碩士 / 國立臺灣師範大學 / 生物研究所 / 88 / In cyanobacterium Synechococcus RF-1 a 97-kDa protein was identified by 45Ca2+ autoradiography being the most predominant calcium-binding protein. This protein was extractable in both the SDS sample buffer and the Tween-20 buffer. Under the implemented culture condition, the abundance of the 97-kDa protein in Synechococcus RF-1 remained constant but was reduced upon depletion of calcium ion in the medium. With the presence of EGTA, the 97-kDa calcium-binding protein migrated into a reduced isoform and an oxidized isoform in a native gel. With the absence of EGTA, the 97-kDa calcium-binding protein was separated into several distinct bands in the native gel, suggesting that it might reside multiple calcium binding sites. To identify the gene encoding the 97-kDa protein, the Synechococcus RF-1 genomic DNA was restriction enzyme-digested and analyzed by Southern blotting using a degenerated oligonucleotide probe corresponding to the N —terminus amino acid sequence of the 97-kDa protein. A 2.2 kb EcoRI-HindIII cut DNA fragment has been cloned and sequenced. It was identified to contain the DNA sequence of the first 108 amino acids from the N-terminus of the protein. Attempts to clone a 6 kb Xba I cut fragment of I cut DNA fragments into the expression vector of pUC19 and lZAPII have not been successful and this is plausibly incurred by the lethal effect of the expressed calcium binding protein to the host cells. So another strategy was used. Based on the restriction map, the DNA sequence of the complete gene can be identified by cloning two overlap DNA fragments. A 3 kb HindIII-cut DNA fragment was cloned into pUC19, and one clone containing the target DNA has been identified. However, besides the target DNA fragment, the clone seemed to contain a different insert DNA of 3 kb. The suggestion was proved when the insert DNA was subcloned into a non-expression vector pBR322. When the target DNA fragment is sequenced, it will be used as a probe to clone the C-terminal region of the calcium-binding protein gene. Further sequencing experiment would help unravel the complete genetic sequence of the 97-kDa calcium binding protein.
| Identifer | oai:union.ndltd.org:TW/088NTNU0112006 |
| Date | January 2000 |
| Creators | chen-yen Lee, 李珍燕 |
| Contributors | Hsueh-Mei Chou, 周雪美 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 73 |
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