Expression of human chromosome 2 ornithine decarboxylase gene in ornithine decarboxylase-deficient chinese hamster ovary cells

Jer Tsong Hsieh, Mitchell F. Denning, Shawn M. Heidel, Ajit K. Verma

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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 × 105 plaques using a human ODC complementary DNA probe, a typical clone with a 5.4-kilobase insert was isolated and then cloned into the Hind III 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 (pbSV2B1-neo) and several G-418-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.

Original languageEnglish (US)
Pages (from-to)2239-2244
Number of pages6
JournalCancer Research
Volume50
Issue number8
StatePublished - Apr 15 1990

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Ornithine Decarboxylase
Chromosomes, Human, Pair 2
Human Chromosomes
Cricetulus
Ovary
Genes
Clone Cells
Restriction Mapping
Polyadenylation
Pseudogenes
Putrescine
DNA Probes
Multigene Family
Growth
Sequence Homology
Gene Library

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Expression of human chromosome 2 ornithine decarboxylase gene in ornithine decarboxylase-deficient chinese hamster ovary cells. / Hsieh, Jer Tsong; Denning, Mitchell F.; Heidel, Shawn M.; Verma, Ajit K.

In: Cancer Research, Vol. 50, No. 8, 15.04.1990, p. 2239-2244.

Research output: Contribution to journalArticle

Hsieh, Jer Tsong ; Denning, Mitchell F. ; Heidel, Shawn M. ; Verma, Ajit K. / Expression of human chromosome 2 ornithine decarboxylase gene in ornithine decarboxylase-deficient chinese hamster ovary cells. In: Cancer Research. 1990 ; Vol. 50, No. 8. pp. 2239-2244.
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abstract = "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 × 105 plaques using a human ODC complementary DNA probe, a typical clone with a 5.4-kilobase insert was isolated and then cloned into the Hind III 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 (pbSV2B1-neo) and several G-418-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.",
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