STRUCTURAL STUDIES ON THE PHOTORECEPTOR PHYTOCHROME: REEVALUATION OF THE EPITOPE FOR MONOCLONAL ANTIBODY Z-3B1

The photoreceptor phytochrome is widely distributed in the plant kingdom from angiosperms to ferns, mosses and algae. The epitope for the monoclonal antibody Z-3B1 which exhibits wide-ranging cross-reactivity with phytochromes from higher and lower plants was mapped by the combination of several methods: by Western blot with proteolytic fragments of known localization, by sequence comparison of phytochromes from various plants, and by production of overlapping fusion proteins. The only sequence which is common to all positively-reacting fusion proteins is the sequence A-830 to R-859. This sequence must contain the Z-3B1 epitope. The best candidate is suggested to be the T-cell antigenic sequence K-Y-V/I-E-A/C-L-L-T (= K-848 to T-855). The significance of the highly conserved epitope in all phytochromes is discussed.     

EXPRESSION OF OAT phyA cDNA IN THE MOSS Ceratodon purpureus

The possibility of transforming Ceratodon purpureus protoplasts by PEG-mediated direct DNA uptake was tested. Transformation with a plasmid carrying a kanamycin-resistance gene resulted in kanamycin-resistant colonies of C. purpureus protonemata. A full-length cDNA clone coding for oat phyA phytochrome was isolated. The clone HM4.1 which is 3.7-kb long exhibits about 99% nucleotide sequence identity to the known phytochrome clone AP3. The expression of HM4.1 in C. purpureus protonemata was tested. A construct with the 35S-promotor and the structural gene of HM4.1 was cotransformed with the plasmid containing the kanamycin-resistance. Kanamycin-resistant colonies were tested for the presence of HM4.1 sequences in a genomic Southern experiment. Two out of 19 kanamycin-resistant colonies reacted positively with a HM4.1 specific probe. The expression of phyA in the positive colonies was examined with monoclonal antibodies specific for oat phytochrome. The Western blot experiment with protein extracts of the two positive colonies grown in the dark revealed clear signals at 124-kDa which were not detected in control plants. These data demonstrate the possibility of expressing oat phyA-apoprotein in C. purpureus protonemata. The transgenic moss protonemata did not show phenotypical alterations in response to the foreign phytochrome polypeptide; it is not known at the moment if the tetrapyrole chromophore is attached to the oat polypeptide in the protonemata or not.