The cloning and reintroduction into animal cells of a functional CAD gene, a dominant amplifiable genetic marker.
Rodent cells resistant to PALA, a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional CAD protein, overproduce CAD as a result of amplification of the CAD gene. We cloned a functional CAD gene from Syrian hamster cells using a cosmid vector. Two independently isolated cosmids containing CAD genes have inserts 40 and 45 kb long. We introduced the cloned genes into CAD-deficient Chinese hamster ovary (CHO) cell mutants by fusing them with protoplasts of Escherichia coli containing the cosmids. We also introduced the cloned genes into wild-type CHO cells by selecting cells that became resistant to high concentrations of PALA following protoplast fusion. The transformants of the mutant and wild-type CHO cells contain multiple active copies of the donated Syrian hamster CAD genes. The cloned genes in three independent transformants are integrated into host-cell chromosomes at single locations identified by in situ hybridization. In two of these transformants, the genes are located in one X chromosome or in a chromosome resembling the X. In the third case, the genes are located in a small metacentric or rearranged chromosome.
D000818:Animals; D001221:Aspartate Carbamoyltransferase; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D004274:DNA, Recombinant; D004080:Dihydroorotase; D004351:Drug Resistance; D005260:Female; D005784:Gene Amplification; D005799:Genes, Dominant; D005819:Genetic Markers; D005822:Genetic Vectors; D009097:Multienzyme Complexes; D010053:Ovary; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D011506:Proteins / Q000235:genetics*; D014170:Transformation, Genetic; D014529:Uridine / Q000378:metabolism
C033626:CAD trifunctional enzyme; D004274:DNA, Recombinant; D005819:Genetic Markers; D009097:Multienzyme Complexes; D011506:Proteins; D001224:Aspartic Acid; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D010746:Phosphonoacetic Acid; D014529:Uridine