GENERATION OF CYSTATIN CDNA PROBE OF RAINBOW-TROUT (ONCORHYNCHUS-MYKISS) USING MIXED PRIMER PCR WITH HIGHLY DEGENERATE CODONS CORRESPONDINGTO CONSERVED AMINO-ACID-SEQUENCES

Cystatins are a superfamily of cysteine proteinase inhibitors found in both plants and animals. We were interested in the molecular cloning of rainbow trout (Oncorhynchus mykiss) cystatin, but were unable to id entify cystatin-related sequences using a mammalian cystatin probe. In a second approach, highly degenerate oligonucleotide primers for poly merase chain reaction were designed from corresponding conserved amino acid sequences of cystatin family 2. First-strand cDNA was made from total RNA of rainbow trout liver, A prominent band of the predicted si ze was observed on agarose gel following electrophoresis of products g enerated by reverse transcription polymerase chain reaction, The amino acid sequence deduced from the corresponding cDNA sequence of this PC R product is 98% identical to the corresponding cystatin amino acid se quence of chum salmon (Oncorhynchus keta).