Using the BLAST Tool to Find Sequences Homologous to the Mitochondrial NADH Dehydrogenase, Subunit 8 Gene

We will now repeat this procedure, using a different mitochondrial gene of R. americana. First, this will be an opportunity to review the entire process of finding, importing and aligning amino acid sequences. Secondly, it will show that the homology seen between bacterial and eukaryotic mitochondrial genes is not limited to one gene.

1. From the Protein Tools homepage, select the Ndjinn ¡V Multiple Database Search (no sequences should be check-marked) and click "Run".

2. "Search" the GBINV database for the "Reclinomonas americana NADH dehydrogenase" gene.
Once again you are faced with the problem that the NADH dehydrogenase (NADH deH) gene sequence is not available as an individual sequence. As before, the sequence has to be isolated from the entire R. americana mitochondrial genome and be re-entered into the Workbench using the "Add New Protein Sequence" tool.

3. "Show Record(s)" for the "Reclinomonas americana mitochondrial DNA, complete genome" sequence (GBINV: 2258325).

4. From the Edit menu of your browser, select "Find" and search for "NADH dehydrogenase" or simply scroll down the Records window until you locate the sequence.

5. Once again several subunits are present. Find and "copy" (by highlighting the sequence and selecting Copy from the Edit menu of the browser) the amino acid sequence of subunit 8 (do not include quotation marks).

6. "Return" to the Protein Tools homepage (the Return button is at the bottom of the window), "Run" the "Add New Protein Sequence" tool.

7. Paste (Edit, Paste) the sequence into the appropriate space and label it "Reclinomonas americana NADH dehydrogenase, subunit 8". "Save" the sequence.
You should now be back at the Protein Tools homepage, with three sequences in the storage area.



8. The mutant sequence (the one on the bottom) has ONLY beta sheets (shown in blue letters). The mutant sequence contains NO alpha helices.



9. Select the "SwissProt" database and Submit your search



Once again, you should see several prokaryotic sequences amongst the closest matches (the six with check-marks next to them in the figure above). The first four (RHOCA or Rhodobacter capsulatus, RICCN or Rickettsia conorii, RICPR or Rickettsia prowazekii and PARDE or Paracoccus denitrificans) are all alpha-Proteobacteria. Once again, this suggests that the alpha-Proteobacteria are probably the evolutionary forerunners of mitochondria. The other two bacterial sequences are of a different class. It is interesting to note that there is a significant drop off in the E-Value, immediately after the alpha-Proteobacteria sequences (from a range of e-66 to the e-27 range ¡V a significant difference). Now, we will see just how similar the R. americana NADH deH subunit 8 sequence is to an alpha-proteobacterial sequence.

10. "Import" the selected sequences and "Return" to the Protein Tools homepage. Select the first alpha-proteobacterial sequence which is Rhodobacter capsulatus (SWISSPROT:NUOI_RHOCA) by checking the box. Also check the R. americana NADH deH subunit 8 sequence and select the CLUSTALW tool. Click "Run". Click "Submit" on the next screen and wait for your results.



As you will see, there is extensive homology between the R. americana mitochondrial sequence and that of the alpha-proteobacterium R. capsulatus. You can try aligning the sequences from one of the other classes of bacteria to see the drop in homology. For example, the alignment below is that of R. americana with the Thermus aquaticus (THETH) bacterial sequence. While homology is still evident, it is nowhere near as impressive as the homology seen with the alpha-proteobacterial sequence (shown above).



Our analysis of the succinate deH and NADH deH genes, suggests that R. americana, a very simple eukaryote, has mitochondrial genes that are highly homologous with those of alpha-Proteobacteria, the presumed evolutionary predecessors of mitochondria. We will demonstrate this relationship further by constructing a phylogenetic tree in "Part VI" of this tutorial.

You can test other R. americana mitochondrial genes by repeating the steps outlined in this section. Some genes that yield interesting results are: ribosomal protein S12, succinate ubiquinone oxidoreductase subunit 2, elongation factor TU, apocytochrome c, and ATP synthase F1 subunit alpha. When blasting these and others, notice how many times the Rickettsia species (RICCN or RICPR) or other alpha-Proteobacteria appear in the top ten matches.