Characterization of Genes for a Pollen Allergen, Cry j 2, of <i>Cryptomeria japonica</i>

Abstract

Background: Cry j 2 is one of the major pollen allergens of Cryptomeria japonica. The polymorphism of Cry j 2 isoforms and the conservation of the structure of Cry j 2 in coniferous species remain to be analyzed. Methods:A cDNA library derived from the pollen of C. japonica was screened using a fragment of Cry j 2 cDNA. Restriction fragment length polymorphism analysis was performed to examine the diversity of Cry j 2 genes. The promoters of Cry j 2 genes were isolated with a commercially available cloning kit. Clonal variations in the expression of Cry j 2 in pollen were examined by RNA gel blot analysis, and the conservation of the structure of the Cry j 2 gene in coniferous species was evaluated by DNA gel blot analysis. Results: We isolated three cDNA clones encoding novel isoforms of Cry j 2. We also sequenced a total of 16 promoter regions from 10 specimens. The sequences of promoter regions of Cry j 2 genes were highly divergent. The amount of Cry j 2 mRNA also varied considerably. The Cry j 2 gene was found to be conserved among species belonging to Taxodiaceae and Cupressaceae but to vary between Taxodiaceae and Pinaceae. Conclusions: The coding and promoter regions of Cry j 2 genes contain large numbers of polymorphisms. Our analysis revealed large variations in the expression of Cry j 2 at the transcriptional level, and we suggest that conserved homologs of Cry j 2 confer cross-allergenicity among Taxodiaceae and Cupressaceae.

Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili

“…To confirm the addition of G at the 5' end of full-length cDNA clones, we compared the 5'-terminal sequences of cDNA clones that encoded Cry j 2 with genomic sequences, including the promoter and coding region of Cry j 2 , that we determined in a previous study [ 22 ]. We found seven clones that encoded Cry j 2 among the 5'-end sequences of 18,843 clones.…”

“…We found seven clones that encoded Cry j 2 among the 5'-end sequences of 18,843 clones. All seven of them were 5'-extended cDNAs, as compared with three Cry j 2 cDNAs that we generated previously [ 22 ]. Five of the seven had one G, one clone had two Gs and one clone had TG at its 5' terminus; these nucleotides were not present in the corresponding region of Cry j 2 genes (data not shown).…”