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.
Surrey
S
S
Delgrosso
K
K
Malladi
P
P
Schwartz
E
E
D001483:Base Sequence; D003001:Cloning, Molecular; D004720:Endonucleases / Q000378:metabolism; D005914:Globins / Q000235:genetics*; D006367:HeLa Cells; D006720:Homozygote; D006801:Humans; D009154:Mutation; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013789:Thalassemia / Q000235:genetics*; D014158:Transcription, Genetic
D005914:Globins; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases