Glutamate dehydrogenase (GDH)-synthesized RNA, a nongenetic code-based RNA is suitable for unraveling the structural constraints imposed on the regulation (transcription, translation, siRNA etc.) of metabolism by genetic code. GDH-synthesized RNAs have been induced in whole plants to knock out target mRNA populations thereby producing plant phenotypes that are allergen-free; enriched in fatty acids, essential amino acids, shikimic acid, resveratrol etc. Methods applied hereunder for investigating the structural properties of GDH-synthesized RNA included purification of GDH isoenzymes, synthesis of RNA by the isoenzymes, reverse transcription of the RNA to cDNA, sequencing of the cDNA, computation of the G+C-contents, profiling the stability through PCR amplification compared with genetic code-based DNA; and biochemical characterization of the RNAs synthesized by individual hexameric isoenzymes of GDH. Single product bands resulted from the PCR amplification of the cDNAs of GDH-synthesized RNA, whereas several bands resulted from the amplification of genetic code-based DNA. The cDNAs have wide G+C-contents (35% to 59%), whereas genetic code-based DNA has narrower G+C-contents (50% to 60%). The GDH β6 homo-hexameric isoenzyme synthesized the A+U-rich RNAs, whereas the a6, and α6 homo-hexameric isoenzymes synthesized the G+C-rich RNAs. Therefore, the RNA synthesized by GDH is different from genetic code-based RNAs. In vitro chemical reactions revealed that GDH-synthesized RNA degraded total RNA to lower molecular weight products. Therefore, GDH-synthesized RNA is RNA enzyme. Dismantling of the structural constraints imposed on RNA by genetic code liberated RNA to become an enzyme with specificity to degrade unwanted transcripts. The RNA enzyme activity of GDH-synthesized RNA is ubiquitous in cells; it is readily induced by treatment of plants with mineral nutrients etc. and may simplify experimental approaches in plant enzymology and molecular biology research projects.
Glutamate dehydrogenase (GDH EC 1.4.1.2) is a multi-subunit enzyme that polymerizes ribo-nucleoside triphosphates to produce RNA independent of any template [
Following the biotechnological applications of GDH-synthesized RNA as enzyme, there is need to characterize their chemical properties as compared with genetic code-based RNA. There are many contrasts in the chemistry of coding
RNA and non-coding RNA [
Peanut (Arachis hypogeae Floor Runner cv.) seeds were sterilized in 5% alcohol solution for 10 min, rinsed with deionized water, and planted on moistened filter paper in five replicate petri dishes. The compositions of the mineral nutrients were: N+N+N+K+S+S (1 L of 75 mM NH4Cl, 4 mM KCl, 100 mM Na2SO4); N+P+K+K+K (1 L of 25 mM NH4Cl, 20 mM Na3PO4, 12 mM KCl); N+P+S+S+S+S (1 L of 25 mM NH4Cl, 20 mM Na3PO4, 200 mM Na2SO4); N+P+P+P (1 L of 25 mM NH4Cl, 60 mM Na3PO4). The control was moistened with distilled water. About 8 - 10 seeds were planted per petri dish and allowed to germinate in the greenhouse temperature of 24˚C - 28˚C, and relative humidity of 70% - 80% under Texas month of May temperature and sunlight conditions. The greenhouse was shaded 50% with a shade cloth. Filter papers were changed and re-wetted with fresh mineral solutions daily. The applied mineral salt compositions were based on stoichiometric combinations to mimick the binomial subunit polypeptide compositions of GDH isoenzymes [
Total RNA was extracted from peanut seedlings using the acidic phenol/chloroform (pH 4.5) method [
GDH was purified from the N+N+N+K+S+S-treated peanut seedlings (20 g) by homogenization at 4˚C with 100 mL of buffer [
RNA was synthesized in the amination direction in cocktails containing solution of 0.87 mM NH4Cl, 3.5 mM CaCl2, 10.0 mM α-ketoglutarate (α-KG), 0.23 mM NADH, 0.6 mM each riboNTP, 5 U of RNase inhibitor, 5 U of DNase 1, and 5.0 μg actinomycin D. The reaction was started by adding 0.2 mL of cryo-electrophoretically purified GDH charge isomers containing about 500 μg protein per mL as described before [
cDNAs were synthesized with 2 μg of each product RNA synthesized by the GDH charge isomers purified from the N+N+N+K+S+S-treated seedlings, using random hexamer primers. The product cDNA (5 μg) was digested with Taq 1 restriction enzyme (5 Units) for 2 h at 65˚C. In order to compare with the chemistry of genetic code-based DNA, pCR4-TOPO vector DNA (5 μg) was similarly digested in another micro tube with Taq 1 restriction enzyme (5 Units). Adaptors, 32P-labeled extension primers, and the selective display PROBE combination (Display Systems Biotech, Vista, CA USA) were ligated to the ends of the restriction fragments. The nucleotide sequences of the adapters, extension primers, and display PROBEs are among the proprietary information of their manufacturer, Display Systems Biotech, Vista, CA USA.
