Biomedical Engineering Reference
In-Depth Information
genomic DNA and agarose/sepharose beads. Coprecipitated genomic DNA is
resolved from histones by incubation in a high salt buffer and purifi ed by phenol/
chloroform treatment and ethanol precipitation.
Depending on the question addressed, the precipitated genomic DNA can be
further analyzed by quantitative PCR, real-time PCR, next-generation sequencing,
microarray, or other techniques. In a related microarray-based method, ChIP-on-
chip, the precipitated DNA is labeled and hybridized to a variety of high-resolution
microarrays.
10.10
Conclusion
All methods mentioned in this chapter have been modifi ed from classical molecular
biological and biochemical experiments and can be applied to any species of any
insects. Honeybees, jewel wasps (
Nasonia vitripennis
),
Drosophila
, ants
(
Pogonomyrmex barbatus
,
Linepithema humile
, and
Solenopsis invicta
), mosquitos
(
Aedes aegypti
and
Anopheles gambiae
), pea aphids (
Acyrthosiphon pisum
), and red
fl our beetles (
Tribolium castaneum
) are insect species whose entire genome has
been sequenced. Of these insect species, honeybees and ants have sophisticated and
complex social behaviors. However, the epigenetic mechanisms involved in these
behaviors remain unclear. The contribution of DNA methylation in the regulation of
gene expression in insect taxa has yet to be elucidated, and detailed investigations
combining standard and state-of-the-art methods, such as “next-generation”
sequencing, are needed.
Recently, a species of sea slug
Aplysia
was shown to be useful for analyzing
DNA methylation status at the single cell level (Moroz
2011
; Moroz et al.
2011
).
The large diameter of molluscan neurons allows the copy number of specifi c
mRNAs to be specifi cally measured by real-time PCR and pyrosequencing
(Sadamoto et al.
2004
; Hatakeyama et al.
2006
; Moroz and Kohn
2010
).
Unfortunately, honeybees and other insects do not have such large neurons in their
central nervous systems. Although molluscs do not display social behaviors like
insects, the analysis of DNA methylation status in large single cells may spur the
development of methods of investigating DNA methylation with ultrasmall amounts
of genomic DNA.
References
Alexandrov A, Chernyakov I, Gu W, Hiley SL, Hughes TR, Grayhack EJ, Phizicky EM (2006)
Rapid tRNA decay can result from lack of nonessential modifi cations. Mol Cell 21:87-96
Araujo FD, Knox JD, Ramchandani S, Pelletier R, Bigey P, Price G, Szyf M, Zannis-Hadjopoulos
M (1999) Identifi cation of initiation sites for DNA replication in the human dnmt1 (DNA-
methyltransferase) locus. J Biol Chem 274:9335-9341
