1. Analysis: Discovery of coregulated genes What follows is a simulation of the proposed graphical interface. As you go through the simulation please consider what capabilities you would want to serve your research and annotation interests. A narrative to help you go through the simulation appears in a red-bordered box, such as the one below. To begin: 1. Click on Slide Show, (on the upper toolbar) 2. Click View Show 3. Click Continue button Continue Scenario 4

2. Gene present in Prochlorococcus MED4 MED4 is naturally adapted to grow in high light. How do cells control response to light? Ortholog absent in Prochlorococcus MIT9313 MIT9313 is naturally adapted to grow in low light Ortholog present in Synechocystis PCC 6803 Reason will become apparent in a moment Synechocystis PCC 6803 ortholog responds to high light Gene turns on by factor > 2 in response to high light What genes are related to the adaptation to high light? Look for: Continue

3. Display set VariableDataOperationFunctionDone Build setCancelHELPSet operation Click to start building a new set Click to see set you or someone else made Click to see statements to manipulate sets Click to give a value to a new or old variable Click to access results of experiments Click to see list of available statements Click to see list of available manipulations Click any red button to get help Variable Data Function Operation Set operation Display set Build set Click Build Set to begin finding orfs with the desired specifications

4. Display set VariableDataOperationFunctionDone Build setCancelHELPSet operation Click to see statements to manipulate sets Click to give a value to a new or old variable Click to access results of experiments Click to see list of available statements Click to see list of available manipulations Click any red button to get help Variable Data Function Operation Set operation You want to go through all MED4 ORFS. Click Operation to see how to do that.

5. Display set VariableData Operation FunctionDone Build setCancelHELPSet operation Consider X in set... (loop) IF... THEN... OTHERWISE Consider X in set... (loop) You want to go consider each ORF in the set of all all MED4 ORFS. Click Consider X in set… Click to go through each element of a set Click to perform actions only under certain conditions Click any red button to get help Consider X in set... (loop) IF... THEN... OTHERWISE

6. Display set VariableDataOperationFunctionDone Build setCancelHELPSet operation Consider each item in All nucleotides of All open reading frames of All amino acid sequences of All intergenic regions of Human-annotated orfs of Private set Public set All open reading frames of Choose set type Arthrobacter platensis Gloeobacter violaceus Microcystis aeruginosa Nostoc punctiforme Nostoc PCC 7120 Prochlorococcus MED4 Prochlorococcus MIT9313 Prochlorococcus S120 Synechococcus PCC6301 Synechococcus PCC7942 Synechococcus WH Synechocystis PCC 6803 Thermosynechococcus Trichodesmium Unicellulular Filamentous All Choose database Choose ORFs as the set type and Prochlorococcus MED4 as the database.

7. Display set VariableDataOperationFunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofArthrobacter platensis Gloeobacter violaceus Microcystis aeruginosa Nostoc punctiforme Nostoc PCC 7120 Prochlorococcus MED4 Prochlorococcus MIT9313 Prochlorococcus S120 Synechococcus PCC6301 Synechococcus PCC7942 Synechococcus WH Synechocystis PCC 6803 Thermosynechococcus Trichodesmium Unicellulular Filamentous All Prochlorococcus MED4 Choose database Choose ORFs as the set type and Prochlorococcus MED4 as the database.

8. Display set VariableDataOperationFunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : Click to give a value to a new or old variable Click to access results of experiments Click to see list of available statements Click to see list of available manipulations Click to see statements to manipulate sets Variable Data Function Operation Set operation You want to consider this MED4 ORF only if an ortholog in MIT9313 doesn’t exist.. Click Operation and choose If… then…

9. Display set VariableData Operation FunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : Consider X in set... (loop) IF... THEN... OTHERWISE KEEP item IF... THEN... OTHERWISE You want to this MED4 ORF only if an ortholog in MIT9313 doesn’t exist.. Click Operation and choose If… then… Click to go through each element of a set Click to perform actions only under certain conditions Click to add item to set Click any red button to get help Consider X in set... (loop) IF... THEN... OTHERWISE Keep item

10. Display set VariableDataOperationFunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Click or to begin specifying condition to be met VariableDataFunction Your condition is that the ortholog of the item in MIT9313 doesn’t exist. Ortholog of… is a function.

