Yeast diversity in open agave fermentations across Mexico.

Yeasts are a diverse group of fungal microorganisms that are widely used to produce fermented foods and beverages. In Mexico, open fermentations are used to obtain spirits from agave plants. Despite the prevalence of this traditional practice throughout the country, yeasts have only been isolated and studied from a limited number of distilleries. To systematically describe the diversity of yeast species from open agave fermentations, here we generate the YMX-1.0 culture collection by isolating 4524 strains from 68 sites with diverse climatic, geographical, and biological contexts. We used MALDI-TOF mass spectrometry for taxonomic classification and validated a subset of the strains by ITS and D1/D2 sequencing, which also revealed two potential novel species of Saccharomycetales. Overall, the composition of yeast communities was weakly associated with local variables and types of climate, yet a core set of six species was consistently isolated from most producing regions. To explore the intraspecific variation of the yeasts from agave fermentations, we sequenced the genomes of four isolates of the nonconventional yeast Kazachstania humilis. The genomes of these four strains were substantially distinct from a European isolate of the same species, suggesting that they may belong to different populations. Our work contributes to the understanding and conservation of an open fermentation system of great cultural and economic importance, providing a valuable resource to study the biology and genetic diversity of microorganisms living at the interface of natural and human-associated environments.

Expression attenuation as a mechanism of robustness against gene duplication.

Gene duplication is ubiquitous and a major driver of phenotypic diversity across the tree of life, but its immediate consequences are not fully understood. Deleterious effects would decrease the probability of retention of duplicates and prevent their contribution to long-term evolution. One possible detrimental effect of duplication is the perturbation of the stoichiometry of protein complexes. Here, we measured the fitness effects of the duplication of 899 essential genes in the budding yeast using high-resolution competition assays. At least 10% of genes caused a fitness disadvantage when duplicated. Intriguingly, the duplication of most protein complex subunits had small to nondetectable effects on fitness, with few exceptions. We selected four complexes with subunits that had an impact on fitness when duplicated and measured the impact of individual gene duplications on their protein-protein interactions. We found that very few duplications affect both fitness and interactions. Furthermore, large complexes such as the 26S proteasome are protected from gene duplication by attenuation of protein abundance. Regulatory mechanisms that maintain the stoichiometric balance of protein complexes may protect from the immediate effects of gene duplication. Our results show that a better understanding of protein regulation and assembly in complexes is required for the refinement of current models of gene duplication.

Nitrogen coordinated import and export of arginine across the yeast vacuolar membrane.

The vacuole of the yeast Saccharomyces cerevisiae plays an important role in nutrient storage. Arginine, in particular, accumulates in the vacuole of nitrogen-replete cells and is mobilized to the cytosol under nitrogen starvation. The arginine import and export systems involved remain poorly characterized, however. Furthermore, how their activity is coordinated by nitrogen remains unknown. Here we characterize Vsb1 as a novel vacuolar membrane protein of the APC (amino acid-polyamine-organocation) transporter superfamily which, in nitrogen-replete cells, is essential to active uptake and storage of arginine into the vacuole. A shift to nitrogen starvation causes apparent inhibition of Vsb1-dependent activity and mobilization of stored vacuolar arginine to the cytosol. We further show that this arginine export involves Ypq2, a vacuolar protein homologous to the human lysosomal cationic amino acid exporter PQLC2 and whose activity is detected only in nitrogen-starved cells. Our study unravels the main arginine import and export systems of the yeast vacuole and suggests that they are inversely regulated by nitrogen.

Synthetic negative genome screen of the GPN-loop GTPase NPA3 in Saccharomyces cerevisiae.

