Mohan K Naga, Chandra H Sharat
Centre for Human Genetics, G 04, International Technology Park, Whitefield Road, Bangalore 560066, India.
Mol Genet Genomics. 2005 Dec;274(6):557-68. doi: 10.1007/s00438-005-0004-9. Epub 2005 Nov 9.
Genomic libraries of Planococcus lilacinus, a mealybug in which paternal chromosomes are facultatively heterochromatic and inactive in sons but not in daughters, were probed with subtraction probes in order to estimate the number of sequences displaying sex-specific cytosine methylation in CpG dinucleotides. Sequences showing male-specific methylation were found to occur approximately 2.5 times more often than those showing female-specific methylation. In order to directly isolate sequences showing sex-specific CpG methylation, we employed methylation-specific arbitrarily primed (MS-AP) polymerase chain reaction (PCR) and identified 72 sex-specific products, of which 51 were from males and 21 from females. Amplification of bisulfite-modified DNA and subsequent Southern hybridization showed that in 33 out of these 72 sex-specific products, there was differential methylation of homologous sequences; i.e., both methylated and unmethylated copies of the same sequence occurred in one sex whereas only unmethylated copies were present in the opposite sex. Sequencing of bisulfite-modified DNA showed an interspersion of CpG and non-CpG methylation among the sex-specifically methylated sequences. Sequences showing male-specific CpG methylation are organized as transcriptionally silent chromatin in males but not in females, whereas those showing female-specific CpG methylation are organized as transcriptionally silent chromatin in females but not in males. The sequences identified in this study that show differential methylation in males, but are unmethylated in females, may prove useful in the study of imprinting in the mealybug system.
淡紫平粉蚧是一种粉蚧,其雄性染色体在子代中为兼性异染色质且无活性(在雄性子代中无活性,在雌性子代中有活性)。为了估计在CpG二核苷酸中显示性别特异性胞嘧啶甲基化的序列数量,我们用消减探针探测了淡紫平粉蚧的基因组文库。结果发现,显示雄性特异性甲基化的序列出现的频率大约是显示雌性特异性甲基化序列的2.5倍。为了直接分离显示性别特异性CpG甲基化的序列,我们采用了甲基化特异性任意引物(MS-AP)聚合酶链反应(PCR),并鉴定出72个性别特异性产物,其中51个来自雄性,21个来自雌性。对亚硫酸氢盐修饰的DNA进行扩增并随后进行Southern杂交分析表明,在这72个性别特异性产物中的33个中,同源序列存在差异甲基化;也就是说,同一序列的甲基化和未甲基化拷贝在一个性别中都存在,而在相反性别中只存在未甲基化拷贝。对亚硫酸氢盐修饰的DNA进行测序表明,在性别特异性甲基化序列中,CpG甲基化和非CpG甲基化相互交错。显示雄性特异性CpG甲基化的序列在雄性中被组织成转录沉默染色质,而在雌性中则不是;而显示雌性特异性CpG甲基化的序列在雌性中被组织成转录沉默染色质,而在雄性中则不是。本研究中鉴定出的在雄性中显示差异甲基化但在雌性中未甲基化的序列,可能在粉蚧系统的印记研究中证明是有用的。