Analysis of enhancer mutants of polyoma virus which can grow in undifferentiated mouse cells.

野生型多瘤病毒可以在分化的小鼠细胞如3T3细胞中生长，但不能在未分化的细胞如F9细胞中生长，所述未分化的细胞对应于小鼠胚胎发育的早期阶段的细胞。已经分离出几种能够在F9细胞中生长的F9突变体，并显示其在病毒基因组的增强子区域中具有序列改变。F441是F9突变体之一，在增强子区域中具有单个碱基变化。合成野生型寡核苷酸和跨越点突变位点的F441序列，并检查其生物活性。凝胶迁移试验表明，存在一种与F441序列强烈结合但不与野生型序列结合的细胞因子，并且该因子存在于未分化和分化的F9细胞以及几种其他细胞系中，而与其种属或组织来源无关。结合位点是包括突变的8个碱基对长的区域。我们还研究了这些寡核苷酸对基因表达的影响。将其中寡核苷酸连接到氯霉素乙酰转移酶（CAT）基因的重组质粒转染到F9细胞中，并测量由质粒表达的CAT活性。发现F441寡核苷酸使CAT基因表达增加为野生型寡核苷酸的三倍。当F441寡核苷酸以二聚体形式与CAT基因连接时，其作用进一步增强。基于上述结果，可以得出结论，F441点突变产生了一个结合位点或增加了与细胞正调控因子的亲和力，并且F441突变体的表型是通过突变序列与因子的缔合而实现的。对多瘤病毒增强子的其他部分的分析正在继续理解为什么野生型增强子在F9细胞中不起作用。

Wild type polyoma virus can grow in differentiated mouse cells such as 3T3 cells, but not in undifferentiated cells such as F9 cells which correspond to the cells at early stages of mouse embryo development. Several F9 mutants, capable of growing in F9 cells, have been isolated and shown to posess sequence alterations in the enhancer region of the viral genome. F441, one of the F9 mutants, harbors a single base change in the enhancer region. Oligonucleotides of wild type and F441 sequences spanning the site of the point mutation were synthesized and examined for its biological activity. Gel shift assay revealed that there was a cellular factor(s) which bound strongly to the F441 sequence but not to the wild type sequence and that the factor was present both in undifferentiated and differentiated F9 cells, as well as in several other cell lines irrespective of their origin of species or tissues. The binding site was a region of 8 base pair long including the mutation. We also examined the effect of these oligonucleotides on gene expression. Recombinant plasmids in which the oligonucleotides were linked to the chloramphenicol acetyl transferase (CAT) gene were transfected to F9 cells and CAT activities expressed by the plasmids were measured. The F441 oligonucleotide was found to increase the CAT gene expression three times as much as the wild type oligonucleotide. The effect of the F441 oligonucleotide was further strengthened when linked to the CAT gene as a dimer.Based on the above results, it was concluded that the F441 point mutation created a binding site or increased the affinity to a cellular positive regulatory factor(s) and that the phenotype of F441 mutant was achieved by association of the mutated sequence and the factor. Analysis of other parts of the polyoma virus enhancer is continuing to understand why wild type enhancer does not function in F9 cells.