The GPN-loop GTPase Npa3 is encoded by an essential gene in the yeast Saccharomyces cerevisiae. Npa3 plays a critical role in the assembly and nuclear accumulation of RNA polymerase II (RNAPII), a function that may explain its essentiality. Genetic interactions describe the extent to which a mutation in a particular gene affects a specific phenotype when co-occurring with an alteration in a second gene. Discovering synthetic negative genetic interactions has long been used as a tool to delineate the functional relatedness between pairs of genes participating in common or compensatory biological pathways. Previously, our group showed that nuclear targeting and transcriptional activity of RNAPII were unaffected in cells expressing exclusively a C-terminal truncated mutant version of Npa3 (npa3∆C) lacking the last 106 residues naturally absent from the single GPN protein in Archaea, but universally conserved in all Npa3 orthologs of eukaryotes. To gain insight into novel cellular functions for Npa3, we performed here a genome-wide Synthetic Genetic Array (SGA) study coupled to bulk fluorescence monitoring to identify negative genetic interactions of NPA3 by crossing an npa3∆C strain with a 4,389 nonessential gene-deletion collection. This genetic screen revealed previously unknown synthetic negative interactions between NPA3 and 15 genes. Our results revealed that the Npa3 C-terminal tail extension regulates the participation of this essential GTPase in previously unknown biological processes related to mitochondrial homeostasis and ribosome biogenesis.

Npa3/ScGpn1 carboxy-terminal tail is dispensable for cell viability and RNA polymerase II nuclear targeting but critical for microtubule stability and function.

Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

Adaptation to High Ethanol Reveals Complex Evolutionary Pathways.

Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts.

Increased rates of protein evolution and asymmetric deceleration after the whole-genome duplication in yeasts.

BACKGROUND: Whole-genome duplication (WGD) events have shaped the genomes of eukaryotic organisms. Relaxed selection after duplication along with inherent functional constraints are thought to determine the fate of the paralogs and, ultimately, the evolution of gene function. Here, we investigated the rate of protein evolution (as measured by dN/dS ratios) before and after the WGD in the hemiascomycete yeasts, and the way in which changes in such rates relate to molecular and biological function. RESULTS: For most groups of orthologous genes (81%) we observed a change in the rates of evolution after genome duplication. Genes with atypically-low dN/dS ratio before the WGD were prone to increase their rates of evolution after duplication. Importantly, the paralogs were often different in their rates of evolution after the WGD (50% cases), however, this was more consistent with an asymmetric deceleration in the protein-evolution rates, rather than an asymmetric increase of the initial rates. Functional-category analysis showed that regulatory proteins such as protein kinases and transcription factors were enriched in genes that increase their rates of evolution after the WGD. While changes in the rate of protein-sequence evolution were associated to protein abundance, content of disordered regions, and contribution to fitness, these features were an attribute of specific functional classes. CONCLUSIONS: Our results indicate that strong purifying selection in ancestral pre-duplication sequences is a strong predictor of increased rates after the duplication in yeasts and that asymmetry in evolution rate is established during the deceleration phase. In addition, changes in the rates at which paralogous sequences evolve before and after WGD are different for specific protein functions; increased rates of protein evolution after duplication occur preferentially in specific protein functions.

Natural selection drove metabolic specialization of the chromatophore in Paulinella chromatophora.

BACKGROUND: Genome degradation of host-restricted mutualistic endosymbionts has been attributed to inactivating mutations and genetic drift while genes coding for host-relevant functions are conserved by purifying selection. Unlike their free-living relatives, the metabolism of mutualistic endosymbionts and endosymbiont-originated organelles is specialized in the production of metabolites which are released to the host. This specialization suggests that natural selection crafted these metabolic adaptations. In this work, we analyzed the evolution of the metabolism of the chromatophore of Paulinella chromatophora by in silico modeling. We asked whether genome reduction is driven by metabolic engineering strategies resulted from the interaction with the host. As its widely known, the loss of enzyme coding genes leads to metabolic network restructuring sometimes improving the production rates. In this case, the production rate of reduced-carbon in the metabolism of the chromatophore. RESULTS: We reconstructed the metabolic networks of the chromatophore of P. chromatophora CCAC 0185 and a close free-living relative, the cyanobacterium Synechococcus sp. WH 5701. We found that the evolution of free-living to host-restricted lifestyle rendered a fragile metabolic network where >80% of genes in the chromatophore are essential for metabolic functionality. Despite the lack of experimental information, the metabolic reconstruction of the chromatophore suggests that the host provides several metabolites to the endosymbiont. By using these metabolites as intracellular conditions, in silico simulations of genome evolution by gene lose recover with 77% accuracy the actual metabolic gene content of the chromatophore. Also, the metabolic model of the chromatophore allowed us to predict by flux balance analysis a maximum rate of reduced-carbon released by the endosymbiont to the host. By inspecting the central metabolism of the chromatophore and the free-living cyanobacteria we found that by improvements in the gluconeogenic pathway the metabolism of the endosymbiont uses more efficiently the carbon source for reduced-carbon production. In addition, our in silico simulations of the evolutionary process leading to the reduced metabolic network of the chromatophore showed that the predicted rate of released reduced-carbon is obtained in less than 5% of the times under a process guided by random gene deletion and genetic drift. We interpret previous findings as evidence that natural selection at holobiont level shaped the rate at which reduced-carbon is exported to the host. Finally, our model also predicts that the ABC phosphate transporter (pstSACB) which is conserved in the genome of the chromatophore of P. chromatophora strain CCAC 0185 is a necessary component to release reduced-carbon molecules to the host. CONCLUSION: Our evolutionary analysis suggests that in the case of Paulinella chromatophora natural selection at the holobiont level played a prominent role in shaping the metabolic specialization of the chromatophore. We propose that natural selection acted as a "metabolic engineer" by favoring metabolic restructurings that led to an increased release of reduced-carbon to the host.

