Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YHR180W-A (YHR180W-A)

What is YHR180W-A and what are its basic structural characteristics?

YHR180W-A is a putative uncharacterized protein in Saccharomyces cerevisiae with a UniProt ID of P0C5N8. It is a small protein consisting of 60 amino acids with the sequence: MTYCHTDVSYFEIQHSCIFSLLSLVVERCTCNAKVASSILAGGIIPVLFFFPLFLFLYHL . The protein is also known as smORF288 and has been identified as a de novo gene that overlaps with the retrotransposon Ty3LTR . The small size and specific sequence characteristics suggest it may function in membrane-associated processes, given its hydrophobic C-terminal region.

How is YHR180W-A classified in evolutionary genomics?

YHR180W-A represents an interesting case study in evolutionary genomics as it has been identified as a de novo gene, meaning it originated from previously non-coding DNA sequences rather than through duplication or horizontal gene transfer mechanisms. Research has shown that YHR180W-A is among the 84 de novo genes identified in S. cerevisiae S288C since its divergence from sister groups . Its association with a retrotransposon element (Ty3LTR) suggests potential mobility-related origins, which researchers have noted as a reasonable speculation for its emergence .

What experimental evidence confirms YHR180W-A expression and translation?

Transcriptome and ribosome profiling data have revealed that numerous de novo genes in S. cerevisiae, including potentially YHR180W-A, demonstrate condition-specific expression and translation. Research indicates that approximately 10% of de novo genes are expressed and 33% are translated only under specific conditions . DNA microarray data has shown that approximately 87% of de novo genes are regulated during various biological processes, including nutrient utilization and sporulation . This contextual expression pattern is critical for researchers to consider when designing experiments to detect and study YHR180W-A.

What expression systems are optimal for producing recombinant YHR180W-A?

For laboratory research purposes, E. coli expression systems have proven effective for producing recombinant YHR180W-A. Commercial providers typically use E. coli to express the full-length protein (1-60aa) fused to an N-terminal His tag . This approach allows for straightforward purification while maintaining the protein's structural integrity. Alternative expression systems such as yeast-based expression might provide advantages for certain applications, particularly when post-translational modifications are of interest, though this requires careful optimization of codon usage and growth conditions.

What purification strategies yield the highest purity of recombinant YHR180W-A?

The most effective purification strategy involves immobilized metal affinity chromatography (IMAC) utilizing the N-terminal His tag. Standard protocols can achieve greater than 90% purity as determined by SDS-PAGE . For enhanced purity, researchers should consider:

• Using a gradient elution with imidazole to minimize non-specific binding

• Implementing secondary purification steps such as size exclusion chromatography

• Optimizing buffer conditions to reduce protein aggregation

For applications requiring higher purity levels, ion exchange chromatography as a polishing step may be necessary.

What are the optimal storage conditions to maintain YHR180W-A stability?

Recombinant YHR180W-A should be stored as a lyophilized powder at -20°C to -80°C upon receipt. For working with the protein, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended . To prevent protein degradation during storage:

• Add 5-50% glycerol (final concentration) before aliquoting for long-term storage

• Store aliquots at -20°C/-80°C to avoid repeated freeze-thaw cycles, which significantly decrease protein stability

• For short-term use, working aliquots can be stored at 4°C for up to one week

The standard storage buffer composition is Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 .

What experimental approaches are most effective for determining YHR180W-A function?

Given the uncharacterized nature of YHR180W-A, a multi-pronged approach to functional analysis is recommended:

• Phenotypic screening: Utilize the S. cerevisiae genome deletion mutant library to screen for sensitivity or resistance to various conditions, similar to methods used in antimicrobial peptide studies . This approach can reveal potential biological processes involving YHR180W-A.

• Transcriptomic analysis: Employ microarray or RNA-seq to analyze differential gene expression in YHR180W-A deletion or overexpression strains under various conditions. Gene Ontology (GO) term analysis can then identify biological processes potentially associated with YHR180W-A .

• Protein localization: Use fluorescent protein tagging to determine the subcellular localization of YHR180W-A, which can provide insights into its potential function.

