Recombinant Gracilaria tenuistipitata var. liui Probable 30S ribosomal protein 3, chloroplastic (ycf65)

What is Gracilaria tenuistipitata var. liui and why is it important in scientific research?

Gracilaria tenuistipitata var. liui is a variety of red algae (Rhodophyta) that was first described for specimens cultured in a pond in Haikou, Hainan Island, China. It is morphologically characterized by percurrent axes bearing numerous, delicate, and short to long flagelliform lateral branchlets . This species is significant in scientific research due to its economic importance in agar production, its adaptation to brackish water environments, and its value as a model organism for genetic and phylogenetic studies. G. tenuistipitata was the first red algae to have its complete chloroplast genome published, making it a crucial reference organism for comparative genomic studies .

What is the chloroplastic 30S ribosomal protein 3 (ycf65) and what is its function?

The chloroplastic 30S ribosomal protein 3, encoded by the ycf65 gene, is a component of the small subunit of the chloroplast ribosome in Gracilaria tenuistipitata. This protein plays a crucial role in translation within the chloroplast, contributing to the assembly and function of the ribosomal complex that synthesizes proteins encoded by the chloroplast genome. The "ycf" designation stands for "hypothetical chloroplast open reading frame," indicating that its function was initially predicted based on sequence homology rather than direct experimental evidence.

How does the ycf65 gene in G. tenuistipitata var. liui compare with similar genes in other red algae?

The ycf65 gene in G. tenuistipitata var. liui is part of the chloroplast genome, which has been completely sequenced. Comparative analyses have shown that G. tenuistipitata shares its most recent common ancestor with the South American G. chilensis . The conservation of chloroplast genes, including ycf65, varies across red algal lineages. While specific information about ycf65 conservation is not provided in the search results, genetic studies have shown that G. tenuistipitata has relatively low nucleotide diversity (π = 0.00243 ± 0.00020) but moderate haplotype diversity (Hd = 0.725 ± 0.030) , which may impact the conservation of genes like ycf65.

What are the optimal protocols for isolating chloroplast DNA from G. tenuistipitata var. liui?

For isolating chloroplast DNA from G. tenuistipitata var. liui, researchers should follow these methodological steps:

• Sample collection and preparation:

  • Collect fresh algal material and clean thoroughly to remove epiphytes

  • Store in silica gel for DNA preservation if immediate extraction is not possible

  • Fragment the cleaned specimens for efficient DNA extraction

• DNA extraction protocol:

  • Use a modified CTAB (cetyltrimethylammonium bromide) method optimized for red algae

  • Include β-mercaptoethanol in the extraction buffer to prevent oxidation of polyphenols

  • Perform RNase treatment to remove RNA contamination

  • Use multiple chloroform:isoamyl alcohol (24:1) extractions to remove proteins and polysaccharides

  • Precipitate DNA with cold isopropanol and wash with 70% ethanol

  • Resuspend in TE buffer or nuclease-free water

• Chloroplast DNA enrichment:

  • Perform CsCl density gradient centrifugation to separate chloroplast DNA

  • Alternatively, use long-range PCR with chloroplast-specific primers to amplify regions of interest

Quality control should include spectrophotometric analysis (A260/A280 ratio) and gel electrophoresis to confirm DNA integrity.

What expression systems are most effective for producing recombinant ycf65 protein?

For recombinant expression of the chloroplastic 30S ribosomal protein 3 (ycf65) from G. tenuistipitata var. liui, the following expression systems have proven effective:

• Bacterial expression systems:

  • E. coli BL21(DE3) with pET vector systems offers high yield but may require codon optimization

  • Cold-inducible systems (e.g., pCold vectors) can improve folding of chloroplast proteins

  • Fusion tags such as His6, MBP, or SUMO can enhance solubility and facilitate purification

• Eukaryotic expression systems:

  • Yeast systems (Pichia pastoris) may provide better post-translational modifications

  • Cell-free expression systems can overcome toxicity issues sometimes encountered with ribosomal proteins

Expression optimization parameters:

What are the recommended methods for purifying recombinant ycf65 protein while maintaining its structural integrity?

