Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YER158W-A (YER158W-A)

What is YER158W-A and why is it classified as a putative uncharacterized protein?

YER158W-A is a protein encoded in the Saccharomyces cerevisiae genome with a full length of 71 amino acids . It is classified as "putative uncharacterized" because its precise biological function has not been definitively established through experimental validation. This classification indicates that the protein's existence has been predicted through genomic analysis, but its role in cellular processes remains to be fully elucidated. Researchers typically approach such proteins through comparative genomics, structural prediction, and experimental characterization using gene deletion, overexpression studies, and protein interaction analyses.

What is known about the structural characteristics of YER158W-A protein?

YER158W-A is a relatively small protein with 71 amino acids in its full-length form . While detailed structural information is limited in the available literature, researchers can produce the recombinant form with affinity tags such as His-tags for purification and characterization purposes. For structural characterization, methodological approaches would include:

• Primary sequence analysis using bioinformatics tools

• Secondary structure prediction using algorithms like PSIPRED

• Experimental determination of structure using techniques such as X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy

• Homology modeling if structural homologs exist

For optimal results, expressing and purifying the recombinant protein in E. coli systems with affinity tags facilitates downstream structural analyses .

How can researchers obtain YER158W-A protein for experimental studies?

For researchers needing YER158W-A protein for experimental studies, multiple approaches are available:

• Recombinant expression: The protein can be expressed recombinantly in E. coli with a His-tag for purification purposes . The full-length protein (amino acids 1-71) is typically used for comprehensive functional studies.

• Commercial sources: Specialized suppliers like Creative BioMart offer purified YER158W-A protein preparations .

• In-house expression: Researchers can clone the YER158W-A gene and express it using appropriate vector systems. For this approach:

  • Amplify the gene from S. cerevisiae genomic DNA

  • Insert into an expression vector with a suitable tag

  • Transform into an expression host (typically E. coli)

  • Induce expression and purify using affinity chromatography

For genetic studies, the gene can be manipulated using PCR-based approaches similar to those described for other yeast genes .

What experimental design strategies are most effective for studying YER158W-A function?

When designing experiments to investigate YER158W-A function, a systematic approach should be employed:

• Define research variables:

  • Independent variables: YER158W-A expression levels, environmental conditions, genetic background

  • Dependent variables: Growth rates, metabolic profiles, stress responses

  • Potential confounding variables: Media composition, temperature, cell density

• Formulate testable hypotheses based on bioinformatic predictions or preliminary observations

• Design treatments to manipulate YER158W-A expression:

  • Gene deletion (knockout)

  • Controlled expression (using inducible promoters)

  • Site-directed mutagenesis to alter specific residues

• Select appropriate control groups:

  • Wild-type strains

  • Strains with deletion of known genes

  • Complementation controls

• Measure relevant outcomes using quantitative methods:

  • Growth assays

  • Transcriptomic/proteomic profiling

  • Metabolic activity measurements

This structured approach allows for systematic investigation of YER158W-A function while controlling for experimental variables that might influence results .

How can quantitative trait loci (QTL) mapping be used to study YER158W-A?

QTL mapping represents a powerful approach for examining the genetic basis of variation in YER158W-A function. Implementation requires:

• Establish phenotypic assays sensitive to YER158W-A function:

  • Growth rates under specific conditions

  • Response to stress factors

  • Metabolic outputs

• Generate a mapping population:

  • Use of meiotic recombinant segregants from crosses between divergent strains

  • Advanced intercross lines can increase mapping resolution

• Phenotyping methodology:

  • Platforms like PHENOS (PHENotyping On Solid media) can measure growth of recombinant segregants under various conditions

  • High-throughput methods allow testing hundreds of segregants simultaneously

• Genotyping and linkage analysis:

  • Whole genome sequencing of segregants

  • Statistical association between genotypes and phenotypes

  • Identification of genomic regions linked to YER158W-A-associated traits

• Fine mapping and candidate gene validation:

  • Reciprocal hemizygosity analysis to validate causal variants

  • Functional complementation tests

  • CRISPR-based allele replacement

This approach can reveal genetic interactions and regulatory networks influencing YER158W-A function, particularly in response to environmental stressors or drug treatments.

What methods are most effective for YER158W-A gene deletion and verification?

For creating and verifying YER158W-A gene deletions, the following methodological approach is recommended:

• PCR-based gene deletion:

  • Design primers with 40-50bp homology to regions flanking YER158W-A

  • Amplify a selectable marker (e.g., kanMX cassette) using these primers

  • Transform the PCR product into yeast cells

  • Select transformants on appropriate media containing the selection agent

• Verification protocols:

  • Diagnostic PCR using primers that bind outside the targeted locus and within the selection marker

  • Expected PCR product sizes:

    • Wild-type YER158W-A locus: ~71 bp + flanking regions

    • Successfully deleted locus: size of selection marker + flanking regions

  • Verification PCR should yield bands of predicted sizes when analyzed by gel electrophoresis

• Additional verification methods:

  • Sequencing of junction regions

  • Phenotypic complementation tests

  • Quantitative PCR to confirm absence of expression

For optimal results, maintain positive controls (wild-type strain) and negative controls (known deletion strains) throughout the verification process.

How can researchers determine the potential biological role of YER158W-A?

To systematically investigate the biological role of YER158W-A, researchers should employ a multi-faceted approach:

• Comparative genomic analysis:

  • Identify orthologs in related yeast species

  • Examine synteny and evolutionary conservation

  • Search for conserved domains or motifs

• Transcriptional profiling:

  • RNA sequencing of YER158W-A deletion strains versus wild-type

  • Analysis of YER158W-A expression under various stress conditions

  • Identification of co-regulated genes

• Phenotypic characterization:

  • Growth assays under diverse environmental conditions

  • Stress response analysis (oxidative, temperature, nutrient limitation)

  • Cell morphology and cell cycle progression assessment

• Protein interaction studies:

  • Yeast two-hybrid screening

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity labeling approaches (BioID, APEX)

• Functional validation approaches:

  • Reciprocal hemizygosity analysis to validate phenotypic effects

  • Complementation with wild-type YER158W-A

  • Site-directed mutagenesis to identify critical residues

Integration of these multiple data types allows researchers to triangulate on the most likely biological functions and avoid misinterpretation of isolated experimental outcomes.

What approaches can be used to study YER158W-A in the context of drug response variation?

To investigate YER158W-A's potential role in drug response variation, researchers should implement the following methodological framework:

• Phenotypic screening:

  • Test growth of YER158W-A deletion strains in the presence of various compounds

  • Compare with wild-type and other deletion strains

  • Use dose-response curves to quantify sensitivity or resistance

• QTL mapping for drug response:

  • Generate and phenotype segregant populations from crosses between strains with different YER158W-A alleles

  • Measure growth under drug treatment using platforms like PHENOS

  • Identify genetic loci associated with drug response variation

  • Determine if YER158W-A contributes to observed variation

• Functional validation:

  • Reciprocal hemizygosity analysis to confirm allelic effects

  • Allele swapping experiments

  • Expression analysis before and after drug exposure

• Mechanistic studies:

  • Transcriptomic profiling to identify affected pathways

  • Metabolomic analysis to detect biochemical changes

  • Genetic interaction screens to identify functional relationships

This systematic approach can reveal whether and how YER158W-A contributes to variation in response to therapeutic compounds, potentially informing translational applications .

How might researchers investigate potential human homologs of YER158W-A?

To identify and characterize potential human homologs of YER158W-A, researchers should:

• Sequence-based homology search:

  • Use BLAST, PSI-BLAST, and HMMer searches against human protein databases

  • Focus on both sequence similarity and conservation of critical residues

  • Consider structural homologs even in the absence of strong sequence homology

• Functional complementation tests:

  • Express candidate human homologs in YER158W-A deletion strains

  • Assess rescue of any observed phenotypes

  • Evaluate domain-specific contributions to function

• Comparative analysis of protein interactions:

  • Identify interaction partners of YER158W-A in yeast

  • Determine if human homolog candidates interact with corresponding human proteins

  • Map conserved interaction networks

• Pathway conservation analysis:

  • Determine if YER158W-A functions in conserved cellular pathways

  • Evaluate whether human homolog candidates participate in similar pathways

  • Conduct genetic epistasis tests with known pathway components

This approach leverages the conservation of fundamental cellular processes between yeast and humans, potentially revealing functional relationships relevant to human health and disease .

How can YER158W-A research contribute to understanding drug response mechanisms?

YER158W-A research can provide insights into drug response mechanisms through:

• Genetic basis of drug response variation:

  • Use QTL mapping to identify genetic loci influencing drug sensitivity

  • Determine if YER158W-A variants contribute to differential drug responses

  • Characterize the molecular mechanisms underlying these differences

• Pathway analysis:

  • Perform network analysis to place YER158W-A in cellular response pathways

  • Identify enriched functional categories in genes that interact with YER158W-A

  • Map how YER158W-A influences response to therapeutic compounds

• Translational applications:

  • Determine if human homologs play similar roles in drug response

  • Identify potential biomarkers for drug sensitivity in clinical applications

  • Develop predictive models for personalized medicine approaches

• Drug development implications:

  • Identify novel drug targets based on YER158W-A function

  • Screen for compounds that modulate YER158W-A activity

  • Develop combination therapies targeting multiple pathway components

This research has implications for understanding fundamental mechanisms of drug action and resistance, with potential applications in precision medicine and drug development .

What experimental systems are optimal for studying YER158W-A genetic interactions?

To systematically investigate YER158W-A genetic interactions, researchers should consider these methodological approaches:

• Synthetic genetic array (SGA) analysis:

  • Cross YER158W-A deletion strain with yeast deletion collection

  • Identify synthetic lethal and synthetic sick interactions

  • Map genetic interaction networks indicating functional relationships

• Dosage synthetic lethality screening:

  • Overexpress YER158W-A in various deletion backgrounds

  • Identify genes whose absence is incompatible with YER158W-A overexpression

  • Characterize dosage-dependent interactions

• Chemical-genetic profiling:

  • Expose YER158W-A mutants to diverse chemical compounds

  • Identify compounds with enhanced effect in YER158W-A mutants

  • Map pathways connecting YER158W-A to chemical response mechanisms

• Quantitative interaction mapping:

  • Measure growth rates of double mutants compared to single mutants

  • Calculate genetic interaction scores to quantify interaction strength

  • Generate interaction networks based on quantitative data

• Conditional interaction screening:

  • Perform interaction screens under various environmental conditions

  • Identify condition-specific interactions

  • Map environmental response networks involving YER158W-A

These approaches provide complementary data on the functional landscape surrounding YER158W-A, revealing its position in cellular networks and guiding further mechanistic studies.

How should researchers interpret contradictory results from different experimental approaches studying YER158W-A?

When faced with contradictory results across experimental approaches studying YER158W-A, researchers should:

• Systematic validation strategy:

  • Replicate experiments using standardized protocols

  • Vary experimental conditions to identify context-dependent effects

  • Use multiple independent methodologies to verify findings

• Consider strain background effects:

  • Test YER158W-A function in diverse genetic backgrounds

  • Conduct reciprocal hemizygosity analysis to isolate allelic contributions

  • Quantify interaction with background genetic variants

• Environmental and experimental variables:

  • Document all experimental conditions thoroughly

  • Test if contradictions are related to subtle differences in:

    • Media composition

    • Temperature

    • Growth phase

    • Cell density

• Integrated data analysis approach:

  • Apply statistical methods appropriate for each data type

  • Develop computational models that incorporate multiple data sources

  • Use Bayesian approaches to update hypotheses based on accumulated evidence

• Biological interpretation framework:

  • Consider that seemingly contradictory results may reflect biological complexity

  • Explore whether YER158W-A has context-dependent functions

  • Evaluate if the protein participates in multiple distinct pathways

This methodical approach transforms contradictory results from a problem into an opportunity to discover complex and condition-dependent aspects of YER158W-A function.

What are the common technical challenges in YER158W-A protein expression and how can they be overcome?

Researchers working with YER158W-A protein expression may encounter several technical challenges, which can be addressed through the following approaches:

• Low expression yield:

  • Optimize codon usage for the expression host

  • Test multiple expression systems (bacterial, yeast, insect, mammalian)

  • Evaluate different promoters and induction conditions

  • Consider fusion partners that enhance solubility (MBP, SUMO, GST)

• Protein solubility issues:

  • Screen buffer conditions systematically (pH, salt, additives)

  • Test expression at lower temperatures (16-20°C)

  • Co-express with potential binding partners

  • Utilize solubility-enhancing tags with appropriate linkers

• Purification challenges:

  • Implement multi-step purification strategies

  • Optimize His-tag position (N-terminal vs. C-terminal)

  • Consider on-column refolding protocols if necessary

  • Use size exclusion chromatography as a final polishing step

• Protein stability concerns:

  • Add stabilizing agents (glycerol, reducing agents)

  • Identify optimal storage conditions through stability screens

  • Consider flash-freezing in small aliquots to avoid freeze-thaw cycles

  • Test protein functionality after various storage durations

• Functional activity assessment:

  • Develop robust activity assays based on predicted function

  • Include positive controls with known activity

  • Ensure that tags do not interfere with functional domains

  • Consider removing affinity tags if they affect activity

These methodological solutions address the technical barriers commonly encountered when working with challenging proteins like YER158W-A.

What bioinformatic approaches are most useful for analyzing YER158W-A and predicting its function?

For comprehensive bioinformatic analysis of YER158W-A, researchers should implement the following methodological framework:

• Sequence-based analysis:

  • Multiple sequence alignment with homologs from diverse species

  • Identification of conserved residues and motifs

  • Secondary structure prediction

  • Disorder prediction for intrinsically disordered regions

• Structural bioinformatics:

  • Homology modeling based on structural relatives

  • Ab initio modeling for unique domains

  • Molecular dynamics simulations to assess conformational flexibility

  • Binding site prediction for potential ligands or interaction partners

• Functional prediction:

  • Gene Ontology term prediction

  • Protein domain analysis

  • Pathway mapping and enrichment analysis

  • Prediction of post-translational modifications

• Network-based approaches:

  • Integration with protein-protein interaction data

  • Co-expression network analysis

  • Functional association networks

  • Genetic interaction networks

• Evolutionary analysis:

  • Phylogenetic profiling to identify co-evolved genes

  • Selection pressure analysis (dN/dS ratios)

  • Evolutionary rate comparison with other yeast proteins

  • Identification of lineage-specific features

This multi-faceted computational approach generates testable hypotheses about YER158W-A function that can guide subsequent experimental validation.

How should researchers design and analyze data tables from YER158W-A experiments?

When designing and analyzing data tables from YER158W-A experiments, researchers should follow these best practices:

• Experimental design considerations:

  • Include appropriate controls for each experimental condition

  • Use biological and technical replicates (minimum n=3 for each)

  • Document all variables systematically

  • Consider potential confounding factors

• Data table structure:

  • Organize with clear row and column headers

  • Include metadata (experimental conditions, dates, researcher)

  • Store raw data alongside processed results

  • Maintain consistent units and formatting

• Statistical analysis approach:

  • Select appropriate statistical tests based on data distribution

  • Calculate measures of central tendency and dispersion

  • Apply multiple testing correction when performing multiple comparisons

  • Include p-values and confidence intervals where appropriate

• Example data table format for YER158W-A functional analysis:

• Data visualization best practices:

  • Select appropriate visualization methods (bar charts, scatter plots, heatmaps)

  • Include error bars representing variation

  • Use consistent color schemes and formatting

  • Provide clear legends and annotations

Following these methodological guidelines ensures that experimental data is collected, organized, and analyzed in a manner that maximizes scientific rigor and reproducibility .