Investigation of the priming activity of the tpg gene of Streptomyces linear plasmid SLP2

• Main Authors: Fang-Shyi Lue, 呂芳錫
• Other Authors: Chien-Chin Yang
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/2hexr5

碩士 === 中原大學 === 化學研究所 === 93 === The termenial protein gene of streptomyces had been found and named tpgC ( from S. coelicolor), tpgL (from S. lividans) and tpgR (from S. rochi). The amino acid sequence of tpgC is the same as tpgL, but the DNA sequence owned two different nucleotides. Two tpg homologues, tpgSLP2 and pseudo-otpg are founded on the linear plasmid SLP2 of Streptomyces lividans. The tpgSLP2 located at 6 kb from the left end, has the homology of tpgC is 74 percentage. The pseudo-tpg located at the right end 8 kb, has the homology of tpgC is 54 percentage. Streptomyces termenial protein as primer for replication of linear plasmid is similar to that of phageψ29. Although it was suggested that the DNA end is binding with threonine of TpgC, the accurate site is not determined. To investigate a series of Tp’s primer activity, we use the Streptomyces lividans stain MR04 as host which is tpgL- and only maintains circular plasmids. Transfering the linearized plasmids containing various form of tpg gene to MR04 and examination it’s stability of linear plasmids to determine the primer activitying of Tp. The results display: (1)The capability of wild type tpgSLP2 to maintain linear plasmids in MR04 indicateds that tpgSLP2 is functional as tpgC. (2)The incapability of wild type pseudo-tpg to maintain linear plasmids in MR04 displays that pseudo-tpg is non functional. (3)The mutated of TpgSLP2 at the 114th threonine to serine, can maintain linear plasmid in MR04. So do mutated TpgC at the 101th、108th、114th、123th and 176th threonine to serine. But the mutated of TpgC at the 114th threonine to cysteine is non functional, this result suggested that the linkage site might be at the 114th. (4)The C-terminal egfp-fused tpgC can maintain linear plasmid in the MR04, indicateds that is functional. (5)The tpgC fused to tapC can’t maintain linear plasmid.