Recombinant Carassius auratus NADH-ubiquinone oxidoreductase chain 3 (MT-ND3)
Description
Genetic Context and Mitochondrial Positioning
The MT-ND3 gene in Carassius auratus is located within the mitochondrial genome, which is a circular double-stranded DNA molecule approximately 16,576 bp in length in related species such as Carassius auratus var. pingxiangnensis . This gene is one of 13 protein-coding genes in the mitochondrial genome, which also contains 22 transfer RNA genes, 2 ribosomal RNA genes, and a non-coding control region . The mitochondrial genome of Carassius auratus has a base composition estimated to be 29.70% A, 26.74% C, 15.35% G, and 28.21% T, reflecting the typical nucleotide bias seen in fish mitochondrial DNA .
Genomic Organization and Sequence Features
In the mitochondrial genome of Carassius species, MT-ND3 exhibits several distinctive organizational features. The gene demonstrates overlaps with adjacent genes, a common characteristic in the compact mitochondrial genome. Specifically, there is an overlap between ND3 and tRNAArg in Carassius auratus var. pingxiangnensis, indicating the economical use of genetic material in mitochondrial DNA . This overlapping arrangement is part of a broader pattern within the mitochondrial genome, where genes overlap by a total of 40 bp in 11 different locations ranging from 1 to 14 bp in length .
Protein Structure and Transmembrane Configuration
The ND3 protein encoded by MT-ND3 is characterized by its highly hydrophobic nature, making it one of the most hydrophobic subunits of Complex I . This hydrophobicity reflects its primary function as part of the core transmembrane region of the NADH dehydrogenase complex. The structure of Complex I is L-shaped, with a long hydrophobic transmembrane domain (where ND3 is located) and a hydrophilic domain forming the peripheral arm that contains the redox centers and NADH binding site .
Role in Mitochondrial Energy Production
The ND3 protein, as a component of Complex I (NADH dehydrogenase), plays a critical role in cellular energy production through oxidative phosphorylation. This complex is responsible for catalyzing the transfer of electrons from NADH to ubiquinone, coupled with proton translocation across the inner mitochondrial membrane . This proton pumping contributes to the electrochemical gradient that drives ATP synthesis, making ND3 an essential component of cellular energy metabolism in Carassius auratus.
Evolutionary Significance in Cyprinid Fishes
MT-ND3 has significant evolutionary importance in Carassius auratus and related species. Analysis of mitochondrial DNA, including MT-ND3, has been used to determine phylogenetic relationships among different Carassius species and to understand evolutionary divergence patterns . Research on goldfish-like hybrid lineages has shown that mtDNA variations, including those in ND3, can provide evidence of hybridization events and the subsequent generation of new lineages .
In studies of mitochondrial genome evolution, comparative analysis of MT-ND3 and other mitochondrial genes has revealed that goldfish (GF) might have diverged from red crucian carp (RCC) after RCC diverged from koi carp . This finding, supported by genetic distance calculations based on mtDNA control region sequence comparisons, highlights the importance of MT-ND3 in understanding the evolutionary history of Carassius species.
Research Applications
Recombinant Carassius auratus MT-ND3 serves as a valuable tool for various research applications. The availability of purified recombinant protein facilitates studies on:
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Structure-function relationships of mitochondrial complex I components
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Comparative analysis of ND3 across different fish species
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Investigation of the role of specific amino acid residues in protein function
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Development of antibodies against ND3 for immunological studies
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In vitro reconstitution experiments to understand complex I assembly
Evolutionary and Phylogenetic Studies
The MT-ND3 gene has proven useful in evolutionary studies of Carassius species. By analyzing variations in MT-ND3 sequences, researchers have gained insights into:
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Divergence times between related cyprinid species
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Patterns of mitochondrial DNA inheritance and potential paternal leakage
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Evidence of hybridization events between fish species
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Mutation rates and selection pressures on mitochondrial genes
For example, studies on red crucian carp-like fish lineage and goldfish-like fish lineage have utilized MT-ND3 and other mitochondrial genes to identify heritable chimeric DNA fragments and mutant loci, providing evidence that hybridizations might lead to changes in mtDNA and the subsequent generation of new lineages .
Organization Within the Mitochondrial Genome
In Carassius auratus and related species, MT-ND3 is encoded on the heavy strand (H-strand) of the mitochondrial genome, as are most mitochondrially encoded genes . Within the mitochondrial genome of Carassius auratus var. pingxiangnensis, only ND6 and eight tRNA genes are encoded on the light strand (L-strand) . This genomic organization reflects the typical pattern seen in vertebrate mitochondrial genomes.
Sequence Conservation and Variation
Comparative analysis of MT-ND3 across different cyprinid species has revealed patterns of sequence conservation and variation that reflect both functional constraints and evolutionary divergence. The gene contains regions that are highly conserved, particularly those encoding functionally critical portions of the protein, while other regions show greater variability .
Analysis of the central conserved blocks and other conserved blocks within the mitochondrial control region, which influences the expression of genes including MT-ND3, has shown similarities among Carassius auratus var. pingxiangnensis and six other cyprinids with different ploidies . This conservation suggests functional importance of these regulatory regions in the expression of mitochondrial genes.
Reconstitution and Handling
When working with lyophilized recombinant Carassius auratus MT-ND3, proper reconstitution is critical for maintaining protein activity. Commercial preparations recommend brief centrifugation prior to opening to ensure all material is at the bottom of the vial . The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and for long-term storage, addition of glycerol (5-50% final concentration) is recommended .
Product Specs
Note: While we prioritize shipping the format currently in stock, we are happy to accommodate your specific format requirements. Please indicate your preference in the order notes section, and we will do our best to fulfill your request.
Note: All protein shipments are standardly packaged with blue ice packs. If you require dry ice for shipping, please inform us in advance, as additional fees may apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. Lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
Q&A
How does the structure of Carassius auratus MT-ND3 compare to its homologs in other species?
When comparing MT-ND3 across species, researchers should examine:
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Sequence homology through multiple sequence alignments
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Conservation of functional domains
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Structural prediction using hydrophobicity plots
Studies comparing mitochondrial genomes have shown that while MT-ND3 is highly conserved among vertebrates, there are species-specific variations that may correlate with environmental adaptations. For example, specific SNPs in MT-ND3 have been associated with high-altitude adaptation in Tibetan yaks and cattle, with mutations m.9893 A>G, m.9932 A>C, and m.10155 C>T showing negative associations with high-altitude adaptation (p < .003), while m.10073C>T showed positive association (p < .0006) .
What are the challenges in expressing and purifying recombinant Carassius auratus MT-ND3?
Expressing and purifying recombinant MT-ND3 presents several challenges:
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High hydrophobicity causing potential aggregation
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Maintaining proper folding in heterologous expression systems
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Preserving functional integrity during purification
Methodology recommendation: Express the protein with an N-terminal His-tag in E. coli, followed by lyophilization to increase stability. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage at -20°C/-80°C . Avoid repeated freeze-thaw cycles as this significantly reduces protein activity.
What are the optimal conditions for storage and handling of recombinant Carassius auratus MT-ND3?
Based on experimental data, the following protocol is recommended:
| Parameter | Recommended Condition |
|---|---|
| Storage temperature | -20°C to -80°C |
| Buffer | Tris/PBS-based buffer, 6% Trehalose, pH 8.0 |
| Form | Lyophilized powder |
| Reconstitution | In deionized sterile water (0.1-1.0 mg/mL) |
| Stabilizer | 5-50% glycerol (final concentration) |
| Working aliquots | Store at 4°C for up to one week |
| Freeze-thaw cycles | Minimize; repeated cycles significantly reduce activity |
Prior to opening, centrifuge the vial briefly to bring contents to the bottom. After reconstitution, aliquot the protein to avoid repeated freeze-thaw cycles .
What techniques are most effective for studying MT-ND3 mutations and their functional implications?
For comprehensive analysis of MT-ND3 mutations, a multi-faceted approach is recommended:
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Genetic screening: Whole-genome sequencing (WGS) and Sanger sequencing of mitochondrial DNA from skeletal muscle and other tissues.
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Heteroplasmy quantification: Last-cycle hot PCR to determine levels of mutated mtDNA in different tissues.
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Functional assessment:
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Respiratory chain activity measurements
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ATP production assays using substrates specific to Complex I
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Cellular oxygen consumption rate (OCR) measurements
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Morphological investigations: Electron microscopy to identify mitochondrial abnormalities such as enlarged mitochondria with paracrystalline inclusions .
These approaches are particularly important when investigating novel mutations, as demonstrated in cases where MT-ND3 mutations were linked to sensorimotor axonal polyneuropathy .
How can researchers effectively assess the functional impact of MT-ND3 variants in model systems?
A comprehensive functional assessment should include:
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In vitro enzyme activity assays:
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Measure Complex I activity using isolated mitochondria
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Quantify NADH:ubiquinone oxidoreductase activity
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Compare ATP production rates with different substrates
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Cellular models:
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Generate cell lines expressing MT-ND3 variants using CRISPR/Cas9
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Assess mitochondrial membrane potential
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Measure reactive oxygen species (ROS) production
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Evaluate cellular respiration using Seahorse XF analyzers
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Organismal models:
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Create transgenic zebrafish (a close relative to goldfish) expressing MT-ND3 variants
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Assess swimming behavior and endurance
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Measure oxygen consumption in different tissues
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Data interpretation should consider the heteroplasmy level, as the phenotypic expression of mitochondrial mutations often depends on the proportion of mutated mtDNA present in tissues .
How does MT-ND3 variation contribute to evolutionary adaptation in Carassius auratus and related species?
MT-ND3 shows evidence of adaptive evolution across species, particularly in response to environmental pressures:
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In high-altitude adapted species, specific haplotypes (H1 and H5) in MT-ND3 show positive associations with high-altitude adaptability, while others (H3) show negative associations (p < .0014) .
