Biomedical subjects

J S McCaskill

Publications and source records attributed to J S McCaskill.

12 recordsLinked to original sources

PubMed2000

End-specific covalent photo-dependent immobilisation of synthetic DNA to paramagnetic beads.

A novel approach for light-dependent covalent immobilisation of synthetic DNA oligomers to amino-coated paramagnetic beads is described. A hetero-bifunctional photo-reactive cross-linking chemical, 4-nitrophenyl 3-diazopyruvate, is applied to attach 5' amino-modified DNA to both silica and polystyrene paramagnetic beads. The coupling yields are comparable with similar methods in which no photo-reactive chemicals are used. The immobilised DNA on the polystyrene and silica beads was used efficiently in hybridisation experiments. An extension of this approach to light-directed immobilisation of specific DNA to beads, located at different positions in micro-flow reactors, opens up a range of integrated applications to complex diagnostics, evolutionary biotechnology and novel areas such as DNA computing.

Chromatography, High Pressure Liquid↗

PubMed1998

In vitro evolution of molecular cooperation in CATCH, a cooperatively coupled amplification system.

BACKGROUND: One of the key issues in the investigation of evolution is how complex systems evolved from simple chemical replicators. Theoretical work proposed several models in which complex replicating systems are kinetically stabilized. The development of powerful isothermal amplification technique allows complex nucleic acid based evolving in vitro systems to be set up, which may then serve to verify experimentally current theories of evolution. Recently such a system based on the 3SR (self-sustained sequence replication) reaction has been established to investigate the evolution of cooperation: the trans-cooperatively coupled CATCH (cooperative amplification by cross hybridization). RESULTS: Over four rounds of serial transfer, the cooperatively coupled two species CATCH system evolved into a more complex cooperative four species system, which then was overgrown by CATCH-derived RNA-Z-like hairpin species. In contrast to the classical RNA-Z species, these molecules have complementary loop sequences and self-amplify using a dual mechanism that includes concentration-dependent phases of noncooperative and cooperative amplification. CONCLUSIONS: The evolution of a cooperative system, under conditions that were alternately unfavorable and favorable for cooperative amplification, led to a system showing facultative cooperation. This principle of facultative cooperation preserves the complexity of the system investigated and could have general implications for the evolution and stabilization of cooperation under oscillating reaction conditions.

Alfalfa mosaic virus↗

PubMed1997

Monitoring the amplification of CATCH, a 3SR based cooperatively coupled isothermal amplification system, by fluorimetric methods.

Three different types of fluorescence detection methods were employed to monitor amplification of a previously established isothermal cooperatively coupled amplification system as it can serve as a tool for the investigation of fundamental issues in evolutionary optimization. By using 5'IRD-41 fluorescent labeled primers, the intercalating dye TOPRO-1 and a 5'fluorescin/3'DABCYL 4-(4-dimethylamino-phenylazo)benzoic acid labeled ss 24 nt DNA, evolving molecular cooperation is accessible, sequence specifically as well as non-sequence-specifically without using radioactivity.

DNA↗

PubMed1997

Spatially resolved in vitro molecular ecology.

Sensitive CCD-based fluorescence detection has made spatially resolved studies of evolving cell-free molecular systems possible. In recent years our attention has focussed on making the transition to open and interacting spatially-resolved amplification systems using silicon microreactor technology and on providing a hardware platform for individual based simulation of such systems. Significant progress has been achieved in this direction. Open microflow reactors have been realized in zero (well-mixed), one and two dimensions with volumes small enough to allow long-time studies with limited biochemical materials. The primer directed 3SR reaction (amplifying DNA and RNA) has been used as a basis for constructing interacting model systems with both predator-prey and cooperative amplification character. Theoretical work has demonstrated the need for individual based modeling of such systems: a significant fraction of the population consists of distinct sequence polymers in any case. A massively parallel processor-configurable computer NGEN has been designed and constructed which allows the high speed simulation in hardware of relatively large populations of locally interacting individual strings of chosen length (e.g. up to 2000*2000 for 64 bases), in addition to its application as an evolvable hardware machine. Simulations show self-replicating spots to stabilize the cooperative amplification in evolving systems (a mechanism proposed by the author in 1994). Both oscillatory kinetics and pattern formation are expected in the experimental model systems under investigation which profoundly affect the course of evolution. Such in vitro model systems serve both to test current theories of cooperative evolution and provide clues for optimisation strategies in molecular biotechnology.

