Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
 2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The TK6 human B lymphoblastoid cell line contains two easily and widely used selectable markers: the X-linked, hemizygous hprt locus, and the heterozygous tk locus on chromosome 17q. In this study, rare APRT heterozygotes were directly isolated from the TK6 population by clonal selection in cell culture medium supplemented with 5 micrograms/ml of 8-azaadenine. One of nine isolated heterozygotes, AZH1, was characterized extensively. APRT- mutants can be recovered from AZH1 at a mutation rate of 1.5 x 10(-7), similar to rates previously determined for the selection of TK- and HPRT- mutants from TK6. A unique sequence alteration was identified in the non-functional aprt allele at position 1930. A G:C to A:T transition at this site alters the canonical AG splice acceptor dinucleotide in exon 3, and also results in the destruction of a Stul recognition sequence. This polymorphism was used to analyze loss of heterozygosity in a set of 32 spontaneous APRT- mutants by restriction analysis following PCR amplification. Analysis of flanking microsatellite dinucleotide polymorphisms demonstrated that LOH occurring in spontaneous APRT- mutants is nearly always a multi-locus event extending at least 7.5 cM along chromosome 16q. This pattern of LOH among APRT- mutants differs from extensive LOH in spontaneous, normal-growth TK- mutants derived from TK6 cells (p < 0.0001), and suggests that cis-acting factors may be equally important in shaping the mutational spectrum as trans-acting factors such as cellular apoptotic capacity.
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PMID:Isolation of an APRT heterozygote from TK6 human lymphoblasts: predominance of multi-locus loss of heterozygosity among spontaneous APRT-mutants. 921 76

The feasibility of introducing point mutations in vivo using single-stranded DNA oligonucleotides (ssON) has been demonstrated but the efficiency and mechanism remain elusive and potential side effects have not been fully evaluated. Understanding the mechanism behind this potential therapy may help its development. Here, we demonstrate the specific repair of an endogenous non-functional hprt gene by a ssON in mammalian cells, and show that the frequency of such an event is enhanced when cells are in S-phase of the cell cycle. A potential barrier in using ssONs as gene therapy could be non-targeted mutations or gene rearrangements triggered by the ssON. Both the non-specific mutation frequencies and the frequency of gene rearrangements were largely unaffected by ssONs. Furthermore, we find that the introduction of a mutation causing the loss of a functional endogenous hprt gene by a ssON occurred at a similarly low but statistically significant frequency in wild type cells and in cells deficient in single strand break repair, nucleotide excision repair and mismatch repair. However, this mutation was not induced in XRCC3 mutant cells deficient in homologous recombination. Thus, our data suggest ssON-mediated targeted gene repair is more efficient in S-phase and involves homologous recombination.
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PMID:Specific targeted gene repair using single-stranded DNA oligonucleotides at an endogenous locus in mammalian cells uses homologous recombination. 1985 87