Human eosinophil major basic protein, a mediator of allergic inflammation, is expressed by alternative splicing from two promoters.
Human eosinophil major basic protein, a mediator of allergic inflammation, is expressed by alternative splicing from two promoters.
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人嗜酸性粒细胞主要碱性蛋白是过敏性炎症的介质，通过两个启动子的选择性剪接表达。
DOI：
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发表时间：
1995
影响因子：
4.1
通讯作者：
Christopher J. F. Spry
中科院分区：
文献类型：
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作者：
Ming;Li Sun;Takahiro Satoh;L. Fisher;Christopher J. F. Spry
人嗜酸性粒细胞主要碱性蛋白（MBP）是过敏性炎症中寄生虫和组织损伤的主要介质之一。MBP储存在嗜酸性粒细胞晶体颗粒中，并在嗜酸性粒细胞作用期间与其他颗粒成分一起释放。先前的研究已经鉴定了MBP基因启动子，其产生编码MBP前原蛋白的1.0kb mRNA转录物，所述MBP前原蛋白经历加工以成熟储存形式。为了研究MBP基因是如何调控的，我们研究了前体细胞和血液嗜酸性粒细胞中MBP转录物的身份和水平。发现该基因从两个上游启动子表达，除了先前描述的启动子P2之外，还有远端启动子P1。第二个启动子的证据最初是通过从人HL-60白血病细胞cDNA文库中分离出一个新的1.6 kb MBP cDNA而提供的，该cDNA不同于已知的1.0 kb cDNA。测定了1.6kbcDNA的完整核苷酸序列，结果表明两个cDNA具有相同的编码区和3'非翻译区，但5'端序列不同。通过从11号染色体文库中分离MBP基因组克隆并测序，证明MBP基因由9个上游外显子和5个编码外显子组成。1.6和1.0 kb的cDNA分别由启动子P1和P2的交替MBP转录物的差异剪接产生，位于基因组DNA中相距32 kb。引物延伸分析确定了两个转录起始位点在P1，既不与一个典型的TATA盒基序。北方印迹和反转录PCR分析表明，1.0 kb的mRNA在包括HL-60和骨髓细胞在内的未成熟细胞中的水平高于1.6 kb的mRNA。相比之下，低水平的1.6 kb的mRNA转录占主导地位的分化的血液嗜酸性粒细胞。结果是相容的差异使用P1和P2启动子作为一种机制，在嗜酸性粒细胞成熟过程中的MBP表达的调节。
Human eosinophil major basic protein (MBP) is one of the principal mediators of injury to parasites and tissues in allergic inflammation. MBP is stored in eosinophil crystalloid granules and released with other granule constituents during eosinophil action. Previous studies have identified an MBP gene promoter that generates a 1.0 kb mRNA transcript encoding MBP preproprotein which undergoes processing to the mature storage form. To investigate how the MBP gene is regulated, we have examined the identity and levels of the MBP transcripts both in precursor cells and in blood eosinophils. It was found that the gene was expressed from two upstream promoters, a distal promoter P1 in addition to the previously described promoter P2. Evidence for the second promoter was initially provided by isolation from a human HL-60 leukaemic cell cDNA library of a novel 1.6 kb MBP cDNA that was distinct from the known 1.0 kb cDNA. The complete nucleotide sequence of the 1.6 kb cDNA was determined, and showed that the two cDNAs had identical coding and 3' untranslated regions but differed in their 5' sequences. By isolating and sequencing MBP genomic clones from an arrayed chromosome 11 library, it was demonstrated that the MBP gene is composed of nine upstream exons and five coding exons. The 1.6 and 1.0 kb cDNAs arise by differential splicing of alternate MBP transcripts from promoters P1 and P2 respectively, located 32 kb apart in the genomic DNA. Primer extension analysis identified two transcription start sites at P1, neither associated with a typical TATA box motif. Northern blotting and reverse-transcription PCR analysis showed that the 1.0 kb mRNA was present at higher levels than the 1.6 kb species in immature cells including HL-60 and bone-marrow cells. By contrast, low levels of 1.6 kb mRNA transcripts predominated in differentiated blood eosinophils. The results are compatible with differential use of P1 and P2 promoters as a mechanism for regulation of MBP expression during eosinophil maturation.