VARIABLE EXPRESSION OF THE HERPES-SIMPLEX VIRUS THYMIDINE KINASE GENEIN NICOTIANA-TABACUM AFFECTS NEGATIVE SELECTION

The potentials and limitations of negative-selection systems based on the human herpes simplex virus thymidine kinase type-1 (HSVtk) gene, w hich causes sensitivity to the nucleoside analog ganciclovir, were exa mined in tobacco as a model system. There were great differences betwe en individual HSVtk(+) transgenic plants in ganciclovir sensitivity. I nhibition of growth while under selection correlated with HSVtk-transc ript levels. Negative selection against HSVtk(+) transformants at the level of Agrobacterium-mediated transformation using a ganciclovir/kan amycin double-selection medium (the positive selection marker neomycin phosphotransferase-II gene was in the transformation vector) resulted in a three- to six-fold reduction in the frequency of kanamycin-resis tant shoots. The efficiency of negative selection in this case was lim ited due to the great variation in HSVtk expression, i.e., the frequen tly occurring transformants with low, or no, ganciclovir sensitivity e scaping negative selection. Two independently constructed HSVtk genes showed the same variability of the phenotype in Nicotiana tabacum tran sformants. Distinct phenotypes, ranging from no regeneration through a bnormal or delayed regeneration, were observed when leaf segments were placed on shoot-inducing medium supplemented with 10(-6)-10(-3) M gan ciclovir. The highest HSVtk mRNA and ganciclovir sensitivity levels we re observed in plants which were transformed with the pSLJ882 chimeric construct. The pSLJ882 plant expression vector carried the coding seq uence of HSVtk, whereas plasmid pCX305.1 carried an HSVtk construct re taining the untranslated 5 leader and viral 3 regions. The pCX305.1 tr ansformants showed, at most, a delayed formation of shoots with thin s tems and very narrow leaves, Ganciclovir sensitivity showed typical Me ndelian segregation. A gene-dosage effect was also seen at the seedlin g level in the progeny of two transgenic lines.