Functional analysis of a beta-globin gene containing a TATA box mutation from a Kurdish Jew with beta thalassemia.

We recently reported a TATA box mutation (ATAAAA to ATACAA) in a cloned beta-globin gene from a Kurdish Jew with homozygous beta thalassemia (Poncz, M., Ballantine, M., Solowiejczyk, D., Barak, I., Schwartz, E., and Surrey, S. (1982) J. Biol. Chem. 257, 5994-5996). We have now introduced this gene into HeLa cells after CaPO4 precipitation of the DNA and studied expression by analyzing globin-gene transcripts with a novel S1 nuclease mapping assay. Quantitative and qualitative comparison with the normal beta-globin gene revealed a promoter-down phenotype in the TATA box mutant, with normal RNA processing, and a normal start site for initiation of the primary transcript. Decreased transcriptional efficiency was confirmed directly by analysis of run-off transcripts using assays in vitro. The patient's phenotype of beta thalassemia major is probably the result of two different mutations since haplotype analysis of the beta-like globin gene clusters in genomic DNA from this patient shows heterozygosity for the Mediterranean-type haplotypes I and VII, with the TATA box mutation on a haplotype I chromosomal background.