How old is the genetic code? Statistical geometry of tRNA provides an answer.

The age of the molecular organization of life as expressed in the genetic code can be estimated from experimental data. Comparative sequence analysis of transfer RNA by the method of statistical geometry in sequence space suggests that about one-third of the present transfer RNA sequence divergence was present at the urkingdom level about the time when archaebacteria separated from eubacteria. It is concluded that the genetic code is not older than, but almost as old as our planet. While this result may not be unexpected, it was not clear until now that interpretable data exist that permit inferences about such early stages of life as the establishment of the genetic code.

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.

Template-directed and template-free RNA synthesis by Q beta replicase.

In the absence of extraneously added templates, Q beta replicase produces different RNA species after long lag times spontaneously in vitro. The sequences of the spontaneous products are short (30 to 45 nucleotides) and bear little sequence relation to one another and no detectable sequence homology to Q beta virus RNA or to the host. Their replication rates are much lower than those of optimized products. Incubation without template in long closed capillaries produces after long lag times many separate RNA growth foci with heterogeneous kinetics. The template-free reaction is strongly dependent on the conditions: lowering the enzyme or the triphosphate concentrations abolishes the template-free RNA synthesis without affecting the template-dependent synthesis. An explanation of the emerging RNA species in template-free reactions by residual RNA contaminants in the incubation mixture is very unlikely in the light of the experimental evidence; however, the experimental evidence is fully compatible with a de novo mechanism (which may include instruction by non-replicatable oligonucleotides).

The origin of genetic information: viruses as models.

A living entity can be described as a complex adaptive system which differs from any, however complex, chemical structure by its capability of functional self-organization based on the processing of information. If one asks, where does this information come from and what is its primary semantics, the answer is: information generates itself in feedback loops via replication and selection, the objective being 'to be or not to be'. This paper describes the theoretical framework of information-generating systems and provides experimental clues for some basic forms of genetic organization, such as molecular quasi-species, hypercyclic and compartmentalized RNA-protein assemblies. The results are primarily obtained with RNA viruses and virus-like systems. The experiments are carried out with the help of automated, computer-controlled bioreactors, called 'evolution machines', that may form the basis of a new 'evolutionary biotechnology'.

Multichannel PCR and serial transfer machine as a future tool in evolutionary biotechnology.

As an improved strategy for producing functional macromolecules by in vitro evolutionary optimization, we propose an automated machine that can process up to 960 samples in parallel. It consists of a 960-well PCR machine with special sealed plastic reaction vessels and appropriate handling devices. We show that the heat-sealing technique does not significantly affect the activity of Taq DNA Polymerase or that of the temperature-sensitive Q beta RNA polymerase but avoids cross-contamination and evaporation of the samples. Initial experiments demonstrate the suitability of the apparatus to uniformly process the samples and to perform the thermocycling. Serial transfer of reaction products into fresh reaction solution was used to initiate further rounds of amplification as a typical experimental setup for an evolutionary biotechnology application.

Analysis of the envelope region of the highly divergent HIV-2ALT isolate extends the known range of variability within the primate immunodeficiency viruses.

HIV-2ALT is a highly divergent HIV-2-related isolate that is genetically equidistant to the prototypic HIV-2 strains, defined by HIV-2ROD, and to the simian immunodeficiency viruses SIVmac and SIVsm. We have now cloned and sequenced the envelope region of HIV-2ALT, thus completing the analysis of the whole viral genome. The sequences of env and nef and of the second exons of tat and rev were compared with those of the other viruses of the HIV-2/SIVsm/SIVmac group. Despite of the high degree of variation of HIV-2ALT, functional domains of the genes are conserved. Although in env, the overall pattern of constant and variable domains is maintained, many single amino acid exchanges exist at positions previously thought to be constant in HIV-2 strains. In addition, when compared with a broader spectrum of immunodeficiency viruses, which includes SIVMND from mandrill and SIVAGM from African green monkey, HIV-2ALT Env has a high percentage of amino acid exchanges, which are unique to this strain. This underlines the separate branch of HIV-2ALT within the phylogenetic tree and makes obvious the inclusion of such divergent strains in preventive and therapeutic programs.

Selforganization in molecular and cellular networks.

