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
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/27207 |
| Date | 25 February 1997 |
| Creators | Li, Fugen |
| Contributors | An, Haejung |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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