[TGC Repeats in Intron 2 of the TCF4 Gene have a Good Predictive Power Regarding to Fuchs Endothelial Corneal Dystrophy]. / TGC-Repeats im Intron 2 des TCF4-Gens haben eine große Vorhersagekraft bezüglich Fuchs-Hornhautendotheldystrophie.

BACKGROUND: Fuchs endothelial corneal dystrophy (FECD) is one of the most common indications for corneal transplants. FECD is associated with various genes, e.g., COL8A2 or SLC4A11. Among other things a TGC trinucleotide repeat expansion in intron 2 of the TCF4 gene has been characterised in FECD patients and the allele G of the polymorphism rs613872 in intron 3 of the same gene has been associated with this disease. Our intention was to investigate sources in molecular genetics in the German population and to calculate the odds ratio as indicator for the chance to suffer from FECD. PATIENTS AND METHOD: 42 unrelated FECD patients, 93 unrelated controls and 17 members of a family with four FECD affected patients have been examined for the described changes in the TCF4 gene. After amplification of the TGC repeats with specific PCR the obtained products were electrophoretically divided according to their length and investigated with a triplet-primed PCR. Polymorphism rs613872 was analysed by Sanger sequencing. All coding exons of the adjacent genes TCF4 and LOXHD1 were sequenced in six patients in order to exclude potential disease associated mutations. RESULTS: 33 out of 42 unrelated analysed patients (79 %) had a TGC repeat expansion (> 50 TGC repeats) in intron 2 of the TCF4 gene. Out of 93 controls only 10 (10.8 %) showed an expanded allele. In the family the four diseased and four healthy subjects of the 17 examined family members had an expanded allele. Analysis of the polymorphism rs613872 in intron 3 of the TCF4 gene exhibited 33 of 42 unrelated patients (78.6 %) heterozygous TG and four homozygous GG (9.5 %). 65 of 93 controls were homozygous TT (69.9 %) and only 21 heterozygous TG (22.6 %). Of the 17 family members nine had the genotype TG, including the four FECD patients. Sequencing of the coding exons of TCF4 and LOXHD1 in six patients showed no variant described with FECD. The odds ratio as indicator for being affected by FECD in our data for the expanded TGC allele is 30. The chance of being affected is thus 30 times higher when someone exhibits the expanded allele. For a carrier of the risk allele G the chance is 16.5 times higher. DISCUSSION: An expanded TGC allele with more than 50 TGC repeats in intron 2 and the described risk allele G of the polymorphism rs613872 in intron 3 of the TCF4 gene appear as an association to FECD. The chance to be affected by FECD is up to 30 times higher. With molecular genetics also donors with clinically unknown FECD may be detected.

The stability of the B conformation in wet-spun films of CaDNA: a raman study as a function of water content.

Raman spectra from 600 to 1150 cm(-1) have been collected from wet-spun films of highly oriented CaDNA as a function of relative humidity (rh). Particular attention was paid to the Raman modes at 834 and 807 cm(-1); the presence of these modes indicates that the DNA backbone is in the B or A conformations. Though the B conformation is usually found only under conditions of high hydration in such wet-spun films, CaDNA is found to remain in the B conformation down to 75% rh. These results are consistent with the hypothesis that intermolecular bonds in ordered samples of DNA are critical for the stabilization of the A conformation of DNA.

Phonon dispersion of oriented DNA by inelastic x-ray scattering.