11. Display set VariableDataOperation Function Done Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Ortholog of Protein product of Sequence of Upstream region of Downstream region of Ortholog of Your condition is that the ortholog of the item in MIT9313 doesn’t exist. Ortholog of… is a function.

12. Display set VariableDataOperation Function Done Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item Set Specify Variable Item ( in Ortholog of Arthrobacter platensis Gloeobacter violaceus Microcystis aeruginosa Nostoc punctiforme Nostoc PCC 7120 Prochlorococcus MED4 Prochlorococcus MIT9313 Prochlorococcus S120 Synechococcus PCC6301 Synechococcus PCC7942 Synechococcus WH8102 Synechocystis PCC 6803 Thermosynechococcus Trichodesmium Choose database ) You want the ortholog of the item (the specific ORF of MED4 being considered)…

13. Display set VariableDataOperation Function Done Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Arthrobacter platensis Gloeobacter violaceus Microcystis aeruginosa Nostoc punctiforme Nostoc PCC 7120 Prochlorococcus MED4 Prochlorococcus MIT9313 Prochlorococcus S120 Synechococcus PCC6301 Synechococcus PCC7942 Synechococcus WH8102 Synechocystis PCC 6803 Thermosynechococcus Trichodesmium Choose database ) Prochlorococcus MIT9313... And you want the ortholog of the item in Prochlorococcus MIT9313

14. Display set VariableData Operation FunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 ) =  exists doesn’t exist Click specific operation to continue specifying the condition to be met or click to save results of function Variable You want the ortholog of the item in Prochlorococcus MIT9313 not to exist.

15. Display set VariableData Operation FunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 ) doesn’t exist AND OR BUT NOT IF... THEN... OTHERWISE Click a logical operation to continue specifying the condition to be met or IF... THEN... to end the condition Let’s pause to see where we are in the task at hand (Click to proceed)

16. Gene present in Prochlorococcus MED4 MED4 is naturally adapted to grow in high light. How do cells control response to light? Ortholog absent in Prochlorococcus MIT9313 MIT9313 is naturally adapted to grow in low light Ortholog present in Synechocystis PCC 6803 Reason will become apparent in a moment Synechocystis PCC 6803 ortholog responds to high light Gene turns on by factor > 2 in response to high light What genes are related to the adaptation to high light? Look for: √ √ Continue

17. Display set VariableData Operation FunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 ) doesn’t exist AND OR BUT NOT IF... THEN... OTHERWISE AND Click a logical operation to continue specifying the condition to be met or IF... THEN... to end the condition There are more conditions to fulfill, so click AND.

18. Display set VariableDataOperationFunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 ) doesn’t exist AND Click or to continue specifying condition to be met VariableDataFunction Your condition now is that the ortholog of the item in Synechocystis does exist. Ortholog of… is a function.

19. Display set VariableDataOperation Function Done Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 ) doesn’t exist AND Ortholog of Protein product of Sequence of Upstream region of Downstream region of Ortholog of Your condition now is that the ortholog of the item in Synechocystis does exist. Ortholog of… is a function.

20. Display set VariableDataOperation Function Done Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item Set Specify Variable Item ( in Ortholog of Arthrobacter platensis Gloeobacter violaceus Microcystis aeruginosa Nostoc punctiforme Nostoc PCC 7120 Prochlorococcus MED4 Prochlorococcus MIT9313 Prochlorococcus S120 Synechococcus PCC6301 Synechococcus PCC7942 Synechococcus WH8102 Synechocystis PCC 6803 Thermosynechococcus Trichodesmium Choose database ) You want the ortholog of the item (the specific ORF of MED4 being considered)…

21. Display set VariableDataOperation Function Done Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Arthrobacter platensis Gloeobacter violaceus Microcystis aeruginosa Nostoc punctiforme Nostoc PCC 7120 Prochlorococcus MED4 Prochlorococcus MIT9313 Prochlorococcus S120 Synechococcus PCC6301 Synechococcus PCC7942 Synechococcus WH8102 Synechocystis PCC 6803 Thermosynechococcus Trichodesmium Choose database ) Synechocystis PCC6803 You want the ortholog of the item in Synechocystis PCC 6803

22. Display set VariableData Operation FunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) =  exists doesn’t exist Click specific operation to continue specifying the condition to be met or click to save results of function Variable You want the ortholog of the item in Synechocystis PCC 6803, this time to exist... but first you need to save the ortholog for later. To do this click Variable.

23. Display set Variable DataOperationFunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog Type variable name ) Give the ortholog a logical name so that you can refer to it later (for now, just click on the box and I’ll do the typing)

24. Display set VariableData Operation FunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) Click specific operation to continue specifying the condition to be met or click to save results of function Variable =  exists doesn’t exist exists Now you can demand that the ortholog of the item in Synechocystis PCC 6803 exists.

25. Display set VariableData Operation FunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists Click a logical operation to continue specifying the condition to be met or IF... THEN... to end the condition AND OR BUT NOT IF... THEN... OTHERWISE AND Still one more condition (2x expression) so click AND.

26. Display set VariableDataOperationFunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND Click or to continue specifying condition to be met VariableDataFunction This time the condition to be met concerns data from a microarray experiment. Click Data.

27. Display set Variable Data OperationFunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for Item 6803 ortholog Specify Variable in Microcystis aeruginosa Nostoc punctiforme Nostoc PCC 7120 Prochlorococcus MED4 Prochlorococcus MIT9313 Prochlorococcus S120 Synechococcus PCC6301 Synechococcus PCC7942 Synechococcus WH8102 Synechocystis PCC 6803 Choose organism used 6803 ortholog The data desired concerns the 6803 ortholog and an experiment using Synechocystis PCC6803

28. Display set Variable Data OperationFunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Microcystis aeruginosa Nostoc punctiforme Nostoc PCC 7120 Prochlorococcus MED4 Prochlorococcus MIT9313 Prochlorococcus S120 Synechococcus PCC6301 Synechococcus PCC7942 Synechococcus WH8102 Synechocystis PCC 6803 Choose organism used Synechocystis PCC 6803 The data desired concerns the 6803 ortholog and an experiment using Synechocystis PCC6803

29. Display set Variable Data OperationFunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Synechocystis PCC 6803microarray 2D gel Choose type microarray It’s a microarray experiment…

30. Display set Variable Data OperationFunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Synechocystis PCC 6803microarray Hihara1 Suzuki1 Yoshimura1 Choose expt Hihara1 High light vs low light experiment It’s a microarray experiment, Hihara et al, you think… Mouse over that experiment to see (here click on it)

31. Display set VariableData Operation FunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Synechocystis PCC 6803microarray Hihara1 < < or = =  > or = > exists doesn’t exist > You want the experimental condition (high light) to be greater than the control…

32. Display set VariableData Operation FunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Synechocystis PCC 6803microarray Hihara1 Value +2 > You want the experimental condition (high light) to be greater than the control by a factor of 2 (I’ll type it).

33. Display set VariableData Operation FunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Synechocystis PCC 6803microarray Hihara1 Value +2 > Click a logical operation to continue specifying the condition to be met or IF... THEN... to end the condition AND OR BUT NOT IF... THEN... OTHERWISE (Let’s pause again)

34. Gene present in Prochlorococcus MED4 MED4 is naturally adapted to grow in high light. How do cells control response to light? Ortholog absent in Prochlorococcus MIT9313 MIT9313 is naturally adapted to grow in low light Ortholog present in Synechocystis PCC 6803 Reason will become apparent in a moment Synechocystis PCC 6803 ortholog responds to high light Gene turns on by factor > 2 in response to high light What genes are related to the adaptation to high light? Look for: √ √ √ √ Continue

35. Display set VariableData Operation FunctionDone Build setCancelHELPSet operation Consider each item in All open reading frames ofProchlorococcus MED4 : IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Synechocystis PCC 6803microarray Hihara1 Value +2 > Click a logical operation to continue specifying the condition to be met or IF... THEN... to end the condition AND OR BUT NOT IF... THEN... OTHERWISE That’s it! We’ve specified all the conditions, so end the IF segment and specify what do do if all the conditions are met.

