The cloning and reintroduction into animal cells of a functional CAD gene, a dominant amplifiable genetic marker

Cell. 1981 Dec;27(2 Pt 1):267-77. doi: 10.1016/0092-8674(81)90410-4.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Aspartate Carbamoyltransferase
  • Aspartic Acid / analogs & derivatives
  • Aspartic Acid / pharmacology
  • Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)*
  • Cell Line
  • Cloning, Molecular / methods*
  • Cricetinae
  • Cricetulus
  • DNA, Recombinant*
  • Dihydroorotase*
  • Drug Resistance
  • Female
  • Gene Amplification
  • Genes, Dominant
  • Genetic Markers
  • Genetic Vectors
  • Multienzyme Complexes*
  • Ovary
  • Phosphonoacetic Acid / analogs & derivatives
  • Phosphonoacetic Acid / pharmacology
  • Proteins / genetics*
  • Transformation, Genetic
  • Uridine / metabolism


  • CAD trifunctional enzyme
  • DNA, Recombinant
  • Genetic Markers
  • Multienzyme Complexes
  • Proteins
  • Aspartic Acid
  • sparfosic acid
  • Aspartate Carbamoyltransferase
  • Dihydroorotase
  • Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)
  • Phosphonoacetic Acid
  • Uridine