2010 PCB 5530 Class Projects

2016 PCB 5530 Class Projects and Grading ● Background The projects take cutting-edge comparative genomics research into the classroom, to bring to life what you are learning. They will train you to reason and to integrate transcriptomics and genomics data. The class is split into four groups, each coordinated by a postdoctoral from our project. Groups are: 1. Folate 2. Pyridoxine 3. Pantothenate, CoA 4. Riboflavin, FMN, FAD Antje Thamm Tom Niehaus Jiayi Sun Guillaume Beaudoin [email protected] [email protected] [email protected] [email protected] Ann C Bernert Lorenzo N Bizzio Francesco Cappai Nguyet-Minh Dao Shaheen Bibi Edgar Sierra Yongduo Sun Jianxin Zhao Drake M Garner Qingyuan Xiang Zhuolun Wang Kamal Bansal

Each group will: - Become familiar with the assigned B vitamin/cofactor biosynthesis and salvage pathways in plants, and with the types of enzymes that utilize the cofactor. - Use BlastP to find Arabidopsis orthologs of 10 candidate maize transporters for the assigned vitamin (the maize candidates were identified based on co-expression with vitamin synthesis/salvage genes). - Use ATTED and GeneMANIA tools to view (i) the co-expression networks around the Arabidopsis orthologs, and (ii) the list of the top 300 genes that are co-expressed with the orthologs. - Search the co-expression networks and top 300 list for genes that synthesize or salvage the assigned vitamin/cofactor, or use the cofactor. Such genes would reinforce the evidence from maize for a functional association between the transporter and the vitamin/cofactor. - Search for bacterial homologs of the maize transporter. If homologs are found, use those from genomes in STRING and PubSEED to find genes that consistently cluster on bacterial chromosomes with the transporter gene, or are associated in other ways. If such genes include genes for synthesis or salvage of the assigned B vitamin/cofactor, or use a cofactor derived from it, this would be further evidence that the transporter has a function related to the vitamin. Each group will meet several times, under the supervision of the postdoctoral mentor, to decide the work to be done, to review progress, and to plan and prepare a single report in the format below. Project reports. Reports should be submitted to Dr. Andrew Hanson [email protected] as an electronic file and a good quality hard copy, by 5 pm on Friday, November 18, 2016. Grading. For each student, 45% of the grade will be based on the performance of their group as a whole as judged from the project report. Another 45% will be based on the postdoctoral instructor’s and my assessment of that student’s individual contribution to the group effort (independent of the group size). The individual contribution assessment will emphasize creativity, initiative, and quality of work, and will also take account of the amount of work done. The final 10% of the grade will be based on contributions in class, e.g. on questions asked or answered by the student. Outcomes. In the best case, the groups’ predictions would form part of a publication in a peer-reviewed journal, in which case the group members would be included as authors.

● Report format Reports should be arranged in three sections: 1. Pathways and cofactor-dependent enzymes a. A diagram showing the metabolites, enzymes, compartmentation, and known or inferred transport steps in the plant synthesis and salvage pathways. Use a format like that on p. 2. b. A table listing the names and EC numbers of the synthesis and salvage enzymes, and giving the AGI codes (e.g. At5g14760) for the enzymes in Arabidopsis. 1 c. A table listing the individual enzymes (for folate) or classes of enzymes (for other cofactors) that use the cofactor. For instance, for the cofactor pyridoxal 5’-phosphate some classes of enzymes are aminotransferases and amino acid decarboxylases. 2. Transporter candidate genes: a. A table listing the 10 maize transporter candidates and - The AGI code of the Arabidopsis protein most similar to each of the maize candidates

- The identifier, Blast score and percent identity of a strong bacterial homolog of each maize candidate (if one exists); these homologs should come from a genome in STRING and SEED b. TMHMM plots for each of the Arabidopsis proteins 3. Functional prediction: For one candidate transporter gene (the strongest candidate if there is more than one), a 200-300 word summary of the transport function you predict. Include screenshots of evidence for this function (e.g. ATTED or GeneMANIA co-expression networks, STRING interaction diagram or Predicted Functional Partners table, SEED Compare Regions display). Also include evidence that this protein has not yet been assigned any functional annotation, or has been incompletely annotated or mis-annotated. Cite appropriate literature. Literature citations must be in the following format: Zheng M, Wang X, LaRossa RA, Storz G (2001) DNA microarray-mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide. J Bacteriol 183: 4562-4570

● Report format – example of pathway diagram

2 ● Recommendations Start by identifying the relevant plant synthesis and salvage pathways. Resources include PlantSEED http://pubseed.theseed.org/seedviewer.cgi?page=PlantGateway, AraCyc http://pmn.plantcyc.org/, and KEGG http://www.genome.jp/kegg/pathway.html#cofactor. Then draw the summary pathway diagram. Construct (i) the table listing Arabidopsis synthesis and salvage genes and (ii) the table listing the individual enzymes (for folate) or classes of enzymes (for other cofactors) that use the cofactor. Expasy Enzyme has information on enzymes that use each cofactor. The supplied maize expression datasheet lists, for each B vitamin, the ten highest correlations between synthesis/salvage genes (column B) and candidate transporter genes (column A). Note that more than one transporter gene may be correlated with a single synthesis/salvage gene. For each enzyme, use the HTML Tables from http://pubseed.theseed.org/seedviewer.cgi?page=PlantGateway to check that the spreadsheet vitamin enzyme annotation (column J) is correct and complete (some are incomplete). Use BlastP to find the Arabidopsis ortholog(s) of the candidate maize transporters, and use their AGI numbers to construct ATTED and GeneMANIA co-expression networks. Search these networks for genes whose products synthesize or salvage the vitamin/cofactor, or that use the cofactor. Use BlastP to search for the best bacterial homologs of the candidate maize transporters, and use these as entry points to search STRING and SEED for associations between these homologs and genes whose products synthesize or salvage the vitamin/cofactor, or that use the cofactor. Marking scheme:

Pathway figure and tables 10 Arabidopsis synthesis/salvage gene table 5 Arabidopsis cofactor utilization table 5 Maize candidates & Arabidopsis & bacterial ortholog table 3 TMHMM plots of Arabidopsis ortholgs 2 Functional prediction 20