Computing with Pathway/Genome
Databases
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Motivations for Understanding
Pathway Tools Schema
 When
writing complex queries to PGDBs, those
queries must refer to classes and slots within the
schema
 Queries using Lisp, Perl, Java APIs
 Queries using Query Page
 Queries using Structured Advanced Query Form
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Motivations for Understanding
Schema
 Pathway
Tools visualizations and analyses
depend upon the software being able to find
precise information in precise places within a
Pathway/Genome DB
A
Pathway/Genome Database is a web of
interconnected objects; each object represents a
biological entity
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Pathway Tools Implementation Details
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
Platforms:
 Macintosh, PC/Linux, and PC/Windows platforms

Same binary can run as desktop app or Web server

Production-quality software
 Version control
 Two regular releases per year
 Extensive quality assurance
 Extensive documentation
 Auto-patch
 Automatic DB-upgrade

420,000 lines of Lisp code
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More Information
 Pathway


http://bioinformatics.ai.sri.com/ptools/
http://bioinformatics.ai.sri.com/ptools/examples.lisp
 PerlCyc


& JavaCyc API , includes some relationships
http://www.arabidopsis.org/tools/aracyc/perlcyc/
http://www.arabidopsis.org/tools/aracyc/javacyc/
 Pathway

Tools Web Site, Tutorial Slides
Tools User’s Guide
Appendix: Guide to the Pathway Tools Schema
 Curator's

Guide
http://bioinformatics.ai.sri.com/ptools/curatorsguide.pdf
 aic/pathway-tools/nav/12.0/lisp/relationships.lisp
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References
 Ontology
Papers section of
http://biocyc.org/publications.shtml
 "An Evidence Ontology for use in Pathway/Genome
Databases"
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
"An ontology for biological function based on molecular
interactions"

"Representations of metabolic knowledge: Pathways"

"Representations of metabolic knowledge"
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Data Exchange


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APIs: Lisp API, Java API, and Perl API : read & modify
Cyclone

Export to files
 BioPAX Export: since Pathway Tools 9.0
Biopax.org
 Export PGDB genome to Genbank format
 Export entire PGDB as column-delimited and attribute-value file formats
 Export PGDB reactions as SBML -- sbml.org
 Import/Export of Pathways: between PGDBs
 Import/Export of Selected Frames, for Spreadsheets
 Import/Export of Compounds as Molfile, CML
 Registering/Publishing PGDBs on WWW

BioWarehouse : Loader for Flatfiles, SQL access
 http://bioinformatics.ai.sri.com/biowarehouse/
 BMC Bioinformatics 7:170 2006
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Programmatic Access to BioCyc

Common LISP
•
Native language of Pathway Tools
•
Interactive & Mature Environment
•
Full Access to the Data & Many Utility Functions
•
Source code is available for academics

PerlCyc
•
API of Functions, Exposed to Perl
•
Communication through UNIX Socket

JavaCyc
•
API of Functions, Exposed to Java
•
Communication through UNIX Socket
•
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Cyclone
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Cyclone
 Developed
by Schachter and colleagues from
Genoscope
 http://nemo-cyclone.sourceforge.net/archi.php
 Cyclone
is a Java-based system that:
 Extracts data from a Pathway Tools PGDB
 Converts it to an XML schema
 Maps the data to Java objects and to a relational database
 Changes made to the data on the Java side can be
committed back to a Pathway Tools PGDB
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Pathway Tools Data Model


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PGDBs are object-oriented databases
Frame Representation System, named Ocelot

Frame data model
 PGDB = Knowledge base = KB = Database = DB
 Frames
 Slots

PGDBs are stored in three possible ways
•
Preloaded into binary executable
•
Ocelot file: single-user
•
RDBMS: MySQL-4 or Oracle-10 : multi-user, change-logging

Query API: GFP (Generic Frame Protocol)
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Frames
Entities
with which facts are associated
Kinds


of frames:
Classes: Genes, Pathways, Biosynthetic Pathways
Instances (objects): trpA, TCA cycle
Classes:

