PCR Amplification, Cloning, Sequence Determination, and Bioinformatics Analyses of Novel Plant GAPDH Genes from Cyperus alternifolius, Schefflera actinophylla and Tropical Flora Endemic to Puerto Rico<br />Lydia E. Cortes, Dr. Michael Rubin. University of Puerto Rico at Cayey<br />Biographical Sketch (CV)<br />Lydia E. Cortes<br />Education and Honors: <br />University of Puerto Rico at Cayey<br />Bachelor degree in Natural Sciences, Concentration in Biology<br />Actual GPA 3.90<br />Professional Experience: <br />Investigator in training <br />RISE Program, University of Puerto Rico at Cayey<br />Exchange student at UMASS Boston <br />Courses about Caribbean history and culture. <br />Certification on PCR <br />University of North Carolina at Chapel Hill <br />Summer Research<br />Leadership Alliance<br />Miller School of Medicine, University of Miami<br />Research Plan<br />Specific Aims<br />The investigation has four principal aims:<br />Transform and clone Cyperus alternifolius and Schefflera actinophylla GAPDH genes into E. Coli JM109<br />Purify and clone Cyperus alternifolius and Schefflera actinophylla GAPDH genes<br />Sequence determination of clones Cyperus alternifolius and Schefflera actinophylla GAPDH genes<br />Bioinformatics analysis of Cyperus alternifolius and Schefflera actinophylla GAPDH genes<br />Background and Significance<br /> GAPDH, or glyceraldehyde-3-phosphate dehydrogenase, is a protein coding gene. The product of this gene catalyzes an energy-yielding step in carbohydrate metabolism, catalyzing the sixth step of glycolysis and thus serving to break down glucose for energy and carbon molecules.In addition, GAPDH has recently been implicated with transcription activation, initiation of apoptosis, and Endoplasmic Reticulum to Golgi vesicle shuttling.The enzyme of GAPDH exists as a tetramer of identical chains, each subunit having an active site. The reaction catalyzed by GAPDH is:<br />Glyceraldehyde-3-phosphate+ NAD+ + Pi —>1,3-bisphosphoglycerate+ NADH +H+<br />The GAPDH gene has been found and sequenced in various organisms such as humans and Arabidopsis Thaliana; but other organisms have not been sequenced for this gene. The purpose of this investigation is to sequence the GAPDH gene from various plants endemic to Puerto Rico. The hypothesis is that the two plants studied in this investigation will have some segments with a similar sequence, but other sequences will be different.<br />GAPDH is one of many genes called housekeeping genes. These genes are really important for the scientists since they code for proteins that are expressed at a relatively constant rate.Housekeeping genes provides a reference against which to compare a protein (or RNA) of interest. Lately, research has proven that the proteins coded from GAPDH are not expressed constantly, so some scientists do not consider GAPDH as a housekeeping gene anymore.<br /> The role of GAPDH in cell death or apoptosis is the principal factor studied from this gene. According to past research, GAPDH appears to contribute to cell death triggered by a nitric oxidecascade; this is by functioning in the nucleus to stimulate the acetyltransferase activity of p300/CBP, leading to the activation of p53 and proapoptotic gene expression. Burke et al.(1996) postulated that the diseases characterized by the presence of an expanded CAG repeat may share a common metabolic pathogenesis involving GAPDH as a functional component. Observations made by Myers et al. (2002), Li et al. (2004), and other scientist raised the possibilities that the GAPDH genes are Alzheimer disease risk factors, a hypothesis that is consistent with the role of GAPDH in neuronal apoptosis. GAPDH gene has been related with other diseases such as Cancer and Huntington disease.<br /> Sequencing and studies of the GAPDH gene have been developing for a long time. Li et al. (2004) located a GAPDHpseudogene on chromosome 12q of the human. Arabidopsis thaliana is a plant studied extensively and used as the control for various research. A. thaliana’s genome has been sequence completely, including the GAPDH gene. The sequence of A. thaliana’s GAPDH gene is presented in figure 1.A. thaliana has eight GAPDH genes, some of them are: GAPC (Cytosolic), GAPCP ( Plastid), GAPA (Chloroplast), GAPB (Chloroplast) and GAPN (Cytosolic, nonphosphorylating). In table 1 is seen the reaction that catalyzes each enzyme.Even when a lot of information is available about the GAPDH gene of various organisms, several other organisms have not been sequenced for the gene. For example, most of the plants native from Puerto Rico have not been sequenced for the GAPDH gene.