1. Cloning and Bioinformatics Analyses of Novel Plant GAPDH Genes
Valeria Rivera and Dr. Michael Rubin
RISE Program, Department of Biology,
University of Puerto Rico at Cayey
ABSTRACT
GAPDH stands for Glyceraldehyde 3-phosphate dehydrogenase. This gene
codes for a protein that catalyzes a step of an important pathway known as
glycolysis. And which is involved in energy production in carbohydrate metabolism.
It is important to learn more about this gene responsible for energy production.
One reason is because the gene is expressed in 21 types of cancers sand associated
with neuronal diseases like Alzheimer’s. The problem to address is, “Do GAPDH
proteins have conserved amino acids related to the active site involved in catalytic
function? Previously, the lab team performed a series of techniques and found that
two of the three plants studied (O.corniculata, and P.amboinicus) were successfully
cloned. The new stage of this ongoing research will start with midi-preps followed
by BGLII digestion to then do a bioinformatics analysis of O.corniculata and
P.amboinicus.
Purpose of the study genome has an orthologous GAPDH gene.
It is important to learn more about this
This will be an ongoing semester gene responsible for energy production.
research intended to clone GAPDH genes The gene is expressed in 21 types of
from various plants to study differences cancers sand associated with neuronal
in the amino acids that compose the diseases (Sirover, 1999; Altenberg and Greulich,
protein the gene is expressing. 2004; Kim and Dang, 2005 in Lau J, and Robinson D,
Bioinformatics tools will be used to Researchers think the enzyme
2009).
analyze gene sequences. This project will GAPDH takes part in DNA replication and
result in the production of two unique repair, cytoskeletal organization, and
DNA sequences that could be published phosphotransferase activity (Tatton et al.,
2000 in Lau J. 2009).
in GenBank.
Background and Significance The significance of this current
research includes the gain of new
knowledge through the study of important
GAPDH stands for Glyceraldehyde
genes involved in energy production in
3-phosphate dehydrogenase. This gene
plants. Also, compare GAPDH genes from
codes for a protein that catalyzes a step
the selected plants. Analyze unique DNA
of an important pathway known as
sequences that could be published in
glycolysis. And which is involved in
GenBank and introduce important
energy production in carbohydrate
bioinformatics techniques in an educational
metabolism. For example, human setting.

2. Energy is the ability to do work; it Preliminary Studies
is present in everything we do on a daily
basis. Plants use light energy, carbon This is part of an ongoing
dioxide, and water to make sugar energy research project, which began in 2009 by
thorough photosynthesis releasing oxygen. studying the GAPDH gene on Oxalis
The production of energy in plants follows a corniculata, Plectranthus amboinicus, and
specific pathway. First, energy from the Myrtaceae psidium. The techniques used
sun is converted to adenosine triphosphate in this research involved DNA isolation,
(ATP) a usable chemical energy in the Polymerase Chain Reaction (PCR), gel
chloroplast that contains the pigment electrophoresis, and cloning and
chlorophyll. Then, ATP is obtained from bacterial culture. They were performed
glycolysis that is the oxidation of glucose to in the following sequence: PCR, cloning
pyruvate. Glycolysis begins by adding two and bacteria culture, mini-preps, and
molecules of ATP to glucose making it digestion with enzyme BGlII. The results
fructose 1,6 biphosphate. Finally, it is split of the electrophoresis are visible in figure
to glyceraldehyde 3 phosphates that are then 1. They are mentioned here because
oxidized to pyruvic acid yielding ATP and their results helped refocus this study by
NADH. With the liberated ATP the cycle eliminating the plant that was not cloned.
begins again.
In figure 1 the agarose gel shows
Throughout this investigation, we first the ladders or markers, then the
will compare GAPDH genes from various negative controls followed by the positive
plants to study their similarities and controls. In the upper area of the gel, are
differences. The problem to address is, the cloning vectors that co-migrate.
“Do GAPDH proteins have conserved Then, there are the three groups of
amino acids related to the active site plants with three samples each. The first
involved in catalytic activity and other DNA sample in the gel is M. psidum,
conserved amino acids related to enzymatic guava. It showed no cloning since not
function?” We hypothesize that GAPDH enough DNA sample was extracted nor
proteins will have conserved amino acids amplified due to the absence of PCR
148, 149, 150, 176, 209, and 231 related to
product band. From O. corniculata, only
the active site involved in catalytic function.
two of the three samples were cloned
Other conserved amino acids include the
whereas in the P. amboinicus samples all
binding of GAP substrate (amino acids 179,
three were successfully cloned. Due to
181, and 231), NAD+ (amino acids 8, 10,
these results M. psidum, guava was
11, 32, 96, and 313), and phosphate binding
eliminated for further study.
(held by amino acids 148, 150, and 208).
And that GAPDH proteins will have
different amino acids at other positions not Figure 1 Mini-prep cloning gel results of
M.psidum, O.corniculata, and P.amboinicus.
related to the active site important for
protein function (BIO RAD, Cloning and
Sequencing Explorer Series).

3. agarose gel through gel electrophoresis
to verify the cloned DNA. Later comes
the measurement of the OD of each
sample and digestion. Gel
electrophoresis will allow the analysis of
the digestion results. Once verified the
region where the GAPDH gene is located,
samples will be prepared for sequencing
reactions, using Sanger Sequencing in the
facilities of Yale University. When the
results are available, the sequence will be
studied performing bioinformatics
analyses using Cap3, VecScreen, and
BLAST.
Research Design and Method
Results
-Preliminary studies methods -Preliminary studies results
Lysis buffer is used for DNA DNA extraction, amplification, was
isolation of the plant species Oxalis a success based on the PCR product for
corniculata, Plectranthus amboinicus, and plants O.corniculata, and P.amboinicus.
Myrtaceae psidium. Then PCR primers Cloning occurred for both plants except
were designed to amplify GAPDH in the for one of the three samples per plant M.
three tropical plants. psidium.
The primers anneal to the region
of the target gene. Next, the PCR product Cited literature
is ligated into plasmid cloning vector
pJET to transform ligation into Lau J &Robinson D. (2009).
competent E. coli. After transformation Effectiveness of a Cloning and
into E. coli the bacteria has to be cultured Sequencing Exercise on Student
before purifying the plasmid DNA with Learning with Subsequent Publication in
Restriction Endonucleases to Identify the National Center for Biotechnology
Cloned PCR Products in a 1% agarose gel. Information GenBank. CBE—Life Sciences
The restriction enzyme used is BGlII. Education Vol. 8, 326–337, Winter 2009.
-Current study methods Cloning and Sequencing Explorer Series
from BIO RAD. Catalog #166-5000EDU,
The ongoing study will begin by explorer.bio-rad.com
using the previously prepared samples
for the midi-prep, large-scale plasmid Acknowledgements
purification. By first growing and
culturing E.coli bacteria with cloned DNA. RISE Program
In second, inoculate bacteria resistant to Dr. Michael Rubin
AMP to then extract the DNA from the Melissa Medina
cultured bacteria. Then digest with Dr. Elena Gonzalez
enzyme BGLII (2ul/sample) on a 1%