Tomio Morino

Construction and characterization of a cosmid of Streptomyces lividans.

SummaryWe constructed a plasmid pR4C1 in which a DNA fragment containing the cohesive ends of an actinophage, R4 was inserted into the ClaI site of plasmid pIJ365. The plasmid pR4C1 was packaged efficiently into R4 phage particles in vivo and therefore transferred by transduction. Southern hybridization analysis of DNA from pR4C1-transducing R4 phage particles indicated that the plasmid DNA was encapsidated as head-to-tail concatemers with the cohesive ends in the termini. We designated the pR4C1 plasmid a cosmid, following the termination of Collins and Hohn (1978).

Construction of a runaway vector and its use for a high-level expression of a cloned human superoxide dismutase gene

SummaryA runaway cloning vector pR3 from plasmid pBEU17 has been constructed by introducing three deletions (ΔI, ΔH and ΔEHi), multiple cloning sites and transcription terminator sequences. Introduction of the deletion ΔH restored a drastic plasmid amplification at high temperature (37° C). This suggested the presence of unknown sequences around the ΔH region which repressed runaway replication. To examine the usefulness of a pR3 runaway vector on the high-level expression of a cloned gene, a pRT8 plasmid containing a cloned human superoxide dismutase (SOD) gene in the pR3 was constructed. A host with tolerance to high copy numbers of pRT8 was selected, and the cultural conditions required for high-level expression were investigated. It was shown that the copy number of pRT8 increased in the stationary phase even at 30° C, and that cells carrying an excess number of plasmid did not support the runaway replication, yielding less SOD protein. SOD synthesis in the production culture lasted for about 24 h after the thermal shift, though viability was soon lost. The maximum yield was determined as approximately 20% of total cellular protein. This was 12-fold higher than that of a vector having the ColE1 plasmid replicon.

Cleavage Analysis of Actinophage R4 and Its Deletion Mutants

We have constructed restriction maps of actinophage R4 for PstI, PvuII, EcoRI, SeaI, KpnI, BglII and BclI. DNA of R4 phage had no cleavage sites for seven restriction endonucleases, BamHI, ClaI, HindIII, SacII, SlaI (XhoI) or XbaI. The entire genome size of the R4 phage was estimated to be 53.7 kilobases. Three deletions of the R4 phage were delimitated on the R4 genome. All three deletions were located on the right hand side of the R4 restriction map.

Interspecific transfer and expression of melanin gene(s) on cosmids in Streptomyces strains

SummaryCosmid pR4C1 has been constructed from a multicopy plasmid pIJ365 and the cohesive ends site of an actinophage R4 (Morino et al. 1985). The cosmid can be transferred to various Streptomyces strains by R4 phage-mediated transduction. Melanin-synthesizing gene(s) originated from Streptomyces antibioticus was inserted into the cosmid and succesfully transferred by tranduction to 7 different strains out of 10 strains examined. The features of melanin-gene(s) expresion were examined in those cells. Tyrosinase activities from the melanin-gene(s) were detected in culture media and/or cell extracts although the extents of mel gene expression and tyrosinase excretion vary strain to strain. The cosmid transfer system described in this paper will be promising to survey suitable hosts for the maximum expression of cloned genes among Streptomyces strains.