<br /> Cyperus alternifolius and Schefflera actinophylla are the two species of plants studied in this investigation. Cyperus alternifolius (umbrella papyrus or umbrella palm) is a grass-like plant in the very large genus Cyperus of the sedge family, Cyperaceae. The sequences for eight genes of Cyperus alternifolius are present in GenBank, but none of them is the GAPDH gene. Schefflera actinophylla (Brassaia actinophylla) is a tree in the Araliaceae family, and only five genes of this plant have been sequenced. The purpose of this investigation is to sequence the GAPDH gene from the plants Cyperus alternifolius and Schefflera actinophylla.<br /> With a wider knowledge about GAPDH variants, it will be possible to study and learn more about the gene. It could be possible to find a way for the GAPDH to still be functional as a housekeeping gene. By sequencing the gene it will be possible too to study its sequence and mutations, information that could be used in future studies.<br />Research Design and Methods<br />Transform and cloneCyperus alternifolius andSchefflera actinophylla GAPDH genes into E. Coli JM109<br /> For transformation, we pipette 5 microliters of DNA from Cyperus alternifolius and Schefflera actinophylla in a microcentrifuge tube. Then added 50 microliters of JM 109 E. Coli competent bacteria (prepared before) to the tubes. Incubate for 30 minutes in ice. Heated each microcentrifuge tube for 45 seconds at 37 °C. Incubated in ice for two minutes. Added 950 microliters of SOC media to each tube. Incubated for 45 minutes at 37°C, while shaking. Plated 5 microliters of cells in LB Amp agar plates. Took the rest 900 microliters, centrifuged and took out the supernatant. Pipette 50 microliters of media and resuspended. Plated the cells. Incubate at 37 °C overnight. <br /> Before the following step, transformed bacterial colonies will need to be grown in liquid culture minipreps. First, we prepared 25 ml of LB Amp broth. Using sterile technique, pipette 18 ml of LB Amp broth into one culture tube. Used a sterile pipette tip to pick a single colony from the LB Amp IPTG plate containing the plated bacteria transformed with the plant gene ligation reaction. Placed the miniprep cultures to grow overnight at 37°C in a shaking incubator. Prepared a 1% agarose gel and electrophoresis running buffer to analyze the plasmid miniprep restriction enzyme digestion. Counted the number of bacterial colonies that grew on the LB Amp IPTG agar plates.<br />Purify and clone Cyperus alternifolius and Schefflera actinophyllaGAPDH genes<br /> Added 100 ml of 95–100% ethanol to the Aurum wash solution and mix well. Transfered 1.5 ml of each miniprep culture into one of the appropriately labeled microcentrifuge tubes by pipetting or decanting. Centrifuged the microcentrifuge tubes for 1 minute at top speed (>12,000 x g) to pellet the bacteria. Located the bacterial pellet and removed the supernatant from each tube using a 1,000 µl pipet or a vacuum source, avoiding the pellet. Resuspended the bacterial pellet in each tube in 250 µl of resuspension solution by pipetting up and down or vortexing. Pipette 250 µl of lysis solution into each tube andl mixed by gently inverting 6–8 times. Within 5 minutes of adding lysis solution, pipette 350 µl of neutralization solution into each tube and mixed by gently inverting 6–8 times. Centrifuged the tubes for 5 minutes at top speed in the microcentrifuge. Decanted or pipette supernatant from the centrifuged tubes onto the appropriately labeled column. Centrifuged the columns in the microcentrifuge for 1minute at top speed. Discarded the flow-though from the collection tube and replaced the column in the collection tube. Pipette 750 µl of wash solution onto each column. Centrifuged columns in the capless collection tubes in the microcentrifuge for 1 minute at top speed. Discarded the flow-though from the collection tube. Replaced columns into collection tubes and centrifuged for an additional 1 minute to dry out the column. Transfered each column to the appropriately labeled capped "