Fluorescently labeled model DNA sequences for exonucleolytic sequencing.

We describe here the enzyme-catalyzed, low-density labeling of DNAs with fluorescent dyes. Firstly, for "natural" template DNAs, dNTPs were partially substituted in the labeling reactions by the respective fluorophore-bearing analogs. The DNAs were labeled by PCR using Taq DNA polymerase. The covalent incorporation of dye-dNTPs decreased in the following order: rhodamine-green-5-dUTP (Molecular Probes, the Netherlands), tetramethylrhodamine-4-dUTP (FluoroRed, Amersham Pharmacia Biotech), Cy5-dCTP (Amersham Pharmacia Biotech). Exonucleolytic degradation by the 3'-->5' exonuclease activity of T7 DNA polymerase (wild type) in the presence of excess reduced thioredoxin proceeded to complete breakdown of the labeled DNAs. The catalytic cleavage constants determined by fluorescence correlation spectroscopy were between 0.5 and 1.5 s(-1) at 16 degrees C, normalized for the covalently incorporated dye-nucleotides. Secondly, rhodamine-green-X-dUTP (Roche Diagnostics), tetramethylrhodamine-6-dUTP (Roche Diagnostics), and Cy5-dCTP were covalently incorporated into the antisense strand of "synthetic" 218-b DNA template constructs (master sequences) at well defined positions, starting from the primer binding site, by total substitution for the naturally occurring dNTPs. The 218-b DNA constructs were labeled by PCR with a thermostable 3'-->5' exonuclease deficient mutant of the Tgo DNA polymerase which we have selected. The advantage of the special, synthetic DNA constructs as compared to natural DNAs lies in the possibility of obtaining tailor-made nucleic acids, optimized for testing the performance of exonucleolytic sequencing. The number of incorporated fluorescent nucleotides determined by complete exonucleolytic degradation and fluorescence correlation spectroscopy were six out of six possible incorporations for rhodamine-green-X-dUTP and tetramethylrhodamine-6-dUTP, respectively. Their covalent and base-specific incorporations were confirmed by the novel analysis methodology of re-sequencing (i.e. mobility-shift gel electrophoresis, reversion-PCR and re-sequencing) first developed in the paper Földes-Papp et al. (2001) and in this paper. This methodology was then used by other groups within the whole sequencing project.

Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy.

Interactions of the peptides melittin and magainin with phospholipid vesicle membranes have been studied using fluorescence correlation spectroscopy. Molecular interactions of melittin and magainin with phospholipid membranes are performed in rhodamine-entrapped vesicles (REV) and in rhodamine-labelled phospholipid vesicles (RLV), which did not entrap free rhodamine inside. The results demonstrate that melittin makes channels into vesicle membranes since exposure of melittin to vesicles causes rhodamine release only from REV but not from RLV. It is obvious that rhodamine can not be released from RLV because the inside of RLV is free of dye molecules. In contrast, magainin breaks vesicles since addition of magainin to vesicles results in rhodamine release from both REV and RLV. As the inside of RLV is free of rhodamine, the appearance of rhodamine in solution confirms that these vesicles are broken into rhodamine-labelled phospholipid fragments after addition of magainin. This study is of pharmaceutical significance since it will provide insights that fluorescence correlation spectroscopy can be used as a rapid protocol to test incorporation and release of drugs by vesicles.

Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy.

BACKGROUND: The accumulation of fibrillar deposits of amyloid beta-peptide (Abeta) in brain parenchyma and cerebromeningeal blood vessels is a key step in the pathogenesis of Alzheimer's disease. In this report, polymerization of Abeta was studied using fluorescence correlation spectroscopy (FCS), a technique capable of detecting small molecules and large aggregates simultaneously in solution. RESULTS: The polymerization of Abeta dissolved in Tris-buffered saline, pH 7.4, occurred above a critical concentration of 50 microM and proceeded from monomers/dimers into two discrete populations of large aggregates, without any detectable amount of oligomers. The aggregation showed very high cooperativity and reached a maximum after 40 min, followed by an increase in the amount of monomers/dimers and a decrease in the size of the large aggregates. Electron micrographs of samples prepared at the time for maximum aggregation showed a mixture of an amorphous network and short diffuse fibrils, whereas only mature amyloid fibrils were detected after one day of incubation. The aggregation was reduced when Abeta was incubated in the presence of Abeta ligands, oligopeptides previously shown to inhibit fibril formation, and aggregates were partly dissociated after the addition of the ligands. CONCLUSIONS: The polymerization of Abeta is a highly cooperative process in which the formation of very large aggregates precedes the formation of fibrils. The entire process can be inhibited and, at least in early stages, partly reversed by Abeta ligands.

