title: Mutant human adenosine deaminase alleles and their expression by transfection into fibroblasts.
abstract: 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.
authors.affiliation: Children's Hospital Research Foundation, Cincinnati, Ohio.
authors.forename: A L
authors.initials: AL
authors.lastname: Akeson
authors.forename: D A
authors.initials: DA
authors.lastname: Wiginton
authors.forename: M R
authors.initials: MR
authors.lastname: Dusing
authors.forename: J C
authors.initials: JC
authors.lastname: States
authors.forename: J J
authors.initials: JJ
authors.lastname: Hutton
mesh_terms: D000243:Adenosine Deaminase / Q000235:genetics*; D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D005784:Gene Amplification; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009700:Nucleoside Deaminases / Q000235:genetics*; D010957:Plasmids; D011487:Protein Conformation; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D004247:DNA; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase