*This page is part of
undergraduate assignment for Molecular Biology at Davidson College
BIO 306: Molecular Biology Course Webpage
By Elizabeth Shafer
Molecular Biology is a 300
level biology course taught at Davidson College, by the illustrious Dr. A.
Macolm Campbell. This course focuses on techniques used in Molecular Biology in
conjunction with providing information on cellular processes. This webpage was
designed to outline lab work I completed during this course on cloning
isocitrate dehydrogenase and to link to a paper I wrote reviewing an article in
nature. The following links will take
you to either project.
NEW: Review
of “A Genomic Regulatory Network for Development” by Davidson et.al.
Cloning Isocitrate Dehydrogenase
Cloning
5 Isoforms of Isocitrate Dehydrogenase
Introduction to Isocitrate Dehydrogenase
As part of my Molecular Biology course this spring, my class is attempting to clone and express the genes that encode the 5 isoforms of the Saccharomyces’ gene isocitrate dehydrogenase: IDH1, IDH2, IDP1, IDP2, and IDP3.
Isocitrate
dehydrogenases are enzymes that oxidize isocitrate and are involved in
glutamate biosynthesis, isocitrate biosynthesis, and the tricarboxylic acid
cycle.
Cloning and Sequence Information about Isocitrate
Dehydrogenase
The
5 isoforms we are interested in differ in DNA sequence, amino acid sequence,
restriction fragment map, the primers they require, the fusion protein they
produce with the plasmid, and the coenzyme that they require to catalyze
reactions.
Each
isoform is described in the following links:
In
our experiment we will isolate each gene, amplify the gene with PCR, and clone
it in to a plasmid pQE
–30UA for expression. To insure that each insert is in the correct
orientation within the plasmid, we will digest the plasmid with a restriction
enzyme that is known to cut at a certain location with in the insert. If the
insert is in the correct orientation then a Southern blot will reveal the
correct size band, if not the Southern blot will yield a band of a different
size. Because each gene varies in sequence and the restriction enzymes that cut
them, we will use a specific enzyme for each gene will expect different size
bands to result for each gene insert. Digestion
Experiment Design.
Resulting Orientation of Inserts of IDP2
Jennifer Madden and I cloned IDP2 into the pQE-30UA plasmid. Of the eight samples we checked only one had an insert and that insert was in the reverse orientation. Because the insert is in the reverse orientation the protein will not be transcribed. We were not the only group that were unable to get inserts in the forward position. Of the five isoforms we have only IDH1, IDH2, and IDP1 in the correct orientation. Of lab groups that have an insert in the forward orientation there was a low ratio of inserts in the forward position to those found in the reverse or without an insert.
This phenomena could be attributed to a number of variables in the
process of isolating, amplifing, and cloning the gene as well as to the
biological function of the gene itself.
Protein and Ortholog Information
Plasmids
which have the insert in the correct position will express the protein encoded
by the insert. The structure of the isocitrate dehyrdogenase proteins provide
insight into its biochemical role within the cell. Orthologs are genes with a similar sequence in differ species.
Information on orthologs reveal how conserved a protein is across species,
thereby suggesting the protein’s universal importance in the cell’s
functioning.
Check
out this links to see more about the protein structure and orthologs of
isocitrate dehydrogenase.
Created by: Elizabeth Shafer. Email questions to lishafer@davidson.edu