The Snakelike Chain Character of Unstructured RNA

In the absence of base-pairing and tertiary structure, ribonucleic acid (RNA) assumes a random-walk conformation, modulated by the electrostatic self-repulsion of the charged, flexible backbone. This behavior is often modeled as a Kratky-Porod “wormlike chain” (WLC) with a Barrat-Joanny scale-dependent persistence length. In this study we report measurements of the end-to-end extension of poly(U) RNA under 0.1 to 10 pN applied force and observe two distinct elastic-response regimes: a low-force, power-law regime characteristic of a chain of swollen blobs on long length scales and a high-force, salt-valence-dependent regime consistent with ion-stabilized crumpling on short length scales. This short-scale structure is additionally supported by force- and salt-dependent quantification of the RNA ion atmosphere composition, which shows that ions are liberated under stretching; the number of ions liberated increases with increasing bulk salt concentration. Both this result and the observation of two elastic-response regimes directly contradict the WLC model, which predicts a single elastic regime across all forces and, when accounting for scale-dependent persistence length, the opposite trend in ion release with salt concentration. We conclude that RNA is better described as a “snakelike chain,” characterized by smooth bending on long length scales and ion-stabilized crumpling on short length scales. In monovalent salt, these two regimes are separated by a characteristic length that scales with the Debye screening length, highlighting the determining importance of electrostatics in RNA conformation.     

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.     

Breakdown of low-level total petroleum hydrocarbons (TPH) in contaminated soil using grasses and willows

A phytoremediation study targeting low-level total petroleum hydrocarbons (TPH) was conducted using cool- and warm-season grasses and willows (Salix species) grown in pots filled with contaminated sandy soil from the New Haven Rail Yard, CT. Efficiencies of the TPH degradation were assessed in a 90-day experiment using 20–8.7–16.6 N-P-K water-soluble fertilizer and fertilizer with molasses amendments to enhance phytoremediation. Plant biomass, TPH concentrations, and indigenous microbes quantified with colony-forming units (CFU), were assessed at the end of the study. Switchgrass grown with soil amendments produced the highest aboveground biomass. Bacterial CFU's were in orders of magnitude significantly higher in willows with soil amendments compared to vegetated treatments with no amendments. The greatest reduction in TPH occurred in all vegetated treatments with fertilizer (66–75%) and fertilizer/molasses (65–74%), followed sequentially by vegetated treatments without amendments, unvegetated treatments with amendments, and unvegetated treatments with no amendment. Phytoremediation of low-level TPH contamination was most efficient where fertilization was in combination with plant species. The same level of remediation was achievable through the addition of grasses and/or willow combinations without amendment, or by fertilization of sandy soil.     

Localization of the 26S proteasome during mitosis and meiosis in fission yeast.

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the localization of this complex in the fission yeast, Schizosaccharomyces pombe. Immunofluorescence microscopy shows a striking localization pattern whereby the proteasome is found predominantly at the nuclear periphery, both in interphase and throughout mitosis. Electron microscopic analysis revealed a concentration of label near the inner side of the nuclear envelope. The localization of green fluorescent protein (GFP)-tagged 26S proteasomes was analyzed in live cells during mitosis and meiosis. Throughout mitosis the proteasome remained predominantly at the nuclear periphery. During meiosis the proteasome was found to undergo dramatic changes in its localization. Throughout the first meiotic division, the signal is more dispersed over the nucleus. During meiosis II, there was a dramatic re-localization, and the signal became restricted to the area between the separating DNA until the end of meiosis when the signal dispersed before returning to the nuclear periphery during spore formation. These findings strongly imply that the nuclear periphery is a major site of protein degradation in fission yeast both in interphase and throughout mitosis. Furthermore they raise interesting questions as to the spatial organization of protein degradation during meiosis.     

Characterizing the pH-dependent stability and catalytic mechanism of the family 11 xylanase from the alkalophilic Bacillus agaradhaerens

The xylanase, BadX, from the alkalophilic Bacillus agaradhaerens was cloned, expressed and studied in comparison to a related family 11 xylanase, BcX, from B. circulans. Despite the alkaline versus neutral conditions under which these bacteria grow, BadX and BcX both exhibit optimal activity near pH 5.6 using the substrate o-nitrophenyl beta-xylobioside. Analysis of the bell-shaped activity profile of BadX yielded apparent pK(a) values of 4.2 and 7.1, assignable to its nucleophile Glu94 and general acid Glu184, respectively. In addition to having an approximately 10-fold higher k(cat)/K(m) value with this substrate at pH 6 and 40 degrees C, BadX has significantly higher thermal stability than BcX under neutral and alkaline conditions. This enhanced stability, rather than a shift in its pH-optimum, may allow BadX to hydrolyze xylan under conditions of elevated temperature and pH.     

