Accession Number : ADA185458

Title :   Molecular Cloning of Adenosinediphosphoribosyl Transferase.

Descriptive Note : Annual rept. 1 Sep 86-31 Aug 87,

Corporate Author : CALIFORNIA UNIV SAN FRANCISCO

Personal Author(s) : Kun, Ernest

PDF Url : ADA185458

Report Date : 08 Sep 1987

Pagination or Media Count : 67

Abstract : The purpose of obtaining the gene of Adenosinediphosphoribosyl Transferase (ADPRT) is: 1) the complete amino acid sequence of this large protein is best determined from the DNA sequence of the gene, 2) isolation of the gene provides gene probes that permit location and quantitation of the gene within genomic DNA, and 3) a variety of biological experiments at the cellular level requires specific gene probes. The DNA-associating enzyme, adenosinediphosphoribosyl transferase, has been isolated from calf thymus by selective precipitation with a solution of dihydroxy-Reactive Red 120, followed by extraction of the enzyme from the precipitate with 2 M KC1 and an on-line train of three successive column chromatographic steps, including a final 3-aminobenzamide-Sepharose-4B affinity chromatography. The method yields 8-9 mg of more than 95% homogeneous enzyme protein per kg starting material and requires about 3 working days. This dye precipitation method is distinct from affinity precipitation, since it involves the binding of the dye to both nonspecific sites and the substrate- and DNA-site of the transferase as indicated by enzyme inhibition by dihydroxy Reactive red 120 at both enzyme sites.

Descriptors :   *PHOSPHORUS TRANSFERASES, *CLONES, GENES, AMINO ACIDS, MAPPING, MOLECULAR STRUCTURE, DEOXYRIBONUCLEIC ACIDS, CHROMATOGRAPHY, THYMUS, PEPTIDES, DYES, BOVINES

Subject Categories : Genetic Engineering and Molecular Biology
      Biochemistry

Distribution Statement : APPROVED FOR PUBLIC RELEASE