Combining the microarray and metabolic capabilities of BioBIKE

1. Combining the microarray and metabolic capabilities of BioBIKE Case Study How is carbon metabolism affected by starving the cyanobacterium Anabaena for nitrogen?* This demonstration is best viewed as a slide show,enabling you to simulate a session and make changes in cursor position more obvious.To do this, click Slide Show on the top tool bar, then View show. *Problem taken from Xu X, Elhai J, Wolk CP (2007). Transcriptional and developmental responses by Anabaena to deprivation of fixed nitrogen. In: The Cyanobacteria: Molecular Biology, Genetics and Evolution (Herrero A, Flores E, eds). Horizon Scientific Press. pp.387-426.

2. Nitrogen deprivation differentiation in Anabaena +N +N Autofluorescence (photosynthetic apparatus) All cells in same state The cyanobacterium Anabaena grows as multicellular filaments, all cells in the same state when nitrogen is plentiful. Anabaena is photosynthetic, so it supplies its own carbon from CO2.

3. Nitrogen deprivation differentiation in Anabaena +N +N Autofluorescence (photosynthetic apparatus) All cells in same state -N When starved for nitrogen, Anabaena differentiates specialized cells, heterocysts, that can utilize atmospheric N2 gas. N2-fixing heterocysts

4. Metabolic correlates to N-deprivation Amino acids Photosynthetic apparatus proteins N2-fixing heterocysts When the supply of fixed nitrogen (e.g. ammonia) dries up, Anabaena faces the necessity of making N2-fixing heterocysts fast. They get the amino acids to make the new protein needed by heterocysts by cannibalizing the biggest source of proteins in the cell – the photosynthetic apparatus.

5. Metabolic correlates to N-deprivation Amino acids Photosynthetic apparatus proteins glycolipid N2-fixing heterocysts Energy Sugar But new cells are not just proteins. The surface is covered with special glycolipid, made in part from sugar. Sugar is also the source of energy that drives the process of differentiation. If photosynthetic capacity is being depleted, then where does this sugar come from?

6. Metabolic correlates to N-deprivation What enzymes of carbon metabolism are affected by N-starvation? RNA -N +N We can address this question by examining which genes involved in carbon metabolism are turned on or turned off by N-starvation. Microarrays can be used to get a snapshot of gene expression under different conditions. Microarray Courtesy of Inst. für Hormon-und Fortpflanzungsforschung, Universität Hamburg

7. Metabolic correlates to N-deprivation What enzymes of carbon metabolism are affected by N-starvation? Two broad sets of enzymes are of interest: - The enzymes that catalyze the dark reactions of photosynthesis - Those that breakdown glycogen and metabolize glucose derivatives.

8. Metabolic correlates to N-deprivation Glycogenmetabolism PentosePhosphatePathway What enzymes of carbon metabolism are affected by N-starvation? Carbon fixation Cyanobacteria use primarily the reactions of the Pentose Phosphate Pathway to break down glucose derivatives. They use carbon fixation reactions to build glucose. These sets overlap a great deal.

9. Metabolic correlates to N-deprivation PLAN - Define genes involved in pentose phosphate pathway - Find from microarray how the levels of expression of these genes are affected by N-deprivation - Sort the results, from lowest expression to highest - Display the results, along with a description of the genes, in an intelligible way Go to the BioBIKE Portalbiobike.csbc.vcu.edu

10. Go to a public site

11. Your name (no spaces) Enter a log in nameClick New Login

12. Function palette Workspace The BioBIKE environment is divided into three areas as shown. You'll bring functions down from the function palette to the workspace, execute them, and note the results in the results window Results window

13. HELP! PROBLEM Two very important buttons on the function palette: On-line help (general) Something went wrong? Tell us!

14. Two very important buttons in the workspace: Undo (return to workspace before last action) Redo (Get back the workspace you undid)

15. PentosePhosphatePathway What now? The first step in exploring the expression of the genes of carbon metabolism is to define each set. In this tour, we'll focus only on the genes of the pentose phosphate pathway, but you will be able to imagine how the analysis could be extended. Mouse over the DEFINITION button

16. … and click DEFINE, to bring down a DEFINE box.

17. DEFINE box? Before going on, let's take a break to look at what boxes mean.

18. Argument(object) Function-name Flag Keyword object General Syntax of BioBIKE The basic unit of BioBIKE is the function box. It consists of the name of a function, perhaps one or more required arguments, and optional keywords and flags. A function may be thought of as a black box: you feed it information, it produces a result.

