Recombinant Schizosaccharomyces pombe Uncharacterized protein C330.19c (SPCC330.19c)

What expression patterns does SPCC330.19c exhibit across different conditions?

Transcriptomic analysis of SPCC330.19c has been conducted using both tiling array and Illumina sequencing technologies across various experimental conditions . These data are available through resources like pombeTV from the Bähler Lab, which hosts expression profiles for numerous S. pombe genes including SPCC330.19c. Researchers can use these expression profiles to identify conditions where SPCC330.19c is differentially regulated, potentially offering clues about its function. When analyzing expression data, it is important to consider both absolute expression levels and relative changes across conditions, as even modest expression changes may be biologically significant for regulatory proteins.

What is the genomic context of SPCC330.19c in S. pombe?

SPCC330.19c is located in a genomic region that has been studied in the context of DNA replication origins. It is positioned near the SPCC330.03c gene, with the nearest predicted Origins of Replication (ORIs) located approximately 32 kb upstream and 10 kb downstream . This genomic region has been experimentally verified to lack a replication origin through two-dimensional electrophoresis . The gene's designation (SPCC330.19c) indicates its location on chromosome II of S. pombe. Understanding the genomic neighborhood of SPCC330.19c is crucial for interpreting its potential functional relationships with nearby genes and its role in chromosome organization and replication timing.

Could SPCC330.19c be regulated by iron availability through the CCAAT-binding factor Php4?

While SPCC330.19c is not explicitly mentioned in the context of iron regulation in the provided search results, multiple S. pombe genes, particularly those involved in the TCA cycle and electron transport chain, are regulated in response to iron availability through the CCAAT-binding factor Php4 . The table below shows examples of genes regulated by iron and Php4:

To determine if SPCC330.19c is similarly regulated, researchers could analyze its promoter region for the presence of CCAAT boxes and experimentally test its expression in wild-type versus php4Δ strains under iron-replete and iron-depleted conditions.

What methodologies are most effective for studying the transcriptional regulation of SPCC330.19c?

For studying the transcriptional regulation of uncharacterized genes like SPCC330.19c, a multi-faceted approach would be most informative:

• RNA-seq or microarray analysis under diverse conditions to identify factors that influence expression, building upon existing tiling array and Illumina sequencing data .

• Promoter analysis to identify potential regulatory elements, such as CCAAT boxes that might indicate regulation by factors like Php4 .

• Chromatin immunoprecipitation (ChIP) experiments to identify transcription factors that bind to the promoter region.

• Reporter gene assays using the SPCC330.19c promoter fused to reporter genes like luciferase or GFP to validate the functionality of potential regulatory elements.

• CRISPR interference (CRISPRi) targeting potential regulatory regions to assess their contribution to gene expression.

Integration of these approaches can provide a comprehensive understanding of the regulatory mechanisms controlling SPCC330.19c expression.

What experimental strategies can researchers employ to determine SPCC330.19c function?

Several complementary approaches can be employed to determine the function of uncharacterized proteins like SPCC330.19c:

• Gene deletion or disruption to assess phenotypic consequences, using PCR-based deletion methods or CRISPR-Cas9 .

• Protein localization studies using fluorescent tags to determine subcellular distribution.

• Protein-protein interaction studies (yeast two-hybrid, co-immunoprecipitation, BioID) to identify interaction partners.

• Expression profiling under various conditions to identify co-regulated genes .

• Comparative genomics to identify orthologs in other species with known functions.

• Phenotypic profiling under diverse stress conditions to identify specific sensitivities.

• Biochemical characterization of the purified recombinant protein to identify potential enzymatic activities or binding properties .

The integration of multiple approaches increases the likelihood of successfully characterizing this uncharacterized protein.

How can researchers produce and purify recombinant SPCC330.19c for functional studies?

Recombinant SPCC330.19c can be produced in E. coli with a His-tag for purification, as indicated by the available commercial product . The optimal protocol would include:

• Cloning the SPCC330.19c coding sequence into an expression vector with an N- or C-terminal His-tag.

• Transforming the construct into an E. coli strain optimized for protein expression (e.g., BL21(DE3)).

• Optimizing expression conditions including temperature (typically 16-30°C), IPTG concentration (0.1-1 mM), and induction time (4-24 hours).

• Cell lysis under native conditions using methods such as sonication or high-pressure homogenization.

• Purification using immobilized metal affinity chromatography (IMAC) with Ni-NTA or Co-NTA resins.

• Additional purification steps such as ion exchange or size exclusion chromatography if higher purity is required.

