The Enrichment of miRNA-Targeted mRNAs in Translationally Less Active over More Active Polysomes.

miRNAs moderately inhibit the translation and enhance the degradation of their target mRNAs via cognate binding sites located predominantly in the 3'-untranslated regions (UTR). Paradoxically, miRNA targets are also polysome-associated. We studied the polysome association by the comparative translationally less-active light- and more-active heavy-polysome profiling of a wild type (WT) human cell line and its isogenic mutant (MT) with a disrupted DICER1 gene and, thus, mature miRNA production. As expected, the open reading frame (ORF) length is a major determinant of light- to heavy-polysome mRNA abundance ratios, but is rendered less powerful in WT than in MT cells by miRNA-regulatory activities. We also observed that miRNAs tend to target mRNAs with longer ORFs, and that adjusting the mRNA abundance ratio with the ORF length improves its correlation with the 3'-UTR miRNA-binding-site count. In WT cells, miRNA-targeted mRNAs exhibit higher abundance in light relative to heavy polysomes, i.e., light-polysome enrichment. In MT cells, the DICER1 disruption not only significantly abrogated the light-polysome enrichment, but also narrowed the mRNA abundance ratio value range. Additionally, the abrogation of the enrichment due to the DICER1 gene disruption, i.e., the decreases of the ORF-length-adjusted mRNA abundance ratio from WT to MT cells, exhibits a nearly perfect linear correlation with the 3'-UTR binding-site count. Transcription factors and protein kinases are the top two most enriched mRNA groups. Taken together, the results provide evidence for the light-polysome enrichment of miRNA-targeted mRNAs to reconcile polysome association and moderate translation inhibition, and that ORF length is an important, though currently under-appreciated, transcriptome regulation parameter.

Comparative Analysis of microRNA Binding Site Distribution and microRNA-Mediated Gene Expression Repression of Oncogenes and Tumor Suppressor Genes.

MicroRNAs (miRNAs) are a family of short, noncoding RNAs that can regulate gene expression levels of over half of the human genome. Previous studies on the role of miRNAs in cancer showed overall widespread downregulation of miRNAs as a hallmark of human cancer, though individual miRNAs can be both tumor suppressive and oncogenic, and cancer genes are speculated to be more targeted by miRNA. However, the extents to which oncogenes and tumor suppressor genes (TSG) are controlled by miRNA have not been compared. To achieve this goal, we constructed lists of oncogenes and TSGs and compared them with each other, and with the whole protein-coding gene population, in terms of miRNA binding sites distribution and expression level changes upon genetic disruption of miRNA production. As expected, the results show that cancer gene mRNAs anchor more miRNA binding sites, and are under a higher degree of miRNA-mediated repression at both mRNA abundance and translation efficiency levels than the whole protein-coding gene population. Importantly, on average, TSG mRNAs are more highly targeted and regulated by miRNA than oncogene mRNAs. To the best of our knowledge, this is the first comparison of miRNA regulation of oncogenes and TSGs.

Uncovering the cellular capacity for intensive and specific feedback self-control of the argonautes and MicroRNA targeting activity.

The miRNA pathway has three segments-biogenesis, targeting and downstream regulatory effectors. We aimed to better understand their cellular control by exploring the miRNA-mRNA-targeting relationships. We first used human evolutionarily conserved sites. Strikingly, AGOs 1-3 are all among the top 14 mRNAs with the highest miRNA site counts, along with ANKRD52, the phosphatase regulatory subunit of the recently identified AGO phosphorylation cycle; and the AGO phosphorylation cycle mRNAs share much more than expected miRNA sites. The mRNAs for TNRC6, which acts with AGOs to channel miRNA-mediated regulatory actions onto specific mRNAs, are also heavily miRNA-targeted. In contrast, upstream miRNA biogenesis mRNAs are not, and neither are downstream regulatory effectors. In short, binding site enrichment in miRNA targeting machinery mRNAs, but neither upstream biogenesis nor downstream effector mRNAs, was observed, endowing a cellular capacity for intensive and specific feedback control of the targeting activity. The pattern was confirmed with experimentally determined miRNA-mRNA target relationships. Moreover, genetic experiments demonstrated cellular utilization of this capacity. Thus, we uncovered a capacity for intensive, and specific, feedback-regulation of miRNA targeting activity directly by miRNAs themselves, i.e. segment-specific feedback auto-regulation of miRNA pathway, complementing miRNAs pairing with transcription factors to form hybrid feedback-loop.

Correction to: Next Generation Sequencing (NGS) Application in Multiparameter Gene Expression Analysis.

Chapter 2 was inadvertently published with the contributing author's name printed as Andrey L. Karamychev, whereas it should have been Andrey L. Karamyshev. This correction has been updated in the book.

Next Generation Sequencing (NGS) Application in Multiparameter Gene Expression Analysis.

Next generation sequencing (NGS) is routinely used in gene expression analyses. In particular, RNA-seq has been the method of choice for highly sensitive genome-wide quantification of RNA expression. The method can be used in a wide variety of model systems, including studies to gain insight into underlying mechanisms of toxicologic processes and disease development induced by environmental toxicants. RNA-seq has also been coupled to many other molecular biology protocols to monitor specific aspects of the gene expression process. Here, we describe two such coupling-(a) global run-on sequencing (GRO-seq) that coupled it to the nuclear run-on (NRO), and (b) polysome profiling that coupled it to sucrose-gradient-based polysome isolation. Simultaneous RNA-seq, GRO-seq, and polysome profiling analyses enabled genome-wide analysis of the mode of stability control of individual RNA molecules.

Monitoring cyanobacterial toxins in a large reservoir: relationships with water quality parameters.

Cyanobacteria are widely distributed in fresh, brackish, and ocean water environments, as well as in soil and on moist surfaces. Changes in the population of cyanobacteria can be an important indicator of alterations in water quality. Metabolites produced by blooms of cyanobacteria can be harmful, so cell counts are frequently monitored to assess the potential risk from cyanobacterial toxins. A frequent uncertainty in these types of assessments is the lack of strong relationships between cell count numbers and algal toxin concentrations. In an effort to use ion concentrations and other water quality parameters to determine the existence of any relationships with cyanobacterial toxin concentrations, we monitored four cyanobacterial toxins and inorganic ions in monthly water samples from a large reservoir over a 2-year period. Toxin concentrations during the study period never exceeded safety limits. In addition, toxin concentrations at levels above the limit of quantitation were infrequent during the 2-year sampling period; non-detects were common. Microcystin-LA was the least frequently detected analyte (86 of 89 samples were ND), followed by the other microcystins (microcystin-RR, microcystin-LR). Cylindrospermopsin and saxitoxin were the most frequently detected analytes. Microcystin and anatoxin concentrations were inversely correlated with Cl-, SO 4 - 2 , Na+, and NH 4 + , and directly correlated with turbidity and total P. Cylindrospermopsin and saxitoxin concentrations in water samples were inversely correlated with Mg+2 and directly correlated with water temperature. Results of our study are expected to increase the understanding of potential relationships between human activities and water quality.

Nanocarrier-Mediated Chemo-Immunotherapy Arrested Cancer Progression and Induced Tumor Dormancy in Desmoplastic Melanoma.

In desmoplastic melanoma, tumor cells and tumor-associated fibroblasts are the major dominators playing a critical role in the fibrosis morphology as well as the immunosuppressive tumor microenvironment (TME), compromising the efficacy of therapeutic options. To overcome this therapeutic hurdle, we developed an innovative chemo-immunostrategy based on targeted delivery of mitoxantrone (MIT) and celastrol (CEL), two potent medicines screened and selected with the best anticancer and antifibrosis potentials. Importantly, CEL worked in synergy with MIT to induce immunogenic tumor cell death. Here, we show that when effectively co-delivered to the tumor site at their optimal ratio by a TME-responsive nanocarrier, the 5:1 combination of MIT and CEL significantly triggered immunogenic tumor apoptosis and recovered tumor antigen recognition, thus eliciting overall antitumor immunity. Furthermore, the strong synergy benefitted the host in reduced drug exposure and side effects. Collectively, the nanocarrier-mediated chemo-immunotherapy successfully remodeled fibrotic and immunosuppressive TME, arrested cancer progression, and further inhibited tumor metastasis to major organs. The affected tumors remained dormant long after dosing stopped, resulting in a prolonged progression-free survival and sustained immune surveillance of the host bearing desmoplastic melanoma.

A Multi-Parameter Analysis of Cellular Coordination of Major Transcriptome Regulation Mechanisms.

To understand cellular coordination of multiple transcriptome regulation mechanisms, we simultaneously measured transcription rate (TR), mRNA abundance (RA) and translation activity (TA). This revealed multiple insights. First, the three parameters displayed systematic statistical differences. Sequentially more genes exhibited extreme (low or high) expression values from TR to RA, and then to TA; that is, cellular coordination of multiple transcriptome regulatory mechanisms leads to sequentially enhanced gene expression selectivity as the genetic information flow from the genome to the proteome. Second, contribution of the stabilization-by-translation regulatory mechanism to the cellular coordination process was assessed. The data enabled an estimation of mRNA stability, revealing a moderate but significant positive correlation between mRNA stability and translation activity. Third, the proportion of mRNA occupied by un-translated regions (UTR) exhibited a negative relationship with the level of this correlation, and was thus a major determinant of the mode of regulation of the mRNA. High-UTR-proportion mRNAs tend to defy the stabilization-by-translation regulatory mechanism, staying out of the polysome but remaining stable; mRNAs with little UTRs largely followed this regulation. In summary, we quantitatively delineated the relationship among multiple transcriptome regulation parameters, i.e., cellular coordination of corresponding regulatory mechanisms.

Perfluoroalkylsulfonic and carboxylic acids in earthworms (Eisenia fetida): Accumulation and effects results from spiked soils at PFAS concentrations bracketing environmental relevance.

Effects of perfluorobutanesulfonic acid (PFBS), perfluorohexanesulfonic acid (PFHxS), perfluorononanoic acid (PFNA), and perfluoroheptanoic acid (PFHpA) on earthworms (Eisenia fetida) in soils contaminated with these compounds at 0.1, 1, 10, 1,000, and 100,000 µg kg-1 dry weight, covering concentration levels found in background, biosolid-amended, and facility-surrounding soils, were investigated. Earthworms were exposed to spiked soil for 21 days. Concentrations of these compounds in earthworms after 21-d exposure ranged from below detection to 127 mg kg-1 wet weight with the rank order of PFNA > PFHxS > PFHpA > PFBS; no mortality of earthworms was observed in all treatments including controls, except PFBS at 1,000 µg kg-1 and all PFASs at 100,000 µg kg-1. The highest weight loss (29%) was observed for earthworms exposed to PFNA at 100,000 µg kg-1, which was significantly different from all other treatments except PFHpA at 100,000 µg kg-1. These results are expected to fill some data gaps in toxicity of PFASs in terrestrial environments and provide helpful information on the potential for trophic transport of PFASs from soil to higher organisms.

Nanoparticle-Mediated Trapping of Wnt Family Member 5A in Tumor Microenvironments Enhances Immunotherapy for B-Raf Proto-Oncogene Mutant Melanoma.