The template DNA (0.5 μg) was amplified according to the ‘touch-down’ restriction fragment double display (RF-DD) PCR methods of Display Systems Biotech, Vista, CA, USA. Initial denaturation was 96˚C for 1 min. For the first 10 cycles: denaturation was at 96˚C for 30 sec., annealing was at 60˚C for 30 sec, for the first cycle, then reduced the annealing temperature 0.5˚C each cycle until an annealing temperature of 55˚C was reached after 10 cycles; extension was at 72˚C for 1 min. PCR was continued another 25 cycles: denaturation (96˚C, 30 sec), annealing (55˚C, 30 sec), extension (72˚C, 1 min); final elongation (72˚C, 5 min). All the 64 display PROBEs in the Display Systems Biotech kit were used in the differential display PCR. The differential bands/products were visualized by autoradiography following polyacrylamide sequencing gel electrophoresis. Selected cDNA, and vector DNA fragments were sub-cloned into pCR4-TOPO vector and transformed into TOP10 One Shot Chemically Competent (non-pathogenic) Escherichia coli (Invitrogen, Carlsbad, CA), followed by overnight growth on kanamycin selective plates. Up to 15 positive transformant colonies were picked per plate and cultured overnight in LB medium containing 50 μg/mL of kanamycin. Plasmids were purified with a plasmid kit (Novagen, Madison, WI), and the insert cDNA or vector DNA fragment of selected recombinant plasmids were sequenced with T7, and T3 primers by MWG Biotech Inc., High Point, North Carolina, USA. The sequenced plasmids were amplified by ‘touch-down’ PCR using M13 primers (Invitrogen Life Technologies, California); separated by agarose gel electrophoresis; the product bands were UV-visualized, and photo-documented.
GDH-synthesized RNA and total RNA preparations were normalized to a concentration of 5 µg/µL in 0.1 M Tris-HCl buffer solution pH 8.0. The reactions were conducted in 8 numbered micro-centrifuge tubes placed on ice. Tube 1 contained 100 µg of total RNA of control peanut, and 100 µg of the GDH-synthesized RNA of N+N +N+K+S+S-treated peanut. Tube 2 contained 200 µg of total RNA of control peanut. Tube 3 contained 100 µg of total RNA of N+P+K+K+K-treated peanut and 100 µg of the GDH-synthesized RNA of N+N+N+K+S+S-treated peanut. Tube 4 contained 200 µg of the GDH synthesized RNA of N+N +N+K+S+S-treated peanut. Tube 5 contained 100 µg of the total RNA of N+P+S+S+S+S-treated peanut and 100 µg of the GDH synthesized RNA of N+N+N+K+S+S-treated peanut. Tube 6 contained 200 µg of the GDH synthesized RNA of N+N+N+K+S+S-treated peanut. Tube 7 contained 100 µg of the total RNA of N+P+P+P-treated peanut and 100 µg of the GDH synthesized RNA of N+N+N+K+S+S-treated peanut. Tube 8 contained 200 µg of the total RNA of control peanut. To each tube, 1 µL of 0.6 mM ribo-NTP mix was added; and the final volume of the reaction was brought to 50 µL with 0.1 M Tris-HCl buffer solution pH 8. Tubes 1, 2, 3, 4, 5, and 7 were thermo-cycled: pre-heat (96˚C, 30 sec). Then 40 cycles of cool (5˚C, 1 min), warm (37˚C, 2 min). Final storage (5˚C). Tubes 6, and 8 were left on ice. The extent of reaction was demonstrated by 2% agarose gel electrophoresis of 7 µL of each reaction solution. The gel was stained with ethidium bromide solution; and photographed. RNA band intensities were digitalized using UN-SCAN-IT gel digitalizing software (Silk Scientific, Inc., Orem, Utah, USA).
