Recombinant Schizosaccharomyces pombe Uncharacterized protein C1739.04c (SPCC1739.04c)

What experimental approaches are recommended for initial characterization of SPCC1739.04c?

For initial characterization, a multi-faceted approach is recommended:

• Localization studies: GFP-tagging at either N- or C-terminus to determine subcellular localization, as performed in the S. pombe nuclear pore complex study . Replace the genomic sequence coding for SPCC1739.04c with a GFP-tagged version expressed under its natural promoter.

• Gene deletion analysis: Generate heterozygous diploid deletion mutants using plasmid-based deletion strategies with long homologous regions as described in the S. pombe genome deletion project . This will help determine if the gene is essential for vegetative growth.

• Protein-protein interaction studies: Perform co-immunoprecipitation experiments or yeast two-hybrid screens to identify interacting partners, as BioGRID data suggests SPCC1739.04c has 4 known protein interactions .

• Phenotypic analysis: Examine phenotypic changes in deletion or conditional mutants under various stress conditions to infer function.

How should I design experiments to determine if SPCC1739.04c is essential for S. pombe growth?

To determine essentiality, follow this experimental design protocol:

• Generate heterozygous diploid deletion mutants: Use a plasmid-based knockout strategy with extended homologous regions flanking the target gene. This approach has proven more efficient than PCR-based methods for difficult-to-delete genes in S. pombe .

• Sporulation and tetrad analysis: Induce sporulation in heterozygous diploid strains and dissect asci on rich medium (YES). Observe colony formation for all four spores.

• Viability assessment:

  Observation	Interpretation
  All four spores form colonies	Gene is non-essential
  Only two spores form colonies	Gene is essential
  Aberrant growth patterns in two spores	Gene affects growth but is not strictly essential

• Confirmation with complementation: Transform the deletion strain with a plasmid expressing SPCC1739.04c to confirm that any observed phenotype is due to the gene deletion.

• Conditional shutdown system: For essential genes, employ a conditional expression system (e.g., nmt1 promoter) to control gene expression and study the terminal phenotype .

This rigorous approach will provide conclusive evidence regarding the essentiality of SPCC1739.04c for vegetative growth in S. pombe.

What are the key considerations for designing experiments to study SPCC1739.04c function during meiosis?

Based on evidence that many non-essential S. pombe nucleoporins are required for normal meiotic progression , consider these methodological aspects when investigating SPCC1739.04c's role in meiosis:

• Induction of synchronous meiosis: Use temperature-sensitive pat1-114 mutants or nitrogen starvation methods to induce synchronous meiosis in deletion or conditional mutant strains.

• Experimental design structure:

  • Independent variable: SPCC1739.04c expression level/presence

  • Dependent variables: Meiotic progression, spore formation, spore viability

  • Control variables: Temperature, media composition, cell density

• Analysis methods:

  • Live imaging with fluorescent markers for meiotic progression

  • DAPI staining for nuclear division assessment

  • Tetrad dissection to quantify spore viability

• Quantification metrics:

  • Percentage of cells completing meiosis I and II

  • Efficiency of spore formation

  • Spore viability (percentage of spores forming colonies)

• Statistical approach: Use between-subjects experimental design with multiple replicates (n ≥ 3) and appropriate statistical tests to determine significance of phenotypic differences .

How can I optimize GFP-tagging of SPCC1739.04c to study its localization while preserving protein function?

When designing GFP-tagging experiments for SPCC1739.04c, consider these methodological approaches:

• Tag position optimization:

  • Test both N- and C-terminal tags, as the C-terminal region may contain a transmembrane domain that could be disrupted by tagging .

  • If terminal tagging disrupts function, consider internal tagging at predicted loop regions.

• Integration method:

  • Use genomic integration rather than plasmid-based expression to maintain endogenous expression levels .

  • Design long homologous regions (>500 bp) flanking the integration site to improve recombination efficiency.

• Functional validation:

  • Confirm that GFP-tagged protein is functional by testing complementation of deletion phenotypes.

  • For essential genes, the viability of strains with GFP-tagged protein as the sole copy indicates functionality.

• Co-localization analysis:

  • Include known marker proteins (e.g., Cut11-mCherry for nuclear envelope) to accurately determine subcellular localization .

  • Implement the control methodology used for Sec13 in S. pombe nuclear pore complex studies, testing localization in wildtype and nup132Δ strains where NPCs cluster on one side of the nucleus .

