Kawame H, Gartler S M, Hansen R S
Department of Medicine, University of Washington, Seattle 98195, USA.
Hum Mol Genet. 1995 Dec;4(12):2287-93. doi: 10.1093/hmg/4.12.2287.
Using a bromodeoxyuridine incorporation method to detect replicated DNA, we studied allele-specific replication of several sites within the human Prader-Willi/Angelman and IGF2/H19 imprinted regions. No obvious allele-specific differences in time of replication were detected at most loci previously reported to replicate asynchronously in the same cell types as determined by a FISH-based replication assay. Our finding of an absence of allelic replication asynchrony may be related to low levels of imprinted gene expression near these loci in the examined cells (lymphocytes, fibroblasts and lymphoblastoid cells). This view is supported by our studies of the imprinted SNRPN gene in that cells with paternal allele-specific expression (lymphocytes and lymphoblasts) replicate SNRPN alleles asynchronously, whereas cells with a low level of expression (HeLa) replicate SNRPN later and with less allelic asynchrony. In lymphoblasts, the early replicating allele of SNRPN was identified as the paternal one based on the properties of maternal allele-specific methylation and paternal allele-specific expression. Our studies suggest that FISH data implying replication asynchrony in nonexpressing cells reflect structural differences between the maternal and paternal alleles rather than differences in replication timing.
我们使用溴脱氧尿苷掺入法检测复制的DNA，研究了人类普拉德-威利/安吉尔曼综合征和IGF2/H19印记区域内多个位点的等位基因特异性复制。通过基于荧光原位杂交的复制分析确定，在先前报道在相同细胞类型中异步复制的大多数位点，未检测到明显的等位基因特异性复制时间差异。我们发现不存在等位基因复制异步现象，这可能与所检测细胞（淋巴细胞、成纤维细胞和淋巴母细胞）中这些位点附近印记基因的低表达水平有关。我们对印记的SNRPN基因的研究支持了这一观点，即具有父本等位基因特异性表达的细胞（淋巴细胞和淋巴母细胞）异步复制SNRPN等位基因，而表达水平低的细胞（HeLa细胞）较晚复制SNRPN且等位基因异步性较小。在淋巴母细胞中，基于母本等位基因特异性甲基化和父本等位基因特异性表达的特性，SNRPN的早期复制等位基因被确定为父本等位基因。我们的研究表明，在非表达细胞中暗示复制异步的荧光原位杂交数据反映了母本和父本等位基因之间的结构差异，而不是复制时间的差异。
