Recombinant Saccharomyces cerevisiae Putative succinate dehydrogenase [ubiquinone] cytochrome b small subunit YLR164W, mitochondrial (YLR164W)

Basic Research Questions

• What is YLR164W and what is its genomic location in Saccharomyces cerevisiae?

  YLR164W is a putative succinate dehydrogenase [ubiquinone] cytochrome b small subunit located on chromosome XII in S. cerevisiae, specifically in the right ribosomal DNA flank region . The predicted protein sequence of YLR164W shows 52% similarity to Sdh4p, a known component of the succinate dehydrogenase complex . Notably, YLR164W contains a histidine residue in a position homologous to SdhD His71 in E. coli SdhD, suggesting potential functional conservation .

  This gene has been studied in the context of DNA double-strand break (DSB) formation and appears to be a site where Pch2-dependent regulation of Spo11-mediated DSB formation occurs during meiosis .

• What experimental approaches are available for manipulating YLR164W in yeast?

  Several molecular techniques can be employed to manipulate YLR164W in S. cerevisiae:

  Technique	Description	Advantages	Reference
  Transformation-associated recombination cloning	Utilizes yeast's homologous recombination machinery to assemble DNA fragments	Efficient for large constructs
  CRISPR/Cas9 genome editing	Precise genome editing using guide RNAs targeting YLR164W locus	High specificity and efficiency
  Epitope tagging	Addition of tags (FLAG, TAP, GFP, etc.) to track protein expression and localization	Enables visualization and purification
  Gene knockout	Complete deletion of YLR164W using selectable markers	Assess loss of function phenotypes

  When designing genetic manipulations, consider using a high-expression locus for integration if overexpression is desired, or maintain the native locus for studying physiological expression patterns .

• How can I confirm successful genetic modification of YLR164W?

  Multiple validation methods should be employed:

  • PCR confirmation using primers flanking the integration site

  • Growth on selective media containing appropriate antibiotics (G418, NAT, HYG) or nutrient deprivation media

  • Western blot analysis using antibodies against epitope tags (if tagged versions were created)

  • Southern blot analysis to verify genomic integration and copy number

  • Functional complementation assays in YLR164W deletion strains to confirm activity

  For example, when verifying epitope-tagged versions, immunoprecipitation followed by western blot analysis can confirm expression, as demonstrated for other yeast proteins: "Co-immunoprecipitation of 3xFlag-Pch2 and 3xFlag-Pch2 243-564 with Orc1–TAP (via α-TAP-IP) during the meiotic prophase (4 h into meiotic program). For α-Flag, short and long exposures are shown. α-Pgk1 is used as a loading control."

• What growth conditions are optimal for studying YLR164W expression and function?

  Based on experimental protocols in the literature, consider the following conditions:

  • Standard yeast-peptone (YP) medium containing 10 g/L yeast extract, 20 g/L bacteriological peptone, and 0.33 g/L L-tryptophan

  • Carbon sources: 2% dextrose (YPD) for standard growth conditions

  • For specialized metabolic studies: 0.5% Type III porcine gastric mucin (YPM)

  • Temperature: Standard cultivation at 30°C, but consider temperature sensitivity experiments at 23°C

  • For meiotic studies, use established meiosis-inducing protocols and collect samples at various time points (e.g., 4h into meiotic program)

Advanced Research Questions

• How can I design experiments to study the role of YLR164W in DNA double-strand break (DSB) formation?

  Design a comprehensive experimental approach based on published methodologies:

  1. Create the following strains:

     • Wild-type control

     • YLR164W deletion (ylr164wΔ)

     • DSB repair-deficient background (e.g., dmc1Δ) to allow DSB accumulation

     • Combined mutants (e.g., ylr164wΔ dmc1Δ)

  2. Synchronize meiotic progression using established protocols

  3. Collect time-course samples after meiotic induction (0h, 2h, 4h, 6h, 8h)

  4. Perform Southern blot analysis of the YLR164W locus and a control DSB region (e.g., YCR047C on chromosome III)

  Expected results table:

  Strain	YLR164W locus DSBs	Control locus DSBs	Interpretation
  Wild-type	Moderate levels	Moderate levels	Normal DSB formation
  dmc1Δ	Accumulation	Accumulation	DSBs form but cannot be repaired
  ylr164wΔ dmc1Δ	Altered pattern	Normal accumulation	YLR164W affects local DSB formation
  Complemented strain	Rescue of wild-type pattern	Normal pattern	Confirms YLR164W function

  Remember that "Southern blot analysis of YLR164W locus (right ribosomal DNA flank; chromosome XII) and YCR047C locus (control DSB region; chromosome III), in dmc1Δ, pch2Δ dmc1Δ backgrounds" has been successfully used to detect meiotic DSBs .

