Mutant human adenosine deaminase alleles and their expression by transfection into fibroblasts

• Published in: The Journal of biological chemistry 1988-11, Vol.263 (31), p.16291-16296
• Main Authors: Akeson, A L, Wiginton, D A, Dusing, M R, States, J C, Hutton, J J
• Format: Article
• Language: English
• Subjects: Adenosine Deaminase - genetics; Alleles; Amino Acid Sequence; Base Sequence; Biological and medical sciences; Cell Line; Cloning, Molecular; DNA - genetics; Fibroblasts - enzymology; Fundamental and applied biological sciences. Psychology; Gene Amplification; Gene expression; Genes; Humans; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutation; Nucleoside Deaminases - genetics; Plasmids; Protein Conformation; Transcription, Genetic; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)37591-4

Adenosine deaminase (ADA) deficiency in humans is one cause of severe combined immunodeficiency disease. Single base mutations affecting the ADA protein have been identified for both alleles of the ADA-deficient cell line GM2606 and for one allele of the ADA-deficient cell line GM2825A. One allele of GM2606 has a mutation altering amino acid 101 from Arg to Trp, and the other allele has a mutation altering amino acid 211 from Arg to His. As previously reported, one ADA allele of GM2825A has a single base mutation changing Ala-329 to Val-329, and the other allele has a mutation which eliminates exon 4 from the mature mRNA. Sequence analysis of polymerase chain reaction-amplified GM2825A DNA showed a single base change of A to G within the invariant bases of the 3′ splice site of intron 3 that can account for the mis-splicing of exon 4. To test the effect on ADA catalytic activity of these mutations and the mutations previously found in the ADA-deficient line GM2756, expression vectors containing normal and mutant ADA-coding sequences under transcriptional regulation of the Rous sarcoma virus long terminal repeat were constructed and transfected into human fibroblasts. All transfected cells had levels of ADA mRNA 15-25 times higher than the endogenous ADA message. Yet, cells transfected with the normal ADA-coding sequences had ADA enzymatic levels 40 times higher than cells transfected with any of the mutant ADA sequences. This analysis demonstrates that while the mutant ADA-coding sequences are transcribed, they do not encode a functional ADA protein.