Recombinant Neophocaena phocaenoides Sex-determining region Y protein (SRY)

What is the SRY gene and what role does it play in Neophocaena phocaenoides?

The SRY (Sex-determining Region on the Y chromosome) gene is a critical genetic factor responsible for male sex determination in mammals, including the finless porpoise Neophocaena phocaenoides. This gene functions by initiating a cascade of gene expression that leads to testis development and subsequent male phenotype expression. In Neophocaena phocaenoides and other cetaceans, the SRY gene exhibits a strict paternal mode of inheritance, making it particularly valuable for tracking paternal lineages in population studies . The gene encodes a testis-specific transcript and contains a DNA-binding motif that is present in nuclear high-mobility-group proteins (HMG1 and HMG2) . This conservation across mammals indicates its fundamental role in sex determination mechanisms.

What is the structural organization of the SRY gene in finless porpoise?

The SRY gene in Neophocaena phocaenoides, like in other cetaceans, consists of approximately 600 base pairs spanning from the initiation codon (ATG) to the stop codon (TAG). The gene contains three primary structural regions: the N-terminal region (approximately 156 bp), the highly conserved HMG (High Mobility Group) box (231 bp), and the C-terminal region (variable length between 216-261 bp) . The HMG box functions as a DNA binding motif and is the most conserved portion of the gene across cetacean species. Additionally, the 5' flanking region contains regulatory elements, including a "GGGGGCGG" sequence that represents the consensus motif for the Sp1 (Specificity Protein 1) transcription factor, which is likely involved in gene expression regulation .

How does the SRY protein structure relate to its function in sex determination?

The SRY protein's functional activity is primarily attributed to its HMG box domain, which consists of approximately 78 amino acids. This domain enables the protein to bind and bend DNA, facilitating the regulation of target genes involved in testis development. In cetaceans including Neophocaena phocaenoides, this HMG box is highly conserved, reflecting its critical role in protein function . The N-terminal and C-terminal regions show greater variability, with the C-terminal region in the superfamily Delphinoidea (which includes Neophocaena phocaenoides) exhibiting a frame-shift due to a base insertion at the end of the HMG box . This variation suggests different selective pressures act on different regions of the protein, with the HMG box under strong negative selection (dN/dS ratio of 0.15) while terminal regions experience relaxed selective pressures or potentially weak positive selection .

What are the best approaches for isolating and amplifying the SRY gene from Neophocaena phocaenoides samples?

For successful isolation and amplification of the SRY gene from Neophocaena phocaenoides samples, researchers should implement PCR-based methodologies using sex-specific primers. As demonstrated in previous studies with cetaceans, PCR amplification of the SRY gene yields a single band product in males but not in females, serving as a reliable sex identification method . When designing primers, researchers should target conserved regions flanking the SRY coding sequence, taking advantage of the gene's relatively small size (approximately 600 bp). For verification, amplified products should be sequenced and compared to known SRY sequences through database searches such as BLAST to confirm identity. Additionally, researchers should be aware that no polymorphism has been detected within species in previous cetacean studies, which may influence experimental design decisions when working with multiple individuals from the same population .

What expression systems are most effective for producing recombinant Neophocaena phocaenoides SRY protein?

While the search results don't specifically address expression systems for recombinant Neophocaena phocaenoides SRY protein, effective systems can be inferred from mammalian protein expression research. For functional studies of DNA-binding proteins like SRY, eukaryotic expression systems such as mammalian cell lines (HEK293, CHO) or insect cell systems (baculovirus) are recommended to ensure proper protein folding and post-translational modifications. Since the SRY protein contains the HMG box DNA binding domain that requires proper folding for functionality, bacterial expression systems might yield improperly folded proteins despite higher production yields. Expression constructs should include the complete coding sequence (approximately 600 bp) with optimization of codon usage for the selected expression system. Additionally, incorporating affinity tags (such as His-tag or GST) facilitates downstream purification while fusion with fluorescent proteins can enable localization studies in cellular contexts.

What methodological considerations are important when studying SRY binding interactions?

When investigating SRY binding interactions, researchers should consider several methodological approaches to characterize both DNA binding specificity and protein-protein interactions. For DNA binding studies, electrophoretic mobility shift assays (EMSA) can determine the binding affinity and specificity of recombinant SRY protein to target DNA sequences. Chromatin immunoprecipitation (ChIP) assays are valuable for identifying in vivo binding sites, though these require specific antibodies against Neophocaena phocaenoides SRY. For structural analysis of binding interactions, X-ray crystallography or NMR spectroscopy can elucidate the three-dimensional conformation of the HMG box when bound to DNA. The highly conserved nature of the HMG box (231 bp) in cetaceans suggests similar binding properties across species , which may allow researchers to extrapolate from studies of other mammalian SRY proteins. Additionally, the dN/dS ratio of 0.15 for the HMG box indicates strong negative selection , emphasizing the importance of maintaining the structural integrity of this domain for proper binding function.

