Recombinant Vampyressa thyone NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is MT-ND4L and what is its function in Vampyressa thyone?

MT-ND4L is a mitochondrially encoded gene that provides instructions for making the NADH dehydrogenase 4L protein. In Vampyressa thyone, as in other mammals, this protein functions as a subunit of respiratory chain Complex I (NADH dehydrogenase/ubiquinone oxidoreductase). This complex is embedded in the inner mitochondrial membrane and plays a crucial role in oxidative phosphorylation by transferring electrons from NADH to ubiquinone in the first step of the electron transport chain, ultimately contributing to ATP production, the cell's primary energy source .

The MT-ND4L protein in V. thyone, like its human counterpart, is likely a highly hydrophobic component forming part of the core transmembrane region of Complex I. The protein is approximately 11 kDa in size and composed of 98 amino acids . Its conservation across species suggests its fundamental importance in cellular energy metabolism.

How is Vampyressa thyone distributed geographically, and how might this affect MT-ND4L research?

Vampyressa thyone is a neotropical frugivorous bat with a distribution ranging from Mexico to Bolivia. Recent research has extended the known distribution range of this species to the state of Guerrero in Mexico, which represents the northwesternmost record, extending the species' distribution limit by more than 153 km .

For MT-ND4L research, this expanded geographical distribution is significant because mitochondrial DNA analysis has revealed three distinct geographic clades within V. thyone. The Mexican specimens, particularly, represent the most divergent cytochrome-b sequences within the species . This geographical genetic divergence suggests potential variation in the MT-ND4L gene across different populations, which researchers should consider when selecting specimens for recombinant protein production or comparative studies.

What techniques are commonly used to express recombinant MT-ND4L?

Recombinant MT-ND4L is typically expressed using E. coli expression systems. The procedure involves:

• Gene synthesis or cloning of the MT-ND4L sequence into a suitable expression vector

• Transformation of E. coli with the construct

• Induction of protein expression

• Purification via techniques such as IMAC (Immobilized Metal Affinity Chromatography)

For instance, commercial recombinant human MT-ND4L is produced with an N-terminal His6-ABP tag in E. coli, resulting in a purified product with >80% purity as determined by SDS-PAGE and Coomassie blue staining . Similar approaches could be applied for V. thyone MT-ND4L, although species-specific codon optimization might be necessary to enhance expression efficiency.

How do the genetic variations in Vampyressa thyone MT-ND4L compare across the three identified geographic clades?

Research using mitochondrial cytochrome-b sequences has identified three distinct genetic-geographic clades within Vampyressa thyone: a clade with specimens from Peru to Panama, a second clade with specimens from Costa Rica to Honduras, and a third lineage represented by Mexican specimens . The genetic divergence between the South and Central American clades was approximately 2.1%, while divergence between the Mexican samples and the South and Central American clades was substantially higher at 4.41% and 4.64%, respectively .

Given that mitochondrial genes tend to evolve together due to their linkage in the mitochondrial genome, it is highly probable that similar levels of divergence exist in the MT-ND4L gene across these clades. Researchers investigating recombinant V. thyone MT-ND4L should therefore carefully consider the source population of their genetic material, as functional or structural differences may exist between proteins derived from different clades.

What are the potential implications of MT-ND4L mutations on mitochondrial function in V. thyone compared to human MT-ND4L pathologies?

In humans, mutations in MT-ND4L have been associated with Leber hereditary optic neuropathy (LHON), particularly the T10663C (Val65Ala) mutation . This raises interesting comparative research questions regarding V. thyone MT-ND4L:

• Do naturally occurring variants in V. thyone MT-ND4L affect mitochondrial function in ways that might provide adaptive advantages in different ecological contexts?

• Could comparison between human pathological mutations and naturally occurring V. thyone variants provide insights into structure-function relationships?

• Are there compensatory mechanisms in V. thyone that might prevent pathological outcomes from MT-ND4L variants that would be deleterious in humans?

Such comparative studies could potentially illuminate both the functional constraints on MT-ND4L and the evolutionary adaptations that allow for genetic diversity without compromising essential functions.

How does the expression of MT-ND4L correlate with other mitochondrially encoded genes in V. thyone, and how does this compare to patterns observed in humans?

Research in human cells has demonstrated that certain mitochondrial genetic polymorphisms, such as the 10398A>G variant, are associated with altered expression of several mitochondrial genes, including MT-ND4L . This coordinated regulation suggests functional interactions between mitochondrial gene products.

In V. thyone, studies examining the co-expression patterns of mitochondrial genes across different tissues or developmental stages could reveal:

• Whether similar coordinated regulation exists

• If the three identified genetic clades show different patterns of mitochondrial gene expression

• If there are bat-specific patterns of mitochondrial gene regulation that differ from humans or other mammals

Such research would require comprehensive transcriptomic studies across multiple tissues and populations of V. thyone.

What is the optimal protocol for isolating and sequencing MT-ND4L from Vampyressa thyone tissue samples?

For researchers working with V. thyone tissue samples, the following methodological approach is recommended:

• Tissue preservation: Fresh tissue samples should be immediately preserved in RNAlater or flash-frozen in liquid nitrogen to prevent degradation of nucleic acids.

• DNA extraction: Total DNA extraction from tissue samples (typically wing membranes, liver, or muscle) can be performed using standard phenol-chloroform extraction or commercial kits designed for mitochondrial DNA isolation.

• PCR amplification: Design primers specific to conserved regions flanking the V. thyone MT-ND4L gene. Based on related bat species, the following primer sets might be effective:

  • Forward: 5'-CTACAAACCCTTATCAGAAAC-3'

  • Reverse: 5'-GTTTTAGGTCTACAAGACGC-3'

• Sequencing: Following PCR amplification, products can be sequenced using Sanger sequencing or next-generation sequencing methods.

For comparison with other mitochondrial genes, researchers might follow protocols similar to those used for cytochrome-b sequencing in V. thyone, which have successfully identified phylogenetic relationships within the species .

What are the critical considerations for expressing functional recombinant V. thyone MT-ND4L?

Expressing functional recombinant MT-ND4L presents several challenges due to its hydrophobic nature and role as a membrane protein. Key considerations include:

• Expression system selection: While E. coli is commonly used for recombinant protein production, membrane proteins often require eukaryotic expression systems for proper folding. Researchers should consider:

  • Baculovirus-insect cell systems

  • Yeast expression systems

  • Mammalian cell culture systems

• Solubilization strategies:

  • Fusion tags: Addition of solubility-enhancing tags such as MBP (maltose-binding protein) or SUMO

  • Detergent selection: Screening various detergents for optimal solubilization

  • Buffer optimization: Testing various pH conditions and salt concentrations

• Purification approach:

  • For His-tagged constructs, IMAC purification under denaturing conditions (with urea or guanidine HCl) may be necessary

  • Size exclusion chromatography for further purification

  • Quality assessment via SDS-PAGE and Western blotting

• Functional verification:

  • Complex I activity assays

  • Membrane incorporation studies

  • Electron transport measurements

The commercial human MT-ND4L recombinant protein is stored in PBS with 1M Urea at pH 7.4 , suggesting that some denaturing agent may be necessary for stability.

How can researchers analyze phylogenetic relationships of V. thyone MT-ND4L across geographical populations?

To analyze phylogenetic relationships based on V. thyone MT-ND4L sequences, researchers should employ the following approach:

• Sequence alignment: Multiple sequence alignment of MT-ND4L sequences from different V. thyone populations using tools like MUSCLE or CLUSTAL.

• Model selection: Determine the appropriate evolution model for MT-ND4L. Previous studies on V. thyone mitochondrial genes have used models such as SYM+I+Γ, HKY+I, and GTR+I for different codon positions .

• Phylogenetic analysis: Construct phylogenetic trees using:

  • Bayesian inference (MrBayes)

  • Maximum likelihood methods (RAxML or MEGA)

  • Parsimony methods for comparison

• Genetic distance calculation: Calculate genetic distances between populations using appropriate models (e.g., Kimura 2-parameter model) .

• Geographic correlation: Map genetic distances to geographical distribution to identify potential barriers to gene flow or historical population events.

Based on cytochrome-b analysis, researchers should expect to find similar phylogenetic patterns in MT-ND4L, with potential divergence between Mexican, Central American, and South American populations of V. thyone .

What statistical approaches are most appropriate for analyzing MT-ND4L expression differences between V. thyone populations?

When analyzing MT-ND4L expression differences between V. thyone populations, researchers should consider:

• Normalization strategies:

  • Use of multiple reference genes for qRT-PCR analysis

  • Total count normalization or TMM (Trimmed Mean of M-values) for RNA-Seq data

  • Consideration of tissue-specific effects

• Statistical tests:

  • ANOVA or Kruskal-Wallis for multiple population comparisons

  • DESeq2 or edgeR for RNA-Seq differential expression analysis

  • Linear mixed models to account for confounding variables

• Multiple testing correction:

  • FDR (False Discovery Rate) correction as used in mitochondrial gene expression studies

  • Bonferroni correction for more stringent control

• Effect size calculation:

  • Log2 fold change for expression differences

  • Cohen's d or Hedges' g for standardized effect sizes

In human studies, significant associations between mitochondrial variants and gene expression have been detected with FDR < 0.005 , suggesting similar statistical thresholds might be appropriate for V. thyone studies.

How can V. thyone MT-ND4L research inform our understanding of human mitochondrial disorders?

Comparative research between V. thyone and human MT-ND4L can provide valuable insights into mitochondrial disorders through:

• Natural variation analysis: V. thyone populations exhibit significant genetic divergence (up to 4.64% between populations) , providing a natural experiment in MT-ND4L variation that can be compared with human pathological variants.

• Structure-function relationships: By comparing amino acid substitutions that are tolerated in V. thyone with known pathogenic mutations in humans, researchers can better understand which regions of MT-ND4L are functionally constrained.

• Adaptive mechanisms: Investigating how V. thyone accommodates MT-ND4L variation without apparent dysfunction may reveal compensatory mechanisms that could inform therapeutic approaches for human mitochondrial disorders.

• Evolutionary medicine insights: Understanding the evolutionary context of MT-ND4L function across species can help distinguish between pathogenic mutations and benign polymorphisms in humans.

The T10663C (Val65Ala) mutation associated with Leber hereditary optic neuropathy in humans provides a specific comparative target for analysis in V. thyone populations.

What unique adaptations might exist in V. thyone MT-ND4L related to the bat's ecological niche?

As frugivorous bats with specific metabolic demands, V. thyone may exhibit unique adaptations in MT-ND4L that relate to their ecological niche:

• Energy metabolism adaptations: Frugivorous bats have distinct metabolic requirements compared to insectivorous or carnivorous bats, potentially reflected in mitochondrial gene adaptations.

• Geographic adaptations: The three distinct genetic clades of V. thyone occupy different climatic regions, which may exert selective pressure on mitochondrial genes involved in metabolism.

• Flight energetics: The high energy demands of flight may have selected for specific adaptations in oxidative phosphorylation efficiency, potentially visible in MT-ND4L sequence or structure.

• Tropical adaptation: As neotropical species, V. thyone may show mitochondrial adaptations to tropical environments that differ from temperate bat species.

Research comparing MT-ND4L sequences and functions across bat species with different ecological niches could reveal how this gene contributes to metabolic adaptation in these mammals.