To assign putative functions to the RNAs synthesized by GDH (RNA enzyme), their cDNA sequences were used as queries to search the NCBI nucleotide-nucleotide (excluding ESTs) BLAST (blastn) for peanut taxid 3818 database (Arachis hypogaea) [
DNA sequences were applied to the Kibbe equations (http://www.basic.northwestern.edu/biotools/oligocalc.html) to count the cytosines and guanines accurately and to calculate the G+C contents, nearest neighbor, and melting temperatures (Tm) taking into account base stacking energy.
Single product bands resulted from the PCR amplification of the insert cDNA of GDH-polymerized RNA (
Plasmid Inserts | Length (base pairs) | GC Content % | Nearest Neighbor ˚C | Tm basic ˚C | Comment |
---|---|---|---|---|---|
1 | 143 | 57.0 | 84.92 | 83.7 | A |
2 | 313 | 35.5 | 79.86 | 77.3 | A |
3 | 153 | 44.0 | 79.98 | 78.5 | A |
4 | 153 | 51.0 | 82.27 | 81.4 | A |
5 | 191 | 54.0 | 83.76 | 83.5 | A |
6 | 243 | 50.0 | 84.49 | 82.5 | A |
7 | 243 | 50.0 | 84.49 | 82.5 | A |
8 | 243 | 50.0 | 84.49 | 82.5 | A |
9 | 261 | 54.0 | 85.39 | 84.5 | A |
10 | 241 | 49.8 | 84.45 | 82.5 | A |
11 | 241 | 49.8 | 84.45 | 84.1 | A |
12 | 334 | 52.0 | 84.98 | 84.1 | A |
13 | 319 | 59.0 | 87.47 | 86.8 | A |
14 | 146 | 59.0 | 86.18 | 84.4 | A |
15 | 308 | 35.0 | 79.70 | 77.0 | A |
16 | 312 | 35.3 | 79.88 | 77.2 | A |
17 | 101 | 54 | 83.39 | 80.6 | A |
18 | 69 | 55 | 78.24 | 77.7 | A |
19 | 341 | 37 | 80.79 | 78.3 | A |
20 | 121 | 55 | 82.99 | 82.0 | A |
21 | 159 | 55 | 84.27 | 83.3 | B |
22 | 302 | 50 | 85.17 | 83.3 | B |
23 | 199 | 59.0 | 87.74 | 85.8 | B |
24 | 365 | 50.7 | 86.28 | 84.0 | B |
25 | 365 | 51.2 | 86.37 | 84.0 | B |
26 | 188 | 59.0 | 87.74 | 85.8 | B |
27 | 292 | 51.0 | 84.67 | 83.5 | B |
28 | 166 | 59.0 | 86.18 | 84.4 | B |
29 | 442 | 60.0 | 89.4 | 87.8 | B |
a = cDNA of GDH-synthesized RNA (nongenetic code-based). b = genetic code-based DNA.
