Chromatin structure studied by linear dichroism at different salt concentrations

We have studied the linear dichroism (LD) of rat liver chromatin oriented by flow. Soluble chromatin, prepared by brief nuclease digestion, is found to exhibit a positive LD at low ionic strength (1 mM NaCl), with a constant LD/A over the absorption band centered at 260 nm (A, isotropic absorbance). Several previous dichroism studies on soluble chromatin have been performed on sonicated materials and have given negative LD, probably due to the presence of uncoiled DNA. The positive dichroism can be interpreted in terms of a supercoil of DNA in chromatin with a pitch angle larger than 55°, and is, for example, consistent with a model where the cylindrical nucleosome core particles are stacked face to face in the chromatin filament. In contrast to the nuclease-digested chromatin, sonicated chromatin was confirmed to exhibit negative LD. This difference can be attributed to a partial uncoiling of the linker regions between the nucleosomes due to the shearing. The structural transition of chromatin to a compact form can be observed as a reduction of the positive LD of the nuclease-digested chromatin to almost zero in 0.1 M NaCl or in 0.1 mM MgCl₂. This transition is due to a decreased electrostatic repulsion between negative phosphate groups on the DNA chain. In the case of Na⁺, this can be explained as a screening effect due to the bulk concentration of Na⁺. With Mg²⁺ a considerably stronger effect may indicate a more localized binding to the phosphates. At ionic strengths higher than 0.5M NaCl, the dissociation of the histones from DNA leads to uncoiling of chromatin. The change in LD during this process shows that histone H1 contributes only to a small degree to the coiling of the DNA chain, whereas histones H3 and H4 play the major role in the coiling.     

Reinterpretation of Linear Dichroism of Chromatin Supports a Perpendicular Linker Orientation in the Folded State

Abstract

We present a reinterpretation of linear dichroism data for the salt induced condensation of chromatin. A conflict between electric and flow linear dichroism data for identical chromatin samples, studied at varying degrees of Mg²⁺ induced folding, can be solved if the orientation in electric fields is mainly determined through the polarization of counter ions along the linker parts, whereas the orientation in flow is governed by the hydrodynamical response of the entire chromatin fiber. The orientation of a chromatin fiber in an electric field would then depend on the linker tilt angle so that at an angle larger than 55° the fiber would tend to orient perpendicular to the applied field. The different orientation distributions obtained with the two methods of alignment may in this way provide extra information about the structure and folding of chromatin.     

Structure of hyperacetylated chromatin: light scattering and flow linear dichroism study

Cation-induced folding of 10 nm chromatin filament to 30 nm fiber was studied with hyperacetylated chromatin using light scattering at 90 degrees and flow linear dichroism. Acetylated chromatin folded in a way indistinguishable from that of the control chromatin: both the compactness of chromatin and the orientation of nucleosomes relative to the fiber axis were identical at a given salt concentration.     

Structural transitions of chromatin at low salt concentrations: a flow linear dichroism study

We have studied the structure of nuclease-solubilized chromatin from Ehrlich ascites cells by flow linear dichroism (LD) using the anisotropic absorption of the DNA bases and of two intercalated dyes, ethidium bromide and methylene blue. It is confirmed that intercalation occurs preferentially in the linker part of the chromatin fiber, at binding ratios (dye/base) below 0.020. Using this information, we determined the orientation of the linker in relation to the average DNA organization in chromatin. The LD measurements indicate that the conformation of chromatin is considerably changed in the ionic strength interval 0.1-10 mM NaCl: with increasing salt concentration, the LD of the intrinsic DNA base absorption changes signs, from negative to positive, at approximately 2.5 mM NaCl. The LD of the intercalated dyes also changes signs, however, at a somewhat higher salt concentration. The results are analyzed in terms of possible allowed combinations of tilt angles of nucleosomes and pitch or tilt angles of linker DNA sections relative to the fiber axis, at different salt concentrations in the interval 0.1-10 mM NaCl. Two models for the salt-induced structural change of chromatin are discussed.     

Concentration of protein in fibrin fibers and fibrinogen polymers determined by refractive index matching

We have used refractive index matching to determine the concentration of protein in the fibers in fibrin clots and of needlelike crystals of native fibrinogen. Our results are in agreement with those of Carr and Hermans [(1978) Macromolecules 11 , 46–50], as determined by light scattering—namely, that protein makes up about 20% of the volume of the fiber. However, we have found that the protein concentration is strongly dependent on ionic strength. An increase in ionic strength caused a substantial drop in the protein concentration. In a buffer containing 100 mM NaCl, the protein concentration was 26.6–29.8 g of protein per 100 cm³ of polymer, and at 200 mM NaCl it was reduced to 22.1–23.1 g/100 cm³.     

