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101.
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Green fluorescent protein as a reporter for macromolecular localization in bacterial cells 总被引:8,自引:0,他引:8
Margolin W 《Methods (San Diego, Calif.)》2000,20(1):62-72
Green fluorescent protein (GFP) is a highly useful fluorescent tag for studying the localization, structure, and dynamics of macromolecules in living cells, and has quickly become a primary tool for analysis of DNA and protein localization in prokaryotes. Several properties of GFP make it an attractive and versatile reporter. It is fluorescent and soluble in a wide variety of species, can be monitored noninvasively by external illumination, and needs no external substrates. Localization of GFP fusion proteins can be analyzed in live bacteria, therefore eliminating potential fixation artifacts and enabling real-time monitoring of dynamics in situ. Such real-time studies have been facilitated by brighter, more soluble GFP variants. In addition, red-shifted GFPs that can be excited by blue light have lessened the problem of UV-induced toxicity and photobleaching. The self-contained domain structure of GFP reduces the chance of major perturbations to GFP fluorescence by fused proteins and, conversely, to the activities of the proteins to which it is fused. As a result, many proteins fused to GFP retain their activities. The stability of GFP also allows detection of its fluorescence in vitro during protein purification and in cells fixed for indirect immunofluorescence and other staining protocols. Finally, the different properties of GFP variants have given rise to several technological innovations in the study of cellular physiology that should prove useful for studies in live bacteria. These include fluorescence resonance energy transfer (FRET) for studying protein-protein interactions and specially engineered GFP constructs for direct determination of cellular ion fluxes. 相似文献
104.
Pei-Chun Chen Yelena N. Kryukova Show-Ling Shyng 《The Journal of biological chemistry》2013,288(47):34098-34109
Pancreatic β-cells secrete insulin in response to metabolic and hormonal signals to maintain glucose homeostasis. Insulin secretion is under the control of ATP-sensitive potassium (KATP) channels that play key roles in setting β-cell membrane potential. Leptin, a hormone secreted by adipocytes, inhibits insulin secretion by increasing KATP channel conductance in β-cells. We investigated the mechanism by which leptin increases KATP channel conductance. We show that leptin causes a transient increase in surface expression of KATP channels without affecting channel gating properties. This increase results primarily from increased channel trafficking to the plasma membrane rather than reduced endocytosis of surface channels. The effect of leptin on KATP channels is dependent on the protein kinases AMP-activated protein kinase (AMPK) and PKA. Activation of AMPK or PKA mimics and inhibition of AMPK or PKA abrogates the effect of leptin. Leptin activates AMPK directly by increasing AMPK phosphorylation at threonine 172. Activation of PKA leads to increased channel surface expression even in the presence of AMPK inhibitors, suggesting AMPK lies upstream of PKA in the leptin signaling pathway. Leptin signaling also leads to F-actin depolymerization. Stabilization of F-actin pharmacologically occludes, whereas destabilization of F-actin simulates, the effect of leptin on KATP channel trafficking, indicating that leptin-induced actin reorganization underlies enhanced channel trafficking to the plasma membrane. Our study uncovers the signaling and cellular mechanism by which leptin regulates KATP channel trafficking to modulate β-cell function and insulin secretion. 相似文献
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Jeffrey T. Lell Michael D. Brown Theodore G. Schurr R. I. Sukernik Yelena B. Starikovskaya Antonio Torroni Lorna G. Moore Gary M. Troup D. C. Wallace 《Human genetics》1997,100(5-6):536-543
We have initiated a study of ancient male migrations from Siberia to the Americas using Y chromosome polymorphisms. The first
polymorphism examined, a C→T transition at nucleotide position 181 of the DYS199 locus, was previously reported only in Native
American populations. To investigate the origin of this DYS199 polymorphism, we screened Y chromosomes from a number of Siberian,
Asian, and Native American populations for this and other markers. This survey detected the T allele in all five Native American
populations studied at an average frequency of 61%, and in two of nine native Siberian populations, the Siberian Eskimo (21%)
and the Chukchi (17%). This finding suggested that the DYS199 T allele may have originated in Beringia and was then spread
throughout the New World by the founding populations of the major subgroups of modern Native Americans. We further characterized
Native American Y chromosome variation by analyzing two additional Y chromosome polymorphisms, the DYS287 Y Alu polymorphic (YAP) element insertion and a YAP-associated A→G transition at DYS271, both commonly found in Africans. We found
neither African allele associated with the DYS199 T allele in any of the Native American or native Siberian populations. However,
we did find DYS287 YAP+ individuals who harbored the DYS199 C allele in one Native American population, the Mixe, and in one
Asian group, the Tibetans. A correlation of these Y chromosome alleles in Native Americans with those of the DYS1 locus, as
detected by the p49a/p49f (p49a,f) probes on TaqI-digested genomic DNA, revealed a complete association of DYS1 alleles (p49a,f haplotypes) 13, 18, 66, 67 and 69 with the
DYS199 T allele, while DYS1 alleles 8 and 63 were associated with both the DYS199 C and T allele.
