1. Anotation: Gene of which little is known What follows is a simulation of an orf page in the proposed graphical interface. The interface does not yet exist. 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 1

2. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: This is an example of an orf that has only automated annotation, except for annotation carried over automatically from its ortholog in Synechocystis PCC 6803. The notation System indicates that the assertion has not been confirmed by a human annotator. Clicking on System brings you to a page that provides the basis for the assertion. Continue A

3. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: Clicking on Chromosome brings you to a zoomable map of the chromosome showing the position of all4312 (see the more interesting case in Scenario 5). A Continue

4. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: Clicking on the coordinates 5166997... brings you to a view of the sequence of the region around all4312 (this is the subject of Scenario 6). Continue A

5. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: Clicking on the length 256 amino acids brings you to the amino acid sequence. Both the DNA and amino acid sequences can be downloaded in this way. Continue A

6. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: There is no experimental data (or any other human annotation) available for all4312. However, there is microarray data available for its ortholog sll1330. Click to show these experiments. A

7. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to contract)Experiment Suzuki et al (2001) Cold shock responseExperiment Hihara et al (2001) low light to high lightExperiment Yoshimura et al (2001) cAMP receptor mutantExperiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: Clicking on the specific experiment gives details concerning protocol and results. Click to contract the display back to its previous form. A

8. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: A mutant exists for the ortholog in Synechocystis. Mouse over* to Experiment to see who made the mutant and whether the work is published. *In real life, information will pop up when you mouse to an informative position. In this simulation you’ll also need to click. A

9. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Presumed inviable (failed to segregate) Experiment Cyanobacterial orthologs: IMP (Inferred from Mutant Phenotype) is a standard Gene Ontology Consortium descriptor, useful for searching (see Scenario 3 for more). Mouse away from the region. *In real life, information will go away when you mouse away from an informative position. In this simulation you’ll also need to click anywhere. IMP: Masahiko Ikeuchi unpublished A

10. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Presumed inviable (failed to segregate) Experiment Cyanobacterial orthologs: Clicking on Experiment would bring you to a page describing the experiment and its results. For now, mouse over* to the Annotator icon to see who made this annotation. *Remember that in this simulation you have to click to pop up information over a region. A

11. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: Clicking on the icon would bring your designated e-mail screen with the annotator’s e-mail address ready to go. For now, mouse away from the region.* *In real life, information will go away when you mouse away from an informative position. In this simulation you’ll also need to click anywhere.. Masahiko Ikeuchi Tokyo University A

12. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: The bottom part of the screen shows the genetic neighborhood of all4312 and its cyanobacterial orthologs. Orfs are color-coded by function. Mouse over* to the gene downstream from all4312 to see its identity. Then mouse away.* *Remember to click to view the popup and click to make it disappear. A ABC transporter ATP-binding protein

13. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: Mouse over to the red motif bar to see the identity of the motif found in the indicated region of all4312. The Pfam number and E-value of the match are given. Mouse away from the motif bar.* *Remember to click to view the popup and click to make it disappear. A Response receiver domain (PF00072): 1.4e-31

14. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 Options Annotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: To predict operon structure, it is useful to visualize potential inverted repeats, which may function as transcriptional termination signals. Operon structure isn’t an issue in this case, but let’s go for it anyway. Click and then Show inverted repeats. A View: graphic Show cb orthologs Show hits: <1e-6 Show neighbors: +/- 1 Show motifs Show alternate starts Show annotated features Show predicted features Show transposon ends Show tandem repeats Show inverted repeats Show dispersed repeats Show private set Show inverted repeats Options

15. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 Options Annotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: There are evidently no features annotated yet for this orf, but there may be some that have been automatically predicted. Click and then Show predicted features. A View: graphic Show cb orthologs Show hits: <1e-6 Show neighbors: +/- 1 Show motifs Show alternate starts Show annotated features Show predicted features Show transposon ends Show tandem repeats Show inverted repeats Show dispersed repeats Show private set Show predicted features Options

16. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: Surprisingly, the same feature has been predicted for the entire set of orthologs. Mouse over to the feature upstream from all4312 to identify it. A

17. Anab7120:all4312 NostPunc:618.077 TricEryt:5.6053 Syny6803:sll1330 TherElon:tlr1330 Anabaena PCC 7120: all4312 OptionsAnnotate Main Menu History Replicon: Chromosome Coordinates: 5166997 (stop) <- 5167767 (start-GTG) System Length = 256 amino acids Strand: Complementary Function: Two-component response regulator System Syny6803:sll1330: Expression data (click to expand)Experiment Mutant: None Syny6803:sll1330: Failed to segregate Experiment Cyanobacterial orthologs: Clicking on the green triangle would lead to a page that describes the motif and how it was identified.* *Participant Jackie Collier and Chip Lawrence are developing a method to predict transcriptional binding sites. While the set does not yet exist, the triangles below represent sites that have actually been identified by a means similar to that described in Scenario 5. A End Lawrence/Collier conserved motif set

18. Anotation: Gene of which little is known Summary The Annotation Page provides links to different views of the region surrounding the gene The page links to other pages that facilitate downloading sequences The page provides experimental data when available regarding the orf and its cyanobacterial orthologs DNA features can be visualized wthin the context of the genetic neighborhood of the orf Scenario 1 A prototype of the Annotation Page exists in CYORF, but the full capabilities described here require the resources we are asking of NSF.