1. Cloning and Bioinformatics Analyses of Novel Plant GAPDH Genes
Valeria Rivera
Dr. Michael Rubin
RISE Program, Department of Biology,
University of Puerto Rico at Cayey
ABSTRACT
The Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene codes for a
protein that catalyzes a reaction of an important pathway known as glycolysis, which is
involved in energy production in carbohydrate metabolism. The protein encoded by this
gene is essential in cellular metabolism and may play a role in disease as it is aberrantly
expressed in certain types of cancers and is also associated with neuronal disorders. We
are interested in determining whether GAPDH protein sequences derived from various
tropical plants have conserved amino acids related to the active site involved in catalytic
function. Previously, genomic DNA was isolated from two tropical plants: Oxalis
corniculata, and Plectranthus amboinicus. Specific nested primers were used to PCR
amplify GAPDH genes from these plants. Amplified PCR products were purified,
exonuclease treated, ligated into the pJET cloning vector, and transformed into competent
E. coli. Plasmid DNA was purified from transformants followed by digestion with the
restriction endonuclease BglII and visualized using gel electrophoresis. The GAPDH
PCR product and cloned insert comigrated on the agarose gel. The GAPDH amplified
PCR product was 1 Kb in size in O. corniculata and 1.5 Kb in size in P. amboinicus.
This semester, we cultivated the transformed bacterial strains, purified plasmid DNA
using the midi prep alkaline lysis procedure, and determined the DNA concentrations
using a spectrophotometer. These plasmid purifications were verified again by digestion
with the restriction endonuclease BglII and visualized using gel electrophoresis. Aliquots
of purified plasmids and sequencing primers were sent to a national center for DNA
sequence determination. We will continue these experiments with bioinformatics
analyses of the GAPDH genes from O. corniculata, and P. amboinicus. The
conservation of amino acids important for protein structure and function will help us to
better understand the evolution of the GAPDH gene. We thank the Biology Department
and the RISE Program (GM59429-13) at UPR-Cayey.
Purpose of the study sequences that will be published in
GenBank at the end of the semester.
This will be an ongoing semester
research intended to do bioinformatics Background and Significance
analyses of cloned GAPDH genes from
various plants to study differences in the GAPDH, Glyceraldehyde 3-
amino acids that compose the protein the phosphate dehydrogenase is a gene that
gene is expressing. This project will result codes for a protein that catalyzes a step of
in the production of two unique DNA an important pathway known as glycolysis,

2. which is involved in energy production in chloroplast that contains the pigment
carbohydrate metabolism. For example, the chlorophyll. Then, ATP is obtained from
human genome has an orthologous GAPDH glycolysis that is the oxidation of glucose to
gene. The gene is highly expressed in pyruvate. Glycolysis begins by adding two
certain types of cancers and is associated molecules of ATP to glucose making it
with neuronal diseases such as Alzheimer’s. fructose 1,6 biphosphate. Finally, it is split
(Sirover, 1999; Altenberg and Greulich, to glyceraldehyde 3 phosphates that are then
2004; Kim and Dang, 2005 in Lau J, and oxidized to pyruvic acid yielding ATP and
Robinson D, 2009). Connections between NADH. With the liberated ATP the cycle
the molecular aspects of GAPDH can be proceeds to the Krebs cycle to produce
made with the biomedical and evolutionary more ATP, water, and oxygen.
significance. Researchers think the enzyme
GAPDH takes part in DNA replication and GAPDH has a large active site
repair, cytoskeletal organization, and cavity to accommodate G3P and NAD+(D.
phosphotransferase activity (Tatton et al., Allan Butterfield et al 2010). Thus, we
2000 in Lau J, and Robinson D, 2009). It think that there will be highly conserved
has also been used to study phylogenetic amino acids among plant species. The
relationships among taxa. problem we are addressing is, “Do GAPDH
proteins have conserved amino acids related
The significance of this research to the active site involved in catalytic
includes the gain of new knowledge through activity and other conserved amino acids
the study of important genes involved in related to protein interactions?” We
energy production in plants. By comparing hypothesize that GAPDH proteins will have
GAPDH genes from the selected plants: conserved amino acids comprising
Oxalis corniculata and Plectranthus functionally important regions such as the
amboinicus. Focusing on conserved amino active site with the following amino acids:
acids in specific regions that are 148, 149, 150, 176, 209, and 231. Other
hypothesized to be important for the involved in the binding of GAP substrate
structure and function of the GAPDH with amino acids 179, 181, and 231, NAD+
protein. This will be carried out through with amino acids 8, 10, 11, 32, 96, and 313,
bioinformatics analyses of the sequenced and phosphate binding held by amino acids
GAPDH PCR products from O.corniculata 148, 150, and 208. Also, we hypothesize
and P.amboinicus genomic DNAs. The that GAPDH proteins will have different
sequences will be published in GenBank to amino acids at some other positions not
contribute to the GAPDH database in a related to protein function (BIO RAD,
collaborative effort. Cloning and Sequencing Explorer Series).
Energy is the ability to do work; it
is present in everything we do on a daily Preliminary Studies
basis. Plants use light energy, carbon
dioxide, and water to make sugar energy This is part of an ongoing research
thorough photosynthesis releasing oxygen. project, which began in 2009 by studying
The production of energy in plants follows a the GAPDH gene on Oxalis corniculata,
specific pathway. First, energy from the Plectranthus amboinicus, and Myrtaceae
sun is converted to adenosine triphosphate psidium. The techniques used in this
(ATP) a usable chemical energy in the research involved DNA isolation,