19. Argument(object) Function-name Flag Keyword object General Syntax of BioBIKE Function boxes contain the following elements: • Function-name (e.g. SEQUENCE-OF or LENGTH-OF) • Argument: Required, acted on by the function • Keyword clause: Optional, more information • Flag: Optional, more (yes/no) information

20. Argument(object) Function-name Flag Keyword object • Option icon: Brings up a menu of keywords and flags • Action icon: Brings up a menu enabling you to execute a function, copy and paste, information, get help, etc • Clear/Delete icon: Removes information you entered or removes box entirely General Syntax of BioBIKE … and icons to help you work with functions:

21. Argument(object) Function-name Flag Keyword object • Option icon: Brings up a menu of keywords and flags • Action icon: Brings up a menu enabling you to execute a function, copy and paste, information, get help, etc • Clear/Delete icon: Removes information you entered or removes box entirely General Syntax of BioBIKE … and icons to help you work with functions: And now back to our show…

22. The DEFINE function takes two arguments: the name of the variable to be defined and its value. We want first to define the set of genes encoding the enzymes of the pentose phosphate pathway. Click the variable argument box.

23. That opens the box for input. Notice that the box is now white and selected (surrounded by red dots). Type the name you want to give the set. The name must not contain spaces, but symbols are OK. I chose pentose-phosphate-genes.

24. The box is still open, and the function will not be able to be executed until it is closed. To close it, you could press Enter on your keyboard. Alternatively (and better in this case), press the Tab key to close the box and move on to the next box.

25. Now the value argument box is open for input. We'd like to put in that box the genes of Anabaena involved in the pentose phosphate pathway. To get a function that will provide those genes, mouse over the Genome button…

26. … and click GENES-IN-PATHWAY/S.

27. This function calls for two arguments: the pathway we're interested in and the organism (or a replicon from the organism). The organism is clear, so let's do that first. Click on the organism-or-replicon argument box. Then, once it's open, mouse over the DATA button to get the organism, Anabaena.

28. Anabaena is a nitrogen-fixing cyanobacterium, so mouse down to that category

29. … and across Anabaena PCC 7120 to…Hmmm. Looking ahead, I realize that the microarray I'll be using considers only chromosomally encoded genes. It will simplify matters if I choose the chromosome of Anabaena. Click that.

30. Now we need to supply the pathway. Eventually it will be possible to choose the pathway from a hierarchical menu in the same way you chose the organism. For now, the task is more complicated. We need to find the pentose phosphate pathway from within a list of pathways and then copy its identification number. To find it, we'll display the list. Go to to the INPUT-OUTPUT button to find the DISPLAY-LIST function.

31. Click DISPLAY-LIST to bring down the function.

32. DISPLAY-LIST formats lists of information to make them easier to read. We're going to use it to examine lists of pathways to help us find the one we want. Click the list argument box, then mouse over the DATA button to get to lists of pathways.

33. Mouse over kegg-pathways, and click *CARBOHYDRATE-METABOLISM*, the most likely category in which to find the pentose phosphate pathway.

34. Carbohydrate metabolism consists of many pathways. We want to display each one, with formatting, so mouse over the EACH prefix option and select it.

35. The function is now complete and ready to be executed. Mouse over to the action icon of DISPLAY-LIST…

36. …and click Execute.

37. A popup window appears, displaying the list of pathways defined by KEGG as related to carbohydrate metabolism. The pentose phosphate pathway has an identification number "00030". That's what we came for. Important displays can be downloaded to your computer through the usual browser controls. This time just X out of the box.

38. With the pathway identification number in hand, click the pathway argument box…

39. … enter "00030", and press the ENTER key. Once you've done that, all the boxes will be filled in and closed. The function is now ready to be executed. Mouse over the Action Icon of the DEFINE function…

40. …and click Execute.

41. Note that a new VARIABLES button appears. We'll use it in a moment. At the same time, a list of genes appears in the Result Window. What is that list of genes? It's supposed to be the genes in the chromosome of Anabaena that encode enzymes of the pentose phosphate pathway. Is that true? To check, go to the GENES-PROTEINS button,…

42. … and click DESCRIPTION-OF.

43. We can use this function to describe the set of genes we just defined. If we recognize within the descriptions names of enzymes of the pentose phosphate pathway, all is OK. The function is asking for something to describe. Click on the entity argument box.

44. The set we just defined has become part of our language, accessible through the Variables button. Mouse over that button…

45. … to find pentose-phosphate-genes. Click on it to bring it down to the argument box.

46. You can execute this function as it stands, but the result will be disappointing (try it!). To make the result more intelligible, mouse over the Option Icon…

47. … and click DISPLAY. Now the results will not only be presented in the Result Window, they will also be displayed in an intelligible form.

48. You might think that now the function is ready, but try it and you'll get a description of the list (it's a list) when what you want is a description of each element of the list. So as before, you need to specify the EACH prefix option.

49. Finally, execute the function.

50. PentosePhosphatePathway Glucose-6-phosphate dehydrogenase (G6PDH), transaldolase... They all seem to be there. You've done the right thing, checking the result. Now kill the box.