What is a quasispecies?

The concept of the quasispecies as a society formed from a clone of an asexually reproducing organism is reviewed. A broad spectrum of mutants is generated that compete one with another. Eventually a steady state is formed where each mutant type is represented according to its fitness and its formation by mutation. This quasispecies has a defined wild type sequence, which is the weighted average of all genotypes present. The quasispecies concept has been shown to affect the pathway of evolution and has been studied on RNA viruses which have a particularly high mutation rate. They (and possibly the majority of other species) operate close to the error threshold that allows maximum exploration of sequence space while conserving the information content of the genotype. The consequences of the quasispecies concept for the new 'evolutionary technology' are discussed.

Dual-color fluorescence cross-correlation spectroscopy for monitoring the kinetics of enzyme-catalyzed reactions.

Dual-color fluorescence correlation spectroscopy is a biophysical technique that enables precise and sensitive analyzes of molecular interactions. It is unique in its ability to analyze reactions in real time at nanomolar substrate concentrations and below, especially when applied to the monitoring of enzyme-catalyzed reactions. Furthermore, it offers a wide range of accessible reactions, restricted only by the prerequisite that a chemical bond or a physical interaction between two spectrally distinguishable fluorophores is established or broken. Recently, the optical setup of dual-color fluorescence correlation spectroscopy has been extended toward two-photon excitation, resulting in several advantages compared with standard excitation, such as lower fluorescence background, an even larger spectrum of potential fluorescence dyes to be used, as well as a more stable and simplified optical setup. So far, the method has been successfully employed to analyze the kinetics of nucleic acid and peptide modifications catalyzed by nucleases, polymerases, and proteases.

Highly efficient single molecule detection in microstructures.

For DNA single molecule sequencing, the complete detection of all dye-labeled monomers which are cleaved off during the sequencing reaction is an essential requirement. In this work we address the feasibility of single molecule detection in microstructures with a confocal multi element set-up. We present statistical data on single molecule recognition and explain a refined data evaluation technique for single molecule burst analysis. From these data the signal-to-noise ratio in microstructures is evaluated as well as the overall detection efficiency. So far, detection efficiencies of single molecule events of up to 60% have been shown in microstructures.

Towards a general procedure for sequencing single DNA molecules.

In this paper we report on the latest technical advances towards single molecule sequencing, a useful method currently developed especially for fast and easy de novo sequencing. Different approaches for complete labeling of DNA with fluorescent dyes are described. In addition, the experimental set-up for the sequencing process is shown. We demonstrate the ability to purify the buffer and enzyme solutions. Inorganic buffers were purified down to at least 20 fM of remaining fluorescent impurities. The exonuclease buffer solution could be cleaned down to 0.8 pM whereby its full activity was kept. Finally, we show a selection procedure for beads and present the data of a model experiment, in which immobilized DNA is degraded by an exonuclease within a polymethylmethacrylate (PMMA) microstructure. Furthermore, the mathematical processing of the obtained raw data is described. A first complete experimental cycle is shown, combining all preparatory steps which are necessary for single molecule sequencing in microstructures.

Natural selection: a phase transition?

Information has two aspects: a quantity to be called 'extent' and a quality which may be termed 'content' since it deals with meaning. The latter originates via selective self-organization, which can be described also in quantitative physical terms. A prerequisite is the reproducibility of the informational substrate forming the basis of selection. This paper focuses on selection being the analogue of a physical phase transition. In Section 1 the criteria for phase transitions are formulated. Section 2 introduces the concept of information space and describes information as selected points or regions in this space. In Section 3 selection is analyzed in terms of the criteria for phase transitions, and in Section 4 the concept is confronted with experimental data. The conclusion is reached that information content is generated via selection, which can be described as a phase transition in information space.

Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets.

A definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) relies on the detection of pathological prion protein (PrP(Sc)). However, no test for PrP(Sc) in cerebrospinal fluid (CSF) has been available thus far. Based on a setup for confocal dual-color fluorescence correlation spectroscopy, a technique suitable for single molecule detection, we developed a highly sensitive detection method for PrP(Sc). Pathological prion protein aggregates were labeled by specific antibody probes tagged with fluorescent dyes, resulting in intensely fluorescent targets, which were measured by dual-color fluorescence intensity distribution analysis in a confocal scanning setup. In a diagnostic model system, PrP(Sc) aggregates were detected down to a concentration of 2 pM PrP(Sc), corresponding to an aggregate concentration of approximately 2 fM, which was more than one order of magnitude more sensitive than Western blot analysis. A PrP(Sc)-specific signal could also be detected in a number of CSF samples from patients with CJD but not in control samples, providing the basis for a rapid and specific test for CJD and other prion diseases. Furthermore, this method could be adapted to the sensitive detection of other disease-associated amyloid aggregates such as in Alzheimer's disease.

Putting prions into focus: application of single molecule detection to the diagnosis of prion diseases.

Prion diseases are characterized by the cerebral deposition of an aggregated pathological isoform of the prion protein (PrP(Sc)) which constitutes the principal component of the transmissible agent termed prion. In order to develop a highly sensitive method for the detection of PrP(Sc) aggregates in biological samples such as cerebrospinal fluid (CSF), we used a method based on Fluorescence Correlation Spectroscopy (FCS), a technique which allows detection of single fluorescently labeled molecules in solution. Within the FCS setup, fluorescent photons emitted by molecules passing an open volume element defined by the beam of an excitation laser focussed into a diffraction-limited spot are imaged confocally onto a single photon counting detector. Aggregates of PrP(Sc) could be labeled by co-aggregation of probe molecules such as monomeric recombinant PrP or PrP-specific antibodies tagged with a fluorescent dye. In addition to slow diffusion, labeled aggregates are characterized by high fluorescence intensity, which allows detection and quantification by analysis of fluorescence intensity distribution. To improve detection of rare target particles, the accessible volume element was increased by scanning for intensely fluorescent targets (SIFT). To further improve sensitivity and specificity, two different probes were used simultaneously in a two-color setup. In a diagnostic model system of CSF spiked with purified prion rods, dual-color SIFT was more sensitive than Western blot analysis. In addition, a PrP(Sc)-specific signal was also detected in a number of CSF samples derived from CJD patients but not in controls.

Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening.

Fluorescence-based assay technologies play an increasing role in high-throughput screening. They can be classified into different categories: fluorescence polarization, time-resolved fluorescence, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy. In this work we present an alternative analytical technique for high-throughput screening, which we call confocal fluorescence coincidence analysis. Confocal fluorescence coincidence analysis extracts fluorescence fluctuations that occur coincidently in two different spectral ranges from a tiny observation volume of below 1 fl. This procedure makes it possible to monitor whether an association between molecular fragments that are labeled with different fluorophores is established or broken. Therefore, it provides access to the characterization of a variety of cleavage and ligation reactions in biochemistry. Confocal fluorescence coincidence analysis is a very sensitive and ultrafast technique with readout times of 100 ms and below. This feature is demonstrated by means of a homogeneous assay for restriction endonuclease EcoRI. The presented achievements break ground for throughput rates as high as 10(6) samples per day with using only small amounts of sample substance and therefore constitute a solid base for screening applications in drug discovery and evolutionary biotechnology.

Two-beam cross-correlation: a method to characterize transport phenomena in micrometer-sized structures.

To determine flow properties, namely, the velocity and angle of the flow in microstructured channels, an experimental realization based on fluorescence correlation spectroscopy is described. For this purpose, two micrometer-sized spatially separated volume elements have been created. The cross-correlation signal from these has been recorded and evaluated mathematically. In addition to previous results, two-beam cross-correlation allows for fast and easy determination of even small (down to 200 µm/s) flow velocities, as well as simultaneous measurement of diffusion properties of single dye molecules within a rather short detection time of 5-100 s and an error rate of less than 20%. The spatial flow resolution is around 1-2 µm, limited by the diameter of the volume element. Furthermore, vectorial flow data can be obtained and evaluated. A discussion of the theoretical background and an experimental verification of the theoretical results is performed. The feasibility of fast and easy data processing is shown if the flow time is the only desired information. Possible applications of this precise and simple method are the determination of transportation effects within artificial microstructures for CE and HPLC, fast chemical kinetics, and high-throughput screening.