Purification of recombinant ycf65 protein requires careful consideration of its structural properties. The following methodological approach is recommended:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Use mild elution conditions with imidazole gradient (50-250 mM)

  • Include reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation

• Intermediate purification:

  • Ion exchange chromatography based on predicted pI of the protein

  • Size exclusion chromatography to separate monomeric from aggregated forms

• Final polishing:

  • Heparin affinity chromatography (if the protein has nucleic acid binding properties)

  • Hydroxyapatite chromatography for removal of endotoxins

• Buffer optimization:

  • Maintain pH between 7.0-8.0

  • Include 10-15% glycerol to enhance stability

  • Consider adding low concentrations of arginine (50-100 mM) to prevent aggregation

• Quality assessment:

  • SDS-PAGE for purity analysis

  • Circular dichroism spectroscopy for secondary structure verification

  • Dynamic light scattering for homogeneity assessment

How should researchers analyze sequence variations in the ycf65 gene across different populations of G. tenuistipitata?

For analyzing sequence variations in the ycf65 gene across different populations of G. tenuistipitata, researchers should implement the following analytical framework:

• Sequence alignment and quality control:

  • Use multiple sequence alignment tools (MUSCLE, MAFFT)

  • Trim low-quality regions and check for sequencing errors

  • Verify open reading frames and annotation

• Population genetic analyses:

  • Calculate nucleotide diversity (π) and haplotype diversity (Hd) as done for other genes in G. tenuistipitata

  • Perform neutrality tests (Tajima's D and Fu's Fs) to detect selection signatures

  • Calculate FST values to assess population differentiation

• Phylogenetic analyses:

  • Construct maximum likelihood and Bayesian inference trees

  • Perform bootstrap analyses (1,000+ replicates) to assess branch support

  • Consider including outgroups such as G. chilensis

• Geographic distribution analysis:

  • Map haplotype distribution patterns similar to those observed for other genes in G. tenuistipitata

  • Perform spatial analysis of molecular variance (SAMOVA)

  • Apply Bayesian skyline plot analysis to infer demographic history

• Statistical validation:

  • Use appropriate software such as Arlequin, BEAST, or DnaSP as employed in previous studies of G. tenuistipitata

  • Apply mismatch distribution analysis with sum of squared deviations (SSD) and Harpending's raggedness index (HRag) to test population expansion hypotheses

What statistical approaches are most appropriate for analyzing structural variations in recombinant ycf65 protein?

When analyzing structural variations in recombinant ycf65 protein, researchers should employ these statistical approaches:

• Structural alignment and comparison:

  • RMSD (Root Mean Square Deviation) calculation for comparing backbone conformations

  • TM-score for measuring global fold similarity

  • DALI Z-scores for structural homology detection

• Variation analysis:

  • Principal Component Analysis (PCA) to identify major modes of structural variation

  • Normal Mode Analysis (NMA) to characterize intrinsic flexibility

  • Clustering algorithms (hierarchical, k-means) to identify conformational states

• Statistical validation:

  • Bootstrap analysis for phylogenetic structural trees

  • Cross-validation for predictive structural models

  • PROCHECK or MolProbity for stereochemical quality assessment

• Visualization and interpretation:

  • Distance-based methods for conformational space mapping

  • Heat maps for residue-specific variation analysis

  • Network analysis for correlated motions

• Integration with sequence data:

  • Structure-based sequence alignment

  • dN/dS ratio analysis mapped onto structural elements

  • Statistical coupling analysis for coevolution detection

How does the genetic variability of the ycf65 gene correlate with environmental adaptations in different populations of G. tenuistipitata var. liui?

• Adaptation to brackish environments:
  G. tenuistipitata is a euryhaline and eurythermal species with wide tolerance ranges for salinity, temperature, and heavy metals . While specific information about ycf65 is not provided in the search results, chloroplast genes are often under selection pressure in different environmental conditions. Researchers should:

  • Compare ycf65 sequences from populations in different salinity gradients (measured using refractometers as mentioned in the methodology )

  • Correlate nonsynonymous substitutions with environmental parameters

  • Perform selection analyses to identify adaptive signatures

• Geographic patterns:
  The search results indicate three distinct haplogroups in G. tenuistipitata (northern Chinese, central Vietnamese, and southern in Malaysia, Singapore, and Thailand) . For ycf65:

  • Analyze whether similar geographic patterns exist specifically for this gene

  • Determine if genetic structuring correlates with ecological regions

  • Assess if divergence times align with geological events

• Demographic history:
  G. tenuistipitata shows evidence of population expansion in the middle Pleistocene without bottlenecks . For ycf65:

  • Determine if selection on this gene correlates with population expansion events

  • Analyze whether functional constraints on ribosomal proteins show different patterns than other genes

  • Assess if ycf65 mutations show temporal clustering corresponding to environmental changes

• Functional implications:

  • Predict structural differences in the protein that may affect ribosomal function

  • Correlate nonsynonymous substitutions with differences in chloroplast efficiency

  • Assess if variations correlate with photosynthetic performance in different environments

What are the functional implications of post-translational modifications in ycf65 protein and how can they be experimentally validated?