high nearest neighbor stacking base interactions, and high G+C compositions. The cDNAs of GDH-synthesized RNA varied their G+C contents broadly covering a range from 35% to 59%, whereas the vector coding DNA fragments varied their G+C contents for a narrower range from 50% to 60% (
The high G+C contents of the vector DNA (
The nearest neighbor interaction is a major factor that affects the stability of nucleic acid, the A.T pairing being always destabilizing [
It has been suggested that the RNA synthesized by GDH might function in the regulation of mRNA abundance through homologous sequence-mediated RNA interference activity [
The differences between the G+C-contents of genetic code-based DNA and nongenetic code-based DNA (
The reactions between total RNA and the RNA synthesized by GDH resulted to total RNA degradation (
Comparison of lanes 3 (
Similarly, reaction 5 (
Again, reaction 7 (
The reactions depicted in lanes 1, 3, 5, and 7 in the absence of protein enzymes suggested that the GDH-synthesized RNA acted as enzyme to degrade transcripts that shared sequence homologies with it because comparison of lanes 2 and 8 showed that the thermal cycling did not degrade total RNA, but lanes 4 and 6 showed that GDH-synthesized RNA was sensitive to thermal cycling, the high molecular weight bands above 26S rRNA in lane 6 being absent after thermal cycling (lane 4) which could have resulted from their thermal instability due to the high A+U content of GDH-synthesized RNA (
The results (
Again, the molecular responses of the different total RNAs of peanut to the same GDH-synthesized RNA (lanes 1, 3, 5, and 7) were different, further suggesting that each mRNA profile of the total RNAs was for a specific metabolic phenotype/variant [
Therefore, the cross-over reactions (
In vitro demonstration of the function of GDH-synthesized RNA as enzyme begets a conversation on the structure of the RNA (
GDH RNA | 19 (β6) | 18 (a6) | 8 | 4 | 13 | 20 | 1 | 5 | 7 | 10 | 9 | 15 | 16 | 2 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
19 (β6) | --- | no match | no match | no match | no match | no match | +/− match | no match | no match | no match | no match | +/+ match | +/− match | +/+ match |
17 (α6) | no match | 3 +/+ matches | 6 +/+ matches | 5 +/+ matches | 4 +/− matches | 3 +/+ matches | 6 +/+ matches | 4 +/+ matches | 4 +/+ matches | 4 +/+ matches | 4 +/+ matches | no match | no match | no match |
18 (a6) | no match | --- | 4 +/+ matches | 4 +/+ matches | 3 +/− matches | 3 +/+ matches | 4 +/+ matches | 3 +/+ matches | 4 +/− matches | 4 +/− matches | 6 +/− matches | no match | no match | no match |
15 | +/+ match | no match | no match | +/− match | ||||||||||
16 | +/− match | no match | no match | |||||||||||
4 | no match | 4 +/+ matches | 4 +/+ matches | |||||||||||
5 | no match | 3 +/+ matches | 3 +/+ matches | |||||||||||
2 | +/+ match | no match | no match | |||||||||||
