Molecular Cloning and Chromosomal Localization of Human Hippocalcin Gene.

Hippocalcin是一种23 kDa的钙结合蛋白，具有三EF-hand结构，主要存在于大鼠海马中。海马蛋白与恢复蛋白序列同源，提示海马蛋白可能调控海马受体分子的磷酸化。为了研究海马体蛋白与人类神经系统疾病的可能关系，我们分析了人类海马体蛋白基因。用大鼠海马蛋白cDNA探针筛选人海马cDNA文库。获得8个独立的cDNA克隆，根据限制性内切酶图谱将其划分为5种（hHLP1-5）。克隆的hHLP1编码了人类hippocalin的整个开放阅读框。预测的氨基酸序列与大鼠希波calcin同源性100%。筛选人类基因组DNA cosmid文库，获得3个独立克隆。这3个克隆均表现出相似的限制性片段模式。人类希波calcin基因全长约15kb，包含3个外显子。人希波calcin基因外显子序列与cDNA序列完全匹配，所有外显子-内含子连接符合GT/AG规则。相对于串联重复的EF-hand结构域的编码区，内含子的位置并不一致。用人希波calcin cDNA探针对人类基因组dna和cosmid克隆进行Southern blot分析表明，人希波calcin基因以单拷贝基因的形式存在于单倍体基因组中。用人脑聚(A) + RNA引物延伸试验确定转录起始位点，定位于腺嘌呤残基-181位。在转录起始位点上游的2kb序列中，没有出现TATA-， GC-和caat -box等哺乳动物启动子的共同基序。通过直接r带荧光原位杂交，将人hippocalcin基因定位在1p34.2-35条带上。

Hippocalcin is a 23 kDa calcium-binding protein with a three EF-hand structure, primarily idenditified in the rat Hippocampus. Hippocalcin has a sequence homology with recoverin, suggesting that hippocalcin may regulate phosphorylation of receptor molecules in the hippocampus. To investigate a possible relationship of hippocalcin to human neurological disorders, we have analyzed the human hippocalcin gene. A human Hippocampus cDNA library was screened with a rat hippocalcin cDNA probe. Eight independent cDNA clones were obtained and classified into five species (hHLP1-5) based on the restriction enzyme maps. The clone hHLP1 encode the entire open reading frame of human hippocalin. The predicted amino acid sequence was 100% homologous to that of rat hippocalcin. After screening a human genomic DNA cosmid libray, we obtained 3 independent clones. All these 3 clones exhibited similar restriction fragment patterns. The human hippocalcin gene spans approximately 15-kb and contains 3 exons. The exon sequence of the human hippocalcin gene perfectly matched its cDNA sequence, and all exon-intron junctions conformed to the GT/AG rule. The intron positions are not consistently placed with respect to the coding regions of the tandemly repeated EF-hand domains. Southern blot analysis of the human genomic DNAs and cosmid clones with a human hippocalcin cDNA probe indicated that the human hippocalcin gene is present as a single copy gene in the haploid genome. The transcription initiation site was determined by primer extension assay with poly (A) + RNA from human brain and was assigned to the adenine residue at the position -181. In 2-kb sequence upstream of the transcription initiation site, no common motifs for mammalian promoters such as TATA-, GC- and CAAT-boxes appear to be present. By direct R-banding fluorescence in situ hybridization, the human hippocalcin gene was assigned to bands 1p34.2-35.