Isolation and analysis of sequences showing sex-specific cytosine methylation in the mealybug Planococcus lilacinus

Original Paper


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


  1. Achwal CW, Ganguly P, Chandra HS (1984) Estimation of the amount of 5-methylcytosine in Drosophila melanogaster by photoacoustic spectroscopy. EMBO J 3:263–266PubMedGoogle Scholar
  2. Andersen CL, Koch J, Kjeldsen E (1998) CpG islands detected by self-primed in situ labeling (SPRINS). Chromosoma 107:260–266CrossRefPubMedGoogle Scholar
  3. Basler J, Hastie ND, Pietras D, Matsui SS, Berg AA, Berezney R (1981) Hybridization of nuclear matrix-attached deoxyribonucleic acid fragments. Biochemistry 20:6921–6929CrossRefPubMedGoogle Scholar
  4. Bongiorni S, Cintio O, Prantera G (1999) The relationship between DNA methylation and chromosome imprinting in the coccid Planococcus citri. Genetics 151:1471–1478PubMedGoogle Scholar
  5. Brown SW (1959) Lecanoid chromosome behavior in three more families of the Coccoidea (Homoptera). Chromosoma 10:360–406CrossRefGoogle Scholar
  6. Brown SW, Chandra HS (1973) Inactivation system of the mammalian X chromosome. Proc Natl Acad Sci USA 70:195–199PubMedCrossRefGoogle Scholar
  7. Brown SW, Chandra HS (1977) Chromosome imprinting and the differential regulation of homologous chromosomes. In: Goldstein L, Prescott DM (eds) Cell biology: a comprehensive treatise (vol 1). Academic, New York, pp 109–189Google Scholar
  8. Brown SW, Nelson-Rees WA (1961) Radiation analysis of a Lecanoid genetic system. Genetics 46:983–1007PubMedGoogle Scholar
  9. Buglia G, Preddazi V, Ferraro M (1999) Cytosine methylation is not involved in the heterochromatization of the paternal genome of mealybug Planococcus citri. Chromosome Res 7:71–73CrossRefPubMedGoogle Scholar
  10. Burgos M, Jimenez R, Sanchez A, Diaz de la Guardia R (1992) Restriction enzyme banding and in situ nick translation of different types of hetero- and euchromatin. Exp Cell Res 202:545–548CrossRefPubMedGoogle Scholar
  11. Chandra HS (1963a) Cytogenetic analysis following high dosage paternal irradiation in the mealybug Planococcus citri. I. Cytology of X1 embryos. Chromosoma 14:310–329CrossRefGoogle Scholar
  12. Chandra HS (1963b) Cytogenetic analysis following high dosage paternal irradiation in the mealybug Planococcus citri. II. Cytology of X1 females and the problem of Lecanoid sex determination. Chromosoma 14:330–346CrossRefGoogle Scholar
  13. Chandra HS (1971) Inactivation of whole chromosomes in mammals and coccids: some comparisons. Genet Res 18:265–276PubMedCrossRefGoogle Scholar
  14. Chandra HS, Brown SW (1975) Chromosome imprinting and the mammalian X chromosome. Nature 253:165–168CrossRefPubMedGoogle Scholar
  15. Cheung VG, Nelson SF (1996) Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA. Proc Natl Acad Sci USA 93:14676–14679CrossRefPubMedGoogle Scholar
  16. Chomczynski P (1992) One hour downward alkaline transfer for blotting of DNA and RNA. Anal Biochem 201:134–139CrossRefPubMedGoogle Scholar
  17. De la Torre J, Sumner AT, Gosalvez J, Stuppia L (1992) The distribution of genes on human chromosomes as studied by in situ nick translation. Genome 35:890–894PubMedGoogle Scholar
  18. Deobagkar DN, Muralidharan K, Devare SG, Kalghatgi K, Chandra HS (1982) The mealybug chromosome system I: Unusual methylated bases and dinucleotides in DNA of Planococcus species. J Biosci 4:513–526CrossRefGoogle Scholar
  19. Deobagkar DN, Shankar V, Deobagkar DD (1986) Separation of 5-methylcytosine-rich DNA using immobilized antibody. Enzyme Microb Technol 8:97–100CrossRefGoogle Scholar
  20. Ehrlich M, Wang RY-H (1981) 5-Methylcytosine in eukaryotic DNA. Science 212:1350–1357PubMedCrossRefGoogle Scholar
  21. Frommer M, McDonald L, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA 89:1827–1831PubMedCrossRefGoogle Scholar
  22. Jones PA, Takai D (2001) The role of DNA methylation in mammalian epigenetics. Science 293:1068–1070CrossRefPubMedGoogle Scholar
