Recombinant Pitymys subterraneus Cytochrome b (MT-CYB)

Basic Research Questions

• What is Pitymys subterraneus Cytochrome b (MT-CYB) and what is its role in molecular biology?

  Cytochrome b (MT-CYB) from Pitymys subterraneus (European pine vole, also classified as Microtus subterraneus) is a mitochondrially encoded protein that functions as a critical component of the respiratory chain complex III. Specifically, it serves as Complex III subunit 3 (ubiquinol-cytochrome-c reductase complex cytochrome b subunit) . The protein contains multiple transmembrane domains and is involved in electron transport and cellular energy production. The amino acid sequence begins with MTIIRKKHPLIKIINHSFIDLPTPSNISSWWNFGSLLGLCLAVQILTGLFLAMHYTSDTATAFSSVAHICRDVNYGWLIRYMHANGASMFFICLFLHVGRGVYYGSYNMIETWNMGIILLFAVMATAFMGYVLP . In research applications, the mitochondrial cytochrome b gene serves as an important phylogenetic marker for species identification and evolutionary studies in rodents .

• How is MT-CYB used in species identification of rodents?

  The mitochondrial cytochrome b gene is widely used as a genetic marker for morphological species confirmation and differentiation among closely related vole species. Researchers typically use PCR with specific primers targeting the mitochondrial cytochrome b gene, followed by sequencing. For example, primers such as Cytb-Uni-fw (5′-TCATCMTGATGAAAYTTYGG-3′) and Cytb-Uni-rev (5′-ACTGGYTGDCCBCCRATTCA-3′) are commonly used to amplify an approximately 1000 bp fragment . The resulting sequences are then compared with reference sequences in databases such as GenBank to confirm species identification. This approach has proven valuable for distinguishing between morphologically similar species like Microtus arvalis and Microtus agrestis, or for confirming the identity of Pitymys subterraneus specimens . The technique is particularly important when differentiating between reservoir species in hantavirus studies .

• What methodologies are used to produce recombinant MT-CYB for research purposes?

  The production of recombinant Pitymys subterraneus MT-CYB typically follows these methodological steps:

  a) Gene cloning and vector construction: The MT-CYB coding sequence is amplified from genomic DNA using specific primers, then inserted into an expression vector.

  b) Expression system selection: The recombinant protein is commonly expressed in either:

  • Saccharomyces cerevisiae (yeast) systems, which have proven effective for expressing functional MT-CYB

  • Baculovirus-infected insect cells, similar to other cytochrome proteins

  c) Protein purification: Typically involves His-tag affinity chromatography, with the protein stored in Tris-based buffer with 50% glycerol at -20°C for stability .

  d) Validation: The recombinant protein is validated through western blotting, mass spectrometry, and functional assays to confirm its identity and activity.

  For example, ELISA test development often uses yeast-expressed recombinant MT-CYB proteins to achieve proper folding and functionality .

Experimental Design Questions

• How should researchers design PCR protocols for amplifying MT-CYB genes from field samples?

  When designing PCR protocols for MT-CYB amplification from field samples, researchers should consider:

  a) Primer selection: Universal cytochrome b primers work well for initial screening. Recommended primers include:

  • Cytb-Uni-fw (5′-TCATCMTGATGAAAYTTYGG-3′)

  • Cytb-Uni-rev (5′-ACTGGYTGDCCBCCRATTCA-3′)

  b) Sample preparation: For field specimens, researchers should:

  • Extract DNA from tissue samples (preferably lung, liver, or ear) using commercial kits

  • Use 400 ng of DNA for PCR amplification

  • Consider using high-fidelity polymerases like Platinum Taq High Fidelity DNA Polymerase

  c) Cycling conditions: Optimize thermal cycling based on primer Tm values and expected amplicon size (~1000 bp for full cytochrome b gene)

  d) Validation: Always include positive controls (known Pitymys/Microtus samples) and negative controls in each PCR run

  e) Sequence analysis: Purify PCR products and sequence directly or after cloning; compare sequences with references in GenBank using BLAST to confirm species identity

  This approach has successfully identified Microtus species in field studies across Europe, with sequence similarity typically ≥98% for correct species assignment .