36. Display set VariableDataOperationFunctionDone Build setCancelHELPSet operation IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Synechocystis PCC 6803microarray Hihara1 +2 > THEN Click to give a value to a new or old variable Click to access results of experiments Click to see list of available statements Click to see list of available manipulations Click to see statements to manipulate sets Variable Data Function Operation Set operation And if they are met, what you want to do is to save the gene in the set you’re building, a Set operation.

37. Display set VariableDataOperationFunctionDone Build setCancelHELP Set operation IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Synechocystis PCC 6803microarray Hihara1 +2 > THEN ADD TO DELETE FROM UNION OF INTERSECTION OF Arithmetic ADD TO In other words, you want to ADD the gene to the growing set.

38. Display set VariableDataOperationFunctionDone Build setCancelHELP Set operation IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Synechocystis PCC 6803microarray Hihara1 +2 > THEN Item 6803 ortholog Specify 6803 ortholog to Add Variable Type name of set Which gene? I could save the Prochlorococcus gene (the item), but I’ll instead save the gene from PCC 6803 (more known about them).

39. Display set VariableDataOperationFunctionDone Build setCancelHELP Set operation IF Item ( in Ortholog of Prochlorococcus MIT9313 doesn’t exist AND Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Synechocystis PCC 6803microarray Hihara1 +2 > THEN 6803 ortholog to Add Light-specific genes Type name of set I need to give the set a logical name (you click, I’ll type).

40. Display set VariableDataOperationFunctionDone Build setCancelHELPSet operation Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Synechocystis PCC 6803microarray Hihara1 +2 > THEN 6803 ortholog to Add Type name of set Light-specific genes Click to give a value to a new or old variable Click to access results of experiments Click to see list of available statements Click to see list of available manipulations Click to see statements to manipulate sets Variable Data Function Operation Set operation If I stop here, then all Prochlorococcus genes will be considered, and if the conditions are met, the 6803 ortholog will be saved. That’s what I want, so click Done.

41. Display set VariableDataOperationFunction Done Build setCancelHELPSet operation Item ( in Ortholog of Synechocystis PCC 6803 ) (assigned to 6803 ortholog ) exists AND data for 6803 ortholog in Synechocystis PCC 6803microarray Hihara1 +2 > THEN 6803 ortholog to Add Type name of set Light-specific genes Save results and script Save only results Save results and script That was a complicated script, so I’ll save it in case I (or someone else) needs to run it again or modify it.

42. Equivalent script that bypasses interface (loop for item in (#^Genes ProcMed4) as all-orthologous = (all-blast-orthologous-geneIDs item stdevalue) as 6803ortholog = (#^Genes Syny6803) :in all-orthologous) as light-specific-genes = nil when (and (there-are-not-any #’member-geneID-of-gene-frames (#^Genes slotv Proc9313) :in all-orthologous)) (there-are-any #'member-geneID-of-gene-frames 6803ortholog) (>= ratio-value (select-matching-geneIDs-from-table Hihara1 item) 2)))) collect light-specific-genes 6803ortholog) This is the script that the interface would have produced. It looks for all the world like a computer program. In fact, it is a computer program, written in BioLingua. Continue

43. Build setDisplay set Set: Light-specific genes Syny6803:sll0990 Formaldehyde dehydrogenase (glutathione dependent) Syny6803:slr1332 fabF beta ketoacyl acyl carrier protein synthase Syny6803:sll0337 Sensor histidine kinase Syny6803:sll0335 Hypothetical Syny6803:sll0789 Response regulator (OmpR) Syny6803:sll0576 Putative epimerase/hydratase DoneHELPSet operation Here are the results of the program. The genes meeting all the conditions are given along with a brief description and a graphic display of the regions surrounding the genes. (Click to proceed)

44. Build setDisplay set Set: Light-specific genes Syny6803:sll0990 Formaldehyde dehydrogenase (glutathione dependent) Syny6803:slr1332 fabF beta ketoacyl acyl carrier protein synthase Syny6803:sll0337 Sensor histidine kinase Syny6803:sll0335 Hypothetical Syny6803:sll0789 Response regulator (OmpR) Syny6803:sll0576 Putative epimerase/hydratase DoneHELPSet operation What can you do with this set? Certainly one thing of interest is the function of the genes. Clicking on a gene name brings you to the annotation page. Try clicking on slr1332.

