Transcript Pyrimidines
Microbial Sequence Analysis Exploring the Pyrimidine Biosynthetic Pathway with NMPDR Katharine Mixter Mayne William Woods University Pierluigi Barone University of Illinois at Urbana-Champaign Remember! Species can be related closely or distantly. How do we measure evolution and evolutionary relatedness in microbes? Morphological features (shape, motility, capsules) Chemical and Physiological features (gram stain, catalase test) Modern biologists have used 16S RNA sequences to completely redraw the evolutionary tree (Carl Woese 1970s ) Why 16S? A feature of cells that is 1. Universally common, 2. Important to viability, 3. Slow to mutate Prokaryotic ribosome: 70s 2 subunits: 50S and 30S 3 primary RNA components: 5S, 28S, 23S 5s RNA is too small (not enough information available), 23s RNA is too large, evolving more rapidly 16s RNA is of manageable size and information content Signature sequences Sequences chosen as primers are virtually unchanged in ALL species. Madigan et al. 2003 How do we use extract and use the information? Extracting the data There are several approaches: one of the most common is to use the DNA (the gene) encoding for the 16 rRNA 1. Extract DNA from the cell 2. Heat to form single stranded DNA 3. Add a primer (a short, known sequence of bases) that will hybridize to the single stranded DNA 4. Using DNA polymerase, synthesize the rest of the sequence. If this is done in a PCR machine, many copies of the gene may be made and copied (millions of copies) 5. Purify, sequence and review sequence analysis Madigan et al. 2003 How do we use extract and use the information? Using the Data: Evolutionary Distance (ED) 1. Align the sequences 2. Find those that differ 3. Develop an estimate of evolutionary distance (un-relatedness) 4. Model all of the possibilities and produce the outcome Madigan et al. 2003 1. From the home page of NMPDR, www.nmpdr.org, select the link for "Subsystems" in the main body of the page This will open a subsystem tree of the available subsystems in NMPDR. Expand the category labeled "Nucleosides and Nucleotides," (click on the plus symbol). Click on the link for De Novo Pyrimidine Synthesis. 2. Wait for the subsystem spreadsheet to load. Once the page is loaded look at the features as described in the hand out. Click in here to see the pathway De Novo Pyrimidine Synthesis: Diagram De Novo Pyrimidine 3. Go back to the subsystem spreadsheet. Scroll WAY DOWN and find the row for E. coli O157:H7. Find the column for your gene and click in it (e.g. pyrAB as shown). 4. Protein page: What does this show? •What is an arrow? •Colors? •Direction? •Click on the “show protein ” button. 5. The Genome Context Table presents the same information as in the graphic, and also provides buttons to start several tools for comparative analysis. The name of the focus gene is highlighted green. In this row, click on the button. A large graphic display will open (be patient) in a new window. 6. Pins Display: What does this page show? Refer to questions in the hand out 7. Scroll all the way to the bottom of the Pins Display and click on the button. This will open a new window (commentary table) that displays information corresponding to genes homologous to your focus gene. Scroll all the way to the bottom of this page, then slowly, find the top of the lower table. Click on the button to Now select two more species beside E. coli O157:H7 (e.g. Bacillus anthracis str. Ames and Streptococcus pyogenes M1) and scroll to the bottom of the page again, and click the button This will redraw the display with only the genome maps you picked with the check box. Describe how well the pattern of genes shown in the 3 genomes match or how different they are. Investigate what are the different genes?