Biological organisation rests on forces among material constituents which manifest themselves in two principal forms, namely as static-conservative and as dynamic-dissipative, the latter being responsible for selforganisational and regulatory behaviour. There is a unifying concept underlying such phenomena as selection, evolution, differentiation, morphogenesis, memory and learning which involes various forms of autocatalysis in open systems far from equilibrium. Examples are the steady turnover of selfreproducing molecules, which is the basis of selection and evolution at the genetic level, or the excitatory and inhibitory interplay of cells allowing for controlled growth and intercellular communication as encountered in the immune system or in nerve cell networks. Recent studies of the dynamics of systems far from equilibrium have revealed deep insights into the nature of biological organisation. This is exemplified by three model systems dealing with The modern understanding of conservative and dissipative structure provide a solid basis for an old morphological concept which - since ancient times - has been intuitively perceived: Gestalt, indeed, is not simply the sum of its parts, even if forces among the constituents - the stoics "pneuma" - are taken into consideration. Gestalt rather is a dynamical state dependent upon initial and boundary conditions of the material system in which it manifests itself.

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.

Specific information concerning taxonomy, pathogenicity and methicillin resistance of staphylococci obtained by a multiplex PCR.

The use of DNA amplification techniques such as the polymerase chain reaction (PCR) in modern diagnostic microbiology not only allows the sensitive and specific identification of micro-organisms but also the detection of specific antibiotic resistance genes. This study describes a multiplex PCR on bacterial colonies picked directly from agar plates without preceding DNA preparation. Eubacteria and staphylococci were identified by, 16S rRNA specific PCR products. In parallel, specific primers were used for the detection of staphylococcal coa and mecA genes. This 4-h multiplex PCR, consisting of four sets of primers, was evaluated for rapid and specific differential diagnosis of methicillin-resistant and methicillin-susceptible strains of Staphylococcus aureus and coagulase-negative staphylococci. To analyse specificity of the amplification products, 100 non-staphylococcal, eubacterial isolates and 20 Candida albicans strains were tested. In a first step, specificity of all four single sets of primers was evaluated before the co-amplification within the multiplex PCR procedure was performed. The results were compared with those of conventional susceptibility and typing methods. The specific 16S rRNA PCR product for eubacterial isolates (n = 786) and staphylococci (686) was found in all strains tested. The coa gene was detected only in S. aureus (488) strains with a specificity of 100%, and was not detected in any of the coagulase-negative staphylococci (198). The mecA gene was detected in 98% of methicillin-resistant staphylococci (393) and in 2% of all methicillin-susceptible staphylococci (293). The multiplex PCR with co-amplification of different determinants provides rapid reliable information on staphylococcal identification and methicillin susceptibility supporting the diagnosis, treatment and control of staphylococcal infections.

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.

Prionics or the kinetic basis of prion diseases.

A comparative kinetic analysis of mechanisms of prion diseases based on the "protein only" hypothesis is presented. The Prusiner mechanism of autocatalytic conversion of a host protein into a genetically identical, but conformationally different, prion state requires cooperativity in order to work, given realistic values of rate parameters. It then becomes phenomenologically indistinguishable from the Lansbury mechanism of plaque formation which is also a form of (passive) autocatalysis. Though the two kinds of mechanisms still may differ on the question which of the two monomeric protein conformations is the favoured equilibrium state they both require an aggregated state as the form that is eventually favored at equilibrium. While these considerations allow for a critical comparison of the mechanisms they do not yet tell us what the actual mechanism of infection is. Experiments rather indicate that the infectious unit in vivo may still differ from an in vitro form of aggregated prion proteins. Hence aggregation of the prionic form is most probably a necessary, but possibly not sufficient, prerequisite of infection. Be that as it may, the premise of a linkage between prion aggregation and infection offers a very sensitive method for diagnosing the disease at a very early stage, using fluorescence cross-correlation analysis. The possible analogies to Alzheimer's disease make such a prospect a "hot topic".

Selection and self-organization of self-reproducing macromolecules under the constraint of constant flux.

We investigate the dynamic behavior of a set of self-reproducing macromolecules (e.g., polynucleotides) under conditions such that the fluxes of all monomer units into the system are kept constant. Such conditions might prevail in an evolution reactor or in certain naturally occurring situations. A general set of equations is developed to describe the behavior of both the macromolecule and the monomer concentrations. The question of how the rate of macromolecule synthesis varies with the monomer levels is discussed briefly. With the help of several physically reasonable approximations, we obtain an exact solution for a simplified constant flux system. Comparison with the corresponding system under the constraint of constant overall organization reveals important similarities, most notably in the existance and composition of quasispecies. Given the same set of physical and chemical parameters, a system subject to constant flux will always evolve toward selective equilibrium more slowly than under the constraint of constant organization.

Determining the base sequence of RNA molecules using continuous degradation kinetics.

