Cloning of the Spinach GAPDH Gene

From One Spinach to Another: Cloning ofthe Spinach GAPDH Gene

 

Introduction

Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene is a highly conserved enzyme that is essential in dinucleotide binding and glycolytic catalysis (Tso, Sun, Kao, Reece & Wu, 1985). Over the past twenty years, GAPDH gene has long been a focus of interest for many scientists (Kosova, Khodyreva & Lavrik, 2016). Research data revealed that the GAPDH gene was capable of multiple distinctive functions unrelated to metabolism, including DNA translocation, transcription, regulation, etc. (Kosova et al., 2016). Specifically, recent investigations showed that the GAPDH gene could also play a role in DNA damage and repair (Kosova et al., 2016).

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GAPDH gene can also provide valuable information for cancer diagnostic and therapeutic research. Tokunaga et al. (1987) discovered that there was strongly enhanced GAPDH expression found in human lung cancer tissues. Similarly, persistent elevation of GAPDH gene was also found in other cell lines of the human body, such as kidney and liver (Tokunaga et al., 1987). Considering their findings, Tokunaga et al. (1987) argued that the dramatic increase of GAPDH expression in organs could be used for cancer screening, and the repression of glycolysis activities could inhibit the tumor cells’ growth.

As a consequence, the ability to correctly isolate, sequence and clone GAPDH gene can be of great significance in fully understanding and exploring the precise molecular structure and comprehensive cellular functions of the GAPDH gene in the human body. Tso et al. (1985) isolated the GAPDH gene in rats and humans and compared the sequences in order to study the molecular revolution. Ercolani, Florence, Denaro and Alexander (1988) isolated and sequenced the intact, functional human GAPDH gene and confirmed with GAPDH cDNA sequences. Kuo, Chou and Huang (2004) cloned the GAPDH gene from an edible type of mushroom, Flammulina velutipes and elucidated the whole DNA sequence of GAPDH gene.

Therefore, the purpose of this report is to clone the GAPDH gene from plant samples through a series of molecular biology techniques, such as DNA extraction, PCR reaction and purification, gel electrophoresis, ligation, transformation, inoculation, plasmid purification and restriction enzyme digest. It is hypothesized that the GADPH gene can be cloned from Spinach via the various techniques mentioned above. If the hypothesis is true, the size of the DNA fragment can be determined based on the final gel analysis and it should be the same as the inserted and ligated Spinach plasmid DNA. Nevertheless, due to the complexity of the molecular biology lab, the outcome of the experiment did not match the hypothesis. The final gel analysis gave no band of DNA fragment from Spinach plasmid.

Materials and Procedures

          GAPDH Gene Cloning, DNA Extraction, and PCR

Micropestles, Lysis buffer w/Dithiotheritol (DTT), Wash Buffer, Analytical Balance, Razor Blades, PCR Master Mix 2X, Initial GADPH PCR Primers-Blue. DNA Extraction and PCR Program were performed based on BISC320L Molecular Biology Laboratory Manual Pages 31-35.

          Spectrophotometric Analysis of DNA and Nested PCR

Spectrophotometer, Disposable Micro UV cuvettes (70μL-550μL), Vortex, Nested 2x PCR Master Mix-Yellow, PCR Tubes, Exonuclease I, Heating Block-80OC. Spectrophotometric Analysis of DNA and Nested PCR Program were performed based on BISC320L Molecular Biology Laboratory Manual Pages 41-43.

          PCR Purification

PCR Kleen Spin Column, Vortex. PCR Purification was performed based on BISC320L Molecular Biology Laboratory Manual Pages 47-48.

          Gel Electrophoresis and Ligation

Horizontal Gel Electrophoresis, Chamber, Power Supply, UView 6x Loading Dye, 1% Agarose Gel w/ SYBR Gold, 1X TAE Buffer, Molecular Ruler, Ligation Reaction Buffer 2x, Proofreading Polymerase, T4 DNA Ligase, pJet 1.2 Blunt Vector. Gel Electrophoresis and Ligation Reaction were performed based on BISC320L Molecular Biology Laboratory Manual Pages 54-58.

          Transformation

Culture Tubes, C-Growth Medium, E.coli HB101 Starter Culture, Transformation Reagent A&B, IPTG, Sterile Inoculating Loops, LB Agar with Ampicillin, Parafilm, Incubator at 37 OC, Vortex, LB Agar IPTG Plates. Competent Cell Preparation and Transformation Reaction were performed based on BISC320L Molecular Biology Laboratory Manual Pages 61-63.

