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首页> 外文期刊>Journal of Cellular Physiology >Differential gene expression in SV40-mediated immortalization of human fibroblasts.
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Differential gene expression in SV40-mediated immortalization of human fibroblasts.

机译：在SV40-mediated差异基因表达不灭的人类成纤维细胞。

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摘要

Normal human diploid fibroblasts (HF) have a limited life span, undergo senescence, and rarely, if ever, spontaneously immortalize in culture. Introduction of the gene for T antigen encoded by the DNA virus SV40 extends the life span of HF and increases the frequency of immortalization; however, immortalization requires both T-dependent and T-independent functions. We previously generated independent SV40-transformed non-immortal (pre-immortal) HF cell lines from which we then obtained immortal sublines as part of a multifaceted approach to identify functions responsible for immortalization. In this study we undertook a search for cellular mRNAs which are differentially expressed upon immortalization. A lambda cDNA library was prepared from a pre-immortal SV40-transformed HF (HF-C). We screened the library with a subtracted probe enriched for sequences present in HF-C and reduced in immortal AR5 cells. A more limited screen was also employed for sequences overexpressed in AR5 using a different strategy. Alterations in the level of mRNAs in AR5 encoding functions relevant to signal transduction pathways were identified; however, most cDNAs encoded novel sequences. In an effort to clarify which of the altered mRNAs are most relevant to immortalization, we performed Northern analysis with RNA prepared from three paired sets of independent pre-immortal and immortal (4 cell lines) SV40-transformants using eight cloned cDNAs which show reduced expression in AR5. Three of these were reduced in additional immortal cell lines as well; one, J4-4 (unknown function) is reduced in all the immortal cell lines tested; a second, J4-3 (possible PP2C type phosphatase) is reduced in 2 of the 3 matched sets; and a third, J2-2 (unknown function) is reduced in 2 unrelated immortal cell lines. Although the roles of these genes are as yet unclear, their further analysis should extend our understanding of the molecular bases for immortalization. In particular, the patterns of expression of J4-4 and J4-3 strongly suggest that they are involved in the process of immortalization and/or can serve as target genes for assessing regulators of gene expression in this process.
机译：正常的人类二倍体成纤维细胞(HF)有限寿命、经历衰老和很少,如果有的话,自发地使不灭文化。编码的DNA病毒SV40扩展了的生活高频的频率和增加不灭;需要T-dependent和T-independent功能。细胞系,然后我们获得不朽不同等级的多方面的方法的一部分负责识别功能不灭。寻找细胞信使rna的差异表达不灭。准备从一个λcDNA图书馆pre-immortal SV40-transformed高频(HF-C)。减去探针筛选库对序列存在于HF-C和丰富在不朽内存中减少AR5细胞。屏幕也用于序列在AR5使用不同的策略。改变的mrna水平AR5编码与信号转导相关的功能路径识别;小说编码序列。这改变了mrna最相关不灭,我们北部进行分析与RNA从三个配对组做好准备独立pre-immortal和不朽(4细胞行)SV40-transformants使用八个克隆互补脱氧核糖核酸在AR5显示简化表达式。这些都是减少额外的不朽的细胞行;减少所有的永生细胞系测试;第二,J4-3(可能PP2C类磷酸酶)减少2 3匹配集;J2-2(未知函数)是减少2无关不朽的细胞系。基因尚不明晰,他们进一步分析应该扩大我们的理解的分子基地不灭。J4-4和J4-3强烈的表达模式表明它们参与的过程不灭和/或可以作为目标基因评估监管机构的基因表达这一过程。

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