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Plant and Cell Physiology, 1986, Vol. 27, No. 6 1101-1108
© 1986

Article

Isolation of cDNA for Pea Phytochrome Using an Expression Vector

Ken-ichi Tomizawa1, Yoshibumi Komeda2, Naoki Sato1, Akira Nagatani3, Tetsuo lino1,2 and Masaki Furuya1,3,4

1Department of Biology, Faculty of Science, University of Tokyo Hongo, Tokyo 113, Japan
2Molecular Genetics Research Laboratory, University of Tokyo Hongo, Tokyo 113, Japan
3Division of Biological Regulation, National Institute for Basic Biology 38 Nishigonaka, Myodaijicho, Okazaki, Aichi 444, Japan

4To whom reprint requests should be addressed.

Partially purified phytochrome mRNA was obtained from etiolated pea epicotyls by polyribosome immunoprecipitation or by size fractionation of total poly(A)+RNA, and used for the synthesis of double-stranded complementary DNA (cDNA). cDNA libraries were constructed using an Escherichia coli expression vector, pUC9, and screened for phytochrome cDNA by colony immunological assay. Nine colonies were found to produce a 27 kDa polypeptide that was reactive to both polyclonal and monoclonal antipea phytochrome antibodies. The plasmids from these colonies contained cDNA inserts of 1.2 or 2.0 kbp. Hybridization-arrest translation assay verified that the cDNA clones contained a sequence coding for phytochrome polypeptide. RNA blot hybridization analysis indicated that the cDNA hybridized to a 4.1 kb poly(A)+RNA in dark-grown pea.

(Received March 22, 1986; Accepted June 13, 1986)
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