How does Neophocaena phocaenoides SRY compare with SRY in other cetacean species?

Comparative analysis of the SRY gene across cetacean species reveals both conservation and divergence patterns that reflect evolutionary relationships. The HMG box region (231 bp) of the SRY gene is highly conserved among all cetacean species, including Neophocaena phocaenoides, indicating strong evolutionary constraints on this functional domain . In contrast, both the N-terminal (156 bp) and C-terminal regions (216-261 bp) show considerable variation, with the C-terminal region exhibiting heterogeneity in length and sequence composition. Notably, members of the superfamily Delphinoidea, which includes the finless porpoise, share a distinctive one-base insertion at the end of the HMG box that results in a frame-shift in the C-terminal region . This characteristic molecular feature supports the phylogenetic grouping of Delphinoidea species. Phylogenetic trees constructed using SRY sequences consistently support the monophyly of major cetacean taxonomic groups, with the topology aligning well with trees derived from other genetic markers such as mitochondrial DNA .

What insights does SRY gene analysis provide about the evolutionary history of Neophocaena phocaenoides?

Analysis of the SRY gene contributes valuable insights into the evolutionary history of Neophocaena phocaenoides within the context of cetacean evolution. The SRY gene's paternal inheritance pattern makes it particularly useful for tracing male-mediated gene flow and evolutionary relationships that may not be evident from maternally inherited markers like mitochondrial DNA. In the case of the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), molecular evidence from various genetic markers suggests a marine origin followed by adaptation to freshwater environments . The very low genetic diversity observed in the Yangtze population (nucleotide diversity 0.0011±0.0002) indicates a founder event in its evolutionary history , which could be further investigated through comparative analysis of SRY sequences between freshwater and marine populations. The frame-shift mutation in the C-terminal region shared among Delphinoidea members represents a synapomorphic character that supports the classification of Neophocaena within this superfamily and provides a timeline for when this mutation arose in cetacean evolution.

What does the selective pressure analysis of different SRY regions tell us about functional constraints in cetaceans?

Selective pressure analysis of the SRY gene reveals differential evolutionary constraints across its structural regions, providing insights into functional importance. The HMG box region exhibits a low dN/dS ratio of 0.15 across cetaceans, indicating strong purifying selection consistent with its critical role in DNA binding and sex determination . This value aligns with the average selective pressure observed in other functionally constrained genes, underscoring the evolutionary importance of maintaining HMG box structure. In contrast, both terminal regions show elevated dN/dS ratios: the N-terminal region in Delphinoidea has a ratio of 1.70, while the C-terminal region in Mysticeti reaches 2.83 . Although these values might suggest positive selection, statistical tests indicate these differences are not significant, pointing instead to relaxed selective pressure rather than adaptive evolution. The C-terminal region's substantial variation, particularly the frame-shift in Delphinoidea, suggests this region may have different or possibly reduced functional constraints compared to the HMG box . This pattern of region-specific selection provides a framework for understanding how evolutionary processes have shaped the SRY gene while maintaining its essential sex-determining function.

How can SRY be used as a genetic marker for population studies in Neophocaena phocaenoides?

The SRY gene serves as a valuable paternal lineage marker for population genetic studies in Neophocaena phocaenoides due to its Y-chromosome location and strict paternal inheritance pattern. Unlike mitochondrial DNA which traces maternal lineages, SRY analysis can reveal male-mediated gene flow and population structure that might otherwise remain undetected. This is particularly relevant for the Yangtze finless porpoise, which exhibits significant population genetic structure (FST = 0.44, P < 0.05; φST = 0.36, P < 0.05) based on mitochondrial markers . Researchers can use SRY sequence data to construct paternal phylogenies that complement maternal lineage studies, providing a more comprehensive understanding of population history and structure. The non-recombining nature of the Y chromosome region containing SRY makes it especially useful for tracking historical male dispersal patterns and detecting founder effects, which appear to be significant in the evolutionary history of the Yangtze finless porpoise .

What methodological approaches are most effective for using recombinant SRY in conservation research?

For conservation research involving recombinant SRY, several methodological approaches can be employed to address critical questions about Neophocaena phocaenoides populations. Developing recombinant SRY-based molecular assays can enable rapid and non-invasive sex identification from small tissue samples or environmental DNA, which is particularly valuable for monitoring endangered populations like the Yangtze finless porpoise. These assays can be implemented through PCR-based methods that yield sex-specific amplification patterns, as demonstrated in previous cetacean studies . Additionally, generating antibodies against recombinant SRY protein can facilitate immunohistochemical studies of reproductive development and potential reproductive abnormalities that might affect population viability. For assessing genetic diversity and population structure, next-generation sequencing of SRY and surrounding Y-chromosome regions can reveal finer-scale paternal lineage relationships that complement mitochondrial DNA findings . When interpreting results, researchers should consider the very low genetic diversity reported in the Yangtze finless porpoise (nucleotide diversity 0.0011±0.0002) , which may also be reflected in Y-chromosome markers.

What insights can SRY sequence analysis provide for the conservation of endangered Neophocaena phocaenoides populations?

SRY sequence analysis can provide crucial insights for conservation efforts targeting endangered Neophocaena phocaenoides populations, particularly the critically endangered Yangtze finless porpoise. By analyzing paternal lineages through SRY variations, researchers can assess male-mediated gene flow between isolated populations, identify genetically distinct management units, and evaluate the genetic consequences of population fragmentation. The statistically significant population genetic structure already documented using mitochondrial markers (FST = 0.44, P < 0.05) suggests that similar structure may exist in paternal lineages, potentially revealing finer population subdivisions relevant for conservation management. Additionally, SRY analysis can contribute to understanding the evolutionary significance of different populations by identifying unique paternal lineages that warrant special conservation attention. For captive breeding programs, SRY-based paternity analysis can help maintain genetic diversity and avoid inbreeding. The very low genetic diversity reported in the Yangtze finless porpoise raises concerns about genetic bottlenecks and inbreeding depression, making genetic management informed by both maternal and paternal markers essential for developing effective conservation strategies.

How can recombinant SRY protein be used to investigate transcriptional regulation in Neophocaena phocaenoides?

Recombinant SRY protein offers valuable tools for investigating transcriptional regulation mechanisms in Neophocaena phocaenoides. Researchers can employ chromatin immunoprecipitation followed by sequencing (ChIP-seq) using recombinant SRY antibodies to identify genome-wide binding sites and target genes regulated by this transcription factor. The presence of the "GGGGGCGG" consensus motif of Sp1 (Specificity Protein 1) in the predicted promoter region of the SRY gene itself suggests complex transcriptional regulation that can be further explored through reporter gene assays. Additionally, electrophoretic mobility shift assays (EMSAs) using recombinant SRY protein can characterize specific DNA-binding properties and identify potential cofactors that modulate its activity. The highly conserved HMG box domain (231 bp) should be the focus of binding studies, as this region mediates DNA interactions. For transcriptional activation studies, researchers can investigate whether the frame-shift in the C-terminal region observed in Delphinoidea, including Neophocaena phocaenoides , affects the protein's ability to recruit transcriptional machinery. These approaches collectively would provide insights into how SRY functions in the context of sex determination and potentially identify species-specific aspects of transcriptional regulation.

What experimental design would be most appropriate for studying the effects of SRY mutations on protein function?

To investigate the functional consequences of SRY mutations, researchers should implement a comprehensive experimental design combining in silico, in vitro, and when possible, ex vivo approaches. Initially, comparative sequence analysis should identify naturally occurring variations between different cetacean species, focusing on the N-terminal region where seven synonymous and 12 nonsynonymous substitutions have been detected, and the C-terminal region which shows heterogeneity in length (216–261 bp) . Using site-directed mutagenesis, researchers can then introduce specific mutations into recombinant expression constructs to generate variant SRY proteins. Functional characterization should include DNA binding assays (EMSAs) to assess whether mutations affect the protein's ability to recognize target sequences, with particular attention to mutations in the highly conserved HMG box region. Reporter gene assays in cell culture systems can evaluate transcriptional activation capabilities of mutant proteins compared to wild-type. Structural analyses using circular dichroism spectroscopy or more advanced techniques like X-ray crystallography can determine how mutations affect protein folding and stability. The differential dN/dS ratios observed across SRY regions (HMG box: 0.15, N-terminal in Delphinoidea: 1.70, C-terminal in Mysticeti: 2.83) provide guidance on which regions might tolerate mutations versus those likely to be functionally compromised when altered.

