Biological activities of oligonucleotides spanning the F9 point mutation within the enhancer region of polyomavirus DNA.

A mutant of polyomavirus, F441, selected to grow in undifferentiated mouse F9 embryonal carcinoma cells, carries a single-base change in the enhancer region at nucleotide (nt) 5233 of the viral genome. Enhancers of most of the F9 mutants have a duplicated segment of viral DNA encompassing nt 5233. The minimum duplicated segment of all the known F9 mutants is from nt 5218 to nt 5239. We prepared oligonucleotides spanning the sequence from nt 5218 through nt 5239 of the genome of the wild type and F441 and examined the biological activities of the oligonucleotides by a transient assay of chloramphenicol acetyltransferase (CAT) gene expression in F9 cells. The oligonucleotide harboring the F441 mutation was shown to increase cat gene expression in F9 cells when linked at an upstream position in both orientations. When dimerized at an upstream position, the F441 oligonucleotide showed even higher cat gene expression enhancing activity. In contrast, no such effects were observed with the oligonucleotide of the wild-type sequence. In addition, the F441 oligonucleotide, but not the wild-type sequence, could inhibit the activity of whole enhancer fragment of F441 when cotransfected into F9 cells in excess amounts. On the basis of the results obtained, we suggest that the segment of F441 enhancer encompassing the point mutation contains a target for a cellular factor(s) which acts in a positive manner to increase the transcription of a gene in undifferentiated mouse F9 cells.

A point mutation in the conserved hexanucleotide at a yeast 5' splice junction uncouples recognition, cleavage, and ligation.

We have constructed an actin-HIS4 gene fusion, such that expression of HIS4 requires proper splicing of the actin intron. Using this chimeric gene in an in vivo screen for splicing mutations, we have isolated a G to A transition in the fifth position of the yeast 5' consensus sequence/GTAPyGT. This mutation still allows the junction to be recognized by the splicing machinery, albeit inefficiently. Surprisingly, the fidelity of the 5' endonucleolytic cleavage is also reduced. This results in an incorrect cleavage 6 nucleotides 5' of the 5' junction, at the dinucleotide/AT. Cleavage at this abnormal site does not lead to the production of mature mRNA, although this species appears to be in a lariat structure. The behavior of this mutant argues that recognition of the 5' junction and subsequent cleavage are separable events and, furthermore, that requirements for 3' endonucleolytic cleavage may be more complex than previously imagined.

On the fidelity of DNA replication: use of synthetic oligonucleotide-initiated reactions.

The phi X174 fidelity system provides a biological assay for quantitating the accuracy of DNA polymerases. Expansion of this system to cell extracts and DNA replication complexes from eucaryotes has been limited by the presence of nucleases in these preparations. We have overcome these limitations by priming the phi X template with a synthetic oligodeoxynucleotide, with its free 3'-hydroxyl terminus only a short distance from the amber locus that is the site for determining the frequency of misincorporation. In this paper, this modified phi X system is characterized and compared to that using defined natural DNA restriction fragments as primers. The modified system has been applied to studies on the fidelity of DNA synthesis using different forms of purified DNA polymerase-alpha from calf thymus, as well as crude extracts from human lymphocytes.