Post-translational modifications (PTMs) of the ycf65 protein may significantly impact its function in chloroplast translation. To understand and validate these modifications:

• Prediction and identification of PTMs:

  • Phosphorylation sites can be predicted using tools like NetPhos and PhosphoSite

  • Methylation, acetylation, and other modifications can be predicted using specialized algorithms

  • Mass spectrometry (LC-MS/MS) analysis should be performed on purified native protein to identify actual PTMs

• Functional implications:

  • Phosphorylation often regulates protein-protein interactions in ribosomal complexes

  • Methylation may affect RNA binding properties

  • Acetylation could influence protein stability and turnover

  • PTMs may regulate ribosome assembly under stress conditions

• Experimental validation approaches:

  PTM Type	Detection Method	Functional Validation
  Phosphorylation	Pro-Q Diamond staining, Phos-tag SDS-PAGE	Site-directed mutagenesis (S/T→A, S/T→D/E)
  Methylation	Antibody detection, MALDI-TOF	Methyltransferase inhibition assays
  Acetylation	Anti-acetyl lysine antibodies	HDAC inhibitors, K→R mutations
  Ubiquitination	Western blot with anti-Ub antibodies	Proteasome inhibition studies

• Structural and functional assessment:

  • Circular dichroism to detect structural changes upon modification

  • Fluorescence anisotropy to measure RNA binding affinity changes

  • In vitro translation assays to measure functional impact

  • Cryo-EM to visualize structural integration in ribosomal complex

• Systems biology approach:

  • Correlation of PTM patterns with environmental stressors

  • Network analysis of PTM crosstalk

  • Integration with transcriptomic and proteomic data sets

How can researchers effectively use ycf65 as a molecular marker for phylogenetic studies of red algae?

To effectively use ycf65 as a molecular marker for phylogenetic studies of red algae, researchers should follow these methodological approaches:

• Marker evaluation:

  • Assess evolutionary rate of ycf65 compared to other chloroplast markers

  • Evaluate conservation level across diverse red algal lineages

  • Compare phylogenetic signal with established markers like COI-5P (used in G. tenuistipitata studies )

  • Determine appropriate taxonomic resolution level (species, genus, family)

• Primer design and optimization:

  • Design universal primers based on conserved regions flanking variable domains

  • Develop taxon-specific primers for difficult groups

  • Optimize PCR conditions for various taxonomic groups

  • Validate primers across diverse red algal lineages

• Phylogenetic analysis workflow:

  • Compare tree topologies generated using maximum likelihood, Bayesian inference, and maximum parsimony

  • Assess node support using bootstrap values (>1,000 replicates) and posterior probabilities

  • Compare with multi-gene phylogenies to evaluate congruence

  • Implement partition models if combining with other markers

• Calibration and dating:

  • Use fossil records where available for node calibration

  • Apply relaxed molecular clock models with appropriate priors

  • Validate substitution rates against those established for red algae (e.g., 1.0 × 10^-9 substitutions site^-1 y^-1)

  • Compare divergence time estimates with geological events

• Application examples:

  • Species delimitation in cryptic complexes

  • Biogeographic pattern reconstruction

  • Comparison with population-level markers for comprehensive evolutionary studies

  • Integration with morphological character evolution

What are the main challenges in expressing functional recombinant ycf65 protein and how can researchers overcome them?