Molecular flexibility of extended and compacted polynucleosomes

We have studied the effects of Na⁺ (5–120 mM) and Mg²⁺ (0–6 mM) on the internal and overall flexibility of polynucleosome fragments from nucleasesolubilized chromatin from Ehrlich ascites cells. The mobility was monitored by the steady-state fluorescence polarization of the intercalated ethidium cation. The internal polynucleosome flexibility decreases continuously as the extended chromatin fragments are being compacted at increasing salt concentrations, and it can be further suppressed at ionic strengths above those where the 30 nm fiber is formed. The effect may be visualized as an initial formation of a loose 30 nm fiber that is further compacted at increasing ionic strengths. We observe several differences in the effects of Na⁺ and Mg²⁺ upon chromatin compaction. First, chromatin compacted by Mg²⁺ is less flexible than that compacted by Na⁺, suggesting a tighter chromatin structure with Mg²⁺. Second, Mg²⁺ affects the internal mobility in polynucleosome fragments shorter than 6–7 nucleosomes, which are too short to be compacted with Na⁺. Third, Mg²⁺ causes extensive macroscopic aggregation at concentrations above 0.2–0.3 mM, but the aggregation is uncorrelated with the intramolecular compaction. A quantitative evaluation of the overall polynucleosome tumbling mobility indicates that the compacted fragments possess more internal flexibility than do corresponding high molecular weight chromatin fibers. Finally, we note a correlation between the ethidium binding constant and the internal chromatin flexibility, possibly arising from lower torsional and unwinding flexibility of the linker DNA segments of compacted chromatin fibers.Abbreviations FPA fluorescence polarization anisotropy - CT calf thymus - HMW high molecular weight - ARF amplitude reduction factor - kbp kilobasepairs This project is supported by the Swedish Natural Research Council. P.E.N. is the recipient of a Hallas-Mølle Fellowship through the NOVO Foundataion     

Reinterpretation of linear dichroism of chromatin supports a perpendicular linker orientation in the folded state

We present a reinterpretation of linear dichroism data for the salt induced condensation of chromatin. A conflict between electric and flow linear dichroism data for identical chromatin samples, studied at varying degrees of Mg2+ induced folding, can be solved if the orientation in electric fields is mainly determined through the polarization of counter ions along the linker parts, whereas the orientation in flow is governed by the hydrodynamical response of the entire chromatin fiber. The orientation of a chromatin fiber in an electric field would then depend on the linker tilt angle so that at an angle larger than 55 degrees the fiber would tend to orient perpendicular to the applied field. The different orientation distributions obtained with the two methods of alignment may in this way provide extra information about the structure and folding of chromatin.     

Structural changes in bacteriorhodopsin induced by electric impulses

Electric impulses of high field intensity (2 × 10⁵ to 3 × 10⁶ Vm^?1, 1 to 20 μs duration) cause transient changes in the optical absorbance of suspended purple membranes of Halobacterium halobium. The electric dichroism at 1 mm NaCL, pH ≈ 6 and at 293K is dependent on field strength, pulse duration and wavelength of the monitoring, plane-polarized light in the range 400 to 650 nm. The optically detected processes are, however, independent of bacteriorhodopsin concentration, of ionic strenght and of the intensity of the monitoring light. These data together with the analysis of time course ands steady state of the reduced dichroism, suggest electric field-sensitive, intramemembraneous structural changes which lead to restricted orientation changes of the chromophore. A thoretical analysis of restricted orientation is developed and applied to the electro-optic data. As a result, it is found that the electric dichroism of purple membrane is associated with a large polarizability anisotropy of 2.4 × 10^?30 Fm² (2.2 × 10^?14 cm³); the electric permanent dipole moment which is involved amounts to 4.7 × 10^?28 Cm(140 Debye). The kinetic data suggest a cyclic reaction scheme with at least five different conformations. The high polarizability is probably due to displaceable ionic groups within the cooperative lattice of bacteriorhodopsin molecules in purple membranes.     

