Chloroplast nucleoids (ct-nucleoids) are DNA/protein complexes involved in compacting the chloroplast genome, and may play a role in regulating DNA replication. Ct-nucleoids were isolated from young soybean plants and separated by 2-D gel electrophoresis. Gel spots were excised and analyzed by MALDI-ToF mass spectrometry, resulting in several protein identifications. The proteins identified all have functions unrelated to DNA replication. While some of these proteins may be due to contamination, it is possible that some of these proteins are dual-functional, playing direct roles in the regulation of DNA replication.
A 28 kDa soybean protein was isolated by sequence specific DNA affinity chromatography from total chloroplast protein isolations. Mass spectrometry analysis revealed that the 28 kDa protein contains some homology within an ssb domain of an Arabidopsis mitochondrial-targeted SSB (mtSSB) of approximately 21 kDa. N-terminal sequencing revealed that the 28 kDa soy protein is identical to a 36 amino acid region at the N-terminus of the Arabidopsis mtSSB. Protein fractions containing the 28 kDa protein shift oriA in electrophoretic mobility shift assays (EMSAs). Arabidopsis mtSSB fails to shift oriA in EMSAs run under identical conditions. Arabidopsis mtSSB causes a shift of ssDNA in EMSAs, while the ability of the 28 kDa soy protein to bind ssDNA is still unclear. Importantly, the 28 kDa soy protein was identified from total protein extracts obtained from intact chloroplasts, while in-vitro targeting experiments suggest that the Arabidopsis mtSSB localizes only to mitochondria and not to chloroplasts. BLAST searches of the available soybean genomic and EST databases do not produce any significant homologies to the 36 amino acid N-terminal sequence.
College and Department
Life Sciences; Microbiology and Molecular Biology
BYU ScholarsArchive Citation
Lassen, Matthew G., "Identification of Proteins Involved in Chloroplast DNA Replication" (2004). Theses and Dissertations. 221.
DNA, replication, chloroplast, plant, protein