• What methods can I use to investigate YLR164W protein interactions and function in the succinate dehydrogenase complex?

  Multiple complementary approaches should be employed:

  Method	Protocol Outline	Applications	Reference
  Yeast two-hybrid	Test direct interaction between YLR164W and other SDH components	Initial screening for interactions
  Co-immunoprecipitation	Epitope-tag YLR164W (e.g., 3xFlag-YLR164W) and potential interactors (e.g., TAP-tagged proteins); perform IP followed by western blot	Validate interactions in vivo
  Pulldown assays	Express and purify recombinant proteins (e.g., His-MBP-YLR164W) from heterologous systems like insect cells; perform amylose-based pulldown	Biochemical verification of direct interactions
  Size exclusion chromatography	Analyze complex formation using purified components	Determine stoichiometry and complex stability
  Cross-linking mass spectrometry	Identify specific residues involved in protein-protein interactions	Detailed molecular interaction mapping

  For pulldown assays, protocols similar to those described for Pch2 can be adapted: "Amylose-based pulldown of His–ORC (His–Orc1-6) purified from insect cells, with His–MBP–Pch2 or His–MBP–Pch2-243-564. Coomassie Brilliant Blue (CBB) staining."

• How can I investigate the potential role of YLR164W in homologous recombination (HR) and genomic stability?

  Recent studies have highlighted HR as an important genetic process that can lead to loss of production capacity in engineered S. cerevisiae strains . To investigate YLR164W's potential role:

  1. Create reporter systems:

     • Integrate fluorescent reporter constructs that activate upon HR events

     • Design systems with direct repeats flanking the YLR164W gene

  2. Engineer strains with variable expression levels:

     • YLR164W deletion

     • YLR164W overexpression

     • YLR164W-fluorescent protein fusions to track expression levels

  3. Measure HR rates in populations:

     • Flow cytometry sorting based on fluorescent reporter activation

     • Compare HR rates in YLR164W high-expressing vs. low-expressing cells

  4. Long-term stability analysis:

     • Conduct extended cultivation experiments (>90 generations)

     • Regularly assess copy number variation and genetic stability

  This approach is based on methods described for studying HR heterogeneity: "The enrichment in Rad52-high cells did not lead to visible phenotypic effects in terms of sugar consumption. Of note, the final OD for Rad52-high populations progressively decreased compared to the Rad52-low and the non-sorted populations, which could be due to high expression of the YFP-tdTomato, high Rad52 expression levels or indirect consequences."

• What metabolic engineering approaches can be used to study YLR164W in relation to succinate dehydrogenase function?

  As a putative succinate dehydrogenase component, YLR164W may play roles in cellular metabolism. Several approaches can be employed:

  1. Knockout strain characterization:

     • Create YLR164W deletion strains

     • Compare growth rates on different carbon sources

     • Measure oxygen consumption rates and respiratory capacity

  2. Multi-gene manipulations:

     • Create strains with combinations of modifications to SDH components

     • Design double knockouts (e.g., YLR164W with other SDH components)

     • Employ codon-optimized synthetic genes for heterologous expression

  3. Metabolomic analysis:

     • Perform targeted metabolomics focusing on TCA cycle intermediates

     • Use GC/MS, LC/MS or NMR to quantify metabolite levels

     • Compare metabolic profiles between wild-type and engineered strains

  4. Carbon flux analysis:

     • Use 13C-labeled glucose to trace carbon flow through central metabolism

     • Quantify flux differences between wild-type and YLR164W-modified strains

  Similar approaches have been successfully applied to study recombinant enzyme production: "GC/MS analysis of culture filtrates of this strain showed 11 times higher response in EIC for the m/z 479 (methyloxime-tetra-TMS derivative of 2-DOI) than the YP-btrC recombinant that has only a single copy of btrC expression cassette integrated into the genomic DNA of the CEN.PK strain."

• How should I design experiments to understand YLR164W's evolutionary significance across yeast species?

