Predicting stochastic gene expression dynamics in single cells

Fluctuations in protein numbers (noise) due to inherent stochastic effects in single cells can have large effects on the dynamic behavior of gene regulatory networks. Although deterministic models can predict the average network behavior, they fail to incorporate the stochasticity characteristic of gene expression, thereby limiting their relevance when single cell behaviors deviate from the population average. Recently, stochastic models have been used to predict distributions of steady-state protein levels within a population but not to predict the dynamic, presteady-state distributions. In the present work, we experimentally examine a system whose dynamics are heavily influenced by stochastic effects. We measure population distributions of protein numbers as a function of time in the Escherichia coli lactose uptake network (lac operon). We then introduce a dynamic stochastic model and show that prediction of dynamic distributions requires only a few noise parameters in addition to the rates that characterize a deterministic model. Whereas the deterministic model cannot fully capture the observed behavior, our stochastic model correctly predicts the experimental dynamics without any fit parameters. Our results provide a proof of principle for the possibility of faithfully predicting dynamic population distributions from deterministic models supplemented by a stochastic component that captures the major noise sources.     

Predicting drug response based on gene expression

Predicting drug response is a challenging problem in oncology. In the 1975-1985 decade, important efforts were devoted to the generation of cellular assays able to predict, on an individual basis, the in vitro response of tumour cells to chemotherapeutic agents, but such methods could not be adopted in routine. Numerous mechanisms of resistance to anticancer agents have been identified in cultured cell lines selected for growth in the presence of infratoxic, increasing doses of anticancer agents. They mainly concern drug transport, drug activation or detoxification, target quantitative or qualitative alterations, DNA repair efficiency, and alterations in signalling and/or execution of cell death programmes. New molecular biology techniques have been developed in order to identify the genes involved in drug resistance; they mainly involve differential expression techniques, but functional approaches may also prove informative. The availability of techniques of gene expression profiling has allowed to establish correlations between gene expression and drug sensitivity of tumour cells or human cancers. This type of approach has been initiated on in vitro systems by the National Cancer Institute (NCI) in the USA and is pursued by a growing number of public and private laboratories around the world. In the clinical setting, a number of genes or proteins have been identified as potential predictive markers of drug activity and their use could be progressively implemented for drug selection in patients receiving chemotherapy, allowing thus more rational and individualised treatments.     

Elevated gene expression levels distinguish human from non-human primate brains

Little is known about how the human brain differs from that of our closest relatives. To investigate the genetic basis of human specializations in brain organization and cognition, we compared gene expression profiles for the cerebral cortex of humans, chimpanzees, and rhesus macaques by using several independent techniques. We identified 169 genes that exhibited expression differences between human and chimpanzee cortex, and 91 were ascribed to the human lineage by using macaques as an outgroup. Surprisingly, most differences between the brains of humans and non-human primates involved up-regulation, with approximately 90% of the genes being more highly expressed in humans. By contrast, in the comparison of human and chimpanzee heart and liver, the numbers of up- and down-regulated genes were nearly identical. Our results indicate that the human brain displays a distinctive pattern of gene expression relative to non-human primates, with higher expression levels for many genes belonging to a wide variety of functional classes. The increased expression of these genes could provide the basis for extensive modifications of cerebral physiology and function in humans and suggests that the human brain is characterized by elevated levels of neuronal activity.     

Comparative analysis of gene expression among low G+C gram-positive genomes

We present a comparative analysis of predicted highly expressed (PHX) genes in the low G+C Gram-positive genomes of Bacillus subtilis, Bacillus halodurans, Listeria monocytogenes, Listeria innocua, Lactococcus lactis, Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Clostridium acetobutylicum, and Clostridium perfringens. Most enzymes acting in glycolysis and fermentation pathways are PHX in these genomes, but not those involved in the TCA cycle and respiration, suggesting that these organisms have predominantly adapted to grow rapidly in an anaerobic environment. Only B. subtilis and B. halodurans have several TCA cycle PHX genes, whereas the TCA pathway is entirely missing from the metabolic repertoire of the two Streptococcus species and is incomplete in Listeria, Lactococcus, and Clostridium. Pyruvate-formate lyase, an enzyme critical in mixed acid fermentation, is among the highest PHX genes in all these genomes except for C. acetobutylicum (not PHX), and B. subtilis, and B. halodurans (missing). Pyruvate-formate lyase is also prominently PHX in enteric gamma-proteobacteria, but not in other prokaryotes. Phosphotransferase system genes are generally PHX with selection of different substrates in different genomes. The various substrate specificities among phosphotransferase systems in different genomes apparently reflect on differences in habitat, lifestyle, and nutrient sources.     

Gene clustering pattern, promoter architecture, and gene expression stability in eukaryotic genomes

A balance between gene expression stability and evolvability is essential for the long-term maintenance of a living system. In this paper, we studied whether the genetic and epigenetic properties of the promoter affect gene expression variability. We hypothesized that upstream distance and orientation (head-to-head or head-to-tail) are important for the promoter architecture and gene expression variability. We found that in budding yeast genes with a short upstream distance tend to have low gene expression variability, and their promoter is flanked by strongly positioned nucleosomes and tends to have low nucleosome occupancy. These observations suggest that in vivo positioning of the flanking nucleosomes facilitates stable nucleosome depletion at the core promoter region and enhances gene expression stability. Head-to-head genes have, on average, lower gene expression variability, greater nucleosome depletion at the core promoter region, and more strongly positioned nucleosomes that flank the core promoter than do head-to-tail genes. These observations hold for diverse eukaryotes. In complex organisms such as mammals, only a small fraction of head-to-tail genes have retained a short upstream distance, probably because the promoter may not be flanked by a strongly positioned nucleosome on the upstream side.     

Blood gene expression signatures predict exposure levels

To respond to potential adverse exposures properly, health care providers need accurate indicators of exposure levels. The indicators are particularly important in the case of acetaminophen (APAP) intoxication, the leading cause of liver failure in the U.S. We hypothesized that gene expression patterns derived from blood cells would provide useful indicators of acute exposure levels. To test this hypothesis, we used a blood gene expression data set from rats exposed to APAP to train classifiers in two prediction algorithms and to extract patterns for prediction using a profiling algorithm. Prediction accuracy was tested on a blinded, independent rat blood test data set and ranged from 88.9% to 95.8%. Genomic markers outperformed predictions based on traditional clinical parameters. The expression profiles of the predictor genes from the patterns extracted from the blood exhibited remarkable (97% accuracy) transtissue APAP exposure prediction when liver gene expression data were used as a test set. Analysis of human samples revealed separation of APAP-intoxicated patients from control individuals based on blood expression levels of human orthologs of the rat discriminatory genes. The major biological signal in the discriminating genes was activation of an inflammatory response after exposure to toxic doses of APAP. These results support the hypothesis that gene expression data from peripheral blood cells can provide valuable information about exposure levels, well before liver damage is detected by classical parameters. It also supports the potential use of genomic markers in the blood as surrogates for clinical markers of potential acute liver damage.     

A second insulin gene in fish genomes

The recent characterization of diverse vertebrate genomes has revealed the importance of gene duplication in vertebrate evolution. Evidence suggests that a genome duplication event occurred on the lineage leading to teleost fish-species that are often used to understand human biology. The existence of a genome duplication event complicates the use of fish as a model for human diseases as there are often two fish homologues for a single copy human gene. Often the second homologue has not been recognized. Our searches of the near complete zebrafish and fugu fish genomes indicate that both species have two insulin genes. Phylogenetic analysis indicates that the two genes are likely the product of the fish-specific genome duplication. The maintenance of two insulin genes within the fish suggests that the two genes have different functions. Thus the well-characterized insulin genes in some fish species may not be complete homologues of the human insulin gene.     

Evaluation of gene expression levels for cytokines in ocular toxoplasmosis

This study evaluated levels for mRNA expression of 7 cytokines in ocular toxoplasmosis. Peripheral blood mononuclear cells (PBMC) of patients with ocular toxoplasmosis (OT Group, n = 23) and chronic toxoplasmosis individuals (CHR Group, n = 9) were isolated and stimulated in vitro with T. gondii antigen. Negative controls (NC) were constituted of 7 PBMC samples from individuals seronegative for toxoplasmosis. mRNA expression for cytokines was determined by qPCR. Results showed a significant increase in mRNA levels from antigen stimulated PBMCs derived from OT Group for expressing IL‐6 (at P < .005 and P < .0005 for CHR and NC groups, respectively), IL‐10 (at P < .0005 and P < .005 for CHR and NC groups, respectively) and TGF‐β (at P < .005) for NC group. mRNA levels for TNF‐α and IL‐12 were also upregulated in patients with OT compared to CHR and NC individuals, although without statistical significance. Additionally, mRNA levels for IL‐27 and IFN‐γ in PBMC of patients with OT were upregulated in comparison with NC individuals. Differences between OT and NC groups were statistically significant at P < .05 and P < .0005, respectively.     

