The role of vanadium as a chemical defense of the solitary tunicate, Phallusia nigra

Ascidians may defend themselves from fish predators, fouling organisms, and bacterial infection by producing secondary metabolites or sequestering acid, but many species also accumulate heavy metals, most notably vanadium. The possible defensive functions of heavy metals in ascidians are unclear. Vanadium is a transition metal with oxidation states ranging from –1 to +5, but under physiological conditions occurs most often between +3 and +5. The black tunicate, Phallusia nigra, sequesters vanadium in blood cells and in the exterior tunic surface. Vanadium content of the whole tunic, the tunic bladder cell layer, whole soft body, and blood were determined by flame atomic absorption spectroscopy. The blood and soft body contain the highest vanadium concentrations; however, vanadium is also concentrated at the tunic surface. The concentration of vanadium within the soft body, blood and tunic exterior was significantly higher than that of the whole tunic. Vanadium accumulation, speciation, chelation environment, oxidation state and storage are associated with pH. The objective of this investigation was to attempt to decouple the defensive properties of vanadium from those of low pH in the solitary tunicate, Phallusia nigra. Results of feeding assays with the blue head wrasse, Thalassoma bifasciatum confirmed outcomes of past studies that demonstrated that vanadyl sulfate (VOSO4·6H2O) and sodium vanadate (Na3VO4) were unpalatable to fish. However, the use of vanadium salts does not accurately reflect the chelation environment or oxidation state of vanadium in situ in P. nigra. The effects of vanadium chelated to naturally occurring compounds found within the blood of P. nigra and crude organic extracts of tunic and soft body tissues were evaluated in assays testing anti-predatory effects with the blue head wrasse, T. bifasciatum. The chelated vanadium compounds and crude organic extracts were also assessed for anti-microbial effects against a panel of 4 marine bacteria known to be pathogens of marine invertebrates: Vibrio parahaemolyticus, V. harveyi, Leucothrix mucor, and Deleya marina. Crude organic extracts of whole tunic and soft body tissues are palatable to T. bifasciatum and do not inhibit the growth of any of the bacterial lines assayed. Non-acidic vanadium (+3) complexes do not deter predation or inhibit microbial growth, whereas acidic aqua vanadium (+3 and +4) complexes were unpalatable to T. bifasciatum and exhibited anti-microbial activity. Difficulties in decoupling acidity from oxidation state and chelation environment of vanadium prevent definitive conclusions regarding the relative importance of low pH and vanadium to the chemical defense of Phallusia nigra

Cell Cycle-Dependent Induction of Homologous Recombination by a Tightly Regulated I-SceI Fusion Protein

Double-strand break repair is executed by two major repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ contributes to the repair of ionizing radiation (IR)-induced double strand breaks (DSBs) throughout the cell cycle, HR acts predominantly during the S and G2 phases of the cell cycle. The rare-cutting restriction endonuclease, I-SceI, is in common use to study the repair of site-specific chromosomal DSBs in vertebrate cells. To facilitate analysis of I-SceI-induced DSB repair, we have developed a stably expressed I-SceI fusion protein that enables precise temporal control of I-SceI activation, and correspondingly tight control of the timing of onset of site-specific chromosome breakage. I-SceI-induced HR showed a strong, positive linear correlation with the percentage of cells in S phase, and was negatively correlated with the G1 fraction. Acute depletion of BRCA1, a key regulator of HR, disrupted the relationship between S phase fraction and I-SceI-induced HR, consistent with the hypothesis that BRCA1 regulates HR during S phase

Differential Regulation of Short- and Long-Tract Gene Conversion between Sister Chromatids by Rad51C

The Rad51 paralog Rad51C has been implicated in the control of homologous recombination. To study the role of Rad51C in vivo in mammalian cells, we analyzed short-tract and long-tract gene conversion between sister chromatids in hamster Rad51C(−/−) CL-V4B cells in response to a site-specific chromosomal double-strand break. Gene conversion was inefficient in these cells and was specifically restored by expression of wild-type Rad51C. Surprisingly, gene conversions in CL-V4B cells were biased in favor of long-tract gene conversion, in comparison to controls expressing wild-type Rad51C. These long-tract events were not associated with crossing over between sister chromatids. Analysis of gene conversion tract lengths in CL-V4B cells lacking Rad51C revealed a bimodal frequency distribution, with almost all gene conversions being either less than 1 kb or greater than 3.2 kb in length. These results indicate that Rad51C plays a pivotal role in determining the “choice” between short- and long-tract gene conversion and in suppressing gene amplifications associated with sister chromatid recombination