45. Synechocystis PCC 6803: slr1332 Replicon: Chromosome Coordinates: 1670650 (start-ATG) 1671864 (stop)Human Length = 404 amino acids Strand: Direct Gene name(s): fabF or fabJ Function: beta-ketoacyl-acyl carrier protein synthaseHuman Activity: In vivo activity: existsExperiment Cyanobacterial orthologs: Syny6803 Nost7120 NostPun OptionsAnnotate Main Menu History More A A A HELP You can find out more about this kind of page from Scenarios 1 – 3. For now, click to return to the Set Display page.

46. Build setDisplay set Set: Light-specific genes Syny6803:sll0990 Formaldehyde dehydrogenase (glutathione dependent) Syny6803:slr1332 fabF beta ketoacyl acyl carrier protein synthase Syny6803:sll0337 Sensor histidine kinase Syny6803:sll0335 Hypothetical Syny6803:sll0789 Response regulator (OmpR) Syny6803:sll0576 Putative epimerase/hydratase DoneHELPSet operation Another interesting point of attack is the regulation of this set of genes (after all, they were selected as being coregulated by light). Perhaps the upstream regions share a common motif. (Click to continue)

47. Build setDisplay set Set: Light-specific genes Syny6803:sll0990 Formaldehyde dehydrogenase (glutathione dependent) Syny6803:slr1332 fabF beta ketoacyl acyl carrier protein synthase Syny6803:sll0337 Sensor histidine kinase Syny6803:sll0335 Hypothetical Syny6803:sll0789 Response regulator (OmpR) Syny6803:sll0576 Putative epimerase/hydratase DoneHELPSet operation Unfortunately, in three cases, the genes don’t have an upstream region. Evidently these genes are part of operons. We’d like to consider the upstream regions of the operons by adding the first genes of the operon to the set. Add to set... that’s a Set operation. (Click on that)

48. Build setDisplay set Set: Light-specific genes Syny6803:sll0990 Formaldehyde dehydrogenase (glutathione dependent) Syny6803:slr1332 fabF beta ketoacyl acyl carrier protein synthase Syny6803:sll0337 Sensor histidine kinase Syny6803:sll0335 Hypothetical Syny6803:sll0789 Response regulator (OmpR) Syny6803:sll0576 Putative epimerase/hydratase DoneHELP Set operation Operation ADD TO DELETE FROM UNION OF INTERSECTION OF Arithmetic ADD TO Click ADD TO...

49. Build setDisplay set Set: Light-specific genes Syny6803:sll0990 Formaldehyde dehydrogenase (glutathione dependent) Syny6803:slr1332 fabF beta ketoacyl acyl carrier protein synthase Syny6803:sll0337 Sensor histidine kinase Syny6803:sll0335 Hypothetical Syny6803:sll0789 Response regulator (OmpR) Syny6803:sll0576 Putative epimerase/hydratase DoneHELP Set operation Find specific gene Add set of genes Specify gene Click on icon of gene Add to set Click ADD TO and click on the icon (the short black arrow) upstream from sll0990, the first gene without an upstream region.

50. Build setDisplay set Set: Light-specific genes Syny6803:sll0990 Formaldehyde dehydrogenase (glutathione dependent) Syny6803:slr1332 fabF beta ketoacyl acyl carrier protein synthase Syny6803:sll0337 Sensor histidine kinase Syny6803:sll0335 Hypothetical Syny6803:sll0789 Response regulator (OmpR) Syny6803:sll0576 Putative epimerase/hydratase DoneHELP Set operation Find specific gene Add set of genes Specify gene Click on icon of gene Add to set Syny6803:srl7009 trnR: tRNA Arg (UCU) The short arrow is now named and part of the set. Click on the icon (the long black arrow) upstream from slr1332, the next gene without an upstream region.

51. Build setDisplay set Set: Light-specific genes Syny6803:sll0990 Formaldehyde dehydrogenase (glutathione dependent) Syny6803:slr1332 fabF beta ketoacyl acyl carrier protein synthase Syny6803:sll0337 Sensor histidine kinase Syny6803:sll0335 Hypothetical Syny6803:sll0789 Response regulator (OmpR) Syny6803:sll0576 Putative epimerase/hydratase DoneHELP Set operation Find specific gene Add set of genes Specify gene Click on icon of gene Add to set Syny6803:srl7009 trnR tRNA Arg (UCU) Syny6803:slr1331 Processing protease slr1331 is now part of the set. Click on the icon upstream from sll0789, the third gene without an upstream region.

