Clark L Scott, Harrington-Brock Karen, Wang Jianyong, Sargent Linda, Lowry David, Reynolds Steve H, Moore Martha M
University of North Carolina Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC, USA.
Mutagenesis. 2004 Jul;19(4):263-8. doi: 10.1093/mutage/geh024.
The mouse lymphoma L5178Y Tk+/- 3.7.2C assay is a well-characterized in vitro system used for the study of somatic cell mutation. It was determined that this cell line has a heterozygous mutation in exon 5 of Trp53. Based on this assumption that the cell line is heterozygous for the Trp53 gene, it was postulated that the small colony thymidine kinase (Tk) mutant phenotype may be due to a newly induced mutation/deletion in both the Trp53 and Tk1 alleles. The resultant Tk-/- mutants would also be Trp53+/0 or Trp53+/+ and would lose their ability to grow at normal rates. Subsequently, we published our evaluation of the Trp53 status in L5178Y cells. This analysis included sequencing of Trp53 exon 4 and determined that the mouse lymphoma cell line has a mutation in both of the Trp53 alleles and, therefore, no wild-type Trp53 allele in either Tk+/- cells or Tk-/- mutants. Because the cells have no wild-type Trp53, it is not possible that the small colony phenotype results from a newly induced loss of both functional Trp53 and Tk. To determine whether small colonies might, however, include the deletion of both Trp53 and Tk we evaluated, using microsatellite marker analysis, a series of small colony mutants. We also utilized in situ hybridization to determine that the Trp53 alleles are, in fact, in their normal chromosome 11 location in Tk+/- 3.7.2C mouse lymphoma cells. From all of these analyses we can conclude that the small colony mutant phenotype is not caused by deletion of both Trp53 and Tk1.
小鼠淋巴瘤L5178Y Tk+/- 3.7.2C检测是一种用于体细胞突变研究的特征明确的体外系统。已确定该细胞系在Trp53基因的外显子5中存在杂合突变。基于该细胞系Trp53基因为杂合的这一假设，推测小集落胸苷激酶（Tk）突变表型可能是由于Trp53和Tk1等位基因中均出现了新诱导的突变/缺失。产生的Tk-/-突变体也将是Trp53+/0或Trp53+/+，并将失去以正常速率生长的能力。随后，我们发表了对L5178Y细胞中Trp53状态的评估。该分析包括对Trp53外显子4进行测序，并确定该小鼠淋巴瘤细胞系在两个Trp53等位基因中均存在突变，因此，在Tk+/-细胞或Tk-/-突变体中均不存在野生型Trp53等位基因。由于细胞中不存在野生型Trp53，小集落表型不可能是由功能性Trp53和Tk均新诱导缺失导致的。然而，为了确定小集落是否可能包括Trp53和Tk的缺失，我们使用微卫星标记分析评估了一系列小集落突变体。我们还利用原位杂交确定Trp53等位基因实际上位于Tk+/- 3.7.2C小鼠淋巴瘤细胞的正常11号染色体位置。从所有这些分析中我们可以得出结论，小集落突变表型不是由Trp53和Tk1均缺失引起的。