DNA orientation in shear flow

The linear dichroism (LD) has been measured for DNA molecules 239–164,000 base pairs long oriented in shear flow over a large range of velocity gradients (30–3,000 s ^?1) and ionic strengths (2–250 mM). At very low gradients, the degree of DNA orientation increases quadratically with the applied shear as predicted by the Zimm theory [J. Zimm, (1956) Chemical Physics, Vol. 24, p. 269]. At higher gradients, the orientation of fragments ≥ 7 kilobase pairs (kbp) increases linearly with increasing shear, whereas the orientation of fragments ≥ 15 kbp shows a more complicated dependence. In general, the orientation decreases with increasing ionic strength throughout the studied ionic strength interval, owing to a decrease in the persistence length of the DNA. The effect is most dramatic at ionic strengths below 10 mM, and is more pronounced for longer DNA fragments. For fragments ≥ 15 kbp and velocity gradients ≥ 100 s^?1, the orientation can be adequately described by the empirical relation: LD^r= –(k₁-G)/(k₂ + G), where k₁is a linear function of the square root of the ionic strength and k₂ depends on the DNA contour length. Since the DNA persistence length can be represented as a linear function of the reciprocal square root of the ionic strength [D. Porschke, (1991) Biophysical Chemistry, Vol. 40, p. 169], extrapolation of the empirical relation provides information about the stiffness of the DNA fibers. © 1993 John Wiley & Sons, Inc.     

Membrane properties of identified embryonic nerve and glial cells of the leech central nervous system

Summary The membrane potential of identified nerve (Retzius) cells and neuropil glial cells from 11 (±1) day-old embryos of the leechHirudo medicinalis was recorded using conventional intracellular microelectrodes. At this stage all ganglia of the segmental nervous system are formed. The membrane potential of Retzius cells was –68±4 mV (±SD,n=8), and showed a slope of 42 mV between 10 mM and 100 mM external K concentration. Retzius cells were able to fire action potentials which had a fast Na-dependent component, and, under appropriate conditions, also generated slow Ca (Ba) action potentials. The mean membrane potential of the neuropil glial cell at physiological K concentration (4 mM) was –83±5 mV (±SD,n=10), and showed a dependence of 56 mV for a tenfold change in the external K concentration (> 4mM). Neuropil glial cells showed no signs of voltage-activated excitability, but they repeatedly depolarized in the presence of 0.1 mM 5-HT.     

Lactate: a substrate for reptilian muscle gluconeogenesis following exhaustive exercise

Summary The pattern of lactate and glycogen metabolism in red and white muscle fibers was examined in fasted, cannulated lizards (Dipsosaurus dorsalis) run on a treadmill to exhaustion. The white and red portions of the iliofibularis (wIF, rIF) muscle of the hindlimb were analyzed post-exercise and at intervals over 120 min of recovery for lactate and glycogen changes. Five min of exercise resulted in lactate concentrations of from 35 mM (rIF) to 48 mM (wIF) while blood lactate concentrations were elevated to 21 mM from resting levels of 1.8 mM. Glycogen depletion was significant (p<0.05) in whole hindlimb (–30%) and in wIF (–42%) but not in rIF (–25%). Metabolite changes were consistent with a pattern of fiber type recruitment favoring fast-twitch glycolytic (FG) fibers during high intensity locomotion. Glycogen replenishment during recovery was fiber typespecific. After 2 h recovery, whole hindlimb glycogen concentration had increased 24% above pre-exercise levels (p<0.05). Rates of glycogen resynthesis during recovery were significant only in oxidative fibers of the red iliofibularis. Animals were infused with either [U-¹⁴C]-lactate or [U-¹⁴C]-glucose at the point of exhaustion, and label incorporation into muscle glycogen was used to estimate the substrate preference for glycogenesis during recovery. Lactate uptake and incorporation occurred in both wIF and rIF. Glucose uptake and incorporation into glycogen was greatest in the rIF, where it equalled 9% of the rate of lactate incorporation. The rate of lactate incorporation could account for 67% of the rate of glycogen synthesis that occurred in oxidative fibers of the rIF. The data indicate that in contrast to mammalian muscle, reptilian muscle replenishes glycogen while it removes lactate, utilizing lactate directly as a gluconeogenic substrate. The data also suggest that lactate produced by FG fibers during exercise is utilized by oxidative fiber types post-exercise to synthesize glycogen in excess of pre-exercise levels.Abbreviations wIF, rIF white, red portions of iliofibularis muscle - FG fast-twitch, glycolytic muscle fiber - FOG fast-twitch, oxidative, glycolytic muscle fiber - HPLC high performance liquid chromatography - SA specific activity - [LA] lactate concentration - GLU glucose - ANOVA analysis of variance - C.I. confidence interval     

