Recombinant Geophagus steindachneri Cytochrome b (mt-cyb)

What is Geophagus steindachneri and why is its cytochrome b gene significant for research?

Geophagus steindachneri (commonly known as Red Hump Eartheater) is a South American cichlid species first described in 1910 by Eigenmann and Hildebrand. The species is taxonomically complex, with the name often appearing in quotes ('Geophagus' steindachneri) because it doesn't represent true Geophagus species and will eventually be assigned to its own genus . The cytochrome b gene (mt-cyb) from this species is significant for research because mitochondrial genes serve as important markers for evolutionary studies, taxonomic classification, and functional studies of oxidative phosphorylation complexes. The mt-cyb gene encodes a core subunit of respiratory complex III, which plays a central role in cellular energy production . Studying recombinant expressions of this protein allows researchers to investigate evolutionary relationships among cichlid fishes and examine functional properties of the mitochondrial respiratory system across different species.

How does Geophagus steindachneri mt-cyb differ from cytochrome b in other fish species?

Geophagus steindachneri mt-cyb exhibits unique sequence variations that reflect its evolutionary history and taxonomic position within the cichlid family. Current molecular phylogenetic studies place 'G. steindachneri' in a variable position, sometimes appearing as a sister to Gymnogeophagus or to Geophagus sensu stricto in phylogenetic trees . Within the Geophagus complex, significant genetic diversity exists, with multiple lineage delineation methods (mPTP, LocMin, bGMYC, and GMYC) identifying between 14-30 distinct lineages across the group . The sequence variations in G. steindachneri mt-cyb, particularly in catalytic domains, may impact protein function and stability, similar to how human mt-cyb variants affect complex III properties . Comprehensive comparative studies are necessary to fully characterize these differences, as they may reflect adaptations to specific environmental conditions or evolutionary constraints unique to this species.

What are the current taxonomic challenges in working with Geophagus steindachneri for mt-cyb studies?

Researchers face several taxonomic challenges when working with Geophagus steindachneri for mt-cyb studies:

• Taxonomic uncertainty: The species is commonly denoted as 'Geophagus' steindachneri with quotation marks indicating its uncertain taxonomic position. It doesn't represent true Geophagus species and is expected to be assigned to a different genus in the future (Weidner, 2000) .

• Misidentification issues: The species is frequently confused with other members of the Red Hump Eartheater complex including 'G. crassilabrus' and 'G. pellegrini', leading to potential sample misidentification .

• Synonymy problems: Historical identification challenges have resulted in synonyms including 'G. hondae' and 'G. magdalenae', creating literature search and database annotation difficulties .

• Phenotypic variability: The species is highly variable throughout its range and potentially represents more than one species, complicating genetic sample attribution .

These taxonomic challenges necessitate careful specimen verification using both morphological and molecular approaches before conducting mt-cyb studies. Researchers should document collection localities precisely and preserve voucher specimens to ensure experimental reproducibility and accurate interpretation of results in the context of ongoing taxonomic revisions.

What are the optimal protocols for cloning and expressing recombinant G. steindachneri mt-cyb?

The optimal protocol for cloning and expressing recombinant G. steindachneri mt-cyb follows established methodologies for mitochondrial proteins with modifications specific to this cichlid species:

Sample Collection and DNA Extraction:

• Obtain tissue samples (preferably fin clips or muscle tissue) from verified G. steindachneri specimens.

• Extract total DNA using standard phenol-chloroform methods or commercial kits optimized for fish tissue.

PCR Amplification and Cloning:

• Design primers flanking the complete mt-cyb coding region based on conserved regions in related cichlids or universal fish mt-cyb primers.

• Amplify the gene (~1140 bp) using high-fidelity PCR with optimized annealing temperatures (typically 52-56°C).

• Clone the amplified product into an appropriate expression vector (e.g., pET series for bacterial expression, yeast vectors like those used in mt-cyb studies) .

Expression Systems:
Based on similar studies with mitochondrial proteins, yeast expression systems provide advantages for functional studies of mt-cyb, as demonstrated with human mt-cyb variants . The Saccharomyces cerevisiae system is particularly valuable because:

• It allows direct mitochondrial transformation

• Can accommodate mutations in the mitochondrial genome

• Provides a eukaryotic environment for proper protein folding

• Enables biochemical and biophysical characterization of complex III function

Purification Strategy:

• Include a histidine tag for affinity purification

• Optimize detergent conditions for membrane protein extraction (typically DDM or digitonin)

• Employ size exclusion chromatography as a final purification step

This methodological approach enables both structural studies and functional characterization of the recombinant protein, providing insights into species-specific properties of G. steindachneri mt-cyb.

