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frames (ORFs), ~800 metabolites] (15),
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
(viii) 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 (16) and figs.
S1 and S2].
Adam formulated and tested 20 hypotheses
concerning genes encoding 13 orphan enzymes
(16) (Table 1). The weight of the experimental
evidence for the hypotheses varied (based on ob-
servations of differential growth), but 12 hypothe-
ses with no previous evidence were confirmed
with P < 0.05 for the null hypothesis.
Because Adam’s experimental evidence for its
conclusions is indirect, we tested Adam’s conclu-
sions with more direct experimental methods. The
enzyme 2-aminoadipate:2-oxoglutarate amino-
transferase (2A2OA) catalyzes a reaction in the
lysine biosynthetic pathways of fungi. Adam hy-
pothesized that three genes (YER152C, YJL060W,
and YGL202W) encode this enzyme and ob-
served results consistent with all three hypotheses
(Table 1). To test Adam’s conclusions, we pu-
rified the protein products of these genes and
used them in in vitro enzyme assays, which
confirmed Adam’s conclusions [supporting on-
line material (SOM)] (Fig. 2).
To further test Adam’s conclusions, we ex-
amined the scientific literature on the 20 genes
investigated (Table 1) (16). 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).
The reason that Adam considered them to be
orphans was due to the use of an incomplete bio-
informatic database. These six genes therefore
constitute a positive control for Adam’s meth-
odology. A possible error was also revealed
(Table 1) (SOM).