We have identified a novel .ca 400 kDa cell-cycle dependent kinetochore associated protein in human cells, designated CENP-F, using human autoimmune serum. Immunofluorescence staining using the native serum, affinity purified antibodies, or antibodies raised against a cloned portion of CENP-F first reveals CENP-F homogeneously distributed throughout the nucleus of HeLa cells in the G2 stage of the cell cycle. Progression into prophase is accompanied by the localization of CENP-F to all the kinetochore regions of the karyotype. Kinetochore association is maintained throughout metaphase, but at the onset of anaphase CENP-F is no longer detected in association with the kinetochore but is found at the spindle mid-zone. By telophase, it is concentrated into a narrow band on either side of the midbody. Studies of the interaction of CENP-F with the kinetochore indicate that this protein associates with the kinetochore independent of tubulin and dissociation is dependent on events connected with the onset of anaphase. Nuclease digestion studies and immunoelectron-microscopy indicate that CENP-F is localized to the kinetochore plates and specifically to the outer surface of the outer kinetochore plate. The distribution of CENP-F closely parallels that of another high molecular weight kinetochore associated protein, CENP-E. Comparative studies indicate that there are antibodies in the CENP-F reactive autoimmune serum that recognize determinants present in the central helical rod domain of CENP-E. Immune depletion experiments confirm that CENP-F exhibits the distribution pattern in cells that was seen with the native autoimmune serum.

Cells of Sphingomonas sp. strain BSAR-1 constitutively expressed an alkaline phosphatase, which was also secreted in the extracellular medium. A null mutant lacking this alkaline phosphatase activity was isolated by Tn5 random mutagenesis. The corresponding gene, designated phoK, was cloned and overexpressed in Escherichia coli strain BL21(DE3). The resultant E. coli strain EK4 overexpressed cellular activity 55 times higher and secreted extracellular PhoK activity 13 times higher than did BSAR-1. The recombinant strain very rapidly precipitated >90% of input uranium in less than 2 h from alkaline solutions (pH, 9 ؎ 0.2) containing 0.5 to 5 mM of uranyl carbonate, compared to BSAR-1, which precipitated uranium in >7 h. In both strains BSAR-1 and EK4, precipitated uranium remained cell bound. The EK4 cells exhibited a much higher loading capacity of 3.8 g U/g dry weight in <2 h compared to only 1.5 g U/g dry weight in >7 h in BSAR-1. The data demonstrate the potential utility of genetically engineering PhoK for the bioprecipitation of uranium from alkaline solutions.Environmental metal pollution is a serious problem, and treatment/recovery of desired metals from such wastes is a major challenge. Effective immobilization of radionuclides of metals is critical in order to prevent groundwater contamination (17). Bioremediation of the toxic metal wastes by microbes offers a relatively inexpensive and ecofriendly alternative to commonly used physical and chemical methods (7,19,26). In particular, enzymatic bioprecipitation of heavy metals as metal phosphates is very attractive, since it can recover metals from very low concentrations not amenable to chemical techniques (18). Successful bioprecipitation of metals, such as uranium and cadmium, using acid phosphatase from naturally occurring bacteria, such as Citrobacter sp. (19), has been reported. The uranium bioprecipitation potentials of Bacillus sp., Rahnella sp. (5, 20), Pseudomonas sp. (22), and Salmonella sp. (27) in an acidic-to-neutral pH range have also been explored. Genetic engineering of the radio-resistant bacterium Deinococcus radiodurans R1 by using a nonspecific acid phosphatase, PhoN, for the biorecovery of uranium from dilute acidic/neutral wastes was reported by our laboratory recently (2).Based on the process used, uranium mining and processing generate large quantities of dilute acidic and alkaline nuclear waste containing uranium, which are dumped as mill tailings. Alkaline wastes containing traces of uranium also arise from nuclear reactors and power plants using uranium as fuel. In nature, uranium (VI) forms highly soluble carbonate complexes, such as [UO 2 (CO 3 ) 2 ] Ϫ2 and [UO 2 (CO 3 ) 3 ] Ϫ4 , at alkaline pH levels (9). This leads to increase in mobility and availability of uranium to groundwater and soil from the dumped nuclear wastes, leading to health hazards. Nearly 130 million liters of alkaline nuclear wastes containing uranium carbonate awaits disposition at the Savannah River Site, Aiken, SC, alone (9). In order to extend microbial ...

