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A Closer Look at Griffith’s Research

A Closer Look at Griffith’s Research. Karen A. Curto Team Curto. Prior Knowledge. Recall Bioinformatics lab Examined the similarities between Aquaporin protein in humans and Drosophila Learned about (Basic Local Alignment Search Tool) BLAST Protein Data Bank (PDB). Prior Knowledge.

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A Closer Look at Griffith’s Research

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  1. A Closer Look at Griffith’s Research Karen A. Curto Team Curto

  2. Prior Knowledge • Recall • Bioinformatics lab • Examined the similarities between Aquaporin protein in humans and Drosophila • Learned about • (Basic Local Alignment Search Tool) BLAST • Protein Data Bank (PDB)

  3. Prior Knowledge • Recall • Griffith’s and Avery’s research on DNA • What did they do? • What did the data suggest? • Do you recall what organism Griffith (& Avery) used that wasn’t a mouse? • Talk about bacteria (prokaryote refresher)

  4. What was the difference between S and R? • S contained a polysaccharide capsule and R did not • How did this capsule protect S? • Mouse immune cells unable to engulf & destroy • What was the term used to describe the change in R mixed with heat killed S? • transformation

  5. Using Bioinformatics to Explain the Difference between R and S • Polysaccharide Coat • Biosynthetic pathway for polysaccharide capsule • Collection of enzymes that • regulates production of sugars, • links then together • Facilitates transport to surface

  6. Check Out These Two Sequences • I will provide you with two sequences labeled A and B* • A is in our synthetic pathway, B just outside it • Enter database IMG JGI • Paste the sequences into box (one at a time) • Select four strains • S. pneumoniae R6, S. pneumoniae D39, another S. pneumoniae and S. pyrogensis • A = cps 2A part of pathway which is truncated in R • B = cps 2I outside of deleted section

  7. Comparing Results • When compare for A (cps 2A) only get two hits • Examine gene ortholog neighborhoods • From what strains are these?

  8. Now examine B (cps 2I) • Examine gene ortholog neighborhoods • How many strains are listed ? • Drag pointer over gene for cps 2I • What is its size? • 469aa(2),478aa,428aa

  9. Size Discrepancy • Using this same group • Drag pointer over gene for cps 2A • What is its size in two strains that have it? • R is truncated 481 aa 330aa

  10. Do multiple sequence alignment for these 2 • Use MUSCLE • Shows initial identity for two and then loss >D39 MSRRFKKSRSQKVKRSVNIVLLTIYLLLVCFLLFLIFKYNILAFRYLNLVVTALVLLVAL VGLLLIIYKKAEKFTIFLLVFSILVSSVSLFAVQQFVGLTNRLNATSNYSEYSLSVAVLA DSEIENVTQLTSVTAPTGTDNENIQKLLADIKSSQNTDLTVNQSSSYLAAYKSLIAGETK AIVLNSVFENIIELEYPDYASKIKKIYTKGFTKKVEAPKTSKNQSFNIYVSGIDTYGPIS SVSRSDVNILMTVNRDTKKILLTTTPRDAYVPIADGGNNQKDKLTHAGIYGVDSSIHTLE NLYGVDINYYVRLNFTSFLKMIDLLGGVDVHNDQEFSALHGKFHFPVGNVHLDSEQALGF VRERYSLADGDRDRGRNQQKVIVAILQKLTSTEALKNYSTIINSLQDSIQTNVPLETMIN LVNAQLESGGNYKVNSQDLKGTGRMDLPSYAMPDSNLYVMEIDDSSLAVVKAAIQDVMEG R >R6 MSRRFKKSRSQKVKRSVNIVLLTIYLLLVCFLLFLIFKYNILAFRYLNLVVTALVLLVAL VGLLLIIYKKAEKFTIFLLVFSILVSSVSLFAVQQFVGLTNRLNATSNYSEYSLSVAVLA DSEIENVTQLTSVTAPTGTDNENIQKLLADIKSSQNTDLTVNQSSSYLAAYKSLIAGETK AIVLNSVFENIIELEYPDYASKIKKIYTKGFTKKVEAPKTSKNQSFNIYVSGIDTYGPIS SVSRSDVNILMTVNRDTKKILLTTTPRDAYVPIADGGNNQKDKLTHAGIYGVDSSIHTFS E---------------------------------------SRWTFPI------------- ----YIISN-------------ICLL-----------YNT-------------------- ------------------------------------------------------------ -

  11. Pfam analysis • Look at Pfam information • Do any of these functions fit into our interest in R and S? • Pfam description • A regulatory protein involved in exocellular polysaccharide synthesis

  12. Compare Total Genome Size of Strains • IMG JGI • Look for total length • Compare D39 to R6 • D39 = 2046115 • R6 = - 2038615 7500

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