiolabeled primer positioned 128 nt upstream of the translation start site. Reaction products (2) were run on a polyacrylamide gel in parallel with radiolabeled size marker (1). (B) Nucleotide sequence of the 5′-flanking region of the gilz gene. The sequence is numbered with the first nucleotide of the transcription start site indicated as +1 corresponding to position 6536 of GenBank accession number AL590423.14. The putative cis-regulatory elements present in the gilz promoter are represented in gray boxes and named accordingly. Circled letters indicate the TATA box. The translation start site is underlined +1. (C) Gilz promoter activity in CTLL-2 cells. A reporter construct containing the –1940 to +19 bp of the 5′ flanking sequence from the gilz gene was generated and transfected into CTLL-2 cells. Cells were then cultured with or without IL-2 for 8 hours. Results are expressed as normalized (Firefly/Renilla) relative luciferase units (RLU) and represent the mean ± SEM (error bars) of 3 independent experiments performed in duplicate.