A
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Superclass(es), Subclass(es), Instance(s)
symbolic frame name (id, key) uniquely identifies each frame
 Examples: EG10223, TRP, Proteins
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Slots
Encode
attributes and properties of a frame
Represent

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relationships between frames
The value of a slot is the identifier of another frame
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Slots

Number of values
 Single valued
 Multivalued: sets, bags

Slot values
 Any LISP object: Integer, real, string, symbol (frame name)

Every slot is described by a “slot frame” in a KB that
defines meta information about that slot
 Datatype, classes it pertains to, constraints
 Two slots are inverses if they encode opposite relationships


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Slot Product in class Genes
Slot Gene in class Polypeptides
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Pathway Tools Ontology / Schema
 Ontology
classes: 1621
 Datatype classes: Define objects from genomes to pathways
 Classification systems for pathways, chemical compounds,
enzymatic reactions (EC system)
 Protein Feature ontology
 Controlled vocabularies:


Cell Component Ontology
Evidence codes
 Comprehensive
set of 248 attributes and
relationships
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Root Classes in the Pathway Tools
Ontology

Chemicals
Polymer-Segments
Protein-Features
Paralogous-Gene-Groups

Organisms

Generalized-Reactions
Enzymatic-Reactions
Regulation
-- Reactions and pathways
-- Link enzymes to reactions they catalyze
-- Regulatory interactions
CCO
Evidence
-- Cell Component Ontology
-- Evidence ontology
Notes
Organizations
People
Publications
-- Timestamped, person-stamped notes




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

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-- All molecules
-- Regions of polymers
-- Features on proteins
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Use GKB Editor to Inspect the
Pathway Tools Ontology
 GKB
Editor = Generic Knowledge Base Editor
 Type in Navigator window: (GKB)
or
 [Right-Click] Edit->Ontology Editor
 View->Browse
Class Hierarchy
 [Middle-Click] to expand hierarchy
 To view classes or instances, select them and:
 Frame -> List Frame Contents
 Frame -> Edit Frame
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Schema Overview
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Principal Classes

Class names are capitalized, plural, separated by dashes

Genetic-Elements, with subclasses:
 Chromosomes
 Plasmids
Genes
Transcription-Units
RNAs
 rRNAs, snRNAs, tRNAs, Charged-tRNAs
Proteins, with subclasses:
 Polypeptides
 Protein-Complexes




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Principal Classes
 Reactions,
with subclasses:
 Transport-Reactions
 Enzymatic-Reactions
 Pathways
 Compounds-And-Elements
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Principal Classes
 Regulation
Regulation-of-Enzyme-Activity
 Regulation-of-Transcription

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Regulation-of-Transcription-Initiation
Transcriptional-Attenuation
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Example of a Single GFP Call
The General Pattern:
gfp-function(frame-ID slot-ID value ...)
(gfp-function frame-ID slot-ID value …)

LISP
(get-slot-values 'TRYPSYN-RXN 'LEFT)
==> (INDOLE-3-GLYCEROL-P SER)

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Architecture of the API server –
PerlCyc and JavaCyc
 Works
on Unix (Solaris or Linux) only
 Start up Pathway Tools with the –api arg
 Pathway Tools listens on a Unix socket – perl
program communicates through this socket
 Supports both querying and editing PGDBs
 Must run perl or java program on the same
machine that runs Pathway Tools
 This is a security measure, as the API server has no built-in
security
 Can only handle one connection at a time
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Obtaining PerlCyc and JavaCyc
Download from
http://www.sgn.cornell.edu/downloads/
PerlCyc written and maintained by Lukas Mueller at
Boyce Thompson Institute for Plant Research.
JavaCyc written by Thomas Yan at Carnegie
Institute, maintained by Lukas Mueller.
Easy to extend…
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Examples of PerlCyc, JavaCyc
Functions
GFP
functions (require knowledge of Pathway Tools
schema):
 getSlotValues
 get_slot_values
 getClassAllInstances
 get_class_all_instances
 putSlotValues
 put_slot_values
Pathway Tools functions (described at
http://bioinformatics.ai.sri.com/ptools/ptools-fns.html):
 genes_of_reaction
 genesOfReaction
 find_indexed_frame
 findIndexedFrame
 pathways_of_gene
 pathwaysOfGene
 transport_p
 transportP
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Writing a PerlCyc or JavaCyc program



