Human ornithine decarboxylase(ODC)-encoding gene: cloning and expression in ODC-deficient CHO cells.
We have cloned a full-length human ornithine decarboxylase (ODC)-encoding gene from a genomic library of human myeloma cells which overproduce ODC due to a selective gene amplification. Correct expression of the cloned gene was assessed by transfecting it into a Chinese hamster ovary (CHO) cell mutant devoid of ODC activity. Transfection with a 10-kb BamHI DNA fragment of the genomic clone, conferred ODC activity to the recipient cells and relieved them of dependence on exogenous polyamines for growth. A set of 40 transformants was isolated, eight of which were further characterized. The transfected ODC gene appeared to be hypomethylated at the cytosine residues in the sequence CpG. The transfectants were all responsive to serum stimulation, but showed different levels of ODC expression depending on both copy number and integration site of the transfected ODC gene. ODC serum induction in the transfectants was sensitive to cycloheximide and polyamine additions, and the half-life of the enzyme was very short, like that in normal CHO cells. These results suggest that the human ODC gene we transfected contains all the elements needed for normal control of ODC expression.
Hölttä
E
E
Department of Pathology, University of Helsinki, Finland.
Hirvonen
A
A
Wahlfors
J
J
Alhonen
L
L
Jänne
J
J
Kallio
A
A
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D008745:Methylation; D009955:Ornithine Decarboxylase / Q000235:genetics*; D015183:Restriction Mapping; D014162:Transfection
D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes; D009955:Ornithine Decarboxylase