碩士 / 國立臺灣海洋大學 / 食品科學系 / 92 / DNA mismatches occur because of incorporation of
noncomplementary, Watson-Crick bases opposite each other in the DNA
helix during replication or recombination. Transition mispairs (G-T or
A-C) are repaired by the mismatch repair process more efficiently than
transversion mispairs (G-G, A-A, G-A, C-C, C-T, and T-T).In this study, a
G-T probe was used to detect mismatch binding proteins.
DNA mismatch binding activities in the extracts of the unicellular
alga Chlorella pyrenoidosa were examined by electrophoretic mobility
shift assay(EMSA). A protein concentration-dependent binding study
revealed the presence of mismatch binding activities having low or no
affinity for homoduplex DNA. Addition of 0.3 and 1.0 mM ATP to EMSA
mixtures induced the formation of some high-shifting complexes
reflecting the regulation of ATP on DNA mismatch recognition.
Two polypeptides about 62 and 48 kDa estimated by SDS-PAGE
were found to bind with high specificity to a biotin-labeled G-T probe
immobilized on streptavidin-conjugated agarose beads and a few
62-kDa G-T binding polypeptides possessing pIs ranging from 5.4 to 5.8
were identified by two-dimensional gel electrophoresis after silver
staining. Staining of affinity-captured proteins on a SDS-polyacrylamide
gel by fluorescent SYPRO Ruby dye, however, detected a 13-kDa and a
weak 48-kDa GT binding polypeptide. Two 13-kDa G-T binding
polypeptides were found by fluorescence staining of 2-D gels. Peptide
mass fingerprinting (PMF) of the 13-kDa polypeptide with pI5.3 matched
best to a arabidopsis thaliana Serine/threonine protein phosphatase. The
other polypeptide with pI5.5 matched best to a yeast ATP-dependent
RNA helicase. The exact identities of the two polypeptides need to be
detected.
| Identifer | oai:union.ndltd.org:TW/092NTOU5253004 |
| Date | January 2004 |
| Creators | Kai-Ning Chang, 張凱甯 |
| Contributors | 吳清熊 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 61 |
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