Shuster M, Dhar M S, Olins A L, Olins D E, Howell C Y, Gollin S M, Chaillet J R
Department of Human Genetics, University of Pittsburgh, Graduate School of Public Health, Pennsylvania, USA.
Dev Genet. 1998;23(4):275-84. doi: 10.1002/(SICI)1520-6408(1998)23:4<275::AID-DVG3>3.0.CO;2-#.
Molecular features of imprinted genes include differences in expression, methylation, and the timing of DNA replication between parental alleles. Whereas methylation differences always seem to be associated with differences in expression, differences in the timing of replication between parental homologs are not always seen at imprinted loci. These observations raise the possibility that differences in replication timing may not be an essential feature underlying genomic imprinting. In this study, we examined the timing of replication of the two alleles of the imprinted RSVIgmyc transgene in individual embryonic cells using fluorescence in situ hybridization (FISH). The cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. Thus, allele-specific differences in replication, if they indeed govern RSVIgmyc imprinting, should be found in RSVIgmyc sequences. We found that the parental alleles of RSVIgmyc, which exhibit differences in methylation, replicated at the same time. Synchronous replication was also seen in embryonic cells containing a modified version of RSVIgmyc that exhibited parental allele differences in both methylation and expression. These findings indicate that maintenance of expression and methylation differences between alleles does not require a difference in replication timing. The differences in replication timing of endogenous imprinted alleles detected by FISH might therefore reflect structural differences between the two alleles that could be a consequence of imprinting or, alternatively, could be unrelated to imprinting.
印记基因的分子特征包括亲本等位基因在表达、甲基化以及DNA复制时间上的差异。虽然甲基化差异似乎总是与表达差异相关，但亲本同源染色体之间复制时间的差异在印记位点并不总是能观察到。这些观察结果提出了一种可能性，即复制时间的差异可能不是基因组印记的一个基本特征。在本研究中，我们使用荧光原位杂交（FISH）在单个胚胎细胞中检测了印记RSVIgmyc转基因两个等位基因的复制时间。RSVIgmyc印记的顺式作用信号在RSVIgmyc本身内部。因此，如果复制的等位基因特异性差异确实控制着RSVIgmyc印记，那么在RSVIgmyc序列中应该能发现这种差异。我们发现，甲基化存在差异的RSVIgmyc亲本等位基因同时进行复制。在含有RSVIgmyc修饰版本的胚胎细胞中也观察到同步复制，该修饰版本在甲基化和表达方面都表现出亲本等位基因差异。这些发现表明，等位基因之间表达和甲基化差异的维持并不需要复制时间的差异。因此，通过FISH检测到的内源性印记等位基因复制时间的差异可能反映了两个等位基因之间的结构差异，这可能是印记的结果，或者也可能与印记无关。
