Scanning force microscopy reveals ellipsoid shape of chicken erythrocyte nucleosomes.

Scanning force microscopy was used to investigate the conformation of hypotonic spread chicken erythrocyte nucleosomes. Nucleosomal chains were prepared in low-salt conditions and fixed before centrifugation onto glass coverslips and air drying. The images of single nucleosomes were isolated by image processing, and the height and geometry of the resulting three-dimensional structures were investigated. An average nucleosome height of 4.2 +/- 1.1 nm was determined. A virtual cross section at half-maximum height of the nucleosome structure was used for a characterization of the nucleosome geometry. The shape of this cross section was best described by an ellipse with an aspect ratio (major/minor axis) of approximately 1.30.     

Mutagen sensitivity and suppression of position-effect variegation result from mutations in mus209, the Drosophila gene encoding PCNA.

The mus209B1 mutant of Drosophila melanogaster exhibits a complex pleiotropy of temperature-sensitive (ts) lethality, hypersensitivity to DNA-damaging agents such as ionizing radiation and methyl methanesulfonate, suppression of position-effect variegation (PEV), and female sterility. Our discovery that mus209 encodes proliferating cell nuclear antigen (PCNA), which is an indispensable component of the DNA replication apparatus, suggests that alterations to chromosome replication may underlie that pleiotropy. Nine lethal mutations, three of them ts, genetically define the Pcna locus. Temperature shift studies reveal that the vital function of PCNA is required throughout virtually all stages of fly development, and that maternally encoded PCNA is essential for embryogenesis. All three ts mutants strongly suppress PEV, which suggests a role for PCNA in chromatin assembly or modification.     

An NMR study of pyridine associated with DMPC liposomes and magnetically ordered DMPC-surfactant mixed micelles.

With molecular dynamics simulations of phospholipid membranes becoming a reality, there is a growing need for experiments that provide the molecular details necessary to test these computational results. Pyridine is used here to explore the interaction of planar aromatic groups with the water-lipid interface of membranes. It is shown by magic angle spinning 13C nuclear magnetic resonance (NMR) to bind between the glycerol and choline groups of dimyristoylphosphatidylcholine (DMPC) liposomes. The axial pattern for the 31P NMR spectrum of DMPC liposomes is preserved even with more than half of the interfacial sites occupied, indicating that pyridine does not disrupt the lamellar phase of this lipid. 2H NMR experiments of liposomes in deuterium oxide demonstrate that pyridine might promote greater penetration of water into restricted regions in the interface. Magnetically oriented DMPC/surfactant micelles were investigated as a means for improving resolution and sensitivity in NMR studies of species bound to bilayers. The quadrupolar splittings in the 2H NMR spectra of d5-pyridine in DMPC liposomes and magnetically oriented DMPC/Trixon X-100 micelles indicate a common bound state for the two bilayer systems. The well resolved quadrupolar splittings of d5-pyridine in oriented micelles were used to establish the tilt of the pyridine ring relative to the bilayer plane.     

The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent.

The highly conserved zinc fingers in retroviral nucleocapsid (NC) proteins have the general structure Cys-(X)2-Cys-(X)4-His-(X)4-Cys. Human immunodeficiency virus type 1 (HIV-1) contains two Zn2+ fingers, and mutants were constructed in which the native sequence of each Zn2+ finger was maintained but their positions in the NC protein were changed. Mutants had either two first-finger sequences (pNC1/1), two second-finger sequences (pNC2/2), or reversed first- and second-finger sequences (pNC2/1). Cells transfected with mutant or wild-type clones produced similar levels of Tat, Gag, Pol, and Env proteins, formed syncytia, and shed viruslike particles that were indistinguishable by electron microscopy. However, the pNC2/1 and pNC2/2 mutants were inefficient in packaging genomic RNA (less than 15% of wild-type levels), whereas the pNC1/1 mutant packaged approximately 70% of wild-type levels of RNA. No infectious virus could be detected with either the pNC2/1 or pNC2/2 mutants, whereas the pNC1/1 mutant appeared to sustain a low level of replication and reverted to a competent wild-type-like viral species after a 2- to 4-week lag period. The data strongly suggest that the two Zn2+ fingers of HIV-1 are not functionally equivalent and that the first Zn2+ finger in the Gag precursor plays a more prominent role in RNA selection and packaging. The data also indicate that both Zn2+ fingers in the mature NC protein play as yet unknown roles in viral assembly or the early stages of the viral infection process.     

Electron diffraction analysis of structural changes in the photocycle of bacteriorhodopsin.

Structural changes are central to the mechanism of light-driven proton transport by bacteriorhodopsin, a seven-helix membrane protein. The main intermediate formed upon light absorption is M, which occurs between the proton release and uptake steps of the photocycle. To investigate the structure of the M intermediate, we have carried out electron diffraction studies with two-dimensional crystals of wild-type bacteriorhodopsin and the Asp96-->Gly mutant. The M intermediate was trapped by rapidly freezing the crystals in liquid ethane following illumination with a xenon flash lamp at 5 and 25 degrees C. Here, we present 3.5 A resolution Fourier projection maps of the differences between the M intermediate and the ground state of bacteriorhodopsin. The most prominent structural changes are observed in the vicinity of helices F and G and are localized to the cytoplasmic half of the membrane.     