Effect of sucrose,inorganic salts,inositol, and thiamine on protease excretion during pineapple culture in temporary immersion bioreactors

Summary Although pineapple plants have been found to produce proteases ex vitro, most of the biotechnological investigations of this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore, we decided to modify the culture medium composition of this phase looking for an increase in protease excretion. Four independent experiments were performed to evaluate the effects of different levels of sucrose (0–350.4 mM), inorganic salts [0–200% Murashige and Skoog (MS) salt strength], inositol (0–2.20 mM), and thiamine (0–1.2μM). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. Specific protease activity, the most important indicator recorded, was highest with 262.8 mM sucrose, 100% MS salt strength, 0.3 μM thiamine and no inositol. Results shown here demonstrate that conditions adequate for propagation purposes (87.6 mM sucrose, 100% MS salt strength, 0.55 mM inositol, 0.3 μM thiamine) are not always adequate for protease excretion.     

Elastin as a random-network elastomer: A mechanical and optical analysis of single elastin fibers

The physical properties of single, 5–8-μm diameter, water-swollen elastin fibers have been investigated on a microtest apparatus attached to a polarizing microscope. Analysis of the mechanical and optical properties at extensions below 100% indicate that the elastic modulus (G) has a value of 4.1 × 10⁵ N m^?2, the average molecular weight of chains between crosslinks is in the range of 6000–7100, and the stress optical coefficient (C′) is 1 × 10^?9 m² N^?1 at 24°C. Analysis of the temperature dependence of the stress optical coefficient indicates that the polarizability of the random link decreases with increasing temperature, with an apparent activation energy for this process of the order of 1.6 kcal/mol. Analysis of the non-Gaussian mechanical and optical properties at extensions above about 100% suggest that the chains between crosslinks contain approximately 10 “effective” random links, with each link consisting of 7–8 amino acid residues. These parameters for the random chains in the elastin network have been used to predict the dimensions of other random proteins. The close correlation of these predictions with published values for the dimensions of a series of proteins in solution in 6M guanidinium hydrochloride provides an independent test of the appropriateness of our analysis.     

Antigen-induced changes in lymphoid cell histones. IV. Changes in solubility of isolated chromatin

In this study we have examined the solubility of deoxyribonucleoprotein (DNP) isolated from control and antigen-affected thymocytes. 2-M sodium chloride extracts containing the DNP of rat thymus glands were serially diluted. A comparison was made of the effect of dilution on fiber formation in the control and test series. Fiber formation is usually complete for the control material at a salt concentration between 0.63 and 0.57 M. The test material shows some fiber formation within this range. However, a significant portion of the DNP is precipitated at dilutions of 0.54–0.48 M. Ammoniacal silver (A-S) stains the control fibers a characteristic yellowish color. With the test material, those fibers formed within the control range tended to be stained yellowish brown by A-S, whereas those formed only after greater dilution stained blackish. These data, coupled with our previous observations on altered A-S staining, clearly demonstrate an antigen-induced physical and/or chemical alteration of the histone or histone-DNA complex of lymphoid cell chromatin.     

Mechanochemical study of conformational transitions and melting of Li-, Na-, K-, and CsDNA fibers in ethanol–water solutions