Received: 18 November 1996 / Accepted: 19 May 1997 相似文献
108.
Corwin, L. M. (Walter Reed Army Institute of Research, Washington, D.C.), G. R. Fanning, F. Feldman, and P. Margolin. Mutation leading to increased sensitivity to chromium in Salmonella typhimurium. J. Bacteriol. 91:1509-1515. 1966.-Certain deletion mutants including the tryptophan operon in Salmonella typhimurium are unable to utilize several sugars as carbon sources in solid media, although they are able to grow in liquid media with these sugars. The addition of citrate or washing the agar with ethylenediaminetetraacetic acid permits growth on solid media. Analysis of the agar revealed that Fe(3+) and Cr(3+) were present at concentrations of 22 and 75 mum, respectively. The addition of Fe(3+) to liquid media in 0.5 mm concentrations did not inhibit the wild type or the mutants. A similar concentration of Cr(3+) did not inhibit the wild type, but concentrations as low as 0.01 to 0.05 mm inhibited the deletion mutants. Other metals were inhibitory at various concentrations, but none showed any significant differential effects on the mutants and the wild type. The increased sensitivity of the mutants to chromium may be due either to an increased permeability to Cr(3+), resulting in higher effective intracellular concentrations and inhibition of one or more metabolic functions, or to a binding of Cr(3+) to an altered cell wall, resulting in decreased permeability of required substrates. 相似文献
109.
Rod-shaped bacteria such as Escherichia coli accurately maintain their shape from generation to generation. The cytoskeletal proteins MreB and FtsZ, which respectively guide parallel growth of the sidewall and perpendicular growth of the division septum, are important to maintain a straight sidewall and uniformly rounded cell poles. FtsZ normally assembles into a ring at the cell midpoint, called the Z ring, which is oriented perpendicular to the cell's axis and is thus in perfect position to guide growth of a perpendicular septum. In this issue of Molecular Microbiology, Potluri et al. show that low molecular weight penicillin binding proteins, particularly PBP5, have a role in maintaining the perpendicular geometry of the Z ring and subsequent septum in E. coli. When these factors are absent or perturbed, division septa are readily deformed, which results in abnormal cell poles that often bifurcate over time to generate branches. The data suggest that cellular branching in E. coli is specifically induced by aberrant septation events caused by mis-oriented Z rings and not by deformation of a growing cell pole or emergence of new tips from the sidewall, which are likely mechanisms of branching in other bacterial families. 相似文献
110.
A comparison of three techniques for quantitative analysis of galactosylation present on immunoglobulins is described. ESIMS, MALDI-TOF MS, and anion-exchange chromatography with fluorescence detection were evaluated in terms of repeatability, limit of quantitation, selectivity, and linearity. A recombinant monoclonal IgG was enzymatically modified in vitro to produce essentially completely galactosylated and degalactosylated forms of the immunoglobulin. Samples of known galactosylation levels were prepared by mixing the modified forms with the native form of the immunoglobulin. Good repeatability and linearity were demonstrated for all three assays (RSDs<1.0%, correlation coefficients>0.99). Differences in selectivity, sensitivity, and other performance aspects of the three techniques are also discussed in this paper. 相似文献