Looking at Chloroplasts
Now that we have demonstrated the relatedness between bacterial genes and eukaryotic mitochondrial genes, suggesting a bacterial origin for mitochondria, we are going to try to make a similar case for the origin of chloroplasts. The chloroplast gene that we will be working with encodes a highly conserved ribosomal protein in the primitive plant Porphyra purpurea. To start this new search, you need to "Return" to the Protein Tools homepage. You are now ready to conduct your search:

1. Select "Ndjinn-Multiple Database Search" and click "Run". In the search box, type "Porphyra purpurea ribosomal protein S12" and select 10 in the drop-down "Hits per page" menu. Select the database "SwissProt". As mentioned earlier, this database is very useful when searching for protein sequences because it contains extensive lists of both eukaryotic and prokaryotic sequences.

2. Click on "Search". At the time of writing this tutorial only one match was obtained. However many results you obtain, the sequence to look for is "Chloroplast 30S ribosomal protein S12". Since this is a single sequence for the ribosomal protein S12 gene, it is not necessary to go through the process described in previous sections to "Add Protein Sequences". Instead:

3. Select the RR12_PORPU sequence, click on "Import Sequence", and "Return" to the Protein Tools homepage.

4. Check-mark the "Porphyra purpurea S12 sequence", select BLASTP and click "Run". Once you get to the BLAST page, select the SwissProt and GenBank Bacterial Sequences databases. Leave all of the default settings on this page as is and click "Submit".

Once again the closest match, as expected, is the very same sequence that was blasted. Among the twelve closest-matching sequences nine are bacterial sequences. This indicates that the P. purpurea chloroplast ribosomal S12 sequence is more closely related to some bacterial ribosomal protein S12 sequences than it is to other plant sequences. The bacterial sequences with highest homology (Synechococcus, Synechocystis, and Spirulina platensis) all belong to the class of bacteria known as Cyanobacteria. Thus, in the same way that the mitochondrial genes of R. americana that we worked with earlier were especially homologous to sequences of alpha-Proteobacteria, the chloroplast S12 ribosomal gene of P. purpurea, shows highest homology specifically with genes of the Cyanobacteria group of bacteria.







5. Return to the results page (by clicking on the "Back" button in Internet Explorer or by simply closing the "Show Records" window in Netscape), select the closest matching bacterial sequence ("SwissProt:RS12_SYNP6") and click on "Import Sequence". You will be returned to the Protein Tools homepage.
Now we are going to use the CLUSTALW tool to view the level of homology between the chloroplast S12 ribosomal gene of P. purpurea and the ribosomal protein S12 of Synechococcus.

6. Check-mark the two sequences, select CLUSTALW and click "Run". On the next page click "Submit".



The protein sequence, although very short, is very highly conserved between Porphyra and Synechococcus (blue letters indicate identical amino acids!). Scroll down past the aligned sequences to locate the alignment Score (percent identity); you should see that it is 87, an extremely good indicator of evolutionary relatedness.

Up to this point you have used various bioinformatics tools to obtain supportive evidence for the theory that prokaryotic organisms are the evolutionary predecessors of the mitochondria and chloroplasts of eukaryotic cells. While constructing our argument, we noticed that:
a. Mitochondrial sequences are more closely related to alpha-Proteobacteria than to other classes of bacteria.
b. Chloroplast sequences are more closely related to Cyanobacteria than to other classes of bacteria. To further analyze the relatedness of mitochondria and chloroplasts to specific classes of bacteria, you are going to use another tool of the Biology Workbench to construct phylogenetic trees.