Recombinant Phoca largha Sex-determining region Y protein (SRY)

What is the genomic organization of Phoca largha SRY and how does it compare to other mammals?

The Sex-determining Region Y (SRY) gene in mammals encodes a transcription factor containing a conserved High Mobility Group (HMG) box domain responsible for DNA binding. While specific information on Phoca largha SRY genomic organization is limited, research on human SRY shows that the protein contains three primary domains: the N-terminal domain, the central HMG box domain, and the C-terminal domain . The DNA binding activity of SRY is essential for sex determination, as demonstrated by studies showing that mutations in the HMG box region can lead to sex development disorders .

In comparative analyses, the HMG box is typically highly conserved across mammalian species, while the N-terminal and C-terminal domains show greater variation. Research methodologies to characterize Phoca largha SRY would likely include PCR amplification of the gene from male genomic DNA, followed by sequencing and comparative analysis with other pinniped and mammalian SRY sequences.

What expression systems are most effective for producing recombinant Phoca largha SRY protein?

• Prokaryotic systems (E. coli): Most commonly used for basic structural studies, DNA-binding assays, and antibody production. These systems typically produce higher yields but may lack post-translational modifications.

• Eukaryotic systems: Consider mammalian (HEK293, CHO) or insect cell (Sf9) systems when post-translational modifications such as acetylation are important, as these modifications have been shown to regulate SRY activity .

• Cell-free systems: Useful for rapid small-scale production when testing multiple constructs or mutations.

When designing expression constructs, researchers should include affinity tags (His, GST, or MBP) for purification, with consideration for tag position to minimize interference with DNA binding activity. Given that human SRY can interact with proteins like p300 , expression systems preserving these interaction capabilities would be advantageous for functional studies.

How can the purity and activity of recombinant Phoca largha SRY protein be assessed?

Multiple complementary approaches should be employed:

Purity Assessment:

• SDS-PAGE with Coomassie or silver staining (expect a band at approximately 20-27 kDa depending on the construct)

• Western blotting using anti-SRY antibodies (though cross-reactivity with Phoca largha SRY would need validation)

• Mass spectrometry for precise molecular weight determination and protein identification

Activity Assessment:

• Electrophoretic Mobility Shift Assay (EMSA) to verify DNA binding capability to the consensus sequence AACAAAG, as identified in human SRY research

• Circular dichroism spectroscopy to assess proper folding, particularly of the HMG box domain

• Functional assays measuring transcriptional activation of known SRY target genes in cell-based reporter systems

The DNA binding activity is particularly critical, as research has shown this functionality is required for proper sex determination . When establishing activity assessment protocols, researchers should consider that wild Phoca largha may exhibit different physiological characteristics than captive specimens, which could potentially extend to differences in protein function .

How does acetylation affect Phoca largha SRY protein function and what methods can be used to study this regulation?

Based on human SRY research, acetylation is a critical post-translational modification that regulates SRY subcellular distribution and activity . While specific data on Phoca largha SRY acetylation is not available, researchers can apply the following methodological approaches:

• Detection of acetylation:

  • Immunoprecipitation with anti-SRY antibodies followed by western blotting with anti-acetyllysine antibodies

  • Mass spectrometry to identify specific acetylated lysine residues

• Functional analysis:

  • Site-directed mutagenesis of predicted acetylation sites (lysine to arginine or glutamine)

  • Subcellular localization studies using fluorescently-tagged SRY variants

  • Co-immunoprecipitation assays to identify interactions with acetyltransferases like p300, which has been shown to associate with human SRY in cells and in vitro

• Regulatory mechanisms:

  • Chromatin immunoprecipitation (ChIP) assays to assess how acetylation affects DNA binding in a cellular context

  • Reporter gene assays to determine transcriptional activity changes upon acetylation

Research has demonstrated that human SRY can associate with p300 acetyltransferase both in vivo and in vitro . This interaction suggests that "acetylation and deacetylation of SRY may be important mechanisms for regulating SRY activity during mammalian sex determination" .

What are the challenges in designing CRISPR-Cas9 experiments to study Phoca largha SRY mutations, and how can they be addressed?