1 | +/− match | 4 +/+ matches | 4 +/+ matches | |||||||||||
20 | no match | 3 +/+ matches | 7 +/+ matches | |||||||||||
10 | no match | 4 +/− matches | no match |
β6 homo-hexameric GDH isomer selected ribo-ATP and UTP preferentially as substrates; whereas the α6, a6 and combinations thereof selected ribo-GTP and ribo-CTP preferentially as substrates. These biochemical considerations give strength to the repeatedly published over whelming preponderance of G+C-rich regions as against the lesser frequency of the A+T-rich regions in the genome [
The stoichiometric mineral solutions applied (N+N+N+K+S+S; N+P+K+K+K; N+P+S+S+S+S; and N+P+P+P) in peanut germination were selected because they were among those that did not delay the emergence of seedling radicle compared with water treatment. There are 299 stoichiometric combinations of mineral macronutrients that mimic the hexameric subunit structure of GDH. We have planted peanut seeds in the month of May in the tissue culture chamber,
Plasmid 1: cDNA insert Arachis hypogaea oxalate oxidase (OxOxl) mRNA, EU024476.1 TGAGTCCTGACCGATAGCGCCAATGCGTTGAGTACCTTCAACGCCAAGAACGTCAACTACCAGCGCACGCCGCACTTCAAGAACAAACCCGGCACGCGGCATAGCGATGAGTCCTGACCGGGTACGCAGTCTACGAGACCAGTA. |
---|
Plasmid 2: cDNA insert Arachis hypogaea profilin (Ara h5) mRNA AF059616.1 GATTTTATTTAGGAGGTATTGGGAACGAATTGGAATGTAATAATATTGATTCATAGAGATCCAGAAGAAAAAGAATAATCTTCTACTTTGAGAATAATAAAAAAAGAAAAGTGTTCAATTGGAACATGAAAACGTGACCTGACTGAATACTGGTCTCGTAGACTGCGTACCCGGTCAGGACTCATCGCTACGTTAGCGTCTCTGAGGCGCGCTATTCTACAATCTTAAAAACCCCTGTCAACCCTTTAAATTGCTTTTAAGACAATGATTTGCGCTTCTTTCTGATTTCTTCTTGGGGAGAAGAAACCCGTGGGCTGACGTTGCTGCGGGGCGCACTTTACAAGCCTTTGCCTTACAGTTCAACGCCCTATCGGTCAGGACTCATAAGGGC |
Plasmid 3: cDNA insert Arachis hypogaea ubiquitin-conjugating enzyme (UBC) mRNA, AY769917.2 GAGTCCTGACCGATAGTGAATAGGTCGTTGTGTTTCATGAGGCCTCCTTGATACTCATGAACTACAGATATTTGACGTCAAAAATAATTCAAATAAGTTGTCCGACAATGCTGATGAGTCCTGACCGGGTACGCAGTCTACGAGACCAGTAAG |
Plasmid 4: cDNA insert Arachis hypogaea type 2 metallothionein (MT2d) mRNA, DQ665256.1 GAGTCCTGACCGATAGGAGATCAAGGCACCCCATGTCTTGAGGGTGGGACGGTTATTTGCTCAGGATAATAAAGGGCGGTTTCAGTTCAAAGTGCCTGAGCTTAGTATAGCGATGAGTCCTGACTGGGTACGCAGTCTACGAGACCAGTAAGG |
Plasmid 5: cDNA insert Arachis hypogaea mRNA for ABA 8'-hydroxylase (CYP707A2 gene), cultivar Yueyou 7, mRNA HG764751.1 GAGTCCTGACCGATAGCGGCCTGCATGCTCATGTTGCCAGTCTTGCCACCAGTACCCGTTCCAGTGTCAGGAGCCGGGAACTGACCTACGCCATTTTTGTAAACACCGGTAGAAGAGGAATAAGGACTCCCGGAAGTGTGCCAAGTCACCAAGGTTTCAACTTCGGGTACGCAGTCTACGAGACCAGTAAG |
Plasmid 8: cDNA insert Arachis hypogaea cultivar fuhua 8 glutamate dehydrogenase 1 (GDH1) mRNA, KT933119.1 ACTGGTCTCGTAGACTGCGTACCCGGTCAGGACTCATCGCTACGTTAGCGTCTCTGAGGCGCGCTATTCTACAATCTTAAAAACCCCTGTCAACCCTTTAAATTGCTTTTAAGACAATGATTTGCGCTTCTTTCTGATTTCTTCTTGGGGAGAAGAAACCCGTGGGCTGACGTTGCTGCGGGGCGCACTTTACAAGCCTTTGCCTTACAGTTCAACGCCCTATCGGTCAGGACTCATAAGGGC |