Development of an effective treatment against advanced tumors remains a major challenge for cancer immunotherapy. Approximately 50% of human melanoma is driven by B-Raf proto-oncogene mutation (BRAF mutant). Tumors with such mutation are desmoplastic, highly immunosuppressive, and often resistant to immune checkpoint therapies. We have shown that immunotherapy mediated by low-dose doxorubicin-induced immunogenic cell death was only partially effective for this type of tumor and not effective in long-term inhibition of tumor progression. Wnt family member 5A (Wnt5a), a signaling protein highly produced by BRAF mutant melanoma cells, has been implicated in inducing dendritic cell tolerance and tumor fibrosis, thus hindering effective antigen presentation and T-cell infiltration. We hypothesized that Wnt5a is a key molecule controlling the immunosuppressive tumor microenvironment in metastatic melanoma. Accordingly, we have designed and generated a trimeric trap protein, containing the extracellular domain of Fizzled 7 receptor that binds Wnt5a with a Kd ∼ 278 nM. Plasmid DNA encoding for the Wnt5a trap was delivered to the tumor by using cationic lipid-protamine-DNA nanoparticles. Expression of Wnt5a trap in the tumor, although transient, was greater than that of any other major organs including liver, resulting in a significant reduction of the Wnt5a level in the tumor microenvironment without systematic toxicity. Significantly, combination of Wnt5a trapping and low-dose doxorubicin showed great tumor growth inhibition and host survival prolongation. Our findings indicated that efficient local Wnt5a trapping significantly remodeled the immunosuppressive tumor microenvironment to facilitate immunogenic cell-death-mediated immunotherapy.

The Pattern of microRNA Binding Site Distribution.

Micro-RNA (miRNA or miR) regulates at least 60% of the genes in the human genome through their target sites at mRNA 3'-untranslated regions (UTR), and defects in miRNA expression regulation and target sites are frequently observed in cancers. We report here a systematic analysis of the distribution of miRNA target sites. Using the evolutionarily conserved miRNA binding sites in the TargetScan database (release 7.1), we constructed a miRNA co-regulation network by connecting genes sharing common miRNA target sites. The network possesses characteristics of the ubiquitous small-world network. Non-hub genes in the network-those sharing miRNA target sites with small numbers of genes-tend to form small cliques with their neighboring genes, while hub genes exhibit high levels of promiscuousness in their neighboring genes. Additionally, miRNA target site distribution is extremely uneven. Among the miRNAs, the distribution concentrates on a small number of miRNAs, in that their target sites occur in an extraordinarily large number of genes, that is, they have large numbers of target genes. The distribution across the genes follows a similar pattern; the mRNAs of a small proportion of the genes contain extraordinarily large numbers of miRNA binding sites. Quantitatively, the patterns fit into the P(K) ∝ K-α relationship (P(K): the number of miRNAs with K target genes or genes with K miRNA sites; α: a positive constant), the mathematical description of connection distribution among the nodes and a defining characteristic of the so-called scale-free networks-a subset of small-world networks. Notably, well-known tumor-suppressive miRNAs (Let-7, miR-15/16, 26, 29, 31, 34, 145, 200, 203-205, 223, and 375) collectively have more than expected target genes, and well-known cancer genes contain more than expected miRNA binding sites. In summary, miRNA target site distribution exhibits characteristics of the small-world network. The potential to use this pattern to better understand miRNA function and their oncological roles is discussed.

R-Spondin1/LGR5 Activates TGFß Signaling and Suppresses Colon Cancer Metastasis.

Leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5), an intestinal stem cell marker, is known to exhibit tumor suppressor activity in colon cancer, the mechanism of which is not understood. Here we show that R-spondin 1 (RSPO1)/LGR5 directly activates TGFß signaling cooperatively with TGFß type II receptor in colon cancer cells, enhancing TGFß-mediated growth inhibition and stress-induced apoptosis. Knockdown of LGR5 attenuated downstream TGFß signaling and increased cell proliferation, survival, and metastasis in an orthotopic model of colon cancer in vivo Upon RSPO1 stimulation, LGR5 formed complexes with TGFß receptors. Studies of patient specimens indicate that LGR5 expression was reduced in advanced stages and positively correlated with markers of TGFß activation in colon cancer. Our study uncovers a novel cross-talk between LGR5 and TGFß signaling in colon cancer and identifies LGR5 as a new modulator of TGFß signaling able to suppress colon cancer metastasis. Cancer Res; 77(23); 6589-602. ©2017 AACR.

Tyrosine phosphorylation regulates ERß ubiquitination, protein turnover, and inhibition of breast cancer.