Archaeological Central American maize genomes suggest ancient gene flow from South America

Maize (Zea mays ssp. mays) domestication began in southwestern Mexico ∼9,000 calendar years before present (cal. BP) and humans dispersed this important grain to South America by at least 7,000 cal. BP as a partial domesticate. South America served as a secondary improvement center where the domestication syndrome became fixed and new lineages emerged in parallel with similar processes in Mesoamerica. Later, Indigenous cultivators carried a second major wave of maize southward from Mesoamerica, but it has been unclear until now whether the deeply divergent maize lineages underwent any subsequent gene flow between these regions. Here we report ancient maize genomes (2,300–1,900 cal. BP) from El Gigante rock shelter, Honduras, that are closely related to ancient and modern maize from South America. Our findings suggest that the second wave of maize brought into South America hybridized with long-established landraces from the first wave, and that some of the resulting newly admixed lineages were then reintroduced to Central America. Direct radiocarbon dates and cob morphological data from the rock shelter suggest that more productive maize varieties developed between 4,300 and 2,500 cal. BP. We hypothesize that the influx of maize from South America into Central America may have been an important source of genetic diversity as maize was becoming a staple grain in Central and Mesoamerica.

Modern maize (Zea mays ssp. mays) is the world’s most productive staple crop (1) with over 1,100 million tons grown in 2018 (https://knoema.com/). Molecular data indicate that maize evolved from the annual grass teosinte (Zea mays ssp. parviglumus, hereafter parviglumus) in southwestern Mexico, and that the first steps toward domestication occurred ∼9,000 calendar years before present (cal. BP) (2, 3). Starch grain and phytolith data from archeological sites in the Balsas region of southwestern Mexico confirm the early use of maize by ∼8,700 cal. BP (4). Archaeobotanical evidence also indicates dispersal of maize out of the Balsas through Central America by ∼7,500 cal. BP (5) and into South America by ∼7,000 cal. BP (6, 7) and ultimately into North America starting around 4,000 cal. BP (8).Global dispersal of this productive crop beyond the Americas started with European contact with Native Americans in the 15th and 16th centuries (9). In addition to thousands of landraces developed by Indigenous cultivators in the Americas (10–15), experimentation worldwide has led to a staggering array of morphological diversity and adaptations to a wide range of geographic and climatic conditions. This diversity results from human-mediated selection, reproductive isolation from parviglumus (and related Zea mays ssp. mexicana, hereafter mexicana), secondary improvement, and the potential reintroduction of new varieties back to original homelands. A contemporary concern is the backflow of transgenic or commercial hybrid maize varieties to the Mexican heartland because gene flow with extant landraces can result in the loss of genetic diversity and cultural knowledge (10, 16). However, precolonial backflow of divergent maize varieties into Central and Mesoamerica during the last 9,000 y remains understudied, and could have ramifications for the history of maize as a staple in the region.Morphological evidence from ancient maize found in archaeological sites combined with DNA data confirms a complex and extended domestication history. The earliest maize cobs found in the highlands of Oaxaca, Mexico dating to 6,250 cal. BP are small, and have two nondisarticulating vertical rows of alternating seeds (17, 18) indicating that Indigenous cultivators were controlling plant reproduction. Early four-row cobs from Mexico’s Tehuacán Valley (5,300-4,950 cal. BP) are also small, and show comparable evidence for nondisarticulating seeds consistent with domestication (19, 20). Experimental work has shown that the lower atmospheric CO₂ and temperatures in the Late Pleistocene and Early Holocene may have favored phenotypic expression of maizelike inflorescence and seed architecture—nonbranching stalk architecture and naked grains—which could have been promoted and fixed by human-mediated selection (21, 22). Ancient DNA data from Tehuacán cobs (San Marcos Cave) dating between 5,300 and 4,950 cal. BP have alleles comparable to modern maize for inflorescence and seed architecture (td1 – tassel dwarf1; tb1 – teosinte branced1, ba1 – barren stalk1), circadian clock and flowering time (zmg1), and glycogen biosynthesis (bt2 – brittle endosperm2) (23, 24). However, some alleles controlling ear shattering and starch biosynthesis (zag1 – MADS-box gene; su1 – sugary 1, and wx1 – waxy 1) were more teosintelike at that point—four millennia after the onset of domestication. Introgression with mexicana favored adaptation to drier and cooler conditions in the Mexican highlands, and most modern landraces from highland Mexico and Central America carry strong signals of postdomestication mexicana admixture (13, 25). Continued introgression within the natural range of parviglumus and mexicana may also explain the gradual changes in cob size evident in the Tehuacán Valley (19).The initial first wave of maize dispersal (7,500–7,000 cal. BP) (5) through Central and South America likely occurred when maize was partially domesticated, and before the domestication syndrome—the suite of characters setting a domesticated species apart from its wild counterpart—was fixed (26). Multiple waves of dispersal brought maize out of southwestern Mexico into South America (26, 27), and may have episodically increased admixture and diversity. There is also evidence that deeply structured maize lineages in South America underwent independent fixation of the domestication syndrome and secondary improvement outside the range of parviglumus and mexicana (26). Reduced crop-wild gene flow outside the range of parviglumis and mexicana also enhanced selection for increased cob and seed size (6), and was instrumental in the development of more productive staple grain varieties and greater consumption in Central America starting after 4,700 cal. BP (28, 29). Furthermore, adaptations in the US Southwest—outside the domestication center—resulted in the shorter growing season varieties with earlier flowering times required for dispersal through more temperate parts of North America after 2,000 cal. BP (30). In total, it is clear from the available data that maize diversity and biogeography is complex, and resulted from multiple episodes of human-mediated selection and dispersal throughout the Americas.Research has focused on the outward dispersal of maize from the original domestication center in southwestern Mexico. However, crop movements were complex, and the archaeological record shows clear evidence of two-way movements of plants and people lasting millennia between Central and South America. It is reasonable to suspect that maize, the most widespread crop species of the precolonial Americas, traveled back toward the domestication center in the hands of skilled farmers as part of this complex history. Here, we sequenced maize genomes from three archaeological samples from El Gigante rock shelter in western Honduras dating to between 2,300 and 1,900 cal. BP and compared these data to published modern landraces of maize and archaeological samples from North, Central, and South America. We use these genomes as a temporal anchor to test the hypotheses that humans moved maize from South America into Central America. In this scenario, the reintroduced germplasm may have been impactful for the development of highly productive varieties. Isotopic evidence from Central America demonstrates substantial maize consumption as a staple grain beginning between 4,700 and 4,000 cal. BP (29). Finally, we use morphological comparisons within the El Gigante maize assemblage to help constrain the timing of this gene flow.     