Fluorescence correlation spectroscopy of enzymatic DNA polymerization.

We show that fluorescence correlation spectroscopy (FCS) can be used as a reliable, simple, and fast tool for detecting products of the polymerase chain reaction (PCR). By use of autocorrelation experiments, it is demonstrated that fluorescent 217-bp DNA fragments can be detected at very low initial ss M13mp18(+) DNA and tetramethylrhodamine-4-dUTP concentrations and that these polymers are cleaved by the chosen restriction enzymes. A FCS calibration curve is presented, where the translational diffusion times of different size DNA fragments are plotted versus the number of base pairs they contain. At zero and very low template concentrations a large "background" species emerges, which is a reflection of the experimental conditions chosen and the extremely high sensitivity of FCS. The relative amount of nonspecific product formation is less than 1%. The ease by which a FCS measurement can be performed (a few minutes at most) also enables the technique to be used as an effective screening method.

Time-resolved fluorescence studies of the molten globule state of apomyoglobin.

We have investigated the structure of the molten globule state of horse heart apomyoglobin by energy transfer experiments. The tyrosine residue at position 146 was converted into 3-nitro-tyrosine and distances between this side-chain and the two tryptophanyl side-chains were obtained from the time-resolved tryptophanyl fluorescence decay curve. Since both Trp residues are located in the N-terminal A-helix and the modified Tyr residue is located in the C-terminal H-helix, these measurements give information about this helix-helix distance. The energy transfer experiments provide direct evidence for a close contact between the A-helix and the H-helix in the molten globule state. This gives a very strong indication of the presence of a single near-native hydrophobic cluster in this state, as previously proposed by other authors. The distance distribution suggests that the fluctuations in the compact states have correlation times shorter than 1 ns. The experiments also show larger fluctuations, both in the native state and in the molten globule state. In addition, the tryptophanyl fluorescence anisotropy decay curves have been measured. The results suggest loose tertiary contacts in the molten globule state, which is in good agreement with earlier studies. For the denatured state of apomyoglobin, both techniques indicate an extended random coil structure.

Rotational and translational motions of human spermatozoa: angle dependence of dynamic laser light scattering.

We have studied how the dynamic components of laser light scattered from human spermatozoa depend on the scattering angle. This was done by investigating the halfwidth of the intensity autocorrelation function. A model of the spermatozoa as freely rotating and translating linear objects was adequate to describe the scattered light. Rotational motions determined the halfwidth of the intensity autocorrelation function at very small scattering angles and contribution from translational motions was dominant at scattering angles larger than 20 degrees. The contribution from translational motions increased with increasing scattering angle. We found a nearly linear relationship between the translation speed and the rotation frequency. However, the ratio between the two properties varied more than expected from the methodological error. Therefore we introduced a propelling efficacy as a concept to describe the swimming efficiency. This property might contain important information about the swim characteristics.

Distribution of cholesterol between vesicles and micelles in human gallbladder bile: influence of treatment with chenodeoxycholic acid and ursodeoxycholic acid.