How can researchers verify the authenticity and functionality of recombinant G. steindachneri mt-cyb?

Verification of recombinant G. steindachneri mt-cyb requires multiple analytical approaches to confirm both molecular identity and functional integrity:

Molecular Authentication:

• Sequencing validation: Complete sequencing of the cloned gene to confirm identity with reference sequences.

• Western blot analysis: Using antibodies against conserved cytochrome b epitopes or targeting fusion tags.

• Mass spectrometry: Peptide mass fingerprinting and MS/MS analysis to confirm protein identity.

Functional Verification:

• Spectral analysis: Cytochrome b exhibits characteristic absorption spectra (peaks at 562, 532, and 429 nm in reduced state).

• Enzyme activity assays: Measure ubiquinol-cytochrome c reductase activity of complex III containing the recombinant mt-cyb.

• Complementation studies: Ability to restore respiratory function in yeast strains with deletion or mutation of endogenous cytochrome b.

Structural Integrity Assessment:

• Circular dichroism to assess secondary structure composition

• Thermal stability assays to determine protein stability

• Binding studies with known complex III inhibitors (e.g., antimycin, myxothiazol)

A comprehensive verification approach, similar to those used for human mt-cyb variants in yeast studies , should include parallel analysis of wild-type and recombinant variants to detect any functional differences that might arise from the expression system or protein modifications.

What are the key considerations when designing mutation studies for G. steindachneri mt-cyb?

When designing mutation studies for G. steindachneri mt-cyb, researchers should consider several critical factors:

Target Selection Strategy:

• Focus on conserved catalytic sites: Target mutations in the Qi and Qo binding sites that are critical for electron transfer and inhibitor binding.

• Phylogenetically informative sites: Select positions that differ between G. steindachneri and related species.

• Structural motifs: Consider mutations in transmembrane helices and functional loops.

Mutagenesis Approach:

• Site-directed mutagenesis for specific amino acid changes

• Domain swapping between G. steindachneri and other species to identify functional regions

• Alanine scanning of potentially important regions to systematically map functional residues

Functional Evaluation Parameters:

• Growth phenotypes in complementation systems (yeast growth curves)

• Complex III enzyme kinetics (Vmax, Km, catalytic efficiency)

• Inhibitor sensitivity profiles (IC50 values for various complex III inhibitors)

• Reactive oxygen species production measurements

Experimental Controls:

• Include wild-type G. steindachneri mt-cyb as positive control

• Use well-characterized mutations known to affect function (from human studies) as reference points

• Include mt-cyb from closely related species for comparative analyses

Based on human mt-cyb studies, a table of targeted mutation regions could be organized as follows:

This approach aligns with the methods employed in human mt-cyb variant studies while focusing on the specific characteristics of G. steindachneri .

How should researchers approach phylogenetic analysis using G. steindachneri mt-cyb sequences?

Researchers conducting phylogenetic analyses with G. steindachneri mt-cyb should implement a comprehensive methodological framework:

Sequence Acquisition and Verification:

• Generate high-quality bidirectional sequences of the complete mt-cyb gene (~1140 bp).

• Verify taxonomic identity of specimens using both morphological characters and molecular markers.

• Cross-reference with established databases (GenBank, BOLD) for sequence confirmation.

Multiple Sequence Alignment Strategy:

• Align G. steindachneri mt-cyb with sequences from multiple representatives of:

  • Other 'Geophagus' species (with particular attention to the Red Hump Eartheater complex)

  • True Geophagus sensu stricto species

  • Other geophagine genera

  • Appropriate outgroups from other cichlid subfamilies

• Use alignment algorithms specifically optimized for coding sequences (e.g., MUSCLE, MAFFT).

• Manually inspect alignments to correct frame shifts or alignment errors, particularly around indels.

