Molecular cloning and functional analysis of the CD33 promoter
Molecular cloning and functional analysis of the CD33 promoter
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DOI：
10.1046/j.1365-2141.1998.00863.x
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发表时间：
1998-09-01
影响因子：
6.5
通讯作者：
Hart, DNJ
中科院分区：
文献类型：
--
作者：
Bodger, MP;Hart, DNJ
CD 33是一种局限于血液和骨髓中髓系细胞的白细胞分化抗原。在白血病细胞系中观察到编码CD 33的两种mRNA转录物。较小的1.5 kb转录本的大小与分离的CD 33 cDNA相当，但较大的1.8 kb转录本的来源尚不清楚。为了研究人CD 33表达的调控，分离了来自CD 33基因的5'基因组克隆并研究了启动子活性。该克隆虽然缺乏TATAA盒，但表现出启动子的其他序列特征。鉴定了两个转录起始位点，ATG起始密码子5'端的414和527个核苷酸，表明这些位点用于产生在CD 33（+）细胞系中观察到的1.8kb转录物。CD 33基因组序列指导髓样细胞系中荧光素酶报告基因的高表达。使用启动子序列的缺失突变体，最大表达定位于ATG起始密码子的前220 bp 5'。在该区域内的Sp-I和PU. 1结合位点的定点诱变显示PU. 1而不是Sp-I对于髓系中的CD 33表达是关键的。由于造血细胞上CD 33的表达有限，因此鉴定CD 33启动子可能有助于研究在早期髓系分化过程中调节基因表达的转录因子。
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-l and PU.1 binding site within this region showed that the PU.1, but not Sp-l, 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.