The present study aimed at determining the relative distribution of cholesterol between the vesicular and micellar phases in gallbladder bile of gallstone patients (n = 23) and gallstone-free subjects (n = 7). Nine of the gallstone patients were treated with chenodeoxycholic acid and seven were treated with ursodeoxycholic acid, 15 mg/kg/day, for 3 wk before cholecystectomy. The vesicular and micellar fractions in bile were separated by sucrose density gradient ultracentrifugation, and a clear separation between the two phases was obtained. The vesicles were further identified by quasielastic light scattering spectroscopy and appeared to be of a uniform size with a mean hydrodynamic radius of 760 A. The proportion of cholesterol in the vesicular fraction was significantly higher in the untreated gallstone group (40% +/- 4%) compared with the gallstone-free (28% +/- 3%), ursodeoxycholic acid (28% +/- 3%) and chenodeoxycholic acid (18% +/- 4%) groups. Despite a low cholesterol saturation of bile in the latter three groups (88% +/- 12%, 51% +/- 9% and 65% +/- 5%, respectively), a considerable part of the biliary cholesterol was carried in the vesicular fraction. The cholesterol/phospholipid ratio in the vesicular fraction averaged between 0.49 and 0.58 in the gallstone, gallstone-free and chenodeoxycholic acid groups, whereas the ursodeoxycholic acid group had a significantly lower ratio of 0.24. The nucleation time of bile from the gallstone group was short (2 +/- 1 days) compared with the gallstone-free, chenodeoxycholic acid and ursodeoxycholic acid groups (23 +/- 3, 24 +/- 6 and 14 +/- 3 days, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic laser light scattering compared with video micrography for analysis of sperm velocity and sperm head rotation.

A new method which allows separation of the rotational and translational components of human sperm motility, based on the angular dependence of the dynamic laser light scattering (DLS) has been developed. The technique was used in a clinical study and was compared with an independent evaluation by video micrography. A good correlation was found between the two techniques when applied on different semen samples (r = 0.90; P less than 0.01 and r = 0.96; P less than 0.01 for rotation and translation, respectively) and when applied on the same semen sample at different temperatures. The rapid evaluation of these parameters using DLS technique opens the possibility to study large number of semen samples under physiological and pathological conditions at a lower cost.

Biophysical models of ciliary activity: Gaussian frequency distributions.

A model of a freely rotating extended scatterer is proposed to describe light scattering from beating cilia. Gaussian rotation frequency distributions, characterized by a mean angular frequency and a standard deviation, are introduced in order to simulate intensity autocorrelation functions and to fit the model to experimental data. Thus the ciliary beats are characterized by a mean beat frequency and a standard deviation of the beat frequency distribution. The standard deviation influences the damping of the intensity autocorrelation function of light scattered from cilia. The calculated intensity autocorrelation function shows a more prominent oscillating behaviour the smaller the standard deviation of the beat frequency. The validity of the model is supported by experimental data in two ways: 1) The model fits very well to experimental data in computer evaluations, 2) Neither the model nor information obtained from measurements are dependent on the measuring angle.

Laser light scattering spectroscopy: a new method to measure tracheobronchial mucociliary activity.

Laser light scattering spectroscopy is based on the evaluation of the frequency shift of coherent light scattered by moving particles. This makes it particularly suitable for use in light guiding systems. In this study laser light scattering spectroscopy was assessed for its ability to provide information on the motility of respiratory cilia. In vitro and in vivo measurements were undertaken with animal tracheal mucosa. The intensity fluctuations of laser light scattered from moving cilia were analysed in terms of their autocorrelation functions to provide information on the frequency and synchrony of beating cilia. In vitro measurements were performed on fresh bovine trachea to estimate a safe laser power level for mucosal exposure and to test the method by defining the temperature dependence of the ciliary beat frequency. Power densities not exceeding 0.3 kW/cm2 were found to be the upper limit for long term exposure of the mucosa in vitro. Ciliary beat frequency showed a pronounced temperature dependence, ranging from 5.8 to 28.3 Hz over the temperature range 20-43.5 degrees C. A maximum frequency was found at 41.5 degrees C. In vivo measurements of ciliary activity were performed in six pigs by means of optical fibres for light transmission combined with fibreoptic bronchoscopy. A ciliary beat frequency of 5 Hz was obtained; heart and breathing frequencies could be separated and identified. These results suggest that laser light scattering spectroscopy might provide a convenient method of studying the mucociliary system of the lower airways.

Rotational and translational swimming of human spermatozoa: a dynamic laser light scattering study.

The rotational swimming motion of human spermatozoa is evaluated from measurements of depolarized dynamic laser light scattering at zero angle. The analysis is based on a Maxwellian angular velocity distribution and yields a rotational frequency of about 4 Hz that is ascribed to the rotation of the sperm head. From comparison with the translational swimming motion, a propelling efficiency of about 10 micron per turn is deduced. This parameter describes the linkage between the rotational and translational swimming motion and is likely to be discriminatory in the analysis of physiological and pathological sperm motions.