Recombinant Strongylocentrotus purpuratus NADH-ubiquinone oxidoreductase chain 4L (ND4L)

What is the structure and function of NADH-ubiquinone oxidoreductase chain 4L in Strongylocentrotus purpuratus?

NADH-ubiquinone oxidoreductase chain 4L (ND4L) in Strongylocentrotus purpuratus is a small hydrophobic protein component of the mitochondrial respiratory chain complex I (NADH dehydrogenase). The protein consists of 97 amino acids with the sequence: MALLIVILSMFYLGLMGILLNRLHFLSILLCLELLLISLFIGIAIWNNNTGVPQNTTFNLFVLTLVACEASIGLSLMVGLSRTHSSNLVGSLSLLQY .

Functionally, ND4L contributes to the electron transfer from NADH to ubiquinone in the respiratory chain, where ubiquinone is believed to be the immediate electron acceptor for the enzyme . This protein is encoded by the mitochondrial genome and, like its homologs in other species, is a multi-pass membrane protein located in the inner mitochondrial membrane .

How does S. purpuratus ND4L differ from homologous proteins in other species?

When comparing S. purpuratus ND4L with homologous proteins from other species, several structural and functional differences become apparent:

• Sequence conservation: Comparative analyses of mitochondrial genomes show that ND4L is moderately conserved across species. In echinoids, the percent similarity of nucleotide sequences for ND4L between closely related species (like Acanthaster planci and A. brevispinus) is approximately 85-90% .

• Evolutionary rate: Among the 13 mitochondrial protein-coding genes, ND4L shows an intermediate rate of evolution compared to the most conserved (CO1 at 99.2% conservation) and the least conserved (ATP8 at 85.2% conservation) genes in related species .

• Membrane topology: While the basic membrane topology is similar across species (multi-pass membrane protein), small variations in transmembrane domains may exist between S. purpuratus and other marine invertebrates.

What are the primary molecular characteristics of recombinant S. purpuratus ND4L?

The recombinant Strongylocentrotus purpuratus ND4L protein has the following molecular characteristics:

For optimal stability, repeated freezing and thawing should be avoided, and working aliquots may be stored at 4°C for up to one week .

What are the most effective methods for extracting and purifying mitochondrial DNA containing the ND4L gene from S. purpuratus samples?

For effective extraction and purification of mitochondrial DNA containing the ND4L gene from S. purpuratus samples, the following methodological approach has shown good results:

• Tissue selection: Gonad tissue generally provides the best results for DNA extraction from S. purpuratus, offering better resolution for subsequent analyses compared to digestive gland, tube feet, or muscle of Aristotle's lantern .

• DNA extraction protocol: The Chelex 100 method has been successfully employed for extracting mtDNA from sea urchin gonad tissue . The protocol follows standard procedures:

  • Tissue samples are processed soon after collection or temporarily stored in flowing seawater aquaria

  • Tissues are separated before freezing at -70°C

  • The Chelex 100 extraction method follows the Walsh et al. (1991) protocol

• PCR amplification: For amplifying the ND4L region, PCR can be performed using:

  • Standard reaction volume of 50 μl

  • Magnesium chloride concentration of 2 mM

  • Thermocycle profile: 94°C for 60s; 50°C for 60s; 72°C for 90s (35 cycles), followed by 5 min at 72°C

  • PCR products purified using spin filters like Microcon 100

• Purification: PCR products should be purified before sequencing or further analysis, typically using spin column methods or commercial purification kits.

How can researchers design effective primers for amplifying the ND4L gene from S. purpuratus?

When designing effective primers for amplifying the ND4L gene from S. purpuratus, researchers should consider the following methodological approach:

• Reference sequence identification: Use the complete S. purpuratus mtDNA sequence as a reference. The complete mitochondrial genome has been sequenced and is available in public databases.

• Primer design considerations:

  • Target conserved regions flanking the ND4L gene

  • Optimal primer length is typically 18-25 nucleotides

  • Aim for GC content between 40-60%

  • Avoid secondary structures and primer-dimer formation

  • Check for specificity against other regions of the S. purpuratus genome

• Example approach: Following the methodology used in similar studies with other echinoderm species, primers can be designed based on the complete mtDNA sequence. For example, in the Acanthaster planci study, researchers designed primers for COI gene amplification based on the complete mtDNA sequence (Jacobs et al. 1988): primer COIC (5'-TCGTCTGATCCGTCTTTGTCAC-3', Positions 6335 to 6356) and primer COIJ (5'-CAATACCTGTGAGTCCTCCTA-3', Positions 6833 to 6853) .