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In goldfish-like hybrids, mtDNA recombination and sequence variations have been identified, particularly in triploid fish derived from distant hybridization .
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Comparative analysis of genetic distances based on mtDNA control region sequences shows:
These variations likely reflect adaptations to different energetic demands across environments, suggesting MT-ND3's role in metabolic adaptation.
What methods are most appropriate for studying MT-ND3 in the context of hybrid species and mitochondrial heteroplasmy?
For studying MT-ND3 in hybrid species and cases of heteroplasmy, researchers should employ:
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Deep sequencing approaches:
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Ultra-deep sequencing of mitochondrial DNA
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Single-cell sequencing to detect heteroplasmy at the cellular level
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Quantitative PCR methods:
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Last-cycle hot PCR for heteroplasmy quantification
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Digital droplet PCR for absolute quantification of variant frequencies
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Restriction fragment length polymorphism (RFLP) analysis:
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Phylogenetic analysis:
These methods are particularly valuable when investigating mitochondrial inheritance in hybrids, as seen in goldfish-like hybrid lineages where paternal mtDNA fragments were found to be stably embedded in the mtDNAs, forming chimeric DNA fragments .
How can Carassius auratus MT-ND3 be used as a model for understanding mitochondrial diseases in humans?
Carassius auratus MT-ND3 provides a valuable model for understanding human mitochondrial diseases for several reasons:
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Functional conservation: The core function of Complex I is highly conserved between fish and humans, making functional studies translatable.
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Disease modeling opportunities:
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Experimental advantages:
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Fish models offer unique advantages for visualizing mitochondrial dynamics in vivo
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Transparency of fish embryos allows real-time imaging of mitochondrial function
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Higher throughput than mammalian models for drug screening
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Researchers should incorporate the following methodological approaches:
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CRISPR/Cas9-mediated introduction of disease-associated mutations
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Behavioral testing to assess neurological function
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Histological assessment of affected tissues
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Biochemical measurement of respiratory chain complex activities
What are the key considerations when using recombinant MT-ND3 to study complex I deficiency disorders?
When using recombinant MT-ND3 to study complex I deficiency disorders, researchers should consider:
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Mutation selection: Choose mutations that correspond to known pathogenic variants in humans, such as those identified in Leigh Syndrome or sensorimotor axonal polyneuropathy .
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Protein stability and folding: MT-ND3 mutations may affect protein stability and incorporation into complex I. Assess:
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Protein half-life using pulse-chase experiments
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Assembly into complex I using blue native PAGE
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Structural changes using circular dichroism spectroscopy
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Functional assays:
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Complex I activity measurements in reconstituted systems
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ATP production assays with specific substrates
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ROS production as a marker of electron leakage
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Tissue specificity:
This multi-faceted approach is essential as no single assay can fully characterize the impact of MT-ND3 variants on complex I function.
How can recombinant Carassius auratus MT-ND3 be applied in environmental toxicology research?
Recombinant Carassius auratus MT-ND3 offers unique applications in environmental toxicology:
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Biomarker development:
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MT-ND3 expression and modification patterns can serve as biomarkers for mitochondrial toxicity
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Changes in complex I activity can indicate exposure to specific environmental toxicants
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Mechanistic toxicology studies:
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Direct binding assays between recombinant MT-ND3 and suspected toxicants
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Structure-activity relationship studies for compounds that inhibit complex I
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In vitro screening platforms:
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Development of high-throughput assays using purified recombinant MT-ND3
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Competitive binding assays to identify compounds that displace natural substrates
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Aquatic ecotoxicology applications:
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Goldfish are common aquatic test organisms for environmental monitoring
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MT-ND3 functionality can be linked to whole-organism responses to pollutants
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Methodological approach: Combine in vitro biochemical assays with in vivo exposures, correlating molecular changes with physiological outcomes at the organism level.
What role does MT-ND3 play in adaptation to extreme environments, and how can this be studied experimentally?
MT-ND3 has been implicated in adaptation to extreme environments, particularly high-altitude conditions:
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Genetic evidence:
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Experimental approaches:
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Biochemical characterization of MT-ND3 variants under different oxygen tensions
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Respirometry studies comparing complex I efficiency at varying oxygen concentrations
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Measurement of ROS production as an indicator of electron transport chain efficiency
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Comparative studies:
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Analyze MT-ND3 sequences across related species adapted to different environments
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Correlate sequence variations with environmental parameters (altitude, temperature, etc.)
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Assess convergent evolution patterns in MT-ND3 across independent lineages
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Functional genomics:
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Generate transgenic models expressing MT-ND3 variants from high-altitude adapted species
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Assess phenotypic responses to hypoxic conditions
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Measure metabolic parameters under environmental stress conditions
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This research has broader implications for understanding the molecular basis of adaptation to challenging environments across species, including potential applications to conservation biology and climate change research .