Create a PerlCyc, JavaCyc object:
perlcyc -> new (“ORGID”)
new Javacyc (“ORGID”)
Call PerlCyc, JavaCyc functions on this object:
my $cyc = perlcyc -> new (“ECOLI”);
my @pathways = $cyc -> all_pathways ();
Javacyc cyc = new Javacyc(“ECOLI”);
ArrayList pathways = cyc.allPathways ();
Functions return object IDs, not objects.
 Must connect to server again to retrieve attributes of an object.
foreach my $p (@pathways) {
print $cyc -> get_slot_value ($p, “COMMON-NAME”);}
for (int i=0; I < pathways.size(); i++) {
String pwy = (String) pathways.get(i);
System.out.println (cyc.getSlotValue (pwy, “COMMON-NAME”); }
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Sample PerlCyc Query
 Number
of proteins in E. coli
use perlcyc;
my $cyc = perlcyc -> new (“ECOLI”);
my @proteins = $cyc->
get_class_all_instances("|Proteins|");
my $protein_count = scalar(@proteins);
print "Protein count: $protein_count.\n";
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Sample PerlCyc Query
 Print
IDs of all proteins with molecular weight
between 10 and 20 kD and pI between 4 and 5.
use perlcyc;
my $cyc = perlcyc -> new (“ECOLI”);
foreach my $p ($cyc->get_class_all_instances("|Proteins|")) {
my $mw = $cyc->get_slot_value($p, "molecular-weight-kd");
my $pI = $cyc->get_slot_value($p, "pi");
if ($mw <= 20 && $mw >= 10 && $pI <= 5 && $pI >= 4) {
print "$p\n";
}
}
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Sample PerlCyc Query
 List
all the transcription factors in E. coli, and the
list of genes that each regulates:
use perlcyc;
my $cyc = perlcyc -> new (“ECOLI”);
foreach my $p ($cyc->get_class_all_instances("|Proteins|")) {
if ($cyc->transcription_factor_p($p)) {
my $name = $cyc->get_slot_value($p, "common-name");
my %genes = ();
foreach my $tu ($cyc->regulon_of_protein($p)) {
foreach my $g ($cyc->transcription_unit_genes($tu)) {
$genes{$g} = $cyc->get_slot_value($g, "common-name");
}
}
print "\n\n$name: ";
print join " ", values %genes;
}
}
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Sample Editing Using PerlCyc
 Add
a link from each gene to the corresponding
object in MY-DB (assume ID is same in both
cases)
use perlcyc;
my $cyc = perlcyc -> new (“HPY”);
my @genes = $cyc->get_class_all_instances (“|Genes|”);
foreach my $g (@genes) {
$cyc->add_slot_value ($g, “DBLINKS”, “(MY-DB \”$g\”)”);
}
$cyc->save_kb();
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Sample JavaCyc Query:
Enzymes for which ATP is a regulator
import java.util.*;
public class JavacycSample {
public static void main(String[] args) {
Javacyc cyc = new Javacyc("ECOLI");
ArrayList regframes =
cyc.getClassAllInstances("|Regulation-of-Enzyme-Activity|");
for (int i = 0; i < regframes.size(); i++) {
String reg = (String)regframes.get(i);
boolean bool = cyc.memberSlotValueP(reg, “Regulator", "ATP");
if (bool) {
String enzrxn = cyc.getSlotValue (reg, “Regulated-Entity”);
String enzyme = cyc.getSlotValue (enzrxn, “Enzyme”);
System.out.println(enz); } } } }
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Simple Lisp Query Example:
Enzymes for which ATP is a regulator
(defun atp-inhibits ()
(loop for x in (get-class-all-instances '|Regulation-of-Enzyme-Activity|)
;; Does the Regulator slot contain the compound ATP, and the mode
;; of regulation is negative (inhibition)?
when (and (member-slot-value-p x ‘Regulator 'ATP)
(member-slot-value-p x ‘Mode “-”) )
;; Whenever the test is positive, we collect the value of the slot Enzyme
;; of the Regulated-Entity of the regulatory interaction frame.
;; The collected values are returned as a list, once the loop terminates.
collect (get-slot-value (get-slot-value x ‘Regulated-Entity) ‘Enzyme) )
)
;;; invoking the query:
(select-organism :org-id 'ECOLI)
(atp-inhibits)
(get-slot-values 'TRYPSYN-RXN 'LEFT)
==> (INDOLE-3-GLYCEROL-P SER)
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Simple Perl Query Example:
Enzymes for which ATP is a regulator
use perlcyc;
my $cyc = perlcyc -> new("ECOLI");
my @regs = $cyc -> get_class_all_instances("|Regulation-of-EnzymeActivity|");
## We check every instance of the class
foreach my $reg (@regs) {
## We test for whether the INHIBITORS-ALL
## slot contains the compound frame ATP
my $bool1 = $cyc -> member_slot_value_p($reg, “Regulator", "Atp");
my $bool2 = $cyc -> member_slot_value_p($reg, “Mode", “-");
if ($bool1 && $bool2) {
## Whenever the test is positive, we collect the value of the slot
ENZYME .
## The results are printed in the terminal.
my $enzrxn = $cyc -> get_slot_value($reg, “Regulated-Entity");
my $enz = $cyc -> get_slot_value($enzrxn, "Enzyme");
print STDOUT "$enz\n";
}
}
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Getting started with Lisp





