Procedure:
1. Using your favorite web bmwscr, connect to 2 wchsiles in

2 different Tabs. 2. Open wyn“trbi.nlin.nilloon “maimed in one tab and open www gcnume.intionlidclustalw/ in another tab.

3. In the ncbi tab. change “PubMed” to “nucleotide” by clicking on arrows and selecting “nucleotide”. A. In the search box. enter the Accexsion.version number for the Enterobacteriaceae you have identified from experiment 12. Hit the “search” button or hit the “enter” key. B. Click on “FASTA” under the name of the organism and copy the nucleotide sequence starting from the ->” smbol.

4. Go to the Clusta1W tab.

A. Change the setting from protein to DNA B. Paste the nucleotide sequence inside the box and then hit the “enter” or “return” key

5. Go hack to the ncbi window and type in the search box the accession.version number for the first bacterium listed in Figure 15.3 (Afethanococcus voltae) and hit “search”.

6. Click on “FASTA” under the name of the organism and copy the nucleotide sequence starling from the “>” symbol.

7. Go to the ClustalW tab and paste nucleotide sequence. Hit the “return” or “enter” key.

8. C70 back to the ncbi window and type in the accession.vcrsion number for the next organism listed. 9. Repeat these steps until you have copied all the nucleotide sequences of all the organisms in the phenotypic nee.

You will need to find the accession numbers for Neurospora crassu, Candida albicans and Saccharotnyces cerevisiae on your own. Do this by searching for the organisms name along with “185 ribosomal RNA” as additional key words in the search box.

Choose the first result that appears, click on “PASTA” and the accession number will be located immediately after the ” > ” symbol. Write this num down for identification of organism in the phylogenetic tree. 10. After pasting all nucleotide sequences in ClustalW, click on the “Execute Multiple Alignment” button underneath the box. 11. To generate phylogenetic tree, A. Select “Rooted phylogenetic tree (UPGMA)” from the select tree menu.

B. Hit “Exec” button C. Click on PostScript file or PDF file to save the phylogenetic tree. D. Print out the phylogenetic tree and relabel the accession numbers with the corresponding name of organisms E. Answer the post lab questions and turn in answers with the printed phylogenetic tree.

Post-lab questions: {20 points)

I. Print out the phylogenctic tree and turn it in with the answers to the following questions. 16 points]

2. Print out the alignment of the 16s rRNA sequences of your assigned bacterium and the 4 most closely related bacteria. I low many nucleotide changes distinguish each of these organisms? What are the major biochemical differences between These organisms? [a rows]

3. Do the relationships of the taxa in the phylogenetic tree agree with those predicted from a tree based strictly on phenotypes? Describe two ways in which the relationships arc likely to be similar and two ways in which they are likely to differ.12 points]

4. Even with the limited number of sequences involved, the unroofed phylogenetic tree allows the recognition of three large groups of organisms. These groups are called Domains. On your phylogenetic tree, circle each Domain and give each its correct name. p points’

5. Predict where each of the following taxa would be located in your unroofed tree: Homo sapiens (#337376) and Candida alhicans (you will have to look up this GenBank Accession number yourself). Check your predictions by adding the indicated sequences to your list and redrawing the phylogenctic tree. Turn in the revised tree with your answers. Re sure to use 185 ribosomal RNA when you search these sequences. p points] , •

6. It is possible to identify uncultured microbes in environmental samples by using PCR printers that amplify the 16$ rR.NA. sequencing the amplified DNA fragment, and using the sequence to place the organism on the phylogenic tree. What does the placement of the 16s sequence AJ606250 tell you about the organism? l2 Pints]

7. All but one of the prokaryotes without peptidoglycan in their cell walls are Archac. What group is the exception and why? it point]