How might comparative studies between different porpoise species inform our understanding of SRY evolution and function?

Comparative studies between different porpoise species can provide significant insights into SRY evolution and function through multiple analytical approaches. By examining sequence variations across species with different evolutionary histories and ecological adaptations, researchers can identify correlations between molecular changes and functional adaptations. The frame-shift mutation in the C-terminal region observed in Delphinoidea represents a notable example of evolutionary change that could be investigated for functional consequences. Researchers should compare freshwater populations (like the Yangtze finless porpoise) with marine populations to determine whether habitat-specific selective pressures have influenced SRY evolution. Transcriptome analysis from testicular tissue across species could reveal differences in downstream gene regulation despite the conserved HMG box. The low genetic diversity observed in the Yangtze finless porpoise (nucleotide diversity 0.0011±0.0002) provides an opportunity to study how population bottlenecks and genetic drift affect functional genes under selection. Additionally, differences in mating systems and reproductive strategies among porpoise species may correlate with specific SRY variants, potentially revealing connections between molecular evolution and behavioral adaptations. These comparative approaches would extend beyond simple phylogenetic reconstruction to address fundamental questions about how a critical developmental gene evolves while maintaining its essential function.

What are the main challenges in expressing and purifying recombinant Neophocaena phocaenoides SRY protein?

The expression and purification of recombinant Neophocaena phocaenoides SRY protein presents several technical challenges that require specific methodological solutions. The DNA-binding nature of the SRY protein, conferred by its HMG box domain , can lead to toxicity issues in bacterial expression systems due to non-specific binding to host DNA. To address this, researchers should consider inducible expression systems with tight regulation or the use of eukaryotic expression systems that can better tolerate transcription factor expression. Solubility challenges may arise from the hydrophobic regions within the protein structure, particularly in the variable C-terminal region. This can be mitigated by expressing the protein as a fusion with solubility-enhancing tags such as MBP (maltose binding protein) or SUMO, followed by tag removal using specific proteases. The heterogeneity in the C-terminal region length (216–261 bp) must be considered when designing expression constructs, with researchers potentially needing to create multiple constructs to represent the natural variation. For purification, DNA contamination is a common issue with DNA-binding proteins; this can be addressed through high-salt washes during affinity chromatography steps and additional purification techniques such as ion exchange or size exclusion chromatography.

What approaches can resolve contradictory results when comparing SRY functional data across different analytical methods?

When contradictory results arise from different analytical methods examining SRY function, researchers should implement a systematic troubleshooting approach. First, examine the specific protein regions being tested in each assay, as the functional properties of the conserved HMG box (231 bp) likely differ from those of the more variable N-terminal and C-terminal regions . Consider whether the DNA binding properties observed in vitro through EMSAs accurately reflect the in vivo situation, which may require validation through chromatin immunoprecipitation or reporter gene assays in relevant cell types. Additionally, the frame-shift in the C-terminal region observed in Delphinoidea may cause functional differences that appear as contradictory results when comparing across species. Researchers should assess whether different post-translational modifications affect protein activity in different experimental systems; this is particularly relevant when comparing bacterial expression systems (lacking modifications) with eukaryotic systems. Statistical analysis should be applied to determine whether apparent contradictions reflect true biological differences or experimental variation. When conflicting results persist, consider employing orthogonal techniques that measure different aspects of the same function, or developing experimental conditions that more closely mimic the physiological environment in which SRY naturally functions.

How should researchers address the limited availability of Neophocaena phocaenoides samples for SRY research?

The endangered status of Neophocaena phocaenoides, particularly the Yangtze finless porpoise population , creates significant ethical and practical constraints on sample availability that researchers must address through alternative methodological approaches. Non-invasive sampling should be prioritized, including the collection of environmental DNA (eDNA) from water samples in habitats where these porpoises are present, which can be used for PCR-based detection of SRY sequences. When tissue samples are available, typically from stranded or deceased animals, researchers should implement nucleic acid extraction protocols optimized for small sample quantities and potentially degraded material. The development of highly sensitive PCR methods, including nested PCR or digital PCR approaches, can maximize data generation from limited samples. Collaborative research networks should be established to facilitate sample sharing between institutions, reducing the need for multiple sample collections from these endangered populations. Additionally, researchers can consider studying SRY from closely related species as models, justified by the relatively conserved nature of the gene across cetaceans, particularly in the functionally critical HMG box region . For functional studies requiring protein, recombinant expression based on the gene sequence remains the most ethical approach, eliminating the need for additional tissue samples once the sequence has been determined.