Plasmid 9: cDNA insert. Arachis hypogaea strain E2-4-83-12 delta-12 fatty acid desaturase (FAD2B) gene, JN544190.1 TGAGTCCTGACCGATAGCCTGCCTAAACCTTCTTGAAGTAGTGGCGGCGGTCGTTTTCGGTGACTGTCTGCTGGAAAATGTCCGTCCAGAAATCCCGCTCCATTACGTCCTGGTGAAACATCACCCCGCAGATAACCTCCATCGGGTTGCACTTCAAAAGCTCGGCAACCTTCACGGCCTGCTTGACGCTCATTTCATGTTTTCCCGCCTTCTGTAGCGATGAGTCCTGACCGGGTACGCAGTCTACGAGACCAGTAAGGG |
Plasmid 10 cDNA Insert. Arachis hypogaea cultivar fuhua 8 glutamate dehydrogenase 1 (GDH1) mRNA, Sequence ID: KT933119.1 CTGGTCTCGTAGACTGCGTACCGGGTCAGGACTCATCGCTACGTTAGCGTCTCTGAGGCGCGCTATTCTACAATCTTAAAAACCCCTGTCAACCCTTTAAATTGCTTTTAAGACAATGATTTGCGCTTCTTTCTGATTTCTTCTTGGGGAGAAGAAACCCGTGGGCTGACGTTGCTGCGGGGCGCACTTTACAAGCCTTTGCCTTACAGTTCAACGCCCTATCGGTCAGGACTCATAAGGG |
Plasmid 11: cDNA insert. Arachis hypogaea cultivar JL24 phenylalanine ammonia-lyase (PAL) mRNA, GU477587.1 ACTGGTCTCGTAGACTGCGTACCCGACTAGTGAGCTATTACGCTTTCTTTAAAGGGTGGCTGCTTCTAAGCCAACCTCCTAGCTGTCTAAGCCTTCCCACATCGTTTCCCACTTAACCATAACTTTGGGACCTTAGCTGACGGTCTGGGTTGTTTCCCTTTTCACGACGGACGTTAGCACCCGCCGTGTGTCTCCCATGCTCGGCACTTGTAGGTATTCGGAGTTTGCATCGGTTTGGTAAGTCGGGATGACCCCCTAGCCGAAACAGTGCTCTACCCCCTACAGTGATACATGAGGCGCTACCTAAATAGCTATCGGTCAGGACTCATAAGGG |
Plasmid 12: cDNA insert Arachis hypogaea cultivar JL24 phenylalanine ammonia-lyase (PAL) mRNA,ID: GU477587.1 TGGTCTCGTAGACTGCGTACCCGACTAGTGAGCTATTACGCTTTCTTTAAAGGGTGGCTGCTTCTAAGCCAACCTCCTAGCTGTCTAAGCCTTCCCACATCGTTTCCCACTTAACCATAACTTTGGGACCTTAGCTGACGGTCTGGGTTGTTTCCCTTTTCACGACGGACGTTAGCACCCGCCGTGTGTCTCCCATGCTCGGCACTTGTAGGTATTCGGAGTTTGCATCGGTTTGGTAAGTCGGGATGACCCCCTAGCCGAAACAGTGCTCTACCCCCTACAGTGATACATGAGGCGCTACCTAAATAGCTATCGGTCAGGACTCATAAGGG |
Plasmid 13: cDNA insert Arachis hypogaea beta-ketoacyl-ACP synthetase II-like (KASII) mRNA, FJ358425.1 CTGGTCTCGTAGACTGCGTACCCGACTAACCCATGTGCAAGTGCCGTTCACATGGAACCTTTCCCCTCTTCGGCCTTCAAAGTTCTCATTTGAATATTTGCTACTACCACCAAGATCTGCACCGACGGCCGCTCCGCCCGGGCTCGCGCCCCAGGTTTTGCAGCGACCGCCGCGCCCTCCTACTCATCGCGGCATAGCCCTTGCCCCGACGGCCGGGTATAGGTCACGCGCTTCAGCGCCATCCATTTTCGGGGCTAGTTGATTCGGCAGGTGAGTTGTTACACACTCCTTAGCGGATGTCGGTCAGGACTCATAAGGG |
---|
Plasmid 14: vector DNA insert gnl|uv|U37573.1:2706-4083 Phagemid vector pBK-CMV (+/-homology) GGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGC. |
Plasmid 15:cDNA insert Arachis hypogaea profilin (Ara h 5) mRNA: AF059616.1 GGATTTTATTTAGGAGGTATTGGGAACGAATTGGAATGTAATAATATTGATTCATAGAGATCCAGAAGAAAAAGAATAATCTTCTACTTTGAGAATAATAAAAAAAGAAAAGTGTTCAATTGGAACATGAAAACCGTGACCTGACTGAATTAGTTCTCGTTATTTTTAGGGAAGGAGTGGAGATTATCGAACGAAGGATCCAATTACTTCGAAAGAATTGAACGAGGAGCCGTATGAGGTGAAAATCTCATGTACGGTTCTGTAGAGTGGCAGTAAGGATGACTTATCTGTCAACTTTTCCACTATTA |
Plasmid 16: cDNA insert Arachis hypogaea mitogen-activated protein kinase 2 mRNA, DQ068453.1 TAATAGTGGAAAAGTTGACAGATAAGTCATCCTTACTGCCACTCTACAGAACCGTACATGAGATTTTCACCTCATACGGCTCCTCGTTCAATTCTTTCGAAGTAATTGGATCCTTCGTTCGATAATCTCCACTCCTTCCCTAAAAATAACGAGAACTAATTCAGTCAGTCACGTTTTCATGTTCCAATTGAACACTTTTCTTTTTTTATTATTCTCAAAGTAGAAGATTATTCTTTTTCTTCTGGATCTCTATGAATCAATATTATTACATTCCAATTCGTTCCCAATACCTCCTAAATAAAATCCAAGGGC |
