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The Expression, Purification, and Characterization of a Ras Oncogene (Bras2) in Silkworm (Bombyx mori)
Zhengbing Lv1, Tao Wang, Wenhua Zhuang
1College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China ; Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Hangzhou 310018, China.
Abstract:
The Ras oncogene of silkworm pupae (Bras2) may belong to the Ras superfamily. It shares 77% of its amino acid identity with teratocarcinoma oncogene 21 (TC21) related ras viral oncogene homolog-2 (R-Ras2) and possesses an identical core effector region. The mRNA of Bombyx mori Bras2 has 1412 bp. The open reading frame contains 603 bp, which encodes 200 amino acid residues. This recombinant BmBras2 protein was subsequently used as an antigen to raise a rabbit polyclonal antibody. Western blotting and real-time PCR analyses showed that BmBras2 was expressed during four developmental stages. The BmBras2 expression level was the highest in the pupae and was low in other life cycle stages. BmBras2 was expressed in all eight tested tissues, and it was highly expressed in the head, intestine, and epidermis. Subcellular localization studies indicated that BmBras2 was predominantly localized in the nuclei of Bm5 cells, although cytoplasmic staining was also observed to a lesser extent. A cell proliferation assay showed that rBmBras2 could stimulate the proliferation of hepatoma cells. The higher BmBras2 expression levels in the pupal stage, tissue expression patterns, and a cell proliferation assay indicated that BmBras2 promotes cell division and proliferation, most likely by influencing cell signal transduction.
Insights
The silkworm Ras oncogene (Bras2) is similar to human Ras proteins and promotes cell proliferation. Its expression is highest in pupae and specific tissues, suggesting a role in cell division and signal transduction.
Area of Science:
- Molecular Biology
- Oncogene Research
- Insect Genetics
Background:
- The Ras superfamily of oncogenes plays critical roles in cellular signaling pathways.
- Understanding novel Ras homologs in invertebrates can provide insights into conserved biological functions.
- The silkworm Bombyx mori offers a model system for studying gene function in insects.
Purpose of the Study:
- To characterize the silkworm Ras oncogene (Bras2) and investigate its expression and function.
- To determine the potential role of BmBras2 in cell proliferation and signal transduction.
Main Methods:
- Sequence analysis and comparison with known Ras proteins.
- mRNA and protein expression analysis using real-time PCR and Western blotting.
- Subcellular localization studies and cell proliferation assays.
Main Results:
- Bombyx mori Bras2 (BmBras2) shares high amino acid identity with human R-Ras2 and has an identical core effector region.
- BmBras2 is expressed across developmental stages, with peak levels in pupae, and is abundant in head, intestine, and epidermis.
- BmBras2 localizes primarily to the nucleus and stimulates hepatoma cell proliferation.
Conclusions:
- BmBras2 is a functional Ras homolog in silkworms with a role in promoting cell division and proliferation.
- Its expression patterns suggest involvement in key developmental and physiological processes.
- BmBras2 likely influences cell signal transduction pathways, similar to other Ras proteins.