Films of oriented deoxyribonucleic acid (DNA), prepared by the wet spinning method, have been studied using inelastic x-ray scattering. Spectra were recorded within the range of energy transfers -30< homega <30 meV at momentum transfers homegaQ ranging from 2.5 to 30 nm(-1) whereby the direction of Q essentially coincided with the helical axis. Measurements at ambient temperature cover samples in the A, B, C, and D conformations of DNA. Within the limits of the instrumental resolution, the spectra were analyzed by the response of a damped harmonic oscillator delivering dispersion and damping of modes having displacements with nonzero projections onto Q, i.e., essentially the compression waves traveling along the helical axis. The longitudinal speed of sound resulting from the sinusoidal dispersion varies only weakly with conformation. Our sound speed values are compared to results from Brillouin spectroscopy. The dispersion curves exhibit a minimum at about the inverse rise per residue, which -- together with strong elastic scattering -- reflect the large degree of disorder. Overdamping of the modes is observed for Q>5 nm(-1). The possibility that the observed large damping parameters are due to several contributing modes is discussed in terms of a simple model calculation for an idealized double helix. Whereas the quasicrystalline approximation for an effective disordered chain could well describe the sinusoidal dispersion, it fails to reproduce the observed damping by one order of magnitude. Our results indicate that the high-frequency dynamics of DNA is liquidlike and is most appropriately described by instantaneous normal modes of short correlation length.

A Raman study of the hydration of wet-spun films of Li-hyaluronate.

Raman spectroscopy between 620 and 1700 cm(-1) has been performed on wet-spun films of Li-hyaluronate as a function of relative humidity between 54 and 93% at room temperature. The observed vibrational modes show no significant dependence on water content, suggesting that the molecule does not undergo any conformational transitions in this humidity range.

Chromosome banding in amphibia. XXIII. Giant W sex chromosomes and extremely small genomes in Eleutherodactylus euphronides and Eleutherodactylus shrevei (Anura, Leptodactylidae).

Highly differentiated, heteromorphic ZZ female symbol /ZW male symbol sex chromosomes were found in the karyotypes of the neotropical leptodactylid frogs Eleutherodactylus euphronides and E. shrevei. The W chromosomes are the largest heterochromatic, female-specific chromosomes so far discovered in the class Amphibia. The analyses of the banding patterns with AT- and GC base-pair specific fluorochromes show that the constitutive heterochromatin in the giant W chromosomes consists of various categories of repetitive DNA sequences. The W chromosomes of both species are similar in size, morphology and banding patterns, whereas their Z chromosomes exhibit conspicuous differences. In the cell nuclei of female animals, the W chromosomes form very prominent chromatin bodies (W chromatin). DNA flow cytometric measurements demonstrate clear differences in the DNA content of male and female erythrocytes caused by the giant W chromosome, and also shows that these Eleutherodactylus genomes are among the smallest of all amphibian genomes. The importance of the heteromorphic ZW sex chromosomes for the study of Z-linked genes, the similarities and differences of the two karyotypes, and the significance of the exceptionally small genomes are discussed.

Hydration of biological molecules: lipids versus nucleic acids.

We used FTIR spectroscopy to comparatively study the hydration of films prepared from nucleic acids (DNA and double-stranded RNA) and lipids (phosphatidylcholines and phosphatidylethanolamines chosen as the most abundant ones) at room temperature by varying the ambient relative humidity in terms of solvent-induced structural changes. The nucleic acids and phospholipids both display examples of polymorphism on the one hand and structural conservatism on the other; even closely related representatives behave differently in this respect. DNA undergoes a hydration-driven A-B conformational transition, but RNA maintains an A-like structure independently of the water activity. Similarly, a main transition between the solid and liquid-crystalline phases can be induced lyotropically in certain phosphatidylcholines, while their phosphatidylethanolamine counterparts do not exhibit chain melting under the same conditions. A principal difference concerning the structural changes that occur in the studied biomolecules is given by the relevant water-substrate stoichiometries. These are rather high in DNA and often low in phospholipids, suggesting different mechanisms of action of the hydration water that appears to induce structural changes on global- and local-mode levels, respectively.

Experimental studies on the nature of bonding of DNA*bipyridyl-(ethylenediamine)platinum(II) and DNA*netropsin complexes in solution and oriented wet-spun films.

