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991.
Sequence-dependent enhancement of hydrolytic deamination of cytosines in DNA by the restriction enzyme PspGI 总被引:6,自引:5,他引:1
Hydrolytic deamination of cytosines in DNA creates uracil and, if unrepaired, these lesions result in C to T mutations. We have suggested previously that a possible way in which cells may prevent or reduce this chemical reaction is through the binding of proteins to DNA. We use a genetic reversion assay to show that a restriction enzyme, PspGI, protects cytosines within its cognate site, 5′-CCWGG (W is A or T), against deamination under conditions where no DNA cleavage can occur. It decreases the rate of cytosine deamination to uracil by 7-fold. However, the same protein dramatically increases the rate of deaminations within the site 5′-CCSGG (S is C or G) by ~15-fold. Furthermore, a similar increase in cytosine deaminations is also seen with a catalytically inactive mutant of the enzyme showing that endonucleolytic ability of the protein is dispensable for its mutagenic action. The sequences of the mutants generated in the presence of PspGI show that only one of the cytosines in CCSGG is predominantly converted to thymine. Our results are consistent with PspGI ‘sensitizing’ the cytosine in the central base pair in CCSGG for deamination. Remarkably, PspGI sensitizes this base for damage despite its inability to form stable complexes at CCSGG sites. These results can be explained if the enzyme has a transient interaction with this sequence during which it flips the central cytosine out of the helix. This prediction was validated by modeling the structure of PspGI–DNA complex based on the structure of the related enzyme Ecl18kI which is known to cause base-flipping. 相似文献
992.
993.
The transmembrane (TM) domain of the M2 channel protein from influenza A is a homotetrameric bundle of α-helices and provides a model system for computational approaches to self-assembly of membrane proteins. Coarse-grained molecular dynamics (CG-MD) simulations have been used to explore partitioning into a membrane of M2 TM helices during bilayer self-assembly from lipids. CG-MD is also used to explore tetramerization of preinserted M2 TM helices. The M2 helix monomer adopts a membrane spanning orientation in a lipid (DPPC) bilayer. Multiple extended CG-MD simulations (5 × 5 μs) were used to study the tetramerization of inserted M2 helices. The resultant tetramers were evaluated in terms of the most populated conformations and the dynamics of their interconversion. This analysis reveals that the M2 tetramer has 2× rotationally symmetrical packing of the helices. The helices form a left-handed bundle, with a helix tilt angle of ∼16°. The M2 helix bundle generated by CG-MD was converted to an atomistic model. Simulations of this model reveal that the bundle's stability depends on the assumed protonation state of the H37 side chains. These simulations alongside comparison with recent x-ray (3BKD) and NMR (2RLF) structures of the M2 bundle suggest that the model yielded by CG-MD may correspond to a closed state of the channel. 相似文献
994.
Greg A. Ludvigson Brian J. Witzke Luis A. Gonz lez Scott J. Carpenter Chris L. Schneider Franciszek Hasiuk 《Palaeogeography, Palaeoclimatology, Palaeoecology》2004,210(2-4):187-214
Five positive carbon isotope excursions are reported from Platteville–Decorah strata in the Upper Mississippi Valley. All occur in subtidal carbonate strata, and are recognized in the Mifflin, Grand Detour, Quimbys Mill, Spechts Ferry, and Guttenberg intervals. The positive carbon isotope excursions are developed in a Platteville–Decorah succession in which background δ13C values increase upward from about −2‰ at the base to about 0‰ Vienna Pee Dee belemnite (VPDB) at the top. A regional north–south δ13C gradient, with lighter values to the north and heavier values to the south is also noted. Peak excursion δ13C values of up to +2.75 are reported from the Quimbys Mill excursion, and up to +2.6 from the Guttenberg excursion, although there are considerable local changes in the magnitudes of these events. The Quimbys Mill, Spechts Ferry, and Guttenberg carbon isotope excursions occur in units that are bounded by submarine disconformities, and completely starve out in deeper, more offshore areas. Closely spaced chemostratigraphic profiles of these sculpted, pyrite-impregnated hardground surfaces show that they are associated with very abrupt centimeter-scale negative δ13C shifts of up to several per mil, possibly resulting from the local diagenetic effects of incursions of euxinic bottom waters during marine flooding events. 相似文献
995.
One popular and fruitful approach to understanding what influences the decision of where to look next has been to present targets in a series of trials either to the right or left of a central fixation point and examine sequential effects on saccadic latency. However, there is a problem with this paradigm: Every saccade to a target is necessarily followed by an equal and opposite movement back to the center, yet the potentially confounding influence of this refixation saccade is rarely considered. Here, we introduce a novel random-walk paradigm that eliminates this difficulty. Each successive target appears to the left or right of the previous one, allowing us to study long sequences of saccades uncontaminated by refixations. This exposes a new stimulus-history effect, which is remarkably prolonged and relates primarily to movement direction: A saccade reduces the latency for subsequent movements made in the same direction and retards those in the opposite direction. Although in conventional refixation paradigms this effect cancels out, it is of particular significance in the real world--where our fixation point shifts constantly with the object of interest--and reflects a prediction of the way that real objects typically move. 相似文献
996.