Pro-sterol carrier protein-2: role of the N-terminal presequence in structure, function, and peroxisomal targeting

Although the 20-amino acid presequence present in 15-kDa pro-sterol carrier protein-2 (pro-SCP-2, the precursor of the mature 13-kDa SCP-2) alters the function of SCP-2 in lipid metabolism, the molecular basis for this effect is unresolved. The presequence dramatically altered SCP-2 structure as determined by circular dichroism, mass spectroscopy, and antibody accessibility such that pro-SCP-2 had 3-fold less alpha-helix, 7-fold more beta-structure, 6-fold more reactive C terminus to carboxypeptidase A, 2-fold less binding of anti-SCP-2, and did not enhance sterol transfer from plasma membranes. These differences were not due to protein stability since (i) the same concentration of guanidine hydrochloride was required for 50% unfolding, and (ii) the ligand binding sites displayed the same high affinity (nanomolar K(d) values) in the order: cholesterol straight chain fatty acid > kinked chain fatty acid. Laser scanning confocal microscopy and double immunofluorescence demonstrated that pro-SCP-2 was more efficiently targeted to peroxisomes. Transfection of l-cells or McAR7777 hepatoma cells with cDNA encoding pro-SCP-2 resulted in 45% and 59% of SCP-2, respectively, colocalizing with the peroxisomal marker PMP70. In contrast, l-cells transfected with cDNA encoding SCP-2 exhibited 3-fold lower colocalization of SCP-2 with PMP70. In summary, the data suggest for the first time that the 20-amino acid presequence of pro-SCP-2 alters SCP-2 structure to facilitate peroxisomal targeting mediated by the C-terminal SKL peroxisomal targeting sequence.     

The effect of ethanol on the phase transition temperature and the phase structure of monounsaturated phosphatidylcholines

Previous studies from our laboratories have delineated the relationship between the acyl chain asymmetry of mixed-chain phosphatidylcholines, C(X):C(Y)PC, and the effect of ethanol concentration, [EtOH], on the main phase transition temperature, T(m), and the phase structure of the lipid bilayer composed of C(X):C(Y)PC using differential scanning calorimetry and X-ray diffraction techniques [Huang and McIntosh, Biophys. J. 72 (1997) 2702--2709]. In the present work, we have extended these studies to characterize the effect of [EtOH] on the T(m) and the phase structure of the lipid bilayer composed of sn-1 saturated/sn-2 monounsaturated phosphatidylcholines with various positions of the cis double bond. Specifically, five positional isomers of 1-eicosanoyl-2-eicosenoyl-sn-glycero-3-phosphocholines, C(20):C(20:1 Delta(n))PC with n=5, 8, 11, 13 and 17, were synthesized and studied. For C(20):C(20:1 Delta(n))PC with n=5 and 8, results from the calorimetric experiments showed that in response to various concentrations of ethanol, the change in T(m) of the lipid bilayer composed of monounsaturated lipids was characterized by a sigmoidal or biphasic profile in the plot of T(m) versus [EtOH]. In contrast, a continuous depression of the T(m) by ethanol was observed calorimetrically for C(20):C(20:1 Delta(n))PC with n> or =11. The X-ray diffraction experiments further demonstrated that C(20):C(20:1 Delta(5))PC and C(20):C(20:1 Delta(8))PC can undergo the ethanol-induced gel-to-fully interdigitated phase transition at T相似文献   

Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching

The diffusion coefficient of tubulin has been measured in the cytoplasm of eggs and embryos of the sea urchin Lytechinus variegatus. We have used brain tubulin, conjugated to dichlorotriazinyl-aminofluorescein, to inject eggs and embryos. The resulting distributions of fluorescence were perturbed by bleaching with a microbeam of light from the 488-nm line of an argon ion laser. Fluorescence redistribution after photobleaching was monitored with a sensitive video camera and photography of the television-generated image. With standard photometric methods, we have calibrated this recording system and measured the rates of fluorescence redistribution for tubulin, conjugated to dichlorotriazinyl-aminofluorescein, not incorporated into the mitotic spindle. The diffusion coefficient (D) was calculated from these data using Fick's second law of diffusion and a digital method for analysis of the photometric curves. We have tested our method by determining D for bovine serum albumin (BSA) under conditions where the value is already known and by measuring D for fluorescein-labeled BSA in sea urchin eggs with a standard apparatus for monitoring fluorescence redistribution after photobleaching. The values agree to within experimental error. Dcytoplasmtubulin = 5.9 +/- 2.2 X 10(-8) cm2/s; DcytoplasmBSA = 8.6 +/- 2.0 X 10(-8) cm2/s. Because DH2OBSA = 68 X 10(-8) cm2/s, these data suggest that the viscosity of sea urchin cytoplasm for protein is about eight times that of water and that most of the tubulin of the sea urchin cytoplasm exists as a dimer or small oligomer, which is unbound to structures that would impede its diffusion. Values and limitations of our method are discussed, and we draw attention to both the variations in D for single proteins in different cells and the importance of D for the upper limit to the rates of polymerization reactions.