Quantitative modeling of stochastic systems in molecular biology by using stochastic Petri nets

An integrated understanding of molecular and developmental biology must consider the large number of molecular species involved and the low concentrations of many species in vivo. Quantitative stochastic models of molecular interaction networks can be expressed as stochastic Petri nets (SPNs), a mathematical formalism developed in computer science. Existing software can be used to define molecular interaction networks as SPNs and solve such models for the probability distributions of molecular species. This approach allows biologists to focus on the content of models and their interpretation, rather than their implementation. The standardized format of SPNs also facilitates the replication, extension, and transfer of models between researchers. A simple chemical system is presented to demonstrate the link between stochastic models of molecular interactions and SPNs. The approach is illustrated with examples of models of genetic and biochemical phenomena where the UltraSAN package is used to present results from numerical analysis and the outcome of simulations.

Quantitative, chemically specific imaging of selenium transformation in plants

Quantitative, chemically specific images of biological systems would be invaluable in unraveling the bioinorganic chemistry of biological tissues. Here we report the spatial distribution and chemical forms of selenium in Astragalus bisulcatus (two-grooved poison or milk vetch), a plant capable of accumulating up to 0.65% of its shoot dry biomass as Se in its natural habitat. By selectively tuning incident x-ray energies close to the Se K-absorption edge, we have collected quantitative, 100-μm-resolution images of the spatial distribution, concentration, and chemical form of Se in intact root and shoot tissues. To our knowledge, this is the first report of quantitative concentration-imaging of specific chemical forms. Plants exposed to 5 μM selenate for 28 days contained predominantly selenate in the mature leaf tissue at a concentration of 0.3–0.6 mM, whereas the young leaves and the roots contained organoselenium almost exclusively, indicating that the ability to biotransform selenate is either inducible or developmentally specific. While the concentration of organoselenium in the majority of the root tissue was much lower than that of the youngest leaves (0.2–0.3 compared with 3–4 mM), isolated areas on the extremities of the roots contained concentrations of organoselenium an order of magnitude greater than the rest of the root. These imaging results were corroborated by spatially resolved x-ray absorption near-edge spectra collected from selected 100 × 100 μm2 regions of the same tissues.

Quantitative analysis of mRNA amplification by in vitro transcription

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.

Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice.

The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regulation in hepatocytes where, in presence of dox, less than one molecule of luciferase was detected per cell. By contrast, a more than 10(5)-fold activation of the luciferase gene was observed in the absence of the antibiotic. This regulation was homogeneous throughout but stringently restricted to hepatocytes. These results demonstrate that both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants.

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.

Microevolutionary responses in experimental populations of plants to CO2-enriched environments: parallel results from two model systems.

Despite the critical role that terrestrial vegetation plays in the Earth's carbon cycle, very little is known about the potential evolutionary responses of plants to anthropogenically induced increases in concentrations of atmospheric CO2. We present experimental evidence that rising CO2 concentration may have a direct impact on the genetic composition and diversity of plant populations but is unlikely to result in selection favoring genotypes that exhibit increased productivity in a CO2-enriched atmosphere. Experimental populations of an annual plant (Abutilon theophrasti, velvetleaf) and a temperate forest tree (Betula alleghaniensis, yellow birch) displayed responses to increased CO2 that were both strongly density-dependent and genotype-specific. In competitive stands, a higher concentration of CO2 resulted in pronounced shifts in genetic composition, even though overall CO2-induced productivity enhancements were small. For the annual species, quantitative estimates of response to selection under competition were 3 times higher at the elevated CO2 level. However, genotypes that displayed the highest growth responses to CO2 when grown in the absence of competition did not have the highest fitness in competitive stands. We suggest that increased CO2 intensified interplant competition and that selection favored genotypes with a greater ability to compete for resources other than CO2. Thus, while increased CO2 may enhance rates of selection in populations of competing plants, it is unlikely to result in the evolution of increased CO2 responsiveness or to operate as an important feedback in the global carbon cycle. However, the increased intensity of selection and drift driven by rising CO2 levels may have an impact on the genetic diversity in plant populations.