A kinetic approach to sequence analysis is presented. A polymer chain composed of a small number of monomer types is degraded continuously with degradation proceeding in only one direction of the chain. Ordinarily, the direct determination of the sequence from the amount of degraded monomers is hampered by the rapid loss of reaction synchrony. However, it is shown, that if the monomers can be measured reasonably accurately, one can solve a system of linear equations with the unknowns giving the positions of the monomers in the chain, and the syn chrony loss due to the stochastic nature of the degradation process can be accounted for. Model calculations are presented.

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.

Comments on "growth of a hypercycle" by King (1981).

The notion of hypercycles has been introduced (Eigen, 1971) in order to characterize a functional entity which integrates information stored in individual self-replicating elements. Commonly, these self-replicating elements compete, like polynucleotides do in test tube experiments (Spiegelman, 1971; Biebricher et al., 1981) or, like species do in nature. A single species is selected eventually together with its most frequent mutants and the less efficient competitors disappear. In order to suppress competition, specific coupling terms of second order - these are coupling terms which are proportional to the product of two population numbers or concentration - have to be introduced into the kinetic equations such a link has to extend to all members, requiring the formation of a closed catalytic cycle (Eigen and Schuster, 1979). The copying of polynucleotide sequences, like every transfer of information, can only occur with finite accuracy (Eigen et al., 1981). This physical restriction imposes a limit upon the content of information that can be transmitted. A higher content of information requires more precise replication. A more involved replication machinery, however, presupposes more information to build it. Thus, a single autocatalyst will not be able to increase its content of information over the given threshold. How does one escape from this vicious circle? Hypercycles present a solution to the problem: many information carriers coexist and may cooperate to build a more sophisticated and more precisely copying replication machinery. Optimization of growth rates has never been the goal for which hypercycles were conceived. Any first order process can proceed at a higher rate than a second order process if low enough concentrations are chosen. On the other hand, any realistic hypercycle involves both first and second order terms. At low concentrations first order terms provide for accumulation of material while at higher concentration second order terms stabilize cooperation. At which concentration level such a prevalence changes is a matter of quantitative values of rate parameters rather than of basic principle. An autocatalyst may outgrow a hypercycle under certain conditions but it can never solve the problem of integrating information.

Elementary step dynamics of catalytic hypercycles.

Two model systems for hypercyclic organisation constructed from a series of uni- and bimolecular reaction steps were studied under the condition of unlimited growth by means of qualitative analysis and numerical integration of the corresponding differential equations. It is shown that both models lead, within wide ranges of parameter and initial conditions, to the same characteristic dynamical behaviour as the elementary hypercycles and hypercycles with translation introduced in 1978 by Eigen and Schuster.

Toward Bion's starting point: between catastrophe and faith.

Catastrophe and faith are explored as the most basic terms of Bion's work. In doing so it is shown how Bion gets underneath Kleinian concepts to expand the depth phenomenology of a catastrophic sense of origins. For Bion the self is born, evolves and dissolves with a sense of catastrophe. Whether or not Bion is right in various aspects of his account, catastrophe is an elemental term of our existence. For Bion, Faith is a proper primordial and developed response to catastrophe. This paper discusses aspects of the relationship between faith, knowledge, container-contained and catastrophe.

The area of faith in Winnicott, Lacan and Bion.

The area of faith is distinguishable from operations which primarily emphasize ego mastery and introjection-internalization processes. Faith is implicit in Winnicott's transitional experiencing and carried forward in object usage. In Bion faith is linked with openness to O or the (unknowable) ultimate reality of a session and is the operative principle of the psychoanalytic attitude. This paper emphasizes the play of faith in Winnicott's object usage and Bion's O. A certain faith is also required to tolerate the movement of meaning in Lacan's Symbolic order. Faith as a fundamental dimension is in tension with the defensive use of mastery and introjective-projective representational networks. In its various forms this complex tension is part of the structure of human life and constitutes the arena in which faith may evolve.

The differentiation of an androgynous imago.

Global phobic-obsessive states accompanied by androgynous images broke, with treatment, into alternating phobic and obsessive phases characterized by paternal images in the former and maternal images in the latter. The dynamics of the phobic states related to the patient's wish for and dread of the paternal phallus, complicated by the father's death during a transitional anal period. The dynamics of the obsessive states were related to wishes and fears of destructiveness of or by the maternal insides. The maternal insides were also experienced as both dangerous and life-giving to the father. As the patient worked through her paternal and maternal fantasies, especially in the transference, her fears of the outside world (the father) and her need to control the insides of her home (the mother) markedly diminished. Active and passive-responsive tendencies bound up and distorted in her obsessive and phobic states were able to undergo fuller differentiation and development with regard to both the internal maternal and paternal objects and the ego's modes of relating to them.