Researchers face several challenges when expressing functional recombinant ycf65 protein from G. tenuistipitata var. liui. Here are the key challenges and methodological solutions:

• Codon usage bias:

  • Challenge: Algal chloroplast genes may contain codons rarely used in expression hosts

  • Solution: Optimize codons for the expression system while maintaining key structural elements

  • Validation: Compare expression levels and solubility between native and optimized sequences

• Protein solubility and folding:

  • Challenge: Chloroplast ribosomal proteins often aggregate when expressed heterologously

  • Solutions:

    • Use solubility-enhancing fusion partners (SUMO, MBP, TrxA)

    • Express at lower temperatures (16-18°C)

    • Include molecular chaperones (GroEL/ES) as co-expression partners

    • Test various induction conditions (0.1-0.5 mM IPTG)

• Structural integrity:

  • Challenge: Maintaining native conformation without ribosomal RNA partners

  • Solutions:

    • Co-express with interacting RNA or protein partners

    • Include stabilizing agents (glycerol, arginine, low concentrations of detergents)

    • Use circular dichroism to verify secondary structure elements

• Functional validation:

  • Challenge: Assessing whether recombinant protein retains native function

  • Solutions:

    • Develop in vitro translation assays with chloroplast components

    • Measure RNA binding affinity using fluorescence anisotropy

    • Perform complementation assays in model systems

• Post-translational modifications:

  • Challenge: Bacterial expression systems lack eukaryotic PTM machinery

  • Solutions:

    • Use eukaryotic expression systems for certain modifications

    • Employ enzymatic methods for in vitro modification

    • Create phosphomimetic mutants (S/T→D/E) when studying phosphorylation

How can researchers resolve contradictory phylogenetic signals between ycf65 and other molecular markers in red algal studies?

When researchers encounter contradictory phylogenetic signals between ycf65 and other molecular markers in red algal studies, the following methodological approaches can help resolve these discrepancies:

• Evaluate marker characteristics:

  • Compare evolutionary rates between markers

  • Assess saturation levels using transition/transversion plots

  • Test for compositional biases that may affect phylogenetic reconstruction

  • Evaluate the impact of alignment ambiguities on tree topology

• Apply appropriate analytical methods:

  • Use partitioned models that allow different evolutionary parameters for each marker

  • Implement mixture models that accommodate heterogeneous evolutionary processes

  • Apply site-heterogeneous models (CAT, CAT-GTR) for better handling of homoplasy

  • Test alternative tree topologies using approximately unbiased (AU) tests

• Consider biological explanations:

  • Investigate potential horizontal gene transfer events

  • Assess incomplete lineage sorting using coalescent-based methods

  • Examine the possibility of hybridization or introgression

  • Consider heteroplasmy in chloroplast genomes

• Integrate multiple lines of evidence:

  • Combine molecular data with ultrastructural and biochemical characters

  • Implement total evidence approaches using both molecular and morphological data

  • Use species tree methods that accommodate gene tree discordance

  • Apply network-based phylogenetic methods to visualize conflicting signals

• Methodological validation:

  • Perform simulation studies to test method performance under various scenarios

  • Use posterior predictive approaches to assess model adequacy

  • Apply cross-validation techniques to evaluate model fit

  • Implement sensitivity analyses with varying taxon sampling

What are the most effective approaches for studying the interaction of ycf65 with ribosomal RNA and other ribosomal proteins in the chloroplast?

To effectively study the interactions between ycf65 and other components of the chloroplast ribosome, researchers should employ these methodological approaches:

• Structural biology techniques:

  • Cryo-electron microscopy of reconstituted ribosomal subunits

  • X-ray crystallography of protein-RNA complexes

  • NMR spectroscopy for dynamic interaction studies

  • Small-angle X-ray scattering (SAXS) for low-resolution complex structure

• Biochemical interaction analyses:

  • RNA electrophoretic mobility shift assays (REMSA)

  • Filter binding assays for quantitative RNA-protein interaction measurement

  • Surface plasmon resonance (SPR) for binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

• In vivo interaction studies:

  • RNA immunoprecipitation (RIP) to identify bound RNA sequences

  • Cross-linking and immunoprecipitation (CLIP) for precise binding site mapping

  • Fluorescence resonance energy transfer (FRET) for interaction dynamics

  • Bimolecular fluorescence complementation (BiFC) for protein-protein interactions

• Computational approaches:

  • Molecular dynamics simulations of protein-RNA complexes

  • Coevolution analysis to identify interacting residues

  • Homology modeling based on bacterial ribosomal structures

  • Docking simulations to predict binding interfaces

• Functional validation:

  • Site-directed mutagenesis of predicted interface residues

  • In vitro reconstitution of minimal functional complexes

  • Chimeric protein analysis to map functional domains

  • Complementation assays in model systems