CRISPR-Cas9 gene editing has proven valuable for studying SRY mutations in other species, as demonstrated by the mouse model carrying human SRY . When designing similar experiments for Phoca largha SRY, researchers should consider:

Experimental Design Challenges:

• Limited genomic information:

  • Solution: Perform preliminary sequencing of the Phoca largha SRY gene and flanking regions to design precise guide RNAs

  • Utilize comparative genomics with closely related pinniped species to predict conserved regions

• Model system selection:

  • Challenge: Ethical and practical limitations in direct editing of protected Phoca largha

  • Solution: Develop transgenic mouse models expressing Phoca largha SRY as demonstrated with human SRY , or utilize in vitro cell culture systems from available Phoca largha tissues

• Mutation selection strategy:

  • Focus on three domain-specific modifications (N-terminal, HMG box, C-terminal) similar to the approach used for human SRY

  • Prioritize mutations in the HMG box region, as studies with human SRY demonstrate its critical role in DNA binding activity

• Functional assessment:

  • Develop appropriate readouts for sex determination pathway activation

  • Combine molecular (RNA-seq, ChIP-seq) and cellular (immunostaining, reporter assays) approaches

The systematic approach used for human SRY, where "novel genetic modifications in each of SRY's three domains" were generated and subjected to "detailed analysis of their molecular and cellular effects" , provides a valuable template for Phoca largha studies.

How can comparative proteomics approaches enhance our understanding of Phoca largha SRY function in wild versus captive environments?

Recent proteomic research comparing wild and captive Phoca largha pups revealed significant physiological differences that could inform SRY studies . Researchers can leverage these insights through:

• Integrated proteomics approach:

  • Expand comparative proteomics to include sex-specific differences, potentially revealing SRY downstream targets

  • Combine immunoprecipitation with mass spectrometry (IP-MS) to identify SRY-interacting proteins in samples from wild versus captive males

• Physiological context integration:

  • Examine how environmental factors affect SRY expression and function, considering that 51 proteins showed significant expression differences between wild and captive pups

  • Focus on enriched biological pathways identified in previous studies, including "cytoskeleton, phagocytosis, proteolysis, the regulation of gene expression, and carbohydrate metabolism"

• Methodological considerations:

  • Utilize label-free comparative proteomic analysis similar to the approach used for whole blood analysis

  • Apply principal component analysis (PCA) to differentiate SRY-dependent protein expression patterns, as this approach successfully distinguished wild from captive pups with 57.8% explained variation

What are the optimal storage conditions for recombinant Phoca largha SRY protein to maintain stability and activity?

Based on general practices for recombinant DNA-binding proteins and transcription factors:

• Short-term storage (1-2 weeks):

  • Store at 4°C in buffer containing:

    • 20-50 mM Tris-HCl or phosphate buffer (pH 7.5-8.0)

    • 150-300 mM NaCl

    • 1-10% glycerol

    • 1 mM DTT or 0.5-1 mM TCEP (reducing agents)

    • Protease inhibitor cocktail

• Long-term storage:

  • Primary recommendation: Aliquot and store at -80°C in buffer containing 20-50% glycerol

  • Alternative: Lyophilize small aliquots and store at -20°C or -80°C

  • Avoid repeated freeze-thaw cycles, as these can significantly reduce DNA-binding activity

• Activity preservation considerations:

  • Include stabilizing agents such as BSA (0.1-1 mg/ml) when diluting for functional assays

  • Test activity retention after various storage conditions using DNA binding assays

  • Consider the impact of post-translational modifications, particularly acetylation which has been shown to affect human SRY function

• Quality control timeline:

  • Establish a regular testing schedule to verify protein activity

  • Document batch-to-batch variation and storage time effects on functional assays

How can researchers distinguish between direct and indirect effects of Phoca largha SRY on target gene expression?