Unlike estrogen receptor α (ERα) that predominantly promotes hormone-dependent breast tumor growth, ERß exhibits antitumor effects in a variety of cancer types. We recently identified a phosphotyrosine residue in ERß, but not ERα, that dictates ERß transcriptional activity and antitumor function. We show here that this ER isotype-specific phosphotyrosine switch is important for regulating ERß activity in cell proliferation, migration, and invasion. At the mechanistic level, phosphorylated ERß, which recruits transcriptional coactivator p300, is in turn targeted by p300 for ubiquitination and proteasome-dependent protein turnover. Furthermore, ERß-specific agonists such as S-equol enhance ERß phosphorylation, suggesting a crosstalk between ligand- and posttranslational modification-dependent ERß activation. Inhibition of xenograft tumor growth by S-equol is associated with reduced tumor Ki-67 expression and elevated ERß tyrosine phosphorylation. Taken together, our data support the notion that phosphotyrosine-dependent ERß signaling is an attractive target for anticancer treatment.

Relationship between gene duplicability and diversifiability in the topology of biochemical networks.

BACKGROUND: Selective gene duplicability, the extensive expansion of a small number of gene families, is universal. Quantitatively, the number of genes (P(K)) with K duplicates in a genome decreases precipitously as K increases, and often follows a power law (P(k)∝k-α). Functional diversification, either neo- or sub-functionalization, is a major evolution route for duplicate genes. RESULTS: Using three lines of genomic datasets, we studied the relationship between gene duplicability and diversifiability in the topology of biochemical networks. First, we explored scenario where two pathways in the biochemical networks antagonize each other. Synthetic knockout of respective genes for the two pathways rescues the phenotypic defects of each individual knockout. We identified duplicate gene pairs with sufficient divergences that represent this antagonism relationship in the yeast S. cerevisiae. Such pairs overwhelmingly belong to large gene families, thus tend to have high duplicability. Second, we used distances between proteins of duplicate genes in the protein interaction network as a metric of their diversification. The higher a gene's duplicate count, the further the proteins of this gene and its duplicates drift away from one another in the networks, which is especially true for genetically antagonizing duplicate genes. Third, we computed a sequence-homology-based clustering coefficient to quantify sequence diversifiability among duplicate genes - the lower the coefficient, the more the sequences have diverged. Duplicate count (K) of a gene is negatively correlated to the clustering coefficient of its duplicates, suggesting that gene duplicability is related to the extent of sequence divergence within the duplicate gene family. CONCLUSION: Thus, a positive correlation exists between gene diversifiability and duplicability in the context of biochemical networks - an improvement of our understanding of gene duplicability.

Negative elongation factor controls energy homeostasis in cardiomyocytes.

Negative elongation factor (NELF) is known to enforce promoter-proximal pausing of RNA polymerase II (Pol II), a pervasive phenomenon observed across multicellular genomes. However, the physiological impact of NELF on tissue homeostasis remains unclear. Here, we show that whole-body conditional deletion of the B subunit of NELF (NELF-B) in adult mice results in cardiomyopathy and impaired response to cardiac stress. Tissue-specific knockout of NELF-B confirms its cell-autonomous function in cardiomyocytes. NELF directly supports transcription of those genes encoding rate-limiting enzymes in fatty acid oxidation (FAO) and the tricarboxylic acid (TCA) cycle. NELF also shares extensively transcriptional target genes with peroxisome proliferator-activated receptor α (PPARα), a master regulator of energy metabolism in the myocardium. Mechanistically, NELF helps stabilize the transcription initiation complex at the metabolism-related genes. Our findings strongly indicate that NELF is part of the PPARα-mediated transcription regulatory network that maintains metabolic homeostasis in cardiomyocytes.

Duplicate gene enrichment and expression pattern diversification in multicellularity.