  23. Kantheti P (1994) Studies on a female-specific cDNA clone and chromatin organization, in a mealybug, Planococcus lilacinus. PhD Thesis, Indian Institute of Science, BangaloreGoogle Scholar
  24. Kantheti P, Jayarama KS, Chandra HS (1996) Developmental analysis of a female-specific 16S rRNA gene from mycetome-associated endosymbionts of a mealybug, Planococcus lilacinus. Insect Biochem Mol Biol 26:997–1009CrossRefPubMedGoogle Scholar
  25. Khosla SP, Augustus M, Brahmachari V (1999) Sex-specific organisation of middle repetitive DNA sequences in the mealybug Planococcus lilacinus. Nucleic Acids Res 27:3745–3751CrossRefPubMedGoogle Scholar
  26. Loebel DA, Johnston PG (1993) Analysis of DNaseI sensitivity and methylation of active and inactive X chromosomes of kangaroos (Macropus robustus) by in situ nick translation. Chromosoma 102:81–87CrossRefPubMedGoogle Scholar
  27. Lyko F, Ramsahoye BH, Jaenisch R (2000) DNA methylation in Drosophila melanogaster. Nature 408:538–540CrossRefPubMedGoogle Scholar
  28. Mohan KN, Ray P, Chandra HS (2002) Characterization of the genome of the mealybug Planococcus lilacinus, a model organism for studying whole-chromosome imprinting and inactivation. Genet Res 79:111–118CrossRefPubMedGoogle Scholar
  29. Mohandas T, Sparkes RS, Shapiro LJ (1981) Reactivation of an inactive human X chromosome: evidence for X inactivation by DNA methylation. Science 311:393–396CrossRefGoogle Scholar
  30. Muralidharan K (1984) A study of DNA methylation in the mealybug Planococcus lilacinus. PhD Thesis, Indian Institute of Science, BangaloreGoogle Scholar
  31. Newmann B, Barlow DP (1996) Multiple roles for DNA methylation in gametic imprinting. Curr Opin Genet Dev 6:159–163CrossRefPubMedGoogle Scholar
  32. Nur U (1963) Meiotic parthenogenesis and heterochromatization in a soft scale Pulvinaria hydrangeae (Coccoidea: Homoptera). Chromosoma 14:123–139CrossRefGoogle Scholar
  33. Pfeifer GP, Riggs AD (1991) Chromatin differences between active and inactive X chromosomes revealed by genomic foot-printing of permeabilized cells using DNaseI and ligation-mediated PCR. Genes Dev 5:1102–1113PubMedCrossRefGoogle Scholar
  34. Pfeifer GP, Tanguay RL, Steigaerwald SD, Riggs AD (1990) In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. Genes Dev 4:1277–1287PubMedCrossRefGoogle Scholar
  35. Prantera G, Ferraro M (1990) Analysis of methylation and distribution of CpG sequences on human active and inactive X chromosomes by in situ nick translation. Chromosoma 99:18–23CrossRefPubMedGoogle Scholar
  36. Ramsahoye BH, Biniszkiewicz D, Lyko F, Clark V, Bird AP, Jaenisch R (2000) Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc Natl Acad Sci USA 97:5237–5242CrossRefPubMedGoogle Scholar
  37. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  38. Scarbrough K, Hattman S, Nur U (1984) Relationship of DNA methylation level to the presence of heterochromatin in mealybugs. Mol Cell Biol 4:599–603PubMedGoogle Scholar
  39. Singer-Sam J, Grant M, Lebon JM, Okuyama K, Chapman V, Monk M, Riggs AD (1990) Use of HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-inactivation. Mol Cell Biol 10:4987–4989PubMedGoogle Scholar
  40. Tweedie S, Charlton J, Clark V, Bird A (1997) Methylation of genomes and genes at the invertebrate-vertebrate boundary. Mol Cell Biol 17:1469–1475PubMedGoogle Scholar
  41. Viegas-Pequignot E, Dutrillaux B, Thomas G (1988) Inactive X chromosome has the highest concentration of unmethylated HhaI sites. Proc Natl Acad Sci USA 85:7657–7660PubMedCrossRefGoogle Scholar
  42. Wu G, Su S, Bird SC (1994) Optimization of subtractive hybridization in construction of subtractive cDNA libraries. Genet Anal Tech Appl 11:29–33PubMedGoogle Scholar
  43. Xiong Z, Laird PW (1997) COBRA: a sensitive and quantitative methylation assay. Nucleic Acids Res 15:2532–2534CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  1. 1.Centre for Human Genetics, G 04International Technology ParkBangaloreIndia
  2. 2.Department of Microbiology and Cell BiologyIndian Institute of ScienceBangaloreIndia

Personalised recommendations