Moriwaki S, Tarone R E, Kraemer K H
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
J Invest Dermatol. 1994 Jul;103(1):7-12. doi: 10.1111/1523-1747.ep12388847.
The diagnosis of the melanoma-prone disorder dysplastic nevus syndrome (DNS) is based currently on a combination of clinical and histopathologic examinations of patients. To develop a potential laboratory test for DNS, we utilized the observation that an ultraviolet light (UV)-treated mutagenesis plasmid shuttle vector has an abnormally increased frequency of mutations after transfection into lymphoblastoid cells from a patient with familial DNS. pSP189 (containing the bacterial suppressor tRNA gene supF as a marker for mutations and a gene for ampicillin resistance for selection) was treated with UV and transfected into familial DNS, xeroderma pigmentosum complementation group A (XP-A), and normal lymphoblastoid cells by electroporation or diethylaminoethyl (DEAE) dextran. Untreated plasmid pZ189K (containing a gene for kanamycin resistance) was co-transfected as an internal standard to reduce the variability of plasmid survival measurements. After 2 d, plasmids were extracted, used to transform an indicator strain of Escherichia coli, and assayed on plates containing ampicillin or kanamycin. Counting light blue or white colonies (containing mutated supF in the plasmid) and blue colonies (with wild type supF) permitted measurement of the plasmid survival and mutation frequency. Transfection by electroporation or DEAE dextran resulted in abnormally reduced survival of UV-treated plasmid after passage through the XP-A but normal survival in the three DNS lines. Transfection of UV-treated plasmid by DEAE dextran yielded a greater hypermutability with the familial DNS lines than by electroporation. These results suggest that pSP189 UV hypermutability with normal UV survival using DEAE dextran transfection may form the basis of a potential laboratory assay for familial DNS.
易患黑色素瘤的发育异常痣综合征（DNS）目前的诊断是基于对患者的临床和组织病理学检查相结合。为了开发一种针对DNS的潜在实验室检测方法，我们利用了这样一个观察结果：一种经紫外线（UV）处理的诱变质粒穿梭载体转染入家族性DNS患者的淋巴母细胞后，其突变频率异常增加。用紫外线处理pSP189（含有细菌抑制性tRNA基因supF作为突变标记和氨苄青霉素抗性基因用于筛选），并通过电穿孔或二乙氨基乙基（DEAE）葡聚糖转染入家族性DNS、着色性干皮病A互补组（XP - A）和正常淋巴母细胞。未处理的质粒pZ189K（含有卡那霉素抗性基因）作为内标共同转染，以降低质粒存活测量的变异性。2天后，提取质粒，用于转化大肠杆菌指示菌株，并在含有氨苄青霉素或卡那霉素的平板上进行检测。通过计数浅蓝色或白色菌落（质粒中含有突变的supF）和蓝色菌落（含有野生型supF），可以测量质粒的存活和突变频率。通过电穿孔或DEAE葡聚糖转染后，经紫外线处理的质粒在通过XP - A后存活异常降低，但在三个DNS细胞系中存活正常。与电穿孔相比，用DEAE葡聚糖转染经紫外线处理的质粒在家族性DNS细胞系中产生了更高的超突变性。这些结果表明，使用DEAE葡聚糖转染时，pSP189在正常紫外线存活情况下的紫外线超突变性可能构成家族性DNS潜在实验室检测方法的基础。