We report on an experimental investigation of porous silicon layer (PSL) formation and silicon/hydrofluoric acid false(HFfalse) interfaces, using internal‐reflection Fourier‐transform infrared spectroscopy and Fourier‐transform electrochemically modulated infrared spectroscopy. Low‐doping Si samples ( normalp≈3×1015 cm−3 , resistivity ≈4 Ω cm) have been used for PSL growing. The in situ electromodulated spectra during the formation of PSL and the transmission spectra of the PSL in contact with the electrolyte contain only a broad band at around 2100 cm−1, whereas the transmission spectra of the dried PSL give rise to three sharp peaks at around 2085, 2115, and 2140 cm−1. All these vibrational peaks are ascribed to normalsurface‐normalSiH chemical species. The broadening of the normalSiH spectrum in the former cases has been found to be due to interaction of normalSiH species with the solvent. Under cathodic conditions—either for n‐Si in the dark or for p‐Si with illumination—the spectrum of the Si/HF interface exhibits three sharp normalSiH peaks, characteristic of a surface normalSiH in contact with a gas phase. This is attributed to the presence of hydrogen gas bubbles on the Si surface. It is observed that the Si surface is covered with Si‒H bonds in HF solution at all the applied potentials and current densities. An attempt has been made to detect intermediate species such as SiHF3 in the process of anodic dissolution of Si in HF , and it is concluded that if these species exist at all, their lifetime is shorter than 0.3 ms.

Aim: Purification and characterization of a chitinase from Microbispora sp. V2. Methods and Results: The chitinase from Microbispora sp. V2 was purified to homogeneity by gel filtration chromatography with 4AE6% recovery. It had a molecular weight of 35 kDa and showed maximum activity towards p-nitrophenyl-b-D D-N,N¢-diacetylchitobiose, indicating a chitobiosidase activity. The enzyme had a pH optimum of 3AE0 and temperature optimum of 60°C. It was stable in a wide pH range from 3AE0 to 11AE0, retaining 61% activity at pH 3AE0 and 52% activity at pH 11AE0. It retained 71% activity at 30°C and 45% activity at 50°C, up to 24 h. The enzyme activity was not inhibited by any of the metal ions tested except Hg 2+ , in the presence of which only 10% activity was retained. Conclusions: The 35 kDa chitinase from Microbispora sp. V2 has an acidic pH optimum and a high temperature optimum. It is fairly stable and active, and degrades chitin efficiently, although the growth of the culture and enzyme production is slow. Significance and Impact of the Study: This report is the first detailed study of a chitinase from Microbispora sp. V2, isolated from hot springs. The chitinase from Microbispora sp. V2 may have potential applications in the recycling of chitinous wastes, particularly due to its thermophilic and acidophilic character. Studies at molecular level may provide further insight on the chitinolytic system of Microbispora spp. with respect to the number and types of chitinases and their regulation.

Genetic engineering of radiation-resistant organisms to recover radionuclides/heavy metals from radioactive wastes is an attractive proposition. We have constructed a Deinococcus radiodurans strain harboring phoN, a gene encoding a nonspecific acid phosphatase, obtained from a local isolate of Salmonella enterica serovar Typhi. The recombinant strain expressed an ϳ27-kDa active PhoN protein and efficiently precipitated over 90% of the uranium from a 0.8 mM uranyl nitrate solution in 6 h. The engineered strain retained uranium bioprecipitation ability even after exposure to 6 kGy of 60 Co gamma rays. The PhoN-expressing D. radiodurans offers an effective and eco-friendly in situ approach to biorecovery of uranium from dilute nuclear waste.

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We sequenced bacteriophage T4 genes 2 and 3 and the putative C-ial portion of gen 50. We found that the product of the cloned gene 2 can protect T4 DNA double-stranded ends from exonucease V action.Gene 2 codes for a head protein of bacteriophage T4 (7). Gene 2 amber mutants do not make as many heads filled with DNA (10), and only about half the amount of maturable head proteins are cleaved as is the case for wild-type phage (13). In addition, tails do not attach to the heads as well as they do in wild-type phage (10). Nevertheless, completed phage particles with full heads have only cleaved head proteins