Evidence for excision repair in promitochondrial DNA of anaerobic cells of Saccharomyces cerevisiae.

The respiratory adaptation (i.e., essentially mitochondrial biogenesis) in the excision repair-defective rad3-type mutants of Saccharomyces cerevisiae undergoing transition from the anaerobic to the aerobic state is found to be far more sensitive to 254-nm ultraviolet radiation (UV) than that of the RAD wild-type strain. We confirm that mitochondria of aerobic cells of a RAD strain lack the excision repair capacity of UV-induced pyrimidine dimers at all doses tested (1-15 J/m2). In contrast, in promitochondria of anaerobic cells of the wild-type strain excision repair appears to take place. This process is very efficient at low doses (at 0.5-5 J/m2 100% of the UV endonuclease-sensitive sites disappear), whereas at high doses its efficiency is reduced by about 50%. The promitochondrial excision repair of pyrimidine dimers appears to be under nuclear control since it is blocked in the rad2 mutant. Finally photoreactivation is found to be operating in nuclei, mitochondria and promitochondria.     

Can Metal Nanoparticles Be a Threat to Microbial Decomposers of Plant Litter in Streams?

The extensive use of nanometal-based products increases the chance of their release into aquatic environments, raising the question whether they can pose a risk to aquatic biota and the associated ecological processes. Aquatic microbes, namely fungi and bacteria, play a key role in forested streams by decomposing plant litter from terrestrial vegetation. Here, we investigated the effects of nanocopper oxide and nanosilver on leaf litter decomposition by aquatic microbes, and the results were compared with the impacts of their ionic precursors. Alder leaves were immersed in a stream of Northwest Portugal to allow microbial colonization before being exposed in microcosms to increased nominal concentrations of nanometals (CuO, 100, 200 and 500 ppm; Ag, 100 and 300 ppm) and ionic metals (Cu²⁺ in CuCl₂, 10, 20 and 30 ppm; Ag⁺ in AgNO₃, 5 and 20 ppm) for 21 days. Results showed that rates of leaf decomposition decreased with exposure to nano- and ionic metals. Nano- and ionic metals inhibited bacterial biomass (from 68.6% to 96.5% of control) more than fungal biomass (from 28.5% to 82.9% of control). The exposure to increased concentrations of nano- and ionic metals decreased fungal sporulation rates from 91.0% to 99.4%. These effects were accompanied by shifts in the structure of fungal and bacterial communities based on DNA fingerprints and fungal spore morphology. The impacts of metal nanoparticles on leaf decomposition by aquatic microbes were less pronounced compared to their ionic forms, despite metal ions were applied at one order of magnitude lower concentrations. Overall, results indicate that the increased release of nanometals to the environment may affect aquatic microbial communities with impacts on organic matter decomposition in streams.     

Herbicide resistance from a divided EPSPS protein: the split Synechocystis DnaE intein as an in vivo affinity domain

We report that the N- and C-terminal splicing domains of the intein found in the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) are capable of association in vivo and in vitro, even with key splicing residues changed to alanine (Cys(1), Asn(159), and Cys(+1) to Ala). These studies utilized the herbicide resistant form of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Salmonella typhimurium and an Escherichia coli strain with the EPSPS gene deleted from its genome (E. coli strain ER2799). EPSPS was mapped to identify potential split sites using a facile Tn7 linker scanning procedure. Forty positions were found to tolerate a five amino acid insertion while 21 sites did not, as assayed by the rescue of growth of E. coli strain ER2799. Further characterization of these sites by inserting a full length Ssp DnaE intein identified residue 235 of EPSPS as the optimal position. The EPSPS gene was then divided into amino acids 1-235 and 236-427 which were fused to residues 1-123 and 124-159 of a splicing defective Ssp DnaE intein, respectively. Expression of the EPSPS-intein fusions from separate DNA molecules conferred resistance to the herbicide glyphosate, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. As a control the split EPSPS without the intein-affinity domain did not allow cell growth. The use of an intein as an in vivo affinity domain was termed intein-mediated protein complementation (IPC). Intein fragment assembly was verified in vitro by immobilizing the C-terminal splicing domain of the Ssp DnaE intein on a resin and demonstrating that the N-terminal 235 amino acids of EPSPS only bound to the resin when fused to the N-terminal splicing domain of the Ssp DnaE intein. As chloroplast DNA is not transmitted by pollen in plants such as corn and soybean, transgene spread via pollen may be controlled in the future by expressing inactive gene fragments from separate DNA locations, such as the nuclear and chloroplast genome, and using the split intein to generate protein activity.     

