Decreased globin messenger RNA in thalassemia detected by molecular hybridization.
In previous studies of patients with beta thalassemia, mRNA extracted from reticulocytes in peripheral blood when added to cell-free systems reproduces the deficient beta-chain synthesis characteristic of intact cells. The present studies with specific probes for alpha and beta mRNA were designed to decide whether the decreased beta mRNA activity is due to the presence of abnormal or reduced beta globin mRNA in these cells. Purified alpha and beta complementary DNAs (cDNAs) have been synthesized with RNA-instructed DNA polymerase; alpha and beta mRNAs isolated from heavy (beta-producing) and light (alpha-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 alpha cDNA labeled with [(32)P]dCTP and the beta 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 alpha and beta mRNA content in the same annealing mixture. In six nonthalassemic patients, relatively equal amounts of hybridizable alpha and beta mRNA appear to be present. In five of seven patients with beta-thalassemia, significantly decreased amounts of beta mRNA compared to alpha mRNA can be demonstrated. In two patients with Hemoglobin H disease, there is a decreased amount of alpha mRNA compared to beta mRNA.
D000818:Animals; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D005914:Globins / Q000096:biosynthesis*; D006801:Humans; D009693:Nucleic Acid Hybridization; D010759:Phosphorus Isotopes; D011132:Polyribosomes / Q000032:analysis; D012333:RNA, Messenger / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis; D013698:Templates, Genetic; D013789:Thalassemia / Q000097:blood*; D014316:Tritium
D010759:Phosphorus Isotopes; D012333:RNA, Messenger; D014316:Tritium; D005914:Globins; D004247:DNA; D012194:RNA-Directed DNA Polymerase