PCR Analysis of GAPDH Genes in Parsley

PCR GAPDH Genes Parsley

PCR Analysis of GAPDH Genes in Parsley

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The purpose of this review is to consider the structure and the function of the protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC in Petroselinum crispum and Coriandrum sativum cells. For over three decades, GAPDH was studied for its pivotal role in glycolysis. As an abundant cell protein, it proved useful as a model for investigations examining basic mechanisms of enzyme action as well as the relationship between amino acid sequence and protein structure. Further, with the advent of molecular technology, GAPDH, as a putative ‘house-keeping’ gene, provided a model with which to use new methods for gene analysis to advance our understanding of the mechanisms through which cells organize and express their genetic information.

As with many things in life, what is thought to be simple and relatively straight-forward turns out to be quite complex and elaborate. In this regard, a number of studies, accelerating in the last decade, have indicated that GAPDH is not an uncomplicated, simple glycolytic protein. Instead, independent laboratories identified diverse biological properties of the mammalian GAPDH protein. These included roles for GAPDH in membrane transport and in membrane fusion, microtubule assembly, nuclear RNA export, protein phosphotransferase/kinase reactions, the translational control of gene expression, DNA replication and DNA repair. Each activity appears to be distinct from its glycolytic function (Sirover, 1999).

The gene that codes for the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is of the same name. GAPDH is a crucial enzyme in glycolysis. The gene is known as a housekeeping gene — a gene that is expressed constitutively and is necessary for cells to survive. Since GAPDH is abundant in cells and can be purified for study, much is known about the protein structure and function. GAPDH consists of four subunits (hence a tetramer) held together through non-covalent attachments. All four subunits may be identical (designated as A4, a homodimer) or they may consist of pairs of slightly different subunits (designated A2B2, a heterodimer). In both cases, each subunit has an active site and can bind one molecule of NAD+ cofactor.

GAPDH protein has two major domains, the amino terminal has an NAD+ binding domain and the carboxy terminal has glyceraldehyde 3′ phosphate dehydrogenase activity. GAPDH protein domain structure. The active cysteine is in the catalytic site. In addition, recent research has found that GAPDH plays many other roles outside of glycolysis. For example, the human GAPDH gene is overexpressed (i.e., expressed at levels much higher than normal) in 21 different classes of cancer (Altenburg and Greulich, 2004). GAPDH has been shown to play roles in membrane fusion, endocytosis, microtubule bundling, and DNA repair. GAPDH is also involved in viral pathogenesis, regulation of apoptosis (programmed cell death), and human neuronal diseases including Alzheimer’s and Huntington’s disease (reviewed in Sirover 1999).

GAPDH catalyzes the sixth reaction of glycolysis, the pathway by which glucose is converted into pyruvate in a series of ten enzymatic reactions. In mammals, most dietary polysaccharides are broken down to glucose in the bloodstream. In plants, glucose is synthesized from carbon dioxide in the Calvin cycle of photosynthesis.

Glycolysis has a number of useable products:

• The production of ATP and NADH during glycolysis, providing energy for the cells

• Pyruvate, the end product of glycolysis, feeds into the citric acid cycle, producing more energy for the cells

• Many of the intermediate compounds of glycolysis are precursors for the formation of other biological molecules. For example, glucose-6-phosphate is a precursor for the synthesis of ADP, NAD+, and coenzyme Q, and phosphoenolpyruvate is a precursor for the synthesis of the amino acids, tyrosine, phenylalanine, and tryptophan.

The reaction catalyzed by GAPDH is:

Glyceraldehyde-3-phosphate + NAD+ + PiAE1,3-bisphosphoglycerate + NADH + H+

GAPDH oxidizes glyceraldehyde-3-phosphate (GAP) by removing a hydrogen ion (H+) and transferring it to the acceptor molecule, NAD+ (NAD+ + H+AENADH). In addition, GAPDH adds a second phosphate group to GAP. This reaction is catalyzed by a cysteine in the active site of the GAPDH protein.

When the source of carbohydrate for glycolysis is a sugar, glycolysis will occur in the cytosol, as it does in animal cells. When the carbohydrate source is starch however, glycolysis can occur in plastids (a group of organelles that includes chloroplasts).

GAPDH Genes in Plants and their Origins

Plants such as Petroselinum Crispum and Coraindrum sativum contain three forms of GAPDH: a cytosolic form which participates in glycolysis and two chloroplast forms which participates in photosynthesis. These three forms are encoded by distinct genes. In plants there are two metabolic pathways for carbohydrates: the Calvin Cycle in chloroplasts and glycolysis in the cytosol. The pathways share some enzymatic reactions (including the reaction catalyzed by GAPDH), but the enzymes in the two pathways are not identical even though they catalyze the same reactions in both pathways. The enzymes in the two pathways are isozymes or isoenzymes, homologous enzymes that catalyze the same reaction but differ in amino acid sequence. A separate gene encodes each isoenzyme, and all of the genes are nuclear (reviewed in Plaxton 1996).

