Transductional selection of cloned bacteriophage phi 105 and SP02 deoxyribonucleic acids in Bacillus subtilis.

The Bacillus subtilis temperate bacteriophages phi 105 and SP02 are incapable of transduction of the small, multicopy drug resistance plasmids pUB110 and pCM194. Cloning endonuclease-generated fragments of phi 105 or SP02 DNA into each of the plasmids renders the chimeric derivatives susceptible to transduction specifically by the phage whose deoxyribonucleic acid is present in the chimera. The majority of phage deoxyribonucleic acid fragments identified that render plasmids transducible by phi 105 or SP02 appear to be internal fragments, not fragments containing the cohesive ends. However, the highest overall transduction frequency was observed in SP02-mediated transduction of a derivative of pUB110 containing a 1.6-megadalton EcoRI fragment that likely contains the SP02 cohesive ends (plasmid pPL1010). The transducing activity present in a phi 105 transducing lysate had a buoyant density slightly greater than infectious particles, whereas the majority of transducing particles in an SP02(pPL1010) transducing lysate had a buoyant density slightly less than infectious particles. Although no detectable change in plasmid structure resulted from transduction by phi 105 or SP02, deoxyribonucleic acid isolated from a purified SP02(pPL1010) transducing lysate contained no detectable monomeric pPL1010, but did contain a form of pPL1010 of higher molecular weight than the monomer.     

High-frequency elimination of SP02 prophage from Bacillus subtilis by plasmid transformation

Transformation of competent Bacillus subtilis lysogenic for SP02 with any of three plasmids (pCM194, pUB110, pAM77) generates drug-resistant transformants of which 5 to 20% have lost the infectivity and immunity associated with the SP02 prophage. Such cured derivatives can be again lysogenized with SP02 and again cured by introduction of a different plasmid. Elimination of the SP02 prophage was not detected when plasmids were introduced by PBS1 transduction or by transformation of protoplasts. Similarly, transformants of B. subtilis selected for chromosome markers retained the prophage. The phi 105 prophage was not eliminated from competent B. subtilis transformed with plasmids.     

Functional activity of rat testicular cells in culture

Testicular cells from adult hypophysectomized rats were cultured for 10 or 12 days, and the effect of treatment with hCG (10 ng/ml) on testosterone and progesterone production and the activity of the Leydig cell enzyme, 3 beta-hydroxysteroid dehydrogenase, were studied. Regardless of hormone treatment, on 4th day in culture a decline in the steroidogenic activity of cultured cells could be observed. Treatment with hCG resulted in stimulation of steroidogenesis on days 6 to 10 in culture, as measured by testosterone and progesterone production. Hormone treatment stimulated or inhibited the enzyme activity depending on the presence or absence in the culture medium of 10(-6) M spironolactone, an inhibitor of 17 alpha-hydroxylase, or an anti-androgen, cyproterone acetate.     

Cloning of the Bacillus subtilis recE+ gene and functional expression of recE+ in B. subtilis.

By use of the Bacillus subtilis bacteriophage cloning vehicle phi 105J23, B. subtilis chromosomal MboI fragments have been cloned that alleviate the pleiotropic effects of the recE4 mutation. The recombinant bacteriophages phi 105Rec phi 1 (3.85-kilobase insert) and phi 105Rec phi 4 (3.3-kilobase insert) both conferred on the recE4 strain YB1015 resistance to ethylmethane sulfonate, methylmethane sulfonate, mitomycin C, and UV irradiation comparable with the resistance observed in recE+ strains. While strain YB1015 (recE4) and its derivatives lysogenized with bacteriophage phi 105J23 were not transformed to prototrophy by B. subtilis chromosomal DNA, strain YB1015 lysogenized with either phi 105Rec phi 1 or phi 105Rec phi 4 was susceptible to transformation with homologous B. subtilis chromosomal DNA. The heteroimmune prophages phi 105 and SPO2 were essentially uninducible in strain YB1015. Significantly, both recombinant prophages phi 105Rec phi 1 and phi 105Rec phi 4 were fully inducible and allowed the spontaneous and mitomycin C-dependent induction of a coresident SPO2 prophage in a recE4 host. The presence of the recombinant prophages also restored the ability of din genes to be induced in strains carrying the recE4 mutation. Finally, both recombinant bacteriophages elaborated a mitomycin C-inducible, 45-kilodalton protein that was immunoreactive with Escherichia coli recA+ gene product antibodies. Collectively, these data demonstrate that the recE+ gene has been cloned and that this gene elaborates the 45-kilodalton protein that is involved in SOB induction and homologous recombination.     

Synthetic menthyl α/β-(1→6)-diglucopyranosides-induced cell death in human leukemia cells is dependent on caspases

A series of alkyl α/β-(1→6)-diglucopyranosides 1-12 were synthesized and assessed for cytotoxicity against HL-60, U937, Molt-3 and MCF-7 cancer cell lines. The menthyl derivatives displayed strong cytotoxic properties showing IC(50) values between 6 and 16 μM. Furthermore, we demonstrated that the selected synthetic (+)-menthyl β-(1→6)-diglucopyranoside 5 induces apoptotic cell death in human leukemia cells through a mechanism that involves activation of multiple caspases. Cell death was completely prevented by the non-specific caspase inhibitor z-VAD-fmk and found to be associated with the release of cytochrome c, an increase in the expression of Bax levels and a decrease in the generation of reactive oxygen species.     

Cells expressing murine RAD52 splice variants favor sister chromatid repair

The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.