Assay of hybrid ribonuclease using a membrane filter-immobilized synthetic hybrid: application to the human leukemic cell.
A method for assaying hybrid ribonuclease has been devised which utilizes as substrate the synthetic hybrid [3H]polyriboadenylic acid [poly(rA)]:polydeoxythymidylic acid [poly(dT)] immobilized on the solid matrix of nitrocellulose filters. The hybridization on filter of [3H]poly(rA) to poly(dT) has been explored in terms of efficacy of the process and the response of the product to RNase H. A pulse of uv irradiation of poly(dT) while in dry state on the filter increased its firm binding to the filter in a concentration-dependent manner, resulting in a concomitant increase of the yield of hybrid formation. The filter-immobilized hybrid was 95% resistant to RNase A but sensitive to RNase H. When stored in toluene in the cold the hybrid maintained its stability for over 6 months, as judged by its resistance to RNase A. The method offers a number of advantages over assays that use solution hybrids as substrates and was readily applicable in the screening of leukemic patients, in the leukocytes of which it has demonstrated increased RNase H levels.
Papaphilis
A D
AD
Kamper
E F
EF
D000818:Animals; D004722:Endoribonucleases / Q000032:analysis* / Q000097:blood; D005374:Filtration; D006801:Humans; D007938:Leukemia / Q000201:enzymology*; D007962:Leukocytes / Q000201:enzymology; D009693:Nucleic Acid Hybridization; D011061:Poly A / Q000378:metabolism* / Q000528:radiation effects; D011071:Poly T / Q000378:metabolism* / Q000528:radiation effects; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D051381:Rats; D016914:Ribonuclease H; D012259:Ribonuclease, Pancreatic / Q000032:analysis; D013379:Substrate Specificity
D011089:Polydeoxyribonucleotides; D011061:Poly A; D011071:Poly T; C008950:poly A-T; D004722:Endoribonucleases; D016914:Ribonuclease H; D012259:Ribonuclease, Pancreatic