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.     

Optical Anisotropy of Chromatin. Flow Linear Dichroism and Electric Dichroism Studies

Abstract

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentraitons by the use of flow linear dichroism (LD) and electric dichroism.

We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA—2 mM NaCl and close positive values in the range of 2–100 mM NaCl. Mg²⁺ cations, in contrast to Na⁺ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5–10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers.

The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA—for chromatins with the linker DNA of 10–30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible.

Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin.

Presented resutls explain the contradictory data obtained by electrooptical and hydrooptical methods.     

Retinol Transport and Regeneration of Human Cone Photopigment

A PUZZLING characteristic of fundus reflectometry data is that cone photopigment regeneration occurs more rapidly after a brief full bleach than after a prolonged full bleach^1,2. The hypothesis that the relative slowness of recovery from extended photolysis results from a reduction in the store of 11-cis retinal available for photopigment synthesis¹ has been found unsatisfactory². We present here an alternative explanation which allows physical interpretation and quantitative characterization of the observed dependence of recovery rate on illuminance history.     

Coenzyme Reorientations in the Active Sites of Aspartate Aminotransferase Isoenzymes Studied by Linear Dichroism Method

Abstract

Cytosolic and mitochondrial pig heart aspartate aminotransferases (cAspAT and mAspAT) and chicken heart cAspAT have been oriented in a compressed slab of polyacrylamide gel and their linear dichroism LD spectra have been recorded. The coenzyme's tilt angles in the active sites of chicken cAspAT and pig mAspAT and their quasisubstrate complexes imitating catalytic intermediates have been computed. The computations are based on reduced linear dichroism values (ΔA/A), the known directions of the transition dipole moments in the coenzyme ring and atomic coordinates of the coenzyme obtained by X-ray crystallography. It has been found that formation of the enzyme complex with glutarate and protonation of the internal pyridoxal-lysine aldimine induce reorientations of the coenzyme. As a result of protonation, the coenzyme ring tilts by 27° in cAspAT and 13° in mAspAT. Formation of the external aldimine with 2-mehtylaspartate is accompanied by tilting of the coenzyme ring by 44° in cAspAT and 39° in mAspAT. For the quinonoid complex with erythro-3-hydroxyaspartate, the tilt angles were found to be 63° in cAspAT and 53° in mAspAT. It is inferred that the basic features of the active site dynamics are similar in the three AspATs studied. The diiferences in the coenzyme tilt angles between cAspAT and mAspAT may be linked to catalytic and structural peculiarities of the isoenzymes.     

Peptide Adsorption to Lipid Bilayers: Slow Processes Revealed by Linear Dichroism Spectroscopy

The adsorption and insertion kinetics for the association of two 34-residue cyclic peptides with phosphocholine membranes have been studied using circular and linear dichroism approaches. The two peptides studied are identical with the exception of two residues, which are both tyrosine in one of the peptides and tryptophan in the other. Both peptides adopt random coil conformations in solution in the absence of membranes and do not aggregate at concentrations below 20 μM. After addition to liposome dispersions, circular dichroism spectroscopy indicated that both peptides undergo an extremely rapid transformation to a β-conformation that remains unchanged throughout the remainder of the experiment. Linear dichroism (LD) spectroscopy was used to study the kinetics of membrane adsorption and insertion. The data were analyzed by nonlinear least squares approaches, leading to identification of a number of bound states and their corresponding LD spectra. Two pseudo-first order processes could be identified that were common to both peptides. The first occurred with a time constant of the order of 1 min and led to a bound state characterized by weak LD signals, with significant bands corresponding to the transitions of aromatic side chains. The second process occurred with an unusually long time constant of between 75 and 100 min, forming a state with considerably stronger positive LD absorbance in the far-ultraviolet region of the spectrum. For the tyrosine-substituted peptide, a third slow process with a long time constant (76 min) could also be delineated and was attributed to rearrangements of the peptide within the membrane.     