• What are the optimal storage conditions for maintaining recombinant MT-CYB stability?

  Based on established protocols for recombinant MT-CYB and similar proteins, the following storage conditions are recommended:

  a) Short-term storage (up to one week): Store working aliquots at 4°C

  b) Medium-term storage: Store at -20°C in a buffer containing 50% glycerol as a cryoprotectant

  c) Long-term storage: For extended storage periods, maintain at -80°C

  d) Buffer composition: Optimal preservation requires:

  • Tris-based buffer optimized for the specific protein

  • 50% glycerol to prevent freeze-thaw damage

  • pH optimization (typically pH 7-8)

  e) Handling precautions: Avoid repeated freeze-thaw cycles which can cause protein degradation and loss of activity. Instead, prepare small single-use aliquots during initial storage

  These storage recommendations ensure protein stability and activity preservation, which is critical for experimental reproducibility when using recombinant MT-CYB in immunological assays or functional studies.

Phylogenetic and Evolutionary Research Applications

• How can MT-CYB sequences be used to reconstruct phylogenetic relationships among vole species?

  MT-CYB sequences serve as powerful markers for phylogenetic reconstruction among vole species due to their appropriate rate of molecular evolution. The methodology involves:

  a) Sequence acquisition: Obtain high-quality cytochrome b sequences (typically 1000+ bp) from target species through PCR amplification and sequencing

  b) Multiple sequence alignment: Align sequences using tools like MUSCLE or CLUSTAL, with manual refinement when necessary

  c) Model selection: Select appropriate nucleotide substitution models (e.g., GTR+I+G) based on likelihood ratio tests

  d) Tree construction methods:

  • Neighbor-joining (NJ) for rapid initial analysis

  • Maximum Likelihood (ML) for more robust evolutionary model-based trees

  • Bayesian inference for probability-based phylogenies

  e) Confidence assessment: Implement bootstrap analysis (typically 1000 replicates) with values ≥70% considered significant support

  f) Divergence time estimation: Calculate pairwise distances and net distances between phylogenetic clusters to estimate nucleotide substitutions on internal branches

  This approach has successfully resolved evolutionary relationships among Microtus species, including identification of distinct lineages within M. arvalis and M. agrestis populations across Europe . For example, cytochrome b analysis confirmed the Central lineage of M. arvalis inhabits most of Central Europe, while M. agrestis populations belong to the Western lineage with a distribution encompassing Western, Central, and Eastern Europe and Scandinavia .

• What insights has MT-CYB analysis provided about the evolutionary diversification of rodent species in Europe?

  MT-CYB analysis has provided significant insights into rodent evolution across Europe:

  a) Glacial refugia and post-glacial recolonization: Cytochrome b studies revealed that separation of populations during glacial cycles led to intraspecific genetic divergence into several evolutionary lineages in species like M. arvalis and M. agrestis

  b) Lineage identification: Major evolutionary lineages identified include:

  • M. arvalis Central lineage (Germany, Denmark, Netherlands, Switzerland)

  • M. arvalis Eastern lineage (Poland, Czech Republic)

  • M. agrestis Western lineage (spanning Western, Central, Eastern Europe and Scandinavia)

  c) Species boundaries assessment: Cytochrome b divergence has raised questions about species status in some cases:

  • The genetic divergence between M. bavaricus and M. liechtensteini is the lowest observed among any two pine vole species, suggesting recent divergence

  • Some researchers question the validity of M. bavaricus as a distinct species based on low cytochrome b divergence

  d) Cryptic diversity detection: MT-CYB analysis has revealed cryptic diversity within morphologically similar vole species, particularly in mountainous regions that may have served as glacial refugia

  These findings demonstrate that cytochrome b provides sufficient resolution to reconstruct the evolutionary history of European rodents, revealing how past climate changes shaped current patterns of genetic diversity and distribution.

Advanced Research Applications

• How has recombinant MT-CYB been used in hantavirus research and diagnostics?

  Recombinant MT-CYB has played a crucial role in hantavirus research through several applications:

  a) Reservoir host identification: Cytochrome b sequencing is used to confirm the morphological determination of rodent species in hantavirus studies, ensuring accurate identification of reservoir hosts . This is particularly important for distinguishing between morphologically similar species like M. arvalis and M. agrestis that may host the same virus .

  b) Serological test development: Yeast-expressed recombinant MT-CYB is used as a component in ELISA tests for detecting hantavirus infections . For example:

  • Detection of Tula virus (TULV)-reactive antibodies employs ELISA protocols using yeast-expressed recombinant N protein of TULV strain Moravia

  • These serological tests have been used to determine infection rates in different rodent populations, with data showing gender-specific differences in seropositivity (Table 1)

  Small Mammal Species	Total Collected	Sex Ratio Male/Female	Number of Positive Samples (Male/Female)	Percentage of Positive Samples [%]
  Microtus arvalis	86	40/46	13 (8/5)	15.1
  Dryomys nitedula	15	7/8	2 (1/1)	13.3

  c) Virus-host co-evolution studies: Combined analysis of hantavirus sequences and host cytochrome b sequences has revealed:

  • Phylogenetic clustering of TULV strains according to geographic origin rather than host species

  • Evidence of frequent cross-species transmission (spillover) of TULV between M. arvalis and M. agrestis

  • Gender-specific differences in susceptibility to spillover infections, with male M. agrestis showing higher rates of TULV infection than females

  These applications have significantly advanced our understanding of hantavirus ecology and transmission dynamics in natural rodent populations.

• What are the methodological challenges in differentiating between closely related vole species using MT-CYB, and how can they be overcome?

  Differentiating closely related vole species using MT-CYB presents several methodological challenges:

  a) Low interspecific variation: Some closely related species show minimal genetic divergence in cytochrome b sequences. For example:

  • M. bavaricus and M. liechtensteini exhibit the lowest cytochrome b divergence observed among any two pine vole species

  • This challenges species determination based solely on this marker

  b) Methodological solutions include:

  • Increasing sequence length: Targeting longer cytochrome b fragments (>1000 bp) provides more informative sites for species discrimination

  • Multi-gene approach: Combining cytochrome b with nuclear markers or whole mitochondrial genome analysis

  • Population-level sampling: Analyzing multiple specimens from different localities to account for intraspecific variation

  • Reference database quality: Using verified reference sequences from taxonomically validated specimens

  c) Integrated approach: For the most reliable species identification, researchers should:

  • Combine morphological classification with molecular identification

  • Use both mitochondrial (cytochrome b) and nuclear markers when species boundaries are unclear

  • Apply appropriate phylogenetic methods with statistical support (bootstrap values ≥70%)

  • Consider geographical origin of samples, as some lineages show strong geographic structure

  d) Case study: The successful resolution of Tula virus reservoir hosts demonstrates the value of this integrated approach. Initial morphological identification of rodents was confirmed by cytochrome b sequencing, revealing that TULV can infect both M. arvalis and M. agrestis in sympatric populations .

• How can researchers interpret genetic variations in MT-CYB in the context of disease susceptibility studies?

  Interpreting MT-CYB genetic variations in disease susceptibility contexts requires sophisticated analytical approaches:

  a) Functional impact assessment: Variations in MT-CYB can significantly alter complex III properties, as demonstrated in studies using yeast models to express human MT-CYB variants . Methodologies include:

  • Introducing specific mutations into yeast mtDNA through mitochondrial transformation

  • Measuring complex III activity through cytochrome c reduction assays

  • Determining drug sensitivity through inhibitor titration studies

  • Assessing respiratory growth in the presence of increasing drug concentrations

  b) Disease association evaluation: Several MT-CYB variants have been linked to human diseases, including:

  • Neurological disorders (e.g., Leber hereditary optic neuropathy, Parkinson's)

  • Metabolic disorders (e.g., diabetes)

  • Various cancers (e.g., glioblastoma)

  c) Functional consequences of specific variations:

  • m.15257G>A (p.Asp171Asn): Increases sensitivity to the antimalarial drug atovaquone

  • m.14798T>C (p.Phe18Leu): Enhances sensitivity to the antidepressant drug clomipramine

