Molecular cloning and functional analysis of the CD33 promoter.
CD33 is a leucocyte differentiation antigen restricted to myeloid cells in blood and bone marrow. Two mRNA transcripts encoding CD33 are observed in leukaemic cell lines. The smaller transcript of 1.5 kb is comparable in size to the isolated CD33 cDNA but the origin of the larger 1.8 kb transcript is unknown. To study the regulation of human CD33 expression, a 5' genomic clone from the CD33 gene was isolated and studied for promoter activity. The clone, although lacking a TATAA box, exhibits other sequences characteristic of a promoter. Two transcriptional start sites were identified, 414 and 527 nucleotides 5' of the ATG initiation codon, suggesting that these sites are used to generate the 1.8 kb transcript observed in CD33+ cell lines. The CD33 genomic sequence directed high expression of a luciferase reporter gene in myeloid cell lines. Using deletion mutants of the promoter sequence, maximal expression was localized to the first 220 bp 5' of the ATG initiation codon. Site-directed mutagenesis of an Sp-1 and PU.1 binding site within this region showed that the PU.1, but not Sp-1, was critical for CD33 expression in myeloid lines. Given the restricted expression of CD33 on haemopoietic cells, the identification of the CD33 promoter may be useful for the study of transcription factors that regulate gene expression during early myeloid differentiation.
Bodger
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MP
Department of Pathology, Christchurch School of Medicine, University of Otago, New Zealand.
Hart
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DN
D015703:Antigens, CD / Q000235:genetics*; D015214:Antigens, Differentiation, Myelomonocytic / Q000235:genetics*; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000235:genetics*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D063268:Sialic Acid Binding Ig-like Lectin 3; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D015815:Cell Adhesion Molecules; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D063268:Sialic Acid Binding Ig-like Lectin 3; D014157:Transcription Factors; D008156:Luciferases