The stability of complexes of NaDNA with bipyridyl- (ethylenediamine)platinum(II) (abbreviated [(bipy)Pt(en)](2+)) and with netropsin has been studied using two techniques: (i) ultraviolet (UV) melting experiments were done on NaDNA* [(bipy)Pt(en)](2+), showing that the [(bipy)Pt(en)](2+) ligand stabilizes the DNA double helix structure; and (ii) swelling measurements (via optical microscopy) as a function of relative humidity were done on wet-spun oriented films of NaDNA*[(bipy)Pt(en)](2+) and of NaDNA*netropsin. The swelling data shows that an irreversible transition of the films occurs at high relative humidity, first for the NaDNA*netropsin, then for pure NaDNA, and lastly for the NaDNA*[(bipy)Pt(en)](2+). These results are indicative that the [(bipy)Pt(en)](2+) complex stabilizes the intermolecular bonds which mediate the film swelling characteristics. A model is suggested for the binding of [(bipy)Pt(en)](2+) to DNA to explain why the swelling experiments show this ligand as increasing the intermolecular bond strength between the DNA double helices, while netropsin decreases this degree of stabilization.

Competitive substitution of hexammine cobalt(III) for Na+ and K+ ions in oriented DNA fibers.

Competition of the trivalent cation, Co(NH3)(3+)(6), with K+ and Na+ ions in binding to DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions (65 and 52% v/v EtOH), containing different combinations and concentrations of KCl and NaCl and constant concentration (0.8 mM) of Co(NH3)(6)Cl(3). The degree of Co(NH3)(3+)(6) binding to DNA does not depend significantly on the ethanol concentration or on the kind of univalent cation (Na+ or K+). The ion exchange selectivity coefficient of monovalent-trivalent ion competition, D(1)(c3), increases with the concentration of Me+, C(o)(+), and the monotonic dependence of log D(1)(c3) vs log C(o)(+) has an inflection between 100 and 300 mM that is caused by a structural transformation of DNA from A- to B-form. The ion exchange experimental data are compared with results of grand canonical Monte Carlo (GCMC) simulations of systems of parallel and hexagonally ordered, discretely charged polyions with density and spatial distribution of the charged groups modeling B- and A-forms of DNA. The GCMC method for discretely charged models of the DNA polyion produces a quantitative agreement with experimental data on trivalent-monovalent ion competition in dependence on DNA structural state and salt concentration. Based on this and previous studies it is concluded that the affinity of DNA for the cations decreases in the order Co(NH3)(3+)(6) >> Ca2+ > Mg2+ >> Na+ approximately K+ > Li+. DNA does not exhibit selectivity for Na+ or K+ in ethanol/water solutions either in the absence or in the presence of Co(NH3)(3+)(6), Ca2+, and Mg2+.

NaDNA-bipyridyl-(ethylenediamine)platinum (II) complex: structure in oriented wet-spun films and fibers.

Complexes of NaDNA with bipyridyl-(ethylenediamine)platinum(II) (abbreviated [(bipy)Pt(en)](2+)) in solid, oriented films, prepared with a wet-spinning method, have been studied using x-ray diffraction, elastic neutron scattering, two-dimensional magic-angle-spinning nuclear magnetic resonance (NMR), infrared (IR) linear dichroism, and IR absorption. All of these experiments indicate that the DNA in this complex is in the B conformation. The neutron diffraction experiments reveal that the rise per residue is 3.31 A, indicating that the [(bipy)Pt(en)](2+) molecular ion causes a small distortion of the B conformation. The neutron data in the direction perpendicular to the helical axis are consistent with a centered orthorhombic unit cell with a=22.65 A and b=32.2 A. The NMR and IR experiments show that the orientation of phosphate groups in the DNA small middle dot[(bipy)Pt(en)](2+) complex is the same as that observed for pure DNA in the B conformation. The IR experiments also show that the [(bipy)Pt(en)](2+) molecular ion stabilizes the B conformation of DNA down to 59% relative humidity, a low water activity. Mechanochemical experiments on wet-spun NaDNA fibers in 68% ethanol with and without [(bipy)Pt(en)](2+) reveal a 9% elongation of the DNA fibers as the complex is formed.

