| Standard techniques of molecular biology were used to investigate the mechanism of drug resistance in borrelidin-resistant CHO cell lines, and the possible regulation of threonyl-tRNA synthetase (ThrRS) mRNA levels in wild-type CHO cells. The regional location of the ThrRS structural gene, already assigned to the short arm of human chromosome 5 (Gerken et al., (1986) Somat. Cell. Mol. Genet. 12, 519-522), was also further refined to within 5p13.;A cDNA for human ThrRS was isolated from a human placental cDNA library in ;Methods were developed to quantitate ThrRS messenger RNA levels in CHO cells grown in medium lacking threonine or other essential amino acids, and in medium containing borrelidin. Increases in steady state ThrRS mRNA levels were observed in extracts of wild-type CHO cells starved for threonine, and effect which is reversed by adding threonine back to the growth medium. Over a period of 72 hours ThrRS mRNA levels in CHO cells starved for threonine rise 3- to 4-fold, reaching a maximum at about 24 hours, and then falling back to control levels. When CHO cells are grown in the same medium plus a low concentration of the ThrRS inhibitor borrelidin the increase in mRNA levels is more rapid, reaching a peak of approximately 10-fold in about 24 hours. Exposing CHO cells to increasing concentrations of borrelidin results in increasing levels of ThrRS mRNA, even in medium containing threonine. The accumulation of ThrRS mRNA can be reversed by shifting CHO cells to complete medium. These studies implicate tRNA... |
