BIOCHEM AND CLINICAL APPLICATION OF ACID PHOSPHATASE 5

这是对以前的生物化学研究的继续， 酸性磷酸酶5（AP 5）的临床应用。生物化学研究 将致力于构建AP 5的cDNA克隆，验证 AP 5推定cDNA的真实性，以及其 结构 然后将阳性cDNA克隆用于测量mRNA 在表现出不同酶活性的细胞中， 阐明AP 5基因表达的调控机制。 在实验上， 将测定AP 5的部分氨基酸序列， 将用于合成筛选cDNA克隆的寡核苷酸探针 对于AP 5。 总RNA将从毛细胞中提取，毛细胞产生 高水平的酶，使用SDS -苯酚或盐酸胍在 RNase抑制剂的存在。 将使用以下方法分离含聚腺苷酸的RNA： 寡聚-dT纤维素亲和层析和AP 5的mRNA的存在 将通过体外测定含多聚腺苷酸的RNA组分中的 翻译测定 cDNA将用逆转录酶合成， 克隆到pBR 322中。 含有同工酶5 cDNA序列的质粒将 通过与合成的寡核苷酸杂交来选择，所述合成的寡核苷酸含有 编码AP 5蛋白质的具有最小 密码子分配不明确，单特异性免疫筛选 抗同工酶5抗体和对AP 5特异的比色法 活动 将通过凝胶电泳分析来自阳性克隆的质粒DNA 电泳 含有最长DNA插入片段的克隆将进一步 通过限制性内切酶图谱和DNA测序进行表征。 的 将来自cDNA序列的氨基酸序列与 以确定cDNA克隆的真实性。 AP5 mRNA 将通过北方印迹和斑点印迹杂交测定水平 使用克隆的同工酶5 cDNA进行实验。 为了进一步破译 AP 5基因表达的控制机制，长期研究将包括 基因组克隆的分离和表征，使用表征的 来自表现出不同酶活性的各种细胞类型的cDNA克隆， 活动 将通过限制性内切酶分析阳性基因组克隆 核酸内切酶定位、DNA甲基化模式分析和DNA测序。 既往临床研究的继续将包括（a） 应用DNA克隆来阐明毛细胞的起源，以及 (b)应用血清中的AP 5活性作为标记物，以跟踪 对癌症患者、戈谢病患者和 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.