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FIg. 2. ASsay results Tor CAZUA aCctiv- ity. The proteins encoded by YGL202W, YJLO60W, YER152C, and YDL168W were expressed from OpenBiosystems (www. openbiosystems.com) yeast ORF clones and purified. Activity was tested in an assay of NADPH (reduced form of nico- tinamide adenine dinucleotide phosphate) production based on (22). t-c.-aminoadipic acid and 2-oxoglutarate were provided as substrates and pyridoxal phosphate as cofactor. Glutamate production was assayed by using commercially available yeast glutamate dehydrogenase, which uses NADP as cofactor and deaminates Absorbance at 340nm -¥- control YDL168w 1 -P> no protein added “Ar YGL202w -E YER152C — YJLosow 0 5 10 15 20 Time (min) glutamate, producing ammonia and NADPH and regenerating 2-oxoglutarate (16). Also consister 2A20A activity is experimental evidence indicating a higher activity with -c-aminoadipic acid over alanine or aspartate (26). A major motivation for the formalization of experimental knowledge is the expectation that such knowledge is more easily reused to answer other scientific questions. To test this, we investi- gated whether we could reuse Adam’s functional genomic research (/6). An example question investigated was the relative growth rates (Umax) in rich and defined media of the deletion strains compared with those of the wild type. What was observed, in both media, was a skewed dis- tribution, with a few deletants having a much lower Umax than that of the wild type, but most having a slightly higher Unax. These observations question the common assumption that wild-type S. cerevisiae is optimized for Umax and provide quantitative test data for yeast systems biology models (/9).

Figure 2 ASsay results Tor CAZUA aCctiv- ity. The proteins encoded by YGL202W, YJLO60W, YER152C, and YDL168W were expressed from OpenBiosystems (www. openbiosystems.com) yeast ORF clones and purified. Activity was tested in an assay of NADPH (reduced form of nico- tinamide adenine dinucleotide phosphate) production based on (22). t-c.-aminoadipic acid and 2-oxoglutarate were provided as substrates and pyridoxal phosphate as cofactor. Glutamate production was assayed by using commercially available yeast glutamate dehydrogenase, which uses NADP as cofactor and deaminates Absorbance at 340nm -¥- control YDL168w 1 -P> no protein added “Ar YGL202w -E YER152C — YJLosow 0 5 10 15 20 Time (min) glutamate, producing ammonia and NADPH and regenerating 2-oxoglutarate (16). Also consister 2A20A activity is experimental evidence indicating a higher activity with -c-aminoadipic acid over alanine or aspartate (26). A major motivation for the formalization of experimental knowledge is the expectation that such knowledge is more easily reused to answer other scientific questions. To test this, we investi- gated whether we could reuse Adam’s functional genomic research (/6). An example question investigated was the relative growth rates (Umax) in rich and defined media of the deletion strains compared with those of the wild type. What was observed, in both media, was a skewed dis- tribution, with a few deletants having a much lower Umax than that of the wild type, but most having a slightly higher Unax. These observations question the common assumption that wild-type S. cerevisiae is optimized for Umax and provide quantitative test data for yeast systems biology models (/9).

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train and defined-growth-medium experiments each day (from a selection of thousands of yeast strains), with each experiment lasting up to 5 days. The Jesign enables measurement of ODsos5nm for each experiment at least once avery 30 min (more often if running at less than full capacity), allowing ac- curate growth curves to be recorded (typically we take over a hundred mea- surements a day per well), plus associated metadata. See the supporting online material for pictures and a video of Adam in action. Use of robot scientist enables all aspects ofa scientific investigation to be formalized in logic. For the core organization of this formalization, we used the ontology of scientific experiments: EXPO (//, 12). This ontology formalizes generic knowledge about experiments. For Adam, we developed LABORS, a customized version of EXPO, expressed in the description logic lan- guage OWL-DL (/7). Application of LABORS produces experimental descriptions in the logic- reading frames (ORFs), ~800 metabolites] (5), expressed in the logic programming language Prolog; (ii) a general bioinformatic database of genes and proteins involved in metabolism; (iii) software to abduce hypotheses about the genes encoding the orphan enzymes, done by using a combination of standard bioinformatic software and databases; (iv) software to deduce experi- ments that test the observational consequences of hypotheses (based on the model); (v) software to plan and design the experiments, which are based on the use of deletion mutants and the addition of selected metabolites to a defined growth medium; (vi) laboratory automation software to physically execute the experimental plan and to record the data and metadata in a relational database; (vii) software to analyze the data and metadata (gen- erate growth curves and extract parameters); and (vili) software to relate the analyzed data to the hypotheses; for example, statistical methods are required to decide on significance. Once this in- frastructure is in place, no human intellectual inter- vention is necessary to execute cycles of simple hypothesis-led experimentation. [For more details of the software, and its application to a related functional genomics problem, see (/6) and figs. S1 and 82]. To further test Adam's conclusions, we ex- amined the scientific literature on the 20 genes investigated (Table 1) (76). This revealed the ex- istence of strong empirical evidence for the cor- rectness of six of the hypotheses; that is, the enzymes were not actually orphans (Table 1).