• Protein-protein interaction studies: Implement yeast two-hybrid or affinity purification coupled with mass spectrometry to identify potential interaction partners.

How can YHR180W-A deletion mutants be efficiently generated and verified?

Generation of precise YHR180W-A deletion mutants can be achieved through several approaches:

• Conventional homologous recombination: Using PCR-mediated gene disruption with selectable markers as described in the literature for other S. cerevisiae genes . This approach would utilize strains with deleted marker genes to minimize homology with marker genes in vectors.

• Integrative targeting (IT) method: More recent techniques employ meganuclease I-SceI to induce double-strand breaks at chromosomal target sites, requiring minimal steps and using smaller cassettes (~1.3 kb vs. traditional ~4.6 kb) . This approach allows for precise genomic modifications.

• CRISPR-Cas9 system: For highly efficient gene editing, CRISPR-Cas9 can be employed to create precise deletions with minimal off-target effects.

Verification of successful deletion should include:

• PCR confirmation of the deletion

• Sequencing of the modified genomic region

• RT-PCR or Northern blot analysis to confirm absence of transcript

• If antibodies are available, Western blot to confirm absence of protein

What expression conditions should be considered when studying YHR180W-A?

Research indicates that many de novo genes, potentially including YHR180W-A, exhibit condition-specific expression patterns. Based on microarray data, 87% of de novo genes are regulated during various biological processes such as nutrient utilization and sporulation . Therefore, when studying YHR180W-A expression, researchers should consider:

• Testing multiple growth conditions, particularly those involving stress responses, nutrient limitation, and developmental processes like sporulation

• Examining expression during different growth phases (log phase vs. stationary phase)

• Investigating expression under various environmental stresses (temperature, pH, osmotic stress)

• Analyzing expression during mating and pheromone response

It is critical to note that standard laboratory conditions may not elicit YHR180W-A expression, as approximately 10% of de novo genes are expressed only under specific conditions .

What evidence supports the de novo origin of YHR180W-A?

YHR180W-A has been identified as one of 84 de novo genes in S. cerevisiae S288C that have originated since divergence from sister groups . The evidence supporting its de novo origin includes:

• Absence in outgroup species: Rigorous comparative genomic analyses have confirmed the absence of orthologous sequences in outgroup species.

• Overlap with transposable elements: YHR180W-A has been found to overlap with retrotransposon Ty3LTR, suggesting its potential origin from mobile genetic elements .

• Sequence analysis: High GC content and specific mutational patterns characteristic of de novo gene birth have been observed.

• Transcriptomic evidence: Expression data supports that YHR180W-A, like other de novo genes, has gained transcriptional activity relatively recently in evolutionary time.

Research suggests that several factors contribute to the birth of de novo genes, including SNP/indel mutations, high GC content, and DNA shuffling, while domestication and natural selection drive their spread and fixation .

What methodologies are recommended for studying YHR180W-A evolution across yeast species?

To effectively study YHR180W-A evolution, researchers should employ a comprehensive set of methodologies:

• Comparative genomics: Utilize whole-genome sequencing data from multiple yeast species and strains to identify potential homologs or precursor sequences.

• Synteny analysis: Examine conserved gene order around the YHR180W-A locus to identify orthologous regions even in the absence of sequence conservation.

• Ancestral sequence reconstruction: Apply probabilistic models to infer ancestral sequences and track the evolutionary trajectory of YHR180W-A.

• Population genetics: Analyze polymorphism data from diverse S. cerevisiae isolates to assess selective pressures and fixation patterns.

• Experimental evolution: Implement laboratory evolution experiments to potentially observe real-time changes in YHR180W-A sequence and function.

Interestingly, there is evidence suggesting a possible parallel origin of a de novo gene between S. cerevisiae and Saccharomyces paradoxus , which makes comparative studies particularly valuable.

How might the association with transposable elements influence YHR180W-A function and evolution?

The association of YHR180W-A with the retrotransposon Ty3LTR suggests several important considerations for researchers:

• Mobility-associated origins: The association with transposons suggests potential mobility in the genome, which could influence its regulatory landscape and functional context .