52. Build setDisplay set Set: Light-specific genes Syny6803:sll0990 Formaldehyde dehydrogenase (glutathione dependent) Syny6803:slr1332 fabF beta ketoacyl acyl carrier protein synthase Syny6803:sll0337 Sensor histidine kinase Syny6803:sll0335 Hypothetical Syny6803:sll0789 Response regulator (OmpR) Syny6803:sll0576 Putative epimerase/hydratase DoneHELPSet operation Syny6803:srl7009 trnR tRNA Arg (UCU) Syny6803:slr1331 Processing protease Syny6803:sll0788 Hypothetical protein Find specific gene Add set of genes Specify gene Click on icon of gene Add to set Now the genes presumably at the start of the operons have been added, and it’s time to remove the genes without upstream regions. Click on the radio buttons of the three genes and click Set operation.

53. Build setDisplay set Set: Light-specific genes Syny6803:sll0990 Formaldehyde dehydrogenase (glutathione dependent) Syny6803:slr1332 fabF beta ketoacyl acyl carrier protein synthase Syny6803:sll0337 Sensor histidine kinase Syny6803:sll0335 Hypothetical Syny6803:sll0789 Response regulator (OmpR) Syny6803:sll0576 Putative epimerase/hydratase DoneHELPSet operation Syny6803:srl7009 trnR tRNA Arg (UCU) Syny6803:slr1331 Processing protease Syny6803:sll0788 Hypothetical protein KEEP (delete others) DELETE FROM Click DELETE FROM to remove the three genes from the set.

54. Build setDisplay set Set: Light-specific genes Syny6803:srl7009 trnR tRNA Arg (UCU) Syny6803:sll0337 Sensor histidine kinase Syny6803:sll0335 Hypothetical Syny6803:sll0576 Putative epimerase/hydratase DoneHELPSet operation Syny6803:slr1331 Processing protease Syny6803:sll0788 Hypothetical protein Now you have what you want: a set of genes coregulated by light. The game now is to extract their upstream regions and determine if that set contains a common sequence motif. So you need a new set. Click Build set.

55. Click to see set you or someone else made Click to see statements to manipulate sets Click to give a value to a new or old variable Click to access results of experiments Click to see list of available statements Click to see list of available manipulations Click any red button to get help Variable Data Function Operation Set operation Display set VariableDataOperationFunctionDone Build setCancelHELPSet operation The set will be based on the upstream regions of the set of light-specific genes. Upstream region of... that’s a Function.

56. Display set Variable DataOperation Function Done Build setCancelHELPSet operation Ortholog of Protein product of Sequence of Upstream region of Downstream region of Common sequences (Meme) of Upstream region of The set will be based on the upstream regions of the set of light-specific genes. Upstream region of... that’s a Function.

57. Upstream region of 6803 ortholog Set Specify Choose variable Display set VariableData Build set Operation Function Done CancelHELPSet operation HELP Set ( in Arthrobacter platensis Gloeobacter violaceus Microcystis aeruginosa Nostoc punctiforme Nostoc PCC 7120 Prochlorococcus MED4 Prochlorococcus MIT9313 Prochlorococcus S120 Synechococcus PCC6301 Synechococcus PCC7942 Synechococcus WH8102 Synechocystis PCC 6803 Thermosynechococcus Trichodesmium Choose database ) You want not the upstream region of a variable (6803 ortholog is the only one you’ve made so far) but rather a set.

58. All open reading frames of Human-annotated orfs of Private set Public set Upstream region of Choose set type Display set VariableData Build set Operation Function Done CancelHELPSet operation HELP ( Arthrobacter platensis Gloeobacter violaceus Microcystis aeruginosa Nostoc punctiforme Nostoc PCC 7120 Prochlorococcus MED4 Prochlorococcus MIT9313 Prochlorococcus S120 Synechococcus PCC6301 Synechococcus PCC7942 Synechococcus WH8102 Synechocystis PCC 6803 Thermosynechococcus Trichodesmium Choose database ) Private set You have a couple of premade sets available, but you want your own. Click Private set.

