Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YBR076C-A (YBR076C-A)

What is the current knowledge status of YBR076C-A in Saccharomyces cerevisiae?

YBR076C-A represents one of the uncharacterized open reading frames in the Saccharomyces cerevisiae genome. While specific numerical values associated with YBR076C-A (51.68987815, 142.1093608) have been documented in research contexts, the functional characterization remains incomplete . The protein belongs to the category of putative uncharacterized proteins, indicating that its existence has been predicted through computational analysis of the yeast genome, but experimental verification of its expression and function requires further investigation. As with many uncharacterized proteins, its biological significance might be underestimated despite potentially contributing to important cellular processes.

What are the foundational techniques for confirming the expression of YBR076C-A?

Confirming the expression of putative uncharacterized proteins like YBR076C-A typically employs multiple complementary approaches:

• RT-PCR and qPCR to detect and quantify mRNA transcripts

• Western blotting with specific antibodies or epitope tagging to verify protein expression

• Mass spectrometry-based proteomics to identify the protein in cellular extracts

For epitope tagging approaches, techniques similar to those used in other yeast studies can be applied, such as adding a His-tag to the protein and confirming expression through Western blotting with anti-His antibodies, as demonstrated in studies with other recombinant yeast proteins . The expression vector pGAPZαC has proven effective for such constructs in S. cerevisiae, allowing for selection of transformants using Zeocin resistance .

How can researchers construct a YBR076C-A overexpression strain in S. cerevisiae?

To construct a YBR076C-A overexpression strain, researchers should follow this methodological approach:

• PCR amplification of the YBR076C-A gene with appropriate restriction enzyme sites incorporated into primers

• Inclusion of a Kozak consensus sequence for optimal expression

• Ligation of the PCR product into an expression vector (e.g., pGAPZαC)

• Linearization of the recombinant plasmid with an appropriate restriction enzyme (e.g., AvrII)

• Transformation into S. cerevisiae via electroporation

• Selection of transformants on media containing appropriate antibiotics (e.g., Zeocin)

• Confirmation of successful transformation and expression via PCR and Western blotting

This methodology parallels successful approaches used with other yeast genes, such as ari1, where linearization of the vector with AvrII enzyme followed by electroporation transformation has proven effective .

What techniques are most effective for determining the subcellular localization of YBR076C-A?

For determining subcellular localization of YBR076C-A, researchers should consider implementing:

• Fluorescent protein fusion constructs (GFP, YFP, mCherry) for live-cell imaging

• Immunofluorescence microscopy using antibodies against native protein or epitope tags

• Subcellular fractionation followed by Western blotting

• Co-localization with known organelle markers

These approaches should be combined with controls for validating specific localization patterns. When constructing fusion proteins, both N-terminal and C-terminal tagging should be attempted as protein function may be disrupted differently depending on tag position. Comparing the localization patterns under different growth conditions may provide insights into conditional functionality.

How can researchers assess potential RNA-binding capabilities of YBR076C-A?

Given the context in which YBR076C-A was mentioned alongside RNA-binding proteins , investigating its potential RNA interaction capabilities would be valuable:

• RNA immunoprecipitation (RIP) using tagged YBR076C-A to identify associated RNAs

• Electrophoretic mobility shift assays (EMSA) to detect direct RNA binding

• UV crosslinking and immunoprecipitation (CLIP) to identify in vivo RNA binding sites

• Structural analysis using techniques like NMR or X-ray crystallography to identify potential RNA-binding domains

• Mutational analysis of predicted RNA-binding motifs to confirm functional relevance

Comparative analysis with known RNA-binding proteins from S. cerevisiae, particularly those studied at research centers like CIPF and IBV-CSIC, could provide valuable insights into YBR076C-A's potential role in RNA processing or metabolism .

What phenotypic assays can reveal the function of YBR076C-A?

To investigate YBR076C-A function through phenotypic analysis, researchers should implement:

• Growth curve analysis comparing wild-type and YBR076C-A deletion/overexpression strains under various conditions

• Stress tolerance assays (oxidative, osmotic, temperature, nutrient limitation)

• Cell cycle analysis to detect potential roles in division or morphology

• Metabolic profiling to identify affected biochemical pathways

• Genetic interaction screens to place YBR076C-A in functional networks

When conducting growth assays, researchers should monitor not only maximum OD600 values but also lag phases and growth rates, as these parameters have proven informative in similar studies with engineered S. cerevisiae strains . Documentation of growth patterns at multiple time points (e.g., 24h, 48h, 72h, 96h, 120h, 144h) provides comprehensive characterization of potential phenotypes .