High-resolution profiling of stationary-phase survival reveals yeast longevity factors and their genetic interactions.

Lifespan is influenced by a large number of conserved proteins and gene-regulatory pathways. Here, we introduce a strategy for systematically finding such longevity factors in Saccharomyces cerevisiae and scoring the genetic interactions (epistasis) among these factors. Specifically, we developed an automated competition-based assay for chronological lifespan, defined as stationary-phase survival of yeast populations, and used it to phenotype over 5,600 single- or double-gene knockouts at unprecedented quantitative resolution. We found that 14% of the viable yeast mutant strains were affected in their stationary-phase survival; the extent of true-positive chronological lifespan factors was estimated by accounting for the effects of culture aeration and adaptive regrowth. We show that lifespan extension by dietary restriction depends on the Swr1 histone-exchange complex and that a functional link between autophagy and the lipid-homeostasis factor Arv1 has an impact on cellular lifespan. Importantly, we describe the first genetic interaction network based on aging phenotypes, which successfully recapitulated the core-autophagy machinery and confirmed a role of the human tumor suppressor PTEN homologue in yeast lifespan and phosphatidylinositol phosphate metabolism. Our quantitative analysis of longevity factors and their genetic interactions provides insights into the gene-network interactions of aging cells.

Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae.

Production of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4Δ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4Δ mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of α-IPMS activity in ethanol-grown cultures showed that α-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast α-IPMSs have resulted in a specific regulatory system that controls the leucine-α-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms.

Molecular mechanisms of paralogous compensation and the robustness of cellular networks.

Robustness is the ability of a system to maintain its function despite environmental or genetic perturbation. Genetic robustness is a key emerging property of living systems and is achieved notably by the presence of partially redundant parts that result from gene duplication. Functional overlap between paralogs allows them to compensate for each other's loss, as commonly revealed by aggravating genetic interactions. However, the molecular mechanisms linking the genotype (loss of function of a gene) to the phenotype (genetic buffering by a paralog) are still poorly understood and the molecular aspects of this compensation are rarely addressed in studies of gene duplicates. Here, we review molecular mechanisms of functional compensation between paralogous genes, many of which from studies that were not meant to study this phenomenon. We propose a standardized terminology and, depending on whether or not the molecular behavior of the intact gene is modified in response to the deletion of its paralog, we classify mechanisms of compensation into passive and active events. We further describe three non-exclusive mechanisms of active paralogous compensation for which there is evidence in the literature: changes in abundance, in localization, and in protein interactions. This review will serve as a framework for the genetic and molecular analysis of paralogous compensation, one of the universal features of genetic systems.

Modular epistasis in yeast metabolism.

Epistatic interactions, manifested in the effects of mutations on the phenotypes caused by other mutations, may help uncover the functional organization of complex biological networks. Here, we studied system-level epistatic interactions by computing growth phenotypes of all single and double knockouts of 890 metabolic genes in Saccharomyces cerevisiae, using the framework of flux balance analysis. A new scale for epistasis identified a distinctive trimodal distribution of these epistatic effects, allowing gene pairs to be classified as buffering, aggravating or noninteracting. We found that the ensuing epistatic interaction network could be organized hierarchically into function-enriched modules that interact with each other 'monochromatically' (i.e., with purely aggravating or purely buffering epistatic links). This property extends the concept of epistasis from single genes to functional units and provides a new definition of biological modularity, which emphasizes interactions between, rather than within, functional modules. Our approach can be used to infer functional gene modules from purely phenotypic epistasis measurements.

