Comparison of the promoters of the mouse (APEX) and human (APE) apurinic endonuclease genes.
We investigated the minimal promoter of APEX, which encodes mouse apurinic DNA repair endonuclease. A 1.85-kb fragment with APEX upstream sequences and approximately 290 bp of the transcribed region linked to a chloramphenicol acetyltransferase (CAT) reporter gene was assayed by transient transfection in NIH-3T3 cells. The minimal APEX promoter was comprised of approximately 190 bp of upstream and approximately 170 bp of transcribed DNA (exon 1 and most of intron 1). This approximately 360-bp region contains two CCAAT boxes and other consensus protein binding sites, but no TATA box. Deletion of the 5'-most CCAAT box decreased activity approximately 5-fold. The second CCAAT box (situated in exon 1) may play an independent role in APEX expression. Transcription start sites have been identified downstream of the second CCAAT box, and DNase I footprinting demonstrated NIH-3T3 nuclear proteins binding this region, including an Spl site located between the CCAAT boxes. Electrophoretic mobility-shift assays indicated binding by purified Sp1. Mouse proteins did not bind three myc-like (USF) sites in the APEX promoter, in contrast to the APE promoter. The APEX and APE promoter had similar activity in Hela cells, but in mouse cells, the murine promoter had approximately 5-fold higher activity than did the human promoter. Both the APEX and APE promoters exhibited bidirectional activity in their cognate cells.
Harrison
L
L
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, MA 02115, USA.
Ascione
A G
AG
Takiguchi
Y
Y
Wilson
D M
DM
Chen
D J
DJ
Demple
B
B
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D019757:Carbon-Oxygen Lyases; D002478:Cells, Cultured; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004260:DNA Repair; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003850:Deoxyribonuclease I / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014162:Transfection
D018076:DNA, Complementary; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D003850:Deoxyribonuclease I; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; C486216:Apex1 protein, mouse; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase