Description

1. Introduction

With the emergence of molecular chemistry and the potential of chimerenomics to expand the sciences and technologies of medicine [1], it is now expedient to begin to lay down the foundation, principles, and practices of this innovative research subject. Although the substrate specificities of glutamate dehydrogenase redox cycle hexameric isoenzymes have been identified and optimized [2], large-scale productions of chimerenomic RNAs are needed to support the research and for technology. To optimize the yield of the product chimerenomic RNA, the reacting concentrations of the different GDH hexameric isoenzymes need to be optimized with respect to their respective isoelectric point (pI) values because they are also substrates in the Schiff base reaction, which is the committed step of the chimerenomic RNA synthesis (GDH chigramming). The aim of these research programs was to determine the properties of the chimerenomic pump for the kilogram-scale production of chimerenomic RNAs. No scholars have sought to optimize the Schiff base reaction chemistry [3; 4; 5], and not for the chimerenomic RNA synthetic activities of GDH redox cycle isoenzymes.

2. Methods

2.1. Multi-Dimensional Electrophoretic Purification of GDH Hexameric Isoenzymes

GDH was extracted from cultured cells (130000 cells) of homo sapiens, or from leaves/viable seeds (15 – 30 g) of higher plants [6; 7]. The dialyzed extract was subjected to free solution isoelectric focusing methodology of Bio-Rad [7] to arrange the GDH hexameric isoenzymes in the gradient of their isoelectric point (pI) values. The isoelectric focusing ampholyte was Bio-Rad’s Bio-Lyte 3/10. Ribonuclease A (1 unit per ml of the extraction buffer solution), and deoxyribonuclease 1 (2 Units per ml of the extraction buffer solution) were added to the 0.1 M Tris-HCl pH 8 extraction buffer. After isoelectric focusing, the pH values of the Rotofor fractions were recorded before the fractions were dialyzed to remove the urea and ampholyte.

Aliquots (200 µL) of the dialyzed Rotofor fractions were subjected to native 7.5% polyacrylamide gel (PAG) electrophoresis (Bio-Rad protean II xi cell) in duplicate. After the gel electrophoresis, one gel was stained with the phenazine methosulfate-glutamate-NAD+-tetrazolium bromide solution [7] to locate the positions of the GDH isoenzymes. GDH isoenzyme distribution pattern in the gel landscape was photo-documented. Using the stained gel as guide/template on a lightbox, the location of the GDH isoenzymes was excised from the duplicate electrophoresed gel [7]. If there are no GDH bands in the stained PAG landscape, then major technical errors occurred upstream in the initial steps of the pump, and the whole procedure must be started all over after due trouble shooting process. The GDH isoenzymes were electro-eluted in 0.05 M Tris-HCl solution from the excised piece of gel using Bio-Rad whole gel eluter at −20˚C [7]. The fractions from the whole gel eluter were not combined.

2.2. GDH Chigramming: The Synthesis of Chimerenomic RNA Enzyme

Synthesis of chimeres was conducted in the amination substrate and deamination substrate solutions in separate tubes [8] of the chigrammer/chimmerization cell. The fractions from Bio-Rad whole gel eluter were combined as follows to make 4 groups:

Group 1: acidic isoenzymes (pI 5.1) whole gel fractions 3 and 4.

Group 2: mildly acidic isoenzymes (pI 6.3) whole gel fractions 5 and 6.

Group 3: neutral isoenzymes (pI 7.2) whole gel fractions 7, 8, and 9.

Group 4: mildly alkaline isoenzymes (pI 8.5) whole gel fractions10, and 11.

For demonstrating the arrays of chimeres chigrammed in the amination direction, the substrate solutions were prepared in 0.1 M Tris-HCl buffer (pH 7.5) containing the four ribo-NTPs (0.6 mM each), CaCl₂ (3.5 mM), NH₄Cl (0.875 mM), α-ketoglutarate (10.0 mM), NADH (0.225 mM), 5 Units RNase inhibitor, 10 Units of DNase 1, and 5 µg of actinomycin D. The reaction cocktail (0.4 mL) was pipetted into each of four 15 mL chigrammer tubes on ice bath. The chigrammer tubes were numbered groups 1 to 4 for the GDH isoenzymes. Reaction was started by adding 0.4 mL of whole gel-eluted GDH isoenzymes containing 5 - 11 µg protein per mL to the respective chigrammer tube. The final volume of reaction was adjusted to 1.0 mL with 0.1 M Tris-HCl buffer solution pH 7.5. Reaction was incubated at 16˚C for 60 min in the chimere chigrammer; and stopped by phenol-chloroform (pH 5.5) removal of proteins [6; 7]. The chimerenomic RNA enzyme was precipitated with ethanol, pelleted (6000 g, 15 min), the pellet was air-dried to remove phenol and ethanol, then dissolved in minimum volume of molecular biology quality water; and stored at −20˚C in preparation for analysis. RNA enzyme yield and quality were verified by photometry and by agarose gel electrophoresis using RNA molecular weight markers as standards [6; 7]. The agarose gel was stained with ethidium bromide solution, and the RNA yield/distribution pattern was photo documented. If there are no RNA bands in the ethidium bromide-stained agarose gel landscape, then major human-technical errors occurred upstream in the initial steps of the pump, and the whole procedure must be started all over after due trouble shooting process.

In the deamination direction, the substrate solutions were prepared in 0.1 M Tris-HCl buffer pH 9.0 containing the four ribo-NTPs (0.6 mM each), CaCl₂ (3.5 mM), L-glu (3.23 µM), NAD⁺ (0.375 µM), 5 Units RNase inhibitor, 10 Units DNase 1, and 5 µg of actinomycin D. The reaction cocktail (0.4 mL) was similarly setup in each of four 15 mL chigrammer tubes on ice bath. The chigrammer tubes were labeled groups 1 to 4 for the GDH isoenzymes. Reaction was started by adding 0.4 mL of whole gel-eluted GDH charge isomers containing 5 - 11 µg protein per mL to the respective chigrammer tube. The final volume of reaction in each chigrammer tube was brought to 1.0 mL with 0.1 M Tris-HCL buffer solution pH 9.0. The purified GDH isoenzymes were same as those used for the amination reaction. Reactions was incubated at 16˚C for 60 min in the chimere chigrammer and stopped by phenol-chloroform (pH 5.5) removal of the proteins. The chimerenomic RNA enzyme was precipitated with ethanol, then pelleted (6000 g, 15 min), the pellet was air-dried, and dissolved in minimum volume of molecular biology quality water; and stored at −20˚C before use. The RNA yield and quality were verified by photometry and by agarose gel electrophoresis using RNA molecular weight markers as standards. The agarose gel was stained with ethidium bromide solution, and the RNA yield/distribution pattern was photo documented. If there are no RNA bands in the ethidium bromide-stained agarose gel landscape, then major human-technical errors occurred upstream in the initial steps of the pump, and the whole procedure must be started all over after due trouble shooting process.

2.3. Quality Assurance Determinations of the Chimeres

This was done through conversion of the chimeres to cDNA followed by their restriction enzyme digestion. cDNAs were synthesized with 2 µg of each product chimere synthesized by the whole gel-eluted GDH isoenzymes in the deamination direction, and amination direction, using random hexamer primer. Digestion of the cDNA with taq 1 restriction enzyme; ³²P-labeled adapter and linker ligations to the restriction fragments; restriction fragment PCR amplification; sequencing gel fractionation to remove excess ³²P-labeled adapter and ³²P-labeled NTP [9] were conducted according to the methods of Display Systems Biotech, Vista, CA, USA. The sequencing gel was radio autographed. The resulting images (bands) were scanned and quantified using the Un-Scan-it digitalizing software of Silk Scientific Inc., UT, USA. The band intensities of the chimerenomic RNAs synthesized by the neutral isoenzymes (pI 7.2) in the amination direction were applied as baseline for normalizing and for the calibration of the results.

3.0. Results

3.1. The Yields of Chimerenomic RNA Fragments

The bands in the sequencing gel (Figure 1) were semi-quantitatively normalized with the bands in lane C as baseline, and it was assigned the value of a unit. Chimerenomic RNA chimmerized in the amination direction by the acidic, and mildly acidic GDH isoenzymes (Figure 1, lanes A, and B), were semi-quantitatively higher than those synthesized by the same GDH hexameric isoenzymes in the deamination direction. Also, the yields of RNA fragments synthesized by the mildly alkaline GDH isoenzymes (Figure 1, lane H) in the deamination direction were about three folds higher than those synthesized in the amination direction (Figure 1, lane D).

Figure 1

Legend to Figure 1: The chimerenomic pump. Projection of the outcomes of the integrated free solution isoelectric focusing, and polyacrylamide gel electrophoretic purification of NADH-glutamate dehydrogenase redox cycle hexameric isoenzymes, and chigramming of kilogram yields of plus-chimerenomic RNA in the amination direction and minus-chimerenomic RNA in the deamination direction in an infinitely reciprocating chigramming cycles. The chimeres chigrammed by the acidic isoenzymes (pI 5.1) in the amination direction (lane A), and deamination direction (lane E); mildly acidic isoenzymes (pI 6.3) in the amination direction (lane B), and deamination direction (lane F); neutral isoenzymes (pI 7.2) in the amination direction (lane C), and deamination direction (lane G); mildly alkaline isoenzymes (pI 8.0) in the amination direction (lane D), and deamination direction (lane H) were used for cDNA synthesis. The cDNAs were digested with taq 1 restriction enzyme, the fragments were amplified by Double Differential PCR method, and the products were fractionated on sequencing polyacrylamide gel. Note the differential yields of product chimeres by GDH Shiff bases according to the pI values of the GDH isoenzymes, and amination versus deamination in the chigrammer (Osuji et al., 2021).

These observations suggest that the GDH redox cycle consists of two main cycles of unequal activities in the chigramming (synthesis) of chimerenomic RNA; the acidic, mildly acidic, and neutral GDH isoenzymes being very active in the amination direction, whereas the mildly alkaline isoenzymes were more active in the deamination direction. The RNase and DNase that were added to the GDH extraction buffers hydrolyzed all the genetic code-based nucleic acids and assured that the resulting oligonucleotide products were removed during the multi-dimensional electrophoretic purification of GDH hexameric isoenzymes [9].

3.2. Fractionation on the Sequencing Gel and the Restriction Fragments

GDH amination reaction (left side) and deamination reaction (right side) were molecularly demonstrated in chemistry, visually in real-time (Figure 1). The cDNA bands in Figure 1, lane A were reverse-transcribed from the chimerenomic RNAs chigrammed in the aminating direction by the acidic GDH isoenzymes (pI 5.1); whilst the cDNA bands in Figure 1, lane E were reverse-transcribed from the chimerenomic RNAs chigrammed in the deaminating direction by the same pI 5.1 GDH isoenzymes. In this molecular chemistry approach, GDH amination was physically separated from the deamination reaction without introduction of any inhibitors into the chigramming reactions [10].

The cDNA bands in Figure 1, lane B were reverse-transcribed from the chimerenomic RNAs chigrammed by the mildly acidic GDH isoenzymes (pI 6.3) in the aminating direction; whilst the bands in Figure 1, lane F were reverse-transcribed from the chimerenomic RNAs chigrammed by same mildly acidic GDH isoenzymes (pI 6.3) in the deaminating direction.

The cDNA bands in Figure 1, lane C were reverse-transcribed from the chimerenomic RNAs chigrammed by the neutral GDH isoenzymes (pI 7.2) in the aminating direction; whilst the bands in Figure 1, lane G were reverse-transcribed from the chimerenomic RNAs chigrammed by same pI 7.2 neutral GDH isoenzymes in the deaminating direction.

The cDNA bands in Figure 1, lane D were reverse-transcribed from the chimerenomic RNAs chigrammed by the alkaline GDH isoenzymes (pI 8.5) in the aminating direction; whilst the bands in Figure 1, lane H were reverse-transcribed from the chimerenomic RNAs chigrammed by same pI 8.5 alkaline GDH isoenzymes in the deaminating direction.

GDH hexameric isoenzymes chigrammed plus-RNA enzyme in the amination direction; but minus-RNA enzyme in the deamination direction [6; 7]. These molecular chemistry models (Figure 1) of the opposite chemical differences in chimerenomic RNA primary structure illuminate the hitherto inexplicable phenomena shrouding the reductive amination and the oxidative deamination activities of GDH and are crucially important for understanding the molecular logic of the regulation of metabolism and biological processes in higher organisms. They confirm that the chigramming of chimerenomic RNAs is a cycle [6] of different irreversible chemical reactions driven by NADH/NAD⁺, and α-KG/L-Glu/NH₄⁺ couples rather than a classical reversible oxidation-reduction reaction. The plus/minus chimerenomic RNA chigramming by GDH isoenzymes further confirms the existence of orientation and positionality of the subunit polypeptides in the active hexameric isoenzymes [1], a structure that only the chimerenomic pump preserved. In the GDH cycle model, the plus-RNA is not reversibly oxidized to minus-RNA. Conversely, the minus-RNA is not reduced to plus-RNA. The chimerenomic circulating pump [6] for the chigramming of minus-RNA and plus-RNA is non-reversible. Therefore, the function of NADH/NAD+, and α-KG/L-Glu/ NH₄⁺ in biology is to establish the redox homoeostasis so that GDH can chimmerize plus-RNA and minus-RNA. In a single sweep of the cycle, GDH isoenzymes chimmerize four folds of the quantities of chimerenomic RNA products of opposite primary structures. The chimerenomic pump is akin to a dual cycling infinitely reciprocating machine complex. This made for thermodynamic efficiency with the spinning out of kilogram quantities of chimerenomic RNA, and economy in the utilization of NADH/NAD⁺, and of α-KG/L-Glu/NH₄⁺.

The reciprocity of the GDH chigramming reaction is clearly demonstrated (Figure 1), internally within the amination reaction domain, and again internally within the deamination reaction domain. The chimerenomic RNA band intensities in lanes A and B were approximately double those of lanes C and D. The converse is the case in the deamination reactions because the band intensities in lanes G and H approximately quadrupled those in lanes E and F. Then furthermore, the total band intensity under the aminating reaction approximately tripled that of the deamination reaction, thereby creating several reciprocal relationships in the maximization of the yield of the product chimerenomic RNAs.

The plus-RNA, and minus-RNA chigramming is the core of the molecular chemistry of GDH; ensuring that the chimerenomic RNA enzymes are very complicated in primary and secondary structures to match the equally complex structures of the mRNA they degrade. No other template-independent RNA polymerization apparatus can chigramm two types of opposite polarity complicated RNAs simultaneously with such astonishing cyclical chemical repeatability and fidelity. The chigramming of complicated plus-RNA and minus-RNA by GDH is crucial for understanding the molecular reprogramming of metabolic processes consequent upon the degradation of superfluous total RNA by chimerenomic RNA in response to physicochemical changes in the environmental conditions [1]. Hexameric isoenzymes of GDH may henceforth be assayed comprehensively by monitoring the kinetic and thermodynamic transformations of the NADH/NAD⁺, and/or by the α-KG/L-Glu/NH₄⁺ homeostasis at the protein level.