This methodological question requires multi-layered experimental approaches:

• Direct DNA binding identification:

  • Perform ChIP-seq to identify genome-wide SRY binding sites

  • Validate binding using in vitro methods like EMSA with the consensus sequence AACAAAG identified for human SRY

  • Use DNase I footprinting to precisely map binding regions

• Transcriptional impact assessment:

  • RNA-seq following SRY overexpression or knockdown

  • Time-course experiments to identify early (likely direct) versus late (likely indirect) responsive genes

  • Nascent RNA capture methods (GRO-seq, PRO-seq) to distinguish primary transcriptional effects

• Mechanistic validation:

  • Reporter gene assays with wild-type and mutated target gene promoters

  • Testing SRY variants with mutations in the HMG box domain, as DNA binding ability has been shown to be essential for SRY function

  • CRISPR interference or activation at SRY binding sites to confirm functional relevance

• Protein-protein interaction context:

  • Identify cofactors through IP-MS or yeast two-hybrid screens

  • Assess whether SRY functions as an architectural protein in transcriptional complexes, similar to LEF-1 as suggested for human SRY

  • Map domain-specific interactions, particularly considering that human SRY can interact directly with p300

What are the key considerations when designing antibodies against Phoca largha SRY for research applications?

Developing effective antibodies requires strategic epitope selection and validation:

• Epitope selection strategies:

  • HMG box epitopes: Provide specificity for SRY versus other SOX family proteins

  • N-terminal/C-terminal epitopes: May offer species specificity but could have reduced conservation

  • Consider hydrophilicity, surface accessibility, and secondary structure predictions

  • Avoid regions subject to post-translational modifications like acetylation, which has been documented in human SRY

• Production approaches:

  • Monoclonal antibodies: Offer high specificity but may recognize limited epitopes

  • Polyclonal antibodies: Recognize multiple epitopes but may have higher background

  • Recombinant antibodies: Allow for reproducible production and engineering

• Comprehensive validation protocol:

  • Western blot against recombinant protein and male Phoca largha tissue samples

  • Immunoprecipitation followed by mass spectrometry

  • Immunohistochemistry with appropriate controls (female tissues, competing peptides)

  • Cross-reactivity assessment with SRY from related species

• Application-specific considerations:

  • ChIP-grade antibodies require validation in immunoprecipitation conditions

  • Flow cytometry applications need testing for recognizing native conformations

  • Consider developing phospho-specific or acetylation-specific antibodies based on predicted modification sites

How can recombinant Phoca largha SRY be used to study conservation biology and population genetics in this endangered species?

Phoca largha is critically endangered in China and South Korea , making SRY research valuable for conservation efforts:

• Sex determination and population structure:

  • Develop non-invasive SRY detection methods from environmental samples

  • Use SRY as a male-specific marker to assess sex ratios in wild populations

  • Compare SRY sequence variation across different Phoca largha populations to assess genetic diversity

• Reproductive biology applications:

  • Study SRY expression patterns during development to understand critical timing of sex determination

  • Investigate environmental influences on SRY expression, particularly relevant given the physiological differences observed between wild and captive individuals

  • Develop assays to monitor reproductive health in captive breeding programs

• Evolutionary adaptation analysis:

  • Compare SRY sequence and function between populations under different environmental pressures

  • Assess whether SRY variants correlate with reproductive success or adaptation to specific environments

  • Investigate SRY in the context of the "more powerful immune capacities" observed in wild versus captive pups

• Conservation strategy implications:

  • Guide genetic management of captive populations based on SRY variation data

  • Inform reintroduction programs by understanding how captivity might affect SRY-dependent development

  • Develop molecular tools for monitoring recovery of wild populations

What insights can protein-protein interaction studies of Phoca largha SRY provide about its role in marine mammal sex determination?

Protein interaction studies can reveal evolutionary adaptations in sex determination mechanisms:

• Interactome mapping approaches:

  • Yeast two-hybrid screening using Phoca largha cDNA libraries

  • Proximity labeling methods (BioID, APEX) in cellular models

  • Co-immunoprecipitation with mass spectrometry from gonadal tissues

  • Focus on detecting interactions with proteins like p300, which has been shown to associate with human SRY both in cells and in vitro

• Comparative evolutionary analysis:

  • Compare Phoca largha SRY interactors with those from terrestrial mammals

  • Identify pinniped-specific interactions that might represent adaptations to marine environments

  • Assess conservation of key interactions like those with p300 acetyltransferase that regulate human SRY activity

• Functional characterization of interactions:

  • Mutational analysis to map interaction domains

  • Competitive binding assays to identify regulatory mechanisms

  • Investigate whether environmental factors affecting wild versus captive seals influence these interactions

• Methodological considerations:

  • Develop cell culture systems that approximate the physiological context of developing Phoca largha gonads

  • Consider the impact of post-translational modifications, particularly acetylation which has been shown to affect human SRY localization and function

  • Design experiments accounting for potential differences in wild versus captive animals, given that 51 proteins showed significantly different expression between these groups

How does the differential protein expression observed between wild and captive Phoca largha pups potentially impact SRY function and sex determination?