Quantification of photonic localization properties of targeted nuclear mass density variations: Application in cancer‐stage detection

Light localization is a phenomenon which arises due to the interference effects of light waves inside a disordered optical medium. Quantification of degree light localization in optical media is widely used for characterizing degree of structural disorder in that media. Recently, this light localization approach was extended to analyze structural changes in biological cell like heterogeneous optical media, with potential application in cancer diagnostics. Confocal fluorescence microscopy was used to construct “optical lattices,” which represents 2‐dimensional refractive index map corresponding to the spatial mass density distribution of a selected molecule inside the cell. The structural disorder properties of the selected molecules were evaluated numerically using light localization strength in these optical lattices, in a single parameter called “disorder strength.” The method showed a promising potential in differentiating cancerous and non‐cancerous cells. In this paper, we show that by quantifying submicron scale disorder strength in the nuclear DNA mass density distribution, a wide range of control and cancerous breast and prostate cells at different hierarchy levels of tumorigenicity were correctly distinguished. We also discuss how this photonic technique can be used in examining tumorigenicity level in unknown prostate cancer cells, and potential to generalize the method to other cancer cells.      

Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.     

Monopolar Spindle 1 (MPS1) Protein-dependent Phosphorylation of RecQ-mediated Genome Instability Protein 2 (RMI2) at Serine 112 Is Essential for BLM-Topo III α-RMI1-RMI2 (BTR) Protein Complex Function upon Spindle Assembly Checkpoint (SAC) Activation during Mitosis

Genomic instability and a predisposition to cancer are hallmarks of Bloom syndrome, an autosomal recessive disease arising from mutations in the BLM gene. BLM is a RecQ helicase component of the BLM-Topo III α-RMI1-RMI2 (BTR) complex, which maintains chromosome stability at the spindle assembly checkpoint (SAC). Other members of the BTR complex include Topo IIIa, RMI1, and RMI2. All members of the BTR complex are essential for maintaining the stable genome. Interestingly, the BTR complex is posttranslationally modified upon SAC activation during mitosis, but its significance remains unknown. In this study, we show that two proteins that interact with BLM, RMI1 and RMI2, are phosphorylated upon SAC activation, and, like BLM, RMI1, and RMI2, are phosphorylated in an MPS1-dependent manner. An S112A mutant of RMI2 localized normally in cells and was found in SAC-induced coimmunoprecipitations of the BTR complex. However, in RMI2-depleted cells, an S112A mutant disrupted the mitotic arrest upon SAC activation. The failure of cells to maintain mitotic arrest, due to lack of phosphorylation at Ser-112, results in high genomic instability characterized by micronuclei, multiple nuclei, and a wide distribution of aberrantly segregating chromosomes. We found that the S112A mutant of RMI2 showed defects in redistribution between the nucleoplasm and nuclear matrix. The phosphorylation at Ser-112 of RMI2 is independent of BLM and is not required for the stability of the BTR complex, BLM focus formation, and chromatin targeting in response to replication stress. Overall, this study suggests that the phosphorylation of the BTR complex is essential to maintain a stable genome.     

S-adenosylhomocysteine Hydrolase Participates in DNA Methylation Inheritance

DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for mammalian development and maintenance of DNA methylation following DNA replication in cells. The DNA methylation process generates S-adenosyl-l-homocysteine, a strong inhibitor of DNMT1. Here we report that S-adenosylhomocysteine hydrolase (SAHH/AHCY), the only mammalian enzyme capable of hydrolyzing S-adenosyl-l-homocysteine binds to DNMT1 during DNA replication. SAHH enhances DNMT1 activity in vitro, and its overexpression in mammalian cells led to hypermethylation of the genome, whereas its inhibition by adenosine periodate or siRNA-mediated knockdown resulted in hypomethylation of the genome. Hypermethylation was consistent in both gene bodies and repetitive DNA elements leading to aberrant gene regulation. Cells overexpressing SAHH specifically up-regulated metabolic pathway genes and down-regulated PPAR and MAPK signaling pathways genes. Therefore, we suggest that alteration of SAHH level affects global DNA methylation levels and gene expression.