Fusion with Ribosomal Protein L32 Increased the in vivo Thermostability of Kanamycin Nucleotidyltransferase in Thermus thermophilus
スポンサーリンク
概要
- 論文の詳細を見る
The kanamycin resistance (Km^r) gene widely used as an antibiotic resistance marker in Thermus strains was expressed at temperatures of up to 60℃ in Thermus thermophilus HB27. We isolated a clone from HB27 that exhibited the Km^r phenotype at 70℃. The clone contained a recombinant plasmid pPP442 in which the promoterless Km^r gene was preceded by a 236-bp fragment from HB27 chromosome. The location of the transcriptional start site in the 236-bp fragment was determined. The putative promoter sequence in this fragment exhibited similarity to the consensus promoter sequence of T. thermophilus, but its sequence identities with -35 and -10 consensus sequences were low. The activity of the putative promoter was found not to be very strong based on the results of primer extension. A ribosome binding sequence followed by a truncated open reading frame (ORF) was identified in the 236-bp fragment. The ORF was in frame with the Km^r gene. No mutation was found in the Km^r structural gene. A pPP442 derivative, pPP442-ΔEcUD, was constructed by introducing a frame shift mutation into the ORF. T. thermophilus carrying pPP442-ΔEcUD could not grow at 70℃ in Km-containing medium. The location of the 236-bp fragment on the physical map of T. thermophilus HB27 was determined. The entire ORF was cloned from the genome and the amino acid sequence deduced from the nucleotide sequence showed strong homology to that of ribosomal protein L32 of T. thermophilus HB8.These results suggested that fusion of the ribosomal protein L32 to the N-end of the Km^r gene product increased the thermostability of the Km^r gene product in T. thermophilus.
- 社団法人日本生物工学会の論文
- 1996-12-25
著者
-
Hoshino Takayuki
Institute Of Applied Biochemistry University Of Tsukuba
-
MASEDA HIDEAKI
Institute of Applied Biochemistry, University of Tsukuba
-
Maseda Hideaki
Institute Of Applied Biochemistry University Of Tsukuba
関連論文
- Development of Expression Vectors for Thermus thermophilus
- Role of the DNA Sequence Downstream of the Bacillus subtilis hut Promoter in Regulation of the hut Operon
- Determination of the Transcriptional Initiation Sites of the Kanamycin Resistance Genes of pUB110 and pTHN1
- Purification and Properties of NAD-Linked 1,2-Butanediol Dehydrogenase from Rhodococcus sp. strain TB-42
- Participation of Nitrite Reductase in Conversion of NO_2^- to NO_3^- in a Heterotrophic Nitrifier, Burkholderia cepacia NH-17, with Denitrification Activity
- The Response of Pseudomonas aeruginosa PAO1 Efflux Pump-Defective Mutants to N-Octanoyl-L-Homoserine Lactone
- A Quorum-Sensing Autoinducer Enhances the mexAB-oprM Efflux-Pump Expression without the MexR-Mediated Regulation in Pseudomonas aeruginosa
- Unusual Transcription Regulation of the niaD Gene under Anaerobic Conditions Supporting Fungal Ammonia Fermentation
- The Entire Population of Thermus thermophilus Cells Is Always Competent at Any Growth Phase
- Overproduction of Carotenoids in Thermus thermophilus
- Isolation and Partial Characterization of Carotenoid Underproducing and Overproducing Mutants from an Extremetly Thermophilic Thermus thermophilus HB27
- Fusion with Ribosomal Protein L32 Increased the in vivo Thermostability of Kanamycin Nucleotidyltransferase in Thermus thermophilus
- The α-Aminoadipate Pathway for Lysine Biosynthesis Is Widely Distributed among Thermus Strains
- Cloning and Sequence Analysis of Fumarase and Superoxide Dismutase Genes from an Extreme Thermophile Thermus thermophilus HB27
- Efficient Selection for Thermostable Protease in Themus thermophilus
- Plasmid Marker Rescue Transformation in Thermus thermophilus
- Analysis of the Methionine Biosynthetic Pathway in the Extremely Thermophilic Eubacterium Thermus thermophilus