pathway-tools –lisp
(load “file”) (compile-file “file.lisp”)
Emacs is a useful editor
Pathway Tools source code is available: ask
Lisp resources:
http://bioinformatics.ai.sri.com/ptools/ptools-resources.html
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Query Gotchas
 Study
schema carefully
 :test #’fequal
 Cascade of slot-values: check for NIL
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Semantic Inference Layer
relationships.lisp

Library of functions that encapsulate common query
building blocks and intricacies of navigating the schema

enzymes-of-gene
reactions-of-gene
pathways-of-gene
genes-of-pathway
pathway-hole-p
reactions-of-compound
top-containers(protein)
all-rxns(type) (:metab-smm :metab-all :metab-pathways :enzyme :transport







etc.)
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(all-rxns :metab-pathways)
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Pathway Tools Schema and
Semantic Inference Layer
Genes, Operons, and Replicons
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Representing a Genome
components
genome
ORG
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Gene1
CHROM1
Gene2
CHROM2
Gene3
PLASMID1

product
Classes:
 ORG is of class Organisms
 CHROM1 is of class Chromosomes
 PLASMID1 is of class Plasmids
 Gene1 is of class Genes
 Product1 is of class Polypeptides or RNA
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Product1

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(defun genes-of-chrom (chrom)
(loop for x in (get-slot-values chrom ‘components)
when (instance-all-instance-of-p x ‘|Genes|)
collect x)
)
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Polynucleotides
Review slots of COLI and of COLI-K12
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Genetic-Elements
 Sequence
is stored in a separate file or database
table
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Polymer-Segments
Review slots of Genes
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Complexities of Gene / Gene-Product
Relationships




The Product of a gene can be an instance of Polypeptides
or RNAs
An instance of Polypeptides can have more than one gene
encoding it
Sequence position:
 Nucleotide positions of starting and ending codons specified in Left-EndPosition and Right-End-Position (usually greater, except at origin)
 Transcription-Direction + / Alternative splicing:
 Nucleotide positions of starting and ending codons specified in Left-EndPosition and Right-End-Position
 Intron positions specified in Splice-Form-Introns of gene product
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(200 300) (350 400)
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Gene Reaction Schematic
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Substring Search Example

