Open AccessDecreased Globin Messenger RNA in Thalassemia Detected by Molecular Hybridization
Author(s) -
D. L. Kacian,
Roberto Gambino,
Lois W. Dow,
Elliott B. Grossbard,
Clayton Natta,
Francesco Ramirez,
S. Spiegelman,
Paul A. Marks,
Arthur Bank
Publication year - 1973
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.70.6.1886
Subject(s) - messenger rna , complementary dna , globin , microbiology and biotechnology , polysome , biology , rna , thalassemia , hemoglobin , in situ hybridization , dna , biochemistry , gene , ribosome , genetics
In previous studies of patients with β thalassemia, mRNA extracted from reticulocytes in peripheral blood when added to cell-free systems reproduces the deficient β-chain synthesis characteristic of intact cells. The present studies with specific probes for α and β mRNA were designed to decide whether the decreased β mRNA activity is due to the presence of abnormal or reduced β globin mRNA in these cells. Purified α and β complementary DNAs (cDNAs) have been synthesized with RNA-instructed DNA polymerase; α and β mRNAs isolated from heavy (β-producing) and light (α-producing) polyribosomes of rabbit reticulocytes were used as templates. Each of the cDNAs is more than 80% pure by the criterion of biological activity. The α cDNA labeled with [32 P]dCTP and the β cDNA labeled with [3 H]dCTP have been added simultaneously to reaction mixtures containing various concentrations of mRNA from thalassemic and nonthalassemic subjects. The extent and rate of hybridization were determined, permitting a comparison of relative α and β mRNA content in the same annealing mixture. In six nonthalassemic patients, relatively equal amounts of hybridizable α and β mRNA appear to be present. In five of seven patients with β-thalassemia, significantly decreased amounts of β mRNA compared to α mRNA can be demonstrated. In two patients with Hemoglobin H disease, there is a decreased amount of α mRNA compared to β mRNA.