Tyrosine residues of middle-T and tyrosine phosphorylation are thought to be important in the transformation of cultured rodent cells by polyomavirus. Of the potential tyrosine sites in the carboxyl-terminal half of middle-T, tyrosines 297, 315, and 322 have been studied previously, whereas tyrosine 250 has not. Two mutant plasmids, XD121 and pT250, encode polyomavirus middle-T species in which the tyrosine 250 residue is affected. XD121 is a deletion mutant in which the region encoding tyrosine 250, together with three adjacent amino acids, is deleted, whereas pT250 is a point mutant in which the tyrosine 250 codon has been converted to a phenylalanine codon. The plasmids were handicapped in transforming ability, as judged by focus formation on a monolayer of Rat-1 cells. Both demonstrated a reduction in the number of foci produced and a lag in the time of appearance of foci when compared with wild-type plasmid. The importance of residue 250 in this phenotype was indicated by the observation that plasmids containing multiple mutations proximal to the tyrosine 250 codon were wild type in their transforming ability. Furthermore, a revertant of pT250 (pT250-w.t.), which utilized the alternative tyrosine codon of TAC, was shown to regain full transforming activity. A combined-mutant plasmid, pTH, encodes a middle-T species in which both tyrosines 250 and 315 are converted to phenylalanine. This plasmid was totally defective in the transformation of rodent cells in a focus formation assay; however, it did impart a small measure of anchorage-independent growth when the encoded protein was expressed in NIH 3T3 cells. The in vitro kinase activity and pp60c-src association of the mutant middle-T antigens were examined. These assays demonstrated a reduction in phosphate acceptor activity for the middle-T species encoded by pT250 and pTH. Quantitative kinase assays showed that all of the tyrosine-mutant middle-T species, encoded by pAS131 (containing the tyrosine 315 codon-to-phenylalanine codon mutation), pT250, and pTH, were able to enhance pp60c-src kinase activity but only at levels which were intermediate and which reflected their transforming abilities relative to wild type.

The transforming activity of polyoma virus middle‐T antigen is believed to be dependent on its ability to form a complex with the cellular tyrosine protein kinase, pp60c‐src. This hypothesis is based on observations of mutants of middle‐T which demonstrated a correlation between these two activities. To investigate further the significance of pp60c‐src association in transformation by middle‐T, a series of deletion and point mutants were constructed around the NG59 lesion since this region has been implicated in pp60c‐src binding. Analysis of the middle‐T variants revealed a complete correlation between the presence of associated activated pp60c‐src and the ability to transform. Further, this ability of pp60c‐src to associate with middle‐T may depend on the presence of a beta‐turn between amino acids 177 and 180. The results indicate the NG59 phenotype results from the introduction of an isoleucine residue between amino acids 177 and 178 rather than the transition mutation at 179. The mutant MG1 is a single point mutation (at residue 180) and represents the smallest change in the middle‐T which abolishes both the transformating and kinase activity of middle‐T. Taken together, the data suggest the region surrounding the NG59 lesion is involved in the formation of an active complex between middle‐T and pp60c‐src and strongly suggest that this association is an absolute requirement for polyoma virus‐induced transformation.

A point mutation in the simian virus 40 large-T gene, which was generated by mixed oligonucleotide mutagenesis and resulted in the conversion of Lys 128 to Thr, produced a large-T antigen that was detected in the cytoplasm but not the nucleus of cells. Deletions within the surrounding sequence Lys-128Lys-Lys-Arg-Lys-Val-Glu also produce cytoplasmic large-T and define a region of the protein involved in nuclear location.

An inverted repeat has been created in a plasmid by ligation of two 13 nucleotide synthetic oligonucleotides into the cloning vector pAT153. The resulting recombinant plasmid, pIRbke8, is hypersensitive to cleavage by the single‐strand‐specific nuclease S1, and to modification by the single‐strand‐selective reagent bromoacetaldehyde, when the plasmid is negatively supercoiled. The new inverted repeat is a stronger S1 site than those derived from pBR322, but, in contrast to the ColE1 and phi X174 RF inverted repeats, these repeats share a similar temperature dependence. The kinetics of EcoRI cleavage at the centre of the synthetic inverted repeat have been studied in supercoiled and linear molecules. It is found that in the supercoiled molecule this target is not refractory to EcoRI cleavage to an extent which is greater than the resolution of the experiment. We conclude that in this molecule the cruciform is in a dynamic equilibrium with the regular duplex, in which the cruciform constitutes a relatively small subpopulation of conformational species.

The ability of polyoma virus to transform cells results primarily from the action of one of the virus-coded early proteins, called middle-T antigen. Middle-T has an associated tyrosine-specific protein kinase activity that can be measured in vitro and results in the phosphorylation of middle-T itself. Almost all mutants so far tested that lack the ability to transform cells, also lack associated kinase activity. Attempts to map within middle-T the tyrosine residue(s) that are phosphorylated in vitro suggest that a likely site of phosphorylation is tyrosine 315 (refs 8-10 and unpublished results). The amino acid sequence preceding Tyr 315 includes a tract of six contiguous glutamic acid residues and bears some homology with that preceding the tyrosine phosphorylated in vivo in pp60v-src, the transforming protein of Rous sarcoma virus, and with a region in the polypeptide hormone, gastrin, preceding a tyrosine that is sulphated. Furthermore, although surprisingly large tracts of middle-T may be removed without affecting its transforming activity, mutants that lack the sequences corresponding to amino acids 311-318 inclusive are transformation defective. Because the likely site of phosphorylation, the homology with pp60v-src and gastrin and the sequence apparently required for transformation all overlap, it has generally been accepted that this region of middle-T may form part of an essential region, possibly an active site on the protein. Here we have used techniques of site-directed and site-specific mutagenesis to probe the sequence requirements in more detail. Contrary to expectation, the results obtained strongly suggest that Tyr 315 and conservation of the surrounding amino acid sequence are not essential for transformation.