Hall A V, Antoniou H, Wang Y, Cheung A H, Arbus A M, Olson S L, Lu W C, Kau C L, Marsden P A
Renal Division, St. Michael's Hospital, University of Toronto, Ontario, Canada.
J Biol Chem. 1994 Dec 30;269(52):33082-90.
Neuronal nitric oxide (NO) synthase, localized to human chromosome 12, uniquely participates in diverse biologic processes; neurotransmission, the regulation of body fluid homeostasis, neuroendocrine physiology, control of smooth muscle motility, sexual function, and myocyte/myoblast biology, among others. Restriction enzyme mapping, subcloning, and DNA sequence analysis of bacteriophage- and yeast artificial chromosome-derived human genomic DNA indicated that the mRNA for neuronal NO synthase is dispersed over a minimum of 160 kilobases of human genomic DNA. Analysis of intron-exon splice junctions predicted that the open reading frame is encoded by 28 exons, with translation initiation and termination in exon 2 and exon 29, respectively. Determination of transcription initiation sites in brain poly(A) RNA with primer extension analysis and RNase protection revealed a major start site 28 nucleotides downstream from a TATA box. Sequence inspection of 5'-flanking regions revealed potential cis-acting DNA elements: AP-2, TEF-1/MCBF, CREB/ATF/c-Fos, NRF-1, Ets, NF-1, and NF-kappa B-like sequences. Diversity appears to represent a major theme apparent upon analysis of human neuronal NO synthase mRNA transcripts. A microsatellite of the dinucleotide variety was detected within the 3'-untranslated region of exon 29. Multiple alleles were evident in normal individuals indicating the existence of allelic mRNA sequence variation. Characterization of variant human neuronal NO synthase cDNAs indicated the existence of casette exon 9/10 and exon 10 deletions as examples of structural mRNA diversity due to alternative splicing. The latter deletion of a 175-nucleotide exon introduces a frame-shift and premature stop codon indicating the potential existence of a novel NH2 terminus protein. In summary, analysis of the human neuronal NO synthase locus reveals a complex genomic organization and mRNA diversity that is both allelic and structural.
神经元型一氧化氮（NO）合酶定位于人类12号染色体，独特地参与多种生物学过程；包括神经传递、体液稳态调节、神经内分泌生理学、平滑肌运动控制、性功能以及心肌细胞/成肌细胞生物学等。对噬菌体和酵母人工染色体衍生的人类基因组DNA进行限制性酶切图谱分析、亚克隆和DNA序列分析表明，神经元型NO合酶的mRNA分散在至少160千碱基的人类基因组DNA上。对内含子-外显子剪接接头的分析预测，开放阅读框由28个外显子编码，翻译起始和终止分别在外显子2和外显子29。用引物延伸分析和核糖核酸酶保护法测定脑多聚腺苷酸（poly(A)）RNA中的转录起始位点，发现一个主要起始位点位于TATA框下游28个核苷酸处。对5'侧翼区域的序列检查揭示了潜在的顺式作用DNA元件：AP-2、TEF-1/MCBF、CREB/ATF/c-Fos、NRF-1、Ets、NF-1和NF-κB样序列。多样性似乎是分析人类神经元型NO合酶mRNA转录本时明显的一个主要主题。在外显子29的3'非翻译区域检测到一个二核苷酸类型的微卫星。正常个体中存在多个等位基因，表明存在等位基因mRNA序列变异。对变异的人类神经元型NO合酶cDNA的表征表明，存在盒式外显子9/10和外显子10缺失，这是由于选择性剪接导致的结构mRNA多样性的例子。后者缺失一个175个核苷酸的外显子会引入移码和提前终止密码子，表明可能存在一种新的NH2末端蛋白。总之，对人类神经元型NO合酶基因座的分析揭示了一个复杂的基因组组织和mRNA多样性，包括等位基因和结构多样性。
