Expression of human chromosome 2 ornithine decarboxylase gene in ornithine decarboxylase-deficient Chinese hamster ovary cells.
Ornithine decarboxylase (ODC) belongs to a multigene family and some of these may very well be nonfunctional (pseudogenes). We isolated an ODC gene from a human chromosome 2-specific library and transfected the gene into ODC-deficient Chinese hamster ovary cells to directly demonstrate that this ODC gene is functional and ODC is essential for cell proliferation. After screening 2.5 X 10(5) plaques using a human ODC complementary DNA probe, a typical clone with a 5.4-kilobase insert was isolated and then cloned into the HindIII site of the pGem-1 vector. One (phODC 2B1) of these clones containing a 5.4-kilobase ODC gene insert was identified. Restriction enzyme analysis and partial sequencing data revealed that phODC 2B1 contained the full length protein-coding sequences but lacked first exon and 3'-polyadenylation sequences. Primer extension analysis indicated that human ODC mRNA has homologous sequences with the ODC gene from human chromosome 2. To determine that the chromosome 2 ODC gene is functional, ODC-deficient Chinese hamster ovary cells were transfected with the ODC expression vector (phSV2B1-neo) and several G418-resistant transfectants were isolated which expressed 70- to 400-fold more ODC activity than parental or wild-type Chinese hamster ovary cells. Furthermore, these stable transfectants exhibited a higher growth rate than wild-type cells. These results indicate that the ODC gene from human chromosome 2 encodes functional ODC protein, and ODC (and its product putrescine) is required for cell growth.
Hsieh
J T
JT
Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison 53792.
Denning
M F
MF
Heidel
S M
SM
Verma
A K
AK
D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D002455:Cell Division; D002460:Cell Line; D002889:Chromosomes, Human, Pair 2; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D015870:Gene Expression; D005796:Genes; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D015345:Oligonucleotide Probes; D009955:Ornithine Decarboxylase / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D010053:Ovary; D010957:Plasmids; D011073:Polyamines / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D015345:Oligonucleotide Probes; D011073:Polyamines; D009955:Ornithine Decarboxylase