Cloning of a functional Burkitt's lymphoma polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) gene promoter by the polymerase chain reaction, and its interaction with inducible cellular factors.
The promoter of the human gene encoding the stress-responsive protein polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) was isolated from Burkitt's lymphoma cells by PCR. This promoter DNA segment (termed BiP670) or one of its 5' deletion derivatives was fused to the bacterial chloramphenicol acetyltransferase gene and introduced into HeLa cells for transient expression. BiP670 retained transcriptional activity at both the basal and Ca2+ ionophore A23187-inducible levels. However, there was no significant increase in promoter activity following a 5 h induction with 7 microM-A23187, and less than 5-fold induction at 15 h. In contrast, the steady-state mRNA level was induced by 18-fold at 5 h. The in vivo transactivation assays with BiP670 5' deletion derivatives indicate that the putative A23187-inducible element is located within a 70 bp DNA segment (i.e. spanning -39 to -107 bp upstream of the transcriptional initiation site). Using an in vitro gel mobility shift assay, A23187-inducible nuclear factors were identified from HeLa cell extracts. DNA-binding competition experiments also suggest that the 70 bp DNA segment contains a potential sequence motif for the binding of the A23187-inducible nuclear factors.
Chao
C C
CC
Department of Biochemistry, Chang Gung Medical College, Taoyuan, Taiwan, Republic of China.
Yam
W C
WC
Chen
L K
LK
Lin-Chao
S
S
D001483:Base Sequence; D001667:Binding, Competitive; D002051:Burkitt Lymphoma; D000001:Calcimycin / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006360:Heat-Shock Proteins; D006801:Humans; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009841:Oligonucleotides; D000001:Calcimycin; D012313:RNA; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase