Cloning and direct examination of a structurally abnormal human beta 0-thalassemia globin gene.

Abstract

Restriction endonuclease mapping permitted identification of a form of .beta.0-thalassemia in which a partial deletion of the .beta.-globin structural gene occurred. To analyze its structure more directly, this abnormal human gene was cloned in bacteriophage .lambda.gtWES. Restriction mapping of the cloned gene and of a normal .beta.-globin gene contained in the phage H.beta.G1 confirmed previous findings regarding the presence of a deletion toward the 3'' end of the gene but could not establish its position more accurately. EM of the hybrid of the .beta.-thalassemia gene with globin RNA (R-loop analysis) provided unequivocal evidence for a deletion within the .beta.-globin structural gene. Hybridization of restriction fragments of the mutant gene with homologous fragments of H.beta.G1 (heteroduplex analysis) revealed a continuous, internal deletion of about 0.6 kilobase of DNA in the mutant gene and permitted its localization within the .beta.-globin gene region. This deletion removed the terminal third of the large intervening sequence within the .beta.-globin gene, the entire 3'' coding block (extending from codon 105 to the end of the gene), and approximately 150 base pairs of DNA past the end of the normal globin gene.