Plasmid 17 cDNA insert Arachis hypogaea ethylene-responsive element binding factor 1 (ERF1) mRNA, JQ048930.1 GAGTCCTGACCGAGAACGGCATTGATAGCGATGAGTCCTGACCGACAACGGCATTGATAGCGATGAGTCCTGACCGGGTACGCAGTCTACGAGACCAGTAA |
Plasmid 18 cDNA insert Arachis hypogaea triacylglycerol lipase 1 mRNA, GU902981.1: TGAGTCCTGACCGACAACGGCATTGATAGCGATGAGTCCTGACCGGGTACGCAGTCTACGAGACCAGTA |
Plasmid 19 cDNA insert Arachis hypogaea mitogen-activated protein kinase 2 mRNA: DQ068453.1GATTTTATTTAGGAGGTATTGGGAACGAATTGGAATGTAATAATATTGATTCATAGAGATCCAGAAGAAAAAGAATAATCTTCTACTTTGAGAATAATAAAAAAAGAAAAGTGTTCAATTGGAACATGAAAACGTGACTGACTGAATTAGTTCTCGTTATTTTTAGGGAAGGAGTGGAGATTATCGAACGAAGGATCCAATTACTTCGAAAGAATTGAACGAGGAGCCGTATGAGGNGAAAATCTCATGTACGGTTCTGTACAGTGGCAGTAAGGATGACTTATCTGTCAACTTTTNCACTATTACAAGGGCNAATTCGCGGCCNGTNAATCCAATTCGCC |
Plasmid 20: cDNA insert Arachis hypogaea arachin Ahy-3 mRNA, AY722687.1 GTCCTGACCGAGAACGGCATTGATAGCGATGAGTCCTGACCGACAACGGCATTGATAGCGATGAGTCCTGACCGACAACGGCATTGATAGCGATGAGTCCTGACCGGGTACGCAGTCTACG |
Plasmid 21: vector DNA insert gnl|uv|J01636.1:1-7477 E.coli lactose operon with lacI, lacZ, lacY and lacA genes. CGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTATACGTACGGCAGTTTAAGGTTTACACCTATA |
Plasmid 22: Vector DNA insert gnl|uv|J01749.1:1-4361-49 Cloning vector pBR322 TAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACNGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAG |
Plasmid 23: Vector DNA insert gnl|uv|U37573.1:2706-4083 Phagemid vector pBK-CMV CTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACNGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGC |
---|
Plasmid 24: Vector DNA insert gnl|uv|AF028837.1:1577-1628 Cloning vector pKILHIS-1 TGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCCTTGGCGGCGAATGGGCNTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAA |
Plasmid 25: Vector DNA insert gnl|uv|X65311.2:2262-2409 Cloning vector pGEM-7Zf(-) GCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATT |
Plasmid 27: Vector DNA insert gnl|uv|NGB00350.1:415-785 Invitrogen pZErO-2 vector multiple cloning site TCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCGGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCATGAGATTATCAAAAAGG |
Plasmid 29: vector DNA insert gnl|uv|U37573.1:2706-4083 Phagemid vector pBK-CMV GGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTC |
greenhouse, field plots, and greenhouse raised beds, and treated them with stoichiometric macro-mineral nutrient solutions; and obtained consistent good results on the RNA enzyme activity of GDH-synthesized RNA since 1998 [
Possibility that the repeated plus/plus and plus/minus sequence matches among the GDH-synthesized oligonucleotide RNAs (
The DNA insert of plasmid 14 shared sequence match with a section of gnl|uv|U37573.1 phagemid vector pBK-CMV, a component of TOPO TA cloning vector.
