Abken H, Hegger R, Bützler C, Willecke K
Abteilung Molekulargenetik, Universität Bonn, Germany.
Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6518-22. doi: 10.1073/pnas.90.14.6518.
Cytoplasts of mouse L929 and Ehrlich ascites tumor cells harbor DNA sequences that induce unlimited proliferation ("immortalization") of human lymphocytes after transfection in vitro. By equilibrium centrifugation of cytoplasmic lysates in a neutral CsCl gradient, the immortalizing activity was recovered together with extramitochondrial fractions at high salt densities (1.85-1.87 g/cm3). Unexpectedly, these fractions contain linear DNA molecules of 50-500 bp in length. In contrast, cytoplasts of primary, senescent cells (mouse embryo fibroblasts, human lymphocytes) do not harbor DNA in the corresponding fractions. Cytoplasmic DNA isolated from high-density fractions of mouse tumor cells was cloned in subset libraries, and of 45 DNA sequences we identified 2 clones--one from L929 cytoplasts (203 bp) and another one from the cytoplasm of Ehrlich ascites cells (372 bp)--that induce unlimited proliferation of human lymphocytes in vitro. Immortalized lymphoid cells harbor 1-5 copies of transfected DNA integrated into chromosomal DNA, whereas about 100 copies were found as episomal DNA in the cytoplasmic fraction. No immortalization could be induced by transfection of nuclear DNA randomly fragmented to 200-500 bp. Although the cloned DNA sedimented at 1.70 g/cm3, after transient transfection into lymphocytes, these DNA sequences form salt-stable complexes that sediment in fractions at the same high density (1.82-1.88 g/cm3) from which they were originally cloned. The high-density banding of these cytoplasmic DNA sequences may be due to association with RNA and/or with (metallo-) proteins in vivo. Since both cloned DNA sequences with immortalizing activity have stop codons for protein translation in all possible reading frames, immortalization may be induced by insertional inactivation or functional suppression of genes that are needed to be expressed during cellular senescence or programmed cell death.
小鼠L929细胞和艾氏腹水瘤细胞的胞质体含有DNA序列，这些序列在体外转染后可诱导人淋巴细胞无限增殖（“永生化”）。通过在中性CsCl梯度中对细胞质裂解物进行平衡离心，永生化活性与线粒体外部组分一起在高盐密度（1.85 - 1.87 g/cm³）下回收。出乎意料的是，这些组分包含长度为50 - 500 bp的线性DNA分子。相比之下，原代衰老细胞（小鼠胚胎成纤维细胞、人淋巴细胞）的胞质体在相应组分中不含DNA。从小鼠肿瘤细胞的高密度组分中分离出的细胞质DNA被克隆到亚组文库中，在我们鉴定的45个DNA序列中，有2个克隆——一个来自L929胞质体（203 bp），另一个来自艾氏腹水细胞的细胞质（372 bp）——可在体外诱导人淋巴细胞无限增殖。永生化的淋巴细胞含有1 - 5份整合到染色体DNA中的转染DNA拷贝，而在细胞质组分中发现约100份作为游离DNA。随机片段化为200 - 500 bp的核DNA转染不能诱导永生化。尽管克隆的DNA在1.70 g/cm³处沉降，但在瞬时转染到淋巴细胞后，这些DNA序列形成盐稳定复合物，它们在与最初克隆时相同的高密度（1.82 - 1.88 g/cm³）组分中沉降。这些细胞质DNA序列的高密度条带可能是由于在体内与RNA和/或（金属）蛋白质结合。由于具有永生化活性的两个克隆DNA序列在所有可能的阅读框中都有蛋白质翻译的终止密码子，永生化可能是由细胞衰老或程序性细胞死亡期间需要表达的基因的插入失活或功能抑制诱导的。