Refusing to twist: demonstration of a line hexatic phase in DNA liquid crystals

We report conclusive high resolution small angle x-ray scattering evidence that long DNA fragments form an untwisted line hexatic phase between the cholesteric and the crystalline phases. The line hexatic phase is a liquid-crystalline phase with long-range hexagonal bond-orientational order, long-range nematic order, but liquidlike, i.e., short-range, positional order. So far, it has not been seen in any other three dimensional system. By line-shape analysis of x-ray scattering data we found that positional order decreases when the line hexatic phase is compressed. We suggest that such anomalous behavior is a result of the chiral nature of DNA molecules.

Competitive binding of Mg2+, Ca2+, Na+, and K+ ions to DNA in oriented DNA fibers: experimental and Monte Carlo simulation results.

Competitive binding of the most common cations of the cytoplasm (K(+), Na(+), Ca(2+), and Mg(2+)) with DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions (65 and 52% v/v EtOH) containing different combinations and concentrations of the counterions. The affinity of DNA for the cations decreases in the order Ca > Mg >> Na approximately K. The degree of Ca(2+) and/or Mg(2+) binding to DNA displays maximum changes just at physiological concentrations of salts (60-200 mM) and does not depend significantly on the ethanol concentration or on the kind of univalent cation (Na(+) or K(+)). Ca(2+) is more tightly bound to DNA and is replaced by the monovalent cations to a lesser extent than is Mg(2+). Similarly, Ca(2+) is a better competitor for binding to DNA than Mg(2+): the ion exchange equilibrium constant for a 1:1 mixture of Ca(2+) and Mg(2+) ions, K(c)(Ca)(Mg), changes from K(c)(Ca)(Mg) approximately 2 in 65% EtOH (in 3-30 mM NaCl and/or KCl) to K(c)(Ca)(Mg) approximately 1.2-1.4 in 52% EtOH (in 300 mM NaCl and/or KCl). DNA does not exhibit selectivity for Na(+) or K(+) in ethanol/water solutions either in the absence or in the presence of Ca(2+) and/or Mg(2+). The ion exchange experimental data are compared with results of grand canonical Monte Carlo (GCMC) simulations of systems of parallel and hexagonally ordered, uniformly and discretely charged polyions with the density and spatial distribution of the charged groups modeling B DNA. A quantitative agreement with experimental data on divalent-monovalent competition has been obtained for discretely charged models of the DNA polyion (for the uniformly charged cylinder model, coincidence with experiment is qualitative). The GCMC method gives also a qualitative description of experimental results for DNA binding competitions of counterions of the same charge (Ca(2+) with Mg(2+) or K(+) with Na(+)).

Mediation of a phase transition in hyaluronate films by the counterions Li, Cs, Mg and Ca as observed by infrared spectroscopy, optical microscopy and optical birefringence.

Infrared (IR) spectroscopy and optical microscopy have been performed as a function of relative humidity (rh) on wet-spun oriented films of hyaluronate (HA) prepared with various counterions. Complete swelling measurements have been obtained through optical microscopy for films of Cs-, Mg-, and CaHA. IR spectroscopy of Cs-, Mg-, Ca-, and LiHA films was performed for skeletal vibrations (800-1000 cm(-1)) and for vibrational modes (1150-1300 cm(-1)) attributed to C-C and C-O stretching modes and C-C-H and C-O-H bending modes. These techniques reveal evidence of a counterion-dependent phase transition occuring at high relative humidities. Optical birefringence measurements on the polycrystalline samples showed order before and disorder after the transition from lower to higher humidity.

Low-frequency Raman spectra of lysozyme crystals and oriented DNA films: dynamics of crystal water.

