Analysis of co-transfected expression vectors amplified by methotrexate selection in rat-1 cells.

In an earlier investigation of the influence of high level expression of p21H-ras, rat-1 cells were co-transfected with a selectable vector (pSV2Neo), an amplifiable vector (encoding dihydrofolate reductase; DHFR) and an H-ras expression vector. In this study we have analyzed the gene dose and expression levels of the three co-transfected plasmid vectors in cell lines that had been selected and isolated at different methotrexate concentrations. Growth of the cells in the absence of selection and Southern blot analyses indicate that the transfected vectors are stably co-integrated into the host genome. High expression levels from all three co-transfected vectors were evident at both the mRNA and protein levels, indicating that they are tightly linked in the host genome. The presence of a large amount of unspliced H-ras mRNA in cells expressing high levels of H-ras p21 indicates that processing of mRNA may be rate-limiting. Comparison of the gene dose and expression levels shows that the resistance of cells to increased methotrexate concentrations can occur by different mechanisms. It is concluded that co-transfection of individual plasmid vectors into rat-1 cells, followed by methotrexate selection, is an effective manner of achieving high level expression of proteins in cultured cells.

Positive and negative modulation of H-ras transforming potential by mutations of phenylalanine-28.

Conserved amino-acids of H-ras from residues 25 to 34 were mutated in human H-ras cDNA with a pre-existing valine-12 activating mutation ([V12]p21), and built into SV40-driven expression vectors. The influence of the introduced mutations was initially screened by transfection of Rat-1 cells to score foci of transformed cells. Non-conservative mutations of amino-acids 25 (tryptophan for glutamine), 27 (asparagine for histidine) and 34 (alanine for proline) did not abrogate the transforming potential of [V12]p21. The conservative mutation of phenylalanine-28 to tryptophan ([V12W28]p21) was also still transforming. Significantly, in the absence of the valine-12 activating mutation, tryptophan-28-ras ([W28]p21) was weakly transforming while, in contrast, [V12D28]p21 was unable to transform Rat-1 cells and retarded cell growth. Analysis of the binding and dissociation of GTP and GDP to normal and mutated p21 expressed in Escherichia coli showed that [V12D28]p21 and [D28]p21 do not bind GTP. The dissociation rate of both GTP and GDP bound to [W28]p21 is increased, suggesting a mechanism for its transforming potential in Rat-1 cells. These studies illustrate the importance of phenylalanine-28 in guanine nucleotide binding by p21H-ras. The mutations described could be valuable tools in investigations of cellular signal transduction involving small GTP-binding proteins.