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Proposal march 2012

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  • 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 semesterresearch intended to do bioinformatics Background and Significanceanalyses of cloned GAPDH genes fromvarious plants to study differences in the GAPDH, Glyceraldehyde 3-amino acids that compose the protein the phosphate dehydrogenase is a gene thatgene is expressing. This project will result codes for a protein that catalyzes a step ofin the production of two unique DNA an important pathway known as glycolysis,
  • 2. which is involved in energy production in chloroplast that contains the pigmentcarbohydrate metabolism. For example, the chlorophyll. Then, ATP is obtained fromhuman genome has an orthologous GAPDH glycolysis that is the oxidation of glucose togene. The gene is highly expressed in pyruvate. Glycolysis begins by adding twocertain types of cancers and is associated molecules of ATP to glucose making itwith 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 then2004; Kim and Dang, 2005 in Lau J, and oxidized to pyruvic acid yielding ATP andRobinson D, 2009). Connections between NADH. With the liberated ATP the cyclethe molecular aspects of GAPDH can be proceeds to the Krebs cycle to producemade with the biomedical and evolutionary more ATP, water, and oxygen.significance. Researchers think the enzymeGAPDH takes part in DNA replication and GAPDH has a large active siterepair, cytoskeletal organization, and cavity to accommodate G3P and NAD+(D.phosphotransferase activity (Tatton et al., Allan Butterfield et al 2010). Thus, we2000 in Lau J, and Robinson D, 2009). It think that there will be highly conservedhas also been used to study phylogenetic amino acids among plant species. Therelationships 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 catalyticincludes the gain of new knowledge through activity and other conserved amino acidsthe study of important genes involved in related to protein interactions?” Weenergy production in plants. By comparing hypothesize that GAPDH proteins will haveGAPDH genes from the selected plants: conserved amino acids comprisingOxalis corniculata and Plectranthus functionally important regions such as theamboinicus. Focusing on conserved amino active site with the following amino acids:acids in specific regions that are 148, 149, 150, 176, 209, and 231. Otherhypothesized to be important for the involved in the binding of GAP substratestructure 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 acidsGAPDH PCR products from O.corniculata 148, 150, and 208. Also, we hypothesizeand P.amboinicus genomic DNAs. The that GAPDH proteins will have differentsequences will be published in GenBank to amino acids at some other positions notcontribute 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; itis present in everything we do on a daily Preliminary Studiesbasis. Plants use light energy, carbondioxide, and water to make sugar energy This is part of an ongoing researchthorough photosynthesis releasing oxygen. project, which began in 2009 by studyingThe production of energy in plants follows a the GAPDH gene on Oxalis corniculata,specific pathway. First, energy from the Plectranthus amboinicus, and Myrtaceaesun 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), gelelectrophoresis, and cloning and bacterialculture. They were performed in thefollowing sequence: DNA isolation, PCR,cloning and bacteria culture, mini-preps,and digestion with enzyme BGlII. Theresults of the electrophoresis are visible infigure 1. They are mentioned here becausetheir results helped refocus this study byeliminating the plant that was not cloned. In figure 1 the agarose gel showsfirst the ladders or markers, then thenegative controls followed by the positivecontrols. In the upper area of the gel, are the Research Design and Methodcloning vectors that co-migrate. We havefirst the negative control that is jus the -Preliminary studies methodsplasmid alone, followed by the positivecontrol that is Arabidopsis one of the plants Lysis buffer is used for DNAthat is genome is fully sequenced. Then, isolation of the plant species Oxalisthere are the three groups of plants with corniculata, Plectranthus amoboinicus, andthree samples each. The first DNA sample Myrtaceae psidium. Then PCR primersin the gel is M. psidum, guava. It showed no were designed to amplify GAPDH in thecloning since not enough DNA sample was three tropical plants.extracted nor amplified due to the absenceof PCR product band. For O. corniculata, The primers anneal to the region ofonly two of the three samples were cloned the target gene. Next, the PCR product isbeing the same size as the PCR product ligated into plasmid cloning vector pJET toband whereas for P. amboinicus all three transform ligation into competent E. coli.were successfully cloned. Due to these After transformation into E. coli the bacteriaresults M. psidum, guava was eliminated for has to be cultured before purifying thefurther study. plasmid DNA with Restriction Endonucleases to Identify Cloned PCRFigure 1 Mini-prep cloning gel results of Products in a 1% agarose gel. TheM.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 orderto confirm that the DNA was cloned we did  DNA extraction, amplification, wasa digest with enzyme BGLII (2ul/sample) a success based on the PCR productand was analyzed on a 1% agarose gel for plants O.corniculata, andthrough gel electrophoresis. Gel P.amboinicus.electrophoresis allows the analysis of the  Amplified PCR products weredigestion results. Once verified the region ligated to the pJET plasmid vectorwhere the GAPDH gene is located, samples and transformed into E. coli.were prepared for sequencing reactions,  Midi-Prep plasmid DNAs wereusing Sanger Sequencing and sent to the purified and quantified for clonedfacilities of Yale University. The sequences GAPDH PCR products from O.were returned and roughly analyzed using corniculata and P. amboinicusCap3, VecScreen, and BLAST. This genomic DNAs.resulted in both plant sequences being  DNA samples were digested withGAPC for O.corniculata and GAPDH for the restriction endonuclease BglIIP.amboinicus. and visualized using agarose gel electrophoresis to confirm that theCurent 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 forO.corniculata, and P.amboinicus genomic cloned GAPDH PCR products fromDNAs using bioinformatics tools such as O.corniculata and P. amboinicus.Cap3, VecScreen, and BLAST. This will be  Preliminary Bioinformatics analysesdone following the protocol for the NCBI revealed the cloned genes to be:site database search found in The Complete GAPC (O. corniculata) and GAPDHCloning and Sequencing Explorer Series (P. amboinicus)Curriculum by BIO-RAD. First, do a Blastsearch of the Cap sequence for both plantsequences. In second, comes a contig Cited literaturesequence 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 SequencingCompare the sequencing alignments and Exercise on Student Learning withdetermine intron and exon boundaries. Subsequent Publication in the NationalWhen the whole analysis is complete and Center for Biotechnology Informationverified, the sequences will be submitted to GenBank. CBE—Life Sciences Educationthe DNA database in GenBank. Vol. 8, 326–337, Winter 2009.Furthermore, we can do phylogeneticanalysis of plant GAPDH genes and do a 2) Cloning and Sequencing Explorer Seriesstructural 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 toNeuron Degeneration.AcknowledgementsRISE ProgramDr. Michael RubinMelissa MedinaDr. Elena GonzalezYadira Ortiz