• Testing and validation: New primers should be validated by:

  • In silico analysis using primer design software

  • Experimental validation on known S. purpuratus samples

  • Sequencing of amplicons to confirm target specificity

What are the optimal storage conditions for recombinant S. purpuratus ND4L to maintain biological activity?

For optimal storage of recombinant S. purpuratus ND4L to maintain biological activity, the following evidence-based protocol should be followed:

• Storage buffer composition:

  • Tris-based buffer with 50% glycerol, optimized for this specific protein

  • The buffer should be formulated to maintain protein stability and prevent degradation

• Temperature considerations:

  • Short-term storage: -20°C

  • Extended storage: -20°C to -80°C

  • Working aliquots: 4°C for up to one week

• Handling recommendations:

  • Avoid repeated freezing and thawing cycles, which can significantly decrease protein activity

  • Prepare small working aliquots to minimize freeze-thaw cycles

  • When thawing, keep the protein on ice to prevent thermal denaturation

• Quality control measures:

  • Periodically validate protein activity using appropriate functional assays

  • Monitor for signs of degradation through SDS-PAGE analysis

  • Consider adding protease inhibitors if degradation is observed

How has ND4L been used to study population structure in S. purpuratus and other marine invertebrates?

The ND4L gene has been instrumental in population genetic studies of marine invertebrates, including S. purpuratus, due to its mitochondrial origin and specific evolutionary characteristics:

• Population structure analysis: In studies of the purple sea urchin S. purpuratus, mitochondrial DNA analysis (including genes like ND4L) has revealed significant genetic subdivision among locations, despite the species' high potential for dispersal. Research covering geographic locations along the coast of California and Baja California showed that genetic differentiation over short geographic distances could exceed differentiation over much larger distances .

• Demographic history inference: In similar studies with other marine invertebrates, ND4L along with other mitochondrial genes has been used to infer demographic history. For example, in Branchiostoma belcheri, mitochondrial genome analysis revealed population expansion during the Greenlandian stage of the current geological epoch, following population reductions during glaciation periods .

• Methodological application: In the blue riffle goby (Stiphodon caeruleus), researchers used ND4L together with ND4 to evaluate population genetic structure and demographic history. Their analysis of 876 base pairs from 208 specimens across 15 localities revealed 23 haplotypes with diversity indices of h = 0.489±0.043 and π = 0.097±0.012% .

• Quantitative findings: Studies of S. purpuratus revealed significant allozyme differentiation among subpopulations of adults (FST=0.033) and among subpopulations of recruits (FST=0.037) . These values provide important quantitative measures of population subdivision that can be compared across species and regions.

What insights has ND4L sequence analysis provided about the evolutionary history of echinoids?

ND4L sequence analysis has provided several significant insights into the evolutionary history of echinoids:

• Phylogenetic relationships: Mitochondrial genes including ND4L have been used to establish evolutionary relationships among echinoids. Bayesian analysis of mitochondrial genome sequences has generated trees with high confidence (posterior probabilities at all bifurcating nodes of 100% in some studies), providing robust phylogenetic frameworks .

• Molecular evolution rates: Studies have shown varying rates of evolution across mitochondrial genes in echinoids. Pairwise comparisons of evolutionary rates (ω) across 13 coding sequences (CDS) including ND4L have revealed patterns of selection and adaptation. The highest ω values were consistently observed within the genus Strongylocentrotus, specifically when S. purpuratus was compared to its congeners S. pallidus and S. intermedius .

• Divergence timing: Analysis of mitochondrial genomes including ND4L has been used to establish timeframes for the genesis of different echinoid lineages. For example, calibration points derived from these analyses have helped propose a timeframe for the genesis of the Superfamily Odontophora .

• Demographic expansion evidence: In S. purpuratus and other echinoids, ND4L sequence data has contributed to the identification of demographic expansions following glacial periods, providing insights into how historical climate changes have shaped current population structures .

How does ND4L sequence variation correlate with geographic distribution in S. purpuratus populations?

ND4L sequence variation in S. purpuratus shows specific patterns related to geographic distribution:

• Genetic mosaic pattern: Research on S. purpuratus populations along the California and Baja California coast revealed a genetic mosaic, where genetic differentiation over short geographic distances sometimes exceeded differentiation over much larger distances. This pattern was observed in both nuclear (allozyme) and mitochondrial DNA analyses, which would include the ND4L gene .

• Location-specific genetic structure: Ten geographic locations studied along this coastal region showed significant genetic subdivision. The standardized variance (FST) values of 0.033 among subpopulations of adults and 0.037 among subpopulations of recruits indicate moderate but significant genetic structure .

• Larval recruitment patterns: The data suggests that different cohorts (adults vs. recruits) at the same location can show significant genetic differentiation, indicating temporal variation in larval recruitment sources and challenging the assumption that different age classes from the same location represent a single deme .

• Geographic barriers: While some studies of related species found no mitochondrial DNA differentiation over large regions (e.g., Washington State to southern California), finer-scale analyses have revealed subtle population structures potentially related to oceanographic features and coastal topography that influence larval dispersal patterns .

What controls should be included when studying recombinant S. purpuratus ND4L in experimental settings?

When designing experiments with recombinant S. purpuratus ND4L, the following controls should be incorporated:

• Negative controls:

  • Buffer-only controls to establish baseline measurements

  • Irrelevant protein of similar size/structure to control for non-specific effects

  • Heat-denatured ND4L to confirm activity is dependent on native protein structure

• Positive controls:

  • Well-characterized homologous proteins from related species (if available)

  • Previously validated batches of recombinant S. purpuratus ND4L

  • Native ND4L isolated from S. purpuratus mitochondria (if feasible)

• Experimental validation controls:

  • Concentration gradient to establish dose-dependent effects

  • Time-course experiments to determine optimal reaction conditions

  • pH and ionic strength variations to establish optimal buffer conditions

• Expression system controls:

  • Empty vector controls when using recombinant expression systems

  • Host cell extract controls to identify potential contaminating activities

  • Tag-only controls if the recombinant protein includes affinity tags

How can researchers address the challenge of sequence polymorphism when analyzing ND4L in natural populations?

Addressing sequence polymorphism challenges in ND4L analysis requires rigorous methodological approaches:

• Sampling strategy:

  • Collect statistically sufficient sample sizes (n>30 per population is often recommended)

  • Include geographic replicates to account for spatial heterogeneity

  • Sample different age classes to account for temporal genetic variation (as demonstrated in the S. purpuratus study where adults and recruits showed significant genetic differentiation)

• Sequencing approach:

  • Use high-fidelity polymerases to minimize PCR-induced errors

  • Perform bidirectional sequencing to verify ambiguous positions

  • Consider next-generation sequencing for detecting rare variants

• Data analysis methods:

  • Calculate standard diversity indices (h and π) to quantify genetic diversity

  • Employ appropriate statistical tests (e.g., FST, AMOVA) to assess population structure

  • Apply neutrality tests to distinguish selection from demographic effects

• Bioinformatic solutions:

  • Use appropriate algorithms for sequence alignment that can handle polymorphisms

  • Apply phylogenetic methods that account for intrapopulation variation

  • Consider haplotype network analyses to visualize relationships among variants

For example, in the study of Stiphodon caeruleus, researchers analyzed 876 base pairs from 208 specimens and recovered 23 haplotypes with diversity indices of h = 0.489±0.043 and π = 0.097±0.012%. The median-joining network of haplotypes was star-like in formation with no genetic structure. AMOVA results showed that 99% of the genetic variation was found within populations rather than between streams or islands .

What statistical approaches are most appropriate for analyzing ND4L sequence data in population genetics studies?

