Cloning and sequence analysis of the gene encoding the guamerin from hirudo nipponia

Abstract

Guamerin, a cysteine-rich elastase inhibitor of the Korean leech, Hirudo nipponia, is a specific inhibitor of elastase. A genomic DNA clone encoding guamerin was isolated from a λ BlueSTAR phage library constructed with Sau3AI partial digests of the chromosomal DNA of leeches, by screening with an guamerin cDNA as a probe. The gene of guamerin is about 1.2 kb long and composed of four exons and three introns. At the 5`` end of the coding region, there is a sequence that encodes a hydrophobic region of 19 amino acids, which could form a signal peptide. Primer extension analysis showed the transcription start point (tsp) was at A, 95 bp upstream from the start codon (ATG). The putative two CAAT boxes and the TATA box were found in the 5``-upstream region of the tsp. The 3``-flanking region contains three putative polyadenylation signals (AATAAA) 64 bp, 147 bp, and 232 bp downstream from the stop codon. From the pattern of the genomic Southern-blot analysis using a genomic DNA probe, the gene is considered to be at least two copy genes in the genome of the leech.