• Quantitative analysis:

  • Measure fluorescence intensity relative to other known proteins to estimate abundance .

  • Use standardized exposure settings across different strains for accurate comparisons.

What experimental approaches should be used to identify and validate the protein interaction network of SPCC1739.04c?

For comprehensive protein interaction network analysis, implement these methodological strategies:

• Primary interaction screening:

  • Yeast two-hybrid (Y2H): Use both N- and C-terminal fusions to DNA-binding and activation domains to minimize false negatives.

  • Affinity purification-mass spectrometry (AP-MS): Tag SPCC1739.04c with tandem affinity purification (TAP) tag or FLAG-tag for protein complex isolation.

• Binary interaction validation:

  • Co-immunoprecipitation: Confirm direct interactions using reciprocal co-immunoprecipitation with epitope-tagged proteins.

  • Bimolecular Fluorescence Complementation (BiFC): Visualize interactions in living cells by fusing protein pairs to complementary fragments of a fluorescent protein.

• Interaction mapping:

  • Domain mapping: Create truncation mutants to identify interaction domains.

  • Site-directed mutagenesis: Test the effect of specific amino acid changes on interactions.

• Quantitative analysis:

  • FRET/FLIM: Measure interaction affinities in vivo using Förster resonance energy transfer.

  • Surface plasmon resonance (SPR): Determine binding kinetics with purified recombinant proteins.

• Network analysis:

  • Compare identified interactors with known protein complexes in S. pombe.

  • Integrate with existing interaction data from BioGRID (currently 4 known interactions) .

How should I analyze and interpret gene expression data for SPCC1739.04c across different experimental conditions?

When analyzing gene expression data for SPCC1739.04c, apply these methodological principles:

• Normalization strategies:

  • Normalize to appropriate housekeeping genes stable under your experimental conditions.

  • Consider using multiple reference genes (e.g., act1, cdc2, and pda1) for more reliable normalization.

  • For RNA-seq data, use standard normalization methods (TPM, RPKM, or DESeq2 normalization).

• Statistical analysis framework:

  • Perform minimum of three biological replicates for statistical validity.

  • Apply appropriate statistical tests based on data distribution (parametric vs. non-parametric).

  • Use multiple testing correction (e.g., Benjamini-Hochberg) when analyzing across multiple conditions.

• Comparative analysis:

  • Compare expression patterns with genes of known function to infer potential roles.

  • Look for co-expression with genes in specific complexes or pathways (e.g., nuclear pore complex components).

• Visualization approaches:

  • Use heatmaps for multi-condition comparisons.

  • Create expression profile clusters to identify genes with similar patterns.

• Validation methods:

  • Confirm key findings with alternative methods (e.g., RT-qPCR, northern blot).

  • Correlate expression changes with phenotypic observations.

What approaches should be used to analyze phenotypic data from SPCC1739.04c deletion or mutation studies?

For rigorous phenotypic data analysis, implement these methodological guidelines:

What are the methodological approaches for determining if SPCC1739.04c functions as part of the nuclear pore complex in S. pombe?

Based on nuclear pore complex (NPC) characterization studies in S. pombe , consider these specialized methodological approaches:

• Colocalization analysis:

  • Test colocalization with established NPC markers (e.g., Cut11-mCherry).

  • Examine whether SPCC1739.04c-GFP clusters with NPCs in nup132Δ strains, which display characteristic NPC clustering on one side of the nucleus .

• Biochemical fractionation:

  • Perform subcellular fractionation to isolate nuclear envelopes.

  • Analyze co-purification with known NPC components by western blotting or mass spectrometry.

• Interaction analysis:

  • Test for direct interactions with known NPC components using Y2H or co-IP.

  • Examine whether loss of SPCC1739.04c affects localization of other nucleoporins.

• Functional assays:

  • Assess nuclear transport efficiency in deletion or conditional mutants.

  • Examine nuclear envelope structure by electron microscopy.

  • Test for genetic interactions with known nucleoporin mutants.

• Comparative analysis across species:

  • Determine if SPCC1739.04c has structural or functional homologs in other organisms.

  • Compare its properties with S. cerevisiae Nup42/Rip1 or human Nlp1, which show sequence similarity to another S. pombe protein (Amo1) associated with the NPC .

How can integrative approaches be used to deduce the function of SPCC1739.04c as an uncharacterized protein?

