Open AccessScreening and analysis of DNA fragments that show promoter activities in Thermus thermophilus
Author(s) -
Maseda Hideaki,
Hoshino Takayuki
Publication year - 1995
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1995.tb07511.x
Subject(s) - thermus thermophilus , ecori , biology , kanamycin , microbiology and biotechnology , dna , gene , thermus , genetics , restriction enzyme , escherichia coli , bacteria , thermophile
We have constructed a Thermus promoter probe vector pPP11 containing the promoterless heat‐stable kanamycin resistance (Km r ) gene. Total DNA of T. thermophilus HB27 was randomly sheared into 100–200 bp fragments and inserted into the Eco RI site of pPP11. About 1000 clones which showed Km r were obtained, and they were classified into three groups according to their Km r levels. The DNA sequences of the inserted fragments of fifteen clones (Km r levels were high for seven, medium for three and low for five clones) were determined. Kanamycin nucleotidyl transferase (KNTase) activities of the cell‐free extracts of the clones were measured. Transcriptional starting points were determined for ten clones which showed a high or medium level of Km r . Amounts of Km r mRNA were also estimated for the ten clones. Amounts of Km r mRNA synthesized in the clones were in good agreement, and KNTase activities were in fairly good agreement with the Km r levels of the clones, respectively. From these results, we propose a possible consensus promoter sequence for T. thermophilus .