How can RNA-Seq be optimized to study the impact of YBR076C-A deletion or overexpression?

RNA-Seq experimental design for studying YBR076C-A should include:

• Multiple biological replicates (minimum 3) of wild-type, deletion, and overexpression strains

• Time-course sampling to capture early, middle, and late transcriptional responses

• Varied growth conditions to identify condition-specific effects

• Strand-specific library preparation to capture antisense transcription

• Deeper sequencing (>30 million reads per sample) to detect subtle expression changes

• Inclusion of spike-in controls for accurate normalization

Analysis should focus on both differential gene expression and alternative splicing patterns, as uncharacterized proteins may play roles in post-transcriptional regulation. Special attention should be given to genes encoding mRNA processing factors, as YBR076C-A might be involved in RNA metabolism pathways .

What synthetic recombinant approaches are most suitable for studying YBR076C-A interactions?

When designing synthetic recombinant populations to study YBR076C-A interactions, researchers should consider:

• Pairwise crossing strategy rather than simple mixing of strains, as this maximizes and maintains greater genetic variation

• Inclusion of 8-12 parental strains to increase genetic diversity, which enhances the potential for detecting genetic interactions

• Selection of diverse genetic backgrounds to capture a wider range of potential phenotypic effects

• Development of inbred lines for fine QTL mapping of YBR076C-A-related traits

Research indicates that "more genetic variation is initially present and maintained when population construction includes a round of pairwise crossing" compared to simple mixing of strains . This approach provides greater adaptive potential and increases the likelihood of detecting meaningful genetic interactions with YBR076C-A.

How can CRISPR-Cas9 technology be applied for precise manipulation of YBR076C-A?

For CRISPR-Cas9 based manipulation of YBR076C-A, researchers should implement:

• Design of specific gRNAs targeting YBR076C-A with minimal off-target effects

• Generation of precise deletions, insertions, or point mutations rather than complete gene deletion

• Creation of conditional alleles using inducible degradation tags

• Introduction of epitope tags at the endogenous locus for studying native expression

• Base editing for introducing specific amino acid changes without double-strand breaks

When designing repair templates, include appropriate homology arms (40-60 bp) flanking the desired modification. For studying potential interactions with other proteins, consider multiplex CRISPR to simultaneously modify YBR076C-A and candidate interacting partners to assess genetic interactions.

What are the most sensitive methods for detecting low-abundance proteins like YBR076C-A?

For detecting potentially low-abundance proteins like YBR076C-A:

• Employ sample fractionation techniques to reduce proteome complexity

• Utilize targeted proteomics approaches like selected reaction monitoring (SRM) or parallel reaction monitoring (PRM)

• Implement protein enrichment strategies such as affinity purification

• Apply state-of-the-art mass spectrometry methods with high sensitivity

• Consider proximity labeling techniques like BioID or APEX to identify proteins in close proximity

When designing experiments, include multiple biological replicates and appropriate controls to distinguish genuine signals from background. Integration of transcriptomic data can help prioritize potential peptides for targeted detection efforts.

How can protein-protein interaction networks involving YBR076C-A be mapped?

To map the protein interaction network of YBR076C-A:

• Tandem affinity purification coupled with mass spectrometry (TAP-MS)

• Yeast two-hybrid screening against a comprehensive S. cerevisiae library

• Proximity-dependent biotinylation (BioID, TurboID) followed by streptavidin pulldown

• Co-immunoprecipitation with epitope-tagged YBR076C-A

• Protein complementation assays (split-GFP, split-luciferase)

The resulting interaction data should be validated using orthogonal methods and analyzed in the context of existing interactome data. Particular attention should be paid to interactions with RNA-binding proteins or components of RNA processing machinery, given the potential connection of YBR076C-A to these processes .

How conserved is YBR076C-A across fungal species, and what does this suggest about its function?

To assess evolutionary conservation of YBR076C-A:

• Perform BLAST and HMM-based searches across fungal genomes

• Conduct phylogenetic analysis to establish orthologous relationships

• Compare sequence conservation patterns to identify functional domains

• Analyze synteny relationships to detect genomic context conservation

• Examine selection pressure through dN/dS ratio analysis

Conservation analysis should extend beyond simple sequence similarity to include structural features and potential functional motifs. The presence or absence of YBR076C-A orthologs in different yeast species adapted to various environments may provide clues about its functional importance in specific ecological niches.

