Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties.

We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus. One of the annotated open reading frames (ORFs) shows homology to T4 RNA ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids. The ORF was cloned, and recombinant protein was expressed, purified and characterized. The recombinant enzyme ligates single-stranded nucleic acids in an ATP-dependent manner and is moderately thermostable. The recombinant enzyme exhibits extremely high activity and high ligation efficiency. It can be used for various molecular biology applications including RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The TS2126 RNA ligase catalyzed both inter- and intra-molecular single-stranded DNA ligation to >50% completion in a matter of hours at an elevated temperature, although favoring intra-molecular ligation on RNA and single-stranded DNA substrates. The properties of TS2126 RNA ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis.

Identification and nucleotide sequence analysis of a cryptic plasmid, pRM21, from Rhodothermus marinus.

Here we report the identification and nucleotide sequence analysis of pRM21, a plasmid isolated from the thermophilic eubacterium Rhodothermus marinus. pRM21 consists of 2935 bp, has a G+C content of 58.2% and one major open reading frame whose deduced product shows significant similarities to RepA proteins from several plasmids, the highest being to the RepA of pSa from Escherichia coli. A region with the characteristics of iteron-containing replicons, three 19 bp repeats, DnaA boxes, an A+T rich region and GATC sequences, was identified. Of 40 additional R. marinus strains screened for plasmids, six (15%) were found to harbour plasmids with the same size and restriction pattern as pRM21.