Cyclic GMP-activated channels of the chick pineal gland: Effects of divalent cations,pH, and cyclic AMP

Chick pineal cells maintained in dissociated cell culture express an intrinsic photosensitive circadian oscillator, but the mechanisms of phototransduction in avian pinealocytes are not fully understood. In this study, we have used inside-out patches to examine the characteristics of cyclic GMP-activated channels of chick pinealocytes in more detail, concentrating on the effects of factors known to modulate the secretion of melatonin and/or the function of circadian pacemakers. In most patches, the predominant conductance state was 19 pS in symmetrical 145 mM NaCl. But in some patches, a second cyclic GMP-activated channel with a unitary conductance of 29 pS was also present. The current flowing through cyclic GMP-activated channels was not affected by application of salines containing 1 M Ca²⁺ to the cytoplasmic face of the patch membrane. By contrast, application of 1 mM Ca²⁺ caused a partial reduction in cyclic GMP-activated current at all membrane potentials. Application of 1–5 mM Mg²⁺ ions caused a virtually complete blockade of current at positive membrane potentials, but caused only a small decrease in current at negative membrane potentials. No obvious differences in the gating of cyclic GMP-activated channels were observed in pH 8.2, 7.4 or 6.2 salines. Application of salines containing 100 M, 500 M, or 1 mM cyclic AMP did not cause activation of the channels, but 5 mM cyclic AMP evoked a low level of channel activity. Application of 5 mM but not 100 M cyclic AMP decreased the probability of channel activation caused by 20–100 M cyclic GMP and also increased the percentage of openings to an 11 pS subconductance state. Thus, cyclic AMP acts as a weak partial agonist. Nevertheless, the gating of these channels does not seem to be controlled directly by physiologically relevant changes in intracellular Ca²⁺, pH, or cyclic AMP.     

Radiolabelling Studies of Lipids in the Marine Cryptomonad Chroomonas salina in Relation to Fatty Acid Desaturation

Cells of Chroomonas salina were exposed to [¹⁴C]acetate, [^l4C]16:0,[¹⁴C]18:0, [¹⁴C]18:1(n-9), [¹⁴C]18:2(n–6) or [¹⁴C]18:3(n–3)for 1 h and then incubated for 24 h in non-radioactive medium.At the end of the pulse period, non-glycolipid polar lipidscontained the highest proportions of radioactivity incorporatedfrom [¹⁴C]acetate and [¹⁴C]18:3(n–3) whereas with [¹⁴C]16:0,[¹⁴C]18:1 and [¹⁴C]18:2(n–6), triacylglycerols were mosthighly labelled. ¹⁴C-18:0 was recovered mainly as non-esterifiedfatty acid. Monogalactosyldiacylglycerol initially contained17% of label incorporated from [¹⁴C]acetate but less than 3%of that from [¹⁴C]fatty acids. With all substrates, excluding[¹⁴C]18:0, a gradual transfer of label from polar lipids totriacylglycerols was observed during the chase period. Saturatesand monoenes synthesised from [¹⁴C]acetate were mostly transferedfrom phospholipids and glycolipids to neutral lipid withoutfurther desaturation. Most of the incorporated ¹⁴C-fatty acidsremained unchanged and only with [¹⁴C]18:3(n–3) was substantialamounts of label recovered in penta- and hexaenoic fatty acids.The results indicate that, under the conditions of the study,lipid synthesis in the algae was heavily dominated by triacylglycerolformation and that the mechanisms of fatty acid desaturationin this species may differ from those in higher plants. (Received December 10, 1991; Accepted March 6, 1992)     

Kinetic studies of electron transfer in the cyt b 5 : metHb system

 The kinetics of methemoglobin reduction by cytochrome b ₅ has been studied by stopped-flow and saturation transfer NMR. A forward rate constant k _f = 2.44×10⁴ M^–1 s^–1 and a reverse rate constant k _b = 540 M^–1s^–1 have been observed at 10 mm, pH 6.20, 25  °C. The ratio k _f/k _b = k _eq = 43.6 is in good agreement with the equilibrium constant calculated from the electrochemical potential between cyt b ₅ and methemoglobin. A bimolecular collisional mechanism is proposed for the electron transfer from cyt b ₅ to methemoglobin based on the kinetic data analysis. The dependence of the rate constants on ionic strengths supports such collisional mechanism. It is also found that the reaction rate strongly depends on the conformations of methemoglobin. Received: 20 February 1996 / Accepted: 4 June 1996     

Isolation and characteristics of a wheatbran-degrading Butyrivibrio from human faeces

Screening over 100 isolates from human faeces for cellulolytic activity led to the isolation of a weakly cellulolytic anaerobic, curved, motile bacterium which produced H₂, lactate and butyrate from wheatbran. The mol% of G + C in the DNA was 39–42. These properties, together with the Gram-positive cell wall ultrastructure and SDS-PAGE profile, are consistent with the genus Butyrivibrio. The isolate is believed to be the most active wheatbran-degrading bacterium so far described.