59. Upstream region of Light-specific genes Choose set Display set VariableData Build set Operation Function Done CancelHELPSet operation HELP assigned to Type set name Light-specific genes ( ) You’ve made only one set. Choose it...

60. Upstream region of Light-specific genes Display set VariableData Build set Operation Function Done CancelHELPSet operation HELP assigned to Upstream light-sp genes Type set name You’ve made only one set. Choose it... and give it a name (I’ll type it).

61. Upstream region of Light-specific genes Display set VariableData Build set OperationFunctionDone CancelHELPSet operation HELP assigned to Upstream light-sp genes Type variable name Click to see set you or someone else made Click to see statements to manipulate sets Click to give a value to a new or old variable Click to access results of experiments Click to see list of available statements Click to see list of available manipulations Click any red button to get help Variable Data Function Operation Set operation Display set You want to run this new set through a filter that will give you conserved motifs. That’s a Function.

62. Upstream region of Light-specific genes assigned to Upstream light-sp genes Display set Variable DataOperation Function Done Build setCancelHELPSet operation Ortholog of Protein product of Sequence of Upstream region of Downstream region of Common sequences (Meme) of The function you want, Meme, analyzes a set of sequences for statistically overrepresented motifs.

63. Common sequences of Light-specific genes Upstream light-sp genes Public set Premade set Choose set Display set VariableData Build set Operation Function Done CancelHELPSet operation HELP assigned to Type variable name Upstream light sp-genes Upstream region of Light-specific genes assigned to Upstream light-sp genes You now have two private sets. You want, of course, the set of upstream light- specific genes.

64. Common sequences of Upstream light-sp genes Display set VariableData Build set Operation Function Done CancelHELPSet operation HELP assigned to Memed light-sp genes Type variable name Upstream region of Light-specific genes assigned to Upstream light-sp genes You click and I’ll type in a logical name.

65. Common sequences of Upstream light-sp genes Display set VariableData Build set OperationFunctionDone CancelHELPSet operation HELP assigned to Memed light-sp genes Type variable name Upstream region of Light-specific genes assigned to Upstream light-sp genes Click to see set you or someone else made Click to see statements to manipulate sets Click to give a value to a new or old variable Click to access results of experiments Click to see list of available statements Click to see list of available manipulations Click any red button to get help Variable Data Function Operation Set operation Display set Click Done to put your plans into action and to display the last defined set.

66. Build setDisplay set Set: Memed upstream light-sp genes Upstream of Syny6803:srl7009638D5096.38e-05 AAATATGGGA GAATGGAA TTGAGTAGCA Upstream of Syny6803:sll0337138D 343.29e-05 AGCTTAAAAA GACTAGAA TTCAATGGGT Upstream of Syny6803:sll0335279D1835.30e-05 AAGGGTTAGC GACTGGAG TTGGCAAAAC Upstream of Syny6803:sll0576 79D 281.31e-04 TTTTTTGCTT TACTGGGA ACGGATATTT DoneHELPSet operation Upstream of Syny6803:slr1331159D1524.38e-05 GGCCCATGGG GACTAGGA Upstream of Syny6803:sll0788221D1841.09e-05 TGCTTTGCCA GACTGGAA TTAGAGAAGG Len Pos E-val Left flank Motif Right flank And there you have it! A conserved octomeric sequence found in front of all six light-regulated genes (and, gratifyingly, all in the same orientation: Direct rather than Reverse). Is the sequence involved in gene regulation? Only experiments will tell...

67. Drawn on complex knowledge base What have we done? Written a computer program Modified set by hand Did it ourselves Combined multiple tools Continue

68. Analysis: Discovery of coregulated genes Summary The graphical interface facilitates searches using functions, loops and Boolean operations, with few of the complexities of most computer languages The graphical interface facilitates the searching through experimental data for orfs with desired properties The script interface permits interaction between the search and display capabilities of the web site and outside resources. End Scenario 4 Reminder: This was a simulation. The underlying language (BioLingua) exists, but not the interface that facilitates access.