Photopigment coexpression in mammals: comparative and developmental aspects

In mammals, each cone had been thought to contain only one single type of photopigment. It was not until the early 1990s that photopigment coexpression was reported. In the house mouse, the distribution of color cones shows a characteristic division. Whereas in the upper retinal field the ratio of short wave to middle-to-long wave cones falls in the usual range (1:10), in the ventral retinal field M/L-pigment expression is completely missing. In the transitional zone, numerous dual cones are detectable (spatial coexpression). In other species without retinal division, dual cones appear during development, suggesting that M/L-cones develop from S-cones. Dual elements represent a transitory stage in M/L-cone differentiation that disappear with maturation (transitory coexpression). These two phenomena seem to be mutually exclusive in the species studied so far. In the comparative part of this report the retinal cone distribution of eight rodent species is reported. In two species dual cones appear in adult specimens without retinal division, and dual elements either occupy the dorsal peripheral retina, or make up the entire cone population. This is the first observation proving that all cones of a retina are of dual nature. These species are good models for the study of molecular control of opsin expression and renders them suitable sources of dual cones for investigations on the role and neural connections of this peculiar cone type. In the developmental part, the retinal maturation of other species is examined to test the hypothesis of transitory coexpression. In these species S-pigment expression precedes that of the M/L-pigment, but dual cones are either identified in a small number or they are completely missing from the developing retina. These results exclude a common mechanism for M/L-cone maturation: they either transdifferentiate from S-cones or develop independently.     

Phycomyces and the biology of light and color

Phycomyces has been in the laboratories for about 140 years, sometimes following trends and fashions, but often anticipating them. Researchers have been attracted by the sensitive and precise responses of Phycomyces to light and other stimuli, coupled with easy manipulations and good adaptation to laboratory life. It is a simple prototype of the many organisms that use light as a source of information but not as a significant source of energy. Growth, development, genetics, and carotene production have been other subjects of pioneering research. Phycomyces was the second organism, after us, known to require a vitamin. It was one of the first organisms in the research on spontaneous mutants and the second, after Drosophila, in which mutations were induced artificially. It was used to coin the concept and the name of heterokaryosis. Phycomyces heterokaryons offer unique experimental possibilities, for instance in the study of gene function in vivo and the causes of cell death. An overall impression of parsimony and combinatorial gene usage arises from the genetic analysis of the complex functions of this fungus. The main subjects of recent attention have been the various reactions to light, gravitropism, and some aspects of metabolism, particularly the production of carotene. Interest in Phycomyces is slacking because of the repeated failures at transforming it stably with exogenous DNA.     

Genomic organization of the fungus Phycomyces

The fungus Phycomyces blakesleeanus has a relatively small genome, 30 megabases (Mb), with a low guanine and cytosine (G+C) content, 35%; the coding sequences cloned to date all have a G+C content of about 50%. In order to investigate the organization of the genome of this fungus, we have cloned and sequenced 251 DNA fragments. One hundred and twenty-six clones were obtained by digestion with MspI (target sequence 5′-CCGG-3′) and 125 random clones were obtained by sonication. The average length of sequence obtained was about 200 base pairs (bp) and the total length was about 50 kilobases (kb). The G + C content is not homogeneous throughout the genome: sequences obtained after digestion with MspI have an average of 5% more G + C content than the random fragments, and are enriched in coding sequences. Fourteen MspI fragments show similarities to known proteins and 21 encode ribosomal RNA (rRNA). By contrast, only three of the random fragments are similar to known proteins and only one to a rRNA. We conclude that the Phycomyces genome is composed of G+C-rich genes surrounded by G+C-poor areas. Two clones have similarities to the transposase of the transposon Tcl from Caenorhabditis elegans. This result suggests the presence of a high copy number of a Tcl-like transposable element in the Phycomyces genome. Another clone was similar to the transposon Txl from Xenopus laevis. A novel repetitive nt sequence has been characterized; about 5% of the total genome is a repetition of any of two consensus sequences of 31 by named PrAI and PrA2.     

