Cloning and sequence analysis of the genomic DNA fragment encoding oryzacystatin

A genomic DNA clone encoding oryzacystatin (Oc), a cysteine proteinase inhibitor (cystatin) of rice, was isolated from a lambda EMBL3 phage library constructed with Sau3AI partial digests of rice chromosomal DNA, by screening with an oc cDNA as a probe. The restriction map of the isolated DNA fragment was consistent with the pattern of the genomic Southern-blot analysis using a cDNA probe, and consequently, the gene is considered to be a single-copy gene. The oc gene is about 1.4 kb long and composed of three exons and two introns. The first intron (336 bp) intervenes between Ala-38 and Asp-39. The second intron (372 bp) exists in the 3'-noncoding region at the G residue next to the stop codon. S1 nuclease mapping showed the major transcription start point (tsp) at A, 104 bp upstream from the start codon (ATG). Typical CAT and TATA box sequences were found in the 5'-upstream region of the tsp. The nucleotide sequences around the TATA box, the tsp, the start codon, and the stop codon essentially matched the consensus sequences of other higher plant genes. The intron boundaries of the oc gene were quite different from those of the human kininogen-encoding gene and the human salivary cystatin (cystatin S)-encoding gene.