3. Polymerase Chain Reaction (PCR), gel
electrophoresis, and cloning and bacterial
culture. They were performed in the
following sequence: DNA isolation, PCR,
cloning and bacteria culture, mini-preps,
and digestion with enzyme BGlII. The
results of the electrophoresis are visible in
figure 1. They are mentioned here because
their results helped refocus this study by
eliminating the plant that was not cloned.
In figure 1 the agarose gel shows
first the ladders or markers, then the
negative controls followed by the positive
controls. In the upper area of the gel, are the Research Design and Method
cloning vectors that co-migrate. We have
first the negative control that is jus the -Preliminary studies methods
plasmid alone, followed by the positive
control that is Arabidopsis one of the plants Lysis buffer is used for DNA
that is genome is fully sequenced. Then, isolation of the plant species Oxalis
there are the three groups of plants with corniculata, Plectranthus amoboinicus, and
three samples each. The first DNA sample Myrtaceae psidium. Then PCR primers
in the gel is M. psidum, guava. It showed no were designed to amplify GAPDH in the
cloning since not enough DNA sample was three tropical plants.
extracted nor amplified due to the absence
of PCR product band. For O. corniculata, The primers anneal to the region of
only two of the three samples were cloned the target gene. Next, the PCR product is
being the same size as the PCR product ligated into plasmid cloning vector pJET to
band whereas for P. amboinicus all three transform ligation into competent E. coli.
were successfully cloned. Due to these After transformation into E. coli the bacteria
results M. psidum, guava was eliminated for has to be cultured before purifying the
further study. plasmid DNA with Restriction
Endonucleases to Identify Cloned PCR
Figure 1 Mini-prep cloning gel results of Products in a 1% agarose gel. The
M.psidum, O.corniculata, and restriction enzyme used is BGlII.
P.amboinicus.
Last semester, fall 2011, I continued
the project by using the previously prepared
samples for the midi-prep, large-scale
plasmid purification. By first growing and
culturing E.coli bacteria with cloned DNA.
In second came the inoculation of bacteria
resistant to AMP to then extract the DNA
from the cultured bacteria. After that, we
measured the Optical Densiry of each
sample to check if the DNA concentration

4. was enough to perform the digest. In order
to confirm that the DNA was cloned we did  DNA extraction, amplification, was
a digest with enzyme BGLII (2ul/sample) a success based on the PCR product
and was analyzed on a 1% agarose gel for plants O.corniculata, and
through gel electrophoresis. Gel P.amboinicus.
electrophoresis allows the analysis of the  Amplified PCR products were
digestion results. Once verified the region ligated to the pJET plasmid vector
where the GAPDH gene is located, samples and transformed into E. coli.
were prepared for sequencing reactions,  Midi-Prep plasmid DNAs were
using Sanger Sequencing and sent to the purified and quantified for cloned
facilities of Yale University. The sequences GAPDH PCR products from O.
were returned and roughly analyzed using corniculata and P. amboinicus
Cap3, VecScreen, and BLAST. This genomic DNAs.
resulted in both plant sequences being  DNA samples were digested with
GAPC for O.corniculata and GAPDH for the restriction endonuclease BglII
P.amboinicus. and visualized using agarose gel
electrophoresis to confirm that the
Curent Study Methods DNAs were cloned. The size for
O.corniculata 1kb and for
This semester we will study and P.amboinicus 1.5 kb.
analyze the sequences for plants  Sequences were determined for
O.corniculata, and P.amboinicus genomic cloned GAPDH PCR products from
DNAs using bioinformatics tools such as O.corniculata and P. amboinicus.
Cap3, VecScreen, and BLAST. This will be  Preliminary Bioinformatics analyses
done following the protocol for the NCBI revealed the cloned genes to be:
site database search found in The Complete GAPC (O. corniculata) and GAPDH
Cloning and Sequencing Explorer Series (P. amboinicus)
Curriculum by BIO-RAD. First, do a Blast
search of the Cap sequence for both plant
sequences. In second, comes a contig Cited literature
sequence using Cap 3. After the contig,
eliminate the cloning vectors from the 1) Lau J &Robinson D. (2009).
sequence and do a blast search once again. Effectiveness of a Cloning and Sequencing
Compare the sequencing alignments and Exercise on Student Learning with
determine intron and exon boundaries. Subsequent Publication in the National
When the whole analysis is complete and Center for Biotechnology Information
verified, the sequences will be submitted to GenBank. CBE—Life Sciences Education
the DNA database in GenBank. Vol. 8, 326–337, Winter 2009.
Furthermore, we can do phylogenetic
analysis of plant GAPDH genes and do a 2) Cloning and Sequencing Explorer Series
structural modeling using novel plant from BIO RAD. Catalog #166-5000EDU,
GAPDH protein sequences. explorer.bio-rad.com
3) Butterfield Allan D. et al (2010)
Oxidatively Modified Glyceraldehyde 3-
Previous Results phosphate Dehydrogenase (GAPDH) and

5. Alzheimer’s Disease: Many Pathways to
Neuron Degeneration.
Acknowledgements
RISE Program
Dr. Michael Rubin
Melissa Medina
Dr. Elena Gonzalez
Yadira Ortiz