Pluth J M, O'Neill J P, Nicklas J A, Albertini R J
Genetic Toxicology Laboratory, University of Vermont, Burlington 05401, USA.
Mutat Res. 1998 Feb 2;397(2):137-48. doi: 10.1016/s0027-5107(97)00200-5.
Recently, we reported that 6 of 84 (7.1%) hprt mutants arising in in vitro malathion-treated human T-lymphocytes were characterized by specific genomic deletions in a 125-bp region of exon 3 (Pluth et al., Cancer Research 56 (1996) 2393-2399. We have now extended study to determine whether additional differences in molecular spectrum at a basepair level exist between control and malathion-treated mutations, and investigated whether there is evidence to support the hypothesis that malathion is an alkylating agent. We analyzed 101 hprt mutants (24 from control and 77 from treated cultures) isolated form six in vitro malathion exposures of T-lymphocytes from four healthy male donors. Analysis consisted of: Southern blotting, genomic multiplex PCR, genomic DNA sequencing and reverse transcription of PCR amplification (RT/PCR) and sequencing of the cDNA product. Mutations at several basepair sites were frequent after malathion exposure and were isolated from treated cells from at least two different individuals. Using a human hprt mutation database for comparison, the frequency of mutations at one of these sites (basepair 134) was found to be significantly elevated in the malathion-treated cell (p < 0.0005). Hprt mutations in malathion-treated cells arose preferentially at G:C basepairs, which is consistent with earlier reports that malathion alkylates guanine nucleotides. Assessing molecular changes at both genomic and cDNA levels in the same mutants revealed that many small, partial exon deletions (< 20 bp) in genomic DNA were often represented in the cDNA at the loss of one or more exons. In addition, It was noted that identical genomic mutations can result in different cDNA products in different T-cell isolates. These observations affirm the importance of genomic sequence analysis in combination with RT/PCR for a more accurate definition of the mutation spectrum.
最近，我们报道称，在体外经马拉硫磷处理的人T淋巴细胞中产生的84个次黄嘌呤磷酸核糖基转移酶（hprt）突变体中有6个（7.1%）的特征是外显子3的125碱基对区域存在特定的基因组缺失（Pluth等人，《癌症研究》56(1996)2393 - 2399）。我们现在扩展了研究，以确定在对照和经马拉硫磷处理的突变之间，在碱基对水平上的分子谱是否存在其他差异，并研究是否有证据支持马拉硫磷是一种烷化剂的假设。我们分析了从四名健康男性供体的T淋巴细胞的六次体外马拉硫磷暴露中分离出的101个hprt突变体（24个来自对照培养物，77个来自处理后的培养物）。分析包括：Southern印迹法、基因组多重聚合酶链反应（PCR）、基因组DNA测序以及PCR扩增的逆转录（RT/PCR）和cDNA产物测序。马拉硫磷暴露后，几个碱基对位点的突变很常见，并且是从至少两个不同个体的处理细胞中分离出来的。使用人类hprt突变数据库进行比较，发现其中一个位点（碱基对134）的突变频率在经马拉硫磷处理的细胞中显著升高（p < 0.0005）。经马拉硫磷处理的细胞中的hprt突变优先出现在G:C碱基对处，这与早期关于马拉硫磷使鸟嘌呤核苷酸烷基化的报道一致。在同一突变体的基因组和cDNA水平上评估分子变化发现，基因组DNA中许多小的、部分外显子缺失（< 20碱基对）在cDNA中通常表现为一个或多个外显子的缺失。此外，还注意到相同的基因组突变在不同的T细胞分离物中可导致不同的cDNA产物。这些观察结果证实了基因组序列分析与RT/PCR相结合对于更准确地定义突变谱的重要性。