The enrichment of duplicate genes, and therefore paralogs (proteins coded by duplicate genes), in multicellular versus unicellular organisms enhances genomic functional innovation. This study quantitatively examined relationships among paralog enrichment, expression pattern diversification and multicellularity, aiming to better understand genomic basis of multicellularity. Paralog abundance in specific cells was compared with those in unicellular proteomes and the whole proteomes of multicellular organisms. The budding yeast, Saccharomyces cerevisiae and the nematode, Caenorhabditis elegans, for which the gene sets expressed in specific cells are available, were used as uni and multicellular models, respectively. Paralog count (K) distributions [P((k))] follow a power-law relationship [Formula in text] in the whole proteomes of both species and in specific C. elegans cells. The value of the constant α can be used as a gauge of paralog abundance; the higher the value, the lower the paralog abundance. The α-value is indeed lower in the whole proteome of C. elegans (1.74) than in S. cerevisiae (2.34), quantifying the enrichment of paralogs in multicellular species. We also found that the power-law relationship applies to the proteomes of specific C. elegans cells. Strikingly, values of α in specific cells are higher and comparable to that in S. cerevisiae. Thus, paralog abundance in specific cells is lower and comparable to that in unicellular species. Furthermore, how much the expression level of a gene fluctuates across different C. elegans cells correlates positively with its paralog count, which is further confirmed by human gene-expression patterns across different tissues. Taken together, these results quantitatively and mechanistically establish enrichment of paralogs with diversifying expression patterns as genomic and evolutionary basis of multicellularity.

Comparing transcription rate and mRNA abundance as parameters for biochemical pathway and network analysis.

The cells adapt to extra- and intra-cellular signals by dynamic orchestration of activities of pathways in the biochemical networks. Dynamic control of the gene expression process represents a major mechanism for pathway activity regulation. Gene expression has thus been routinely measured, most frequently at steady-state mRNA abundance level using micro-array technology. The results are widely used in statistical inference of the structures of underlying biochemical networks, with the assumption that functionally related genes exhibit similar dynamic profiles. Steady-state mRNA abundance, however, is a composite of two factors: transcription rate and mRNA degradation rate. The question being asked here is therefore whether steady-state mRNA abundance or any of two factors is a more informative measurement target for studying network dynamics. The yeast S. cerevisiae was used as model organism and transcription rate was chosen out of the two factors in this study, because genome-wide determination of transcription rates has been reported for several physiological processes in this species. Our strategy is to test which one is a better measurement of functional relatedness between genes. The analysis was performed on those S. cerevisiae genes that have bacterial orthologs as identified by reciprocal BLAST analysis, so that functional relatedness of a gene pair can be measured by the frequency at which their bacterial orthologs co-occur in the same operon in the collection of bacterial genomes. It is found that transcription rate data is generally a better parameter for functional relatedness than steady state mRNA abundance, suggesting transcription rate data is more informative to use in deciphering the logics used by the cells in dynamic regulation of biochemical network behaviors. The significance of this finding for network and systems biology, as well as biomedical research in general, is discussed.

Discrepancy between mRNA and protein abundance: insight from information retrieval process in computers.

Discrepancy between the abundance of cognate protein and RNA molecules is frequently observed. A theoretical understanding of this discrepancy remains elusive, and it is frequently described as surprises and/or technical difficulties in the literature. Protein and RNA represent different steps of the multi-stepped cellular genetic information flow process, in which they are dynamically produced and degraded. This paper explores a comparison with a similar process in computers-multi-step information flow from storage level to the execution level. Functional similarities can be found in almost every facet of the retrieval process. Firstly, common architecture is shared, as the ribonome (RNA space) and the proteome (protein space) are functionally similar to the computer primary memory and the computer cache memory, respectively. Secondly, the retrieval process functions, in both systems, to support the operation of dynamic networks-biochemical regulatory networks in cells and, in computers, the virtual networks (of CPU instructions) that the CPU travels through while executing computer programs. Moreover, many regulatory techniques are implemented in computers at each step of the information retrieval process, with a goal of optimizing system performance. Cellular counterparts can be easily identified for these regulatory techniques. In other words, this comparative study attempted to utilize theoretical insight from computer system design principles as catalysis to sketch an integrative view of the gene expression process, that is, how it functions to ensure efficient operation of the overall cellular regulatory network. In context of this bird's-eye view, discrepancy between protein and RNA abundance became a logical observation one would expect. It was suggested that this discrepancy, when interpreted in the context of system operation, serves as a potential source of information to decipher regulatory logics underneath biochemical network operation.