  To investigate evolutionary aspects of YLR164W:

  1. Comparative sequence analysis:

     • Identify homologs across yeast species using BLAST searches

     • Perform multiple sequence alignments to identify conserved residues

     • Generate phylogenetic trees to visualize evolutionary relationships

  2. Structure-function predictions:

     • Use tools like PSIPRED for secondary structure predictions

     • Generate 3D structural models based on homology to known structures

     • Identify conserved functional domains and motifs

  3. Cross-species functional complementation:

     • Express YLR164W homologs from different species in S. cerevisiae ylr164wΔ strains

     • Test functional rescue of phenotypes

     • Create chimeric proteins with domains from different species

  4. Comparative biochemical analysis:

     • Express and purify homologous proteins from different species

     • Compare biochemical properties and interaction partners

     • Analyze functional differences and similarities

  This approach is supported by successful sequence analysis methods: "Sequence alignments of Pch2 from indicated species, including PSIPRED secondary structure predictions. Boxes indicate key AAA+ features, * indicates generated truncation boundaries. Sequence alignments were generated using MAFFT/L-INS-i. Secondary structure predictions were made using PSIPRED. Alignments were visualized using Jalview."

• What transcriptomic approaches are most effective for studying YLR164W expression regulation?

  To investigate the regulation of YLR164W expression:

  1. Global expression analysis:

     • Perform RNA-Seq or microarray analysis under different conditions

     • Compare YLR164W expression patterns with other genes in related pathways

     • Identify co-regulated gene clusters

  2. Promoter analysis:

     • Identify transcription factor binding sites in the YLR164W promoter

     • Perform ChIP-qPCR to confirm binding of transcription factors

     • Create reporter constructs with wild-type and mutated promoter elements

  3. 3' UTR analysis:

     • Search for conserved octa-nucleotide sequences in the 3' untranslated region (250 nt)

     • Use regulatory sequence analysis tools to identify potential motifs

     • Calculate E-values to assess motif significance

  4. Epigenetic regulation:

     • Investigate chromatin state at the YLR164W locus

     • Analyze histone modifications associated with active/inactive transcription

     • Assess the impact of chromatin modifiers on YLR164W expression

  This approach is supported by established methods: "A search for conserved octa-nucleotide sequences in the 3′ untranslated region (250 nt) was performed using regulatory sequence analysis tools. The occurrence of the discovered motif in the group of genes tested (163 genes) was compared with the expected occurrence of a group of same size randomly picked. The E-value represents the probability of finding the number of patterns with the same level of overrepresentation."

• What statistical approaches are most appropriate for analyzing experimental data related to YLR164W function?

  Selecting appropriate statistical methods is crucial for robust data analysis:

  Method	Application	Minimum Sample Size	Reference
  RIAA (Real Value Iterative Adaptive Approach)	Time-series data analysis, spectral estimation	9 (p<0.05), 14 (p<0.005)
  Lomb-Scargle Periodogram	Non-uniformly sampled time-series data	12 (p<0.05), 19 (p<0.005)
  LSPR (Lomb-Scargle Periodogram Regression)	Combined approach for periodicity detection	12 (p<0.05), 19 (p<0.005)
  Robust Capon Method	Handling corrupted biological data	22 (p<0.05), 26 (p<0.005)
  MAPES Method	Missing data compensation in time-series	20 (p<0.05), 26 (p<0.005)
  Hypergeometric distribution test	Functional category enrichment	Dependent on dataset size

  For expression analysis, proper normalization is essential: "Applying the same formula, the probability was calculated where N is the total number of genes where the TF can bind upstream, M is the total number of genes in the cluster, and G is the total number of gene features on the YG98S array (6383)."

• How can I design a comprehensive experimental approach to characterize YLR164W's role in mitochondrial function?

  As a putative mitochondrial protein, YLR164W may play important roles in mitochondrial processes:

  1. Subcellular localization studies:

     • Create YLR164W-GFP fusions to visualize localization

     • Perform mitochondrial fractionation followed by western blot analysis

     • Use MitoTracker dyes for co-localization experiments

  2. Respiratory function analysis:

     • Compare oxygen consumption rates between wild-type and YLR164W mutant strains

     • Measure growth on fermentable (glucose) vs. non-fermentable (glycerol, ethanol) carbon sources

     • Analyze sensitivity to respiratory inhibitors

  3. Mitochondrial membrane potential:

     • Use potential-sensitive dyes (e.g., TMRM, JC-1) to assess membrane potential

     • Compare potential differences between wild-type and mutant strains

     • Test effects of oxidative stress on membrane potential

  4. Interaction with other mitochondrial proteins:

     • Perform proteomics on purified mitochondria from WT and YLR164W mutants

     • Identify altered protein complexes or abundances

     • Validate changes through co-immunoprecipitation and western blotting

  5. Experimental design considerations:

     • Use between-subjects design comparing different strains

     • Control extraneous variables such as growth phase and media composition

     • Include appropriate controls (wild-type, known mitochondrial mutants)

     • Plan for replicate experiments to ensure statistical validity

  This comprehensive approach will provide insights into YLR164W's role in mitochondrial function while adhering to proper experimental design principles described in the scientific literature .