Biotechnology↗

PubMed1997

Cooperative amplification of templates by cross-hybridization (CATCH).

In vitro amplification systems not only serve as a tool for the processing of DNA, but have also provided important model systems for the investigation of fundamental issues in evolutionary optimization. In this work we present a coupled amplification system based on the self-sustained sequence replication (3SR), also known as nucleic acid sequence-based amplification (NASBA), which allows the experimental investigation of evolving molecular cooperation. The 3SR reaction is an isothermal method of nucleic acid amplification and an alternative to PCR. A target nucleic acid sequence can be amplified exponentially in vitro using two enzymes: reverse transcriptase (RT) and a DNA-dependent RNA polymerase (RNAP). A system has been constructed in which amplification of two molecular species is cooperatively coupled. These species are single-stranded (ss)DNA templates (D1 and D2) of lengths 58 and 68 nucleotides, respectively. Coupling occurs when D1 and D2 anneal to each other via a complementary region (DB and DB') situated at the 3' end of each template. RT elongates the hybridized templates producing a double-stranded (ds)DNA of 106 base pairs (bp). This double strand contains two promoters, which are situated on either side of, and directly adjacent to DB, and which are oriented towards each other. These promoters specify two RNA transcripts encompassing, respectively, the D1 and D2 portion of the dsDNA. After hybridization of two primers (P1 and P2) to the transcripts (R1 and R2) and reverse transcription, the ss templates D1 and D2 are regenerated. Amplification cycles of D1 and D2 are coupled cooperatively via the common dsDNA intermediate. Under optimized batch conditions the system shows the expected growth phases: exponential, linear and saturation phase. The enzymes of the 3SR cycle tend to misincorporate nucleotides and to produce abortive products. In future experiments, we intend to use the system for studies of evolutionary processes in spatially distributed systems where new strategies for optimization at the molecular level are possible.

DNA-Directed RNA Polymerases↗

PubMed1997

A molecular predator and its prey: coupled isothermal amplification of nucleic acids.

BACKGROUND: A novel approach to the study of in vitro evolution is provided by the investigation of continuous, functionally coupled, amplifying systems. To date, in vitro evolution experiments have focused on issues of mutation and selection. Our work contributes to the new field of in vitro molecular ecology studies in which detailed information about the relationship between sequence changes and molecular interactions is obtained. Predator-prey systems are interesting in this context both in terms of evolutionary limits and in terms of the potential kinetic properties of oscillation and spatial pattern formation. Such molecular predator-prey models can be extended to a further negative-interaction mode, viral-host molecular evolution. RESULTS: A simple, nonfunctional predator-prey system based on the self-sustained sequence replication reaction is proposed. Coupling within the system is achieved using the single-stranded DNA intermediate of one cycle, the prey cycle, as primer for the second one, the predator cycle. Hybridization by complementary base pairing is the second order reaction step underlying the predation. Single steps of the whole reaction system have been investigated by radiolabeling. Each isolated subsystem operates according to the proposed reaction scheme, and evidence for an efficient coupling of both subsystems according to the proposed mechanism was found. CONCLUSIONS: Simple, interacting model systems based on nucleic acids can be designed and constructed for the study of coevolution. The results of studies such as the one described here will provide a basis for the construction of coupled systems of ribozymes, from which point the engineering of catalytic units for applications in biotechnology is feasible.

Base Sequence↗

PubMed1993

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).

Artifacts↗

PubMed1993

Images of evolution: origin of spontaneous RNA replication waves.

Self-replicating molecules set up traveling concentration waves that propagate in an aqueous enzyme solution. The velocity of each wave provides an accurate (+/- 0.1%) noninvasive measure of fitness for the RNA species currently growing in its front. Evolution may be followed from changes in the front velocity, and these differ from wave to wave. Thousands of controlled evolution reactions in traveling waves have been monitored in parallel to obtain quantitative images of the stochastic process of natural selection. An RNA polymerase (RNA-dependent RNA nucleotidyltransferase, EC 2.7.7.6), extracted from bacteria infected by the Q beta RNA virus, catalyzes the replication. The traveling waves that arise spontaneously without added RNA provide a model system for major evolutionary change.

