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，这些细胞对应于小鼠胚胎发育早期的细胞。几个能够在F9细胞中生长的F9突变体已经被分离出来，并被证明在病毒基因组的增强子区域发生了序列变化。F9突变体之一的F441在增强子区域存在单一碱基变化。合成了野生型寡核苷酸和点突变部位的F441序列，并对其生物活性进行了检测。凝胶漂移分析表明，存在一种与F441序列强结合而不与野生型序列结合的细胞因子(S)，该因子存在于未分化和分化的F9细胞中，以及其他几种细胞系中，无论其物种或组织来源如何。结合位点长为8个碱基对，包括突变。我们还检测了这些寡核苷酸对基因表达的影响。将寡核苷酸与氯霉素乙酰转移酶(CAT)基因连接的重组质粒导入F9细胞，检测其表达的CAT活性。研究发现，F441寡核苷酸比野生型寡核苷酸增加CAT基因表达3倍。根据以上结果，F441点突变产生了与细胞正调控因子(S)的结合位点或亲和力增加，F441突变体的表型是通过突变的序列与该因子结合而获得的。对多瘤病毒增强子的其他部分的分析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.