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Updated: May 2, 2026
A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters
Published on: June 13, 2019
Cloning and characterization of an apoptosis-related DNA fragmentation factor (DFF) from oyster, Crassostrea
Zhiming Xiang1, Fufa Qu2, Lin Qi3
1Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China.
Abstract:
Apoptosis plays an important pathophysiological role in the homeostasis of immune systems. DNA fragmentation factors (DFFs) have been shown to be essential for DNA fragmentation, and the resultant DNA fragments follow a laddering pattern during apoptosis in vertebrates. In invertebrates, the functions of the DFF orthologs are not well characterized; therefore, we cloned and characterized a bivalve DFFA ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChDFFA). The full-length cDNA of ChDFFA is 1186 bp in length and encodes a putative protein of 200 amino acids that contains an N-terminal CAD domain and a DFF-C domain at its C-terminus. Real-time RT-PCR results showed that ChDFFA is ubiquitously expressed in several tissues, and its highest expression is in gill. Following a 3- to 48-h challenge by microbial infection, the expression of ChDFFA increased in hemocytes. Using fluorescence microscopy, ChDFFA was localized in nuclei when exogenously expressed in HeLa cells. In addition, over-expression of ChDFFA inhibited the transcriptional activities of p53/p21-Luc reporter genes in HEK293T cells. These results suggest that ChDFFA may be involved in immune response reactions in the Hong Kong oyster C. hongkongensis.
Insights
Researchers identified a DNA fragmentation factor A (DFFA) ortholog in the Hong Kong oyster. This oyster DFFA is expressed in gills and hemocytes, suggesting its role in immune responses.
Area of Science:
- Molecular Biology
- Immunology
- Marine Biology
Background:
- Apoptosis is crucial for immune system homeostasis.
- DNA fragmentation factors (DFFs) are key in apoptosis-induced DNA fragmentation.
- The function of DFF orthologs in invertebrates remains largely uncharacterized.
Purpose of the Study:
- To clone and characterize a DFFA ortholog from the Hong Kong oyster (Crassostrea hongkongensis).
- To investigate the expression patterns and potential immune functions of this oyster DFFA ortholog.
Main Methods:
- Cloning of the full-length cDNA of ChDFFA.
- Real-time RT-PCR for gene expression analysis in various tissues and hemocytes after microbial challenge.
- Fluorescence microscopy to determine subcellular localization in HeLa cells.
- Reporter gene assays in HEK293T cells to assess functional impact.
Main Results:
- The ChDFFA gene encodes a 200-amino acid protein with conserved CAD and DFF-C domains.
- ChDFFA exhibits ubiquitous tissue expression, with highest levels in gills.
- Expression increased in hemocytes following microbial infection.
- ChDFFA localizes to the nucleus and inhibits p53/p21 transcriptional activity when overexpressed.
Conclusions:
- The Hong Kong oyster DFFA ortholog (ChDFFA) is identified and characterized.
- ChDFFA expression is modulated by microbial infection, particularly in hemocytes.
- Findings suggest ChDFFA plays a role in the immune response of Crassostrea hongkongensis.
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