• Quality control assessment using SDS-PAGE, Western blotting, and mass spectrometry.

For small proteins like SPCC330.19c (90 amino acids), special attention should be paid to potential solubility issues and the impact of the affinity tag on protein structure and function.

What bioinformatic approaches can predict potential functions for SPCC330.19c?

Several bioinformatic approaches can generate hypotheses about SPCC330.19c function:

• Sequence homology searches using PSI-BLAST or HHpred to identify distant relatives with known functions.

• Protein domain and motif analysis using Pfam, PROSITE, or InterPro.

• Secondary and tertiary structure prediction using tools like Phyre2, I-TASSER, or AlphaFold2.

• Co-expression network analysis using existing S. pombe transcriptomic datasets .

• Gene neighborhood analysis to identify functionally related genes based on genomic proximity.

• Integration of multiple data types as demonstrated in approaches for identifying genes involved in human mitochondrial disorders .

These computational predictions can guide experimental design by suggesting targeted assays to test specific functional hypotheses.

How might SPCC330.19c be involved in DNA replication or chromatin organization?

The genomic location of SPCC330.19c near potential replication origins warrants investigation of its role in DNA replication or chromatin organization . To explore this possibility:

• Analyze the effects of SPCC330.19c deletion or overexpression on cell cycle progression and DNA replication timing using flow cytometry and DNA combing techniques.

• Perform ChIP experiments to determine if SPCC330.19c associates with specific genomic regions or replication components.

• Investigate potential DNA-binding properties of purified SPCC330.19c using electrophoretic mobility shift assays (EMSA) or DNA footprinting.

• Study the impact of SPCC330.19c on chromatin structure using techniques like ATAC-seq or MNase-seq.

• Analyze the effect of nearby genomic features, such as the identified stretch of 29 A+Ts (29W) mentioned in relation to ARS activity, on SPCC330.19c function .

These experiments could reveal whether SPCC330.19c plays a direct role in DNA replication or indirectly affects chromatin organization.

How can researchers distinguish between direct and indirect effects when studying SPCC330.19c function?

Distinguishing between direct and indirect effects is crucial when studying uncharacterized proteins. For SPCC330.19c, researchers could:

• Use rapid induction or repression systems (e.g., auxin-inducible degron) to observe immediate consequences of protein depletion or induction.

• Employ separation-of-function mutations to disrupt specific interactions or activities without eliminating the entire protein.

• Perform in vitro reconstitution experiments with purified components to demonstrate direct biochemical activities.

• Apply time-course experiments following protein perturbation to establish the sequence of events.

• Combine genetic approaches with biochemical validation to correlate in vivo and in vitro observations.

• Use proximity labeling techniques (BioID, APEX) to identify proteins in the immediate vicinity of SPCC330.19c in vivo.

These approaches help establish causality and mechanism in functional studies, distinguishing primary effects from downstream consequences.

What novel integrative genomics approaches could accelerate functional characterization of SPCC330.19c?

Innovative integrative genomics approaches similar to those used in characterizing human disease genes could accelerate functional discovery for SPCC330.19c :

• Integration of genomic, transcriptomic, and proteomic data to identify correlations that suggest function.

• Network-based approaches that leverage known protein-protein interactions and pathway information.

• Computational prediction of subcellular localization combined with experimental validation.

• Cross-species functional prediction based on evolutionary conservation patterns.

• Systematic genetic interaction mapping using technologies like synthetic genetic array (SGA).

• Analysis of protein structure-function relationships using structural predictions and targeted mutagenesis.

• Machine learning approaches that integrate diverse data types to predict protein function.

These integrative approaches have successfully identified gene functions in complex systems like human mitochondrial disorders and could be adapted for S. pombe proteins .

How does the conservation pattern of SPCC330.19c relate to its potential essentiality?

Research on S. pombe has shown correlations between gene conservation and essentiality, with approximately 17.5% of genes being essential for vegetative growth . Analysis of SPCC330.19c conservation could provide insights into its functional importance:

• Compare SPCC330.19c conservation across fungal species and more distant eukaryotes.

• Determine whether it belongs to the category of ancient genes that have been conserved in multiple lineages.

• Examine whether SPCC330.19c is among the genes that have been lost in the Saccharomyces cerevisiae lineage but retained in S. pombe and other organisms like Caenorhabditis elegans .

• Analyze whether its orthologs in other species are essential when that information is available.

The study of S. pombe genes has revealed that essentiality is correlated with the timing of a gene's appearance on the tree of life and its conservation within all branches of the tree . Non-essential genes in S. pombe may still play important roles under specific conditions not tested in standard laboratory growth assays.