For example, the enzyme hexokinase phosphorylates glucose both in the chloroplast and in the cytosol, but two separate genes in the plant cell nucleus encode cytosolic hexokinase and chloroplastic hexokinase. Isozymes are very common in plants and animals, and typically result from a gene duplication event that occurred millions of years ago. Sometimes the gene duplication event occurred within the nucleus itself. There are also genes located on chromosomal DNA that appear to have been transferred there from mitochondrial or plastid DNA. One of the observations about mitochondria and plastids that led to the endosymbiotic theory of evolution (that these organelles exist due to an ancient symbiotic event between prokaryotes and eukaryotes) is the fact that they contain DNA that is similar to bacterial DNA.

It was more than one billion years ago that photosynthetic cyanobacteria were engulfed by eukaryotic cells, becoming the antecedents of modern plastids. The resulting sub-cellular organelles, plastids, have taken over many reactions for their host cells, including photosynthesis, carbohydrate metabolism, amino acid synthesis, lipid production, photorespiration, and nitrogen/sulfur reduction. At the same time, plastids still have their own DNA, as well as the machinery for replication, transcription, and translation. However, plastids retain only a fraction of the genome that their ancestors had. The plastid genome encodes between 120 — 135 genes (Lopez-Juez 2007), whereas the closest living relative to the plastid ancestor, cyanobacteria of the genus Nostoc (Martin et al., 2002), have between 3,000 — 7,000 genes.

Most genes originally found in the symbiotic cyanobacteria are now found in the plant cell nucleus. Martin et al. (2002) report that about 18% of the protein-coding genes in Arabidopsis thaliana derive from cyanobacteria. However, the gene transfer was not one way. Genes that pre-existed in the nuclear genome have also been transferred to the plastid genome, but gene expression in the plastid is under nuclear control and most plastid proteins are encoded by nuclear DNA. All GAPDH isozymes found in eukaryotes are nuclear-encoded and are believed to have originated in cyanobacteria (Martin et al., 2002). The duplication of GAPDH genes that gave rise to the chloroplastid form is believed to have occurred during the period when land plants first emerged (Teich et al., 2007), and subsequent gene duplications resulted in the multiple forms now present in modern plants.

Analyzing the GAPDH gene of organisms can reveal differences between them that are important for plant evolution and survival. Plastids, the light-harvesting organelles of plants and algae, are the descendants of cyanobacterial endosymbionts that became permanent fixtures inside nonphotosynthetic eukaryotic host cells. The structural, functional and molecular diversity of plastids in the context of current views on the evolutionary relationships among the eukaryotic hosts in which they reside can be revealed through differences in GAPDH. Green algae, land plants, red algae and glaucophyte algae harbor double-membrane-bound plastids whose ancestry is generally believed to trace directly to the original cyanobacterial endosymbiont, like GAPDH. In contrast, the plastids of many other algae, such as dinoflagellates, diatoms and euglenids, are usually bound by more than two membranes, suggesting that these were acquired indirectly via endosymbiotic mergers between nonphotosynthetic eukaryotic hosts and eukaryotic algal endosymbionts. An increasing amount of PCR analysis of GAPDH genes from diverse photosynthetic taxa has made it possible to test specific hypotheses about the evolution of photosynthesis in eukaryotes and, consequently, improve our understanding of the genomic and biochemical diversity of modern-day eukaryotic phototrophs (Kim and Archibald, 2009).

Works Cited

Kim, E. And Archibald, J. (2009) Diversity and Evolution of Plastids and Their Genomes. Plant Cell Monograph. 1-39.

Lopez-Juez, E. 2007. Plastid biogenesis, between light and shadows. J. Exper. Bot. 58: 11 — 26.

Martin, W., Rujan, T., Richly, E., Hansen, A., Cornelsen, S., Lins, T., Leister, D., Stoebe, B., Hasegawa, M, & Penny, D. 2002. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastic phylogeny and thousands of cyanobacterial genes in the nucleus. PNAS 99: 12246 — 12251.

Plaxton, W.C. 1996. The organization and regulation of plant glycolysis. Annu. Rev. PlantPhysiol. Plant Mol. Biol. 47: 185 — 214.

Sirover, M.A. 1999. New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Bioch. Biophys. Acta 1432: 159 — 184.

Teich, R., Grauvogel, C., & Petersen, J. 2007. Intron distribution in Plantae: 500 million years of stasis during land plant evolution. Gene 394: 96 — 104.

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