Evaluate your trees • bootstrap analysis • shuffling order or other online resources • Use Primer3 to design primers to study this gene in populations.


Using bioinformatics to characterize the solute carrier proteins Background The SLC26 transporters are a class of membrane proteins found in eukaryotes that are responsible for exchange of different anions into the cytosol. Among the various ions that can be transported by these molecules is the small organic acid oxalate. When an excess of oxalate is found in the blood, it tends to electrostatically interact with calcium ions and form hard crystals, which can grow into kidney stones (renal lithiasis). A potential strategy to treat individuals with chronic renal lithiasis could be to selectively inhibit some members of the SLC26 family from binding oxalate (thus preventing excessive oxalate absorption in the small intestine). In order to accomplish this task, one must know how similar the various SLC26s are to each other as well as other members of the SLC family. For your project, we want you to use all of the techniques covered in your assignments to investigate the sequence similarity and evolution of the SLC26 family of proteins. Pick one member of the SLC26 family (SLC26A1 – SLC26A11) and run it through the entire suite of tools you have been using for your assignments. Please note that this project is not an assignment; here we expect you to apply what you have learned and choose the best strategy to accomplish the task at hand. We also expect your report to include figures and tables as well as a narrative description of the results. You will most likely need to read a number of papers on the SLC transporters in order to accomplish this task effectively. Instructions • Use BLAST or FASTA to find a number of closely related homologues within the class of SLC26 proteins. Use UniProtKB to find out more about these proteins. • Use BLAST or FASTA to find 10 more distantly related homologues (members of the SLC family but not SLC26). • Use PSI-BLAST or COBBLER to find 10 even more distant homologues (proteins that are not members of the SLC family). • Characterize the domain structure of your SLC26 protein using BLOCKMAKER, PFSCAN, PFAM, etc. • In order to rationally design a drug, we usually need to have a crystal structure. Use VAST, DALI, or PdbEfold to find genes related by structure. Include images showing the structural alignment. • Use ClustalW and other multiple sequence alignments (T-COFFEE, MAFFT, MUSCLE) to find and highlight conserved regions using the amino acid sequence. Use at least 10 amino acid sequences from 10 different organisms. • Use PHYLIP or MEGA5 to construct phylogenies using these multiple alignments: • One based on distance matrix methods • One based on maximum parsimony • One based on maximum likelihood (you may need to use DNA sequences for this) • Evaluate your trees • bootstrap analysis • shuffling order or other online resources • Use Primer3 to design primers to study this gene in populations. • Use CODEHOP to design primers to fish out related genes from mRNA of an organism that you haven’t previously studied. • Design a protocol to clone the entire coding sequence (i.e. from start to stop codon) of a homologous gene related to your assigned gene but from a different organism into Bluescript • Identify ORF • Design primers that bind as close to start & stop as possible, and evaluate them using primer finder • Describe procedure for performing the cloning, including restriction enzymes used • print map of recombinant plasmid • print 50 bp of sequence at the junctions between the plasmid and cloned DNA Report Prepare a summary in which you first state which gene you started out with, then provide a paragraph for each test in which you summarize the results, discuss why you think that you obtained them and try to explain any unexpected results. • For BLAST/FASTA tell us how many significant results were found, and which sequences were most closely related and who they came from. Are any of your homologues associated with diseases? What ligands do your proteins bind? Identify and try to explain any unexpected similarities and any differences between the searches using RNA versus amino acid sequences. How did you have to modify your search strategy to find more distant homologues (SLC that are not members of SLC26)? What sorts of ligands do your proteins bind in this broader group? • For PSI-BLAST etc. tell us how many more distant relatives were found, what sorts of organisms they came from, and what sorts of proteins were related. Did you find any bacterial anion transporters? If so what organisms do they come from? • For Blockmaker, PFSCAN etc tell us how many motifs were found, what parts of the protein they came from, and what other proteins contain these motifs. Identify and try to explain any unexpected results. How do these results compare to what you found using BLAST/FASTA? Are there specific domains that are more common among your hits? • For VAST (or the other structural alignment programs) tell us what sorts of proteins you found, and whether you found any new ones missed by the sequence-based approaches. Discuss any differences from the sequence-based approaches, and identify and try to explain any unexpected results. What parts of your SLC26 protein seem to have structural homologs with crystal structures? How does this agree with the domains you identified above? • For Clustal W describe the relationships that were identified, and what parts of the protein were related. Identify and try to explain any unexpected results. • For Phylip explain who the closest relatives are, and comment on whether this was expected or a surprising result. Then, if the three methods come up with different trees, try to explain why this might have happened and which tree is most likely to be correct. • For the tree evaluation explain what your results mean in terms of the reliability of the trees. • For part 8 just list the primers that were designed. • For part 9 just list the primers that were designed. • For part 10 first state where the ORF started and finished, then list the sequence of the primers and state where they bound. Next describe which restriction enzymes you will use, print a map of the recombinant plasmid and print 50 bp of sequence at each junction between the plasmid and cloned DNA. Organize your results into sections where everything you present is clearly explained and annotated. Provide a conclusion section wherein you summarize all of your results in narrative form. In order to properly interpret your results and explain their biological significance, you will need to read about your protein, the SLC26 class of proteins and the SLC family.

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