Find all genes that contain a given substring within their
common name or synonym list.
(defun find-gene-by-substring (substring)
(let (result)
(loop for g in (get-class-all-instances '|Genes|)
do
(loop for name in (get-slot-values g 'names)
when (search substring name :test #'string-equal)
do (pushnew g result)
))
result
))
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Proteins
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Proteins and Protein Complexes
 Polypeptide:
the monomer protein product of a
gene (may have multiple isoforms, as indicated at
gene level)
 Protein
complex: proteins consisting of multiple
polypeptides or protein complexes
 Example:
DNA pol III
 DnaE is a polypeptide
 pol III core is DnaE and two other polypeptides
 pol III holoenzymes is several protein complexes combined
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Protein Complex Relationships
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Slots of a protein (DnaE)
 catalyzes
 Is
it a regulator/reactant/etc?
 comment
 component-of
 dblinks
 features (edited in feature editor)
 Many
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other attributes possible
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A complex at the frame level (pol III)
 Most
of the same attributes as polypeptide frame
 component-of
and components
 note coefficients
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Protein Complex Relationships
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Relationships are Defined in Many
Places
 component-of
comes from creating a complex
 appears-in-left-side-of
comes from defining a
reaction (as do modified forms)
 regulates
comes from an enzymatic reaction or
TU
 can
only edit dna-footprint if protein has been
associated with a TU
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Semantic Inference Layer
 Reactions-of-protein
(prot)
 Returns a list of rxns this protein catalyzes
 Transcription-units-of-proteins(prot)
 Returns a list of TU’s activated/inhibited by the given protein
 Transporter? (prot)
 Is this protein a transporter?
 Polypeptide-or-homomultimer?(prot)
 Transcription-factor? (prot)
 Obtain-protein-stats
 Returns 5 values
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Length of : all-polypeptides, complexes, transporters, enzymes, etc…
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Example
 Find
all enzymes that use pyridoxal phosphate as
a cofactor or prosthetic group

(loop for protein in (get-class-all-instances ‘|Proteins|)
for enzrxn = (get-slot-value protein ‘enzymatic-reaction)
when (and enzrxn
(or (member-slot-value-p enzrxn ‘cofactors ‘pyridoxal_phosphate)
(member-slot-value-p enzrxn ‘prosthetic-groups
‘pyridoxal_phosphate))
collect protein)
(member-slot-value-p frame slot value) : T if Value is one of the values of
Slot of Frame.
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Sample
 Find
all proteins without
a comment anywhere
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RNAs
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RNAs
 PGDBs
only represent RNAs that are “terminal
gene products”
 tRNAs
 rRNAs
 Regulatory RNAs
 Miscellaneous small RNAs
 Slots
similar to proteins
 tRNAs
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can have an anticodon
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The RNA Ontology
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Compounds / Reactions / Pathways
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Compounds / Reactions / Pathways
 Think
of a three tiered structure:
 Reactions built on top of compounds
 Pathways built on top of reactions
 Metabolic network defined by reactions alone;
pathways are an additional “optional” structure
 Some reactions not part of a pathway
 Some reactions have no attached enzyme
 Some enzymes have no attached gene
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Compounds
 Relatively
few aspects of a compound defined
within the compound editor
 MW, formula calculated from edited structure
 Most
aspects defined in other editors
 “Pathway reactions” comes from reaction editing followed by
pathway editing
 Activator, etc come from the protein editor
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(print-frame ‘TRP)
-- Instance TRP --Types: |Amino-Acid|, |Aromatic-Amino-Acids|, |Non-polar-amino-acids|
APPEARS-IN-LEFT-SIDE-OF: RXN0-287, TRANS-RXN-76, TRYPTOPHAN-RXN,
TRYPTOPHAN--TRNA-LIGASE-RXN
APPEARS-IN-RIGHT-SIDE-OF: RXN0-2382, RXN0-301, TRANS-RXN-76, TRYPSYN-RXN
CHEMICAL-FORMULA: (C 11), (H 12), (N 2), (O 2)
COMMON-NAME: "L-tryptophan"
DBLINKS: (LIGAND-CPD "C00078" NIL |kaipa| 3311532640 NIL NIL),
(CAS "6912-86-3"), (CAS "73-22-3")
NAMES: "L-tryptophan", "W", "tryptacin", "trofan", "trp", "tryptophan",
"2-amino-3-indolylpropanic acid"
SMILES: "c1(c(CC(N)C(=O)O)c2(c([nH]1)cccc2))"
SYNONYMS: "W", "tryptacin", "trofan", "trp", "tryptophan",
"2-amino-3-indolylpropanic acid"
____________________________________________
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Semantic Inference Layer
 Reactions-of-compound
(cpd)
 Pathways-of-compound (cpd)
 Is-substrate-an-autocatalytic-enzyme-p (cpd)
 Activated/inhibited-by? (cpds slots)
 Returns a list of enzrxns for which a cpd in cpds is a
modulator (example slots: activators-all, activators-allosteric)
 All-substrates (rxns)
 All unique substrates specified in the given rxns
 Has-structure-p (cpd)
 Obtain-cpd-stats
 Returns two values:

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Length of :all-cpds, cpds with structures
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Queries with Multiple Answers

Navigator queries:
 Example: Substring search for “pyruvate”
 Selected list is placed on the Answer list
 Use “Next Answer” button to view each one of them

Lisp queries:
Example : Find reactions involving pyruvate as a substrate

(get-class-all-instances ‘|Compounds|)
(loop
for rxn in (get-class-all-instances ‘|Reactions|)
when (member ‘pyruvate (get-slot-values rxn ‘substrates)
collect rxn)
(replace-answer-list * )
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Reactions
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Reactions
 Represents
information about a reaction that is
independent of enzymes that catalyze the reaction
 Connected
to enzyme(s) via enzymatic reaction
frames
 Classified
with EC system when possible
2.7.7.7 – DNA-directed DNA
polymerization
 Carried out by five enzymes in E. coli
 Example:
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Reaction Ontology
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Where is 2.7.7.7 in the Ontology?
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Slots of Reaction Frames
 Balance-state
 EC-number
 Enzymatic-reaction
Generated in protein or reaction editor
 In-pathway
 Generated in pathway editor
 Left and Right (reactants / products)
 Can include modified forms of proteins, RNAs, etc here
 Not all reactants/products need to be frames
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Enzymatic Reactions (DnaE and
2.7.7.7)
A
necessary bridge between enzymes and
“generic” versions of reactions
 Carries information specific to an
enzyme/reaction combination:
 Cofactors and prosthetic groups
 Alternative substrates
 Links to regulatory interactions
 Frame
is generated when protein is associated
with reaction (via protein or reaction editor)
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Regulation of Enzyme Activity
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Semantic Inference Layer
Genes-of-reaction
(rxn)
Substrates-of-reaction (rxn)
Enzymes-of-reaction (rxn)
Lacking-ec-number (organism)
 Returns list of rxns with no ec numbers in that database
Get-reaction-direction-in-pathway (pwy rxn)
Reaction-type(rxn)
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Indicates types of Rxn as: Small molecule rxn, transport rxn, protein-small-molecule rxn
(one substrate is protein and one is a small molecule), protein rxn (all substrates are
proteins), etc.
All-rxns(type)
 Specify the type of reaction (see above for type)
Obtain-rxn-stats
 Returns six values
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Length of : all-rxns, transport, non-transport, etc…
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Find all small-molecule reactions that have no enzyme but are not
spontaneous (“orphan” reactions)
(defun orphan-reactions (&optional (verbose? t))
(loop for r in (all-rxns :small-molecule)
when (and (not (slot-has-value-p r 'enzymatic-reaction))
(not (get-slot-value r 'spontaneous?)))
collect r)
)
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Reaction Direction
 Left/Right
reflect direction of reaction as written
by Enzyme Commission
 Reflects systematic direction for different reaction classes
 Left/Right do not necessarily correspond to
physiological direction of a reaction
 Get-rxn-direction(rxn)
Returns :L2R or :R2L or :BOTH or NIL
 Integrates all available info about direction of this reaction
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Direction(s) it occurs in all pathways in the PGDB
Direction(s) as specified in Enzymatic-Reactions
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Pathways
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What is a Pathway?
 An
ordered set of interconnected, directed
biochemical reactions
 Reactions form a coherent unit, e.g.
 Regulated as a single unit
 Evolutionarily conserved across organisms as a single unit
 When combined, perform a single cellular function
 Historically grouped together as a unit
 Includes metabolic pathways and signalling
pathways
 Evidence for all reactions in a single organism
 Pathways can be linear, cyclical, branched, or
some combination
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Internal Representation of Pathways
 REACTION-LIST:
unordered list of reactions that
comprise the pathway
 PREDECESSORS: list of reaction pairs that define
ordering relationships between reactions.
E.g.
R1
R2
C
A
B
R3
D
