Cloning of cDNA sequences of human adenosine deaminase.
- 1 December 1983
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 80 (24), 7481-7485
- https://doi.org/10.1073/pnas.80.24.7481
Abstract
Cloned c[complementary]DNA sequences of human adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) were isolated from a cDNA library constructed in bacteriophage .lambda.gt 10. The cDNA for the library was prepared from poly(A)+ RNA isolated from a human T-lymphoblast cell line, CCRF-CEM. The library was initially screened by differential plaque hybridization to labeled cDNA prepared from human T- and B-lymphoblast cell lines with a 21-fold difference in levels of translatable ADA mRNA. Two recombinants containing cloned cDNA sequences for ADA were identified by hybridization-selected translation. Both recombinants contained approximately 1600 base pairs of inserted human DNA. Restriction maps of the two inserts were not identical. One contained approximately 40 base pairs of additional DNA toward the center of the cDNA. The cloned cDNA specifically hybridized to 5 fragments generated by HindIII digestion of human genomic DNA. It also hybridized to human lymphoblast RNA species 1.6 and 5.8 kilobases in length. The cDNA was used as a probe to estimate ADA mRNA levels in human lymphoblast cell lines. ADA mRNA levels correlate closely with levels of ADA catalytic activity and ADA protein in cell lines containing structurally normal ADA. A leukemic T-lymphoblast line produced 6-9 times as much ADA protein and ADA mRNA as transformed B-lymphoblast lines. Two mutant B-lymphoblast lines from patients with hereditary ADA deficiency contained unstable ADA protein but had 3-4 times the normal level of ADA mRNA.This publication has 26 references indexed in Scilit:
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