          Inoculation

15 mL Culture Tubes, Inoculation Loop, LB Amp Broth, pJET Transformed Cells, 37OC Incubator with shaking. Inoculation Reaction was performed based on BISC320L Molecular Biology Laboratory Manual Pages 67-68.

          Plasmid Isolation and Restriction Enzyme Digest

Aurum Plasmid Mini Columns, Aurum Resuspension Solution, Aurum Lysis Solution, Aurum Neutralization Solution, Aurum Wash Solution, Aurum Elution Solution, Vortex, Bgl II Restriction Enzyme, 10x Bgl II Reaction Buffer, UView Loading Dye and Stain 6x, Molecular Weight Ruler. Plasmid Purification and Restriction Enzyme Digestion were performed based on BISC320L Molecular Biology Laboratory Manual Pages 72-75.

          Final Analysis of Cloning Project

FlashGel Electrophoresis Dock, Power Supply, FlashGel Loading Dye, 500 b.p. Molecular Ruler. FlashGel Electrophoresis was performed based on BISC320L Molecular Biology Laboratory Manual Pages 77-78.

Results

As shown in Table 1, the quantity and purity of genomic DNA present in each extracted sample were obtained by spectrophotometry.

Table 1. Data on plant extraction and DNA extraction

Spinach 1

Spinach 2

Sample Weight (mg)

97

95

DNA Concentration (mg/ml)

0.00115

0.545

A260 (A)

0.023

0.218

A280 (A)

0.021

0.086

A260/A280

1.095

2.535

During gel electrophoresis, dyed (blue) samples were loaded at the cathode, migrated through the gel to the anode. When the electrophoresis was complete, the gel image was shown as Graph 1. Based on the 500 b.p. DNA Ladder, it can be seen that the molecular weights of each DNA fragment from Lane 2 to Lane 8 are 1000 b.p., 750 b.p., 1500 b.p., 1250 b.p., 1000 b.p., 1000 b.p. and 1500 b.p., respectively.

Graph 1. PCR gel electrophoresis

Note: The eight lanes (Lane 1 to Lane 8) labeled in the graph from left to right were 500 b.p. DNA Ruler, Romaine Lettuce 1 Purified, Romaine Lettuce 1 Nested PCR, Romaine Lettuce 2 Purified, Romaine Lettuce 2 Nested PCR, Arabidopsis Purified, Arabidopsis Nested PCR and Negative Control (Water) Purified, respectively.

Through transformation reaction, several colonies were observed in each agar plate. The number of transformation colonies formed for each sample was reported in Table 2.

Table 2. Summary of Transformation results

Spinach 1

Spinach 2

Number of transformation colonies

70

80

PCR Band Size (b.p.)

N/A

N/A

Restriction Enzyme Band Size (b.p.)

N/A

N/A

During FlashGel gel electrophoresis, dyed (blue) samples traveled on the cassette. When the power supply was turned off, the picture of the cassette was shown as Graph 2, where no band of DNA fragment was observed for the undigested or digested samples.

Graph 2. Final gel analysis

Note: The six lanes (Lane 1 to Lane 6) labeled in the graph from left to right were DNA Ladder, Undigested Spinach 1, Digested Spinach 1, Undigested Spinach 2, Digested Spinach 2 and DNA Ladder, respectively.

Discussion

As it can be seen in Table 1, both the DNA concentration, A260, A280 and A260/A280 measurements for Spinach 1 are too low to be considered normal. The low absorbance and DNA concentration indicate a low quantity of nucleic acids present in Spinach 1. In other words, the extracted sample from Spinach 1 was not pure. This contamination could be caused by the residual chemicals (ethanol), proteins, carbohydrates (polysaccharides) and lipids mixing with genomic DNA, which would interfere with DNA extraction. Deficient lysis buffer and wash buffer added could also lead to low DNA yield, since lysis buffer helped to break open the membrane enclosing the cell and release the DNA, and wash buffer helped to remove residual contaminants left in the column.

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Graph 1 was borrowed from our lab mates. The entire gel electrophoresis preparation was repeated twice, yet in both cases, the comb failed to make corresponding wells in the agarose gel. As a result, without appropriately loading each sample into its well, the gel electrophoresis experiment couldn’t be conducted. Several reasons might account for this phenomenon. First, during the entire 20-30 minutes’ cooling, the melted agarose gel was not able to fully cool down and solidify. Second, the comb was either posited in the opposite direction or didn’t reach the very bottom of the chamber. Third, when taking the comb out of the agarose gel, it was not lifted straight up and thus the already formed wells could have been destroyed. Lastly, it was highly likely that the comb was defective and was unable to penetrate the agarose gel in the first place. Therefore, no wells would take shape in regards to the reasons stated above.

