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 330aa 481 aa

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