Decreased Globin Messenger RNA in Thalassemia Detected by Molecular Hybridization

• Published in: Proceedings of the National Academy of Sciences - PNAS 1973-06, Vol.70 (6), p.1886-1890
• Main Authors: Kacian, D. L., Gambino, R., Dow, L. W., Grossbard, E., Natta, C., Ramirez, F., Spiegelman, S., Marks, P. A., Bank, A.
• Format: Article
• Language: English
• Subjects: Animals; Banks; Biological Sciences: Medical Sciences; Cell free system; Complementary DNA; Cots; DNA; DNA - biosynthesis; DNA - metabolism; DNA probes; Globins - biosynthesis; Humans; Messenger RNA; Nucleic Acid Hybridization; Phosphorus Isotopes; Polyribosomes; Polyribosomes - analysis; Rabbits; Reticulocytes; Reticulocytes - analysis; RNA, Messenger - blood; RNA, Messenger - isolation & purification; RNA, Messenger - metabolism; RNA-Directed DNA Polymerase - metabolism; Templates, Genetic; Thalassemia; Thalassemia - blood; Tritium
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.70.6.1886

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 [32P]dCTP and the β cDNA labeled with [3H]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.