train and defined-growth-medium experiments each day (from a selection of thousands of yeast strains), with each experiment lasting up to 5 days. The Jesign enables measurement of ODsos5nm for each experiment at least once avery 30 min (more often if running at less than full capacity), allowing ac- curate growth curves to be recorded (typically we take over a hundred mea- surements a day per well), plus associated metadata. See the supporting online material for pictures and a video of Adam in action. Use of robot scientist enables all aspects ofa scientific investigation to be formalized in logic. For the core organization of this formalization, we used the ontology of scientific experiments: EXPO (//, 12). This ontology formalizes generic knowledge about experiments. For Adam, we developed LABORS, a customized version of EXPO, expressed in the description logic lan- guage OWL-DL (/7). Application of LABORS produces experimental descriptions in the logic- reading frames (ORFs), ~800 metabolites] (5), expressed in the logic programming language Prolog; (ii) a general bioinformatic database of genes and proteins involved in metabolism; (iii) software to abduce hypotheses about the genes encoding the orphan enzymes, done by using a combination of standard bioinformatic software and databases; (iv) software to deduce experi- ments that test the observational consequences of hypotheses (based on the model); (v) software to plan and design the experiments, which are based on the use of deletion mutants and the addition of selected metabolites to a defined growth medium; (vi) laboratory automation software to physically execute the experimental plan and to record the data and metadata in a relational database; (vii) software to analyze the data and metadata (gen- erate growth curves and extract parameters); and (vili) software to relate the analyzed data to the hypotheses; for example, statistical methods are required to decide on significance. Once this in- frastructure is in place, no human intellectual inter- vention is necessary to execute cycles of simple hypothesis-led experimentation. [For more details of the software, and its application to a related functional genomics problem, see (/6) and figs. S1 and 82]. To further test Adam's conclusions, we ex- amined the scientific literature on the 20 genes investigated (Table 1) (76). This revealed the ex- istence of strong empirical evidence for the cor- rectness of six of the hypotheses; that is, the enzymes were not actually orphans (Table 1).
of metabolites tested. Existing annotation is the summary from the Saccharomyces Genome Database of the annotation of the ORF. Dry is the summary of whether the annotated function is the same as predicted by Adam. If a gene already has an associated function, we do not consider this to be contradictory to Adam's conclusions unless this function is capable of explaining the observed growth phenotype, for example, BCY1. ida indicates inferred from direct assay and iss, inferred from sequence or structural similarity (5). Wet is the result of our manual enzyme assays. See (16) for details. Table 1. The orphan enzymes and Adam's hypotheses. The hypothesized genes are those which Adam abduced encoded an orphan enzyme. Prob. is Adam's Monte Carlo estimate of the probability of obtaining the observed discrimination accuracy or better with a random labeling of replicates. The discrimination is between the differences in growth curves observed with the addition of specified metabolites to the wild type and the deletant. Acc. is the highest accuracy for a metabolite species in discriminating between the growth curves observed with the addition of specified metabolites to the wild type and the deletant. No. is the number
of metabolites tested. Existing annotation is the summary from the Saccharomyces Genome Database of the annotation of the ORF. Dry is the summary of whether the annotated function is the same as predicted by Adam. If a gene already has an associated function, we do not consider this to be contradictory to Adam's conclusions unless this function is capable of explaining the observed growth phenotype, for example, BCY1. ida indicates inferred from direct assay and iss, inferred from sequence or structural similarity (5). Wet is the result of our manual enzyme assays. See (16) for details. Table 1. The orphan enzymes and Adam's hypotheses. The hypothesized genes are those which Adam abduced encoded an orphan enzyme. Prob. is Adam's Monte Carlo estimate of the probability of obtaining the observed discrimination accuracy or better with a random labeling of replicates. The discrimination is between the differences in growth curves observed with the addition of specified metabolites to the wild type and the deletant. Acc. is the highest accuracy for a metabolite species in discriminating between the growth curves observed with the addition of specified metabolites to the wild type and the deletant. No. is the number
It could be argued that the scientific knowl- edge “discovered” by Adam is implicit in the formulation of the problem and is therefore not novel. This argument that computers cannot originate anything is known as Lady Lovelace’s objection (20): “The Analytical Engine has no pretensions to originate anything. It can do whatever we know how to order it to perform” (her italics). We accept that the knowledge automatically generated by Adam is of a modest kind. However, this knowledge is not trivial, and in the case of the genes encoding 2A20A, it sheds light on, and perhaps solves, a 50-year-old puzzle (2/).