Biological Evolution↗

PubMed1993

RNA multi-structure landscapes. A study based on temperature dependent partition functions.

Statistical properties of RNA folding landscapes obtained by the partition function algorithm (McCaskill 1990) are investigated in detail. The pair correlation of free energies as a function of the Hamming distance is used as a measure for the ruggedness of the landscape. The calculation of the partition function contains information about the entire ensemble of secondary structures as a function of temperature and opens the door to all quantities of thermodynamic interest, in contrast with the conventional minimal free energy approach. A metric distance of structure ensembles is introduced and pair correlations at the level of the structures themselves are computed. Just as with landscapes based on most stable secondary structure prediction, the landscapes defined on the full biophysical GCAU alphabet are much smoother than the landscapes restricted to pure GC sequences and the correlation lengths are almost constant fractions of the chain lengths. Correlation functions for multi-structure landscape exhibit an increased correlation length, especially near the melting temperature. However, the main effect on evolution is rather an effective increase in sampling for finite populations where each sequence explores multiple structures.

Base Composition↗

PubMed1992

Replication of viruses in a growing plaque: a reaction-diffusion model.

An understanding of the viral replication process commonly referred to as "plaque growth" is developed in the context of a reaction-diffusion model. The interactions among three components: the virus, the healthy host, and the infected host are represented using rates of viral adsorption and desorption to the cell surface, replication and release by host lysis, and diffusion. The solution to the full model reveals a maximum in the dependence of the velocity of viral propagation on its equilibrium adsorption constant, suggesting that conditions can be chosen where viruses which adsorb poorly to their hosts will replicate faster in plaques than those which adsorb well. Analytic expressions for the propagation velocity as a function of the kinetic and diffusion parameters are presented for the limiting cases of equilibrated adsorption, slow adsorption, fast adsorption, and large virus yields. Hindered diffusion at high host concentrations must be included for quantitative agreement with experimental data.

Bacterial Physiological Phenomena↗

PubMed1989

Traveling waves of in vitro evolving RNA.

Populations of short self-replicating RNA variants have been confined to one side of a reaction-diffusion traveling wave front propagating along thin capillary tubes containing the Q beta viral enzyme. The propagation speed is accurately measurable with a magnitude of about 1 micron/sec, and the wave persists for hundreds of generations (of duration less than 1 min). Evolution of RNA occurs in the wavefront, as established by front velocity changes and gel electrophoresis of samples drawn from along the capillary. The high population numbers (approximately equal to 10(11], their well-characterized biochemistry, their short generation time, and the constant conditions make the system ideal for evolution experiments. Growth is monitored continuously by excitation of an added RNA-sensitive fluorescent dye, ethidium bromide. An analytic expression for the front velocity is derived for the multicomponent kinetic scheme that reduces, for a high RNA-enzyme binding constant, to the Fisher form v = 2 square root of kappa D, where D is the diffusion constant of the complex and kappa is the low-concentration overall replication rate coefficient. The latter is confirmed as the selective value-determining parameter by numerical solution of a two-species system.

Biological Evolution↗

PubMedDate not supplied

The equilibrium partition function and base pair binding probabilities for RNA secondary structure.

A novel application of dynamic programming to the folding problem for RNA enables one to calculate the full equilibrium partition function for secondary structure and the probabilities of various substructures. In particular, both the partition function and the probabilities of all base pairs are computed by a recursive scheme of polynomial order N3 in the sequence length N. The temperature dependence of the partition function gives information about melting behavior for the secondary structure. The pair binding probabilities, the computation of which depends on the partition function, are visually summarized in a "box matrix" display and this provides a useful tool for examining the full ensemble of probable alternative equilibrium structures. The calculation of this ensemble representation allows a proper application and assessment of the predictive power of the secondary structure method, and yields important information on alternatives and intermediates in addition to local information about base pair opening and slippage. The results are illustrated for representative tRNA, 5S RNA, and self-replicating and self-splicing RNA molecules, and allow a direct comparison with enzymatic structure probes. The effect of changes in the thermodynamic parameters on the equilibrium ensemble provides a further sensitivity check to the predictions.

Animals↗