Khattar N H, Cooper G E, DiMartino D L, Bishop P L, Turker M S
Department of Microbiology and Immunology, University of Kentucky College of Medicine, Lexington 40536.
Biochem Genet. 1992 Dec;30(11-12):635-48. doi: 10.1007/BF02399812.
A mouse embryonal carcinoma cell line isolated for resistance to the adenine analogue 2,6-diaminopurine (DAP) was found to have near-wild-type levels of adenine phosphoribosyltransferase (APRT) activity in a cell-free assay. This DAP-resistant (DAPr) cell line, termed H29D1, also exhibited near-wild-type levels of adenine accumulation and the ability to grow in medium containing azaserine and adenine. Growth in this medium requires high levels of intracellular APRT activity. Using the polymerase chain reaction (PCR) and the dideoxy chain termination sequencing technique, an A-->G transition was discovered in exon 3 of the aprt gene in H29D1. This mutation resulted in an Arg-to-Gln change at amino acid 87 of the APRT protein that, in turn, resulted in a decreased affinity for adenine. An increased sensitivity of APRT to inhibition by AMP was observed when comparing H29D1 to P19, the parental cell line. Using a transgene containing the A-->G mutation, we demonstrated that this mutation is responsible for the biochemical and cellular phenotypes observed for the H29D1 cell line. The approach used in this study provides a definitive method for linking a mutation to a specific cellular phenotype.
一株因对腺嘌呤类似物2,6 - 二氨基嘌呤（DAP）具有抗性而分离出的小鼠胚胎癌细胞系，在无细胞检测中被发现具有接近野生型水平的腺嘌呤磷酸核糖转移酶（APRT）活性。这株对DAP有抗性（DAPr）的细胞系，称为H29D1，还表现出接近野生型水平的腺嘌呤积累以及在含有重氮丝氨酸和腺嘌呤的培养基中生长的能力。在这种培养基中生长需要高水平的细胞内APRT活性。利用聚合酶链反应（PCR）和双脱氧链终止测序技术，在H29D1细胞系的aprt基因外显子3中发现了一个A→G的转换。这种突变导致APRT蛋白第87位氨基酸由精氨酸变为谷氨酰胺，进而导致对腺嘌呤的亲和力降低。与亲本细胞系P19相比，观察到H29D1细胞系中APRT对AMP抑制的敏感性增加。利用含有A→G突变的转基因，我们证明了这种突变是导致H29D1细胞系所观察到的生化和细胞表型的原因。本研究中使用的方法为将突变与特定细胞表型联系起来提供了一种确定的方法。