Characterizing the Human Cone Photoreceptor Mosaic via Dynamic Photopigment Densitometry

Densitometry is a powerful tool for the biophysical assessment of the retina. Until recently, this was restricted to bulk spatial scales in living humans. The application of adaptive optics (AO) to the conventional fundus camera and scanning laser ophthalmoscope (SLO) has begun to translate these studies to cellular scales. Here, we employ an AOSLO to perform dynamic photopigment densitometry in order to characterize the optical properties and spectral types of the human cone photoreceptor mosaic. Cone-resolved estimates of optical density and photosensitivity agree well with bulk estimates, although show smaller variability than previously reported. Photopigment kinetics of individual cones derived from their selective bleaching allowed efficient mapping of cone sub-types in human retina. Estimated uncertainty in identifying a cone as long vs middle wavelength was less than 5%, and the total time taken per subject ranged from 3–9 hours. Short wavelength cones were delineated in every subject with high fidelity. The lack of a third cone-type was confirmed in a protanopic subject. In one color normal subject, cone assignments showed 91% correspondence against a previously reported cone-typing method from more than a decade ago. Combined with cone-targeted stimulation, this brings us closer in studying the visual percept arising from a specific cone type and its implication for color vision circuitry.     

Uninucleate spores of Phycomyces

Summary Under most culture conditions only 0.3% of the vegetative spores of Phycomyces blakesleeanus are uninucleate. On an acidified minimal medium, the uninucleate fraction can be raised up to 4.5% of the spores. The spore population can be fractionated in a gradient under gravity (1xg) yielding fractions that contain over 80% uninucleate spores. These uninucleate spores are fully viable. When the spores to be fractionated are obtained from a heterokaryotic mycelium, the uninucleate fraction produces homokaryotic mycelia.     

Absorption and Screening in Phycomyces

In vivo absorption measurements were made through the photosensitive zones of Phycomyces sporangiophores and absorption spectra are presented for various growth media and for wavelengths between 400 and 580 mµ. As in mycelia, β-carotene was the major pigment ordinarily found. The addition of diphenylamine to the growth media caused a decrease in β-carotene and an increase in certain other carotenoids. Growth in the dark substantially reduced the amount of β-carotene in the photosensitive zone; however, growth on a lactate medium failed to suppress β-carotene in the growing zone although the mycelia appeared almost colorless. Also when diphenylamine was added to the medium the absorption in the growing zone at 460 mµ was not diminished although the colored carotenoids in the bulk of the sporangiophore were drastically reduced. Absorption which is characteristic of the action spectra was not found. Sporangiophores immersed in fluids with a critical refractive index show neither positive nor negative tropism. Measurements were made of the critical refractive indices for light at 495 and 510 mµ. The critical indices differed only slightly. Assuming primary photoreceptors at the cell wall, the change in screening due to absorption appears too large to be counterbalanced solely by a simple effect of the focusing change. The possibility is therefore advanced that the receptors are internal to most of the cytoplasm; i.e., near the vacuole.     

Molecular Orientation in Quantasomes: III. A Flow Dichroism Apparatus and Its Application to the Study of the Structure of Spinach Quantasomes

A new apparatus is described for measuring dichroism spectra with very high sensitivity for macromolecular structures oriented in a hydrodynamic gradient. The method has been used to explore the dichroism spectrum of quantasome aggregates isolated from spinach chloroplasts. The quantasome flow dichroism resembles qualitatively that observed previously using electric field orientation, in that a pigment absorbing at wavelengths longer than 680 mμ exhibits appreciably greater dichroism than those absorbing at shorter wavelengths. It is shown that the absorption oscillator for this long wavelength absorption lies parallel to the streamlines of the sheer gradient, which is assumed to be the direction in which the planes of the chloroplast lamellae are oriented.     

