Tucker K L, Talbot D, Lee M A, Leonhardt H, Jaenisch R
Whitehead Institute for Biomedical Research, Cambridge, MA, USA.
Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12920-5. doi: 10.1073/pnas.93.23.12920.
Previous attempts to express functional DNA cytosine methyltransferase (EC 2.1.1.37) in cells transfected with the available Dnmt cDNAs have met with little or no success. We show that the published Dnmt sequence encodes an amino terminal-truncated protein that is tolerated only at very low levels when stably expressed in embryonic stem cells. Normal expression levels were, however, obtained with constructs containing a continuation of an ORF with a coding capacity of up to 171 amino acids upstream of the previously defined start site. The protein encoded by these constructs comigrated in SDS/PAGE with the endogenous enzyme and restored methylation activity in transfected cells. This was shown by functional rescue of Dnmt mutant embryonic stem cells that contain highly demethylated genomic DNA and fail to differentiate normally. When transfected with the minigene construct, the genomic DNA became remethylated and the cells regained the capacity to form teratomas that displayed a wide variety of differentiated cell types. Our results define an amino-terminal domain of the mammalian MTase that is crucial for stable expression and function in vivo.
以往尝试在转染了现有DNA胞嘧啶甲基转移酶（EC 2.1.1.37）cDNA的细胞中表达功能性该酶，但收效甚微或毫无成功。我们发现，已发表的Dnmt序列编码的是一种氨基末端截短的蛋白质，当在胚胎干细胞中稳定表达时，其耐受水平极低。然而，使用包含在先前定义的起始位点上游具有多达171个氨基酸编码能力的开放阅读框延续片段的构建体，可获得正常表达水平。这些构建体编码的蛋白质在SDS/PAGE中与内源性酶共迁移，并在转染细胞中恢复了甲基化活性。这通过对Dnmt突变胚胎干细胞的功能拯救得以证明，这些细胞含有高度去甲基化的基因组DNA且无法正常分化。当用小基因构建体转染时，基因组DNA重新甲基化，细胞恢复了形成畸胎瘤的能力，畸胎瘤中呈现出多种分化细胞类型。我们的结果确定了哺乳动物甲基转移酶的一个氨基末端结构域，该结构域对于体内稳定表达和功能至关重要。