Could SPCC330.19c be involved in mitochondrial function or the electron transport chain?

While SPCC330.19c is not directly linked to mitochondrial function in the provided search results, the regulation of many S. pombe genes involved in the TCA cycle and electron transport chain by factors like the CCAAT-binding factor Php4 suggests this as a possibility worth investigating . To explore potential mitochondrial functions:

• Check for mitochondrial targeting sequences using prediction tools like MitoFates or TargetP.

• Perform subcellular localization studies with fluorescent reporters to determine if SPCC330.19c localizes to mitochondria.

• Examine phenotypes of SPCC330.19c deletion mutants under conditions that stress mitochondrial function.

• Look for co-expression with known mitochondrial genes in existing transcriptomic datasets.

• Apply integrative genomics approaches similar to those used in identifying genes involved in cytochrome c oxidase deficiency .

The importance of mitochondrial proteins in cellular metabolism and their conservation across eukaryotes makes this a promising avenue for investigation.

What structural biology approaches would be most suitable for a small protein like SPCC330.19c?

For a small protein like SPCC330.19c (90 amino acids), several structural biology approaches would be particularly suitable:

• Nuclear Magnetic Resonance (NMR) spectroscopy, which excels at determining structures of small proteins in solution.

• X-ray crystallography, if the protein can be crystallized, potentially in complex with interaction partners.

• Cryo-electron microscopy (cryo-EM), especially if SPCC330.19c functions as part of a larger complex.

• Computational structure prediction using tools like AlphaFold2, which has shown remarkable accuracy for small proteins.

• Circular dichroism (CD) spectroscopy to determine secondary structure content and thermal stability.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify flexible regions and potential binding interfaces.

Structural information can provide critical insights into function, especially for uncharacterized proteins, by revealing potential binding sites, catalytic residues, or structural homology to proteins of known function.

How can knowledge about SPCC330.19c contribute to our understanding of conserved eukaryotic pathways?

Research on uncharacterized proteins like SPCC330.19c in model organisms like S. pombe can advance our understanding of conserved eukaryotic pathways:

• S. pombe serves as an excellent model for studying conserved cellular processes, with approximately 50% of its genes having human homologs.

• Discovery of SPCC330.19c function could reveal novel components of fundamental pathways like DNA replication, transcription regulation, or mitochondrial function.

• Comparative studies between S. pombe, S. cerevisiae, and higher eukaryotes can highlight evolutionary conservation and divergence of protein functions.

• If SPCC330.19c belongs to the set of ancient genes retained in S. pombe but lost in S. cerevisiae, its characterization could fill knowledge gaps about ancestral eukaryotic functions .

• Findings in S. pombe often translate to human biology, potentially informing our understanding of human disease mechanisms, as demonstrated by studies of mitochondrial disorders .

The compact genome and experimental tractability of S. pombe make it an ideal system for functional characterization of conserved uncharacterized proteins.

What are the potential applications of recombinant SPCC330.19c protein beyond basic research?

While primarily a tool for basic research, recombinant SPCC330.19c protein could have broader applications:

• Development of antibodies for detecting and studying the endogenous protein.

• Screening for small molecule modulators of protein function once its activity is characterized.

• Structural studies to advance our understanding of protein folding and evolution.

• Use as a control or reference protein in proteomic and biochemical studies.

• Educational applications in teaching protein purification, characterization, and functional analysis techniques.

The availability of high-quality recombinant protein enables a wide range of experimental approaches that can accelerate functional characterization and potentially reveal unexpected applications.

What emerging technologies would be most valuable for comprehensive characterization of SPCC330.19c?

Several cutting-edge technologies could significantly advance our understanding of SPCC330.19c:

• CRISPR-based technologies for precise genome editing and transcriptional modulation in S. pombe.

• Single-cell transcriptomics to identify cell-to-cell variation in SPCC330.19c expression and potential heterogeneous functions.

• Proximity labeling methods like TurboID for identifying protein interaction networks in living cells.

• Advanced imaging techniques such as lattice light-sheet microscopy for studying protein dynamics with minimal phototoxicity.

• Cryo-electron tomography for visualizing proteins in their native cellular context.

• Integrative structural biology approaches combining multiple experimental and computational methods.

• Machine learning-based functional prediction algorithms trained on diverse data types.

These technologies, applied in combination with traditional approaches, can provide unprecedented insights into the function, regulation, and interactions of previously uncharacterized proteins like SPCC330.19c.