Need-based up-regulation of protein levels in response to deletion of their duplicate genes.

Many duplicate genes maintain functional overlap despite divergence over long evolutionary time scales. Deleting one member of a paralogous pair often has no phenotypic effect, unless its paralog is also deleted. It has been suggested that this functional compensation might be mediated by active up-regulation of expression of a gene in response to deletion of its paralog. However, it is not clear how prevalent such paralog responsiveness is, nor whether it is hardwired or dependent on feedback from environmental conditions. Here, we address these questions at the genomic scale using high-throughput flow cytometry of single-cell protein levels in differentially labeled cocultures of wild-type and paralog-knockout Saccharomyces cerevisiae strains. We find that only a modest fraction of proteins (22 out of 202) show significant up-regulation to deletion of their duplicate genes. However, these paralog-responsive proteins match almost exclusively duplicate pairs whose overlapping function is required for growth. Moreover, media conditions that add or remove requirements for the function of a duplicate gene pair specifically eliminate or create paralog responsiveness. Together, our results suggest that paralog responsiveness in yeast is need-based: it appears only in conditions in which the gene function is required. Physiologically, such need-based responsiveness could provide an adaptive mechanism for compensation of genetic, environmental, or stochastic perturbations in protein abundance.

The protein-protein interaction landscape of transcription factors during gynoecium development in Arabidopsis.

Flowers are composed of organs whose identity is defined by the combinatorial activity of transcription factors (TFs). The interactions between MADS-box TFs and protein complex formation have been schematized in the floral quartet model of flower development. The gynoecium is the flower's female reproductive part, crucial for fruit and seed production and, hence, for reproductive success. After the establishment of carpel identity, many tissues arise to form a mature gynoecium. TFs have been described as regulators of gynoecium development, and some interactions and complexes have been identified. However, broad knowledge about the interactions among these TFs and their participation during development remains scarce. In this study, we used a systems biology approach to understand the formation of a complex reproductive unit-as the gynoecium-by mapping binary interactions between well-characterized TFs. We analyzed almost 4500 combinations and detected more than 250 protein-protein interactions (PPIs), resulting in a process-specific interaction map. Topological analyses suggest hidden functions and novel roles for many TFs. In addition, we observed a close relationship between TFs involved in auxin and cytokinin-signaling pathways and other TFs. Furthermore, we analyzed the network by combining PPI data, expression, and genetic data, which helped us to dissect it into several dynamic spatio-temporal subnetworks related to gynoecium development processes. Finally, we generated an extended PPI network that predicts new players in gynoecium development. Taken together, all these results serve as a valuable resource for the plant community.

Systematic profiling of subtelomeric silencing factors in budding yeast.

Subtelomeric gene silencing is the negative transcriptional regulation of genes located close to telomeres. This phenomenon occurs in a variety of eukaryotes with salient physiological implications, such as cell adherence, virulence, immune-system escape, and ageing. The process has been widely studied in the budding yeast Saccharomyces cerevisiae, where genes involved in this process have been identified mostly on a gene-by-gene basis. Here, we introduce a quantitative approach to study gene silencing, that couples the classical URA3 reporter with GFP monitoring, amenable to high-throughput flow cytometry analysis. This dual silencing reporter was integrated into several subtelomeric loci in the genome, where it showed a gradual range of silencing effects. By crossing strains with this dual reporter at the COS12 and YFR057W subtelomeric query loci with gene-deletion mutants, we carried out a large-scale forward screen for potential silencing factors. The approach was replicable and allowed accurate detection of expression changes. Results of our comprehensive screen suggest that the main players influencing subtelomeric silencing were previously known, but additional potential factors underlying chromatin conformation are involved. We validate and report the novel silencing factor LGE1, a protein with unknown molecular function required for histone H2B ubiquitination. Our strategy can be readily combined with other reporters and gene perturbation collections, making it a versatile tool to study gene silencing at a genome-wide scale.

Draft Genome Sequence of a Kazachstania humilis Strain Isolated from Agave Fermentation.