Given that no visible band was obtained using FlashGel gel electrophoresis, the following issues which could have great influence on the final results should be considered. First, the purified plasmid DNA was actually not pure enough as contaminants found in DNA could work as repressors of endonuclease activity, resulting in a greater mixture of impurities with the plasmid DNA. The column might not be spun or dried for long enough, so all the buffer solutions used, i.e. lysis solution, neutralization solution and wash solution would remain in the plasmid DNA supernatant. Second, insufficient amount of plasmid DNA was added into each well. As it can be seen in Graph 2, the color of each well from Lane 2 to Lane 5 were relatively light, suggesting that the volume and the concentration for the final purified plasmid DNA were too minimal to be detected by the FlashGel. Third, the prepared miniprep cultures were not single, isolated colonies containing only the plant plasmid DNA. It was very likely that satellite colonies which contained multiple plasmids except for the plasmid of interest were picked incorrectly. As a consequence, no band was generated by the FlashGel.

The final result does not coincide with the hypothesis since the FlashGel gel electrophoresis failed to provide the plasmid DNA fragments separating on the cassette in accordance with their respective band length. However, it will be unreasonable to negate the entire hypothesis just because no final results were achieved. In fact, part of the results presented above can be deemed as reasonable. For example, the data of DNA concentration and absorbance for Spinach 2 fall within the proper range and can signify good amounts of purified nucleic acids present in that sample. The decent number of transformation colonies found in each sample also demonstrates the success of transformation reactions. From the results obtained, DNA extraction and purification, PCR reaction, ligation, and transformation can be considered effective and efficient techniques in GAPDH gene cloning; whereas there is still much to be improved for the specific operations of gel electrophoresis, inoculation, plasmid purification, restriction enzyme digest and FlashGel gel electrophoresis.

Conclusion

Although the final results failed to prove the validity of the hypothesis, from the experiments completed, it can still be concluded that GAPDH gene can be extracted and purified from Spinach, and E.coli HB101 is competent to undergo the transformation reaction of GAPDH gene through heat shock. Successful GAPDH gene cloning will make the replacement of ineffective and mutated GAPDH genes possible and ensure that the cloned GAPDH gene is able to work functionally as an efficient GAPDH gene.

Future directions of research should focus on the multifunctional roles of GAPDH gene involved in the nuclear pathways. By internally controlling the GAPDH gene’s interactions with a number of DNA binding proteins, we can better regulate DNA translocation, transcription, and modification (Sirover, 2005). Furthermore, monitoring of the GAPDH gene expression in different precancerous tissues can be used as a diagnosis for malignant tumor cells. Down regulation of the abnormally expressed GAPDH gene is regarded as a viable solution for preventing human tissues from progressing into tumors (Ramos et al., 2015).

References

Tso, J.Y., Sun, X.H., Kao, T.H., Reece, K.S., & Wu, R. (1985). Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Research, 13, 2485-2502.

Kosova, A.A., Khodyreva, S.N., & Lavrik, O.I. (2016). Role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in DNA repair. Biochemistry, 82, 859-872.

Tokunaga, K., Nakamura, Y., Sakata, K., Fujimori, K., Ohkubo, M., Sawada, K., & Sakiyama, S. (1987). Enhanced Expression of a Glyceraldehyde-3-phosphate Dehydrogenase Gene in Human Lung Cancers. Cancer Research, 47, 5616-5619.

Ercolani, L., Floerence B., Denaro M., & Alexander M. (1988). Isolation and Complete Sequence of a Functional Glyceraldehyde-3-phosphate Dehydrogenase Gene. The Journal of Biological Chemistry, 263, 15335-15341.

Ramos, D., Carcelen, A.P., Agusti, J., Murgui, A., Jorda, E., Pellin, A., & Monteagudo, C. (2015). Deregulation of Glyceraldehyde-3-Phosphate Dehydrogenase Expression During Tumor Progression of Human Cutaneous Melanoma. Anticancer Research, 35, 439-444.

         Kuo, C.Y., Chou, S.Y., & Huang, C.T. (2004). Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes. Applied Microbiology and Biotechnology, 65, 593-599.

         Sirover, M.A. (2005). New nuclear functions of the glycolytic protein, glyceraldehyde‐3‐phosphate dehydrogenase, in mammalian cells. Journal of Cellular Biochemistry, 95, 45-52.