Visualizing high error levels during gene expression in living bacterial cells

To monitor inaccuracy in gene expression in living cells, we designed an experimental system in the bacterium Bacillus subtilis whereby spontaneous errors can be visualized and quantified at a single-cell level. Our strategy was to introduce mutations into a chromosomally encoded gfp allele, such that errors in protein production are reported in real time by the formation of fluorescent GFP molecules. The data reveal that the amount of errors can greatly exceed previous estimates, and that the error rate increases dramatically at lower temperatures and during stationary phase. Furthermore, we demonstrate that when facing an antibiotic threat, an increase in error level is sufficient to allow survival of bacteria carrying a mutated antibiotic-resistance gene. We propose that bacterial gene expression is error prone, frequently yielding protein molecules that differ slightly from the sequence specified by their DNA, thus generating a cellular reservoir of nonidentical protein molecules. This variation may be a key factor in increasing bacterial fitness, expanding the capability of an isogenic population to face environmental challenges.     

Predicting the clinical status of human breast cancer by using gene expression profiles

Prognostic and predictive factors are indispensable tools in the treatment of patients with neoplastic disease. For the most part, such factors rely on a few specific cell surface, histological, or gross pathologic features. Gene expression assays have the potential to supplement what were previously a few distinct features with many thousands of features. We have developed Bayesian regression models that provide predictive capability based on gene expression data derived from DNA microarray analysis of a series of primary breast cancer samples. These patterns have the capacity to discriminate breast tumors on the basis of estrogen receptor status and also on the categorized lymph node status. Importantly, we assess the utility and validity of such models in predicting the status of tumors in crossvalidation determinations. The practical value of such approaches relies on the ability not only to assess relative probabilities of clinical outcomes for future samples but also to provide an honest assessment of the uncertainties associated with such predictive classifications on the basis of the selection of gene subsets for each validation analysis. This latter point is of critical importance in the ability to apply these methodologies to clinical assessment of tumor phenotype.