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

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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.