This web page was created as an assignment for an undergraduate course at Davidson College.
Orthologs for IDH2
IDH2 is the gene in yeast that encodes the IDH2 subunit of the NAD(+)-dependent isocitrate dehydrogenase. This protein complex is found in many organisms and can thus be compared across species lines. Orthologs are genes in different species that evolved from a common ancestral gene by speciation and retained the same function in the course of evolution (Chen et al., 2000). In order to find orthologs of a specific sequence, I used the Protein BLAST program sponsored by NCBI.
How does BLAST work?
BLAST, the Basic Local Alignment Search Tool, is designed to search all sequence databases. In this case, I specifically used the program that searches only protein sequence databases. For more information on the algorithm that BLAST uses or for other information, click here. Once I get to the pBLAST page, I must insert the sequence I wish to compare. I already knew the sequence of the translation product of the Saccharomyces cerevisiae IDH2 gene. By copying and pasting this sequence into the search field, the program does the rest of the work. The full report of my query can be found here.
Below is a list of some orthologs for the IDH2 gene. Their coding sequences and amino acid sequences can be seen by clicking on each link.
Kluyveromyces lactis is a fungus similar to Saccharomyces cerevisiae. The IDH2 protein that composes the NAD(+)-dependent isocitrate dehydrogenase subunit 2 in this species. The IDH2 gene in this case seems to encode a protein similar in function to the yeast IDH2 gene.
Mus musculus This ortholog is the protein product of the mouse gene ICD3A that encodes the NAD(+)-dependent isocitrate dehydrogenase subunit 3A.
Homo sapiens This human ortholog is the protein product of a gene that encodes the alpha subunit of the NAD(+)-dependent isocitrate dehydrogenase.
Bos taurus This is a cow ortholog, producing a protein similar to that produced by both humans and mice.
E. coli The ortholog that closely resembles the yeast IDH2 protein is actually a protein encoded by the E. coli gene ICDA. This gene encodes an NADP(+)-dependent isocitrate dehydrogenase subunit A-- a protein product with a different function. *Chen et al. published a paper discussing orthologs of specific NAD(+) and NADP(+) genes and how it is likely that these genes descend from the same ancestor, even though now they encode proteins with separate functions. With E. coli, it is easy to believe that this may be the case because one of its sequences that closely resembles the yeast IDH2 sequence, produces a protein with a different function.
In my molecular biology lab, I am cloning IDH2 into an E. coli expression vector (see Experimental Design ). It is important to have this sequence in a "forward" orientation so that the bacterial machinery can translate the sequence correctly. Because the IDH2 gene sequence from yeast and the ICDA gene sequence from the bacteria are so similar (note that these sequences are similar because the protein sequences are similar, as illustrated above), the cells may have trouble distinguishing between the two, leading to very little expression of the plasmid. This then could have been a problem throughout the experiment. Luckily, however, my lab partner and I were able to clone at least one IDH2 gene in the correct forward orientation in the plasmid. Our expression experiments can thus commence.
© Copyright 2002 Department of Biology, Davidson College, Davidson NC 28035
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