BIOCHEM AND CLINICAL APPLICATION OF ACID PHOSPHATASE 5

这是之前关于生物化学和生物化学研究的继续 酸性磷酸酶5(AP5)临床应用生化研究 将致力于构建AP5的cdna克隆，验证 AP5基因全长cDNA的真实性及其鉴定 结构。然后用阳性的cdna克隆来检测mrna。 显示不同酶活性的细胞中的水平 阐明AP5基因表达的调控机制。从实验上看， 将确定AP5的部分氨基酸序列，并提供信息 将用于合成用于筛选cdna克隆的寡核苷酸探针。 适用于AP5。总RNA将从毛细胞中提取，这将产生一种 高水平的酶，使用十二烷基硫酸钠-苯酚或盐酸胍 核糖核酸酶抑制剂的存在。含有Poly A的RNA将用以下方法分离 寡聚脱氧核糖核酸纤维素亲和层析及AP5mRNA的存在 在含聚A的RNA组分中将通过体外测定 翻译试验。将用逆转录酶合成cDNA，并 克隆到pBR322中。含有同工酶5 cdna序列的质粒将 通过与含有以下成分的合成寡核苷酸杂交进行选择 编码AP5蛋白区域的可能序列 密码子分配的歧义性、单特异性免疫筛查 AP5特异性抗同工酶5抗体及比色法 活动。阳性克隆的质粒DNA将用凝胶进行分析 电泳法。包含最长DNA插入片段的克隆将进一步 以限制性内切酶图谱和DNA测序为特征。这个 将从该cdna序列得到的氨基酸序列与 AP5的同源性，以确定克隆的真实性。AP5基因 表达水平将通过Northern印迹和斑点杂交来确定 实验使用克隆的同工酶5cDNA。为了进一步破译 AP5基因表达的调控机制，长期研究将包括 利用鉴定的基因组克隆分离和鉴定基因组克隆 来自不同细胞类型的具有不同酶活性的cDNA克隆 活动。阳性基因组克隆将通过限制性内切酶进行分析 核酸内切图谱、DNA甲基化模式分析和DNA测序。 继续进行先前的临床研究将包括：(A) 应用DNA克隆阐明毛细胞的来源，以及 (B)应用血清中AP5活性作为跟踪 癌症患者、高雪病患者和癌症患者的治疗效果 骨质疏松。

This is a continuation of the previous study on the biochemistry and clinical application of acid phosphatase 5 (AP5). The biochemical studies will be directed toward constructing cDNA clones for AP5, verfication of the authenticity of the putative cDNA for AP5, and determination of its structure. The positive cDNA clones will then be used to measure mRNA levels in cells exhibiting different enzyme activities in order to elucidate the regulatory mechanism of AP5 gene expression. Experimentally, partial amino acid sequence of AP5 will be determined and the information will be used to synthesize oligonucleotide probes for screening cDNA clones for AP5. Total RNA will be extracted from hairy cells, which produce a high level of the enzyme, using SDS -phenol or guanidine-HCL in the presence of RNase inhibitors. Poly A-containing RNA will be isolated using oligo-dT cellulose affinity chromatography and the presence of mRNA for AP5 in the poly A-containing RNA fraction will be determined by in vitro translation assay. cDNA will be synthesized with reverse transcriptase and cloned into pBR 322. Plasmids harboring the isoenzyme 5 cDNA sequence will be selected by hybridization with synthetic oligonucleotides containing possible sequences coding for regions of the AP5 protein with a minimal ambiguity in codon assignment, immunological screening with monospecific anti isoenzyme 5 antibody and colorimetric method specific for AP5 activity. Plasmid DNAs from the positive clones will be analyzed by gel electrophoresis. Clones containing the longest DNA inserts will be further characterized by restriction endonuclease mapping and DNA sequencing. The amino acid sequence derived from the cDNA sequence will be compared with that of AP5 to establish the authenticity of the cDNA clones. AP5 mRNA levels will be determined by Northern blot and dot-blot hybridization experiments using the cloned isoenzyme 5 cDNAs. To further decipher the control mechanism of AP5 gene expression, longer term studies will include isolation and characterization of genomic clones, using the characterized cDNA clones, from the various cell types exhibiting different enzymatic activities. Positive genomic clones will be analyzed by restriction endonuclese mapping, DNA methylation pattern analysis and DNA sequencing. The continuation of the previous clinical study will include (a) application of the DNA clones to elucidate the origin of hairy cells, and (b) application of AP5 activity in the serum as a marker to follow the therapeutic effectiveness in cancer patients, Gaucher patients and Osteoporosis.