Rare loss of heterozygosity of the MTS1 and MTS2 tumor suppressor genes in differentiated human thyroid cancer.

Loss of heterozygosity (LOH) of the MTS1 (p16) tumor suppressor gene has been reported to occur frequently in thyroid cancer cell lines. In order to determine the frequency of LOH for these multiple tumor suppressor genes, we used microsatellite markers IFNA and D9S171 to perform differential quantitative polymerase chain reaction. Tumor DNA was isolated from native sections of tumor tissue. Control DNA was isolated from blood. PCR products were separated on 6% polyacrylamide sequencing gels and quantified according to peak height and area. Analysis was informative in 70% of cases for both markers, and in 88% for at least one out of both. LOH was found in 3 out of 35 informative patients (8.6%) with papillary thyroid cancer, in 1 out of 7 patients with follicular thyroid cancer (14.2%), and in 0 out of 18 medullary cancers (0%). No LOH was found in 11 informative patients with multinodular goitre, 7 with follicular adenoma, 4 with Graves' disease, and 6 with other thyroid disease. 75% of LOH was found in T1 and T2 stages, it was not more frequent in patients with lymphonodular metastasis. The low frequency of LOH in these types of thyroid cancer argues against a role of loss of heterozygosity at the MTS 1 and 2 gene locus in the development of differentiated thyroid neoplasia.

Regulation of the HGF-receptor c-met in the thyroid gland.

HGF (hepatocyte growth factor) has been characterised as an important mitogen and motogen in many epithelial cells. The biological and clinical significance of HGF and its receptor c-met in the thyroid is currently under study. Overexpression of c-met is an important feature of papillary thyroid cancer. We developed a quantitative differential RT-PCR method in order to examine HGF-receptor regulation using RNA of about 50 cells per analysis. Experiments were performed in three spontaneously transformed follicular thyroid cancer (FTC) cell lines FTC-133, 236, and 238, 7 primary cultures derived from multinodular goitres, one from Graves disease and 1 from papillary thyroid cancer (PTC). TGF-alpha and to a minor degree HGF were shown to induce a marked up-regulation of the receptor whereas bovine TSH, NaI, dbcAMP or basic FGF had no apparent effect. We conclude that expression of the HGF-receptor is under control of paracrine growth factors activating tyrosine-kinase-dependent pathways. We could demonstrate a minor stimulation of thyroid cell proliferation by HGF in presence but not in absence of 10% fetal calf serum.

Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy.

A method for sensitively monitoring enzyme kinetics and activities by using dual-color fluorescence cross-correlation spectroscopy is described. This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two fluorophores that can be discriminated spectrally. In this work, a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and fluorophores at each 5' end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored on-line at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a KM of 14 +/- 1 nM and a kcat of 4.6 +/- 0.2 min-1. In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color fluorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.

Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: high throughput screening for enzyme activity.

Dual-color fluorescence cross-correlation spectroscopy (dual-color FCS) has previously been shown to be a suitable tool not only for binding but also for catalytic rate studies. In this work, its application as a rapid method for high-throughput screening (HTS) and evolutionary biotechnology is described. This application is called RAPID FCS (rapid assay processing by integration of dual-color FCS) and does not depend on the characterization of diffusion parameters that is the prerequisite for conventional fluorescence correlation spectroscopy. Dual-color FCS parameters were optimized to achieve the shortest analysis times. A simulated HTS with homogeneous assays for different restriction endonucleases (EcoRI, BamHI, SspI, and HindIII) achieved precise yes-or-no decisions within analysis times of about 1 s per sample. RAPID FCS combines these short analysis times with the development of fast and flexible assays resulting in sensitive, homogeneous fluorescence-based assays, where a chemical linkage between different fluorophores is either cleaved or formed, or where differently labeled molecules interact by noncovalent binding. In principle, assay volumes can be reduced to submicroliters without decreasing the signal strength, making RAPID FCS an ideal tool for ultra-HTS when combined with nanotechnology. RAPID FCS can accurately probe 10(4) to 10(5) samples per day, and possibly more. In addition, this method has the potential to be an efficient tool for selection strategies in evolutionary biotechnology, where rare and specific binding or catalytic properties have to be screened in large numbers of samples.