Phylogenetic Analysis Methods:
Implement multiple analytical approaches to ensure robust results:

• Maximum Likelihood (ML) analysis with appropriate substitution models

• Bayesian Inference (BI) methods

• Maximum Parsimony (MP) as a complementary approach

• Distance-based methods (Neighbor-Joining) for comparison

Model Selection and Testing:

• Determine the best-fit nucleotide substitution model using AIC, BIC, or similar criteria

• Test for saturation in the dataset

• Consider partitioning strategies (by codon position)

• Implement appropriate rate heterogeneity parameters

Support Assessment:

• Bootstrap resampling (1000+ replicates) for ML and MP analyses

• Posterior probabilities for Bayesian analyses

• Calculate Bremer support indices for critical nodes

Comparative Analysis with Other Markers:
For robust phylogenetic placement, complement mt-cyb data with:

• Other mitochondrial markers (COI, ND4, 16S)

• Nuclear markers (RAG2, Tmo-M27, Tmo-4C4)

• Morphological data when available

This approach follows methodological practices established in geophagine systematics studies , which have previously used phylogenetic frameworks to resolve relationships within this complex group. Particular attention should be paid to the placement of 'G. steindachneri', which has been variably positioned in previous analyses .

What are the best approaches for analyzing selective pressure on G. steindachneri mt-cyb?

To analyze selective pressure on G. steindachneri mt-cyb, researchers should implement a multi-tiered approach combining various selection detection methods:

Sequence Dataset Preparation:

• Compile aligned mt-cyb sequences from G. steindachneri populations across its geographic range

• Include sequences from closely related species for comparative analysis

• Ensure high-quality alignments with no frameshifts or pseudogenes

Neutrality Tests:

• Tajima's D test to detect deviations from neutral evolution

• Fu and Li's F and D statistics

• McDonald-Kreitman test comparing polymorphism and divergence

Selection Analysis at Codon Level:

• Calculate nonsynonymous (dN) to synonymous (dS) substitution ratios

  • dN/dS < 1 indicates purifying selection

  • dN/dS > 1 suggests positive selection

  • dN/dS ≈ 1 implies neutral evolution

• Implement site-specific models (PAML, HyPhy package):

  • Site models (M0, M1a, M2a, M7, M8) to detect sites under selection

  • Branch models to test for selection in specific lineages

  • Branch-site models to identify sites under selection in specific lineages

Mapping Selection to Functional Domains:
Analyze selection patterns in relation to:

• Catalytic sites (Qi and Qo binding pockets)

• Transmembrane domains

• Interaction surfaces with other complex III subunits

• Regions implicated in inhibitor binding

Structural and Functional Correlates:

• Map sites under selection onto 3D protein structure models

• Correlate selection patterns with functional studies of variant effects

• Compare with selection patterns observed in other fish mt-cyb genes

Population-Level Selection Analysis:

• Test for evidence of local adaptation across different habitat types

• Analyze selection in relation to environmental variables (temperature, dissolved oxygen, etc.)

• Investigate potential selection differences between populations

This methodological framework will provide insights into the evolutionary forces shaping G. steindachneri mt-cyb and may reveal adaptive molecular evolution patterns relevant to the species' ecological niche and diversification history.

How can researchers effectively compare mt-cyb variability between G. steindachneri and related species?

Effective comparison of mt-cyb variability between G. steindachneri and related species requires a structured analytical approach:

Sampling Strategy:

• Obtain representative samples of G. steindachneri across its geographic range

• Include samples from all members of the Red Hump Eartheater complex

• Sample true Geophagus sensu stricto species (G. altifrons, G. brokopondo, G. dicrozoster)

• Include appropriate outgroups from other geophagine genera

Sequence Acquisition and Processing:

• Generate complete mt-cyb sequences (1140 bp) using standardized protocols

• Ensure bidirectional sequencing to verify sequence accuracy

• Process chromatograms with quality control measures (trim low-quality ends, verify base calls)

• Create high-quality multiple sequence alignments

Variability Metrics and Analyses:

• Calculate sequence diversity indices:

  • Nucleotide diversity (π)

  • Haplotype diversity (Hd)

  • Number of polymorphic sites (S)

  • Average number of nucleotide differences (k)

• Conduct population genetics analyses:

  • FST and other fixation indices between populations/species

  • AMOVA to partition genetic variance

  • Genetic distance measures (p-distance, K2P, TN93)

• Implement lineage delineation methods:

  • mPTP (multi-rate Poisson Tree Processes)

  • LocMin (Localized Posterior-Probability Minima)

  • bGMYC (Bayesian General Mixed Yule Coalescent)

  • GMYC (General Mixed Yule Coalescent)

Visualization and Interpretation:

• Construct haplotype networks to visualize relationships

• Create heat maps of genetic distances between populations/species

• Map geographic distribution of genetic variation

• Compare patterns of variability with other mitochondrial and nuclear markers

Comparative Analysis with Published Data:
Based on available data from Geophagus studies , a comparative framework might include:

This approach aligns with established methods in Geophagus diversity studies and provides a comprehensive framework for evaluating mt-cyb variability in an evolutionary and taxonomic context.

How can G. steindachneri mt-cyb be used to study drug sensitivity in complex III?

G. steindachneri mt-cyb can serve as a valuable model for studying drug sensitivity in complex III through heterologous expression and comparative pharmacological analysis:

Experimental System Development:

• Express recombinant G. steindachneri mt-cyb in a yeast (Saccharomyces cerevisiae) system lacking endogenous cytochrome b

• Generate a panel of site-directed mutants targeting residues in drug-binding domains

• Create chimeric proteins with domains from human or other species' mt-cyb to map binding determinants

Drug Sensitivity Profiling:

• Test sensitivity to clinically relevant complex III inhibitors:

  • Antimalarial compounds (atovaquone, similar to human mt-cyb position 171 studies)

  • Antidepressants (clomipramine, similar to position 18 studies in human mt-cyb)

  • Antifungal agents (strobilurins)

  • Other Q-site inhibitors (QoIs and QiIs)

• Determine inhibitory parameters:

  • IC50 values (concentration causing 50% inhibition)

  • Ki values (inhibition constants)

  • Binding kinetics (association/dissociation rates)

Comparative Analysis Framework:
Compare G. steindachneri mt-cyb drug responses to:

• Human mt-cyb (wild-type and variants)

• Other fish species mt-cyb

• Pathogen mt-cyb (Plasmodium, fungal pathogens)

Structure-Function Analysis:

• Map amino acid differences at drug-binding sites between G. steindachneri and other species

• Correlate sequence variations with differential drug sensitivity

• Model structural interactions using computational approaches

Example Data Table Format:

This approach parallels successful methodologies used in human mt-cyb variant studies but focuses on the unique properties of G. steindachneri mt-cyb, potentially revealing species-specific drug interaction patterns that could inform pharmacological development and evolutionary understanding of complex III.

What are the most effective approaches to study the impact of environmental factors on G. steindachneri mt-cyb function?

To study environmental impacts on G. steindachneri mt-cyb function, researchers should implement a multi-faceted approach combining field observations, laboratory experiments, and molecular analyses:

Field-Based Environmental Assessment:

• Sample G. steindachneri populations across environmental gradients:

  • Temperature regimes (seasonal and geographical variations)

  • Dissolved oxygen levels

  • pH and water chemistry parameters

  • Altitude gradients

• Measure in situ mitochondrial function using tissue biopsies and portable respirometry

• Document natural mt-cyb sequence variations across these environmental gradients

Laboratory Experimental Design:

• Acclimation studies:

  • Expose G. steindachneri to controlled environmental conditions

  • Measure changes in complex III activity and mt-cyb expression levels

  • Assess mitochondrial respiration rates and efficiency

• Acute challenge experiments:

  • Subject specimens to environmental extremes (temperature, hypoxia, pH)

  • Measure immediate effects on complex III function

  • Assess reactive oxygen species (ROS) production

Molecular and Biochemical Analyses:

• Quantify mt-cyb expression levels using qPCR under different conditions

• Measure complex III enzyme kinetics parameters (Vmax, Km) across environmental gradients

• Assess post-translational modifications of mt-cyb protein under environmental stress

• Analyze mitochondrial membrane composition changes that may affect mt-cyb function

Recombinant Protein Approaches:

• Express G. steindachneri mt-cyb variants (from different environments) in yeast systems

• Compare functional properties under controlled conditions

• Test thermal stability and pH sensitivity of variants

Integrative Data Analysis:
Correlate environmental parameters with functional outcomes using multivariate statistical approaches:

This comprehensive approach can reveal how environmental factors influence the function of G. steindachneri mt-cyb and provide insights into the adaptive capacity of this species to respond to changing environmental conditions, including potential climate change impacts.

