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Journal of Electron Microscopy Advance Access published online on September 5, 2005
Journal of Electron Microscopy, doi:10.1093/jmicro/dfi058
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© The Author 2005. Published by Oxford University Press on behalf of Japanese Society of Microscopy. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received February 17, 2005
Accepted August 1, 2005

Full-length paper

Guanine is indispensable for immunoglobulin switch region RNA-DNA hybrid formation

Ryushin Mizuta 1*, Midori Mizuta 1, and Daisuke Kitamura 2

1 Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan
2 Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan; Genome and Drug Research Centre, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan

* To whom correspondence should be addressed.
Ryushin Mizuta, E-mail: mizuta{at}rs.noda.tus.ac.jp


  Abstract

It is suggested that the formation of the switch (S) region RNA-DNA hybrid and the subsequent generation of higher-order chromatin structures including R-loop initiate a class switch recombination of the immunoglobulin gene. The primary factor of this recombination is the S-region derived noncoding RNA. However, the biochemical character of this guanine-rich (G-rich) transcript is poorly understood. The present study was performed to analyze the structure of this G-rich RNA using atomic force microscope (AFM). The in vitro transcribed S-region RNA was spread on a mica plate, air-dried and observed by non-contact mode AFM in air. The G-rich transcripts tend to aggregate on the template DNA and to generate a higher-order RNA-DNA complex. However, the transcripts that incorporated guanine analogues as substitutes for guanine neither aggregated nor generated higher-order structures. Incorporation of guanine analogues in transcribed RNA partially disrupts hydrogen bonds related to guanine, such as Watson-Crick GC-base pair and Hoogsteen bond GG-base pair. Thus, aggregation of S-region RNA and generation of the higher-order RNA-DNA complex are attributed to hydrogen bonds of guanine.

Keywords: immunoglobulin gene; class switch recombination; atomic force microscope; Hoogsteen bond; RNA-DNA hybrid.
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