Cosmid library of the marine macroalga Bryopsis maxima (Bryopsidales, Ulvophyceae)

Authors: Inokuchi R.; Motojima K.; Okada M.

Source: Phycological Research, Volume 50, Number 3, September 2002 , pp. 209-215(7)

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Abstract:

Summary

The purpose of this study was to construct a cosmid library from chromosomal DNA of a marine macroalga, Bryopsis maxima Okamura ex Segawa (Bryopsidales, Ulvophyceae), in a rapid, simple and inexpensive manner. In the DNA purification, polysaccharides were removed by covalently binding them to resin particles containing free boric acid groups. The DNA yield was 20 µg g^-1 of B. maxima fresh weight. This DNA was 100-200 kb in length, and its A₂₆₀/A₂₈₀ and A₂₃₀/A₂₆₀ ratios were 1.8 and 0.4, respectively. It was of sufficient quality for molecular research. The cloning procedures were carried out in the following steps: controlled partial shearing of purified DNA through a microsyringe, optimal size separation of the DNA by biased sinusoidal field gel electrophoresis, ligation of the DNA to the cosmid vector in the gel, and in vitro packaging into the lambda phage. The library consisted of 2.0 × 10³ independent clones with an average insert size of 40 kb. The fragment amplified by polymerase chain reaction in the library was hybridized with a DNA fragment (328 bp) encoding B. maxima glutamate dehydrogenase under high-stringency conditions by Southern blot analysis, thus demonstrating that the library contained B. maxima chromosomal DNA. This cosmid library is the first to be constructed for any species of marine macroalgae.

Keywords: biased sinusoidal field gel electrophoresis; Bryopsis maxima; cosmid library; DNA purification; green alga

Document Type: Research article

DOI: http://dx.doi.org/10.1046/j.1440-1835.2002.00275.x

Affiliations: 1: Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi, Chiba 274-8510 and

Publication date: 2002-09-01