Human Adenine Phosphoribosyl Transferase: Molecular Characterization Of The Normal And Deficiency States.

Adenine phosphoribosyltransferase (APRT), a salvage enzyme involved in purine metabolic pathways, has been widely studied and utilized in genetics, biology, and medicine. In spite of its utility, however, little structural information is known about the APRT protein or gene, nor is there any understanding of the abnormal molecular events resulting in the enzyme deficiency state in humans. For this reason we have undertaken a more thorough characterization of human APRT at both the protein and nucleic acid levels. The primary structure of APRT has been determined by well-established protein sequencing methods. It consists of 179 residues and the NH(,2)-terminal alanine is acetylated. Secondary structure predictions define a region of structural homology common to other phosphoribosyltransferase enzymes. Using a cross-hybridizing hamster cDNA as a probe, human cDNA and genomic clones have been isolated. Sequence analysis of restriction fragments of these clones have: (1) defined an open reading frame of 540 nucleotides and predicted an amino acid sequence identical to that determined empirically, (2) identified a rare polyadenylation signal, (3) determined all 4 introns of the gene, and (4) documented sequence features in the 5' non-coding region typical of those found in other housekeeping genes. These latter include the existence of multiple transcription initiation sites, the lack of TATA and CCAAT sequences, and the existence of CCGCCC repeats. Lymphoblastoid cell lines have been established from activity-deficient subjects and have been characterized at both the protein and nucleic acid levels. Evidence from such studies suggest the deficiency state in those cell lines examined is likely due to expression of an abnormal gene product. We are currently involved in efforts which will allow us to determine specific point mutations in the genes of deficient individuals. We have thus gained a great deal of structural information about the normal APRT protein and gene and have begun to utilize this information in our attempt in understanding the molecular pathology underlying APRT deficiency in man.Ph.D.BiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128026/2/8712186.pd