Examining the architecture of cellular computing through a comparative study with a computer.

The computer and the cell both use information embedded in simple coding, the binary software code and the quadruple genomic code, respectively, to support system operations. A comparative examination of their system architecture as well as their information storage and utilization schemes is performed. On top of the code, both systems display a modular, multi-layered architecture, which, in the case of a computer, arises from human engineering efforts through a combination of hardware implementation and software abstraction. Using the computer as a reference system, a simplistic mapping of the architectural components between the two is easily detected. This comparison also reveals that a cell abolishes the software-hardware barrier through genomic encoding for the constituents of the biochemical network, a cell's "hardware" equivalent to the computer central processing unit (CPU). The information loading (gene expression) process acts as a major determinant of the encoded constituent's abundance, which, in turn, often determines the "bandwidth" of a biochemical pathway. Cellular processes are implemented in biochemical pathways in parallel manners. In a computer, on the other hand, the software provides only instructions and data for the CPU. A process represents just sequentially ordered actions by the CPU and only virtual parallelism can be implemented through CPU time-sharing. Whereas process management in a computer may simply mean job scheduling, coordinating pathway bandwidth through the gene expression machinery represents a major process management scheme in a cell. In summary, a cell can be viewed as a super-parallel computer, which computes through controlled hardware composition. While we have, at best, a very fragmented understanding of cellular operation, we have a thorough understanding of the computer throughout the engineering process. The potential utilization of this knowledge to the benefit of systems biology is discussed.

Systematic trans-genomic comparison of protein kinases between Arabidopsis and Saccharomyces cerevisiae.

The genome of the budding yeast (Saccharomyces cerevisiae) provides an important paradigm for transgenomic comparisons with other eukaryotic species. Here, we report a systematic comparison of the protein kinases of yeast (119 kinases) and a reference plant Arabidopsis (1,019 kinases). Using a whole-protein-based, hierarchical clustering approach, the complete set of protein kinases from both species were clustered. We validated our clustering by three observations: (a) clustering pattern of functional orthologs proven in genetic complementation experiments, (b) consistency with reported classifications of yeast kinases, and (c) consistency with the biochemical properties of those Arabidopsis kinases already experimentally characterized. The clustering pattern identified no overlap between yeast kinases and the receptor-like kinases (RLKs) of Arabidopsis. Ten more kinase families were found to be specific for one of the two species. Among them, the calcium-dependent protein kinase and phosphoenolpyruvate carboxylase kinase families are specific for plants, whereas the Ca(2+)/calmodulin-dependent protein kinase and provirus insertion in mouse-like kinase families were found only in yeast and animals. Three yeast kinase families, nitrogen permease reactivator/halotolerance-5), polyamine transport kinase, and negative regulator of sexual conjugation and meiosis, are absent in both plants and animals. The majority of yeast kinase families (21 of 26) display Arabidopsis counterparts, and all are mapped into Arabidopsis families of intracellular kinases that are not related to RLKs. Representatives from 11 of the common families (54 kinases from Arabidopsis and 17 from yeast) share an extremely high degree of similarity (blast E value < 10(-80)), suggesting the likelihood of orthologous functions. Selective expansion of yeast kinase families was observed in Arabidopsis. This is most evident for yeast genes CBK1, HRR25, and SNF1 and the kinase family S6K. Reduction of kinase families was also observed, as in the case of the NEK-like family. The distinguishing features between the two sets of kinases are the selective expansion of yeast families and the generation of a limited number of new kinase families for new functionality in Arabidopsis, most notably, the Arabidopsis RLKs that constitute important components of plant intercellular communication apparatus.