Highly oriented fibers of Li-, Na-, K-, and CsDNA were prepared with a previously developed wet spinning method. The procedure gave a large number of equivalent fiber bundle samples (reference length, L₀, typically = 12–15 cm) for systematic measurements of the fiber length L in ethanol–water solutions, using a simple mechanochemical set up. The decrease in relative length L/L₀ with increasing ethanol concentration at room temperature gave evidence for the B-A transition centered at 76% (v/v) ethanol for NaDNA fibers and at 80 and 84% ethanol for K- and CsDNA fibers. A smaller decrease in L/L₀ of LiDNA fibers was attributed to the B-C transition centered at 80% ethanol. In a second type of experiment with DNA fibers in ethanol–water solutions, the heat-induced helix–coil transition, or melting, revealed itself in a marked contraction of the DNA fibers. The melting temperature T_m, decreased linearly with increasing ethanol concentration for fibers in the B-DNA ethanol concentration region. In the B-A transition region, Na- and KDNA fibers showed a local maximum in T_m. On further increase of the ethanol concentration, the A-DXA region followed with an even steeper linear decrease in T_m. The dependence on the identity of the counterion is discussed with reference to the model for groove binding of cations in B-DNA developed by Skuratovskii and co-workers and to the results from Raman studies of the interhelical bonds in A-DNA performed by Lindsay and co-workers. An attempt to apply the theory of Chogovadze and Frank-Kamenetskii on DNA melting in the B-A transition region to the curves failed. However, for Na- and KDNA the T_m dependence in and around the A-B transition region could be expressed as a weighted mean value of T_m of A- and B-DNA. On further increase of the ethanol concentration, above 84% ethanol for LiDNA and above about 90% ethanol for Na-, K-, and CsDNA, a drastic change occurred. T_m increased and a few percentages higher ethanol concentrations were found to stabilize the DNA fibers so that they did not melt at all, not even at the upper temperature limit of the experiments (～ 80°C). This is interpreted as being due to the strong aggregation induced by these high ethanol concentrations and to the formation of P-DNA. Many features of the results are compatible with the counterion–water affinity model. In another series of measurements, T_m of DNA fibers in 75% ethanol was measured at various salt concentrations. No salt effect was observed (with the exception of LiDNA at low salt concentrations). This result is supported by calculations within the Poisson–Boltzmann cylindrical cell model. © 1994 John Wiley & Sons, Inc.     

Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl),pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65°C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasingpH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55°C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5°C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.     

Production of alkaline protease with Bacillus licheniformis in a controlled fed-batch process

Summary A production method for alkaline serine protease with Bacillus licheniformis in a synthetic medium was developed. Employing closed-loop control of oxygen, nitrogen and carbon source the pO₂ was held at 5%, the ammonium concentration kept below 1 mM and the glycerol concentration was maintained between 20 and 100 mM. Protease production was monitored by flow injection analysis. Thus, in a fed-batch procedure production could be increased 4.6-fold in comparison to an uncontrolled batch process. Offprint requests to: G. Bierbaum     

Linear relaxation analysis of the mechanochemical transformation of collagen fibers

The isometric tensile stress generation observed when collagen fibers are immersed in aqueous solutions of lithium bromide ranging in molar concentration up to 7 was studied at 23°C. The reverse process, namely, isometric stress relaxation of the fiber occurring by subsequent immersion in distilled water, was also studied. We find that the data in the region of LiBr concentration up to about 2.5 moles/liter are adequately represented by a superposition integral where σ(t) is the time-dependent stress generated by the collagen fiber held at fixed length, c(t) is the history of LiBr molar concentration, and K(t) is the isometric contractility function, expressed as stress per unit salt concentration. We conclude that, within a limited range of salt concentration, a collagen fiber in a LiBr bath behaves as if it were a linear, time-invariant system defined mechanochemically by a single function K(t) which depends on the structural characteristics of the fiber while being independent of salt concentration. An analysis is presented of isometric mechanochemical data obtained under conditions of equilibrium by other workers who studied the behavior of collagen fibers in aqueous solutions either of urea, LiBr, or KCNS. The analysis shows that these independent (equilibrium) data confirm the linarity of the relation between isometric contractile stress and salt concentration on which our superposition integral representation is based. We also find that the asymptotic (infinite-time) value of the isometric stress is linearly related to the chemical potential of the salt as well, in agreement with the equilibrium thermodynamic treatment of mechanochemical processes by Katchalsky and Oplatka.     

Higher order structure of chromatin: orientation of nucleosomes within the 30 nm chromatin solenoid is independent of species and spacer length

We have used electric dichroism to study the arrangement of nucleosomes in 30 nm chromatin solenoidal fibers prepared from a variety of sources (CHO cells, HeLa cells, rat liver, chicken erythrocytes, and sea urchin sperm) in which the nucleosome spacer length varies from approximately 10 to approximately 80 bp. Field-free relaxation times are consistent only with structures containing 6 +/- 1 nucleosomes for every 11 nm of solenoidal length. With very few assumptions about the arrangement of the spacer DNA, our dichroism data are consistent with the same orientation of the chromatosomes for every chromatin sample examined. This orientation, which maintains the faces of the radially arranged chromatosomes inclined at an angle between 20 degrees-33 degrees to the solenoid axis, thus appears to be a general structural feature of the higher order chromatin fiber.