M. Czako et al., VARIABLE EXPRESSION OF THE HERPES-SIMPLEX VIRUS THYMIDINE KINASE GENEIN NICOTIANA-TABACUM AFFECTS NEGATIVE SELECTION, Theoretical and Applied Genetics, 91(8), 1995, pp. 1242-1247
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.