Abstract
Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with a severe defect in T-lymphocyte function. The mutant gene was cloned from one PNP-deficient patient who was the offspring of a consanguineous mating. The exons and intron/exon boundaries of the mutant PNP gene were sequenced and compared with the wild-type cDNA sequence. A single base difference was found in the coding region of the mutant gene, a G to A transition in the third exon. This single base mutation alters the codon at position 89 from Glu to Lys, a result which is consistent with previously published peptide mapping data. The patient was demonstrated to be autozygous for the single base mutation on the basis of hybridization of synthetic oligomers to genomic DNA digests. A mammalian expression vector was constructed containing the entire mutant gene under the transcriptional regulation of its own promoter. In another construction, the single base mutation was reverted to the wild-type sequence by in vitro mutagenesis. An isoelectric focusing gel containing extracts of the cells transfected with the mutant and reverted PNP gene was stained histochemically for PNP activity. The proteins from a similar gel were blotted on a nitrocellulose membrane, and immunoreactive human PNP protein was visualized. Cells transfected with the mutant gene contained no human PNP activity, but expressed immunoreactive PNP which focused at an abnormally alkaline pI. Cells transfected with the reverted gene expressed human PNP activity which co-focused with human PNP from a HeLa cell control, proving that the observed single base change was responsible for the loss of catalytic function.
Highlights
Cellstransfected with the mutant gene Two-dimensional tryptic peptide analysisof *251-labeledpepcontained no human Purine nucleoside phosphorylase (PNP) activity, but expressed im- tides revealed that the mutant subunits containedtwo addimunoreactive P N P whichfocused at an abnormally tional peptides andlacked one normal peptide(6). alkaline PI
To gain further insights into the molecular structure and function of purine nucleoside phosphorylase, we studied the defective gene from a patient with an inherited deficiency of the enzyme
She was the product of a consanguineous matingand itwaslikely that bothalleles of her PNP genewould he identical, facilitating genetic analyses
Summary
Genomic Library Construction-Genomic DNA was prepared from the postmortem liver of Patient L. Subcloning into Plasmid Vectors-Phage DNA was prepared from liquid cultures and digested with XbaI. The digests were electrophoresed in preparative agarose gels, and the inserts were electroeluted from the gel and cloned into XbaI-digested, dephosphorylated pUC19 (14).Miniscreen DNA wasprepared from the transformants. Itwas determined that there are no SalIsites in the PNP gene Both 4.8-kb and 7.6-kb XbaI fragments from the mutant PNPgene were subcloned into the XbaI site of the plasmid vector pUC19 (14). The 4.8-kb and 7.6-kb fragments from the digests were purified on agarose gels, electroeluted, ligated together with SalI-digested, dephosphorylated pUC19, and used to transform E. coli[294] cells. Transformants were screened by colony hybridization using a 19base synthetic oligonucleotide probe (5’ dCACATGTATGAAGGGTACC) and washed with tetramethylammonium chloride as described above. P N P Activity Assay-The specific activity of PNP in cell extracts was determined by using the spectrophotometric assay of Kalckar (26)as described (22).Protein was determined by using the Coomassie Blue procedure as formulated by Bio-Rad Laboratories
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
