ISOLATION AND CHARACTERIZATION OF A CDNA FROM FLOWERS OF CYNARA-CARDUNCULUS ENCODING CYPROSIN (AN ASPARTIC PROTEINASE) AND ITS USE TO STUDYTHE ORGAN-SPECIFIC EXPRESSION OF CYPROSIN

Poly(A)(+) RNA isolated from flower buds of Cynava cardunculus has bee n used to prepare a cDNA library. Screening of the cDNA after expressi on of cloned DNA with antibodies raised against the large subunit of c yprosin 3 resulted in the isolation of six positive clones. One of the se clones (cyprols; a 1.7 kb Eco RI fragment) codes for cyprosin. The nucleotide sequence contain a 1419 bp open reading frame coding for 47 3 amino acids (aa) including a putative full-length mature protein (44 0 aa) and a partial prosequence (33 aa). Cyprols contains a 162 bp 3' non-coding region followed by a poly(A) tail. The deduced amino acid s equence shows high homology to other plant aspartic proteinases. The h omology to mammalian and microbial aspartic proteinases is somewhat lo wer. Plant aspartic proteinases contain an insert of around 100 aa. We are modelling where this plant-specific insert will appear in the str ucture of cyprosin. Using cyprols as a probe in northern blot analysis , the expression of cyprosin in developing flowers and other tissues h as been studied. The signal on the northern blot increased for RNA sam ples from early (flower buds 6 mm in length) to later stages of floral development (flower buds up to 40 mm in length). In late stages of fl oral development (open flowers 50 mm in length and styles from such fl owers) no hybridization signal was visualized showing that the synthes is of mRNA encoding the cyprosin starts in early stages of floral deve lopment and switches off at maturation of the flower. Southern blot an alysis of genomic DNA showed 4-5 strong hybridizing bands and several minor bands indicating that the cyprosin genes are organized as a mult i-gene family in C. cardunculus.