Protein Fiber Linear Dichroism for Structure Determination and Kinetics in a Low-Volume, Low-Wavelength Couette Flow Cell

High-resolution structure determination of soluble globular proteins relies heavily on x-ray crystallography techniques. Such an approach is often ineffective for investigations into the structure of fibrous proteins as these proteins generally do not crystallize. Thus investigations into fibrous protein structure have relied on less direct methods such as x-ray fiber diffraction and circular dichroism. Ultraviolet linear dichroism has the potential to provide additional information on the structure of such biomolecular systems. However, existing systems are not optimized for the requirements of fibrous proteins. We have designed and built a low-volume (200 μL), low-wavelength (down to 180 nm), low-pathlength (100 μm), high-alignment flow-alignment system (couette) to perform ultraviolet linear dichroism studies on the fibers formed by a range of biomolecules. The apparatus has been tested using a number of proteins for which longer wavelength linear dichroism spectra had already been measured. The new couette cell has also been used to obtain data on two medically important protein fibers, the all-β-sheet amyloid fibers of the Alzheimer's derived protein Aβ and the long-chain assemblies of α₁-antitrypsin polymers.     

Photopigment and receptor properties in Drosophila compound eye and ocellar receptors

A review of the spectral sensitivity and the rhodopsin and metarhodopsin characteristics in three compound eye receptor types (R1–6, R7, and R8) and ocellar receptors is presented (Fig. 1). Photopigment properties were determined from measures of conversion efficiency. The photopigments of R1–6 were studied using in vivo microspectrophotometry in the deep pseudopupil of white-eyed flies. These studies yielded a refined estimate of the R1–6 metarhodopsin spectrum (Fig. 2). The quantum efficiency relative to the spectral sensitivity estimate of the rhodopsin spectrum was factored out. The quantum efficiency of rhodopsin is about 1.75 times that of metarhodopsin. The peak absorbance of metarhodopsin was estimated to be about 2.6 times that of rhodopsin. The mechanism of the two-peaked R1–6 spectral sensitivity and metarhodopsin spectrum is discussed in terms of evidence that there is only one rhodopsin in R1–6 and that vitamin A deprivation preferentially lowers ultraviolet sensitivity. The prolonged depolarizing afterpotential is reviewed from the standpoint of the internal transmitter hypothesis of visual excitation. A careful comparison of the intensity-responsivity for photopigment conversion and its adaptional consequences is made (Fig. 3).     

Photopigment spectral absorbance of Lake Malaŵi cichlids

To predict spectral sensitivity, microspectrophotometry (MSP) was used to measure absorbance of photoreceptor cells from 15 species of Lake Malaŵi cichlids. Each fish had one rod and at least three cone pigments. UV‐sensitive pigments were common, but spectral sensitivity did not clearly correlate with feeding mode or habitat.     

Photopigment and receptor properties in Drosophila compound eye and ocellar receptors

A review of the spectral sensitivity and the rhodopsin and metarhodopsin characteristics in three compound eye receptor types (R1-6, R7, and R8) and ocellar receptors is presented (Fig. 1). Photopigment properties were determined from measures of conversion efficiency. The photopigments of R1-6 were studied using in vivo microspectrophotometry in the deep pseudopupil of white-eyed flies. These studies yielded a refined estimate of the R1-6 metarhodopsin spectrum (Fig. 2). The quantum efficiency relative to the spectral sensitivity estimate of the rhodopsin spectrum was factored out. The quantum efficiency of rhodopsin is about 1.75 times that of metarhodopsin. The peak absorbance of metarhodopsin was estimated to be about 2.6 times that of rhodopsin. The mechanism of the two-peaked R1-6 spectral sensitivity and metarhodopsin spectrum is discussed in terms of evidence that there is only one rhodopsin in R1-6 and that vitamin A deprivation preferentially lowers ultraviolet sensitivity. The prolonged depolarizing afterpotential is reviewed from the standpoint of the internal transmitter hypothesis of visual excitation. A careful comparison of the intensity-responsivity for photopigment conversion and its adaptional consequences is made (Fig. 3).