We observed low-frequency Raman spectra of tetragonal lysozyme crystals and DNA films, with varying water content of the samples. The spectra are fitted well by sums of relaxation modes and damped harmonic oscillators in the region from approximately 1 cm(-1) to 250 cm(-1). The relaxation modes are due to crystal water, and the distribution of relaxation times is determined. In wet samples, the relaxation time of a small part of the water molecules is a little longer than that of bulk water. The relaxation time of a considerable part of the crystal water, which belongs mainly to the secondary hydration shell, is an order of magnitude longer than that of bulk water. Furthermore, the relaxation time of some water molecules in the primary hydration shell of semidry samples is shorter than we expected. Thus we have shown that low-frequency Raman measurements combined with properly oriented samples can give specific information on the dynamics of hydration water in the ps range. On the other hand, we concluded, based on polarized Raman spectra of lysozyme crystals, that the damped oscillators correspond to essentially intramolecular vibrational modes.

Conformational transitions of the phosphodiester backbone in native DNA: two-dimensional magic-angle-spinning 31P-NMR of DNA fibers.

Solid-state 31P-NMR is used to investigate the orientation of the phosphodiester backbone in NaDNA-, LiDNA-, MgDNA-, and NaDNA-netropsin fibers. The results for A- and B-DNA agree with previous interpretations. We verify that the binding of netropsin to NaDNA stabilizes the B form, and find that in NaDNA, most of the phosphate groups adopt a conformation typical of the A form, although there are minor components with phosphate orientations close to the B form. For LiDNA and MgDNA samples, on the other hand, we find phosphate conformations that are in variance with previous models. These samples display x-ray diffraction patterns that correspond to C-DNA. However, we find two distinct phosphate orientations in these samples, one resembling that in B-DNA, and one displaying a twist of the PO4 groups about the O3-P-O4 bisectors. The latter conformation is not in accordance with previous models of C-DNA structure.

Stabilization of the B conformation in unoriented films of calf thymus DNA by NaCl: a Raman and IR study.

Unoriented films of calf thymus NaDNA with either 3.0 or 5.0 NaCl per base pair were prepared by dehydrating unstressed gels. These films were studied by Raman and ir spectroscopy. The 5.0 samples showed very strong vibrational modes characteristic of the B conformation at relative humidities (RH) as low as 30%, indicating that those samples were entirely in the B conformation. The 3.0 samples showed weaker features: some of the DNA in these samples were in the B conformation at 80% RH while the DNA is essentially in a disordered phase at 30% RH.

Differential scanning calorimetric and X-ray study of the binding of the water of primary hydration to calf-thymus DNA.

Differential scanning calorimetry has been used to study the thermal properties of hydrated films of calf-thymus Na-, K- and CsDNA between 20 and 320 degrees C. A broad endothermic transition near 75 degrees C and a sharp exothermic transition near 240 degrees C are observed. The broad transition is due to the dehydration of the DNA, while the exothermic transition is due to pyrolysis of the sample. the peak temperatures of both transitions increase as the scan rate is increased. Based on a Kissinger analysis, the net activation energy for the desorption of the primary water of hydration is about 0.6 eV while that for the pyrolysis is about 1.9 eV. X-ray diffraction patterns suggest that heating the DNA films to 180 degrees C once does not, but thrice does, destroy their structural ordering.

A Raman study of the time variability for the A-to-B transition in wet-spun films of calf-thymus DNA.

The time evolution of the A-to-B transition has been monitored by Raman spectroscopy and found to vary significantly for different samples. Though all samples were prepared in the identical fashion, some samples completed the transition on the time scale of several hours while other samples took days. The shortest time required for the A conformation to disappear was about 2 hours, as determined by the disappearance of the A-form Raman band at 807 cm-1. For these fastest transforming samples, the B-form Raman band at 835 cm-1 was clearly evident after about 5 hours. These data are consistent with the hypothesis that the A conformation of DNA is stabilized by intermolecular interactions.

Bond orientational order, molecular motion, and free energy of high-density DNA mesophases.

