Evaluation of the expression of the neutrophil antibacterial defensin genes

中性粒细胞在颗粒中含有抗微生物防御素（阳离子肽）。在这项研究中，了解防御素基因的表达，cDNA和基因克隆防御素的分离和分析。从cDNA文库中分离得到3种不同类型的cDNA，序列分析表明，它们的同源性高达99%，其中2种编码防御素-1（defensin-1），1种编码防御素-2（defensin-2），说明防御素-1/-2是由相似但不同的基因编码的。与此相一致，从基因组文库中分离出防御素基因的四个不同克隆（两个代表防御素01基因的等位基因，另外两个代表防御素-2基因的等位基因）。转染分析表明，启动子均位于两个基因转录起始位点上游400 bp以内，且防御素-1基因启动子活性是防御素-2基因的2倍。原位杂交结果显示，防御素mRNA在早幼粒细胞和中幼粒细胞等中性粒细胞前体细胞中表达，而在成熟的中性粒细胞、单核细胞、巨噬细胞、嗜酸性粒细胞和淋巴细胞中不表达。总之，这些观察结果表明，防御素是由相似但不同的基因编码的，并且防御素基因的表达可能在骨髓中的转录水平上在嗜中性粒细胞成熟的有限时期内受到调节。

Neutrophils contain the antimicrobial defensins (cationic peptides) in the granules. IN this study, to understand the defensin gene expression, cDNA and gene clones for defensins were isolated and analyzed. Furthermore, the expression of defensin gene was evaluated in various types of leukocytes using in situ hybridization.Three kinds of cDNAs were isolated from a cDNA library, and sequence analysis revealed that these cDNAs were highly homologous (99%), and that two encoded defensin-1 and one encoded defensin-2, suggesting that defensin-1/-2 are encoded by the similar but different genes. Consistent with this, four different clones for defensin genes (two representing the alleles of defensin01 gene, and other two representing the alleles of defensin-2 gene) were isolated from a genomic library. Transfection analysis demonstrated that the promoters were located within 400 bp upstream of the transcription start sites of both genes, and that the promotor activity of defensin-1 was 2-fold higher than that of defensin-2 gene. In situ hybridization demonstrated that defensin mRNA was expressed in the neutorophil precursor cells, such as promyelocytes and myelocytes, but waas not detected in mature neutrophils, monocytes, macrophages, eosinophils and lymphocytes. Together these observations indicate that defensins are encoded by the similar but different genes, and that the expression of the defensin genes are likely regulated during a limited period of neutrophil maturation at the level of transcription in the bone marrow.