Wang Y, Goligorsky M S, Lin M, Wilcox J N, Marsden P A
Renal Division and Department of Medicine, St. Michael's Hospital, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
J Biol Chem. 1997 Apr 25;272(17):11392-401.
mRNA diversity represents a major theme of neuronal nitric-oxide synthase (nNOS) gene expression in somatic cells/tissues. Given that gonads often express unique and biologically informative variants of complex genes, we determined whether unique variants of nNOS are expressed in the testis. Analysis of cDNA clones isolated from human testis identified a novel, testis-specific nNOS (TnNOS) mRNA transcript. A predicted 3294-base pair open reading frame encodes an NH2-terminal truncated protein of 1098 amino acids. Measurement of calcium-activated L-[14C]citrulline formation and nitric oxide release in CHO-K1 cells stably transfected with the TnNOS cDNA indicates that this protein is a calcium-dependent nitric-oxide synthase with catalytic activity comparable to that of full-length nNOS. TnNOS transcripts exhibit novel 5' mRNA sequences encoded by two unique exons spliced to exon 4 of the full-length nNOS. Characterization of the genomic structure indicates that exonic regions used by the novel TnNOS are expressed from intron 3 of the NOS1 gene. Although lacking canonical TATA and CAAT boxes, the 5'-flanking region of the TnNOS exon 1 contains multiple putative cis-regulatory elements including those implicated in testis-specific gene expression. The downstream promoter of the human nNOS gene, which directs testis-specific expression of a novel NH2-terminal truncated nitric-oxide synthase, represents the first reported example in the NOS gene family of transcriptional diversity producing a variant NOS protein.
信使核糖核酸（mRNA）多样性是体细胞/组织中神经元型一氧化氮合酶（nNOS）基因表达的一个主要特征。鉴于性腺常常表达复杂基因的独特且具有生物学信息价值的变体，我们确定了nNOS的独特变体是否在睾丸中表达。对从人睾丸中分离的互补DNA（cDNA）克隆进行分析，鉴定出一种新的、睾丸特异性的nNOS（TnNOS）mRNA转录本。一个预测的3294个碱基对的开放阅读框编码一个由1098个氨基酸组成的氨基末端截短蛋白。对稳定转染了TnNOS cDNA的中国仓鼠卵巢（CHO-K1）细胞中钙激活的L-[14C]瓜氨酸形成和一氧化氮释放的测量表明，该蛋白是一种钙依赖性一氧化氮合酶，其催化活性与全长nNOS相当。TnNOS转录本展示了由两个独特外显子编码的新的5' mRNA序列，这些外显子与全长nNOS的外显子4拼接。基因组结构的特征表明，新的TnNOS所使用的外显子区域是从一氧化氮合酶1（NOS1）基因的内含子3表达的。尽管缺乏典型的TATA盒和CAAT盒，但TnNOS外显子1的5'侧翼区域包含多个推定的顺式调控元件，包括那些与睾丸特异性基因表达有关的元件。人类nNOS基因的下游启动子指导一种新的氨基末端截短的一氧化氮合酶的睾丸特异性表达，这代表了一氧化氮合酶基因家族中首个报道的转录多样性产生变体一氧化氮合酶蛋白的例子。