3.3. Effects of the Reaction pH and the pI of the GDH Schiff Base Complex on the Yield Efficiency of Chimerenomic Pump.

The two reactions occurring during the production of chimerenomic RNAs were neatly separated in the radioautographs (Figure 1). The first reaction was the GDH-linked Schiff base formation (GDH-chigramming) which is a base or acid catalyzed reaction [5] that is very important in the synthesis of L-glu by GDH. The second reaction was the GDH-Schiff base dependent polymerization [3] (GDH-chimmerization) of ribo-NTPs (monomers) to give chimerenomic RNA. Because the first reaction requires the GDH-Schiff base to be protonated, the neutral pH of the reaction buffer solution together with the low pI values of the GDH hexameric isoenzymes moderately promoted both the Schiff base formation {chigramming) and moderate yields of the product chimerenomic RNA. Conditions in the deamination reaction were the exact opposite because the moderately higher pH value of the buffer solution made for incomplete protonation of the Schiff base thereby enhancing the polymerization (chimmerization) reaction step to achieve the extraordinarily optimal yields of product chimerenomic RNA of higher molecular weight under lane H (Figure 1) by the alkaline (pI 8.2) GDH hexameric isoenzymes. The Schiff base reaction mechanism incorporates an initial condensation/addition step that is followed by the polymerization (chimmerization) of activated monomers [3]. The GDH isoenzymes in lanes E, F, and G being mildly acidic were inefficient in the chimmerization reaction step. The above is the rugged fail-proof 8-cylinder (lanes A, B, C, D, E, F, G, H) biological engine (Figure 1) that supports the production of chimerenomic RNA enzyme by the chimerenomic pump.

Wherefore, chimerenomic pump consists of free solution IEF cell, polyacrylamide gel electrophoresis cell, whole gel eluter, and chigramming/chimmerization cell seamlessly integrated via electronic automation.

Appendix:

New Terminologies: Chimerenomic glossary for the new terminologies in life sciences.

1. These new terminologies in the life sciences describe the discovery of template-independent RNA processes that control the survival of cells and organisms other than genes. The building blocks of this life regulatory system are made up of a unique type of RNA that are not coded nor synthesized through the genetic code. These template-independent RNAs are called chimerenomic RNAs.

2. Chimerenomics: Is the study of everything (NTintis, chigramming, GDH-chimmerization, chimere etc) about chimerenomic RNAs. Chimerenomics confers molecular chemistry pluripotency and totipotency to all cells and tissues. Chimerenomics are the processes by which whole organisms, cells and tissues differentiate, develop and grow by chigramming chimerenomic RNAs that interact with the changing physicochemical internal and external environmental conditions thereby reprogramming and optimizing those metabolic reactions that assure the continued survival of the organism.

3. Chimere: Is the minimum length of nucleotide sequence that can degrade homologous mRNA and other genetic code-based RNAs. Chimere is the active segment of NTinti.

4. NTinti: This is the chimerenomic RNA molecule chigrammed (synthesized) by NADH-glutamate dehydrogenase hexameric redox cycle isoenzymes (GDH) in response to a specific environmental change. NTinti is also synthesized naturally in vivo during normal tissue differentiation, growth and development. Therefore, NTinti can be cell or tissue specific. One NTinti has more than one chimere.

5. Chigramming: This is the process of synthesis of chimeres and NTintis by GDH. It is a spontaneous process. It is the conversion of the electromagnetic changes in the environmental conditions of cells, tissues, whole organisms to the nucleotide sequences of chimerenomic RNAs.

6. GDH-Chimmerization: This is the formation of chemical Schiff base of GDH isoenzymes in response to a new environment leading to new hexameric isoenzyme complexes. This is the initiation process for chigramming.

7. Functional chimerenomics: This is the study of the biological functions of chimere and NTinti.

8. Differential chimerenomics: This is the comparison study of the chimeres and NTintis from same or different tissues under various environmental conditions.

9. Other terminologies

a). Clinical chimerenomics: The application of chimerenomics to clinical studies.

b). Chimerenomic Medicine: This is the application of chimerenomics to primary care in the prevention and treatment of human disorders, diseases and wellness conditions in humans.

c). Chimerenomic Chemistry

d). Chimerenomic Physiology

e). Molecular Chimerenomics

f). Chimerenomic Biology

g). Chimerenomic Pharmacology etc.