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Molecular characterization of the MX genes of rainbow trout (Oncorhynchus mykiss)Trobridge, Grant David 30 April 1996 (has links)
Graduation date: 1997
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Cloning, heterologus expression, and characterization of the cytochrome P450 monooxygenases in rainbow trout (Oncorhynchus mykiss)Yang, Yea-Huey 12 December 1997 (has links)
Graduation date: 1998
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Rainbow trout cystatin C : gene expression, heterologous production and characterizationLi, Fugen 17 July 1998 (has links)
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
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Molecular cloning and sequence analysis of cystatin from rainbow trout (Oncorhynchus mykiss)Li, Fugen 25 February 1997 (has links)
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
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Cloning, sequencing and aflatoxin B��� metabolism by multiple rainbow trout CYP1A cDNAs expressed in yeastYou, Lijing 20 May 1997 (has links)
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
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Characterization of Pacific whiting proteinase P-II and partial cloning of cathepsins L and K cDNA from rainbow trout liverNickel, Xianbin F. 25 April 1996 (has links)
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
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