In vivo and in vitro recombination techniques were used to construct a new cloning vector, pHY300PLK (4.7 kb) from the shuttle vector pHY460 (7 kb). The newly derived shuttle vector can replicate and express the tetracycline resistance gene (TcR) in both Escherichia coli and Bacillus subtilis. pHY300PLK contains the TcR gene, the ampicillin resistance gene (ApR), two replication origins for EK coli and B. subtilis and a polylinker derived from ~AN7. The unique cloning sites are BalI,

The whole nucleotide sequence of pHY300PLK (Ishiwa and Shibahara 1985a) has been determined, pHY300PLK consists of 4870 by and contains an RNA primer for on-177 and three open reading frames corresponding to TcR, ApR and Rep-al. Twenty-three unique restriction endonuclease recognition sites were found. We have confirmed that ORF al of pHY300PLK is the plasmid replication gene. A rescue phenomenon has been found with respect to multiple infection by pHY300.2PLK (rep-) and pUB110. A series of experiments indicated that the oligomer form, pHY300.2PLK, was rescued by the monomeric form, pUB110. Additionally, some useful information for using pHY300PLK as a shuttle vector in molecular cloning studies is presented.

The possibility of incomplete maternal transmission of mitochondrial DNA (mtDNA) in Drosophila, previously suggested by the presence of heteroplasmy, was examined by intra- and interspecific backcrosses of Drosophila simulans and its closest relative, Drosophila mauritiana. mtDNAs of offspring in these crosses were characterized by Southern hybridization with two alpha-32P-labeled probes that are specific to paternal mtDNAs. This method could detect as little as 0.03% paternal mtDNA, if present, in a sample. Among 331 lines that had been backcrossed for ten generations, four lines from the interspecific cross D. simulans (female) x D. mauritiana (male) showed clear evidence for paternal leakage of mtDNA. In three of these the maternal type was completely replaced while the fourth was heteroplasmic. Since in this experiment the total number of fertilization is known to be 331 x 10 = 3310, the proportion of paternal mtDNA per fertilization was estimated as about 0.1%. The mechanisms and evolutionary significance for paternal leakage are discussed in light of this finding.

Tetracycline and erythromycin resistance plasmids were found in Lactobacillus fermentum, strain LF601. They had molecular weights of 10.1+0.03 x 106 and 37.5±0.2 x 106, respectively. Cryptic plasmids with molecular weights of 8.5+0.4 x 106 and 16.7+0.2 x 106 were also observed. This paper provides the first description of drug resistance plasmids in lactobacilli. By a very rare chance, several drug resistant lactobacilli were isolated in our laboratory from healthy human feces (IsHIwA et al., unpublished data). One of them, a tetracycline and erythromycin resistant strain, L. fermentum LF601, was chosen for this investigation.To isolate the isogenic clones, LF601 (Lab. No. Ku-X-5; ISHIWA et a!., unpublished data) was treated with 1µg/ml of acriflavine for 40 hr at 37° in ILS broth of EFTHYMIOU and HANSEN (1). Most of the treated cells (95-99 %) proved to be tetracycline sensitive, while their erythromycin resistance remained unaltered under these conditions. The erythromycin resistance of the original strain isolated from human feces was unstable in broth culture; about 1 of the cells spontaneously became erythromycin sensitive. By a combination of acriflavine treatment and the spontaneous events, four kinds of individual clones were established with respect to drug sensitivity. These were designated as LF601 (tetracycline and erythromycin resistant), LF604 (tetracycline resistant), LF605 (erythromycin resistant), and LF609. Their minimum inhibitory concentrations are listed in Table ).The bacteria were grown for 4 to 5 hr at 37°, in 2l of ILS medium. The spheroplasts were prepared by the method of BARKER and THORNE (2). The cells were collected by centrifugation and suspended gently in 20 ml of cold 10 mM Tris-HC1 (pH 8.0) with 5 mM EDTA. Most of the cells were lysed by this step, and the remaining cells were completely lysed by the addition of 0.4 ml of 25 % sodium dodecyl sulfate, followed by incubation for 5 min at 37°. Cleared lysate (20 ml) was obtained by centrifugation at 30,000 rpm for 30 min 71

SUMMARYStrains of lactobacilli were examined for lysogeny after treatment with mitomycin C. Forty strains belonging to 7 species out of 148 strains of 15 species were lysed by mitomycin C. Lysis was strongly dependent upon the concentration of mitomycin C, temperature and the age of the cultures.Phage-like particles were observed in 3 I out of 40 lysates by electron microscopy. Particles from ten lysates -four from Lactobacillus casei B, five from L. casei C and one from L. acidophilus -produced plaques when plated on suitable indicator strains and were thus considered to be active phages. The remaining 21 lysates that contained phage-like particles failed to produce plaques but four of them inhibited the growth of other lactobacilli. It was concluded that the particles in these lysates were defective phages.Activities, shapes, dimensions and serological properties of the newly induced phage-like particles were also studied.