Structure and Orthologs

"This web page was produced as an assignment for an undergraduate corse at Davidson College."

This webpage is an extension of the previous webpage I made discussing my Molecular Biology's class project this semester. Click on the following link to learn more about the first part of the project to clone the five yeast IDH genes using PCR and to verify the orientation of the inserts. My partner and I were assigned the IDH2 gene and over the past few weeks we performed experiments to verify that we had inserts for the IDH2 gene in the pQE-30 UA Vector. As can be determined from gel electrophoresis following restriction enzyme digestion, I found that I had inserts in two colonies out of four. Then we performed another restriction enzyme digestion to test the orientation and I had one colony in the forward orientation and one in the reverse orientation. We are currently working on a Southern blot to verify that our PCR products are really the genes that we think they are. We used our PCR product as a probe and are hybridizing the blots currently. Click here to see our laboratory outline.

This page contains Chime Images for two forms of IDH, one that binds to NAD+ and one that binds to NADP+. The cDNA and amino acid sequences for 5 orthologs of IDH are also below as is more information about the role of IDH in the cell. Isocitrate Dehydrogenase is a protein that catalyzes a reaction in the Krebs Cycle and my gene, IDH2, has NAD+ as a cofactor. IDH2 is the gene in yeast that encodes the IDH2 catalytic subunit of the NAD(+)-dependent isocitrate dehydrogenase (Chen et al, 2000).

Click here to read a GREAT summary of the role of IDH with NAD+ vs. IDH with NADP+!!

Chime Images

For instructions on viewing Rasmol images go to the following webpage or here.

If you don't have Rasmol, click here.

Chime 1:

Fig. 1: Isocitrate Dehydrogenase from E. coli Complexed With NADP+

This figure was taken from (http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&db=t&Dopt=s&uid=3431). Go to this webpage for more information and options.

Figure 1 is a single chain protein image of Isocitrate Dehydrogenase complexed with NADP+ from Escherichia coli. Click on the Rasmol figure to change the chain colors and for different model types or go to the NCBI structure homepage.

Hold down the mouse button or right click to bring up the menu option. Click on Color, then chain to see the IDH group with the NADP+ complex.

Chime 2:

Fig. 2: Bacillus Stearothermophilus Glycerol Dehydrogenase Complex With NAD+

This figure was taken from (http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&db=t&Dopt=s&uid=17260). Go to this webpage for more information and options.

Figure 2 is an A chain protein image of Glycerol Dehydrogenase complexed with NAD+ from Bacillus Stearothermophilus. Click on the Rasmol figure to change the chain colors and for different model types. No isocitrate dehydrogenase proteins that complex with NAD+ have been isolated. Therefore, I used Glycerol Dehydrogenase which is in the same family of enzymes as isocitrate dehydrogenase. Click on the following link to view pages with information on Glycerol Dehydrogenase or try here.

Hold down the mouse button or right click to bring up the menu option. Click on Color, then chain to see the glycerol dehydrogenase complex with the NAD+ complex.

cDNA and amino acid sequences for 5 Orthologs of IDH Genes

Orthologs are genes in different species that are similar in sequence and function to another gene (Chen et al, 2000).

The above sequences were obtained from using a BLAST sequence similarity search (http://www.ncbi.nlm.nih.gov/BLAST ). Click here for information on using this search.

IDH Gene Homepage