Biomedical subjects
M Eigen
Publications and source records attributed to M Eigen.
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.
Sorting single molecules: application to diagnostics and evolutionary biotechnology.
A method is described that provides for detection and identification of single molecules in solution. The method is based on fluorescence correlation spectroscopy, which records spatio-temporal correlations among fluctuating light signals, coupled with devices for trapping single molecules in an electric field. This technique is applied to studies of molecular evolution, where it allows fast screening of large mutant spectra in which targets are labeled by specific fluorescent ligands. The method expands the horizon in molecular diagnostics by making it possible to monitor concentrations down to (less than) 10(-15) M without any need for amplification.
Selection and the origin of information.
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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'.
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 fifth Paul Ehrlich Lecture, Virus strains as models of molecular evolution.
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Optimization by hierarchical mutant production.
Inspired by the successful description of the first steps of molecular evolution by the quasispecies theory and the successful application of quasispecies-like algorithms to optimization problems, we propose a hierarchically organized algorithm. This new algorithm is able to solve a spin glass and a travelling salesman problem using only point mutations. Furthermore, it performs better under comparable circumstances than the ordinary quasispecies algorithm. Depending on the structure of the fitness landscape of the examined problem under consideration the hierarchically organized algorithm proves to be much more suitable than a simple quasispecies algorithm, especially in clustered landscapes. Tuning the error rates reveals the critical minimum copy fidelity necessary to guarantee optimization. We propose to incorporate hierarchical concepts into optimization algorithms inspired by biological evolution, such a genetic algorithms.
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.
The hypercycle. Coupling of RNA and protein biosynthesis in the infection cycle of an RNA bacteriophage.
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A novel proviral clone of HIV-2: biological and phylogenetic relationship to other primate immunodeficiency viruses.
Infectious molecular clones of the human immunodeficiency virus type 2 (HIV-2) will be valuable tools for the study of regulatory gene functions and the development of an animal model for the human acquired immunodeficiency syndrome (AIDS). To this end, we have cloned and sequenced a novel HIV-2 isolate, HIV-2BEN. One clone, designated MK6, is infectious for various human T-cell lines and for human and macaque peripheral blood lymphocytes (PBL), allowing molecular studies of HIV-2 infection and replication. Since MK6 is highly cytopathic in MT-2 and Molt-4 clone 8 cells, antiviral agents and neutralizing sera may be tested. Cluster analysis of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) env and gag genes revealed that HIV-2BEN yielded the earliest node of phylogenetic divergence for all reported HIV-2 sequences. Noise analysis showed that, with the current data, no specification of any branching order can be made among the four groups of primate lentiviruses, HIV-1, HIV-2/SIVSMM/MAC, SIVAGM, and SIVMND.
Statistical geometry on sequence space.
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How old is the immunodeficiency virus?
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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.
The AIDS debate.
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Statistical geometry in sequence space: a method of quantitative comparative sequence analysis.
A statistical method of comparative sequence analysis that combines horizontal and vertical correlations among aligned sequences is introduced. It is based on the analysis mainly of quartet combinations of sequences considered as geometric configurations in sequence space. Numerical invariants related to relative internal segment lengths are assigned to each such configuration and statistical averages of these invariants are established. They are used for internal calibration of the topology of divergence and for quantitative determination of the noise level. Comparison of computer simulations with experimental data reveals the high sensitivity of assignment of basic topologies even if much randomized. In addition, these procedures are checked by vertical analysis of the aligned sequences to allow the study of divergences with positionally varying substitution probabilities.