Chumakov I, Stuhlmann H, Harbers K, Jaenisch R
J Virol. 1982 Jun;42(3):1088-98. doi: 10.1128/JVI.42.3.1088-1098.1982.
The Mov-7 and Mov-9 substrains of mice, carrying Moloney murine leukemia virus (M-MuLV) in their germ line at the Mov-7 locus and Mov-9 locus, respectively, are different with respect to virus activation. Infectious virus appears in all mice carrying the Mov-9 locus but is not activated in animals carrying the Mov-7 locus. Consequently, only Mov-9 mice develop viremia and subsequent leukemia. The endogenous M-MuLV provirus with flanking mouse sequences corresponding to the Mov-7 and Mov-9 loci was molecularly cloned. Detailed restriction maps obtained from the cloned DNAs revealed no detectable differences in the proviral genomes. The flanking mouse sequences, however, were different, confirming that the Mov-7 and Mov-9 loci represent different integration sites of M-MuLV. Both clones induced XC plaques in a transfection assay. The specific infectivity of the clones, however, was different. A total of 10(-5) XC plaques per genome equivalent were induced by the Mov-9 clone, whereas only 10(-9) XC plaques per genome equivalent were induced by the Mov-7 clone. Moreover, NIH 3T3 cells transfected with the Mov-9 clone produced NB-tropic M-MuLV, whereas cells transfected with the Mov-7 clone did not produce infectious virus. The results suggest that M-MuLV integrated at the Mov-7 locus carries a mutation which prevents synthesis of infectious virus but permits XC plaque induction by partial genome expression or synthesis of noninfectious particles. Thus, the pattern of virus expression in Mov-7 and Mov-9 mice correlates with the biological properties of the respective clones. Genomic DNA from Mov-9 mice was not infectious in the transfection assay (specific infectivity < 10(-7) PFU per genome equivalent). As the only difference between the genomic and the cloned Mov-9 DNA appears to be the presence of 5-methylcytosine in CpG sequences, our results suggest that removal of methyl groups by molecular cloning in procaryotes permits genome expression in transfected eucaryotic cells. Our results support the hypothesis that DNA methylation is relevant not only in genome expression in the animal but also in expression of genes transfected into eucaryotic cells.
Mov - 7和Mov - 9小鼠亚系分别在其生殖系的Mov - 7位点和Mov - 9位点携带莫洛尼鼠白血病病毒（M - MuLV），它们在病毒激活方面存在差异。感染性病毒出现在所有携带Mov - 9位点的小鼠中，但在携带Mov - 7位点的动物中未被激活。因此，只有Mov - 9小鼠会发生病毒血症及随后的白血病。与Mov - 7和Mov - 9位点相对应的带有侧翼小鼠序列的内源性M - MuLV前病毒被进行了分子克隆。从克隆的DNA获得的详细限制性图谱显示，前病毒基因组中没有可检测到的差异。然而，侧翼小鼠序列是不同的，这证实了Mov - 7和Mov - 9位点代表M - MuLV的不同整合位点。在转染实验中，两个克隆都诱导了XC噬斑。然而，克隆的特异性感染性是不同的。Mov - 9克隆每基因组当量诱导产生10^(-5)个XC噬斑，而Mov - 7克隆每基因组当量仅诱导产生10^(-9)个XC噬斑。此外，用Mov - 9克隆转染的NIH 3T3细胞产生嗜NB的M - MuLV，而用Mov - 7克隆转染的细胞不产生感染性病毒。结果表明，整合在Mov - 7位点的M - MuLV携带一种突变，该突变阻止感染性病毒的合成，但通过部分基因组表达或非感染性颗粒的合成允许诱导XC噬斑。因此，Mov - 7和Mov - 9小鼠中的病毒表达模式与各自克隆的生物学特性相关。在转染实验中，来自Mov - 9小鼠的基因组DNA没有感染性（特异性感染性<每基因组当量10^(-7) PFU）。由于基因组Mov - 9 DNA与克隆的Mov - 9 DNA之间唯一的差异似乎是CpG序列中5 - 甲基胞嘧啶的存在，我们的结果表明，原核生物中通过分子克隆去除甲基基团可使转染的真核细胞中基因组得以表达。我们的结果支持这样一种假说，即DNA甲基化不仅与动物体内的基因组表达相关，而且与转染到真核细胞中的基因表达相关。