A female-specific cDNA sequence of Schistosoma mansoni encoding an amidase that is expressed in the gastrodermis.

A female-specifically expressed cDNA clone was obtained by screening of a subtractive cDNA library enriched for RNA from Schistosoma mansoni females. The deduced protein shows significant homology to a class of enzymes functioning as amidases. Northern blots revealed a transcript of 4.0 kb which is absent in larval stages, but is expressed in adult female worms. By in situ hybridization, the expression site of the gene was exclusively localized in the gastrodermis of female schistosomes. This is the first report of a female-specifically transcribed sequence of S. mansoni that is not expressed in the reproductive organs.

Rolfing: a somatic approach to the integration of human structures.

This report describes a system of bodywork called Rolfing (Rolf Institute of Structural Integration, Boulder, CO). A review of the historical considerations in the development of the process of Rolfing is discussed as well as a description of Rolfing. The viewpoint that a therapeutic treatment modality is based on supporting health through the organization of body structure makes this narrative of interest to nurse practitioners. Rolfing is a type of treatment that is compatible with nursing principles. This author's vision is that nursing will teach forms of healing and education that support the innate healing intelligence within each of us.

30 years later--a new approach to Sol Spiegelman's and Leslie Orgel's in vitro evolutionary studies. Dedicated to Leslie Orgel on the occasion of his 70th birthday.

The conditions necessary for evolution are amplification, mutagenesis and selection. Here we describe the evolutionary response of an in vitro replicating system to the selection pressure for fast growth and show what happens to the amplified molecules within this replication system. Our emphasis is on methodology, on the monitoring and the automation of experiments in molecular evolution. In order to perform in vitro studies on the evolution of RNA molecules, a modified self-sustained sequence replication (3SR) method was used. In the first step of the 3SR reaction, the RNA template is reversely transcribed by HIV-1 reverse transcriptase, followed by a second strand synthesis and the transcription of the resulting dsDNA by T7 RNA polymerase. The selection pressure (fast growth) was achieved by applying the principle of serial transfer pioneered in the laboratories of Sol Spiegelman and Leslie Orgel. At the end of the exponential growth phase of the 3SR reaction, an aliquot of the reaction mixture is transferred into a new sample containing only buffer, nucleotides and enzymes while RNA template molecules are provided by the transfer. The conditions in the exponential growth phase allow the RNA molecules to be amplified in a constant environment; all enzymes (HIV-1 reverse transcriptase and T7 RNA polymerase) and nucleotides are present in large excess. Therefore, transferring reproducibly within the exponential growth phase is equivalent to selecting for fast growth; those molecules which can replicate faster will displace others after several transfers. The experiments were performed using a serial transfer apparatus (STA) which allows the nucleic acid concentration to be monitored on-line by measuring the laser-induced fluorescence caused by intercalation of thiazole orange monomers into the RNA/DNA amplification products. The serial transfer experiments were carried out with an RNA template (220b RNA) that represents a 220-base segment of the HIV-1 genome and comprises the in vivo primer binding site (PBS) for the HIV-1 reverse transcriptase. It could be shown that after only two serial transfers two RNA species (EP1 and EP2) emerged that were much shorter. EP1 (48b) and EP2 (54b) were formed by deletion mutations within the original 220b RNA template in the very beginning of the serial transfer experiment; due to their higher replication rate (calculated from the growth curves derived on-line) these two deletion mutants displaced the original 220b RNA template in the course of the following thirty transfers. We assume that these two RNA species evolved independently of each other. Their formation was probably induced by a strand-transfer reaction of HIV-1 reverse transcriptase. Sequence analyses of these two evolution products seem to confirm such a presented pathway. 30 years after Spiegelman's experiment, the study described here is another answer to the question he posed: 'How do molecules evolve if the only demand is the biblical injunction: multiply?'. The answer, derived from a modified 3SR amplification system (mimicking a part of the HIV-1 replication cycle in vitro), is the same as thirty years ago: The RNA molecules adapt to the new conditions by throwing away any ballast not needed for fast replication. Clearly, this is only one aspect of molecular evolution; however, it shows that we should be careful in designating unidentified genetic material as 'junk DNA'.