(R2 R1) : Predecessor of R2 is R1
(R3 R1) : Predecessor of R3 is R1
(R1) : R1 has no predecessor (can be omitted)
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What is missing from Pathway
Representation?
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Reaction directions
 Some reactions are unidirectional, but many are reversible – how do we
know in which direction to draw the reaction?
Main vs. side substrates
A
B
C
D
E
F
 Main compounds form the backbone of the pathway
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substrates shared between connecting reactions
major inputs and outputs.
Side compounds omitted from pathway diagrams at low detail levels
Individual reactions do not necessarily have main and side compounds –
a particular substrate may be either a main or a side depending on the
pathway context.
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Computing Directionality and
Mains/Sides
Our philosophy: Enable curator to specify as little as
possible. Compute as much as possible. This reduces
redundancy and potential for inconsistencies.
Example:
Reactions R1: A + B  C + D
R2: B  E
Predecessors: (R2 R1)
 Only substrate overlap is B
 B must be a main substrate
 A must be a side substrate,
 R1 must proceed from right to left
 R2 must proceed from left to right
C+DBE
A
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But…
Unfortunately, mains, sides and reaction directions are
sometimes ambiguous:
 At beginnings and ends of pathways
 Use heuristics to determine main/side substrates at beginnings, ends of
pathways
 Not always what the curator wants
 Substrate overlap with both sides of a reaction,
e.g. A + B  C + D
C+BE
 Solution: Additional slot PRIMARIES, should only be
populated when necessary:
PRIMARIES: (R (A B) (C)) says that for reaction R, A and B
are both main reactants, and C is a main product.
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More Complications…
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ENZYME-USE: a reaction may be catalyzed by multiple
enzymes, but not all the enzymes necessarily participate in
a given pathway
 Not present in the same compartment with rest of pathway enzymes
 Down-regulated or not expressed under conditions in which pathway is
active
 ENZYME-USE slot tells us which enzymes catalyze reaction in pathway, if
not all.
LAYOUT-ADVICE: helps software draw pathway correctly,
e.g. in a cyclical pathway, tells which substrate should be at
the top.
HYPOTHETICAL-REACTIONS: list of reactions in the
pathway that are considered hypothetical (i.e. no direct
experimental evidence)
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Polymerization Pathways
…  X[n]
X[n+1]
X[10]
 POLYMERIZATION-LINKS:
specifies reactions that
should be connected by a polymerization link
(X R1 R1) --- REACTANT-NAME-SLOT: N-NAME
--- PRODUCT-NAME-SLOT: N+1-NAME
 CLASS-INSTANCE-LINKS:
specifies when a link
should be drawn between a substrate class and
some instance of it (necessary only if instance is
not a member of some reaction, so no
predecessor relationship can be defined)
R1 --- PRODUCT-INSTANCES: X[10]
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Super-Pathways
 Collection
of pathways that connect to each other
via common substrates or reactions, or as part of
some larger logical unit
 Can contain both sub-pathways and additional
connecting reactions
 Can be nested arbitrarily
 REACTION-LIST: a pathway ID instead of a
reaction ID in this slot means include all reactions
from the specified pathway
 PREDECESSORS: a pathway ID instead of a tuple
in this slot means include all predecessor tuples
from the specified pathway
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Querying Pathways Programmatically
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See http://bioinformatics.ai.sri.com/ptools/ptools-resources.html
(all-pathways)
(base-pathways)
 Returns list of all pathways that are not super-pathways
(genes-of-pathway pwy)
(unique-genes-of-pathway pwy)
 Returns list of all genes of a pathway that are not also part of other pathways
(enzymes-of-pathway pwy)
(substrates-of-pathway pwy)
(variants-of-pathway pwy)
 Returns all pathways in the same variant class as a pathway
(get-predecessors rxn pwy), (get-successors rxn pwy)
(get-rxn-direction-in-pathway pwy rxn)
(pathway-inputs pwy), (pathway-outputs pwy)
 Returns all compounds consumed (produced) but not produced (consumed) by
pathway (ignores stoichiometry)
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Example Queries
 Find
all genes involved in metabolic pathways:
(remove-duplicates
(loop for p in (all-pathways)
append (genes-of-pathway p)))
 Find
all compounds that are unique to a single
pathway:
(loop for p in (base-pathways)
append
(loop for c in (substrates-of-pathway p)
when (null (remove p (pathways-of-compound c)))
collect (list c p)))
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Regulation
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Regulation
 Reorganization
and expansion of regulation under
way in Pathway Tools
 Initial application to EcoCyc
 Class
Regulation with subclasses that describe
different biochemical mechanisms of regulation
 Slots:
 Regulator
 Regulated-Entity
 Mode
 Mechanism
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Regulation of Enzyme Activity
 Class
Regulation-of-Enzyme-Activity
 Each instance of the class describes one
regulatory interaction
 Slots:
Regulator -- usually a small molecule
 Regulated-Entity -- an Enzymatic-Reaction
 Mechanism -- One of:
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Competitive, Uncompetitive, Noncompetitive, Irreversible, Allosteric,
Other
Mode -- One of: + ,  Physiologically-relevant? – true/false
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Transcription Initiation
 Class
Regulation-of-Transcription-Initiation
 Transcription factor binds to DNA binding site to regulate
transcription initiation from a promoter
 Slots:
Regulator -- instance of Proteins or Complexes (a
transcription-factor)
 Regulated-Entity -- instance of Promoters
 Mode -- One of: + ,  Associated-binding-site – a DNA-Binding-Site
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Attenuation
 Class
Transcriptional-Attenuation
 Several subclasses depending on type of
attenuation
 Slots
common to all:
 Regulator -- Depends on subtype of attenuation
 Regulated-Entity -- instance of Terminators
 Mode -- One of: + , -
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Attenuation Subtypes
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Ribosome-Mediated-Attenuation
 E.g. trp operon – ribosome pauses based on levels of charged tRNA,
determines formation of terminator or antiterminator
RNA-Mediated-Attenuation
 RNA (tRNA or sRNA) binds to transcript, determines formation of
terminator or antiterminator
Protein-Mediated-Attenuation
 Protein binds to transcript, determines formation of terminator or
antiterminator
Small-Molecule-Mediated-Attenuation
 Small molecule binds to transcript, determines formation of terminator or
antiterminator
Rho-Blocking-Antitermination
RNA-Polymerase-Modification
 Regulatory protein binds to site in transcription unit and interacts with
RNA polymerase to determine termination
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Transcriptional Regulation
trp
apoTrpR
site001
rxn001
reg001
TrpR*trp
pro001
trpLEDCBA
trpL
trpE
trpD
trpC
trpB
trpA
term001
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reg002
charged-tRNA*trp
Data Exchange
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Data Exchange
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Java API and Perl API : read & modify
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BioPAX Export: since Pathway Tools 9.0
 Biopax.org
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Export of entire PGDB as Flatfiles
Export of Reactions as SBML -- sbml.org
Import/Export of Pathways: between PGDBs
Import/Export of Selected Frames, for Spreadsheets
Import/Export of Compounds as Molfile, CML
Registering/Publishing PGDBs on WWW
Export PGDB as Genbank
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BioWarehouse : Loader for Flatfiles, SQL access
 http://bioinformatics.ai.sri.com/biowarehouse/
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Dump PGDB into Flatfiles
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Export of entire PGDB as Flatfiles
Format Description: UG v.I section 4.5
 Column delimited: 1 line per frame
 Attribute-value: 1 record per frame
Multiple slot values:
 Column delimited: several values per column
 Attribute-value: several lines for several values
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Frame Import/Export
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Import/Export of Selected Frames, for Spreadsheets
Frame selection, Slot selection GUI
Format Description: UG v.I section 4.6.3
 Column delimited: 1 line per frame
 Attribute-value: 1 record per frame
Multiple slot values:
 Column delimited: several values per column
 Attribute-value: several lines for several values
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