How can researchers use G. steindachneri mt-cyb to investigate mitochondrial disease mechanisms?

G. steindachneri mt-cyb can serve as a comparative model for investigating mitochondrial disease mechanisms through several strategic research approaches:

Comparative Sequence Analysis:

• Identify conserved residues between G. steindachneri and human mt-cyb

• Map known human disease mutations onto the G. steindachneri sequence

• Analyze conservation patterns at disease-relevant sites across vertebrate evolution

• Identify naturally occurring variations in G. steindachneri that correspond to human disease mutations

Heterologous Expression Studies:

• Generate recombinant G. steindachneri mt-cyb with mutations corresponding to human disease variants

• Express these proteins in yeast systems lacking endogenous cytochrome b

• Compare functional consequences with equivalent human mt-cyb mutations

• Identify species-specific differences in mutation tolerance

Functional Characterization:

• Measure complex III enzyme kinetics of wild-type and mutant forms

• Assess electron transfer efficiency and proton pumping capacity

• Quantify reactive oxygen species (ROS) production

• Evaluate protein stability and complex assembly

Potential Disease Models:
Based on human mt-cyb variants studied in yeast , researchers can investigate:

• Variants near position 18 (p.Phe18Leu in humans) which affect drug sensitivity and may impact complex III function

• Variants near position 171 (p.Asp171Asn in humans) which affect Qo site function and are associated with LHON (Leber's Hereditary Optic Neuropathy)

• Other variants associated with cardiomyopathy, neurological disorders, and metabolic diseases

Compensatory Mechanism Investigation:

• Identify differences in compensatory responses between fish and mammalian systems

• Study nuclear-encoded complex III subunits that may interact differently with mt-cyb

• Investigate species-specific differences in mitochondrial quality control mechanisms

Comparative Data Framework:

This approach leverages the unique evolutionary position of G. steindachneri to provide insights into mitochondrial disease mechanisms, potentially revealing how different genetic backgrounds modify the expression of disease-causing mutations and identify novel therapeutic targets.

What are the key challenges in studying G. steindachneri mt-cyb protein-protein interactions within complex III?

Studying protein-protein interactions of G. steindachneri mt-cyb within respiratory complex III presents several significant challenges that require specialized methodological approaches:

Structural Determination Challenges:

• Membrane protein crystallization difficulties

  • Hydrophobic nature of mt-cyb with 8 transmembrane helices

  • Requirement for detergents that may disrupt native interactions

  • Limited stability outside the membrane environment

• Complex assembly dependencies

  • Need to assemble with multiple nuclear-encoded subunits for proper folding

  • Species-specific assembly factors may not be compatible with expression systems

Heterologous Expression System Limitations:

• Compatibility issues between G. steindachneri mt-cyb and host nuclear subunits

• Potential codon usage bias affecting expression efficiency

• Post-translational modification differences between fish and model organisms

• Mitochondrial import challenges for recombinant mt-cyb expressed from nuclear genes

Interaction Analysis Technical Challenges:

• Distinguishing direct from indirect interactions within the multi-subunit complex

• Maintaining functional interactions during purification and analysis

• Limited antibody availability for G. steindachneri mt-cyb and partner proteins

• Quantifying interaction strengths in the lipid bilayer environment

Methodological Solutions:

• Crosslinking mass spectrometry (XL-MS) to capture interaction interfaces

• Blue native polyacrylamide gel electrophoresis (BN-PAGE) to assess complex assembly

• Förster resonance energy transfer (FRET) with fluorescent fusion proteins

• Genetic complementation studies in yeast expression systems

• Nanodiscs or other membrane mimetics to maintain native-like environment

Comparative Approaches:

• Use human complex III structural data as a scaffold for modeling G. steindachneri interactions

• Create chimeric proteins to map interaction domains

• Leverage evolutionary conservation patterns to predict critical interaction surfaces

Anticipated Challenges Based on Human Studies:
Based on human mt-cyb interaction studies, researchers should focus on interactions with:

• Core proteins (UQCRC1, UQCRC2)

• Rieske iron-sulfur protein (UQCRFS1)

• Assembly factors (UQCC1, UQCC2, UQCC3)

• Supercomplexes formation partners (Complex I and IV components)

This approach acknowledges the significant technical challenges while providing practical methodological solutions based on advances in membrane protein interaction studies and experience from human mt-cyb research .