When analyzing ND4L sequence data in population genetics studies, researchers should consider these statistical approaches:

• Genetic diversity metrics:

  • Haplotype diversity (h): Measures the uniqueness of haplotypes in a population

  • Nucleotide diversity (π): Quantifies the average number of nucleotide differences per site

  • Number of segregating sites (S): Counts the number of polymorphic positions

• Population differentiation analyses:

  • FST and its analogs: Measures the proportion of genetic variance due to population differentiation

  • AMOVA (Analysis of Molecular Variance): Partitions genetic variation among hierarchical levels

  • Exact tests of population differentiation: Tests for non-random distribution of haplotypes

• Demographic history inference:

  • Mismatch distribution analysis: Tests for population expansion

  • Tajima's D, Fu's Fs: Tests for departure from neutrality, often used to infer demographic changes

  • Bayesian skyline plots: Reconstructs historical effective population size changes

• Phylogenetic approaches:

  • Maximum likelihood or Bayesian methods for tree construction

  • Median-joining networks: Visualizes relationships among closely related haplotypes

  • Molecular clock analyses: Estimates divergence times

How can recombinant S. purpuratus ND4L be used in functional studies of mitochondrial complex I?

Recombinant S. purpuratus ND4L can be utilized in functional studies of mitochondrial complex I through several methodological approaches:

• Reconstitution studies:

  • Incorporation of recombinant ND4L into liposomes or nanodiscs

  • Measurement of electron transfer activity in reconstituted systems

  • Assessment of proton pumping capacity using pH-sensitive probes

• Interaction analyses:

  • Pull-down assays to identify protein-protein interactions with other complex I subunits

  • Cross-linking studies to map proximity relationships within the complex

  • Surface plasmon resonance or isothermal titration calorimetry to measure binding affinities

• Structural investigations:

  • Site-directed mutagenesis to probe functionally important residues

  • Hydrogen-deuterium exchange mass spectrometry to examine protein dynamics

  • Contributions to cryo-EM structural studies of complex I assembly

• Comparative biochemistry:

  • Functional comparison with homologous proteins from other species

  • Assessment of activity under various physiological conditions relevant to sea urchin biology

  • Investigation of species-specific adaptations in energy metabolism

What emerging technologies are advancing our understanding of ND4L function and evolution?

Emerging technologies are significantly enhancing our understanding of ND4L function and evolution:

• Advanced sequencing technologies:

  • Long-read sequencing: Provides complete mitochondrial genome sequences without assembly errors

  • Single-molecule real-time sequencing: Reveals heteroplasmy and minor variants

  • Nanopore sequencing: Enables direct sequencing of native mitochondrial DNA

• CRISPR-based approaches:

  • Mitochondrial base editors: Allow specific mutations to be introduced in mtDNA

  • MitoTALENs: Enable targeted manipulation of mitochondrial genes

  • Heteroplasmy shifting: Methods to alter the proportion of variant mtDNA molecules

• Advanced imaging techniques:

  • Super-resolution microscopy: Visualizes complex I organization in mitochondria

  • Cryo-electron tomography: Provides structural insights in near-native conditions

  • Live-cell imaging with genetically encoded sensors: Monitors mitochondrial function

• Computational approaches:

  • Molecular dynamics simulations: Models protein movements and interactions

  • Machine learning algorithms: Predicts functional impacts of sequence variations

  • Phylogenetic methods: Reconstructs evolutionary trajectories with increasing accuracy

How can ND4L sequence data contribute to conservation genetics of marine invertebrates?

ND4L sequence data can make significant contributions to conservation genetics of marine invertebrates through several methodological applications:

• Population identification and management:

  • Delineation of evolutionarily significant units (ESUs) based on genetic distinctiveness

  • Identification of management units for conservation planning

  • Assessment of genetic connectivity among populations to inform marine protected area design

• Monitoring genetic diversity:

  • Establishment of baseline genetic diversity measures for vulnerable populations

  • Detection of genetic bottlenecks that may indicate population decline

  • Tracking changes in genetic diversity over time in response to environmental changes

• Species identification and delimitation:

  • Development of molecular markers for rapid identification of larval forms

  • Resolution of cryptic species complexes that may require different conservation approaches

  • Clarification of taxonomic uncertainties to ensure conservation efforts target correct units

• Adaptation and resilience assessment:

  • Identification of locally adapted populations that may respond differently to environmental stressors

  • Assessment of adaptive potential based on genetic diversity

  • Monitoring genetic responses to climate change and anthropogenic impacts

For example, the methodology used to study population genetics in S. purpuratus, where significant genetic subdivision was found despite high dispersal potential, could be applied to conservation-relevant species to understand their population structure and connectivity patterns . Similarly, the approach used to identify Blue Riffle Goby populations could inform conservation of other endemic marine species .