Hsieh J T, Denning M F, Heidel S M, Verma A K
Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison 53792.
Cancer Res. 1990 Apr 15;50(8):2239-44.
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.
鸟氨酸脱羧酶（ODC）属于一个多基因家族，其中一些基因很可能是无功能的（假基因）。我们从一个人2号染色体特异性文库中分离出一个ODC基因，并将该基因转染到ODC缺陷的中国仓鼠卵巢细胞中，以直接证明这个ODC基因是有功能的，且ODC对细胞增殖至关重要。使用人ODC互补DNA探针筛选2.5×10⁵个噬菌斑后，分离出一个典型的插入片段为5.4千碱基的克隆，然后将其克隆到pGem-1载体的HindIII位点。鉴定出其中一个含有5.4千碱基ODC基因插入片段的克隆（phODC 2B1）。限制性内切酶分析和部分测序数据显示，phODC 2B1包含全长蛋白质编码序列，但缺少第一个外显子和3'聚腺苷酸化序列。引物延伸分析表明，人ODC mRNA与来自人2号染色体的ODC基因具有同源序列。为了确定2号染色体ODC基因是有功能的，将ODC表达载体（phSV2B1-neo）转染到ODC缺陷的中国仓鼠卵巢细胞中，并分离出几个对G418有抗性的转染子，这些转染子表达的ODC活性比亲本或野生型中国仓鼠卵巢细胞高70至400倍。此外，这些稳定的转染子比野生型细胞表现出更高的生长速率。这些结果表明，来自人2号染色体的ODC基因编码有功能的ODC蛋白，并且细胞生长需要ODC（及其产物腐胺）。