The ascomycetous yeast Kazachstania humilis is an active species in backslopped sourdough and in the spontaneous fermentation of several traditional foods and beverages. Here, we report the draft genome sequence of a K. humilis strain isolated from agave must from a traditional distillery in Mexico.

Multiple Forms of Multifunctional Proteins in Health and Disease.

Protein science has moved from a focus on individual molecules to an integrated perspective in which proteins emerge as dynamic players with multiple functions, rather than monofunctional specialists. Annotation of the full functional repertoire of proteins has impacted the fields of biochemistry and genetics, and will continue to influence basic and applied science questions - from the genotype-to-phenotype problem, to our understanding of human pathologies and drug design. In this review, we address the phenomena of pleiotropy, multidomain proteins, promiscuity, and protein moonlighting, providing examples of multitasking biomolecules that underlie specific mechanisms of human disease. In doing so, we place in context different types of multifunctional proteins, highlighting useful attributes for their systematic definition and classification in future research directions.

An Optimized Competitive-Aging Method Reveals Gene-Drug Interactions Underlying the Chronological Lifespan of Saccharomyces cerevisiae.

The chronological lifespan of budding yeast is a model of aging and age-related diseases. This paradigm has recently allowed genome-wide screening of genetic factors underlying post-mitotic viability in a simple unicellular system, which underscores its potential to provide a comprehensive view of the aging process. However, results from different large-scale studies show little overlap and typically lack quantitative resolution to derive interactions among different aging factors. We previously introduced a sensitive, parallelizable approach to measure the chronological-lifespan effects of gene deletions based on the competitive aging of fluorescence-labeled strains. Here, we present a thorough description of the method, including an improved multiple-regression model to estimate the association between death rates and fluorescent signals, which accounts for possible differences in growth rate and experimental batch effects. We illustrate the experimental procedure-from data acquisition to calculation of relative survivorship-for ten deletion strains with known lifespan phenotypes, which is achieved with high technical replicability. We apply our method to screen for gene-drug interactions in an array of yeast deletion strains, which reveals a functional link between protein glycosylation and lifespan extension by metformin. Competitive-aging screening coupled to multiple-regression modeling provides a powerful, straight-forward way to identify aging factors in yeast and their interactions with pharmacological interventions.

Functional genomics of dietary restriction and longevity in yeast.

Dietary restriction-limitation of calories or other specific nutrients in the diet-is the sole non-genetic intervention known to extend the lifespan of a wide range of model organisms from yeast to mammals. Cell biology studies on the responses to dietary restriction have provided important clues about the mechanisms of longevity; however, a comprehensive genome-wide description of lifespan by dietary restriction has been mostly absent. Large-scale genetic analysis in the budding yeast Saccharomyces cerevisiae offers a great opportunity to uncover the conserved systems-level mechanisms that give way to longevity in response to diet. Here, we review recent advances in high-throughput phenotyping of the replicative and chronological life spans of yeast cells, which have contributed to our understanding of longevity by dietary restriction and the cellular crosstalks of nutrient-sensing regulation.

The origin of a novel gene through overprinting in Escherichia coli.

BACKGROUND: Overlapped genes originate by a) loss of a stop codon among contiguous genes coded in different frames; b) shift to an upstream initiation codon of one of the contiguous genes; or c) by overprinting, whereby a novel open reading frame originates through point mutation inside an existing gene. Although overlapped genes are common in viruses, it is not clear whether overprinting has led to new genes in prokaryotes. RESULTS: Here we report the origin of a new gene through overprinting in Escherichia coli K12. The htgA gene coding for a positive regulator of the sigma 32 heat shock promoter arose by point mutation in a 123/213 phase within an open reading frame (yaaW) of unknown function, most likely in the lineage leading to E. coli and Shigella sp. Further, we show that yaaW sequences coding for htgA genes have a slower evolutionary rate than those lacking an overlapped htgA gene. CONCLUSION: While overprinting has been shown to be rather frequent in the evolution of new genes in viruses, our results suggest that this mechanism has also contributed to the origin of a novel gene in a prokaryote. We propose the term janolog (from Jano, the two-faced Roman god) to describe the homology relationship that holds between two genes when one originated through overprinting of the other. One cannot dismiss the possibility that at least a small fraction of the large number of novel ORPhan genes detected in pan-genome and metagenomic studies arose by overprinting.