The mouse calretinin gene promoter region: structural and functional components.
The 5' flanking region of the mouse calretinin gene was cloned and a 1.8 kbp region adjacent to exon 1 was sequenced. Putative upstream promoter and enhancer elements were identified, including appropriately positioned TATA and CAAT boxes (positions -50 and -68, respectively). There was considerable sequence and structural homology between mouse and human upstream elements. Neuron-restrictive activity was demonstrated via transfection of calretinin promoter-reporter constructs into primary embryonic mouse brain cultures expressing calretinin. In promoterless reporter constructs, the proximal upstream 1.5 kbp of the mouse calretinin gene boosted luciferase activity (up to 100-fold) exclusively in the neuronal population. Deletion analysis revealed the minimal promoter to be within the 95-bp proximal to the transcription start site. Transfections with SV40 promoter constructs in these cultures resulted in reporter gene expression predominantly in non-neuronal cells. Inserting the proximal 1.5 kbp of mouse calretinin upstream in SV40 promoter-reporter constructs reduced luciferase activity. Thus, calretinin upstream sequences increased reporter expression in cultured neurons and decreased expression from the SV40 promoter in non-neuronal cultured brain cells. The calretinin promoter contained relevant regulatory element consensus motifs and demonstrated in vitro neuron-restrictive bioactivity.
Strauss
K I
KI
NIMH, Laboratory of Clinical Science, Bethesda, MD 20892-1266, USA. kstrauss@thunder.ocis.temple.edu
Kuźnicki
J
J
Winsky
L
L
Kawagoe
J I
JI
Hammer
M
M
Jacobowitz
D M
DM
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D064032:Calbindin 2; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D051379:Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D016716:PC12 Cells; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis* / Q000235:genetics*; D016415:Sequence Alignment; D017384:Sequence Deletion; D016385:TATA Box; D014162:Transfection; D014599:Uterus / Q000166:cytology
C576764:CALB2 protein, human; C576511:Calb2 protein, mouse; C576512:Calb2 protein, rat; D064032:Calbindin 2; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D064030:S100 Calcium Binding Protein G; D008156:Luciferases