Biomedical Engineering Reference
In-Depth Information
Schaefer and Lyko
2007
; Foret et al.
2009
; Lyko et al.
2010
; Gabor Miklos and
Maleszka
2011
). Second, extensive physiological, biochemical, and molecular bio-
logical data are available to understand the mechanisms of honeybee development
and complex behaviors, such as caste system and long-term memory (Müller and
Hildebrandt
2002
; Park et al.
2003
; Locatelli et al.
2005
; Okada et al.
2007
;
Watanabe et al.
2006
; Kamakura
2011
). Finally, suffi cient quantity of genomic
DNA can be purifi ed from a single honeybee brain to investigate epigenetic changes,
which advantageously allows behavioral changes to be linked to epigenetic modula-
tion at the level of individual organisms.
Recently, increasing evidence is available for understanding the molecular mech-
anisms of epigenetic control in honeybees. While
Drosophila
only has a single type
of putative DNA methyltransferase (DNMT) (Tweedie et al.
1999
; Marhold et al.
2004
), honeybees possess the entire components of functionally active vertebrate-
like DNMTs (Wang et al.
2006
) and several isoforms of methyl-CpG-binding
domain proteins (MBDs) (Wang et al.
2006
). Additionally, non-CpG methylation,
which is observed in the
Drosophila
genome, is either extremely rare or nonexistent
in the honeybee genome (Wang et al.
2006
). These data suggest that honeybees have
an active DNA methylation system similar to that found in mammals. Methylated
cytosine residues have been found in several genes in honeybees (Wang et al.
2006
;
Ikeda et al.
2011
; Shi et al.
2011
), and DNA methylation was shown to play crucial
roles in development and caste differentiation (Kucharski et al.
2008
; Elango et al.
2009
; Kim et al.
2009
). In this review, we focus on cytosine methylation of genomic
DNA and describe fundamental techniques for investigating epigenetic status.
10.3
Dissection of Honeybee Brains and Purifi cation
of Genomic DNA
Honeybees were caught, briefl y anesthetized on ice, and decapitated. Honeybee
heads were embedded and fi xed on the surface of melted dental wax (melting
point = ~60 °C), and their brains were isolated. The brains were frozen in liquid
nitrogen immediately after dissection and stored at −80 °C until used for genomic
DNA purifi cation.
Honeybee brains were homogenized in liquid nitrogen and incubated at 55 °C for
2 h in 500
g/mL proteinase K solution diluted with DNA extraction buffer contain-
ing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA (pH 8.0), and 1.5 %
SDS. After gentle treatment twice with phenol/chloroform and once with chloro-
form/isoamyl alcohol, genomic DNA was precipitated with ethanol and treated with
10
μ
g/mL RNase A at 37 °C for 1 h. Genomic DNA was treated again with phenol/
chloroform and chloroform/isoamyl alcohol, precipitated with ethanol, air-dried,
dissolved in 10 mM Tris-HCl (pH 8.5), and stored at −80 °C until use. This proce-
dure can be applied for the purifi cation of genomic DNA from any tissue or cell type
irrespective of the animal species.
μ
