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.
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