How can researchers integrate G. steindachneri mt-cyb studies with broader mitogenomic and ecological research?

Integrating G. steindachneri mt-cyb studies with broader mitogenomic and ecological research requires a multidisciplinary framework that connects molecular mechanisms to ecosystem-level processes:

Integrated Sampling Design:

• Coordinate sampling across multiple research objectives:

  • Collect tissues for both mt-cyb sequencing and whole mitogenome analysis

  • Record detailed ecological parameters at collection sites

  • Document behavioral observations and community interactions

  • Preserve specimens for morphological and dietary analysis

• Implement hierarchical sampling strategies:

  • Within populations (multiple individuals)

  • Across populations (geographic variation)

  • Between closely related species (comparative approach)

  • Across ecological gradients (environmental variation)

Multi-level Data Integration Approaches:

• Genomic-ecological correlations:

  • Associate mt-cyb sequence variation with specific habitat parameters

  • Correlate functional properties with ecological niche characteristics

  • Map geographical distribution of variants onto ecological features

• Phylogeographic analysis with ecological context:

  • Reconstruct historical population movements using mt-cyb and other markers

  • Correlate divergence events with geological and climatic history

  • Test for ecological speciation signals in molecular data

• Functional genomics in ecological context:

  • Measure respiratory performance in different ecological conditions

  • Assess metabolic adaptation to habitat-specific challenges

  • Link mt-cyb variations to thermal tolerance and hypoxia resistance

Analytical Frameworks:

• Ecological niche modeling incorporating genetic data

• Landscape genetics approaches linking mt-cyb diversity to habitat features

• Comparative phylogenetic methods testing for ecological correlates of molecular evolution

• Gene-environment association analysis

Collaborative Research Structure:

• Coordinate field sampling between molecular biologists and ecologists

• Develop shared databases linking molecular and ecological parameters

• Implement standardized protocols across research groups

• Establish interdisciplinary interpretation frameworks

This integration can be visualized in a research framework table:

This framework builds upon approaches used in geophagine diversity studies but extends them to connect molecular mechanisms to broader ecological contexts.

What novel applications might emerge from studying recombinant G. steindachneri mt-cyb in comparison with human variants?

Research comparing recombinant G. steindachneri mt-cyb with human variants could yield several novel applications spanning biomedical science, evolutionary biology, and biotechnology:

Biomedical Applications:

• Comparative pharmacology platforms:

  • Screening drug candidates against fish and human mt-cyb variants simultaneously

  • Identifying compounds with species-specific effects versus broad-spectrum inhibitors

  • Using G. steindachneri mt-cyb as a surrogate for testing toxicity of compounds targeting complex III

• Disease variant interpretation systems:

  • Developing evolutionary models to predict pathogenicity of novel human mt-cyb variants

  • Creating high-throughput functional assays using fish/human chimeric proteins

  • Identifying compensatory mechanisms present in fish but absent in humans

Evolutionary Applications:

• Molecular evolution modeling:

  • Quantifying selection pressures across vertebrate lineages at specific functional domains

  • Testing hypotheses about adaptation to different thermal environments

  • Developing predictive models for protein evolution in changing environments

• Structural biology insights:

  • Identifying conserved functional constraints versus lineage-specific adaptations

  • Mapping co-evolutionary networks within complex III

  • Revealing structure-function relationships through comparative analysis

Biotechnological Applications:

• Engineered bioenergetic systems:

  • Developing complex III variants with enhanced efficiency or stability

  • Creating temperature-adapted versions for biotechnological applications

  • Engineering resistance to specific inhibitors for selective expression systems

• Biosensor development:

  • Creating mt-cyb based biosensors for environmental toxins affecting complex III

  • Developing species-specific inhibitor detection systems

  • Engineering reporter systems for mitochondrial dysfunction

Experimental Data Framework:
Based on previous studies of human mt-cyb variants , a research program might generate data organized as follows:

This comparative approach could yield significant insights into both fundamental questions about mitochondrial evolution and practical applications in medicine and biotechnology, leveraging the evolutionary distance between fish and humans to identify both conserved mechanisms and lineage-specific adaptations in complex III function.