Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YGL165C (YGL165C)

Basic Research Questions

• What experimental techniques are most effective for initial characterization of YGL165C function?

For initial characterization of YGL165C function, implement a systematic approach combining multiple techniques. Begin with gene deletion studies to assess growth phenotypes under various conditions (temperature, carbon sources, stress). Follow with GFP tagging for localization studies using fluorescence microscopy. RNA-seq analysis during different growth phases can reveal expression patterns. For protein-level characterization, purify the recombinant protein using an affinity tag system and perform in vitro biochemical assays. This multi-faceted approach provides complementary data streams that can converge on potential functions.

• How should researchers design experiments to determine if YGL165C is essential for yeast viability?

To determine if YGL165C is essential for yeast viability, implement a methodical experimental design following established principles . First, create a heterozygous diploid strain (YGL165C/ygl165c∆) using homologous recombination with a selectable marker. Induce sporulation and perform tetrad analysis, observing whether all four spores are viable. If YGL165C is essential, only two spores (containing the wild-type gene) will grow. Alternatively, use a conditional expression system where YGL165C is placed under a regulatable promoter in a complete deletion background. Systematically repress expression and monitor growth across multiple replicate cultures compared to control strains.

• What are the best approaches for generating recombinant YGL165C protein for structural studies?

For generating recombinant YGL165C suitable for structural studies, selection of the appropriate expression system is critical. Consider the following expression systems based on your specific requirements:

Design constructs with cleavable affinity tags (His6, GST) positioned to minimize interference with protein folding. Optimize expression conditions through factorial experimental design , systematically varying temperature, induction time, and media composition.

• How can researchers predict potential functions of YGL165C using bioinformatic approaches?

To predict potential functions of YGL165C using bioinformatics, implement a hierarchical analysis workflow. Start with sequence-based approaches: BLAST searches against multiple databases to identify homologs, InterProScan for domain identification, and multiple sequence alignments to identify conserved residues. Progress to structure-based prediction using AlphaFold2 or SWISS-MODEL to generate 3D models, followed by structural comparison using DALI or TM-align to identify proteins with similar folds despite low sequence identity. Analyze genomic context conservation across related yeast species to identify syntenic relationships. Apply machine learning tools that integrate sequence and structural features to predict function. Finally, examine high-throughput datasets including genetic interaction networks and co-expression data to identify functional associations.

Advanced Research Questions

• How can researchers use two-hybrid analysis to identify interaction partners of YGL165C?

For two-hybrid analysis of YGL165C interaction partners, design a systematic screening approach similar to that described for proteasome proteins . Create both DNA-binding domain (BD) and activation domain (AD) fusions of YGL165C, ensuring proper folding by testing multiple truncation variants if necessary. For screening, use a comprehensive AD-fusion yeast library covering the entire S. cerevisiae proteome. Implement a stringent confirmation protocol: test interactions in both orientations (BD-YGL165C × AD-prey and AD-YGL165C × BD-prey), validate with secondary assays such as co-immunoprecipitation, and quantify interaction strength using β-galactosidase assays. Filter false positives by comparing against published interaction databases and control screens .

• What methodologies should be used to investigate YGL165C's potential role in meiotic processes?

To investigate YGL165C's potential role in meiotic processes, employ a systematic approach informed by established meiotic research methodologies . Begin with expression profiling during meiotic progression using synchronized cultures and RT-qPCR or RNA-seq, comparing YGL165C expression patterns with known meiotic genes. Create genomic epitope-tagged strains (YGL165C-FLAG or YGL165C-HA) and perform ChIP-seq analysis during meiosis to identify potential DNA binding sites, comparing these with known recombination hotspots.

Based on research in related yeast proteins, meiotic chromosomes form a characteristic loop-axis structure where chromatin loops emanate from a proteinaceous axis containing components like Rec8, Hop1, and Red1 . In S. cerevisiae, Spo11-DSB formation is influenced by this chromosome organization, with DSBs forming preferentially in gene promoters within the chromatin loops . Analysis shows that axis proteins (Hop1, Red1) colocalize with cohesion subunit Rec8 along chromosomes, except in specific regions where their absence correlates with suppressed recombination .

• How can CRISPR-Cas9 technology be optimized for studying YGL165C function in Saccharomyces cerevisiae?

To optimize CRISPR-Cas9 for studying YGL165C function, design a comprehensive experimental approach following systematic experimental design principles . Key parameters for guide RNA design in S. cerevisiae include:

For precise gene editing, design repair templates with homology arms flanking the desired modification, incorporating silent mutations in the PAM site to prevent re-cutting .

• What experimental design is most appropriate for resolving contradictory findings about YGL165C function?

To resolve contradictory findings about YGL165C function, implement a systematic experimental design based on established methodological principles . First, identify specific variables that might explain discrepancies: strain background differences, growth conditions, assay sensitivities, or genetic interactions. Design factorial experiments that systematically vary these factors while measuring multiple output variables. Implement both between-subjects (comparing different strains) and within-subjects (same strain under different conditions) designs to increase robustness . Control for extraneous variables by standardizing growth media, temperature, and cell density across experiments. For each contradictory finding, design validation experiments using orthogonal techniques that rely on different principles but address the same biological question.

• How should researchers investigate potential post-translational modifications of YGL165C?

To investigate potential post-translational modifications (PTMs) of YGL165C, implement a multi-faceted approach combining computational prediction with experimental validation. Begin with bioinformatic prediction tools specific to each PTM type (phosphorylation, ubiquitination, sumoylation, etc.) to identify candidate sites. Create a genomically integrated tagged version of YGL165C (TAP-tag or FLAG-tag) and purify the protein under native conditions from cells grown in various environmental conditions. Analyze purified protein using high-resolution mass spectrometry with multiple fragmentation methods (CID, ETD, HCD) to maximize PTM detection. Validate identified modifications using site-specific antibodies or by mutating modified residues to non-modifiable amino acids (e.g., S→A for phosphorylation) and assessing functional consequences.

• What approaches should be used to determine if YGL165C is involved in chromosome organization during meiosis?

To determine if YGL165C participates in chromosome organization during meiosis, design a comprehensive experimental approach informed by established meiotic research methodologies . First, analyze YGL165C localization during meiotic progression using chromosomal spreads and immunofluorescence microscopy, comparing its distribution with known axis proteins (Hop1, Red1) and synaptonemal complex components (Zip1) .

Studies in yeasts show that the synaptonemal complex protein Zip1 forms continuous linear structures along paired chromosomes, with an average length of 13.86 micrometers in L. kluyveri compared to 28 micrometers in S. cerevisiae . This ~2-fold difference in synaptonemal complex length correlates with a ~4-fold difference in recombination frequency and differences in chromosome width (1.26 μm in L. kluyveri vs. 0.83 μm in S. cerevisiae), suggesting species-specific regulation of chromatin loop size . Perform Hi-C experiments in wild-type and ygl165c∆ mutants to detect potential alterations in this chromosome organization pattern.

• How can researchers design experiments to determine if YGL165C has an enzymatic function?

To determine if YGL165C has enzymatic function, design a systematic experimental approach combining structural prediction with biochemical characterization. Begin with in-depth structural analysis using AlphaFold2 predictions and comparison to known enzyme structures to identify potential catalytic residues or substrate binding pockets. Generate recombinant YGL165C protein with various purification tags and expression systems to ensure proper folding. Perform broad-spectrum enzyme activity screens testing multiple substrate classes (nucleotides, carbohydrates, lipids, amino acids, etc.) under varying pH and metal ion conditions. For candidate substrates showing activity, determine detailed enzyme kinetics (Km, kcat, inhibition patterns). Create point mutations of predicted catalytic residues and measure their effect on activity.

• What methods should be employed to study YGL165C behavior under environmental stress conditions?

To study YGL165C behavior under environmental stress conditions, implement a multi-level analysis approach following systematic experimental design principles . First, design a factorial experiment testing multiple stress conditions (oxidative, osmotic, temperature, nutrient deprivation) at varying intensities and durations. Monitor transcriptional and translational responses of YGL165C using RT-qPCR and Western blotting with epitope-tagged strains. Track protein localization changes during stress using time-lapse microscopy with GFP-tagged YGL165C. Compare growth and survival phenotypes between wild-type and ygl165c∆ strains across all stress conditions, generating heat maps of condition-specific sensitivity. For mechanistic insights, perform genetic interaction screens under stress conditions to identify synthetic interactions that emerge only under specific stresses.