Isolation and analysis of sequences showing sex-specific cytosine methylation in the mealybug Planococcus lilacinus
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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 ~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.
We thank Mustafa Saifi and Prameela Kantheti for discussion, D. N. Rao for a critical reading of the manuscript, Yogaraje Gowda for assistance in the laboratory and K. Kuppuswamy for mealybug stock maintenance. Some of the DNA sequencing was done at the DBT-funded sequencing facility at the Indian Institute of Science. This work was supported by CSIR and a programme support grant from DBT, New Delhi. The Jawaharlal Nehru Centre for Advanced Scientific Research provided support in the early stages of this work
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