A recent screen of a combinatorial library of fluorescent compounds discovered fluorescent dyes that were able to distinguish myoblasts from differentiated myotubes. New fluorescent dyes that respond to biologically relevant changes in cell state or type are useful as stains in a wide variety of biological experiments, including high-throughput screens for chemical and genetic regulators. Combining this approach with microscopy imaging is likely to be even more powerful and might lead to the discovery of new dyes with interesting and useful properties. 相似文献
997.
Mátrai J Jonckheer A Joris E Krüger P Carpenter E Tuszynski J De Maeyer M Engelborghs Y 《European biophysics journal : EBJ》2008,38(1):13-23
Correlating the experimentally observed kinetics of protein conformational changes with theoretical predictions is a formidable
and challenging task, due to the multitude of degrees of freedom (>5,000) in a protein and the huge gap between the timescale
of the kinetic event of interest (ms) and the typical timescale of computer simulations (ns). In this study we show that using
the targeted molecular dynamics (TMD) method it is possible to simulate conformational changes of the ms time range and to
correlate multiple simulations of single pathways with ensemble experiments on both the structural and energetic basis. As
a model system we chose to study the conformational change of rat-Δα-chymotrypsin from its inactive to its active conformation.
This activation process has been analyzed previously by experimental and theoretical methods, i.e. fluorescence stopped-flow
spectroscopy (FSF), molecular dynamics (MD) and TMD. Inspired by the results of these studies on the wild type (WT) enzyme,
several mutants were constructed to alter the conformational pathway and studied by FSF measurements. In the present work
WT and mutant N18G were subjected to multiple MD and subsequent TMD simulations. We report the existence of two main activation
pathways, a feature of chymotrypsin activation that has been hitherto unknown. A method to correlate the energetics of the
different pathways calculated by TMD and the kinetic parameters observed by experimental methods such as FSF is presented.
Our work is relevant for experimental single molecule studies of enzymes in general.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
998.
The conidiation of the entomopathogenic fungus Beauveria bassiana (Hyphomycete) is a complex process that involves the stage- and cell-type-specific expression of hundreds of genes. The suppression subtractive hybridization method was used to target genes involved in conidiation. Seventeen genes were cloned that potentially were involved in conidia formation. Six of them demonstrated differential expression between conidial and vegetative cultures. Sequence analysis showed three cDNA fragments had similarity to known genes involved in either cellular metabolism or cell regulatory processes. The other cDNA fragments showed low or no similarity to any genes previously described. The full-length cDNA and genomic sequence of a gene designated A43 was isolated. The A43 protein is composed of 180 amino acids and has 34% identity to a RNA-binding region-containing protein. The temporal expression pattern was consistent with the gene being involved in conidiation. The colony morphology of the A43 knock-out mutant had more floccus mycelium than the wild-type and also produced fewer conidia, indicating the A43 gene is involved in B. bassiana conidiation. 相似文献
999.
Polychlorinated biphenyls (PCBs) are known to be carcinogenic, but the mechanisms of this action are uncertain. Most, but not all, studies have concluded that PCBs are not directly mutagenic, and that much if not all of the carcinogenic activity resides in the fraction of the PCB mixture that contains congeners with dioxin-like activity. The present study was designed to determine genotoxic effects of an ortho-substituted, non-coplanar congener, 2,2',5,5'-tetrachlorobiphenyl (PCB 52), and a non-ortho-substituted coplanar congener with dioxin-like activity, 3,3',4,4'-tetrachlorobiphenyl (PCB 77) on cultured human peripheral lymphocytes. DNA damage was assessed by use of the comet assay (alkaline single-cell gel electrophoresis). After cell cultures were prepared, test groups were treated with different concentrations of PCB 52 (0.2 and 1 microM) and PCB 77 (1 and 10 microM) for 1h at 37 degrees C in a humidified carbon dioxide incubator, and compared to a DMSO vehicle control group. The cells were visually classified into four categories on the basis of extent of migration such as undamaged (UD), low damage (LD), moderate damage (MD) and high damage (HD). The highest concentration of PCBs 52 and 77 significantly increased DNA breakage in human lymphocytes (p<0.001). Our results indicate that both the non-coplanar PCB 52 and coplanar PCB 77 cause DNA damage, and that the ortho-substituted congener was significantly more potent than the dioxin-like coplanar congener. 相似文献
1000.
Sarvary MA Bloem KA Bloem S Carpenter JE Hight SD Dorn S 《Journal of economic entomology》2008,101(2):314-324
Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae) is an invasive herbivore that poses a serious risk to Opuntia cacti in North America. Knowledge of the flight behavior of the cactus moth is crucial for a better understanding of natural dispersal, and for both monitoring and control. We used computer-linked flight mills to investigate diel flight activity and flight performance in relation to gender, age, mating status, and body size. Maximal flight activity for both mated and unmated moths occurred during twilight, whereas flight activity was low during photophase. The total distance flown and the number of initiated flights within a diel cycle were higher in both unmated and mated females than in males, but the longest single flight was similar in both genders. These findings suggest that pheromone trap captures of males likely indicate the simultaneous presence of females and that mated females might even be in areas where males are not detected yet. Flight performance heterogeneity was large, with a small portion of the population (both males and females) performing long unbroken flights, whereas the majority made short flights. Females had higher pupal and adult body size and shorter longevity than males. A few individuals, particularly young mated females, flying long distances may be important for active spread of a population and the colonization of new habitats. Implications of this study in the control of the cactus moth by using the sterile insect technique are discussed. 相似文献