By equilibrating condensed DNA arrays against reservoirs of known osmotic stress and examining them with several structural probes, it has been possible to achieve a detailed thermodynamic and structural characterization of the change between two distinct regions on the liquid-crystalline phase diagram: (i) a higher density hexagonally packed region with long-range bond orientational order in the plane perpendicular to the average molecular direction and (ii) a lower density cholesteric region with fluid-like positional order. X-ray scattering on highly ordered DNA arrays at high density and with the helical axis oriented parallel to the incoming beam showed a sixfold azimuthal modulation of the first-order diffraction peak that reflects the macroscopic bond-orientational order. Transition to the less-dense cholesteric phase through osmotically controlled swelling shows the loss of this bond orientational order, which had been expected from the change in optical birefringence patterns and which is consistent with a rapid onset of molecular positional disorder. This change in order was previously inferred from intermolecular force measurements and is now confirmed by 31P NMR. Controlled reversible swelling and compaction under osmotic stress, spanning a range of densities between approximately 120 mg/ml to approximately 600 mg/ml, allow measurement of the free-energy changes throughout each phase and at the phase transition, essential information for theories of liquid-crystalline states.

Aggregated DNA in ethanol solution.

A recently developed mechanochemical method has provided a new, efficient tool for studies on the thermal stability and structure of aggregated DNA in ethanol-water solutions. At low ethanol concentrations DNA is fully soluble and is in the B form. However, with increasing ethanol concentration the melting temperature of DNA, Tm, decreases. At a critical ethanol concentration, dependent on the nature and concentration of the counterion, aggregation of the DNA molecules sets in. This is reflected in a marked increase in Tm indicating that the aggregated DNA molecules are thermally more stable than the dissolved ones. However, they are still in the B form. In general, Tm of aggregated DNA also decreases with further increasing ethanol concentration and is dependent on the nature of the counterion, but Tm is not affected by the concentration of the counterion (excess salt) in the ethanol-water solution. When the ethanol concentration reaches the range of 70-80% (v/v), the B-to-A conformational transition occurs in the case of Na-, K- and CsDNA. Above this transition point the A form is more stable than the B form due to the reduced water activity and to increased interhelical interactions. At very high ethanol concentrations, above 85% and dependent on the nature of the counterion, a drastic change in the thermal behaviour is observed. Apparently such a strong interhelical interaction is induced in the aggregated DNA that the DNA is stabilized and cannot adopt a random coil state even at very high temperatures. This stability of DNA in the P form is fully reversed if the ethanol concentration is lowered and the activity of water, thereby, is restored.

Mechanochemical study of NaDNA and NaDNA-netropsin fibers in ethanol-water and trifluoroethanol-water solutions.

Highly oriented calf-thymus NaDNA fibers, prepared by a wet-spinning method, were complexed with netropsin in ethanol-water and trifluoroethanol (TFE)-water solutions. The relative fiber length, L/L0, was measured at room temperature as a function of ethanol or TFE concentration to obtain information on the B-A conformational transition. The B-A transition point and transition cooperativity of the fibers were calculated. The binding of netropsin to NaDNA fibers was found to stabilize B form and to displace the B-A transition to higher ethanol concentration, as indicated by its elongational effect on the fiber bundles. An increased salt concentration was found to reduce netropsin binding. In netropsin-free ethanol solution, the dissociation of bound netropsin from the DNA fibers was observable. Pure B-NaDNA fibers were found to be more stable in TFE solution than in ethanol solution. This was interpreted as being due to a different steric factor and a larger polarity of TFE compared with ethanol, resulting in its smaller capacity to reduce the water activity and dielectric constant of the medium in the immediate vicinity of DNA fibers. Therefore, the effect of netropsin binding on the B-A transition of NaDNA fibers became less obvious in TFE solution. In another series of experiments, L/L0 was measured as a function of temperature to obtain information on the helix-coil transition, or melting, as well as the B-A transition of NaDNA and NaDNA-netropsin fibers. The melting temperature and helix-coil transition width were calculated from the melting curves. A phenomenological approach was used to describe the melting behavior of the fibers in and around the B-A transition region. The effect of netropsin on the melting of DNA fibers was attributed mainly to the stabilization of B-DNA and to a higher melting cooperativity in the B-DNA region.