For comprehensive functional characterization of SPCC1739.04c, implement this integrative methodological framework:

• Multi-omics integration:

  • Combine transcriptomics (RNA-seq of deletion mutants)

  • Proteomics (interactome analysis)

  • Metabolomics (metabolic changes in mutants)

  • Phenomics (systematic phenotypic analysis)

• Computational prediction and modeling:

  • Perform structural prediction using AlphaFold or similar tools

  • Conduct molecular dynamics simulations to predict functional domains

  • Use machine learning approaches to predict function from sequence and structure

• Systematic genetic interaction analysis:

  • Create double mutants with genes in related pathways

  • Perform synthetic genetic array (SGA) analysis to identify genetic interactions

  • Quantify epistatic relationships to place gene in functional pathways

• Conditional perturbation approaches:

  • Use auxin-inducible degron (AID) system for rapid protein depletion

  • Apply temperature-sensitive alleles for temporal control

  • Implement chemical-genetic approaches to conditionally inhibit function

• Cross-species complementation:

  • Test functional conservation by complementation with homologs from other species

  • Express SPCC1739.04c in other model organisms to observe phenotypes

  • Perform systematic phylogenetic profiling to identify co-evolved genes

By integrating these diverse approaches, researchers can overcome the challenges of characterizing proteins with no known function and develop robust hypotheses about SPCC1739.04c's biological role.

What strategies can address difficulties in generating SPCC1739.04c deletion mutants?

The S. pombe genome deletion project encountered difficulties deleting certain genes using standard PCR-based methods . For challenging deletions like SPCC1739.04c, implement these methodological solutions:

• Enhanced deletion strategies:

  • Use plasmid-based knockout strategy with extended homologous regions (>1kb) flanking the target gene .

  • Consider the pCloneKan1 plasmid system described in the S. pombe genome deletion project update .

  • Implement a two-step deletion process with initial integration of a marker followed by removal of the wild-type allele.

• Heterozygous diploid approach:

  • Generate heterozygous diploid deletion first, then induce sporulation and tetrad analysis.

  • Use pON177 plasmid containing mat1-M sequence to facilitate sporulation .

• Technical optimization:

  • Vary transformation conditions (electroporation vs. lithium acetate).

  • Test different selection markers (kanMX6, natMX4, hphMX4).

  • Ensure the absence of secondary structure in homologous regions that might impair recombination.

• Verification strategies:

  • Use multiple PCR primer pairs to confirm deletion.

  • Perform Southern blot analysis to verify single integration at the correct locus.

  • Use RT-PCR to confirm absence of transcript in deletion strains.

• Alternative approaches if deletion fails:

  • Consider conditional expression systems (nmt1 repressible promoter).

  • Implement RNA interference or CRISPR interference for knockdown.

  • Use N-degron or auxin-inducible degron tags for conditional protein degradation.

What are the methodological considerations for optimizing expression and purification of recombinant SPCC1739.04c protein?

For successful expression and purification of recombinant SPCC1739.04c protein, consider these methodological details:

• Expression system selection:

  • E. coli: Suitable for initial attempts as indicated by successful expression of His-tagged full-length protein .

  • Yeast expression: Consider S. cerevisiae or P. pastoris for eukaryotic post-translational modifications.

  • Insect cells: Baculovirus expression system for higher eukaryotic protein folding environment.

• Construct design optimization:

  • Test different affinity tags (His, GST, MBP) at both N- and C-termini.

  • Consider fusion partners (SUMO, thioredoxin) to enhance solubility.

  • Examine sequence for potential transmembrane domains that might affect solubility.

  • Design constructs with predicted domain boundaries if full-length expression is problematic.

• Expression condition optimization:

  • Temperature: Test reduced temperatures (16-20°C) to improve folding.

  • Induction: Optimize inducer concentration and induction time.

  • Media formulation: Test rich vs. minimal media, supplementation with rare codons.

  • Codon optimization: Adjust codon usage for the expression host.

• Purification strategy development:

  • Implement two-step purification (affinity + size exclusion chromatography).

  • Optimize buffer composition (pH, salt concentration, additives).

  • Test detergents if transmembrane domains are present.

  • Include protease inhibitors to prevent degradation.

• Quality control assessment:

  • Verify protein identity by mass spectrometry.

  • Assess protein folding by circular dichroism or thermal shift assay.

  • Check monodispersity by dynamic light scattering.

  • Validate biological activity through appropriate functional assays.