Can functional information be inferred from comparing YBR076C-A to characterized proteins in other organisms?

For inferring YBR076C-A function through comparative analysis:

• Conduct domain-based searches to identify distant homologs with known functions

• Perform structural prediction and comparison with characterized proteins

• Analyze shared motifs that might indicate similar biochemical activities

• Examine co-evolution patterns with functionally related proteins

• Assess expression correlation patterns across species

This approach may reveal functional connections not apparent from direct sequence comparison. For instance, examining whether YBR076C-A contains features similar to known RNA-binding domains could provide insights, especially given its mention in the context of research on RNA-binding proteins .

What are the optimal conditions for studying YBR076C-A expression patterns?

For comprehensive analysis of YBR076C-A expression:

• Examine expression across different growth phases (lag, log, stationary)

• Test various stress conditions (nutrient limitation, temperature shifts, chemical stressors)

• Compare expression in different carbon sources and media compositions

• Analyze expression during specialized processes (sporulation, mating, biofilm formation)

• Consider time-course experiments to capture dynamic expression changes

Based on studies of other yeast genes, expression levels may vary significantly over time, with peak expression potentially occurring at specific phases of growth. For instance, studies with ari1 showed maximum expression at 120 hours of culturing . Similar temporal dynamics might exist for YBR076C-A.

How should real-time PCR experiments be designed to accurately quantify YBR076C-A expression?

For reliable qPCR analysis of YBR076C-A:

Expression changes should be reported as fold-changes relative to appropriate controls. When studying gene expression in engineered strains, patterns may differ significantly from wild-type strains under stress conditions, as observed with the ari1 gene where expression increased dramatically (up to 74-fold) in engineered strains compared to wild-type under furfural stress .

What strategies can overcome the challenges of studying proteins with no known functional domains?

For characterizing proteins lacking recognized domains:

• De novo structural prediction using methods like AlphaFold2

• Chemical and enzymatic activity screening to identify potential biochemical functions

• Suppressor screens to identify genetic interactors

• Systematic mutagenesis to identify critical residues

• Heterologous expression in different cellular contexts to observe phenotypic effects

These approaches can be particularly valuable for uncharacterized proteins like YBR076C-A, where conventional homology-based functional prediction may be limited. The situation with YBR076C-A parallels challenges faced in the characterization of many uncharacterized human proteins, where despite extensive study of canonical proteins, "there are still hundreds to thousands of uncharacterized canonical and non-canonical isoforms as their biological functions are yet to be revealed" .

How can researchers address data inconsistencies when characterizing YBR076C-A?

When facing inconsistent results:

• Systematically evaluate experimental conditions that might explain variability

• Test multiple genetic backgrounds to identify strain-specific effects

• Consider conditional functionality dependent on specific environmental factors

• Examine potential post-translational modifications affecting protein function

• Investigate potential genetic redundancy that might mask phenotypes

Data inconsistencies should be transparently reported rather than selectively presenting consistent results. Comprehensive documentation of experimental conditions is essential for understanding context-dependent functions, particularly for uncharacterized proteins that may have subtle or condition-specific roles.

What high-throughput approaches could accelerate functional characterization of YBR076C-A?

Advanced high-throughput strategies include:

• Pooled CRISPR screens with various selective conditions

• Systematic genetic interaction mapping using SGA or E-MAP approaches

• Metabolomic profiling under various conditions

• Ribosome profiling to assess translational impacts

• ChIP-seq or CUT&RUN to identify potential DNA-binding activities if nuclear localization is observed

The integration of multiple omics datasets can provide complementary insights that might not be apparent from any single approach. As noted in studies of uncharacterized proteins, "much more collaborative efforts are required to unveil the mysteries of the cellular functions of uncharacterized proteins" .

How might studying YBR076C-A contribute to understanding basic cellular processes in eukaryotes?

Investigation of YBR076C-A may contribute to fundamental knowledge by:

• Revealing novel regulatory mechanisms in gene expression

• Uncovering previously unknown stress response pathways

• Identifying new components of RNA processing machinery

• Elucidating cellular adaptation mechanisms to environmental changes

• Discovering unexpected functional roles of uncharacterized genomic regions

The study of uncharacterized proteins has repeatedly revealed surprising new biological functions and pathways. As demonstrated in research on other previously uncharacterized proteins, seemingly minor proteins can have significant impacts on cellular processes ranging from transcription and translation to stress responses and metabolic regulation .