Exploiting native forces to capture chromosome conformation in mammalian cell nuclei

Mammalian interphase chromosomes fold into a multitude of loops to fit the confines of cell nuclei, and looping is tightly linked to regulated function. Chromosome conformation capture (3C) technology has significantly advanced our understanding of this structure‐to‐function relationship. However, all 3C‐based methods rely on chemical cross‐linking to stabilize spatial interactions. This step remains a “black box” as regards the biases it may introduce, and some discrepancies between microscopy and 3C studies have now been reported. To address these concerns, we developed “i3C”, a novel approach for capturing spatial interactions without a need for cross‐linking. We apply i3C to intact nuclei of living cells and exploit native forces that stabilize chromatin folding. Using different cell types and loci, computational modeling, and a methylation‐based orthogonal validation method, “TALE‐iD”, we show that native interactions resemble cross‐linked ones, but display improved signal‐to‐noise ratios and are more focal on regulatory elements and CTCF sites, while strictly abiding to topologically associating domain restrictions.     

An elevation of the molar growth yield of Zymomonas mobilis during aerobic exponential growth

Elevated values of molar growth yield (Y_x/s = 14–26 g mol^–1) were obtained during exponential growth (μ > 0.4 h^–1) of Zymomonas mobilis ATCC 29191 by using reduced concentrations of glucose (6.25–100 mM) and increased oxygen supply (E _h > 300 mV) in the growth medium, as compared to the Y_x/s of anaerobic exponential growth (8–10 g mol^–1). Aerobically grown cells showed an increased maximum growth rate (μ_max), and a reduced specific glucose consumption rate (q_s), and specific ethanol formation rate (q_p), thus demonstrating a more pronounced energy-coupling growth under oxic conditions. These results can be neither explained by the concept of a solely operating Entner-Doudoroff pathway as an ATP source in aerobically growing cultures of Z. mobilis nor considered to be consistent with existing data on the lack of the Pasteur effect in this bacterium. Therefore, the results rather give evidence for the essential contribution of aerobic ATP generation under the reported conditions. Received: 24 September 1996 / Accepted: 9 December 1996     

Degradation of naphthalene by thermophilic bacteria <Emphasis Type="Italic">via</Emphasis> a pathway,through protocatechuic acid

A number of thermophilic bacteria capable of utilizing naphthalene as a sole source of carbon were isolated from a high-temperature oilfield in Lithuania. These isolates were able to utilize several other aromatic compounds, such as anthracene, benzene, phenol, benzene-1, 3-diol, protocatechuic acid as well. Thermophilic isolate G27 ascribed to Geobacillus genus was found to have a high aromatic compound degrading capacity. Spectrophotometric determination of enzyme activities in cell-free extracts revealed that the last aromatic ring fission enzyme in naphthalene biotransformation by Geobacillus sp. G27 was inducible via protocatechuate 3, 4-dioxygenase; no protocatechuate 4, 5-dioxygenase, protocatechuate 2, 3-dioxygenase activities were detected. Intermediates such as o-phthalic and protocatechuic acids detected in culture supernatant confirmed that the metabolism of naphthalene by Geobacillus sp. G27 can proceed through protocatechuic acid via ortho-cleavage pathway and thus differs from the pathways known for mesophilic bacteria.     

Molar growth yields of Zymomonas mobilis on glucose after the transition from anaerobic to aerobic continuous growth

An increase in the molar growth yield (Y_X/S = 14.3–20.3 g/mol) on glucose (25 mM) was achieved after the transition of Zymomonas mobilis ATCC 29191 from anaerobic to aerobic steady state growth at dilution rates of D = 0.31–0.40 1/h and under oxygen-unlimited conditions. The transfer of anaerobically or aerobically grown steady state cells into a fresh medium resulted in the higher values of Y_X/S. A positive correlation was established between biomass and acetaldehyde yield within the range of 5–9 mM acetaldehyde in the medium. An inhibitory effect of the exogenously added acetaldehyde (K_i = 16.7 ± 2.8 mM) on the ATPase activity was observed in vitro, using cell-free extracts of anaerobically grown Z. mobilis. The results obtained provide evidence that the increased values of biomass yield could be explained by the redirection of ATP usage during aerobic growth of Z. mobilis.     

Catechol 1,2-dioxygenase from α-naphthol degrading thermophilic Geobacillus sp. strain: purification and properties

The purpose of this study was purification and characterization of catechol 1,2-dioxygenase from Geobacillus sp. G27 strain, which degrades α-naphthol by the β-ketoadipate pathway. The catechol 1,2-dioxygenase (C1,2O) was purified using four steps of ammonium sulfate precipitation, DEAE-celullose, Sephadex G-150 and hydroxylapatite chromatographies. The enzyme was purified about 18-fold with a specific activity of 7.42 U mg of protein⁻¹. The relative molecular mass of the native enzyme estimated on gel chromatography of Sephadex G-150 was 96 kDa. The pH and temperature optima for enzyme activity were 7 and 60°C, respectively. A half-life of the catechol 1,2-dioxygenase at the optimum temperature was 40 min. The kinetic parameters of the Geobacillus sp. G27 strain catechol 1,2-dioxygenase were determined. The enzyme had apparent K_m of 29 μM for catechol and the cleavage activities for methylcatechols were much less than for catechol and no activity with gentisate or protocatechuate was detected.