For transformation of Pleurotus ostreatus, two novel vectors, pPhKM1 and pPhKM2, were constructed, using the regulatory sequences of the P. sajor-caju beta-tubulin gene (TUB1) and the ble gene encoding phleomycin binding protein. pPhKM1 contains ble fused to the TUB1 promoter and the Schizophyllum commune GPD terminator. pPhKM2 contains ble fused to the promoter and terminator regions of P. sajor-caju TUB1. To confirm phleomycin-resistance activity, each vector was cotransformed with pTRura3-2 into the P. ostreatus homokaryotic ura - strain. The transforming DNA was stably integrated into the genomic DNA. Subsequently, phleomycin resistance was conferred on wild-type dikaryotic P. ostreatus by transformation with pPhKM1 or pPhKM2. This transformation system generated stable phleomycin-resistant transformants.
Basidiomycota* ; Carrier Proteins ; DNA ; Phleomycins* ; Pleurotus* ; Schizophyllum ; Terminator Regions, Genetic ; Tubulin

The DNA sequence of a plasmid named pHP489 of Helicobacter pylori strain 489 was determined and analyzed to characterize its replication apparatus. The pHP489 plasmid consisted of 1,222 bp and had an overall G+C content of 33.1%. An ORF was predicted to encode the putative protein of 239 amino acid residues (28 kDa). A putative ribosomal binding site and a potential terminator sequence are located upstream and downstream of the ORF, respectively. However, the consensus sequence for a promoter in upstream of ORF was not found. A potential dna A box was found at 317 nt upstream of a start codon and followed by two-57 bp directed repeats and an inverted repeat. The DNA homology was found in the regions of less than 90 bp among pHPK255, pHPM180, and pHel1 of other H. pylori plasmids and Mycoplasma mycoides plasmids. pHP489K that was produced by pHP489 sequence and C. jejuni derived aph(3')-III, was transformed to various H. pylori isolates and were stably maintained in the H. pylori host without the addition of selective antibiotics for the 30-times subcultues. The plasmic vector, in which the ORF region of pHP489 DNA was deleted, could be transformed into H. pylori. However, the plasmid vector, whose the direct repeats region of pHP489 DNA was deleted, failed to be transformed. The direct repeats region of pHP489 DNA was confirmed to be bound with cytosolic factors of H. pylori. These results showed that the direct repeats region of pHP489 DNA is an essential apparatus by which the plasmid could be replicacted in H. pylori. And pHP489 plasmid was supposed to be replicated by host factors rather than plasmic-encoded factors.
Animals ; Anti-Bacterial Agents ; Base Composition ; Base Sequence ; Binding Sites ; Codon, Initiator ; Consensus Sequence ; Cytosol ; DNA ; Ecthyma, Contagious ; Helicobacter pylori* ; Helicobacter* ; Mycoplasma mycoides ; Plasmids* ; Repetitive Sequences, Nucleic Acid ; Sequence Analysis* ; Terminator Regions, Genetic