It could be argued that the scientific knowl- edge “discovered” by Adam is implicit in the formulation of the problem and is therefore not novel. This argument that computers cannot originate anything is known as Lady Lovelace’s objection (20): “The Analytical Engine has no pretensions to originate anything. It can do whatever we know how to order it to perform” (her italics). We accept that the knowledge automatically generated by Adam is of a modest kind. However, this knowledge is not trivial, and in the case of the genes encoding 2A20A, it sheds light on, and perhaps solves, a 50-year-old puzzle (2/).
hole plant immunity, called systemic \ ,\ / acquired resistance (SAR), often de- velops after localized foliar infections by diverse pathogens. In this process, leaves dis- tal to the localized infection become primed to activate a stronger defense response upon sec- ondary infection (/). Leaves infected with SAR- inducing bacteria produce vascular sap, called petiole exudate, which confers disease resistance to previously unexposed (naive) plants (2, 3). This indicates that a mobile systemic signal(s) is involved in SAR (4). Although the hormone jasmonic acid Fig. 1. Azelaic acid specifically confers resistance to Pseudomonas syringae. (A) Azelaic acid-induced resistance is concentration-dependent. Plants were sprayed with 1, 10, 100, and 1000 uM azelaic acid in 5 mM MES (pH 5.6) or 5 mM MES (pH 5.6) alone 2 days before infection with P. syringae pv. maculicola strain PmaDG3 (OD¢o9 = 0.0001). (B) Induced resistance is time-dependent. Plants sprayed with 5 mM MES or 1 mM azelaic acid for the time periods indicated were subsequently inoculated with PmaDG3. (C) 5 mM MES or 1 mM azelaic acid was injected into local leaves. Two days later, either local or systemic leaves were infected with PmaDG3. (D) Dicarboxylic acids (1 mM) of different carbon-chain lengths were applied to Arabidopsis. M, 5 mM Mes; Cg, suberic acid; Co, azelaic acid; C19, sebacic acid. (E) Mobility of deuterium-labeled azelaic acid [HOOC(CD,),COOH] injected into WT leaves. Azelaic acid amounts were determined in petiole exudates (left) and distal leaves (right) after local injection with 1 mM azelaic acid. *P < 0.05; **P < 0. 01; ¢ test. Error bars indicate SE.
hole plant immunity, called systemic \ ,\ / acquired resistance (SAR), often de- velops after localized foliar infections by diverse pathogens. In this process, leaves dis- tal to the localized infection become primed to activate a stronger defense response upon sec- ondary infection (/). Leaves infected with SAR- inducing bacteria produce vascular sap, called petiole exudate, which confers disease resistance to previously unexposed (naive) plants (2, 3). This indicates that a mobile systemic signal(s) is involved in SAR (4). Although the hormone jasmonic acid Fig. 1. Azelaic acid specifically confers resistance to Pseudomonas syringae. (A) Azelaic acid-induced resistance is concentration-dependent. Plants were sprayed with 1, 10, 100, and 1000 uM azelaic acid in 5 mM MES (pH 5.6) or 5 mM MES (pH 5.6) alone 2 days before infection with P. syringae pv. maculicola strain PmaDG3 (OD¢o9 = 0.0001). (B) Induced resistance is time-dependent. Plants sprayed with 5 mM MES or 1 mM azelaic acid for the time periods indicated were subsequently inoculated with PmaDG3. (C) 5 mM MES or 1 mM azelaic acid was injected into local leaves. Two days later, either local or systemic leaves were infected with PmaDG3. (D) Dicarboxylic acids (1 mM) of different carbon-chain lengths were applied to Arabidopsis. M, 5 mM Mes; Cg, suberic acid; Co, azelaic acid; C19, sebacic acid. (E) Mobility of deuterium-labeled azelaic acid [HOOC(CD,),COOH] injected into WT leaves. Azelaic acid amounts were determined in petiole exudates (left) and distal leaves (right) after local injection with 1 mM azelaic acid. *P < 0.05; **P < 0. 01; ¢ test. Error bars indicate SE.
*Present address: Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853-1801, USA. tTo whom correspondence should be addressed. E-mail: jgreenbe@midway.uchicago.edu
*Present address: Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853-1801, USA. tTo whom correspondence should be addressed. E-mail: jgreenbe@midway.uchicago.edu
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