• Co-evolutionary dynamics: YHR180W-A may have co-evolved with transposable elements, potentially serving functions related to transposon regulation or defense.

• Regulatory elements: Transposable elements often contain regulatory sequences that could influence YHR180W-A expression patterns, potentially explaining its condition-specific expression.

• Genomic instability: The association with mobile elements might contribute to genomic plasticity and adaptability in response to environmental pressures.

Researchers should consider these factors when designing experiments to study YHR180W-A function and evolution, particularly in comparative studies across different S. cerevisiae strains with varying transposon activities.

How can genome editing techniques be optimized for studying YHR180W-A?

For precise genome editing of YHR180W-A, researchers can employ several advanced techniques:

• I-SceI-mediated genome editing: Utilize the integrative targeting (IT) method with I-SceI meganuclease for double-strand break induction. This approach uses smaller cassettes (~1.3 kb) compared to traditional methods (~4.6 kb) and requires minimal steps . The IT cassettes carry a single marker (K. lactis URA3) and built-in I-SceI recognition sites at one or both ends.

• Oligonucleotide-directed mutagenesis: For introducing specific mutations, researchers can use oligonucleotide repair templates with the I-SceI system, similar to the approach used for introducing phosphorylation site mutations in SPO12 .

• Ortholog replacement studies: The precise replacement methodology described for essential proteasome genes can be adapted to replace YHR180W-A with putative orthologs or synthetic constructs to assess functional conservation or innovation .

For expression of I-SceI, researchers can use plasmid-borne or integrated constructs with GAL1 or GAL10 promoters, inducing expression in YP/2% raffinose media .

What transcriptomic approaches provide insights into YHR180W-A regulation and expression?

To comprehensively study YHR180W-A regulation and expression, researchers should consider:

• RNA-seq under multiple conditions: Given that approximately 10% of de novo genes are expressed only under specific conditions , RNA-seq should be performed under various growth conditions, stresses, and developmental stages.

• Ribosome profiling: This technique can verify translation of YHR180W-A, particularly important since approximately 33% of de novo genes are translated only under specific conditions .

• ChIP-seq analysis: To identify transcription factors binding to the YHR180W-A promoter region, chromatin immunoprecipitation followed by sequencing can provide valuable insights.

• ATAC-seq: To assess chromatin accessibility around the YHR180W-A locus under different conditions, providing insights into its regulation.

• Single-cell RNA-seq: To determine if YHR180W-A expression varies among individual cells within a population, potentially identifying subpopulations with distinct expression patterns.

The methodological approach should follow established protocols for RNA extraction using TRIzol reagent under liquid nitrogen grinding conditions to ensure high-quality RNA, similar to protocols described for yeast transcriptome studies .

What are the challenges and solutions in functional characterization of uncharacterized proteins like YHR180W-A?

Functional characterization of uncharacterized proteins like YHR180W-A presents several challenges:

• Condition-specific expression: As many de novo genes are expressed only under specific conditions , researchers should:

  • Screen multiple environmental conditions and stresses

  • Examine various developmental stages (e.g., sporulation)

  • Test different nutrient conditions

• Small size and potential low abundance: With only 60 amino acids , YHR180W-A may be difficult to detect:

  • Use highly sensitive detection methods like targeted mass spectrometry

  • Consider epitope tagging for improved detection

  • Implement overexpression systems while monitoring potential artifacts

• Potential moonlighting functions: Like many small proteins, YHR180W-A may have multiple functions:

  • Implement comprehensive phenotypic screening approaches

  • Use global genetic interaction mapping (e.g., synthetic genetic array analysis)

  • Apply metabolomic profiling to identify affected pathways

• Structural characterization challenges: For small membrane-associated proteins:

  • Consider specialized structural biology approaches for small proteins

  • Use computational prediction combined with experimental validation

  • Implement crosslinking mass spectrometry to identify interaction interfaces

By addressing these challenges with appropriate methodological solutions, researchers can make significant progress in understanding the function of this putative uncharacterized protein.