The cDNA insert of plasmid 15 shared a plus/plus sequence match with peanut Ara h5 mRNA, being the second copy of that RNA synthesized by GDH. These are evidence that the synthesis of RNA by GDH is reproducible although it is template-independent. The cDNA insert of plasmid 16 shared two plus/minus sequence matches with the mRNA encoding peanut mitogen-activated protein kinase 2 (DQ068453.1); and with the mRNA encoding peanut mitogen-activated protein kinase 3 (EU182580.2). Therefore, the same fragment of RNA synthesized by GDH shared homology with two different mRNAs. This is another example of the complex mechanism, may be involving silencing RNA mass action effect (organic chemical reaction) on mRNA silencing by GDH-synthesized RNA. The cDNA insert of plasmid 17 shared minus/minus sequence match with −1/−3 frame shift to the mRNA encoding Arachis hypogaeae thylene-responsive element binding factor 1 [
The DNA insert of plasmid 21 shared sequence match with a section of gnl|uv|J01636.1 E. coli lactose operon lacI, lacZ, lacY and lacA genes, a component of PCR 4 TOPO TA cloning vector. The DNA insert of plasmid 22 shared sequence match with a section of gnl|uv|J01749.1 of pBR322, a component of PCR 4 TOPO TA cloning vector. The DNA insert of plasmid 23 shared sequence match with a section of gnl|uv|U37573.1 phagemid pBK-CMV. The DNA insert of plasmid24 shared sequence match with a section of gnl|uv|X65311.2 vector pGEM-7Zf(−), component of TOPO TA cloning vector. The DNA insert of plasmid25 shared sequence match with a section of gnl|uv|X65310.2 vector pGEM-7Zf(+), component of TOPO TA cloning vector. The DNA insert of plasmid 27 shared sequence match with a section of gnl|uv|NGB00350.1 Invitrogen pZErO-2 vector multiple cloning site, component of TOPO TA vector. The DNA insert of plasmid 29 shared sequence match with a section of gnl|uv|U37573.1 phagemid vector pBK-CMV, a component of TOPO TA cloning vector. Cloning vector DNA inserts 14, 23, and 29 are different lengths from the same pBK-CMV section of TOPO TA vector.
Therefore, the GDH-synthesized RNA oligonucleotide units (
In vitro demonstration of the RNA enzyme activity of GDH-synthesized RNA reveals a smart approach to save time, space and effort on basic research experimentations with plants because preliminary surveys of the responses of plant metabolism to the environment could be conducted at reduced scales in growth chambers, greenhouse, and field plots specifically to collect sufficient tissues for total RNA and GDH purifications; the phenotypic responses of the plant to mineral nutrients, biochemical regulators, agro-chemicals and other xenobiotics being conducted and interpreted in biochemical cross-over reactions as in
Dismantling of the structural constraints imposed on RNA by genetic code liberated RNA to become an enzyme with specificity to degrade unwanted transcripts not on base-pairing as in double stranded siRNA, but on the basis of homologous sequence alignment recognition (
GDH also synthesizes unit oligonucleotides that silence its own encoded mRNAs (
The cDNAs of GDH-synthesized RNA are the Northern blot tools for unraveling the molecular mechanisms underlying the multitude of metabolic networks and biochemical regulations in the cell. Since the GDH-synthesized RNA matches transcript sequences in repeated plus/plus, minus/minus, and/or plus/minus homologous orientations [
In the application of algorithm-designed siRNAs for the silencing of transcripts, it has been reported that siRNA efficacy is limited by target transcript abundance and turnover rate [
This research project was supported by the Evans Allen fund made available to Prairie View A & M University, Prairie View, Texas by USDA NIFA. Appreciation to Drs. Peter Ampim, and Aruna Weerasooriya for discussions on the stoichiometric compositions of the mineral salt mixes.
The authors declare no financial competing interests.
Osuji, G.O. and Johnson, P.M. (2018) Structural Properties of the RNA Synthesized by Glutamate Dehydrogenase for the Degradation of Total RNA. Advances in Enzyme Research, 6, 29-52. https://doi.org/10.4236/aer.2018.63004