用多重聚合酶链反应分析HL-60细胞中自发的、γ射线和乙基亚硝基脲诱导的次黄嘌呤-鸟嘌呤磷酸核糖转移酶突变体。

Analysis of spontaneous, gamma ray- and ethylnitrosourea-induced hprt mutants in HL-60 cells with multiplex PCR.

作者信息

Liu Sheng-Xue, Cao Jia, An Hui, Shun Hua-Min, Yang Lu-Jun, Liu Yong

机构信息

Department of Healath Toxicology, Preventive Medical College, Third Military Medical University, Chongqing 400038, China.

出版信息

World J Gastroenterol. 2003 Mar;9(3):578-83. doi: 10.3748/wjg.v9.i3.578.

DOI:10.3748/wjg.v9.i3.578

PMID:12632522

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4621586/

Abstract

AIM

To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (hprt) gene mutation induced by ethyluitrosourea (ENU) and (60)Co gamma-rays.

METHODS

Independent human promyelocytic leukemia cells (HL-60) mutants at the hprt locus were isolated from untreated, ethyluitrosourea (ENU) and (60)Co gamma-ray-exposed cells, respectively, and verified by two-way screening. The genetic changes underlying the mutation were determined by multiplex polymerase chain reaction (PCR) amplification and electrophoresis technique.

RESULTS

With dosage increased, survival rate of plated cell reduced (in the group with dosage of ENU with 100-200 micro g/ml, P<0.01; in the group with dosage of (60)Co gamma-ray with 2-4 Gy, P<0.05) and mutational frequency increased (in the group of ENU 12.5-200.0 micro g/ml, P<0.05; in the group of (60)Co gamma-ray with 1-4 Gy, P<0.05) significantly. In the 13 spontaneous mutants analyzed, 92.3 % of mutant clones did not show any change in number or size of exon, a single exon was lost in 7.7 %, and no evidence indicated total gene deletion occurred in nine hprt exons. However, deletions were found in 79.7 % of ENU-induced mutations (62.5-89.4 %, P<0.01) and in 61.7 % of gamma-ray-induced mutations (28.6-76.5 %, P<0.01). There were deletion mutations in all 9 exons of hprt gene and the most of induced mutations were chain deletion with multiplex exons (97.9 % in gamma-ray-induced mutants, 88.1 % in ENU-induced mutants).

CONCLUSION

The spectra of spontaneous mutations differs completely from that induced by EUN or (60)Co gamma-ray. Although both ENU and gamma-ray can cause destruction of genetic structure, mechanism of mutagenesis between them may be different.

摘要

目的

探讨乙基亚硝基脲（ENU）和钴-60（^60Co）γ射线诱导人次黄嘌呤鸟嘌呤磷酸核糖转移酶（hprt）基因突变的分子谱及机制。

方法

从未经处理、经乙基亚硝基脲（ENU）和钴-60（^60Co）γ射线照射的人早幼粒细胞白血病细胞（HL-60）中分别分离出hprt基因座的独立突变体，并通过双向筛选进行验证。采用多重聚合酶链反应（PCR）扩增和电泳技术确定突变背后的基因变化。

结果

随着剂量增加，接种细胞的存活率降低（ENU剂量为100 - 200μg/ml组，P<0.01；钴-60（^60Co）γ射线剂量为2 - 4 Gy组，P<0.05），突变频率显著增加（ENU组12.5 - 200.0μg/ml，P<0.05；钴-60（^60Co）γ射线组1 - 4 Gy，P<0.05）。在分析的13个自发突变体中，92.3%的突变克隆在外显子数量或大小上未显示任何变化，7.7%丢失单个外显子，在9个hprt外显子中未发现全基因缺失的证据。然而，在79.7%的ENU诱导突变中发现缺失（62.5 - 89.4%，P<0.01），在61.7%的γ射线诱导突变中发现缺失（28.6 - 76.5%，P<0.01）。hprt基因的所有9个外显子均存在缺失突变，且大多数诱导突变是多个外显子的连锁缺失（γ射线诱导突变体中为97.9%，ENU诱导突变体中为88.1%）。