  • Other variants previously considered "silent" have shown significant effects on complex III properties

  d) Host-pathogen interaction insights: In rodent-virus systems, variations in cytochrome b may correlate with:

  • Susceptibility to virus infection (e.g., TULV in different Microtus species)

  • Gender-specific differences in infection rates (males showing higher rates in some species)

  • Age-related variations in seropositivity (positive association between body mass/age and seropositivity)

  e) Analytical framework: Researchers should integrate:

  • Phylogenetic analysis to place variants in evolutionary context

  • Functional assays to assess biochemical consequences

  • Epidemiological data to evaluate disease associations

  • Statistical analyses to account for confounding factors like age, gender, and geographical distribution

  These approaches enable researchers to move beyond simple association studies to develop mechanistic understanding of how MT-CYB variations influence disease susceptibility and progression.

Methodological Questions

• What PCR protocols are most effective for amplifying MT-CYB from degraded field samples?

  When working with degraded field samples, researchers should optimize PCR protocols as follows:

  a) DNA extraction optimization:

  • Use specialized extraction kits designed for degraded samples (e.g., ancient DNA kits)

  • Include carrier RNA to improve recovery of low-concentration DNA

  • Perform extractions in a clean environment to minimize contamination

  b) PCR strategy modifications:

  • Target shorter amplicons (200-400 bp) instead of full-length cytochrome b

  • Design overlapping primer sets to reconstruct the complete sequence

  • Use nested PCR approaches to increase sensitivity

  c) Polymerase selection:

  • Use high-fidelity DNA polymerases resistant to inhibitors (e.g., Platinum Taq High Fidelity)

  • Consider polymerases specifically designed for difficult or degraded templates

  d) Amplification enhancers:

  • Include PCR additives like DMSO (5-10%) or betaine to improve amplification of GC-rich regions

  • Use bovine serum albumin (BSA) to overcome inhibitors often present in field samples

  e) Cycling modifications:

  • Increase cycle numbers (35-45 cycles)

  • Employ touchdown PCR to increase specificity while maintaining sensitivity

  • Consider longer extension times to accommodate damaged templates

  f) Validation approaches:

  • Always run multiple independent PCR reactions from the same extract

  • Sequence both strands of the PCR product

  • Compare results with reference sequences to detect potential errors or contamination

  These modifications have proven successful in amplifying cytochrome b sequences from degraded samples in field studies of rodents and hantavirus research .

• How can researchers effectively use recombinant MT-CYB in developing serological assays for virus detection?

  Development of effective serological assays using recombinant MT-CYB involves several key methodological considerations:

  a) Expression system selection:

  • Saccharomyces cerevisiae (yeast) expression systems have proven highly effective for producing functional MT-CYB proteins for serological applications

  • This system ensures proper protein folding and preservation of antigenic epitopes

  b) Assay development protocol:

  • For indirect ELISA development:

    1. Coat microplates with purified recombinant MT-CYB (typically 1-2 μg/ml)

    2. Block non-specific binding sites with appropriate blocking buffer

    3. Apply test samples (serum, tissue fluid, etc.) at optimized dilutions

    4. Detect bound antibodies using species-specific secondary antibodies

    5. Develop with appropriate substrate and read at 450 nm (reference at 620 nm)

  • For immunoblot assays:

    1. Separate recombinant proteins by SDS-PAGE

    2. Transfer to nitrocellulose or PVDF membranes

    3. Block and apply test samples

    4. Detect using species-specific secondary antibodies and appropriate detection systems

  c) Quality control measures:

  • Include positive controls (e.g., monoclonal antibody 5E11)

  • Use negative controls from confirmed negative individuals of the same species

  • Establish clear cut-off values to distinguish positive from negative results

  d) Cross-reactivity assessment:

  • Test for cross-reactivity with antibodies against related viruses

  • Include samples from animals infected with different virus strains

  • Validate results using alternative methods (e.g., RT-PCR, neutralization assays)

  e) Field validation:

  • In field studies, integrate serological testing with molecular detection methods

  • Collect demographic and ecological data to interpret serological results

  This methodology has been successfully applied in hantavirus research to detect TULV-reactive antibodies in rodent populations, facilitating studies of virus ecology and transmission dynamics .