The proteomics research on Phoca largha reveals significant physiological differences between wild and captive individuals that may extend to sex determination mechanisms:

• Regulatory pathway connections:

  • Several differentially expressed proteins are involved in gene expression regulation, including BANF1, NIF3L1, CARHSP1, EIF5, and XPO1

  • These regulatory differences could affect SRY expression or the expression of its target genes

  • The altered ubiquitin-mediated proteolysis pathway might influence SRY protein stability and turnover

• Cellular environment considerations:

  • Differences in cytoskeletal proteins (FLNA, TUBB, VCL, ANK3) could affect nuclear transport of SRY

  • Variations in cell adhesion proteins could impact cellular organization during gonadal development

  • The nutritional stress observed in wild pups might trigger adaptive responses in developmental pathways including sex determination

• Immune system interactions:

  • Wild pups show "more powerful immune capacities" , which could interact with developmental processes

  • Proteins involved in phagocytosis show higher abundance in wild pups , potentially affecting tissue remodeling during gonadal development

• Methodological approach for investigation:

  • Combine proteomics with transcriptomics during critical windows of sex determination

  • Develop in vitro models that recapitulate wild versus captive cellular environments

  • Use systems biology approaches to model pathway interactions between differentially expressed proteins and sex determination networks

What emerging technologies could advance our understanding of Phoca largha SRY structure-function relationships?

Several cutting-edge approaches show promise for detailed characterization:

• Structural biology advances:

  • Cryo-electron microscopy for SRY-DNA complex visualization

  • NMR spectroscopy to study dynamics of SRY-DNA interactions

  • AlphaFold2 or similar AI prediction tools to model Phoca largha SRY structure based on sequence, particularly valuable given the challenges of experimental structure determination

• Single-molecule techniques:

  • FRET to study conformational changes upon DNA binding

  • Optical tweezers to examine SRY-induced DNA bending mechanics

  • Single-molecule tracking in live cells to study dynamics of SRY localization and binding

• Genomic and epigenomic approaches:

  • CUT&RUN or CUT&Tag for higher resolution mapping of SRY binding sites

  • ATAC-seq to examine chromatin accessibility changes induced by SRY

  • HiChIP to investigate three-dimensional chromatin interactions mediated by SRY

• Functional genomics:

  • CRISPR screens to identify genes affecting SRY function

  • Base editing to introduce precise mutations mimicking natural variants

  • Massively parallel reporter assays to assess the impact of sequence variations on SRY binding sites

These technologies could help clarify how SRY functions as "a chromosomal docking site for auxiliary proteins" and how "target gene specificity would then be the result of SRY–protein interactions in addition to protein–DNA interactions" .

How can mathematical modeling help predict the impact of environmental factors on Phoca largha SRY expression and function?

Mathematical modeling offers powerful tools for understanding complex biological systems:

• Network modeling approaches:

  • Gene regulatory network models incorporating SRY and its targets

  • Bayesian networks to identify conditional dependencies between environmental factors and SRY pathway components

  • Boolean networks to simulate the binary nature of sex determination decisions

• Environmental factor integration:

  • Develop models incorporating temperature, nutritional status, and stress parameters

  • Use machine learning to identify patterns in how these factors correlate with observed physiological differences between wild and captive pups

  • Create pathway models that connect the 51 differentially expressed proteins identified between wild and captive pups to sex determination networks

• Multi-scale modeling considerations:

  • Molecular scale: SRY binding kinetics and protein-protein interactions

  • Cellular scale: Gene expression dynamics during critical developmental windows

  • Tissue scale: Cell-cell interactions during gonadal development

  • Organism scale: Feedback from systemic factors (hormones, immune system)

• Validation approaches:

  • Design targeted experiments to test model predictions

  • Refine models based on new proteomics and transcriptomics data

  • Apply sensitivity analysis to identify key parameters for experimental focus

Such models could help explain how captivity in artificial environments "significantly affect[s] the protein composition and abundance in the whole blood of P. largha pups" and potentially extends to developmental processes.