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Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.  相似文献   
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The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12–35°C) and KCl concentration (0.15–1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼8 kJ mol−1 decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes.  相似文献   
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The contribution of pre-mRNA processing mechanisms to the regulation of immune responses remains poorly studied despite emerging examples of their role as regulators of immune defenses. We sought to investigate the role of mRNA processing in the cellular responses of human macrophages to live bacterial infections. Here, we used mRNA sequencing to quantify gene expression and isoform abundances in primary macrophages from 60 individuals, before and after infection with Listeria monocytogenes and Salmonella typhimurium. In response to both bacteria we identified thousands of genes that significantly change isoform usage in response to infection, characterized by an overall increase in isoform diversity after infection. In response to both bacteria, we found global shifts towards (i) the inclusion of cassette exons and (ii) shorter 3’ UTRs, with near-universal shifts towards usage of more upstream polyadenylation sites. Using complementary data collected in non-human primates, we show that these features are evolutionarily conserved among primates. Following infection, we identify candidate RNA processing factors whose expression is associated with individual-specific variation in isoform abundance. Finally, by profiling microRNA levels, we show that 3’ UTRs with reduced abundance after infection are significantly enriched for target sites for particular miRNAs. These results suggest that the pervasive usage of shorter 3’ UTRs is a mechanism for particular genes to evade repression by immune-activated miRNAs. Collectively, our results suggest that dynamic changes in RNA processing may play key roles in the regulation of innate immune responses.  相似文献   
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Introduction

The disease burden and risk factors for respiratory syncytial virus (RSV) and human metapneumovirus (MPV) infections among children living in remote, rural areas remain unclear.

Materials and Methods

We conducted a prospective, household-based cohort study of children aged <3 years living in remote rural highland communities in San Marcos, Cajamarca, Peru. Acute respiratory illnesses (ARI), including lower respiratory tract infection (LRTI), were monitored through weekly household visits from March 2009 through September 2011. Nasal swabs collected during ARI/LRTI were tested for RSV, MPV, and other respiratory viruses using real-time RT-PCR. Incidence rates and rate ratios were calculated using mixed effects Poisson regression.

Results

Among 892 enrolled children, incidence rates of RSV and MPV ARI were 30 and 17 episodes per 100 child-years, respectively. The proportions of RSV and MPV ARI that presented as LRTI were 12.5% and 8.9%, respectively. Clinic visits for ARI and hospitalizations were significantly more frequent (all p values <0.05) among children with RSV (clinic 41% and hospital 5.3%) and MPV ARI (38% and 3.5%) when compared with other viral infections (23% and 0.7%) and infections without virus detected (24% and 0.6%). In multivariable analysis, risk factors for RSV detection included younger age (RR 1.02, 95% CI: 1.00-1.03), the presence of a smoker in the house (RR 1.63, 95% CI: 1.12-2.38), residing at higher altitudes (RR 1.93, 95% CI: 1.25-3.00 for 2nd compared to 1st quartile residents; RR 1.98, 95% CI: 1.26-3.13 for 3rd compared to 1st quartile residents). Having an unemployed household head was significantly associated with MPV risk (RR 2.11, 95% CI: 1.12-4.01).

Conclusion

In rural high altitude communities in Peru, childhood ARI due to RSV or MPV were common and associated with higher morbidity than ARI due to other viruses or with no viral detections. The risk factors identified in this study may be considered for interventional studies to control infections by these viruses among young children from developing countries.  相似文献   
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Novel simple synthetic media for inducing sexual co-flocculation in a short time after mixing heterothallic fission-yeast (Schizosaccharomyces pombe) cells of h- and h+ were devised; The most effective of these, mannose synthetic medium (MSM), contains 0.4% mannose as a carbon source in addition to galactose, KH2PO4 (pH4.0) and 4 vitamins. The addition of galactose to the medium suppressed the asexual self-flocculation but rather promoted the sexual co-flocculation. By transferring and mixing h- and h+ cells grown in malt-extract broth plus galactose into MSM, these heterothallic strains were revealed to be sexually ready through a long period of the log to stationary phases. Furthermore, a variety of C sources and NH4Cl at various concentrations in various media were examined for their effects upon sexual co-flocculation, conjugation and sporulation; it was found that the sugar concentration strictly affected the progress of the sequence of sexual reproduction at 26°C but not 30°C and that sexual co-flocculation of the heterothallic strains was induced only under lower concentrations of C and N source than that for the homothallic one.  相似文献   
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Magneto-aerotaxis in marine coccoid bacteria.   总被引:10,自引:0,他引:10  
Magnetotactic cocci swim persistently along local magnetic field lines in a preferred direction that corresponds to downward migration along geomagnetic field lines. Recently, high cell concentrations of magnetotactic cocci have been found in the water columns of chemically stratified, marine and brackish habitats, and not always in the sediments, as would be expected for persistent, downward-migrating bacteria. Here we report that cells of a pure culture of a marine magnetotactic coccus, designated strain MC-1, formed microaerophilic bands in capillary tubes and used aerotaxis to migrate to a preferred oxygen concentration in an oxygen gradient. Cells were able to swim in either direction along the local magnetic field and used magnetotaxis in conjunction with aerotaxis, i.e., magnetically assisted aerotaxis, or magneto-aerotaxis, to more efficiently migrate to and maintain position at their preferred oxygen concentration. Cells of strain MC-1 had a novel, aerotactic sensory mechanism that appeared to function as a two-way switch, rather than the temporal sensory mechanism used by other bacteria, including Magnetospirillum megnetotacticum, in aerotaxis. The cells also exhibited a response to short-wavelength light (< or = 500 nm), which caused them to swim persistently parallel to the magnetic field during illumination.  相似文献   
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