Graduation date: 1997

Graduation date: 1998

Rainbow trout cystatin C cDNA has been isolated from trout liver. The full-length cystatin cDNA (674 bp) included the 5'untranslated region and the polyadenylation signal sequence AATAAA in the 3' region. Translation of the cDNA defines 132 amino acid residues. Comparison of the amino acid sequence with those of family 2 cystatins indicates that the 21 amino acids at the N-terminal end is a signal peptide necessary for cystatin secretion, and the remaining 111 amino acids represent mature cystatin. Four cysteine residues in the cystatin may form two disulfide bonds producing a molecule with the properties of a family 2 cystatin. Trout cystatin C gene expression was analyzed by Northern blot. This gene is expressed at various levels in all tissues examined. This difference may reflect differences in degree of regulation of cysteine proteinase activities. A high level of trout cystatin C expressed in trout hepatic tissue or cell cultures suggested that cystatin C expression might be related to tumorigenesis. Southern blot of trout genomic DNA showed that the copy number of the trout cystatin gene is probably one per haploid genome. Trout cystatin C was expressed in E. coli at a yield of 3-5 mg/L culture, but no inhibitory activity was detected for the untreated recombinant protein. However, after refolding, recombinant cystatin C displayed inhibitory activity against papain. The dissociation constant of recombinant cystatin C against papain is 1.2 x 10������ nM, similar to that of human cystatin C. Trout cystatin C was also expressed in yeast cells, but no inhibitory activity was detected either. No cystatin C was secreted in the yeast expression system using either the trout cystatin C secretion signal, or the yeast invertase secretion signal. The expression levels of trout cystatin C in our expression systems are still low for industrial requirements. Therefore, further investigation will be needed to construct more efficient expression systems and vectors for trout cystatin C heterologous production. / Graduation date: 1999

A partial cystatin cDNA from rainbow trout was generated by reverse transcription polymerase chain reaction with two degenerate primers. The partial cystatin PCR product was 168 bp and used to screen trout liver λgt 11 cDNA library. Four positive clones were isolated and designated as cstl, cst2, cst3 and cst4. Only cst2 contained the full-length cystatin cDNA which was 674 bp and included 5' untranslated region and the polyadenylation signal sequence AATAAA in the 3' region. Translation of the cDNA contains 132 amino acid residues. Comparison of the amino acid sequence with those of family II cystatin indicated that the 21 amino acids at N-terminal end is a signal peptide that leads to cystatin secretion, and the 111 amino acids are mature cystatin. Four cysteine residues in the cystatin may form two disulfide bonds for the secondary structure. Cst2 was subcloned into pGEM-3z for Northern and Southern blot experiments. Northern blot indicated that trout cystatin mRNA is about 750 bp. Cystatin is expressed in all tissues examined but at various levels. This difference may reflect the regulation of cysteine proteinase activities. Southern blot of trout genomic DNA showed that the copy number of the trout cystatin gene is probably one per haploid genome. / Graduation date: 1997

Previous reports indicate the presence of multiple CYP1A sequences in rainbow trout, but their functional differences are unknown. This report describes the cloning and partial characterization of four trout CYP1A cDNAs, which are given the tentative designations CYP1A1v2, v3, v4, and v5. Comparison among these four and three previously reported trout CYP1A sequences reveals that all of the nucleotide and translated amino acid sequences all are closely related (96.9-99.4% cDNA identity; 95.2-99.4% amino acid sequence identity) but none are identical. Six of these sequences encode proteins of 522 amino acids, and one encodes a protein of 536 amino acids. Expression vectors containing the cDNAs for CYP1A1v2, v3, and v4 were transformed into yeast, yielding microsomal hemoprotein CYP contents (63, 156, 96 pmol/mg) comparable to those reported for human CYP1A1 (68-156 pmol/mg) expressed in this system (Eugster et al., 1990, Biochem. Biophys. Res. Commun. 737-744). Kinetic analysis of CYP1A1v2 and v3 proteins indicated similar but not identical Michaelis constants (20��3 vs 13��2 ��M) and molar activities (508��47 vs 218��19 pmol/min/nmol P450) for oxidation of aflatoxin B��� (AFB���) to aflatoxin M a reaction characteristic of human CYP1A2. Trout CYP1A1v2 and v3 exhibited lower activity for production of AFB,-8,9-exo-epoxide, also a human CYP1A2 activity. Kinetic data for ethoxyresorufin 0deethylation, a prototypical mammalian CYP1A1 activity, also revealed modest but distinct differences in which CYP1A1v3 was more active for this substrate (Km=0.07 �� 0.01 ��M, Vm=1398 �� 95 pmol/min/nmol P450) than was CYP1A1v2 (Km=0.15 �� 0.03 ��M, Vm=684 �� 83 pmol/min/nmol p450). Interestingly, CYP1A1v4 showed no catalytic activity towards AFB ethoxyresorufin, or 7,12-dimethylbenzanthracene despite formation of a hemoprotein. These results together with previous studies demonstrate the presence in various rainbow trout populations of at least seven CYPIA cDNAs representing gene duplication or allelic variation. Present results show that one of three such cDNA sequences encodes a CYP1A hemoprotein with no apparent catalytic activity, that two of the encoded proteins possess certain catalytic properties common to both human CYP1A1 and CYP1A2, and that the sequence differences, though small, are reflected in enzymic properties that can be distinguished. / Graduation date: 1998

Proteinase P-II purified from parasitized Pacific whiting muscle was previously identified to be one form of cathepsin L. It appeared to be present in three isozymatic forms on non-denaturing PAGE gel stained for activity. Its autolytic degradation was observed on SDS-PAGE gel under its optimum condition, 55°C and pH 5.5, in the absence of substrate. Amino acid composition analysis revealed that this enzyme had a considerably greater proportion of hydrophobic amino acids than cathepsin L from other fish species, and monosaccharide analysis showed it was not glycosylated. The N-terminal amino acid sequence of the enzyme was 60-65% identical with cathepsin L from chicken and mammalian species, but only 39% identical with mammalian cathepsin B. The moderate identity of the N-terminal amino acid sequence of P-II with other cathepsin L revealed that this cysteine proteinase from Pacific whiting might be encoded by a cathepsin L-related gene. Two degenerate primers were designed to clone cathepsins cDNA from rainbow trout. The 500-bp PCR product from rainbow trout liver cDNA contained at least three different cysteine proteinase sequences, referred to as SFL2, SFL5, and SFL17. SFL5 was the partial cDNA of trout cathepsin L, which was over 80% identical with chicken cathepsin L amino acid sequence. SFL5 was labeled with Dig-11-dUTP and used to screen a trout liver cDNA library. One positive clone referred to as LC was identified and contained a 700-bp insertion overlapping with SFL5. By combining the two overlapping sequences, a 895-bp cDNA sequence was identified, which included 88% of the mature enzyme and a 307-bp 3' end untranslated part. Its deduced amino acid sequences had 83% identity, 91% similarity with chicken cathepsin L and 73% identity, 86% similarity with human cathepsin L. SFL2 might be the partial cDNA of a novel cathepsin L-related cysteine proteinase. SFL17 may be the partial cDNA of trout cathepsin K. It had 70% identity and 89% similarity with rabbit and human cathepsin K at the amino acid level. / Graduation date: 1996